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In vitro proliferation of rabbit bone marrow-derived and thymus-derived lymphocytes in response to vaccinia virus
CELLULAK
7, 516-521 (1973)
IMMUNOLOtiY
In Vitro
Proliferation of Rabbit Bone Marrow-Derived Derived Lymphocytes in Response to Vaccinia GERALD
J. ELFENBEIN
AND
GARY
and ThymusVirus
L. ROSENBERG 1
urtd Illfcctiom Disrascs, uMd Lubovatory of Imvwz~~loyy, National Imtitzrte of .4llrr:~y the Laboratory of Microbiolvyy and Immur~oloyy, National Institute of Derttul Research, National Institutes of Health, Bethesda, Marylund 20014 Received
Februury
13, lY73
Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in viva. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.
Reports from several laboratories (l-4) have described two populations of rabbit lymphocytes with distinct differences in antigenic and functional properties, which are thought to represent hone marrow-derived lymphocytes (B cells) and thymus-derived lymphocytes (T cells). In a recent study Rosenberg ef al. (5) demonstrated that lymphocytes from rabbits immunized with vaccinia virus or herpes simplex virus can he stimulated to proliferate in vitro by the immunizing virus. While antigen-induced lymphocyte proliferation in vitro has been considered a correlate of cellular immunity and, therefore, a T cell response (6-S)) the unusually short-lived nature of the lymphocyte proliferative response to specific viral antigen after immunization raises the question of which population of lymphocytes (T or B cell) is responding to antigenic stimulation (5). Recently, Elfenhein et al. (9) h ave described a method for determination of the degree of proliferation of B cells in vitro. This technique has permitted investigation of the proliferative response of guinea pig B cells to specific antigen (9), guinea pig and rabbit B cells to anti-immunoglobulin antisera (4), and guinea pig, mouse, and rabbit B cells to various mitogens (3). Briefly, this method entails simultaneous detection of complement receptor lymphocytes ( CRLs) and uptake of tritiated thymidine ( 3H-TI~Y) in these CRLs by radioautography. CRLs, which have been shown to be B cells (lo), are detected by the formation of rosettes with sheep erythrocytes bearing activated third component of complement (C’3). In 1 Recipient of NIH Dental Research.
special
fellowship
6 F03 516
Copyright All rights
1973 by Academic Press. o9 reproduction in any form
Inc. reserved.
DE53420-01
from
the National
Institute
of
the present study we ha1.e employ~tl this technique to examine the extent of B cell rabbit peripheral blood participation in the proliferative response of sensitized leukocytes (RPBL) to vaccinia antigen. MATERIALS Vims
Pvepurations
und Antibody
AND
AIETHODS
I)ctrrlrl,inations
was prepared and assayed in primary rabbit Vaccinia virus (strain CVI-79) kidney cells grown in the presence of 3?, rabbit serum (I 1). As described in detail elsewhere (5) virus was pl’tially ljurilied by ultracentrifugalion of stock pools of virus and the11 resuspended in phosphate-buffered saline (PBS) to a titer of 5.0 x 10; plaque-forming units (E’FU) per ml. Further purification was accomplished by sucrose density gradient centrifugation of partially purified virus. After dialysis against PBS, density gradient-purified virus was suspended in PBS to a titer of 1.0 x 10R PFU/ml, and inactivated by exposure to ultraviolet light. This inactivated vaccinia preparation (hereafter VACCINIA) was used as a source of viral antigen for in zlitro lymphocyte stimulation studies. Serum neutralizing antibody to vaccinia was measured by the 50% plaque-reduction method using 100 PFU of virus (12). Antibody titers are expressed as the reciprocal of the highest serum dilution which gives the 50% endpoint.
Six- to eight-week-old male New Zealand White rabbits were immunized with a total of 2.5 X lo7 PFU of partially purified infectious vaccinia virus by intradermal lmoculation into four sites. Twelve days later blood was obtained by cardiac puncture and the erythrocytes sedimented with dextran. The RPBL were suspended at a density of 4.0 x lo6 cells/ml in RPM1 1640 medium supplemented with 10% heat-inactivated fetal calf serum, glutamine, and antibiotics as previously described (5). One-milliliter aliquots of cell suspension were cultured with 0.1 ml of PBS or inactivated vaccinia antigen in PBS (VACCINIA). Antigen and control cultures were set up in glass vials in sextuplicate and incubated at 36.5”C. Eighteen hours before harvesting the cultures, 1 pCi ?&THY (6.0 Ci/mmole, Schwarz/Mann, Rockville, RID) was added to each culture vial. At the end of the incubation period, three of each group of six cultures were harvested on hlillipore filters presaturated with unlabeled thymidine by a modification of the method of Robbins et al. (13). After washing with PBS, 10% trichloroacetic acid, and absolute ethanol, filters were placed in scintillation vials with 10 ml Aquasol (l;ew England Nuclear, Boston, MA) and incorporation of 3H-THY determined by liquid scintillation spectrometry. Complement Receptor Lymphocyte nation (of Radioautoyraphs
Rosette Formation
and Preparation
and Exami-
It has been reported that the number of CRLs present in mouse spleen (MSPL) and in RPBL populations falls off sharply after 48 hr in culture with or without stimulants. Even at 48 hr the precentage of CRLs was approximately one-half that present in MSPL and RPRL immediately after preparation (3). Therefore, 48 hr was chosen as the incubation period for these experiments although the degree of lymphocyte stimulation is not maximal at that time (5).
OF RABBIT
1,559
5,523
VACCINIA
3.5
Stimu3H-THY lation incorporation (cpm/4 X lo6 cells) ratio0
PKOLIFEKATION
Saline
Culture stimulant
v&o
BONE
0.4 2,7~ P < O.OOld
10.4”
1
B Graincd cells* (‘;a
LYMPHOCYTES
12.4
A Rosetted cells * (76)
MARROW-DERIVED
1
a Stimulation ratio is defined as the ratio of cpm 3H-THY incorporated in the presence of VACCINIA * Unless otherwise indicated 1000 consecutive mononuclear cells counted. c Two thousand cells counted. d P value obtained from chi-square test for significance (1 degree of freedom) using raw data.
1
Animal number
In
TABLE
to that incorporated
P > 0.9
1
VIRUS
P < 0.01
1
ix Rosetted cells that are also grained ((,T j
in the presence of saline.
TO VACCINIA
C Grained cells that are also rosetted (‘)y)
IN RESPONSE
E
8
As prleviously described (9), cells from the remaining three vials in each group of six, cultured with or without VACCINTA, were pooled, washed and mixed with sheep erythrocytes coated with 19s rabbit antibody (a gift of Dr. M. Frank) and mouse complement. After incubation to allow CKL rosette formation, cell suspensions were puddled onto subbed slides, which were then dipped in Kodak NTB-2 were developed, fixed, emulsion. After 2 weeks’ exposure time, radioautographs and stained with Giemsa. Slides were examined simultaneously for mononuclear cells, rosetted cells (CRLs) , grained cells (proliferating cells), and simultaneously grained and rosetted cells (proliferating CRLs). For each preparation at least 1000 consecutive cells were counted and scored. RESULTS Antibod,y Production in Viva and Peripheral After Imm6~~i,-ation with Vaccinia Virus
Lyvnphocyte
Stinsulation
in Vitro
Twelve days after immunization with vaccinia virus, serum was obtained for antibody determinations, and RPBL cultures were established in the presence or absence of VACCINIA. Serum vaccinia-neutralizing antibody titers for the rabbits in this study were 64, 64, and 128, respectively (geometric mean titer of 81)) thus demonstrating a good humoral immune response to the immunizing antigen. In addition, as illustrated in Table 1, KPBL from each rabbit showed significant stimulation to vaccinia antigen after 48 hr in culture (stimulation ratios of 3.5-6.2). Detection
of Grained
Complement
Receptor
Lgnzphocytes
in Radioautographs
CRI, rosette assay cell suspensions from cultures of RPBL with or without VACCINIA were processed for radioautographic examination. The results are presented in Table 1. It is apparent from the data in Column A that there is essentially no difference in the percentage of CKLs present in antigen-stimulated cultures as compared to control cultures after 48 hr (mean percentage CRLs 11.1 * 2.7 in control cultures and 11.9 2 1.0 in stimulated cultures, P > 0.7 by Student’s t test). Similar results have been obtained with mitogens and anti-immunoglobulin antisera in the rabbit (3, 4). As shown in Column B, vaccinia antigen produces a significant increase in the percentage of grained cells (proliferating cells) above that with stirnulatiun observed in seen in .cotilrol cultures (P i O.OOl), consistent parallel cultures. The percentage of proliferating cells which are CKLs is presented in Column C. The range of values for control cultures is 18.2-33.37 0 and for stimulated cultures 2.5.2-4O.Sp/,. Statistical analysis of raw data for each animal failed to reveal a significant difference in the fraction of grained cells that are rosetted in control and stimulated cultures. The degree of B cell participation in the in vitro proliferative responsl: of sensitized RPBL to vaccinia antigen is shown in Column D, a tabulation of ,the fraction of CRLs which is proliferating. The data show, for all animals studied, a 13- to 15-fold increase in the fraction of CRLs which is proliferating in cultures stimulated by VACCINIA above that in control cultures. DISCUSSION Our studies with vaccinia virus demonstrate that peripheral blood lymphocytes from rabbits immunized with the virus can proliferate in vitro in response to
520
SIIORT
COMMUNICATIONS
vaccinia antigen at a time when antiviral antibody- is being synthesized in v&o. Analysis for cell type in the proliferating population of lymphocytes revealed that a substantial fraction (IX-lO.Sc/o j b ears the receptor for activated third component of coml)lenieiit, wit11 no tliffereiice iii tlie values for control ant1 stimulated cultures for each animal studied. Furthermore, there is a marked (13- to 15-fold) increase in the fraction of CRLs which is proliferating in response to VACCIKIA as compared to control cultures. Among rabbit peripheral blood lymphocytes the number of immunoglobulin-bearing cells exceeds the number of complement receptor lymphocytes (G. Elfenbein, unpublished observations). For this reason the CKL rosette technique gives an underestimate of the degree of participation of B cells in the proliferative response in vitro. However, since one of us has found that T cells stimulated in 7ifro do not acquire a complement receptor (G. Elfenbein, unpublished observations), we feel confident that the complement receptor is a specific marker for R cells even after 48 hr in vitro. Thus, we have shown that there is, at a minimum, a 13- to IS-fold increase in the fraction of B cells which is proliferating in response to VACCINIA as compared to control cultures. Two distinct patterns of lymphocyte subpopulation responses to stimulation in z&o recently have been described. In cultures of RPBL with phytohemagglutinin (PHA) and concanavalin A (Con A), the percentage of proliferating cells which are CRLs is not significantly different from values for control cultures. At the same time, the fraction of CKLs that is proliferating is markedly increased over control levels. Thus, while there is substantial B cell participation in the proliferative response to PHA and Con A, these mitogens predominantly stimulate T cells in mixed T and B cell populations (3). Indeed, it has been adequately demonstrated that T cells are required for B cells to respond to PIIA and Con A (14, 15). In contrast, in response to anti-immunoglobulin antisera (a situation in which B cells proliferate and T cells are not required) there is a dramatic increase in the fraction of proliferating RPBL that is CRLs (mean 74.1% in stimulated cultures versus 11.0% in control cultures). In fact, all of the proliferation induced by anti-immunoglobulin antisera has been shown to involve B cells exclusively (4). In the studies presented in this communication. the pattern of response of RPRT, to VACCTNTA is similar to that seen with PHA and Con A, implying a predominant T cell response with significant proliferation of E cells. Similar results have been obtained upon stimulation of guinea pig lymph node cells by ovalhumin in vitro (9). Indirect evidence also points to the central role that T cells must play in the in vifro proliferative response of RPBL to viral antigens. Namely, there is no precise correlation bctwecn rabbit lymphocyte stimulation by viral antigen and levels of serum antiviral antibody. In fact, rabbits can be immunized with noninfectious herpes simplex virus in a manner such that the in vitro lymphocyte stimulation response to viral antigen is intact while no antibody can be detected in the sera of these animals (16). There is much in the literature to support an important role of cell-mediated (T cell) immunity in host defenses to vaccinia virus, as well as for infections with other viruses (reviewed in 17, 18). In particular, its role appears to be especially prominent in infection with viruses like vaccinia, which can spread from cell to cell prior to cell lysis (19-21) and which, therefore, are not exposed to neutralizing antibody. In addition, cell-mediated immunity may be a significant factor in a
host with defective humoral immunity and in infections with viruses that are poor inducers of interferon or resistant to its action. It must be emphasized that the CKL rosette-radioautography technique onI4 analyzes the proliferating population of lymphocytes. It does not establish whether T cells are required for B cell proliferation. Whether the proliferation of B cells is triggered by specific interaction with vaccinia antigen or represents a response to a nonspecific mediator elaborated by T cells is yet to be determined. IJntil these questions are fully resolved, we can only state with confidence that the in vitro proliferative response of RPBL to VACCINIA is a mixed T and B cell response. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Daguillard, F., and Richter, M., J. Exp. Med. 131, 119, 1970. Fanger, M. W., Pelley, R. P., and Reese, A. L., J. Imm~~m1. 109, 294, 1972. Elfenbein, G. J., Harrison, M. R., and Green, I., J. 1~n?~?fno/.. in press. Elfenbein, G. J., Harrison, M. R., and Mage, R. G., J. Z~m~u~101..in press. Rosenberg, G. L., Farher, P. A., and Notkins, A. L., Proc. Nat. Acad. Sci. CS.4 69, 756, 1972. Mills, J. A., J. Inlnlutzol. 97, 239, 1966. Oppenheim, J. J., Frd. Prnc. 27, 21, 1968. Green, I., Paul, W. E., and Benacerraf, B., J. Exj. Med. 127, 43, 1968. Elfenbein, G. J., Shevach, E. M., and Green, I., J. Illz+n~rlol. 109, 870, 1972. Bianco, L., Patrick, R., and Nussenzweig, V., J. Exp. Med. 132, 702, 1970. 42, 980, 1968. Kempe, C. H., Fulginiti, V., Minamitani, M., and Shinefield, H., Pcdintrics Hampar, B., Notkins, A. L., Mage, M., and Keehn, M. A., J. Imnlunol. 100, 586, 1968. Robbins, J. H., Burk, P. G., and Levis, W. R., Fed. Proc. 28, 363, 1969. Greaves, M. F., and Bauminger, S., 1Vattrre (Lmdm) 235, 67, 1972. Andersson, J., Miiller, G., and Sjijberg, O., Cc/l. In~n~rnol. 4, 381, 1972. Rosenherg, G. L., and Notkins, A. L., Manuscript in preparation. Glasgow, L. A., Arch. Ivhm. Med. 126, 125, 1970. Allison, A. C., and Burns, W. H., In “Immunogenicity” (F. Borek, Ed.), pp. 177-184. North-Holland, Amsterdam, 1972. Stoker, M. G. P., Nature (London) 182, 1525, 1958. Chri’stian, R. T., and Ludovici, P. P., Pror. Sot. Exfi. h’io/. Med. 138, 1109, 1971. Nishmi, M., and Keller, R., Virology 16, 91, 1962.
7, 516-521 (1973)
IMMUNOLOtiY
In Vitro
Proliferation of Rabbit Bone Marrow-Derived Derived Lymphocytes in Response to Vaccinia GERALD
J. ELFENBEIN
AND
GARY
and ThymusVirus
L. ROSENBERG 1
urtd Illfcctiom Disrascs, uMd Lubovatory of Imvwz~~loyy, National Imtitzrte of .4llrr:~y the Laboratory of Microbiolvyy and Immur~oloyy, National Institute of Derttul Research, National Institutes of Health, Bethesda, Marylund 20014 Received
Februury
13, lY73
Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in viva. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.
Reports from several laboratories (l-4) have described two populations of rabbit lymphocytes with distinct differences in antigenic and functional properties, which are thought to represent hone marrow-derived lymphocytes (B cells) and thymus-derived lymphocytes (T cells). In a recent study Rosenberg ef al. (5) demonstrated that lymphocytes from rabbits immunized with vaccinia virus or herpes simplex virus can he stimulated to proliferate in vitro by the immunizing virus. While antigen-induced lymphocyte proliferation in vitro has been considered a correlate of cellular immunity and, therefore, a T cell response (6-S)) the unusually short-lived nature of the lymphocyte proliferative response to specific viral antigen after immunization raises the question of which population of lymphocytes (T or B cell) is responding to antigenic stimulation (5). Recently, Elfenhein et al. (9) h ave described a method for determination of the degree of proliferation of B cells in vitro. This technique has permitted investigation of the proliferative response of guinea pig B cells to specific antigen (9), guinea pig and rabbit B cells to anti-immunoglobulin antisera (4), and guinea pig, mouse, and rabbit B cells to various mitogens (3). Briefly, this method entails simultaneous detection of complement receptor lymphocytes ( CRLs) and uptake of tritiated thymidine ( 3H-TI~Y) in these CRLs by radioautography. CRLs, which have been shown to be B cells (lo), are detected by the formation of rosettes with sheep erythrocytes bearing activated third component of complement (C’3). In 1 Recipient of NIH Dental Research.
special
fellowship
6 F03 516
Copyright All rights
1973 by Academic Press. o9 reproduction in any form
Inc. reserved.
DE53420-01
from
the National
Institute
of
the present study we ha1.e employ~tl this technique to examine the extent of B cell rabbit peripheral blood participation in the proliferative response of sensitized leukocytes (RPBL) to vaccinia antigen. MATERIALS Vims
Pvepurations
und Antibody
AND
AIETHODS
I)ctrrlrl,inations
was prepared and assayed in primary rabbit Vaccinia virus (strain CVI-79) kidney cells grown in the presence of 3?, rabbit serum (I 1). As described in detail elsewhere (5) virus was pl’tially ljurilied by ultracentrifugalion of stock pools of virus and the11 resuspended in phosphate-buffered saline (PBS) to a titer of 5.0 x 10; plaque-forming units (E’FU) per ml. Further purification was accomplished by sucrose density gradient centrifugation of partially purified virus. After dialysis against PBS, density gradient-purified virus was suspended in PBS to a titer of 1.0 x 10R PFU/ml, and inactivated by exposure to ultraviolet light. This inactivated vaccinia preparation (hereafter VACCINIA) was used as a source of viral antigen for in zlitro lymphocyte stimulation studies. Serum neutralizing antibody to vaccinia was measured by the 50% plaque-reduction method using 100 PFU of virus (12). Antibody titers are expressed as the reciprocal of the highest serum dilution which gives the 50% endpoint.
Six- to eight-week-old male New Zealand White rabbits were immunized with a total of 2.5 X lo7 PFU of partially purified infectious vaccinia virus by intradermal lmoculation into four sites. Twelve days later blood was obtained by cardiac puncture and the erythrocytes sedimented with dextran. The RPBL were suspended at a density of 4.0 x lo6 cells/ml in RPM1 1640 medium supplemented with 10% heat-inactivated fetal calf serum, glutamine, and antibiotics as previously described (5). One-milliliter aliquots of cell suspension were cultured with 0.1 ml of PBS or inactivated vaccinia antigen in PBS (VACCINIA). Antigen and control cultures were set up in glass vials in sextuplicate and incubated at 36.5”C. Eighteen hours before harvesting the cultures, 1 pCi ?&THY (6.0 Ci/mmole, Schwarz/Mann, Rockville, RID) was added to each culture vial. At the end of the incubation period, three of each group of six cultures were harvested on hlillipore filters presaturated with unlabeled thymidine by a modification of the method of Robbins et al. (13). After washing with PBS, 10% trichloroacetic acid, and absolute ethanol, filters were placed in scintillation vials with 10 ml Aquasol (l;ew England Nuclear, Boston, MA) and incorporation of 3H-THY determined by liquid scintillation spectrometry. Complement Receptor Lymphocyte nation (of Radioautoyraphs
Rosette Formation
and Preparation
and Exami-
It has been reported that the number of CRLs present in mouse spleen (MSPL) and in RPBL populations falls off sharply after 48 hr in culture with or without stimulants. Even at 48 hr the precentage of CRLs was approximately one-half that present in MSPL and RPRL immediately after preparation (3). Therefore, 48 hr was chosen as the incubation period for these experiments although the degree of lymphocyte stimulation is not maximal at that time (5).
OF RABBIT
1,559
5,523
VACCINIA
3.5
Stimu3H-THY lation incorporation (cpm/4 X lo6 cells) ratio0
PKOLIFEKATION
Saline
Culture stimulant
v&o
BONE
0.4 2,7~ P < O.OOld
10.4”
1
B Graincd cells* (‘;a
LYMPHOCYTES
12.4
A Rosetted cells * (76)
MARROW-DERIVED
1
a Stimulation ratio is defined as the ratio of cpm 3H-THY incorporated in the presence of VACCINIA * Unless otherwise indicated 1000 consecutive mononuclear cells counted. c Two thousand cells counted. d P value obtained from chi-square test for significance (1 degree of freedom) using raw data.
1
Animal number
In
TABLE
to that incorporated
P > 0.9
1
VIRUS
P < 0.01
1
ix Rosetted cells that are also grained ((,T j
in the presence of saline.
TO VACCINIA
C Grained cells that are also rosetted (‘)y)
IN RESPONSE
E
8
As prleviously described (9), cells from the remaining three vials in each group of six, cultured with or without VACCINTA, were pooled, washed and mixed with sheep erythrocytes coated with 19s rabbit antibody (a gift of Dr. M. Frank) and mouse complement. After incubation to allow CKL rosette formation, cell suspensions were puddled onto subbed slides, which were then dipped in Kodak NTB-2 were developed, fixed, emulsion. After 2 weeks’ exposure time, radioautographs and stained with Giemsa. Slides were examined simultaneously for mononuclear cells, rosetted cells (CRLs) , grained cells (proliferating cells), and simultaneously grained and rosetted cells (proliferating CRLs). For each preparation at least 1000 consecutive cells were counted and scored. RESULTS Antibod,y Production in Viva and Peripheral After Imm6~~i,-ation with Vaccinia Virus
Lyvnphocyte
Stinsulation
in Vitro
Twelve days after immunization with vaccinia virus, serum was obtained for antibody determinations, and RPBL cultures were established in the presence or absence of VACCINIA. Serum vaccinia-neutralizing antibody titers for the rabbits in this study were 64, 64, and 128, respectively (geometric mean titer of 81)) thus demonstrating a good humoral immune response to the immunizing antigen. In addition, as illustrated in Table 1, KPBL from each rabbit showed significant stimulation to vaccinia antigen after 48 hr in culture (stimulation ratios of 3.5-6.2). Detection
of Grained
Complement
Receptor
Lgnzphocytes
in Radioautographs
CRI, rosette assay cell suspensions from cultures of RPBL with or without VACCINIA were processed for radioautographic examination. The results are presented in Table 1. It is apparent from the data in Column A that there is essentially no difference in the percentage of CKLs present in antigen-stimulated cultures as compared to control cultures after 48 hr (mean percentage CRLs 11.1 * 2.7 in control cultures and 11.9 2 1.0 in stimulated cultures, P > 0.7 by Student’s t test). Similar results have been obtained with mitogens and anti-immunoglobulin antisera in the rabbit (3, 4). As shown in Column B, vaccinia antigen produces a significant increase in the percentage of grained cells (proliferating cells) above that with stirnulatiun observed in seen in .cotilrol cultures (P i O.OOl), consistent parallel cultures. The percentage of proliferating cells which are CKLs is presented in Column C. The range of values for control cultures is 18.2-33.37 0 and for stimulated cultures 2.5.2-4O.Sp/,. Statistical analysis of raw data for each animal failed to reveal a significant difference in the fraction of grained cells that are rosetted in control and stimulated cultures. The degree of B cell participation in the in vitro proliferative responsl: of sensitized RPBL to vaccinia antigen is shown in Column D, a tabulation of ,the fraction of CRLs which is proliferating. The data show, for all animals studied, a 13- to 15-fold increase in the fraction of CRLs which is proliferating in cultures stimulated by VACCINIA above that in control cultures. DISCUSSION Our studies with vaccinia virus demonstrate that peripheral blood lymphocytes from rabbits immunized with the virus can proliferate in vitro in response to
520
SIIORT
COMMUNICATIONS
vaccinia antigen at a time when antiviral antibody- is being synthesized in v&o. Analysis for cell type in the proliferating population of lymphocytes revealed that a substantial fraction (IX-lO.Sc/o j b ears the receptor for activated third component of coml)lenieiit, wit11 no tliffereiice iii tlie values for control ant1 stimulated cultures for each animal studied. Furthermore, there is a marked (13- to 15-fold) increase in the fraction of CRLs which is proliferating in response to VACCIKIA as compared to control cultures. Among rabbit peripheral blood lymphocytes the number of immunoglobulin-bearing cells exceeds the number of complement receptor lymphocytes (G. Elfenbein, unpublished observations). For this reason the CKL rosette technique gives an underestimate of the degree of participation of B cells in the proliferative response in vitro. However, since one of us has found that T cells stimulated in 7ifro do not acquire a complement receptor (G. Elfenbein, unpublished observations), we feel confident that the complement receptor is a specific marker for R cells even after 48 hr in vitro. Thus, we have shown that there is, at a minimum, a 13- to IS-fold increase in the fraction of B cells which is proliferating in response to VACCINIA as compared to control cultures. Two distinct patterns of lymphocyte subpopulation responses to stimulation in z&o recently have been described. In cultures of RPBL with phytohemagglutinin (PHA) and concanavalin A (Con A), the percentage of proliferating cells which are CRLs is not significantly different from values for control cultures. At the same time, the fraction of CKLs that is proliferating is markedly increased over control levels. Thus, while there is substantial B cell participation in the proliferative response to PHA and Con A, these mitogens predominantly stimulate T cells in mixed T and B cell populations (3). Indeed, it has been adequately demonstrated that T cells are required for B cells to respond to PIIA and Con A (14, 15). In contrast, in response to anti-immunoglobulin antisera (a situation in which B cells proliferate and T cells are not required) there is a dramatic increase in the fraction of proliferating RPBL that is CRLs (mean 74.1% in stimulated cultures versus 11.0% in control cultures). In fact, all of the proliferation induced by anti-immunoglobulin antisera has been shown to involve B cells exclusively (4). In the studies presented in this communication. the pattern of response of RPRT, to VACCTNTA is similar to that seen with PHA and Con A, implying a predominant T cell response with significant proliferation of E cells. Similar results have been obtained upon stimulation of guinea pig lymph node cells by ovalhumin in vitro (9). Indirect evidence also points to the central role that T cells must play in the in vifro proliferative response of RPBL to viral antigens. Namely, there is no precise correlation bctwecn rabbit lymphocyte stimulation by viral antigen and levels of serum antiviral antibody. In fact, rabbits can be immunized with noninfectious herpes simplex virus in a manner such that the in vitro lymphocyte stimulation response to viral antigen is intact while no antibody can be detected in the sera of these animals (16). There is much in the literature to support an important role of cell-mediated (T cell) immunity in host defenses to vaccinia virus, as well as for infections with other viruses (reviewed in 17, 18). In particular, its role appears to be especially prominent in infection with viruses like vaccinia, which can spread from cell to cell prior to cell lysis (19-21) and which, therefore, are not exposed to neutralizing antibody. In addition, cell-mediated immunity may be a significant factor in a
host with defective humoral immunity and in infections with viruses that are poor inducers of interferon or resistant to its action. It must be emphasized that the CKL rosette-radioautography technique onI4 analyzes the proliferating population of lymphocytes. It does not establish whether T cells are required for B cell proliferation. Whether the proliferation of B cells is triggered by specific interaction with vaccinia antigen or represents a response to a nonspecific mediator elaborated by T cells is yet to be determined. IJntil these questions are fully resolved, we can only state with confidence that the in vitro proliferative response of RPBL to VACCINIA is a mixed T and B cell response. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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