- Email: [email protected]
Workshop 7 Leucocyte - virus interactions
Workshop 7 Leucocyte - Virus Interactions HOLGER KIRCHNER and DOUGLAS MCGREGOR
Department of Immunobiology, Sandoz Forschungsinstitut, Vienna, Austria
Complementation of X-linked gene functions regulating virus-induced natural killer cell responses in inbred mice D. ARMERDING
Males and females of the inbred mouse strains SWR/J, AKRlJ and SJLlJ were shown to be low or non-responders with respect to herpes simplex virus type 2 (HSV2) induced NK cell functions. FI hybrids exhibited sex-associated phenotypes: Females generated significantly higher NK cell responses than males. The females were also more resistant to i.p. infection with HSV2 than males. Intercrosses of PI hybrids between AKR and SJL mice resulted in the expected frequency of about 50 % high respectively low NK responders fulfilling the hypothesis of complementing X-linked genes regulating this potential resistance function. Part of the F2 male population (26.2 %), however, also exhibited significant virus-induced NK activities which may reflect the expression of autosomal gene(s) compensating the X-chromosome associated gene defect(s). The observed phenotypes in the F2 generation segregated independently of gene loci of the major histocompatibility complex. The possible relevants of NK cell functions for resistance against HSV infections in the mouse model will be discussed.
Institute for Virus Research, German Cancer Research Center, Heidelberg, FRG
Replication of herpes simplex virus (HSV) in human lymphocytes: identification of the cellular subset replicating the virus R. BRAUN, H. TEUTE, H. KIRCHNER, and K. MUNK The ability of HSV to persist in human ganglia and its neurotropism are well recognized properties of the virus. Nevertheless, the successful isolation of HSV from the leucocytes of acutely infected patients suggests also lymphotropic properties of this virus. Although the in vitro replication of HSV in human lymphocytes appears to be restricted to T cells, not more
15th International Leucocyte Culture Conference, Asilomar· 223 than 1 % of a mitogen stimulated T cell culture is replicating the virus, as assessed by infectious center assays (ICA). Thus, it cannot be excluded that virus replication might also occur in contaminating cells of other than T cell origin. In our studies we have therefore tried to identify the virus replicating lymphocyte subset by various experimental approaches using monoclonal antibodies for identification and preparation of highly enriched lymphocyte subsets. For performance of these experiments panning procedures, complement mediated mass cytolysis, indirect rosetting procedures and affinity chromatography were used. Lymphocyte subpopulations obtained by these methods were shown to have a purity of more than 92 % as assessed by indirect immunofluorescence; depleted populations on the other hand showed no detectable contamination with the subpopulation removed. Infection of these cultures with HSV revealed, that the virus did not replicate in purified populations of B cells, null cells or monocytes, but in helper and suppressor T cells. Nevertheless again only a small percentage of these cells replicated HSV as shown by ICA. Investigations using indirect double immunofluorescence suggested, that virus replication occurred in the Ia positive subset of T lymphocytes. These date were confirmed by further experiments, in which the Ia positive cells were removed from infected cells. In such cultures no virus replication could be observed. Moreover, ICA's with Ia positive and Ia negative T cells showed that a high percentage of Ia positive cells replicated the virus, whereas no replicating cells were found per 103 Ia negative cells. In conclusion, our data show, that the replication of HSV in human lymphocytes is observed both in activated helper and suppressor cells, but among these only in those that express Ia antigen on the surface. The replication of HSV in T cells expressing the products of the immune response region on their surface may have important biological implications.
Departments of Medicine and Veterinary Medicine, Ohio State University, Columbus, Ohio, USA
Suppression of human T lymphocyte proliferation by a structural protein of feline leukemia virus E. COPELAN,
J. RINEHART, M.
LEWIS, L. MATHES, R. OLSEN, and A. SAGONE
A 15,000 dalton envelope protein (pIS) of the oncogenic c-type feline leukemia virus (FeLV) is immunosuppressive in the cat. The proliferative response of human as well as feline peripheral blood mononuclear cells (PBMC) to mitogens and antigens is impaired by pIS. In order to define the mechanism by which FeLV is immunosuppressive, the effects of ultraviolet inactivated FeLV (UV FeLV) and pIS on the PBMC subpopulations responsible for optimal proliferation were studied. The Concanavalin A (Con A) induced proliferation of human PBMC from normal volunteers was impaired by UV FeLV. UV FeLV did not impair monocyte (mo) function since it did not effect mo production of Interleukin 1 (ILl) or impair mo helper cell function. Rather UV FeL V and pIS acted directly on lymphocytes. ILl induced proliferation of 105 purfied T cells activated by Con A was inhibited more than 90 % by 40 Ilg of UV FeLV or 4 Ilg pIS. Moreover, UV FeLV and pIS did not promote suppressor cell formation or cause T cell death. Interleukin 2 (IL 2) production by activated T cells was diminished by more than 90 % and T cell proliferation to IL 2 was decreased by more than 50 % in the presence of UV FeLV. The following are concluded: 1. UV FeLV markedly impaires the proliferative response of human PBMC to Con A; 2. UV FeL V does not interfere with mo helper cell function; 3. IL 1 stimulated proliferation of activated T cells is inhibited by UV FeLV secondary to impaired IL 2 production and decreased IL 2 response of T cells; 4. the anti-proliferative effects of UV Fe LV are mediated by its pIS envelope protein.
224 . 15th International Leucocyte Culture Conference, Asilomar Cancer Research Center. Tufts University School of Medicine, Boston MA 02111, USA
Thymic epithelium is programmed to induce preleukemic changes in retrovirus expression and thymocyte differentiation in leukemia susceptible mice S. K. DATTA,
J. A. NICKLAS, C. C. ZIELINSKI, S. D. WAKSAL, and J. M. COFFIN
In the leukemia-prone AKR thymus, ecotropic and xenotropic-related viruses are expressed which generate leukemogenic recombinant viruses before the onset of leukemia. We have previously shown that (AKRXNZB)Ft hybrid mice do not develop leukemia because they severely restrict the expression of these retroviruses in their thymuses. The thymic microenvironment of the (AKRXNZB)Ft mice appeared to be of particular importance in determining this restriction, which was specified by an NZB-derived genetic influence (1, 2). In the present study we have analyzed reciprocal thymus graft and irradiation bone marrow chimeras to establish that this influence is exerted by thymic reticulo-epithelial cells. Prospective studies with thymic epithelial grafts from young mice have shown that the AKR thymic epithelium can simultaneously induce the amplified expression of retroviral genes, leukemogenic recombinant viruses and changes in patterns of thymocyte differentiation that precede the development of leukemia, whereas the (AKR X NZB) F t thymic epithelium is deficient in this regard. 1. DATTA, S. K., and R. S. SCHWARTZ, 1978. J. Exp. Med. 148: 329. 2. DATTA, S. K., S. D. WAKSAL, and R. S. SCHWARTZ. 1980. Cell 19: 171. Supported by U.S.P.H.S. Grant No. CA-24950 and ACS FRA No. 180 to S.K.D.
Cancer Research Center, Tufts University School of Medicine, Boston MA 02111, USA
Thymic epithelial genotype influences the steps in recombination of viral genomes that lead to leukemogenic retrovirus production in the thymus S. K. DATTA, C. Y. THOMAS,
J. A. NICKLAS, and J. M. COFFIN
The genomes of leukemogenic recombinant (polytropic) viruses that are expressed in the thymus prior to the onset of leukemia, differ from their non-leukemogenic ecotropic precursor viruses in three regions: the 5' portion of gp70 and 3' end of P15E genes that encode viral envelope proteins and the U3 non-coding region which forms the 3' end of the provirus long terminal repeat (LTR). The recombinational events in these three regions may occur independently (1). Here, we analyzed the genomes of retroviruses expressed by thymocytes from irradiated thymic epithelial graft (TE) and bone marrow chimeras by the Tt-oligonucleotide fingerprinting technique. When prothymocytes of leukemia-resistant (AKR X NZB)F t(ANF 1) mice differentiated in syngeneic ANF, TE grafts, they rarely expressed retroviruses (2) and these viruses had the nucleotide maps of non-leukemogenic eco- and xenotropic viruses. When the ANF\ thymocytes differentiated in the TE of another low-leukemia histocompatible strain, C57BR, they expressed ecotropic viruses which on fingerprint analysis were shown to have undergone recombinational changes only in the 5' portion of the gp70 gene. Only when the ANFI thymocytes had differentiated in the TE of leukemia-susceptible AKR mice did the recombinational changes involving all the three regions of the viral genome evolve, to produce
15th International Leucocyte Culture Conference, Asilomar' 225 leukemogenic retroviruses. The ANF j thymocytes also underwent the preleukemic changes in thymocyte differentiation patterns and leukemic transformation only when they differentiated in the young AKR TE grafts. 1. THOMAS, C. Y., and J. M. COFFIN, 1982, J. VIROL. 43, August (in press). 2. DATTA, S. K., S. D. WAKSAL, and R. S. SCHWARTZ, 1980, Cell 19: 171. Supported by U.S.P.H.S. Grant No. CA-24950 and ACS FRA No. 180 to S.K.D.
Department of Medical Microbiology, Military Medical Academy, 90-647 L6di, Poland
Host resistance in influenza virus infected mice A. DENYS,
J.
BIALEK, and A. GRZYBOWSKI
The results of analysis of Cell Mediated Immune (CMI) responses in influenza virus infected mice are reported. Virus antigen instead of living virus was used. CMI is mediated by the activity of thymus-derived lymphocytes and is expressed by the release of lymphokines or by lysis of virus infected target cells. Inhibition of PHA responses in lymphocytes during influenza virus infection and a slight stimulation in the presence of antigen were observed. Leukocytes inhibition against envelope influenza antigen reached the peak value on 6th day and next it declined by 14th day. The inhibition of the antibacterial activity of peritoneal exsudate cells was noticed after 14 days, in the presence of antigen. The results obtained support the hypothesis that T effector cells are induced in the course of influenza virus infection. Depression of granulocytes phagocytic activity is dependent on the circulation of antigen-antibody complexes. Intranasal inoculation of influenza viruses in the mice and a correspondingly <
Dept. of Pathology, UCLA School of Medicine, Los Angeles, CA. 90024, USA
Diminished T cell response to influenza virus in aged mice RITA B. EFFROS and Roy L. WALFORD
It is well established that numerous immune functions decline with age, and furthermore, that infectious agents are a major cause of morbidity and mortality in the elderly. Therefore, we initiated a study of the T lymphocyte response to influenza virus in aged mice in an effort to dissect the factors contributing to the diminished capacity of aged individuals to deal with viral infections. The primary cytotoxic T cell (CTL) response in two long-lived strains (C57BLl6 and Balb/c) immunized intraperitoneally with influenza was examined on day 5, which is the peak of the response in young adult mice. We demonstrate that the response of the 20-24 months old mice is only 20 % of that obtained from 4 months old animals. An examination of the kinetics of the response revealed that in old mice the maximal response is
226 . 15th International Leucocyte Culture Conference, Asilomar shifted to the day 7-10 timepoint, and even at this point is 50 % of the magnitude seen at the peak of the young response. In order to eliminate the possibility of in vivo suppressive mechanisms being responsible for the delay and decrease in the response, we compared the in vitro generation of a secondary influenza CTL response by old and young memory spleens. Here too, the response of the old mice was diminished, exhibiting only 60 % of the killing observed in the response by young spleens. Our data demonstrate a decreased capacity in aged mice to mount a T cell response to influenza virus. Experiments in progress are analyzing whether the old mice show a decrease in absolute frequency of precursor cells or a diminished ability to perform the cytotoxic function. Supported by USPHS Research Grant AG00790.
Centro de Biologia Molecular. CSIC-UAM. Canto Blanco. Madrid-34, Spain
Strategy of African swine fever virus (ASFV) to avoid the immune response (IR) L. ENJUANES, I. CASAL, A. SANZ, B. G. BARRENO, and E. VINUELA
ASFV is an icosahedral cytoplasmic deoxyvirus with envelope, which infects swine, inducing on IR unable to eliminate the virus. ASFV may use at least two strategies to avoid the host IR: infection of cells involved in the IR and antigenic variation. ASFV infected most of the porcine macrophages and a small percentage of granulocytes. The susceptibility to the virus was species-specific and its replication could be inhibited (> 90 %) by stimulating the PBL with PWM. In contrast neither B or T porcine lymphocytes, resting or stimulated with mitogens, were infected by ASFV. Specific hyperimmune antisera neutralized only partially (60 %) the infectivity of ASFV. The addition of complement to ASFV-antibody complexes increased the neutralization of this envelope-containing virus to 90 %. But neither this procedure nor the use of F (ab'h fragments of anti-ASFV antibodies abrogated the infectivity of ASFV-antibody complexes. The lack of a complete neutralization of ASFV could be related to the antigenic variability of ASFV, because, during replication, the virus changes virulence, the capacity to induce hemad sorption and its genome undergoes deletions closed to the ends of the double stranded DNA. To study the degree of antigenic variability we have produced a collection of 65 monoclonal antibodies reacting against the viral proteins. Using a selected panel of these monoclonal antibodies specific for internal (vp156, VP73, VP12) and external (VP32) proteins, we have shown that ASFV clones isolated from different geographical areas are antigenic ally different.
The Wistar Institute of Anatomy and Biology, Philadelphia, USA
B cell regulated expression of endogenous retroviral antigens D. L. EWERT and M. S. HALPERN
B cells of some inbred lines of chickens have been shown to express antigen that is cross reactive with endogenous retroviral envelope glycoprotein. We have isolated this antigen
15th International Leucocyte Culture Conference, Asilomar· 227 and shown that it is structurally homologous to envelope glycoprotein of the Avian Leukosis viruses, RAV -0. Our analysis suggests that the viral envelope glycoprotein exists both as four chain and as two chain structures of equimolar gp85 and gp37 subunits on the cell surface. As assayed by immunofluorescence, most, if not all, plasma cells obtained either from live animals or from cultures of mitogen stimulated lymphocytes express detectable levels of viral envelope glycoprotein. The majority of the plasma cell-associated envelope glycoprotein is present on the cell surface though it is not bound to surface Ig or Ia antigens. Although envelope antigen was not detectable by immunofluorescence on the surface of bursal cells or peripheral B lymphocytes, immunochemical assays established that retroviral envelope glycoprotein is present on the surface of the bursal cells but at much lower levels (> 20 times) than on the surface of the plasma cells. Both bursal and plasma cells synthesize envelope glycoprotein. These findings indicate that an increased level of expression of endogenous retroviral envelope glycoprotein is a concomitant of the differentiation of B lymphocytes to plasma cells in some chickens.
New England Regional Primate Research Center - Harvard Medical School and Massachusetts General Hospital - Harvard Medical School, USA
Long-term propagation of marmoset T -lymphocytes in T-cell growth factor: infection with T -lymphotropic herpesviruses and surface membrane markers L. A. FALK, D. SILVA, R. BYINGTON, and R. SCHOOLEY
Marmoset monkeys (Saguinus and Callithrix sp.) are highly susceptible to leukemia/ lymphoma induction after experimental inoculation with Herpesvirus saimiri (HVS) or Herpesvirus ateles (HVA), T -lymphotropic viruses of squirrel and spider monkeys respectively. To examine more closely virus-T-lymphocyte interactions in vitro, T-lymphocytes from cotton-topped marmosets (Saguinus oedipus) have been propagated continuously with conditioned medium containing T-cell growth factor (TCGF) and infected with HVS or HVA. Viral-specified antigens were detected by immunofluorescence tests in the infected cells within 24 hours postinfection (PI) and at all subsequent test intervals up to 8 months PI. The infected T -lymphocytes were dependent upon TCGF for growth up to 5-7 months PI when 3 of 5 HVS-infected cultures could be propagated without TCGF. Three of 3 HVA-infected cultures were TCGF dependent for only 2 months PI at which time they grew independently of TCGF. The continued presence of HVS or HVA in TCGF-dependent/independent cultures was demonstrated by: 1. presence of viral-specified antigens in about 1-5 % of cells, 2. ability of cells to induce infectious centers on monolayers of permissive cells and 3. production of small amounts of cell-free virus present in culture fluids. Reactivity with monoclonal antibodies to human T-lymphocyte subsets (5 % reactivity taken as significant): HVS-infected, 4/5 OKT4+ (8-33%) 3/5 OKT11a+ (54-89%) and 2/5 OKMl+ (6-49%); these values were comparable to noninfected, marmoset, TCGF-dependent T-lymphocytes. There was a positive correlation with OKTlla+ and ability to form E-rosettes (79-100% E+ cells). These studies show that marmoset T -lymphocytes can be propagated longterm using TCGF and are susceptible target cells for transformation with HVS/HVA; infection/transformation of the Tlymphocytes with HVS/HVA appears to make them independent of TCGF. These studies were supported in part by NIH grants: RROOI68-20, ROI CA26225-03, and AI 17057-02.
228 . 15th International Leucocyte Culture Conference, Asilomar Neuroimmunology Branch, National Institute of Neurological and Communicative Diseases and Stroke, Bethesda, MD, 20205, USA
Cellular regulation of the human proliferative response to measles, mumps, and vaccinia viruses
J.
I. GREENSTEI:-<, E. S. MI NG IOLl, and H . F. McFARLAND
Previous studies of the proliferative response of peripheral blood lymphocytes to measles, mumps, and vaccinia viral-infected monolayers in normal sero-positive adults, measured by thymidine incorporation, demonstrated that only 5 % of individuals respond significantly to measles virus (Stimulation Index [5.1.] > 5), by comparison with the universal responses to mumps (mean 5.1. = 10.6) and vaccinia viruses (mean 5.1. = 12.5). T-cells are responsible for the response in each case. Responsiveness to measles virus is greater during early convalescence from acute infection, but the level of proliferation declines over a period of weeks. Mixing autologous cells from early and late times after infection did not demonstrate suppression as a cause. The decline of responsiveness after acute measles and the low level of proliferation in late convalescence appear, therefore, to represent a relative lack of circulating long-term responder T -cells. To investigate further the cellular basis for the different responses, highly purified ( > 94 % ) OKT4 + and OKT8 + populations from late convalescents were obtained by affinity plating, and studied individually or mixed; with 20 % plastic adherent cells added to the cultures. The responses to mumps and vaccinia viruses, and to measles virus in the small number of responder individuals, occurred primarily in the OKT4 + population ; with generally lower responses to measles than to mumps and vaccinia viruses. Low levels of OKT8+ proliferation occurred to all three viruses, and were only modestly elevated by the addition of Interleukin-2. Mixing the OKT4 + and OKT8 + populations failed to demonstrate suppression of the response to each virus. These findings demonstrate that OKT4 + cells are the predominant cells which proliferate to each of these viruses, but that the responses to measles are consistently lower in normal individuals. Antigen specific T-cell responses to measles virus are found after infection, but the expansion of a long-term OKT4 + responder population appears not to occur. The mechanism involved may relate to the short-term suppressive effects of this virus on the immune response to other antigens.
Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel
Modulation of the splenic antibody response following infection of mice with lactic dehydrogenase virus N . ISAKOV and S. SEGAL
Lactic dehydrogenase virus (LDV) is a silent, non life-threatening virus which produces a stable infection in mice with a continuously high titer of infective virus in the blood. Acute infection with LDV is followed by histological changes in the lymphatic organs and causes alterations of both humoral and cellular immune responses. We found that immunization of mice with SRBC following acute LDV infection resulted in an augmented splenic response, expressed by increased numbers of IgM-and IgG-antibody-producing cells (APC). The response was preferentially directed at the production of IgG 2-APC. To test whether LDV
15th International Leucocyte Culture Conference, Asilomar . 229 specifically activate splenic APC, we performed in vitro sensitization of spleen cells to SRBC. We found that presence of LDV in the spleen cell cultures failed to affect the APC response. To eliminate the possibility that the result is due to lack of splenic environmental components from the dispersed suspension of cells, we performed a similar experiment using spleen fragments in the in vitro sensitization phase. Yet, again, LDV did not affect the APC response. We then tested whether LDV affects trapping and sequestration of lymphoid cells in the spleen. Staining of spleen cells from acute LDV -infected mice with fluoresceinated antisera directed to mouse Ig or to the Thy 1,2 antigen indicated a slight increase in the percentage of B lymphocytes. Responses of the splenic lymphocytes cells from LDV-infected mice to B cell or T cell-specific mitogens, were not significantly different from those of LDV-free spleen cells; however, the responses to PHA were significantly increased on the fourth day following LDV infection, while at the same time the responses to Con A were decreased. These results and results from in vivo experiments which demonastrated increased trapping of radio labeled lymphocytes into the spleen support the assumption that LDV affects splenic responses by changing the trapping and sequestration of lymphoid cells in the spleen and by modifying the relative proportions of distinct T cell subpopulations in this organ.
Institute of Virus Research, German Cancer Research Center, Heidelberg, FRG and "Immunology Department, Sandoz Research Institute, Vienna, Austria
Role of endogenously produced interferon in genetically-determined resistance of mice against infection with herpes simplex virus (HSV) H. KIRCHNER, H. ENGLER, D. ARMERDING", and R.
ZAWATZKY
Resistance of mice against primary infection with HSV is genetically controlled by at least two genes that are not H2-linked. Previous studies of the laboratories participating in this study have shown that both Natural Killer (NK) cells and interferon (IFN) may playa role in resistance, but the relationship between IFN and NK cells has been poorly understood. In the experiments of the present study, HSV was injected intraperitoneally (i.p.) and IFN was assayed directly in the wash-out fluid, whereas NK cell activiry was determined in the washedout peritoneal cell population (PEC). All mouse strains resistant or medium-resistant to HSV showed high and brisk IFN responses in vivo at the infection site, which was absent in susceptible mouse strains and in newborn mice of the resistant strain C57BLl6 (newborn mice of all strains are highly susceptible to HSV infection). Most resistant mouse strains also showed high NK cell responses which may - or may not - be a secondary consequence of previous IFN production. However, there were at least two notable exceptions SJL/2 mice that are defective in their NK cell response have a high IFN response (and are resistant) and SI M mice that have a very high NK cell response, are poor producer of IFN (and are susceptible). Thus, there is an absolute correlation between the magnitude of the early local IFN response and resistance, whereas the correlation between resistance and NK cell activation is less stringent. The HSV-induced NK cells shared all rypical properties of NK cells and they were lysed by treatment with antisera against Qa 5 and by the anti-asialo GM1 (kindly provided by Ko OKUMURA). Experiments were carried out in vitro to determine the nature of the cell in the PEC population that responded to HSV with IFN production. This cell shared all the properties of macrophages and had nothing in common with NK cells. In conclusion, our studies show that in primary resistance against HSV endogenously produced IFN seems to playa critical role. This role is further supported by experiments in which exogenous IFN was administered with protective effect against HSV and by experiments in which resistance was broken by anti-IFN serum (kindly provided by ION GRESSER).
230 . 15th International Leucocyte Culture Conference, Asilomar Central Lab. Blood Transfusion Service, PO Box 9190, 1006 AD Amsterdam, The Netherlands
Induction of measles-specific cytotoxic T cells, natural killer cells and interferon in human peripheral blood lymphocytes in vitro
c. J.
LUCAS
For eradiation of virus-infected cells by cytotoxic mechanism,·tour mechanisms have been described: T cell mediated cytotoxicity, antibody dependent ~ell mediated cytotoxicity, natural cytotoxicity and macrophage mediated cytotoxicity. The relative contributions of these are still unknown. From mouse studies it is known that immunization with a virus leads to the generation of at least four types of T lymphocytes. Delayed-type hypersensitivity-mediating T cells, cytotoxic T cells (CTL), T helper cells and T suppressor cells. In addition a cellular immune response in volves induction of mediators as interferon and IL-2. It is unclear if and how all these mechanisms are interrelated. In humans it is possible to detect cytotoxic T cells, and perhaps T helper cells, with a given viral specificity. We were able to increase a relatively low number of measles virus responders by repeated stimulation in vitro. The addition of IL-2 in vitro to measles virus stimulated cultures did not result in increased sensitivity. By repeated stimulation of PBL from 9 out of 36 healthy individuals measles virus specific CTL could be generated. By the same procedure 5 out of 5 multiple sclerosis patients responded. In measles virus and influenza virus stimulated cultures as well as in cultures stimulated by allogeneic lymphocytes a high level of natural killing is generated. These natural killer (NK) cells do not lyse autologous virus infected lymphocytes. In most of the cultures significant levels of interferon are formed. The titre of interferon, the level of NK and the measles self specific CTL activity do not appear to be related which suggests that all have their own regulatory mechanisms. Interferon levels were also analysed in relation to proliferation. There was no correlation unless IL-2 was present in which case it appeared that interferon induction occurred without exception.
Irvington House Institute, Department of Pathology, New York University Medical Center, New York, New York 10016, USA
Evidence for a major cluster of lymphocyte differentiation antigens on murine chromosome 2. (Mapping of Ly-ll/Ly-6/viral integration/ radiation leukemia) D. MERUELO, M. OFFER, and A. ROSSOMANDO The region of chromosome 2 between H-3 and H-13 has been previously shown by others to contain loci coding for a variety of alloantigens among which are H-3, H-13, Ly-4, Lym-II, ~2-microglobulin and Pgp-l. We report herein that lymphocyte determinants H-30, Ly-6 and Ly-ll are also coded for by genes in the vicinity of H-3, and therefore that this region of chromosome 2 contains the tightly linked loci coding for Ala-I, DAG, H9/2S, Ly-8, and ThB. The most important question raised by these findings is whether the clustering of these loci has any special significance. Relevant to this issue is the finding that several virus susceptibility loci map in this section of chromosome 2. RiI-l, Av-l, and RLViI-l, loci involved in susceptibility to fractionated irradiation-, Abelson virus-, and radiation leukemia virus-induced leukemia,
15th International Leucocyte Culture Conference, Asilomar' 231 respectively map in this region. The Abelson virus related cellular oncogene, c-abl, also maps in chromosome 2. It may be speculated that this segment of chromosome plays a special role in viral infections and the multitude of lymphocyte differentiation loci found therein are related to this special relationship to viruses or viral infection. In particular, it has been suggested that the genetic expression or regulation of the genes of different viruses might be dependent on cellular control elements that are involved in tissue differentiation and which exert their effect by a site-specific or cis acting mechanism. We shall present further data consistent with this hypothesis. Supported in part by NIH Grant CA 22247, ACS Grant IM-163, a grant from the National Leukemia Association and a grant from the Irma T. Hirschi Foundation. D. MERUELO is a Leukemia Society of America Scholar.
University of South Florida College of Medicine, Tampa, FL, USA
Leukemia virus induced suppression of natural killer cell activity D.
J.
MOODY, ST. SPECTER, M. BENDINELLI, and H. FRIEDMAN
Leukocytes in culture or in vivo are altered in their normal immunologic functions after leukemia virus infection. Incubation of normal murine spleen cultures with Friend leukemia virus (FLV) markedly suppresses normal immune capabilities, including antibody formation and cell mediated immune responses. Recent studies in this laboratory have demonstrated that the natural killer (NK) cell activity is also depressed following infection of mice with FLV. The NK activity in FLV infected mice progressively decreases such that by day 18 post infection there was essentially no response from the splenic population. In vitro stimulation of FLV infected splenocytes with interferon was capable of partially reversing the depressed NK activity. Target binding cell analysis of FLV splenocytes indicate that they were as capable of binding to target cells as the cells from uninfected animals. FLV splenocytes were capable of suppressing the NK activity of uninfected animals when the two groups were mixed together. This suppressive ability of FLV splenocytes was partially abrogated after passing the FLV splenocytes over a nylon wool column. These experiments suggest the involvement of nylon wool adherent suppressor cells in the NK depression. Thus, it appears likely that leukemia virus infection besides altering antibody formation and cell mediated immunity, also has an effect on natural «immuno-surveillance» activity mediated by cytolytic NK cells. The implications of these observations in regard to the mechanism of viral oncogenesis is apparent.
"The Cleveland Clinic Foundation, Cleveland, Ohio, and +National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA
Autoaggressive thymocytes in preleukemic Balb/c MuLV-carrier mice M. R. PROFFITT", C. DE LA MOTTE", M.
J.
CAULFIELD", and C. KOZAK+
We have previously reported that C3H/He mice persistently infected with Moloney leukemia virus (MuLV-M-carriers) develop autoreactive thymocytes prior to the onset of
232 . 15th International Leucocyte Culture Conference, Asilomar leukemia. We have extended these observations to BALB/c mice, demonstrating that thymocytes from carrier mice are cytotoxic to syngeneic and to allogeneic fibroblast targets. Conversely, when these targets are infected with MuLV-M, they are spared from the cytotoxicity. Xenogeneic cells are also unaffected. In vivo, the preleukemic thymocytes exhibit a graft vs. host-like reaction as measured in two assays. 1. These cells cause a «runting» syndrome with characteristic pathology when injected into newborn, syngeneic mice, and 2. popliteal lymph node enlargement when they are injected into the footpad of irradiated syngeneic adult mice. When a large number of MuLV-carrier mice were examined for autoreactive thymocytes, it became apparent that the degree of cytotoxicity was correlated with the number of virus-producing cells present in the thymus; however, free MuLV had no effect on the autoreactivity in vitro. In further studies we have examined the role of ecotropic MuLV receptors and MuLV -M gp 70 antigens in these phenomena. Thus, we looked at the cytotoxicity and MuLV-carrier thymyocytes against hybrid cell lines prepared between BALB/c (receptor-positive) and hamster (receptor-negative) fibroblasts. The results indicate that only the hybrid cells that have the receptors are lysed by the carrier thymocytes. Furthermore, the cytotoxicity is blocked in the presence of monoclonal antibodies to epitopes on MuL V-M gp70 indicating a role for gp70 antigens in the autoreactive phenomenon. The results suggest that the cytotoxicity is mediated, in part, by an interaction between MuLV gp70 antigens on the surface of the thymocytes and MuLV-receptors on the target fibroblasts. Virus-infected target fibroblasts may be spared because their ecotropic MuLV-receptors are blocked by autogenously produced MuLV. Supported NCI grant CA20242.
University of Southern California, Los Angeles, USA
Division and migration of hemopoietic sources in mouse leukemia (Friend) L. L.
RIFE
and G. MATIOU
Normal and leukemic growth and differentiation are processes regulated by fields (microenvironments) set up by source cells producing a stimulus for stem cell (S.c.) self renewal. When infected by leukemic viruses, the sources increase stimulus production to a critical level which triggers a «leukemic response » by the dependent S.c. However, if the sources are irradiated at 950 rads prior to infections, their efficiency for stimulus production remains normal even after exposure to a massive dose of leukemogen (e.g., Friend virus). These facts allow one to investigate, within the same animal, the effects of unirradiated marrow districts upon the progression of leukemia at certain other locations (e.g., spleen) irradiated at 950 rads prior to virus injection. S.c. kinetic studies show that leukemic progression in the irradiated spleen is proportional to the volume of unirradiated marrow. In addition, a further reduction of spleen leukemic growth is obtained when the previously unirradiated (femoral) marrow is irradiated at 950 rads. In summary, these experiments detect an «abscopal>, effect on tumoral growth. Such effect is occasionally observed during radiotherapy of cancerous patients, when the local irradiation of a tumoral site results in the reduction of tumoral masses growing in distant locations. The data are discussed in terms of cell-cell interactions and synergetic mechanisms
15th International Leucocyte Culture Conference, Asilomar' 233 operating in differentiating malignancies. It appears that leukemogens induce division of hyperactive sources which stimulate S.c. into a leukemic phase after migrating into separate hemopoietic districts.
University of Pittsburgh, Pittsburgh, PA, USA
Cellular immunity during human cytomegalovirus infection C. RINALDO, M. Ho, W. HAMOUDT, X. GU!, R. DEBlASIO Cytomegalovirus (CMV) infection of normally healthy adults can result in a selflimiting mononucleosis syndrome, and may predispose male homosexuals to severe superinfections. During CMV mononucleosis there is a depression of mitogen (concanavalin A - ConAl induced lymphocyte blastogenesis and interferon production that has previously been related to enhanced suppressor activity of monocytes and increased levels of T suppressor cells. In the .present study, freshly donated blood mononuclear leukocytes (MNL) from acute phase CMV mononucleosis patients had depressed blastogenesis eH-Tdr uptake) and interferon production in response to ConA and viral antigens (CMV, herpes simplex virus, mumps virus). Preculturing of patients' MNL for 18 hr at 37°C prior to treatment led to loss of suppressor activity and enhanced blastogenesis to ConA, in contrast to even more depressed reactivity to antigens. Studies were done to define the role of immunoregulatory cells in this effect. Removal of plastic-adherent cells (monocytes) from freshly donated or precultured MNL led to greater depression of blastogenesis to ConA or antigens. Monoclonal antibody analysis showed an increased percentage of total T (Leu 1 +) and T suppressor (Leu 2a +) cells, with a low T helper (Leu 3a+) to Leu 2a+ cell ratio during CMV mononucleosis as compared with normal donors. Preculturing of patients' MNL resulted in selective decrease in Leu 2a + cells, higher Leu 3a/Leu 2a ratios, and appearance of an unusual, third T cell subset (Leu 1 +, 2a -, 3a-). Baseline and interferon-boosted NK cell activity versus K562 cell targets during CMV mononucleosis did not differ from that of normal donors. Similarly, a longitudinal study of a male homosexual with P. carinii pneumonia and CMV viremia showed progressively depressed blastogenesis and enhanced suppressor cell activity with variable NK cell responses. Interferon was detected in serum, but was poorly inducible in MNL in vitro. Thus certain cellular immune functions are depressed during CMV infection in correlation with increased suppressor activity, whereas others appear functional and may aid resolution of illness.
Department of Pathology, McMaster University, Hamilton, Ontario, Canada, and University of Zurich, Switzerland
Viral persistence in cloned natural killer cells K. 1. ROSENTHAL, H. HENGARTNER, and R. M. ZINKERNAGEL
In order to better understand how virus infection alters immune functions, we have investigated the interaction of various viruses with cloned subpopulations of effector lymphocytes in vitro. We have established cloned lines of interleukin-2 (IL-2) dependent cytotoxic T cells (CTL) specific for vesicular stomatitis virus (VSV). Lysis by these cells is H-2 restricted, virus-specific and no natural killer (NK) cell activity is detectable. We also have clones of NK cells which proliferate in IL-2 containing medium and lyse YAC and other NK-sensitive target
234 . 15th International Leucocyte Culture Conference, Asilomar cells very efficiently. Results indicate that clones of H-2 restricted, VSV-specific CTL are resistant to productive infection by a variety of viruses, including vaccinia, lymphocytic choriomeningitis virus (LCMV), and VSV. In contrast, cloned lines of NK cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral antigen and produce infectious VSV. Persistently infected NK cells showed no marked alteration of cell morphology and continued to function as normal NK cells, i.e., capable of killing NK-sensitive targets. Furthermore, these persistently infected NK cells were resistent to lysis by virus-specific CTL. Thus, these cells may harbor persisting virus and avoid cell-mediated immune destruction in an immunocompetent host. Supported by MRC, Canada, and SNSF, Switzerland
Laboratory of Tumor Cell Biology, NCI, Bethesda, Maryland 20205, USA
Infection and in vitro transformation of fresh human cord blood and adult peripheral blood T -lymphocytes by isolates of human T-cell leukemia-lymphoma virus (HTL V) S. Z. SALAHUDDlN, P. D. MARKHAM, M. POPOVIC, P. SARIN, and R. C. GALLO Evidence suggesting the involvement of HTLV in the development of some types of human T-cell lymphoma has been previously reported. Proteins and nucleic acids related to HTLV structural components are detectable in neoplastic tissues but not in non-involved tissues from the same patient (GALLO et aI., Proc. Natl. Acad. Sci., in press). Serologic studies have suggested an association between HTLV and certain malignancies of mature T -cells (GALLO et aI., submitted). In addition, a significant proportion of relatives of patients with HTLVassociated disease possess HTLV-specific antibodies (ROBERT-GUROFF et al., submitted). HTLV has also been shown to be infectious for fresh human T -lymphocytes (RUSCETTI et aI., submitted; MARKHAM et al., in preparation). Here we confirm and extend observations on the biological activity of HTLV. We demonstrate that HTLV from TCGF-dependentT-lymphocyte cultures initiated from multiple leukemic donors in North Eastern U.S., Israel, the Caribbean, and Japan can productively infect and transform fresh human cord blood and adult T-lymphocytes. These experiments were done by cocultivation with lethally irradiated primary HTLV producer cells or exposure to cell-free virus. Virus passaged several times through cultured T-lymphocytes retain the infectious and transforming property. Cocultivation of fresh cord blood leukocytes with cell lines producing non-infectious HTLV results in the establishment of transformed, TCGF-independent T-lymphocytes in culture. These cells contain only limited amounts of viral nucleic acids and proteins.
UCLA School of Medicine, Los Angeles, CA. 90024, USA
Abnormal T cell subset response to herpes simplex virus antigens in patients with recurrent herpes genitalis C. A. SPINA, T. LARSON, and Y.
J.
BRYSON
Although cell-mediated immunity is indicated as a decisive influence in limiting the severity and duration of herpes simplex virus (HSV) infections in human disease, the involvement of
15th International Leucocyte Culture Conference, Asilomar' 235 specific immune cell components in this process remains largely unknown. A series of experiments were done to test the hypothesis that patients who are subject to severe clinical disease have selective alterations in the type of T cell subset which proliferates in response to specific HSV antigen challenge. Patients with the chronic, recurrent form of genital herpes infection comprised the test group, and disease free, HSV-seropositive donors served as controls. T cell subset analysis was determined using the «Leu» series of murine monoclonal antibodies: Leu-1 (pan T), Leu-2 (T suppressor/cytotoxic associated), Leu-3 (T helper/inducer associated). During episodes of active disease, patients tended to demonstrate an inverted Leu-3: Leu-2 cell ratio in their circulating T lymphocytes. To examine proliferative function, lymphocyte preparations were stimulated in vitro for 6 days with HSV type-specific antigens and candida antigen, and the levels of 3H-thymidine uptake measured. The levels of proliferative response in the patients, both with active and inactive disease, did not differ from those of the control group. However, when the type of T cell subset which was undergoing proliferation in response to these antigens was examined, dramatic differences were seen in the patients' response. The Leu-3: Leu-2 ratios of the large, responding blast cells cultured with HSV were significantly lower in the patient groups as compared to the controls, indicating a greater Leu-2 (Ts associated) cell response. The response to Candida, used as a control antigen, gave high ratios in the blast cells which reflected a normal, predominant Leu-3 (TH associated) cell response in both the patients and controls. These results indicate that patients with recurrent HSV disease have an abnormal T cell subset response which is selective for interaction with herpes virus. Supported in part by USPHS grant AI15332.
Univ. of California, San Diego, USA
In vitro analysis of immunosuppression in rabbits caused by the malignant variant of Shope fibroma virus D. S. STRAYER, ST. SELL, G. F. CABIRAC, EILEEN SKALETSKY, PATRICIA SHARP, and LEIBOWITZ
J.
L.
A malignant variant (MV) strain of a rabbit tumor virus, the Shope fibroma virus (SFV), causes profound immunosuppression both in vivo and in vitro. MV and SFV are antigenically identical, as judged by serological cross-neutralization and in vivo cross-protection. SFV produces a small local tumor which regresses without sequelae. In contrast, MV causes recipient rabbits to develop local tumors followed by extensive metastatic disease, complicated by fatal Gram-negative infection. Rabbits given SFV show normal or slightly increased in vitro blastogenic responses to Band T lymphocyte mitogens (anti-Ig and con A respectively). Lymphocytes from MV recipients respond poorly to these mitogens by 6 days after inoculation, which is before metastatic disease and bacterial infection have become clinically obvious. Specific blastogenic responses in vitro of lymphocytes from SFV recipients to infectious or ultraviolet (uv) light-inactivated SFV are vigorous. Such lymphocytes respond similarly to uvinactivated MV, but weakly or not at all to infectious MV. Lymphocytes from MV-exposed rabbits do not mount in vitro proliferative responses to any of these viral preparations though they produce considerable neutralizing antibody in vivo. In contrast, pharmacologic immunosuppression of rabbits with cortisone inhibits developing cellular and humoral immunity without altering the responsiveness to con A. MV is immunosuppressive in vitro as well. When infectious MV is added to cultures of normal spleen cells, proliferative responses to anti-Ig and con A are virtually abolished. The same suppression occurs when the malignant variant virus is added 24 or 48 hours after mitogen. SFV or inactivated MV added to similar cultures is without effect. MV induces this depression in immune responsiveness without
236 . 15th International Leucocyte Culture Conference, Asilomar decreasing lymphocyte viability significantly. MV grows in rabbit lymphocyte preparations in vitro and its viral antigens may be detected on lymphocyte membranes. Thus, we have isolated a new oncogenic virus which infects rabbits and causes severe dysfunction involving both B and T lymphocytes.
Dept. of Pathology & Laboratory Medicine, Univ. Nebraska Med. Cetr., Omaha, NE 68105, USA, "on leave from the Dept. of Tumor Biology, Karolinska Inst., Stockholm, Sweden
Establishment of permanent cultures from Epstein-Barr virus (EBV)nonsusceptible human lymphocytes D.
J.
VOLSKY, R. W. ANDERSON, C. KUSZYNSKI, and I. M. SHAPIRO'
Human B lymphocyte cultures can be established with remarkable efficiency by transforming cells with EBV in vitro. The majority of healthy individuals have EBV receptor-positive B lymphocytes that can serve as targets for EBV-mediated transformation. Unfortunately, in many cases where immortalized cell lines would be most helpful (e.g., Hodgkin's disease, chronic lymphocytic leukemia and preleukemia), attempts to transform lymphocytes by EBV were unsuccessful. We are currently characterizing lymphocyte defects in preleukemia which could account for the development of this disease (c.f., accompanying paper by ANDERSON et al.). Analysis of preleukemia B lymphocytes for surface markers by cytofluorography revealed a lack of EBV receptors. No EBV-determined antigens were observed after exposure of the cells to EBV. Recently, we have demonstrated that EBV receptor-negative cells can be infected by EBV, if viral DNA is introduced into the cytoplasm. The technique we employed is based on implanting functional EBV receptors onto membranes of receptor-negative cells, thereby converting them temporarily into receptor-positive targets. Fusogenic reconstituted Sendai virus envelopes are used as receptor carriers (VOLSKY et aI., PNAS 77:5453, 1980). When preleukemia lymphocytes were implanted with EBV receptors prior to exposure to the B95-8 substrain of EBV, EB virus nuclear antigen (EBNA) was detected in 2-5 % of cells 3-5 days after infection. Early (EA) and late (VCA) antigens were also detected although at lower levels than EBNA. Subsequent cultivation of the EBV -infected preleukemia lymphocytes resulted in the establishment of a new cell line, JV - t. JV -1 was heterogeneous with respect to the expression of several surface markers as assessed by cytofluorography: 99 % of cells were surface Ig-positive; 50 % were EBV receptor-positive, 10 % expressed complement c3d receptors, and no cells presented T cell markers. The data imply that JV -1 cells are of a B-cell lineage. With respect to their EBV status, all cells expressed EBNA, 4-5 % of cells expressed EA and less than 0.5 % were VCA-positive. The approach described above may be helpful in other cases where establishment of lymphoblastoid cell lines seems impossible.
FDA, Bethesda, MD, USA
Evidence that cytotoxic T lymphocytes not natural killer cells are responsible for resolution of influenza virus pneumonia in mice M. A. WELLS, S. DANIEL, S. C. KILEY ,
J. Y. DJEU, and
F. A. ENNIS
The mechanism of protection conferred by the passive transfer of immune spleen lymphocytes with enhanced cytotoxic T lymphocyte (CTL) activity on the course of influenza virus pneumonia in BALB/c athymic (T cell deficient) nude mice was investigated. Splenocytes
15th International Leucocyte Culture Conference, Asilomar· 237 taken directly from mice or after in vitro culture were assayed for CTL on virus infected BALBlc kidney cells, or for natural killer activity (NK) on sensitive YAC tumor cells, and then were transferred intravenously to nude mice 24 hours after lethal intranasal infection with AIPort Chalmers/1/73 influenza virus. The following populations of splenocytes were transferred: Virus Immunization Lymphocyte Primary Secondary % Lysis Virus ill-VIVO in-vitro Source CTL NK % Survival titer BALBlc
> 30 days > 30 days none 8 days
> 30 days none
5 days none none
100.0 7.8 neg.
24.6 10.0 6.6
72
< 0.5
18 15
4.0 4.0
NA NA NA
22.8 7.4 neg.
16.0 10.8 9.8
18 9 0
4.2 4.2 4.5
NA neg. 26.7 0 4 days 3.9 none NA neg. 5.2 0 4.4 Protection against death (% survival of 10-25 mice/group) and reduction in pulmonary virus titer (JOgIO of the 50 % egg infectious dose) when sampled on day 7 after infection, was only observed in mice receiving the secondary in vitro stimulated BALBlc splenocytes with high CTL activity. No association between viral clearance and NK activity was detected. These results indicate that CTL are associated with the recovery of mice with influenza pneumonia, and that NK activity probably does not contribute to this protection. Nude
Department of Immunobiology, Sandoz Forschungsinstitut, Vienna, Austria
Complementation of X-linked gene functions regulating virus-induced natural killer cell responses in inbred mice D. ARMERDING
Males and females of the inbred mouse strains SWR/J, AKRlJ and SJLlJ were shown to be low or non-responders with respect to herpes simplex virus type 2 (HSV2) induced NK cell functions. FI hybrids exhibited sex-associated phenotypes: Females generated significantly higher NK cell responses than males. The females were also more resistant to i.p. infection with HSV2 than males. Intercrosses of PI hybrids between AKR and SJL mice resulted in the expected frequency of about 50 % high respectively low NK responders fulfilling the hypothesis of complementing X-linked genes regulating this potential resistance function. Part of the F2 male population (26.2 %), however, also exhibited significant virus-induced NK activities which may reflect the expression of autosomal gene(s) compensating the X-chromosome associated gene defect(s). The observed phenotypes in the F2 generation segregated independently of gene loci of the major histocompatibility complex. The possible relevants of NK cell functions for resistance against HSV infections in the mouse model will be discussed.
Institute for Virus Research, German Cancer Research Center, Heidelberg, FRG
Replication of herpes simplex virus (HSV) in human lymphocytes: identification of the cellular subset replicating the virus R. BRAUN, H. TEUTE, H. KIRCHNER, and K. MUNK The ability of HSV to persist in human ganglia and its neurotropism are well recognized properties of the virus. Nevertheless, the successful isolation of HSV from the leucocytes of acutely infected patients suggests also lymphotropic properties of this virus. Although the in vitro replication of HSV in human lymphocytes appears to be restricted to T cells, not more
15th International Leucocyte Culture Conference, Asilomar· 223 than 1 % of a mitogen stimulated T cell culture is replicating the virus, as assessed by infectious center assays (ICA). Thus, it cannot be excluded that virus replication might also occur in contaminating cells of other than T cell origin. In our studies we have therefore tried to identify the virus replicating lymphocyte subset by various experimental approaches using monoclonal antibodies for identification and preparation of highly enriched lymphocyte subsets. For performance of these experiments panning procedures, complement mediated mass cytolysis, indirect rosetting procedures and affinity chromatography were used. Lymphocyte subpopulations obtained by these methods were shown to have a purity of more than 92 % as assessed by indirect immunofluorescence; depleted populations on the other hand showed no detectable contamination with the subpopulation removed. Infection of these cultures with HSV revealed, that the virus did not replicate in purified populations of B cells, null cells or monocytes, but in helper and suppressor T cells. Nevertheless again only a small percentage of these cells replicated HSV as shown by ICA. Investigations using indirect double immunofluorescence suggested, that virus replication occurred in the Ia positive subset of T lymphocytes. These date were confirmed by further experiments, in which the Ia positive cells were removed from infected cells. In such cultures no virus replication could be observed. Moreover, ICA's with Ia positive and Ia negative T cells showed that a high percentage of Ia positive cells replicated the virus, whereas no replicating cells were found per 103 Ia negative cells. In conclusion, our data show, that the replication of HSV in human lymphocytes is observed both in activated helper and suppressor cells, but among these only in those that express Ia antigen on the surface. The replication of HSV in T cells expressing the products of the immune response region on their surface may have important biological implications.
Departments of Medicine and Veterinary Medicine, Ohio State University, Columbus, Ohio, USA
Suppression of human T lymphocyte proliferation by a structural protein of feline leukemia virus E. COPELAN,
J. RINEHART, M.
LEWIS, L. MATHES, R. OLSEN, and A. SAGONE
A 15,000 dalton envelope protein (pIS) of the oncogenic c-type feline leukemia virus (FeLV) is immunosuppressive in the cat. The proliferative response of human as well as feline peripheral blood mononuclear cells (PBMC) to mitogens and antigens is impaired by pIS. In order to define the mechanism by which FeLV is immunosuppressive, the effects of ultraviolet inactivated FeLV (UV FeLV) and pIS on the PBMC subpopulations responsible for optimal proliferation were studied. The Concanavalin A (Con A) induced proliferation of human PBMC from normal volunteers was impaired by UV FeLV. UV FeLV did not impair monocyte (mo) function since it did not effect mo production of Interleukin 1 (ILl) or impair mo helper cell function. Rather UV FeL V and pIS acted directly on lymphocytes. ILl induced proliferation of 105 purfied T cells activated by Con A was inhibited more than 90 % by 40 Ilg of UV FeLV or 4 Ilg pIS. Moreover, UV FeLV and pIS did not promote suppressor cell formation or cause T cell death. Interleukin 2 (IL 2) production by activated T cells was diminished by more than 90 % and T cell proliferation to IL 2 was decreased by more than 50 % in the presence of UV FeLV. The following are concluded: 1. UV FeLV markedly impaires the proliferative response of human PBMC to Con A; 2. UV FeL V does not interfere with mo helper cell function; 3. IL 1 stimulated proliferation of activated T cells is inhibited by UV FeLV secondary to impaired IL 2 production and decreased IL 2 response of T cells; 4. the anti-proliferative effects of UV Fe LV are mediated by its pIS envelope protein.
224 . 15th International Leucocyte Culture Conference, Asilomar Cancer Research Center. Tufts University School of Medicine, Boston MA 02111, USA
Thymic epithelium is programmed to induce preleukemic changes in retrovirus expression and thymocyte differentiation in leukemia susceptible mice S. K. DATTA,
J. A. NICKLAS, C. C. ZIELINSKI, S. D. WAKSAL, and J. M. COFFIN
In the leukemia-prone AKR thymus, ecotropic and xenotropic-related viruses are expressed which generate leukemogenic recombinant viruses before the onset of leukemia. We have previously shown that (AKRXNZB)Ft hybrid mice do not develop leukemia because they severely restrict the expression of these retroviruses in their thymuses. The thymic microenvironment of the (AKRXNZB)Ft mice appeared to be of particular importance in determining this restriction, which was specified by an NZB-derived genetic influence (1, 2). In the present study we have analyzed reciprocal thymus graft and irradiation bone marrow chimeras to establish that this influence is exerted by thymic reticulo-epithelial cells. Prospective studies with thymic epithelial grafts from young mice have shown that the AKR thymic epithelium can simultaneously induce the amplified expression of retroviral genes, leukemogenic recombinant viruses and changes in patterns of thymocyte differentiation that precede the development of leukemia, whereas the (AKR X NZB) F t thymic epithelium is deficient in this regard. 1. DATTA, S. K., and R. S. SCHWARTZ, 1978. J. Exp. Med. 148: 329. 2. DATTA, S. K., S. D. WAKSAL, and R. S. SCHWARTZ. 1980. Cell 19: 171. Supported by U.S.P.H.S. Grant No. CA-24950 and ACS FRA No. 180 to S.K.D.
Cancer Research Center, Tufts University School of Medicine, Boston MA 02111, USA
Thymic epithelial genotype influences the steps in recombination of viral genomes that lead to leukemogenic retrovirus production in the thymus S. K. DATTA, C. Y. THOMAS,
J. A. NICKLAS, and J. M. COFFIN
The genomes of leukemogenic recombinant (polytropic) viruses that are expressed in the thymus prior to the onset of leukemia, differ from their non-leukemogenic ecotropic precursor viruses in three regions: the 5' portion of gp70 and 3' end of P15E genes that encode viral envelope proteins and the U3 non-coding region which forms the 3' end of the provirus long terminal repeat (LTR). The recombinational events in these three regions may occur independently (1). Here, we analyzed the genomes of retroviruses expressed by thymocytes from irradiated thymic epithelial graft (TE) and bone marrow chimeras by the Tt-oligonucleotide fingerprinting technique. When prothymocytes of leukemia-resistant (AKR X NZB)F t(ANF 1) mice differentiated in syngeneic ANF, TE grafts, they rarely expressed retroviruses (2) and these viruses had the nucleotide maps of non-leukemogenic eco- and xenotropic viruses. When the ANF\ thymocytes differentiated in the TE of another low-leukemia histocompatible strain, C57BR, they expressed ecotropic viruses which on fingerprint analysis were shown to have undergone recombinational changes only in the 5' portion of the gp70 gene. Only when the ANFI thymocytes had differentiated in the TE of leukemia-susceptible AKR mice did the recombinational changes involving all the three regions of the viral genome evolve, to produce
15th International Leucocyte Culture Conference, Asilomar' 225 leukemogenic retroviruses. The ANF j thymocytes also underwent the preleukemic changes in thymocyte differentiation patterns and leukemic transformation only when they differentiated in the young AKR TE grafts. 1. THOMAS, C. Y., and J. M. COFFIN, 1982, J. VIROL. 43, August (in press). 2. DATTA, S. K., S. D. WAKSAL, and R. S. SCHWARTZ, 1980, Cell 19: 171. Supported by U.S.P.H.S. Grant No. CA-24950 and ACS FRA No. 180 to S.K.D.
Department of Medical Microbiology, Military Medical Academy, 90-647 L6di, Poland
Host resistance in influenza virus infected mice A. DENYS,
J.
BIALEK, and A. GRZYBOWSKI
The results of analysis of Cell Mediated Immune (CMI) responses in influenza virus infected mice are reported. Virus antigen instead of living virus was used. CMI is mediated by the activity of thymus-derived lymphocytes and is expressed by the release of lymphokines or by lysis of virus infected target cells. Inhibition of PHA responses in lymphocytes during influenza virus infection and a slight stimulation in the presence of antigen were observed. Leukocytes inhibition against envelope influenza antigen reached the peak value on 6th day and next it declined by 14th day. The inhibition of the antibacterial activity of peritoneal exsudate cells was noticed after 14 days, in the presence of antigen. The results obtained support the hypothesis that T effector cells are induced in the course of influenza virus infection. Depression of granulocytes phagocytic activity is dependent on the circulation of antigen-antibody complexes. Intranasal inoculation of influenza viruses in the mice and a correspondingly <
Dept. of Pathology, UCLA School of Medicine, Los Angeles, CA. 90024, USA
Diminished T cell response to influenza virus in aged mice RITA B. EFFROS and Roy L. WALFORD
It is well established that numerous immune functions decline with age, and furthermore, that infectious agents are a major cause of morbidity and mortality in the elderly. Therefore, we initiated a study of the T lymphocyte response to influenza virus in aged mice in an effort to dissect the factors contributing to the diminished capacity of aged individuals to deal with viral infections. The primary cytotoxic T cell (CTL) response in two long-lived strains (C57BLl6 and Balb/c) immunized intraperitoneally with influenza was examined on day 5, which is the peak of the response in young adult mice. We demonstrate that the response of the 20-24 months old mice is only 20 % of that obtained from 4 months old animals. An examination of the kinetics of the response revealed that in old mice the maximal response is
226 . 15th International Leucocyte Culture Conference, Asilomar shifted to the day 7-10 timepoint, and even at this point is 50 % of the magnitude seen at the peak of the young response. In order to eliminate the possibility of in vivo suppressive mechanisms being responsible for the delay and decrease in the response, we compared the in vitro generation of a secondary influenza CTL response by old and young memory spleens. Here too, the response of the old mice was diminished, exhibiting only 60 % of the killing observed in the response by young spleens. Our data demonstrate a decreased capacity in aged mice to mount a T cell response to influenza virus. Experiments in progress are analyzing whether the old mice show a decrease in absolute frequency of precursor cells or a diminished ability to perform the cytotoxic function. Supported by USPHS Research Grant AG00790.
Centro de Biologia Molecular. CSIC-UAM. Canto Blanco. Madrid-34, Spain
Strategy of African swine fever virus (ASFV) to avoid the immune response (IR) L. ENJUANES, I. CASAL, A. SANZ, B. G. BARRENO, and E. VINUELA
ASFV is an icosahedral cytoplasmic deoxyvirus with envelope, which infects swine, inducing on IR unable to eliminate the virus. ASFV may use at least two strategies to avoid the host IR: infection of cells involved in the IR and antigenic variation. ASFV infected most of the porcine macrophages and a small percentage of granulocytes. The susceptibility to the virus was species-specific and its replication could be inhibited (> 90 %) by stimulating the PBL with PWM. In contrast neither B or T porcine lymphocytes, resting or stimulated with mitogens, were infected by ASFV. Specific hyperimmune antisera neutralized only partially (60 %) the infectivity of ASFV. The addition of complement to ASFV-antibody complexes increased the neutralization of this envelope-containing virus to 90 %. But neither this procedure nor the use of F (ab'h fragments of anti-ASFV antibodies abrogated the infectivity of ASFV-antibody complexes. The lack of a complete neutralization of ASFV could be related to the antigenic variability of ASFV, because, during replication, the virus changes virulence, the capacity to induce hemad sorption and its genome undergoes deletions closed to the ends of the double stranded DNA. To study the degree of antigenic variability we have produced a collection of 65 monoclonal antibodies reacting against the viral proteins. Using a selected panel of these monoclonal antibodies specific for internal (vp156, VP73, VP12) and external (VP32) proteins, we have shown that ASFV clones isolated from different geographical areas are antigenic ally different.
The Wistar Institute of Anatomy and Biology, Philadelphia, USA
B cell regulated expression of endogenous retroviral antigens D. L. EWERT and M. S. HALPERN
B cells of some inbred lines of chickens have been shown to express antigen that is cross reactive with endogenous retroviral envelope glycoprotein. We have isolated this antigen
15th International Leucocyte Culture Conference, Asilomar· 227 and shown that it is structurally homologous to envelope glycoprotein of the Avian Leukosis viruses, RAV -0. Our analysis suggests that the viral envelope glycoprotein exists both as four chain and as two chain structures of equimolar gp85 and gp37 subunits on the cell surface. As assayed by immunofluorescence, most, if not all, plasma cells obtained either from live animals or from cultures of mitogen stimulated lymphocytes express detectable levels of viral envelope glycoprotein. The majority of the plasma cell-associated envelope glycoprotein is present on the cell surface though it is not bound to surface Ig or Ia antigens. Although envelope antigen was not detectable by immunofluorescence on the surface of bursal cells or peripheral B lymphocytes, immunochemical assays established that retroviral envelope glycoprotein is present on the surface of the bursal cells but at much lower levels (> 20 times) than on the surface of the plasma cells. Both bursal and plasma cells synthesize envelope glycoprotein. These findings indicate that an increased level of expression of endogenous retroviral envelope glycoprotein is a concomitant of the differentiation of B lymphocytes to plasma cells in some chickens.
New England Regional Primate Research Center - Harvard Medical School and Massachusetts General Hospital - Harvard Medical School, USA
Long-term propagation of marmoset T -lymphocytes in T-cell growth factor: infection with T -lymphotropic herpesviruses and surface membrane markers L. A. FALK, D. SILVA, R. BYINGTON, and R. SCHOOLEY
Marmoset monkeys (Saguinus and Callithrix sp.) are highly susceptible to leukemia/ lymphoma induction after experimental inoculation with Herpesvirus saimiri (HVS) or Herpesvirus ateles (HVA), T -lymphotropic viruses of squirrel and spider monkeys respectively. To examine more closely virus-T-lymphocyte interactions in vitro, T-lymphocytes from cotton-topped marmosets (Saguinus oedipus) have been propagated continuously with conditioned medium containing T-cell growth factor (TCGF) and infected with HVS or HVA. Viral-specified antigens were detected by immunofluorescence tests in the infected cells within 24 hours postinfection (PI) and at all subsequent test intervals up to 8 months PI. The infected T -lymphocytes were dependent upon TCGF for growth up to 5-7 months PI when 3 of 5 HVS-infected cultures could be propagated without TCGF. Three of 3 HVA-infected cultures were TCGF dependent for only 2 months PI at which time they grew independently of TCGF. The continued presence of HVS or HVA in TCGF-dependent/independent cultures was demonstrated by: 1. presence of viral-specified antigens in about 1-5 % of cells, 2. ability of cells to induce infectious centers on monolayers of permissive cells and 3. production of small amounts of cell-free virus present in culture fluids. Reactivity with monoclonal antibodies to human T-lymphocyte subsets (5 % reactivity taken as significant): HVS-infected, 4/5 OKT4+ (8-33%) 3/5 OKT11a+ (54-89%) and 2/5 OKMl+ (6-49%); these values were comparable to noninfected, marmoset, TCGF-dependent T-lymphocytes. There was a positive correlation with OKTlla+ and ability to form E-rosettes (79-100% E+ cells). These studies show that marmoset T -lymphocytes can be propagated longterm using TCGF and are susceptible target cells for transformation with HVS/HVA; infection/transformation of the Tlymphocytes with HVS/HVA appears to make them independent of TCGF. These studies were supported in part by NIH grants: RROOI68-20, ROI CA26225-03, and AI 17057-02.
228 . 15th International Leucocyte Culture Conference, Asilomar Neuroimmunology Branch, National Institute of Neurological and Communicative Diseases and Stroke, Bethesda, MD, 20205, USA
Cellular regulation of the human proliferative response to measles, mumps, and vaccinia viruses
J.
I. GREENSTEI:-<, E. S. MI NG IOLl, and H . F. McFARLAND
Previous studies of the proliferative response of peripheral blood lymphocytes to measles, mumps, and vaccinia viral-infected monolayers in normal sero-positive adults, measured by thymidine incorporation, demonstrated that only 5 % of individuals respond significantly to measles virus (Stimulation Index [5.1.] > 5), by comparison with the universal responses to mumps (mean 5.1. = 10.6) and vaccinia viruses (mean 5.1. = 12.5). T-cells are responsible for the response in each case. Responsiveness to measles virus is greater during early convalescence from acute infection, but the level of proliferation declines over a period of weeks. Mixing autologous cells from early and late times after infection did not demonstrate suppression as a cause. The decline of responsiveness after acute measles and the low level of proliferation in late convalescence appear, therefore, to represent a relative lack of circulating long-term responder T -cells. To investigate further the cellular basis for the different responses, highly purified ( > 94 % ) OKT4 + and OKT8 + populations from late convalescents were obtained by affinity plating, and studied individually or mixed; with 20 % plastic adherent cells added to the cultures. The responses to mumps and vaccinia viruses, and to measles virus in the small number of responder individuals, occurred primarily in the OKT4 + population ; with generally lower responses to measles than to mumps and vaccinia viruses. Low levels of OKT8+ proliferation occurred to all three viruses, and were only modestly elevated by the addition of Interleukin-2. Mixing the OKT4 + and OKT8 + populations failed to demonstrate suppression of the response to each virus. These findings demonstrate that OKT4 + cells are the predominant cells which proliferate to each of these viruses, but that the responses to measles are consistently lower in normal individuals. Antigen specific T-cell responses to measles virus are found after infection, but the expansion of a long-term OKT4 + responder population appears not to occur. The mechanism involved may relate to the short-term suppressive effects of this virus on the immune response to other antigens.
Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel
Modulation of the splenic antibody response following infection of mice with lactic dehydrogenase virus N . ISAKOV and S. SEGAL
Lactic dehydrogenase virus (LDV) is a silent, non life-threatening virus which produces a stable infection in mice with a continuously high titer of infective virus in the blood. Acute infection with LDV is followed by histological changes in the lymphatic organs and causes alterations of both humoral and cellular immune responses. We found that immunization of mice with SRBC following acute LDV infection resulted in an augmented splenic response, expressed by increased numbers of IgM-and IgG-antibody-producing cells (APC). The response was preferentially directed at the production of IgG 2-APC. To test whether LDV
15th International Leucocyte Culture Conference, Asilomar . 229 specifically activate splenic APC, we performed in vitro sensitization of spleen cells to SRBC. We found that presence of LDV in the spleen cell cultures failed to affect the APC response. To eliminate the possibility that the result is due to lack of splenic environmental components from the dispersed suspension of cells, we performed a similar experiment using spleen fragments in the in vitro sensitization phase. Yet, again, LDV did not affect the APC response. We then tested whether LDV affects trapping and sequestration of lymphoid cells in the spleen. Staining of spleen cells from acute LDV -infected mice with fluoresceinated antisera directed to mouse Ig or to the Thy 1,2 antigen indicated a slight increase in the percentage of B lymphocytes. Responses of the splenic lymphocytes cells from LDV-infected mice to B cell or T cell-specific mitogens, were not significantly different from those of LDV-free spleen cells; however, the responses to PHA were significantly increased on the fourth day following LDV infection, while at the same time the responses to Con A were decreased. These results and results from in vivo experiments which demonastrated increased trapping of radio labeled lymphocytes into the spleen support the assumption that LDV affects splenic responses by changing the trapping and sequestration of lymphoid cells in the spleen and by modifying the relative proportions of distinct T cell subpopulations in this organ.
Institute of Virus Research, German Cancer Research Center, Heidelberg, FRG and "Immunology Department, Sandoz Research Institute, Vienna, Austria
Role of endogenously produced interferon in genetically-determined resistance of mice against infection with herpes simplex virus (HSV) H. KIRCHNER, H. ENGLER, D. ARMERDING", and R.
ZAWATZKY
Resistance of mice against primary infection with HSV is genetically controlled by at least two genes that are not H2-linked. Previous studies of the laboratories participating in this study have shown that both Natural Killer (NK) cells and interferon (IFN) may playa role in resistance, but the relationship between IFN and NK cells has been poorly understood. In the experiments of the present study, HSV was injected intraperitoneally (i.p.) and IFN was assayed directly in the wash-out fluid, whereas NK cell activiry was determined in the washedout peritoneal cell population (PEC). All mouse strains resistant or medium-resistant to HSV showed high and brisk IFN responses in vivo at the infection site, which was absent in susceptible mouse strains and in newborn mice of the resistant strain C57BLl6 (newborn mice of all strains are highly susceptible to HSV infection). Most resistant mouse strains also showed high NK cell responses which may - or may not - be a secondary consequence of previous IFN production. However, there were at least two notable exceptions SJL/2 mice that are defective in their NK cell response have a high IFN response (and are resistant) and SI M mice that have a very high NK cell response, are poor producer of IFN (and are susceptible). Thus, there is an absolute correlation between the magnitude of the early local IFN response and resistance, whereas the correlation between resistance and NK cell activation is less stringent. The HSV-induced NK cells shared all rypical properties of NK cells and they were lysed by treatment with antisera against Qa 5 and by the anti-asialo GM1 (kindly provided by Ko OKUMURA). Experiments were carried out in vitro to determine the nature of the cell in the PEC population that responded to HSV with IFN production. This cell shared all the properties of macrophages and had nothing in common with NK cells. In conclusion, our studies show that in primary resistance against HSV endogenously produced IFN seems to playa critical role. This role is further supported by experiments in which exogenous IFN was administered with protective effect against HSV and by experiments in which resistance was broken by anti-IFN serum (kindly provided by ION GRESSER).
230 . 15th International Leucocyte Culture Conference, Asilomar Central Lab. Blood Transfusion Service, PO Box 9190, 1006 AD Amsterdam, The Netherlands
Induction of measles-specific cytotoxic T cells, natural killer cells and interferon in human peripheral blood lymphocytes in vitro
c. J.
LUCAS
For eradiation of virus-infected cells by cytotoxic mechanism,·tour mechanisms have been described: T cell mediated cytotoxicity, antibody dependent ~ell mediated cytotoxicity, natural cytotoxicity and macrophage mediated cytotoxicity. The relative contributions of these are still unknown. From mouse studies it is known that immunization with a virus leads to the generation of at least four types of T lymphocytes. Delayed-type hypersensitivity-mediating T cells, cytotoxic T cells (CTL), T helper cells and T suppressor cells. In addition a cellular immune response in volves induction of mediators as interferon and IL-2. It is unclear if and how all these mechanisms are interrelated. In humans it is possible to detect cytotoxic T cells, and perhaps T helper cells, with a given viral specificity. We were able to increase a relatively low number of measles virus responders by repeated stimulation in vitro. The addition of IL-2 in vitro to measles virus stimulated cultures did not result in increased sensitivity. By repeated stimulation of PBL from 9 out of 36 healthy individuals measles virus specific CTL could be generated. By the same procedure 5 out of 5 multiple sclerosis patients responded. In measles virus and influenza virus stimulated cultures as well as in cultures stimulated by allogeneic lymphocytes a high level of natural killing is generated. These natural killer (NK) cells do not lyse autologous virus infected lymphocytes. In most of the cultures significant levels of interferon are formed. The titre of interferon, the level of NK and the measles self specific CTL activity do not appear to be related which suggests that all have their own regulatory mechanisms. Interferon levels were also analysed in relation to proliferation. There was no correlation unless IL-2 was present in which case it appeared that interferon induction occurred without exception.
Irvington House Institute, Department of Pathology, New York University Medical Center, New York, New York 10016, USA
Evidence for a major cluster of lymphocyte differentiation antigens on murine chromosome 2. (Mapping of Ly-ll/Ly-6/viral integration/ radiation leukemia) D. MERUELO, M. OFFER, and A. ROSSOMANDO The region of chromosome 2 between H-3 and H-13 has been previously shown by others to contain loci coding for a variety of alloantigens among which are H-3, H-13, Ly-4, Lym-II, ~2-microglobulin and Pgp-l. We report herein that lymphocyte determinants H-30, Ly-6 and Ly-ll are also coded for by genes in the vicinity of H-3, and therefore that this region of chromosome 2 contains the tightly linked loci coding for Ala-I, DAG, H9/2S, Ly-8, and ThB. The most important question raised by these findings is whether the clustering of these loci has any special significance. Relevant to this issue is the finding that several virus susceptibility loci map in this section of chromosome 2. RiI-l, Av-l, and RLViI-l, loci involved in susceptibility to fractionated irradiation-, Abelson virus-, and radiation leukemia virus-induced leukemia,
15th International Leucocyte Culture Conference, Asilomar' 231 respectively map in this region. The Abelson virus related cellular oncogene, c-abl, also maps in chromosome 2. It may be speculated that this segment of chromosome plays a special role in viral infections and the multitude of lymphocyte differentiation loci found therein are related to this special relationship to viruses or viral infection. In particular, it has been suggested that the genetic expression or regulation of the genes of different viruses might be dependent on cellular control elements that are involved in tissue differentiation and which exert their effect by a site-specific or cis acting mechanism. We shall present further data consistent with this hypothesis. Supported in part by NIH Grant CA 22247, ACS Grant IM-163, a grant from the National Leukemia Association and a grant from the Irma T. Hirschi Foundation. D. MERUELO is a Leukemia Society of America Scholar.
University of South Florida College of Medicine, Tampa, FL, USA
Leukemia virus induced suppression of natural killer cell activity D.
J.
MOODY, ST. SPECTER, M. BENDINELLI, and H. FRIEDMAN
Leukocytes in culture or in vivo are altered in their normal immunologic functions after leukemia virus infection. Incubation of normal murine spleen cultures with Friend leukemia virus (FLV) markedly suppresses normal immune capabilities, including antibody formation and cell mediated immune responses. Recent studies in this laboratory have demonstrated that the natural killer (NK) cell activity is also depressed following infection of mice with FLV. The NK activity in FLV infected mice progressively decreases such that by day 18 post infection there was essentially no response from the splenic population. In vitro stimulation of FLV infected splenocytes with interferon was capable of partially reversing the depressed NK activity. Target binding cell analysis of FLV splenocytes indicate that they were as capable of binding to target cells as the cells from uninfected animals. FLV splenocytes were capable of suppressing the NK activity of uninfected animals when the two groups were mixed together. This suppressive ability of FLV splenocytes was partially abrogated after passing the FLV splenocytes over a nylon wool column. These experiments suggest the involvement of nylon wool adherent suppressor cells in the NK depression. Thus, it appears likely that leukemia virus infection besides altering antibody formation and cell mediated immunity, also has an effect on natural «immuno-surveillance» activity mediated by cytolytic NK cells. The implications of these observations in regard to the mechanism of viral oncogenesis is apparent.
"The Cleveland Clinic Foundation, Cleveland, Ohio, and +National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA
Autoaggressive thymocytes in preleukemic Balb/c MuLV-carrier mice M. R. PROFFITT", C. DE LA MOTTE", M.
J.
CAULFIELD", and C. KOZAK+
We have previously reported that C3H/He mice persistently infected with Moloney leukemia virus (MuLV-M-carriers) develop autoreactive thymocytes prior to the onset of
232 . 15th International Leucocyte Culture Conference, Asilomar leukemia. We have extended these observations to BALB/c mice, demonstrating that thymocytes from carrier mice are cytotoxic to syngeneic and to allogeneic fibroblast targets. Conversely, when these targets are infected with MuLV-M, they are spared from the cytotoxicity. Xenogeneic cells are also unaffected. In vivo, the preleukemic thymocytes exhibit a graft vs. host-like reaction as measured in two assays. 1. These cells cause a «runting» syndrome with characteristic pathology when injected into newborn, syngeneic mice, and 2. popliteal lymph node enlargement when they are injected into the footpad of irradiated syngeneic adult mice. When a large number of MuLV-carrier mice were examined for autoreactive thymocytes, it became apparent that the degree of cytotoxicity was correlated with the number of virus-producing cells present in the thymus; however, free MuLV had no effect on the autoreactivity in vitro. In further studies we have examined the role of ecotropic MuLV receptors and MuLV -M gp 70 antigens in these phenomena. Thus, we looked at the cytotoxicity and MuLV-carrier thymyocytes against hybrid cell lines prepared between BALB/c (receptor-positive) and hamster (receptor-negative) fibroblasts. The results indicate that only the hybrid cells that have the receptors are lysed by the carrier thymocytes. Furthermore, the cytotoxicity is blocked in the presence of monoclonal antibodies to epitopes on MuL V-M gp70 indicating a role for gp70 antigens in the autoreactive phenomenon. The results suggest that the cytotoxicity is mediated, in part, by an interaction between MuLV gp70 antigens on the surface of the thymocytes and MuLV-receptors on the target fibroblasts. Virus-infected target fibroblasts may be spared because their ecotropic MuLV-receptors are blocked by autogenously produced MuLV. Supported NCI grant CA20242.
University of Southern California, Los Angeles, USA
Division and migration of hemopoietic sources in mouse leukemia (Friend) L. L.
RIFE
and G. MATIOU
Normal and leukemic growth and differentiation are processes regulated by fields (microenvironments) set up by source cells producing a stimulus for stem cell (S.c.) self renewal. When infected by leukemic viruses, the sources increase stimulus production to a critical level which triggers a «leukemic response » by the dependent S.c. However, if the sources are irradiated at 950 rads prior to infections, their efficiency for stimulus production remains normal even after exposure to a massive dose of leukemogen (e.g., Friend virus). These facts allow one to investigate, within the same animal, the effects of unirradiated marrow districts upon the progression of leukemia at certain other locations (e.g., spleen) irradiated at 950 rads prior to virus injection. S.c. kinetic studies show that leukemic progression in the irradiated spleen is proportional to the volume of unirradiated marrow. In addition, a further reduction of spleen leukemic growth is obtained when the previously unirradiated (femoral) marrow is irradiated at 950 rads. In summary, these experiments detect an «abscopal>, effect on tumoral growth. Such effect is occasionally observed during radiotherapy of cancerous patients, when the local irradiation of a tumoral site results in the reduction of tumoral masses growing in distant locations. The data are discussed in terms of cell-cell interactions and synergetic mechanisms
15th International Leucocyte Culture Conference, Asilomar' 233 operating in differentiating malignancies. It appears that leukemogens induce division of hyperactive sources which stimulate S.c. into a leukemic phase after migrating into separate hemopoietic districts.
University of Pittsburgh, Pittsburgh, PA, USA
Cellular immunity during human cytomegalovirus infection C. RINALDO, M. Ho, W. HAMOUDT, X. GU!, R. DEBlASIO Cytomegalovirus (CMV) infection of normally healthy adults can result in a selflimiting mononucleosis syndrome, and may predispose male homosexuals to severe superinfections. During CMV mononucleosis there is a depression of mitogen (concanavalin A - ConAl induced lymphocyte blastogenesis and interferon production that has previously been related to enhanced suppressor activity of monocytes and increased levels of T suppressor cells. In the .present study, freshly donated blood mononuclear leukocytes (MNL) from acute phase CMV mononucleosis patients had depressed blastogenesis eH-Tdr uptake) and interferon production in response to ConA and viral antigens (CMV, herpes simplex virus, mumps virus). Preculturing of patients' MNL for 18 hr at 37°C prior to treatment led to loss of suppressor activity and enhanced blastogenesis to ConA, in contrast to even more depressed reactivity to antigens. Studies were done to define the role of immunoregulatory cells in this effect. Removal of plastic-adherent cells (monocytes) from freshly donated or precultured MNL led to greater depression of blastogenesis to ConA or antigens. Monoclonal antibody analysis showed an increased percentage of total T (Leu 1 +) and T suppressor (Leu 2a +) cells, with a low T helper (Leu 3a+) to Leu 2a+ cell ratio during CMV mononucleosis as compared with normal donors. Preculturing of patients' MNL resulted in selective decrease in Leu 2a + cells, higher Leu 3a/Leu 2a ratios, and appearance of an unusual, third T cell subset (Leu 1 +, 2a -, 3a-). Baseline and interferon-boosted NK cell activity versus K562 cell targets during CMV mononucleosis did not differ from that of normal donors. Similarly, a longitudinal study of a male homosexual with P. carinii pneumonia and CMV viremia showed progressively depressed blastogenesis and enhanced suppressor cell activity with variable NK cell responses. Interferon was detected in serum, but was poorly inducible in MNL in vitro. Thus certain cellular immune functions are depressed during CMV infection in correlation with increased suppressor activity, whereas others appear functional and may aid resolution of illness.
Department of Pathology, McMaster University, Hamilton, Ontario, Canada, and University of Zurich, Switzerland
Viral persistence in cloned natural killer cells K. 1. ROSENTHAL, H. HENGARTNER, and R. M. ZINKERNAGEL
In order to better understand how virus infection alters immune functions, we have investigated the interaction of various viruses with cloned subpopulations of effector lymphocytes in vitro. We have established cloned lines of interleukin-2 (IL-2) dependent cytotoxic T cells (CTL) specific for vesicular stomatitis virus (VSV). Lysis by these cells is H-2 restricted, virus-specific and no natural killer (NK) cell activity is detectable. We also have clones of NK cells which proliferate in IL-2 containing medium and lyse YAC and other NK-sensitive target
234 . 15th International Leucocyte Culture Conference, Asilomar cells very efficiently. Results indicate that clones of H-2 restricted, VSV-specific CTL are resistant to productive infection by a variety of viruses, including vaccinia, lymphocytic choriomeningitis virus (LCMV), and VSV. In contrast, cloned lines of NK cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral antigen and produce infectious VSV. Persistently infected NK cells showed no marked alteration of cell morphology and continued to function as normal NK cells, i.e., capable of killing NK-sensitive targets. Furthermore, these persistently infected NK cells were resistent to lysis by virus-specific CTL. Thus, these cells may harbor persisting virus and avoid cell-mediated immune destruction in an immunocompetent host. Supported by MRC, Canada, and SNSF, Switzerland
Laboratory of Tumor Cell Biology, NCI, Bethesda, Maryland 20205, USA
Infection and in vitro transformation of fresh human cord blood and adult peripheral blood T -lymphocytes by isolates of human T-cell leukemia-lymphoma virus (HTL V) S. Z. SALAHUDDlN, P. D. MARKHAM, M. POPOVIC, P. SARIN, and R. C. GALLO Evidence suggesting the involvement of HTLV in the development of some types of human T-cell lymphoma has been previously reported. Proteins and nucleic acids related to HTLV structural components are detectable in neoplastic tissues but not in non-involved tissues from the same patient (GALLO et aI., Proc. Natl. Acad. Sci., in press). Serologic studies have suggested an association between HTLV and certain malignancies of mature T -cells (GALLO et aI., submitted). In addition, a significant proportion of relatives of patients with HTLVassociated disease possess HTLV-specific antibodies (ROBERT-GUROFF et al., submitted). HTLV has also been shown to be infectious for fresh human T -lymphocytes (RUSCETTI et aI., submitted; MARKHAM et al., in preparation). Here we confirm and extend observations on the biological activity of HTLV. We demonstrate that HTLV from TCGF-dependentT-lymphocyte cultures initiated from multiple leukemic donors in North Eastern U.S., Israel, the Caribbean, and Japan can productively infect and transform fresh human cord blood and adult T-lymphocytes. These experiments were done by cocultivation with lethally irradiated primary HTLV producer cells or exposure to cell-free virus. Virus passaged several times through cultured T-lymphocytes retain the infectious and transforming property. Cocultivation of fresh cord blood leukocytes with cell lines producing non-infectious HTLV results in the establishment of transformed, TCGF-independent T-lymphocytes in culture. These cells contain only limited amounts of viral nucleic acids and proteins.
UCLA School of Medicine, Los Angeles, CA. 90024, USA
Abnormal T cell subset response to herpes simplex virus antigens in patients with recurrent herpes genitalis C. A. SPINA, T. LARSON, and Y.
J.
BRYSON
Although cell-mediated immunity is indicated as a decisive influence in limiting the severity and duration of herpes simplex virus (HSV) infections in human disease, the involvement of
15th International Leucocyte Culture Conference, Asilomar' 235 specific immune cell components in this process remains largely unknown. A series of experiments were done to test the hypothesis that patients who are subject to severe clinical disease have selective alterations in the type of T cell subset which proliferates in response to specific HSV antigen challenge. Patients with the chronic, recurrent form of genital herpes infection comprised the test group, and disease free, HSV-seropositive donors served as controls. T cell subset analysis was determined using the «Leu» series of murine monoclonal antibodies: Leu-1 (pan T), Leu-2 (T suppressor/cytotoxic associated), Leu-3 (T helper/inducer associated). During episodes of active disease, patients tended to demonstrate an inverted Leu-3: Leu-2 cell ratio in their circulating T lymphocytes. To examine proliferative function, lymphocyte preparations were stimulated in vitro for 6 days with HSV type-specific antigens and candida antigen, and the levels of 3H-thymidine uptake measured. The levels of proliferative response in the patients, both with active and inactive disease, did not differ from those of the control group. However, when the type of T cell subset which was undergoing proliferation in response to these antigens was examined, dramatic differences were seen in the patients' response. The Leu-3: Leu-2 ratios of the large, responding blast cells cultured with HSV were significantly lower in the patient groups as compared to the controls, indicating a greater Leu-2 (Ts associated) cell response. The response to Candida, used as a control antigen, gave high ratios in the blast cells which reflected a normal, predominant Leu-3 (TH associated) cell response in both the patients and controls. These results indicate that patients with recurrent HSV disease have an abnormal T cell subset response which is selective for interaction with herpes virus. Supported in part by USPHS grant AI15332.
Univ. of California, San Diego, USA
In vitro analysis of immunosuppression in rabbits caused by the malignant variant of Shope fibroma virus D. S. STRAYER, ST. SELL, G. F. CABIRAC, EILEEN SKALETSKY, PATRICIA SHARP, and LEIBOWITZ
J.
L.
A malignant variant (MV) strain of a rabbit tumor virus, the Shope fibroma virus (SFV), causes profound immunosuppression both in vivo and in vitro. MV and SFV are antigenically identical, as judged by serological cross-neutralization and in vivo cross-protection. SFV produces a small local tumor which regresses without sequelae. In contrast, MV causes recipient rabbits to develop local tumors followed by extensive metastatic disease, complicated by fatal Gram-negative infection. Rabbits given SFV show normal or slightly increased in vitro blastogenic responses to Band T lymphocyte mitogens (anti-Ig and con A respectively). Lymphocytes from MV recipients respond poorly to these mitogens by 6 days after inoculation, which is before metastatic disease and bacterial infection have become clinically obvious. Specific blastogenic responses in vitro of lymphocytes from SFV recipients to infectious or ultraviolet (uv) light-inactivated SFV are vigorous. Such lymphocytes respond similarly to uvinactivated MV, but weakly or not at all to infectious MV. Lymphocytes from MV-exposed rabbits do not mount in vitro proliferative responses to any of these viral preparations though they produce considerable neutralizing antibody in vivo. In contrast, pharmacologic immunosuppression of rabbits with cortisone inhibits developing cellular and humoral immunity without altering the responsiveness to con A. MV is immunosuppressive in vitro as well. When infectious MV is added to cultures of normal spleen cells, proliferative responses to anti-Ig and con A are virtually abolished. The same suppression occurs when the malignant variant virus is added 24 or 48 hours after mitogen. SFV or inactivated MV added to similar cultures is without effect. MV induces this depression in immune responsiveness without
236 . 15th International Leucocyte Culture Conference, Asilomar decreasing lymphocyte viability significantly. MV grows in rabbit lymphocyte preparations in vitro and its viral antigens may be detected on lymphocyte membranes. Thus, we have isolated a new oncogenic virus which infects rabbits and causes severe dysfunction involving both B and T lymphocytes.
Dept. of Pathology & Laboratory Medicine, Univ. Nebraska Med. Cetr., Omaha, NE 68105, USA, "on leave from the Dept. of Tumor Biology, Karolinska Inst., Stockholm, Sweden
Establishment of permanent cultures from Epstein-Barr virus (EBV)nonsusceptible human lymphocytes D.
J.
VOLSKY, R. W. ANDERSON, C. KUSZYNSKI, and I. M. SHAPIRO'
Human B lymphocyte cultures can be established with remarkable efficiency by transforming cells with EBV in vitro. The majority of healthy individuals have EBV receptor-positive B lymphocytes that can serve as targets for EBV-mediated transformation. Unfortunately, in many cases where immortalized cell lines would be most helpful (e.g., Hodgkin's disease, chronic lymphocytic leukemia and preleukemia), attempts to transform lymphocytes by EBV were unsuccessful. We are currently characterizing lymphocyte defects in preleukemia which could account for the development of this disease (c.f., accompanying paper by ANDERSON et al.). Analysis of preleukemia B lymphocytes for surface markers by cytofluorography revealed a lack of EBV receptors. No EBV-determined antigens were observed after exposure of the cells to EBV. Recently, we have demonstrated that EBV receptor-negative cells can be infected by EBV, if viral DNA is introduced into the cytoplasm. The technique we employed is based on implanting functional EBV receptors onto membranes of receptor-negative cells, thereby converting them temporarily into receptor-positive targets. Fusogenic reconstituted Sendai virus envelopes are used as receptor carriers (VOLSKY et aI., PNAS 77:5453, 1980). When preleukemia lymphocytes were implanted with EBV receptors prior to exposure to the B95-8 substrain of EBV, EB virus nuclear antigen (EBNA) was detected in 2-5 % of cells 3-5 days after infection. Early (EA) and late (VCA) antigens were also detected although at lower levels than EBNA. Subsequent cultivation of the EBV -infected preleukemia lymphocytes resulted in the establishment of a new cell line, JV - t. JV -1 was heterogeneous with respect to the expression of several surface markers as assessed by cytofluorography: 99 % of cells were surface Ig-positive; 50 % were EBV receptor-positive, 10 % expressed complement c3d receptors, and no cells presented T cell markers. The data imply that JV -1 cells are of a B-cell lineage. With respect to their EBV status, all cells expressed EBNA, 4-5 % of cells expressed EA and less than 0.5 % were VCA-positive. The approach described above may be helpful in other cases where establishment of lymphoblastoid cell lines seems impossible.
FDA, Bethesda, MD, USA
Evidence that cytotoxic T lymphocytes not natural killer cells are responsible for resolution of influenza virus pneumonia in mice M. A. WELLS, S. DANIEL, S. C. KILEY ,
J. Y. DJEU, and
F. A. ENNIS
The mechanism of protection conferred by the passive transfer of immune spleen lymphocytes with enhanced cytotoxic T lymphocyte (CTL) activity on the course of influenza virus pneumonia in BALB/c athymic (T cell deficient) nude mice was investigated. Splenocytes
15th International Leucocyte Culture Conference, Asilomar· 237 taken directly from mice or after in vitro culture were assayed for CTL on virus infected BALBlc kidney cells, or for natural killer activity (NK) on sensitive YAC tumor cells, and then were transferred intravenously to nude mice 24 hours after lethal intranasal infection with AIPort Chalmers/1/73 influenza virus. The following populations of splenocytes were transferred: Virus Immunization Lymphocyte Primary Secondary % Lysis Virus ill-VIVO in-vitro Source CTL NK % Survival titer BALBlc
> 30 days > 30 days none 8 days
> 30 days none
5 days none none
100.0 7.8 neg.
24.6 10.0 6.6
72
< 0.5
18 15
4.0 4.0
NA NA NA
22.8 7.4 neg.
16.0 10.8 9.8
18 9 0
4.2 4.2 4.5
NA neg. 26.7 0 4 days 3.9 none NA neg. 5.2 0 4.4 Protection against death (% survival of 10-25 mice/group) and reduction in pulmonary virus titer (JOgIO of the 50 % egg infectious dose) when sampled on day 7 after infection, was only observed in mice receiving the secondary in vitro stimulated BALBlc splenocytes with high CTL activity. No association between viral clearance and NK activity was detected. These results indicate that CTL are associated with the recovery of mice with influenza pneumonia, and that NK activity probably does not contribute to this protection. Nude