Cytokine expression by allergen-activated CD4+ and CD8+ cells

420

Cytokines and lymphoid differentiatiorhatration

creased from 25.4% (range X-35.5) prior to rhG-CSF to 7% (range l&11.9. p = 0.0026,) 8% (range 4-12, p = 0.0091)and 15% (range 9-22, p = 0.00.W) on days +2. +4 and +6 of rhG-CSF treatment. Similar changes were observed in concanavalin-A stimulated cultures; conversely, no significant modifications of lymphocyte reactivity to pokeweed mitogen were evident in rhG-CSF-primed lymphocytes compared to untreated samples. S-phase values of lectin-stimulated lymphocytes inversely correlated with neutrophil (Rs = 0.69; p = 0.0075~ and monocyie count (Rs = 0.61; p = 0.005.) RhG-CSF in healthy donors transiently downmodulates lymphocyte proliferative responses to lectin mitogens; rhG-CSF primed neutrophils and monocytes could release immuno-modulating cytokines. critically involved in down-regulation of lymphocyte proliferation.

P.3.11.08

Identlflcatlon of genes controlllng the Tcell prollferatlve response to IL-2 uslng recombinant congenlc stralns

M. Krulov& I, H. Havelkov& ‘, M.Kosaiovti ‘, V. HOI&Y‘, A.A.M. Hart*, P. Demand*, M. Lipoldov& I. ’ Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, *The Netherlands Cancer /nstitute, Amsterdam, The Netherlands

Intmductlon:The induction of the expression of IL-2 and its homologous receptor is one of the major events in ? lymphocyte activation. Lympho&tes of mouse strains BALB/cHeA (BALB/c) and STS/A (STS) differ in IL-2 induced proliferative response, STS being a high and BALB/c a low responder in the range of concentrations 125-2000 IE/ml. We analyzed the genetic basis of this strain difference using the Recombinant Congenic Strains (RCS) of the BALB/c-c-STS/Dem (CcS/Dem) series. MaterMsandMethods:CcS/Dem series comprises 20 homozygous strains all derived from parental background strain BALB/c and parental donor strain STS. Each CcSIDem strain contains different, random set of approximately 12.5% genes of the donor strain STS and approximately 67.5% genes of the background strain BALB/c. Proliferative response was tested by lymphocyte proliferation assay, genolyping of simple sequence length polymorphism (SSLP) was done by PCR. The role of genetic factors in the proliferative response was examined by analysis of valiance (ANOVA, NCSS). Results:We analysed 540 F2 hybrids between one of the high responder RC strains, CcS-4, and the low responder parental strain BALB/c. We found that the response to high IL-2 concentrations is controlled by a loci Cindal (Cytokine induced activation I), on chromosome 11 near the marker DllMil4. The response to a lower dose of IL-2 tested on lymphocytes of the same mice was found to be controlled by another locus, Cinda2, in the centrometic part of chromosome 12 near the marker Dl2Mit37. Con&don: Identification of genetic factors like Cindal and Cinda2, that control T-cells function and understanding their action Is expected to contribute to the efficient analysis of the genetic control of susceptibility to infections and autoimmune diseases as well as testing the possible role of their human homologues in responsiveness to cytokine immunotherapy.

P.3.11.09

Enhanced sensltlvlty of newborn naive T cells to IL-4 in Th cell differentlatlon

E. Rainsford, D.J. Reen. Children’s Research Centre, Our Lady’s Hospital for Sick Children, Crumlin. Dublin 12. Ireland

Introduction: The factors controlling the transition from naive CD4+CD45RA+ T cells to effector or memory cell phenotype, secreting various cytokines, is still poorly understood in humans. Using anti-CD3 and highly purified CDCCD45RA+ T cells from umbilical cord blood and adult peripheral blood, the nature and kinetics of the cytokine response was examined under the influence of various cc-stimulatory molecules, in an APC independent system. Materialeand Methods:CD4+CD45RA+ T cells were isolated bv neoative selection using Dynabeads and Lymphokwik. The purified cells (
25 Jane 1997 - Poster presentations Conclusion: CD4+CD45RA+Tcells isolated from adult and cord populations are capable of producing ThO-type cytokines following ptimary stimulation in a CD3 system. However, the data also suggests that newborn T cells are more readily directed, by 11-4,towards a ThP-type response.

P.3.11 .lO Studying of immunomodulating and antltumor properties of recombinant prothymosln a L.M. Khromykh’, A.V. Khodyakova*, I.P. Brisgalov’, G.D. Kozlovskaya2, A.M. Vasiliev2, T.V. Chemovskaya*, A.B. Vartapityan3, V.M. Abramov*, V.P.Zav’yalov2. 1Instituteof Carcinogenesis, Cancer Research Centce, Moscow, Russia, 9Wtute of Enginekng Immunology state Stock Company. Moscow State University, Russia, 3nBiopreparationn, Ahcow State University; Russia

Introduction: Theattention of many scientists is rivet to prothymosin (I (ProTa), its role as a nucleus protein, its participation in regulator m&haniti of Cell proliferation and other. In the same lime it is known that (ProTa) is a mediator of immune system, factor of differentiation of T-cell chain of immunity. In our previous research we have shown data abilities of recombinant analogue of (ProTa) in vitro. It was established, that (ProTa) induced the differentiation of eadv precursors of bone marrow cells to mature T-cell forms. The previous treaiment of man or mouse thymocytes lead to the ability of this cells to’respond on suboptimal dose of mitogen and alloantigens. Present work is devoted to studying the recombinant (ProTa) in vivo, its abilw in restoration of immune status of immunodeficient (nude) mice. Materialsand Methods:In the work nude mice (H-2d) were used. (ProTa) injected subcutaneously in dose 10, 50, 100 fig/ mouse. The first group of animals was injected for 5 and IO days, and then lymph node and spleen cells were tested on their ability to react on the presence of mitogens -ConA (2.5 &ml) and PHA (IO &ml). B-cell immunity was studied for the ability to genemte the plaque forming cells on T-dependent antigen (SRBC). Second group of animals after injection of (ProTa) for IO days was inoculated allogeneic (H-2b) tumour cells EL-4 (50 103 cells per mouse) and continued the injection of preparation for 15 days. Reeults: The experiments carrying out have shown that (ProTa) induced the ability of lymph node cells of nude mice to react on the presence of mitogens after first 5 injections of the protein. The proliferate level of the lymphocytes increase in 6-10 times compared to control. The analogue testing after IO injections of (PmTa) defined the ability of splenocytes (group 100 &mouse) to respond on the mitogens use. The level of proliferation increase in 6-8 times compared to control. Studying of the humoral immune response on SRBC have shown practically its full restoration compared with normal (Balblc) mice. Studying the process of tumour development have shown, that injections of (ProTa) lead to significance slowing of tumour growth. The volume of tumour knot decrease in 2-3 times compared to control. Conclusion: The recombinant analogue of (PmTa) induced relatively restoration of cellular and humoral immunity of T-immunodeficieni mice and also enhanced the tumour growth resistance of an organism.

P.3.11.11

Cytoklne expression by allergen-activated CD4+ and CD8+ cells

R.J. Dearman. I. Kimber. Zeneca Central T~icotoov <. Aldedev , Park. “. Laboratom Macclesfield, UK Introduction: Prolonged topical exposure of mice to the respiratory allergen trimellitic anhydride (TMA) elicits the production by draining lymph node cells (LNC) of substantial amounts of interieukin 10 (IL-lo) and mitogen-inducible intetfeukin 4 (IL-4). but little of the Type 1 cytokine interferon y (IFN-y). Using complement depletion techniques, we have demonstrated previously that production of the Tvoe 2 cvtokines IL-4 and IL-IO is associated with CD4+ T iymphocytes, wher& the ixclusive source of the IFN-y is CD8+ cells’. We have now examined the influence of accessory cells on cytokine expression by CD4+ and CD8+ cells. Materialsand Methods:BALB/c strain mice received 50 ~1 of 10% TMA dissolved in 4:1 acetone:olive oil vehicle on both shaved flanks on days 0 and 5. Five days later animals received a further 25 ~1 of chemical on the dorsum of both ears daily for three consecutive days. Thirteen days after the initiation of exposure, draining auricular lymph nodes were excised and a single cell suspension oreoared bv mechanical disaaareoation. CD4+ and CDB+ fractions (=-65% purej a;d CW-depleted (
25 June 1997 - Poster presentations

fractions failed to secrete detectable levels of either Type 2 cytokine. Analysis of IFN-y production revealed that unfractionated and CD4 depleted populations elaborated significant levels of this cytokine while CD4+, CD8+ and CD8 depleted populations failed to express measurable levels of IFN-y. Conclusions: The results demonstrate that the expression of Type 2 cytokines by TMA-activated CD4+ cells is independent of the presence of other cells. In contrast, the presence of accessory cells, presumably via their secretion of interleukin 12, is necessary for the production of IFN-y by CD8+ cells. [I] Dearman R.J. Moussavi A., Kemeny D.M. and Kimber I. (1998). Immunology, 89,502-510

Whole-blood method to determine intracellular cytokines by flow-cytometry W.A.C. Sewell. ME. North. A.D.B. Webster. J. Farrant. MRC ~~munodeficiency Research Group, Depa&tent of Clinical Immunology, Royal Free Hospital School of Medicine, London, NW3 2PF; UK Introduotion: We describe a whole-blood, small volume (500 ~1) technique to determine intracellular cytokine production simultaneously with surface markers using 3- and 4colour flow cytometry. Methods: Hepatinised blood (250 ~1) was cultured in the presence or absence of photbol my&ate acetate (PMA, 10 &j/ml) and ionomycin (2 PmoVI). Both cultures had monensin (3 ~mol!l) present to increase signal intensity by blocking export of newly synthesized cytokine from the Golgi. Cells were cultured for various times up to 24 hours, and the kinetics of cytokine production were assessed in normal volunteers. A short (2 hour) culture period was employed, producing optimal levels of the cytokines TNF-(Yand IFN-y. This short term culture also avoided excessive down-regulation of surface CD4 that makes identification of CD4+ calls diicult following long-term activation through PKC. The cells were fixed and permeabilised using Cytoperm Solution A and Sdution B ISerotec. UK), and then stained using directly conjugated anti-cytokine antitiies. Simt&&ous staining of surface-marker& eg.CD4, CD89 was also performed. Cytokine positive CD4+ and CD8+ T cells were identified using logical gates de&d from regions drawn around lymphocyte, CD4+ and CD+ populations of cells using the WinList version 3 flow cytometry analysis package (Veri!y). Unstimulated, but cultured cells are used to define gate settings for cytokine-positive and cytokine-negative cells. Cytokine production from patients with common variable immunodeficiency and normals was compared. Resuh Both IFN-y and TNFa production by CD4+ or CD8+ cells were clearly identified. Kinetic studies showed that 2 hours of stimulation was optimal for these cytokines. With longer culture times, CD4 down-regulation makes 4-wlor flow cytomeby necessary to define CD4+ cells (eg. CD3+8- cells). There were no significant changes in cytokine production at two hours between these two patient groups, however IFN-y production was significantly elevated in CD8+ cells wmpared to cD4+ cells. Conol&lons: The technique is simple, rapid, and accurate at determining intracellular cytokine production. Four-color studies will permit the cytokine production to be determined within lymphocyte subsets (eg CD4+28* cells).

P.3.11 .13

Differences In preferential T cell dlfferentlatlon between three strains of mice Is reflected In their IgE respondershlp

MC. Nawijn, H.F.J. Savelkoul. Dept. Immunology; Erasmus University. Rotter&m, The Netherlends Introduction: For induction of an IgE response, B cells are critically dependent on the cognate interaction with CD4+ T cells. During this interaction, the B cell receives two signals, a CD40 and an IL-4 signal, leading to the switch to IgE production. BA&, SJUJ and SJA9 strains of mice display a differential abil& to mount an IgE response. The BALB/c strain is a typical IgE high responder. The SJUJ strain is a typical low responder, but can be induced to produce high IgE levels by infection with N@postrongylus brasiliensis, or by poiyclonal anti-IgD activation. The SJA9 strain is characterized by a complete absence of the IaE resoonse in viw. even on strono lafinducina stimuli. The B cells of the&mice ‘were analyzed on their ability toswitch to igE production, the T cells were analvzed for their cvtokine production profiles, their expression of CD4OL on actiiation in vitro and on their preferential differentiation to Thl or Th2 phenotypes. Material and Method% SplenicT cells were purified by complement depletion, and activated in vfbu. Cytokine profiles are measured using ELISA and CT.4S Moassay. Surface expression of several markers was measured by flow cytometry. Small msting B cells were purified by complement depletion and discontinuous percoll gradient centrifugation. lsotype production was measured in ELISA at day 7 after stimulation. FACScan analysis of expression of several markers was performed on several days after stimulation. Reeults~ B cells from all three strains of mice could bs induced in vitro to produce IgE by stimulation through CD40 in the presence of rlL-4. Upregulation

Cytokines and lymphoid differentiation/maturation

421

of CD23 and IL-lFi and the amounts of IgE produced in these stimulations were similar in all three strains. The defect of the SJA9 strain in IgE production could be reversed in viw by infusing SJA9 mice with SJUJ T cells or rlL-4. T cells from all three strains were characterized on their cytokine production levels (11-4,11-5,IL-12. IFNy) and on their expression offunctional CD4OLafter stimulation in vitro. Preferential differentiation of CD4+ T cells in these strains into Thl or Th2 phenotypes was analyzed. Conclusions: The differences between the strains in IgE respondership do not reflect a difference in the IgE switching and production potential of the B cells. Rather, the regulation of the B cell switch to IgE by the CD4+ T cell is affected in the low- and non responder strains. The results indicate that the absence of an IgE response in the SJA9 strain is most likely due to a defect in the T cell compartment. So, in general, the IgE respondership of a mouse strain reflects the preferential differentiation of the CD4+ T cells in these mice to Thl or Th2 phenotypes.

P.s.1 1 .I 4

The influence of intact and toxically injured liver cells on lymphocyte prollferatlon and dlfferentijltldn after cyclwxygenase and llpoxygenase lnhlbltlon

S.I. Pavlovich. Department of Immunology, Bcgomoletz InstiMe of Physidogy NAS, Kyiv. Ukraine Introduction: The nom\al and injured liver cells produce the humoral messengers (cytokines, eicosanoids, and other), which can influence immunocompetence cells, particularly lymphocytes. The humoral influence of intact and toxically injured with tetrachlormetane CBA mice liver explants treated with indomethacine (cyclooxygenase inhibitor) and linoleat of hydroxamic acid (LHA. lipoxygenase inhibitor) on lymphocyte proliferation and differentiation was studied. MaterlaIr and Methods: Liver explants cultured in diffusion chambers in vitro were treated with IO-’ or 1O-6 mol either indomethacine or LHA. Cultural supematants were used to influence intact lymph node lymphocytes proliiration and differentiation. Results: Only supematants of the intact liver explants treated with lo-’ mol indomethacine sianificantlv decreased auantitv of large lvmohocvtes and increased quantity of-middle and little lymphocyt&. Treiment .expl&ts with LHA significantly decreased quantity of large lymphocytes and increased quantity of middle and lie limphocytes. The smaller dose stimulated formation of hightly differentiated lymphocytes. Liver explants from injured mice without blockers aoolication stimulated both lvmohocvte oroliferation and differentiation. The effect; of supematants from in&id Ii& &plants treated with low7 mol indomethacine were similar to those of intact liver supematants, but significantly weaker. Treatment of these explants with LHA in&eased the stimulating effect of their supematants on lymphocyte proliferation and differentiation in dosedeoendent manner. Concluelon: Intact and injured liver explants humoral factors produced under condition of cyclooxygenase and lipoxygenase inhibition with IO-’ mol indomethacine or LHAstimulated both lymphocyte proliferation and differentiation.

P.3.11.15

IFNgamma and IL4 production of CD4+ T cell subsets in the rat

Machteld N. Hylkema ‘, Margaretha van der Deen ‘, Jennie M. Pater ‘, Peter H. van der Meide 2, Paul Nieuwenhuis I, Jaap Kampinga 3, Herman Gmen ’ ’ Department of Hisro/cgy and Cell Biology, University of Groningen, Oostersingei 69- 1, 9713 EZ Groningen, The Netherlands, 2BPRC, Lange Kleiweg 115, Rijswijk, The Netherlands, 3Quadtant Research Foundation, Cambridge, UK Introduction: Previous studies have shown that CD4+/CD45RC+ T cells prolifcrate vigorously in c&mediated reactivity assays and that these cells produce high levels of IL-2 and IFNgamma upon activation. CD4+/CD48RC- T cells. on the other hand, have been shown to provide B cell help in secondary antibody responses. CD4+/RT8+T cells have been associated with the suppression of in v&o or in vivo cell-mediated reactivitv and their activitv has been associated with IL-4 production. In line with the above and the known properties of CD4+/CD45RC+/RT6- and CD4+/CD48RC-/RT8+ T cell subsets, we have earlier proposed that these phenotypes may represent Th-1 and Th-2 memory T cells in the rat. In the present study, we analyzed the IFN-gamma and IL-4 production of different T cell subsets.

Materlals and Methods: We have sorted CD4+/CD45RC+IRT6-, CD4+/CD45RC-lRT6+, CD4+/CD45RC+lRT6+ and CD4+lCD45RC-IRT6- T cell populations from peripheral blood, spleen and cervical lymph nodes of thymectomized PVG mts. Thymectomy was performed to avoid contamination with Thy-l+ Recent Thymus Migrants. To measure IFN-gamma and IL-4 at the protein level, we developed an EUSPOT assay to determine frequencies of IFNgamma and IL-4 producing cells. Prior to cytokine measurement, sorted subsets were in vi&o stimulated overnight with PMA and ionomycin.