Cyclosporin A inhibits T cell development in vitro
Eur. J. Immunol. 1990. 20: 753-757
Richard M. SiegelAo, Katsuyuki Yuioo, Drew E. Tenenholzo, Ralph Kubo+ and Mark I. Greenen Division of Immunology, Department of Pathology and Laboratory Medicineo, University of Pennsylvania Medical School, Philadelphia and Department of Medicine+, National Jewish Center for Immunology and Respiratory Medicine, Denver
Inhibition of T cell developmemt in thymic organ culture: implications for the mechanism of action of cyclosporin A* We have examined the effects of the immunosuppressive drug cyclosporin A (CsA) on the phenotypic maturation of Tcells in thymic organ cultures begun at day 16 of gestation. CsA specifically inhibited the generation of cells expressing high levels of a@ TcR/CD3 complexes and a mature phenotype defined by CD4 and CD8 surface markers. Adding interleukin (IL) lfi, IL 2 or IL 4 failed to reverse the effects of CsA, and major histocompatibility complex class I1 expression in the thymic medulla was preserved. Possible mechanisms of CsA-mediated inhibition of T cell development are discussed.
1 Introduction In the past few years, much has been learned about the pathways of intrathymic T cell development, but understanding the molecular mechanisms underlying T cell repertoire selection remains one of the most challenging and central problems in immunology. In the thymus,Tcells bearing TcR a/p and either CD4 or CD8 are thought to be generated from CD4+CD8+ precursors [l]. Observations made with TcR transgenic mice and other experiments suggest that aTcell will not mature past the double-positive CD4+CD8+stage if its TcR is reactive with self antigens in the thymus (negative selection) [2-4]. However, some engagement of antigen receptors with self components must be required (positive selection) since T cells with certain receptors will not mature in a completely allogeneic environment [ 5 , 61. The soluble factors, cellular interactions, and genetic events which regulate these important processes are largely unknown. Our laboratory has studied mutations which disrupt T cell development in order to better understand the requirements for normal differentiation of matureTcells from their precursors [71. The immunosuppressive drug cyclosporin A (CsA) is a pharmacological agent which also has dramatic effects on T cell development and the acquisition of self tolerance. CsA chronically administered to rodents during normal ontogeny or after BM transplantation decreases matureTcells present in the thymus and LN [8]. However, when CsA is withdrawn, organ-specific autoimmune disease results [9, 101.The repertoire of Tcells which matures in the presence of CsA includes T cells bearing potentially self-reactive receptors, although it has not been proven that
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These studies were supported by the Lucille Markey Charitable Trust and grants from the N.I.H., N.E.I. and N.C.I. to M.I.G. Special Fellow of the Leukemia Society of America. Trainee in the Medical Scientist Training Program at the University of Pennsylvania Medical School (N.I.H. grant #T-32-GM-07170).
Correspondence: Mark I. Greene, Department of Pathology, University of Pennsylvania School of Medicine, 36th St. and Hamilton Walk, Philadelphia, PA 19104-6082,USA Abbreviations: CsA: Cyclosporin A 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
these cells actually mediate the post-CsA autoimmune syndrome [ l l , 121. Because of the effects of CsA on both positive and negative selection of the Tcell repertoire, it is important to understand how this drug is acting on the cells and mediators involved in intrathymic T cell maturation. Previous in vivo experiments with CsA have not been able t o determine the primary mechanism by which it inhibitsT cell development. It is possible that CsA is acting only indirectly on the developing thymus through inhibiting development of stem cells in the BM or release of soluble mediators by other organs.To study the effects of CsA onT cell development in more detail, and attempt to identify its mechanism of action, we have investigated the effect of CsA on thymocyte differentiation in fetal thymic organ culture, which is currently the only system in which Tcell development can be studied in vitro. Using multiparameter FCM analysis and immunostaining of tissue sections, we demonstrate that CsA specifically inhibits the maturation of thymocytes in organ culture. Furthermore, we find that this inhibition is not due to the absence of certain lymphokines important for Tcell growth and maturation, nor to the loss of MHC class I1 expression in the thymic medullary epithelium.
2 Materials and methods 2.1 Fetal thymic organ cultures C57BL/6Ca mice were obtained from the National Cancer Institute breeding facility and were bred in our animal colony. The appearance of the vaginal plug was counted as day 0 of gestation. Organ culture was performed basically as described by others [13].Three to six thymic lobes were removed from fetuses at the indicated days of gestation and placed on HAWP 0.5 pm filters (Millipore Corp. Bedford, MA) which had been boiled in distilled water for 30 min and sterilized prior t o use. Filters were placed on gelatin sponges (Gelfoam: Upjohn Co., Kalamazoo, MI) which were immersed in 3 ml of culture medium (RPMI 1640 supplemented with 10% FCS, 10 pg/ml penicillin and streptomycin, 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 m~ nonessential amino acids and 5 X lop52-ME) with or without addition of other agents. Cultures were maintained in a 37 "C,5% COz environment and the medium and additives were changed every 3-4 days 0014-2980/90/0404-0753$02 solo
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of culture. At the end of the culture period, thymocytes were harvested by suspending the filters in 0.1% collagenase (Sigma Co., St. Louis, MO) solution at 37 "C for 30 min with agitation and vigorous pipetting. The collagenase was neutralized by washing the resulting cell suspension in an equal volume of FCS.
2.2 CsA and lymphokines CsA powder was the generous gift of Dr. B. Ryffel, Sandoz, Co.,Geneva, Swizerland. CsA was dissolved in DMSO at a concentration of 1 m g / d and then diluted into culture medium to a final concentration of 100 pg/ml for storage. Control cultures received the amount of DMSO equivalent to the highest dose of CsA used in each experiment. Murine rIL 2 was a generous gift of the Ajinomoto Co,Yokahama, Japan. D10.G4 SN was harvested from bulk cultures of the T H clone ~ D10.G4 stimulated for 3 days with conalbumin and irradiated AKR spleen cells. SN were tested for lymphokine activity by their ability to stimulate growth of the HT2 IL 2/IL 4-dependent cell line in the presence of appropriate blocking antibodies. The concentration of IL 4 in the D10.G4 SN used in these experiments was found to be = 16 U/ml.
Eur. J. Immunol. 1990. 20: 753-757
3 Results 3.1 T cells in day16 organ cultures sequentially aquire differentiation markers We chose to begin cultures at day 16of gestation as this was shown t o be the stage of development immediately preceding surface expression of the TcR a / p heterodimer [151. As defined by double staining with CD4 and CD8 at day 16 of gestation (Fig. l A ) , all populations were present except C D 4 T D 8 - cells, which are the last to appear during ontogeny. Only a small percentage of day-16 thymocytes were CD3+ (Fig. lB), and these cells probably expressed the TcR $6, as they did not stain with a mAb directed against theTcR a / p framework region [16]. After 8 days of organ culture (Fig. lC), large numbers of CD4+CD8- and 4,
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2.3 Immunofluorescence analysis of frozen tissue sections Thymus lobes were snap-frozen in OCT embedding compound and 10-pm cryostat sections were made.The sections were fixed in acetone for 20 min, blocked with 5% normal mouse serum (Pel-Freeze; Brown Deer,WI), and incubated with primary and secondary antibodies for 45 min each with the 3-min washes in between each step. The sections were mounted in 9 : 1glycerol :DABCO (Sigma Co.)in PBS, pH 8.6 to prevent quenching and were photographed with a Nikon photomicroscope. For FCM analysis, cells were resuspendedinFCM buffer (PBS with0.5% BSAandO.l% NaN3) at a concentration of = 1 x lo7 cells/ml and incubated with primary and secondary reagents for 20 min at 4 "C,with 2 washes between each step. After staining, the cells were fixed with 0.5% formaldehyde overnight and then returned to FCM buffer for analysis. FCM was performed on a modified Becton Dickinson (Sunnyvale, CA) FCM IV analyzer. Gates on the two-color analyses were determined by comparison to negative controls. For one-color CD3 staining, the percentage of CD3bn@"cells was determined as described by White [14]by reflecting the peak of the positively stained cell population around its axis after subtracting background fluorescence. The mAb used in these experiments were biotin-conjugated anti-CD8 and R-PE-conjugated anti-CD4 (Becton Dickinson) anti I-Ab (M5/114.15.2), anti-a/p framework region (H57-597), and anti-CD3~(500A2, a gift from Dr. Jim Allison, U.C. Berkeley). For two-color immunofluorescence, biotinylated antibodies were followed by streptavidin-FITC or streptavidin-R-PE (Tag0 Immunochemicals, Burlingame, CA). For double staining with CD4 or CD8 vs. CD3, cells were incubated with anti-CD3 antibody followed by FITCcoupled goat anti-hamster antibody, CD4-PE or CD8biotin was then added with a tenfold excess of rat IgG to prevent cross-reaction of the anti-hamster secondary antibody. CD8 staining was visualized by incubating the cells with streptavidin-PE.
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Figure I. Representative FCM profiles showing the effects of CsA on day-16 thymic lobes cultured for 8 days. Adult and fresh day-16 thymocytes are shown as controls. In the two-color profiles (A, C, E, G) staining was with anti-CD8-biotin followed by streptavidinFITC and anti-CD4-PE.The percentage of cells positive for control staining with streptavidin-FITC alone was subtracted from experimental values. Single-colorstaining (B, D, F, H) was performed by incubation with 1 pg/ml hamster Ig (dotted lines) or SN from the anti-CD3 hybridoma 500A2 (solid lines). Primary staining was followed by goat anti-hamster FITC.
Eur. J. Immunol. 1990. 20: 753-757
Cyclosporin A inhibits T cell development in vitro
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Table 1. Effccts o f CsA on thymocytc development in organ culture 500 ng/ml CsA. Although doublestaining with CD4 vs. CD8 showed little effect of CsA on the CD8+CD4- single-positive subset, this subset is known to contain both mature CD3+ TcR a / p and y/6 Tcells as well as an immature CD3- subset. Maturation of Tcells within
6.24 f 0.38 2.26 f 0.03 2.06 f 0.40 0.82 f 0.23 0.89 f 0.14 0.99 f 0.35 36.48 f 2.24 32.57 k 4.28 13.30 f 4.02
14.33 f 1.35 15.10 f 3.98
2.10
1.on I1.d.
a) Thymic lobes were removed from day-16 C57BL/6 mouse embryos and cultured for 8 days.Values are the mean f SEM, for experiments which were performed more than once. Most values represent the results of at least three separate experiments. Boldface entries indicate values which differ from controls with a significance of p < 0.05 in an unpaired separate variance t-test [26].
this population was affected by CsA, as double-staining with CD8 vs. CD3 showed a twofold decrease in the percentage of CD3+ cells within the CD8+ population in CsA-treated cultures compared with controls (Table 1). FCM analysis of reactivity with anti-CD3 mAb revealed a large decrease in numbers of CD3bnghtcells in CsA-treated cultures when compared to mock-treated controls (Fig. 1D and F). The percentage of cells expressing CD3 in CsAtreated cultures was similar to the adult thymus. In contrast to the adult thymus, however, most of the CD3+ cells developing in the presence of CsA lacked expression of the TcR a/p framework determinant, showing that CsA specifically affects development of these cells (Table 1). One explanation of these effects could be a toxic effect of CsA on matureTcells. However, this possibility was ruled out by the observation that addition of CsA for 3 days to day 16 fetal thymic lobes already cultured for 5 days did not reduce the levels of T cells with mature phenotype (data not shown). Taken together, this data demonstrates a specific and almost complete block in development of phenotypically mature TcR a/p Tcells imposed by CsA in the isolated thymus.
3.3 IL 18, IL 2 or IL 4 cannot reverse the CsA-induced differentiation block Since inhibition of IL 2 production is a major effect of CsA on peripheral Tcells, CsA may be blocking Tcell development in this system by inhibiting secretion of this or other lymphokines essential to Tcell development. To determine if this was the case, we added various sources of lymphokines thought to be important inTcell development to our culture system in the presence and absence of CsA.Without CsA, D10.G4 SN (a rich source of IL4), and rIL2 decreased the total yield of cells but did not affect significantly expression of CD4 or CD8 markers. IL l p increased total cell numbers, particularly those of mature phenotype, indicating that physiological quantities of the lymphokine were reaching the developing thymocytes in this system. However, none of the lymphokines tested,
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Eur. J. Immunol. 1990.20: 753-757
Table 2. UK lp, IL 2 and IL 4 fail to reverse the effects of CsAa)
Conditions
Cell number per lobe: Cells x 10-5 3 pglml CsA and 1 pglml CsA and 10 Ulml IL 1p IL 2 100 Ulml 20% D10 SN
Celldobe X cD4-CD8CD4+CD8+ CD4+CD8CD8+CD4~~3bmi
5.60 (- 0.2%) 5.60 (- 1.4%) 3.08 (-5.0%) 3.58(+31.7%) 0.79 (+ 15.2%) 0.69 (-28.5%) 0.34 (- 15.5%) 0.52 (- 0.25%) 1.26 (-2.9%) 0.67 (- 1.27%) 0.63(+5.8%) 1.22(+ 2.74%)
7.33 (- 2.67%) 2.01 (+3.79%) 2.97 (- 7.08%) 0.97 (+ 3.53%) 1.21 (+0.61%)
1.07(+4.04%)
including rIL l p and IL 2, D10.G4 SN, Con A SN, or the SN of the IL 2-secreting MLA cell line, could diminish the effects of CsA (Table 2). Thus a simple block in the secretion of any one of these lymphokines cannot account for the inhibition of T cell development by CsA.
a) Day-16 fetal lobes were cultured for 8 days in the presence of CsA with or without the indicated lymphokines. Thymocytes were harvested and analyzed by FCM.The numbers given in parenthesesfor each subset indicate the percent change in cell numbers compared to a parallel culture treated with CsA alone.
anti-IAb mAb showed no loss of class I1 expression in the medullary areas of CsA-treated organ-cultured lobes (Fig. 2).
4 Discussion 3.4 Class II-expressing cells are preserved in the medullae of CsA-treated thymic lobes
Medullary atrophy and a decrease in class I1 expression by epithelial cells in the medulla has been suggested to be a major effect of in vivo CsA treatment on the mouse and rat thymus [19]. However,whether or not medullary atrophy is a cause of the T cell maturation defect or a result of long-term treatment with CsA is not known.To resolve this question, we were interested in the effect of CsA on the distribution and density of class I1 expression in CsAtreated thymic lobes in organ culture. Histological analysis of frozen sections of CsA vs. mock-treated lobes did show somewhat smaller medullary areas in CsA-treated lobes. This could be a result of the smaller numbers of thymocytes filling the medullary compartment after CsA treatment, since FCM analysis of CsA-treated lobes revealed lower percentages of thymocytes with a medullary phenotype. More significantly, indirect immunofluorescence with an
The results of these experiments show that within the controlled and isolated environment of fetal thymic organ cultures, CsA specifically blocks the appearance of phenotypically mature CD4 and CD8 Tcells. As observed in the in vivo experiments with CsA,we found a selective effect onT cells expressing the TcR a@. However, sinceTcells 6/y may have already been present at the beginning of our culture period, we cannot make any direct conclusions about the effect of CsA on the development of these cells in organ culture.The fact that CsA blocksTcell development in vitro rules out any indirect effects such as the release of soluble mediators from other organs which may have been playing a role in the in vivo experiments. Our results correspond with a recently published report describing the effects of CsA in FTOC [20]. However, we observe an opposite effect on CD4+CD8+ immature thymocytes, seeing an overall increase in numbers as opposed to a decrease. This is probably because we began our cultures 1day later in gestation, when these cells were already present in the fetal
Figure 2. Immunostaining of frozen sections of cultured fetal thymic lobes. (A) and (C) were from day-16 lobes cultured for 8 days with diluent alone and (B) and (D) were from parallel cultures treated with 3 pglml CsA. (A) and (B) were incubated with 1:50 dilution of anti-LAb (M5/114) ascites and (C) and (D) were incubated with 1 pg/ml of rat IgG as a nonspecific control. Binding of the primar y antibodies was visualized by a second incubation with FITC-conjugated mouse
anti-rat IgG.
Eur. J. Immunol. 1990. 20: 753-757
Cyclosporin A inhibits T cell development in vitro
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thymus. In addition, our data on lymphokine addition and MHC class I1 expression in thymus sections yield novel insights into the possible mechanisms of CsA action in this system.
publication). These mice will enable us to define the conditions required for clonal deletion of these T cells in
The observations that class I1 expression was preserved in CsA-treated thymic lobes strongly argues against a reduction in the density of class I1 expression being a primary cause for the CsA-induced T cell maturation defect. Medullary epithelial cells may be more sensitive to the effects of long-term in v i v o treatment with CsA. Additionally, experimental down-modulation of class I1 expression has been shown to affect development of CD4 T cells only, while we have demonstrated decreases in both CD4 and CD8 subsets of mature T cell with CsA treatment. An earlier report [21] claimed that differentiation of CDVCD4- cells was unaffected by CsA. While this is superficially correct, our results show that the CD8 singlepositive cells which remain after CsA treatment are actually CD3- or TcR y/&expressing cells, neither of which are end products of the classical T cell differentiation pathway. We have not been able to reverse the effects of CsA with preparations containing large amounts of IL 1, 2, or 4, which have been implicated in the growth and differentiation of various immature thymocyte subsets [22,23]. Thus CsA does not appear to exert is effects through inhibiting the secretion of any one of these lymphokines. It is still possible however, that CsA works by inhibiting the secretion of other lymphokines alone or in combination. Experiments are in progress to investigate these possibilities.
We would like to thank Dr. Richard Schmidt for helpful advice, Dr. Allan Pickard for performing the FCM analyses; Drs. Michael R Cancro and Ali Naji for use of equipment; Sara Kinsman, Jennifer Punt, Dr. Lilly Goumnerova, and Dr. Yasu Hashimoto, for critical reading of this manuscript.
Another explanation for the dramatic effects of CsA on T cell development, and the one that is most consistent with our results, stems from recent advances in our knowledge about the effects of this agent on events inTcell activation. CsA specifically interferes with genetic events triggered by occupation of the TcR without affecting the immediate biochemical consequences of TcR cross-linking such as Ca2+ influx [24, 251. It is possible that CsA blocks the genetic events controlling differentiation and clonal deletion which are thought to result from TcR-mediated signaling in these cells. If CsA is acting solely on developing thymocytes in this system, in principle its effects should be reversible as is the inhibition of mature Tcell activation in vitro. In experiments designed to test this possibility, CsA was added to organ cultures and then removed by extensive washing with fresh medium. Remarkably, as little as 3 h of incubation with CsA was enough to reproduce the effects of CsA treatment for the whole culture period. In these experiments however, the possibility of retention of the drug by the organ lobes or filters after removal from the culture medium cannot be ruled out. Because of the relatively small numbers of cells generated in fetal thymic organ cultures it is difficult to examine the effect of CsA and other agents on clonal deletion of Tcells bearing specific Vg segments. However, we have recently created a transgenic mouse line in which almost 100% of T cells express a single Vg gene which confers reactivity against the Mlsa antigen (K. Yui et al., submitted for
vitro.
Received December 9, 1989.
5 References 1 Strominger, J. L., Science 1989. 244: 943. 2 Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M. and Von Boehmer, H., Nature 1988. 333: 742. 3 Kappler, J. W., Roehm, N. and Marrack, P., Cell. 1987. 49: 273. 4 MacDonald, H. R., Schneider, R., Lees, R. K., Howe, R . C., Acha-Orbea, H., Festenstein, H., Zinkernagel, R. M. and Hengartner, H . , Nature 1988. 332: 40. 5 Kisielow, P., Sia Heh, H., Bluthmann, H . and Von Boehmer, H., Nature 1988. 335: 730. 6 Sha, W. C., Nelson, C. A., Newberry, R. D., Kranz, D. M., Russell, J. H. and Loh, D.Y., Nature 1988. 336: 73. 7 Yui, K.,Wadsworth, S.,Yellen, A., Hashimoto,Y., Kokai,Y. and Greene, M. I., Immunol. Rev. 1988. 104: 120. 8 Kosugi, A . , Sharrow, S. 0. and Shearer, G. M., J. Immunol. 1989. 142: 3026: 9 Glazier, A., Tutchka, P.J., Farmer, E. R. and Santos, G.W., J. Exp. Med. 1983. 158: 1. 10 Sakaguchi, S. and Sakaguchi, N., J. Immunol. 1989. 142: 471. 11 Gao, E. K., Lo, D., Cheney, R . , Kanagawa, 0. and Sprent, J., Nature 1988. 336: 176. 12 Jenkins, M. K., Schwartz, R. H. and Pardoll, D. M., Science 1988. 241: 1655. 13 Shipman, €? M., Schmidt, R. R. and Chepenik, K. P., J. Immunol. 1988. 140: 2714. 14 White, J. A., Herman, A . , Pullen, A . M., Kubo, R., Kappler,J. W. and Marrack, P., Cell 1989. 56: 27. 15 Havran,W. L. and Allison, J. P., Nature 1988. 335: 443. 16 Kubo, R. T., Born,W., Kappler, J. W., Marrack, P. and Pigeon, M., J. Immunol. 1989. 142: 2736. 17 Scollay, R. and Shortman, K., in Watson, J., Marbrook, J. (Eds.), Recognition and Regulation in Cell-Mediated Immunity, Marcel Dekker, Inc., New York and Basel 1985, p. 3. 18 Kisielow, I?, Leiserson,W. and Von Boehmer, H., J. Immunol. 1984. 133: 1117. 19 Cheney, R. T. and Sprent, J., Transplant. Proc. 1985. XVII: 528. 20 Kosugi, A . , Zuniga-Pflucker, J. C., Sharrow, S., Kruisbeek, A. and Shearer, G . , J. Immunol. 1989. 143: 3134. 21 Takeuchi,Y., Habu, S., Okumura, K. and Suzuki, G . , Immunology 1989. 66: 362. 22 Raulet, D. H., Nature 1985. 314: 101. 23 Zlotnik, A . , Ransom, J., Frank, G., Fischer, M. and Howard, M., Proc. Natl. Acad. Sci. USA 1987. 84: 3856. 24 Wiskocil, R., Weiss, A . , Imboden, J., Kamin-Lewis, R. and Stobo, J., J. Immunol. 1985. 134: 1599. 25 Crabtree, G . R., Science 1989. 243: 355. 26 Ott, L., An Introduction to Statistical Methods and Data Analysis. 3rd edit., PWS-Kent, Boston 1988, p. 175.
Eur. J. Immunol. 1990. 20: 753-757
Richard M. SiegelAo, Katsuyuki Yuioo, Drew E. Tenenholzo, Ralph Kubo+ and Mark I. Greenen Division of Immunology, Department of Pathology and Laboratory Medicineo, University of Pennsylvania Medical School, Philadelphia and Department of Medicine+, National Jewish Center for Immunology and Respiratory Medicine, Denver
Inhibition of T cell developmemt in thymic organ culture: implications for the mechanism of action of cyclosporin A* We have examined the effects of the immunosuppressive drug cyclosporin A (CsA) on the phenotypic maturation of Tcells in thymic organ cultures begun at day 16 of gestation. CsA specifically inhibited the generation of cells expressing high levels of a@ TcR/CD3 complexes and a mature phenotype defined by CD4 and CD8 surface markers. Adding interleukin (IL) lfi, IL 2 or IL 4 failed to reverse the effects of CsA, and major histocompatibility complex class I1 expression in the thymic medulla was preserved. Possible mechanisms of CsA-mediated inhibition of T cell development are discussed.
1 Introduction In the past few years, much has been learned about the pathways of intrathymic T cell development, but understanding the molecular mechanisms underlying T cell repertoire selection remains one of the most challenging and central problems in immunology. In the thymus,Tcells bearing TcR a/p and either CD4 or CD8 are thought to be generated from CD4+CD8+ precursors [l]. Observations made with TcR transgenic mice and other experiments suggest that aTcell will not mature past the double-positive CD4+CD8+stage if its TcR is reactive with self antigens in the thymus (negative selection) [2-4]. However, some engagement of antigen receptors with self components must be required (positive selection) since T cells with certain receptors will not mature in a completely allogeneic environment [ 5 , 61. The soluble factors, cellular interactions, and genetic events which regulate these important processes are largely unknown. Our laboratory has studied mutations which disrupt T cell development in order to better understand the requirements for normal differentiation of matureTcells from their precursors [71. The immunosuppressive drug cyclosporin A (CsA) is a pharmacological agent which also has dramatic effects on T cell development and the acquisition of self tolerance. CsA chronically administered to rodents during normal ontogeny or after BM transplantation decreases matureTcells present in the thymus and LN [8]. However, when CsA is withdrawn, organ-specific autoimmune disease results [9, 101.The repertoire of Tcells which matures in the presence of CsA includes T cells bearing potentially self-reactive receptors, although it has not been proven that
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These studies were supported by the Lucille Markey Charitable Trust and grants from the N.I.H., N.E.I. and N.C.I. to M.I.G. Special Fellow of the Leukemia Society of America. Trainee in the Medical Scientist Training Program at the University of Pennsylvania Medical School (N.I.H. grant #T-32-GM-07170).
Correspondence: Mark I. Greene, Department of Pathology, University of Pennsylvania School of Medicine, 36th St. and Hamilton Walk, Philadelphia, PA 19104-6082,USA Abbreviations: CsA: Cyclosporin A 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
these cells actually mediate the post-CsA autoimmune syndrome [ l l , 121. Because of the effects of CsA on both positive and negative selection of the Tcell repertoire, it is important to understand how this drug is acting on the cells and mediators involved in intrathymic T cell maturation. Previous in vivo experiments with CsA have not been able t o determine the primary mechanism by which it inhibitsT cell development. It is possible that CsA is acting only indirectly on the developing thymus through inhibiting development of stem cells in the BM or release of soluble mediators by other organs.To study the effects of CsA onT cell development in more detail, and attempt to identify its mechanism of action, we have investigated the effect of CsA on thymocyte differentiation in fetal thymic organ culture, which is currently the only system in which Tcell development can be studied in vitro. Using multiparameter FCM analysis and immunostaining of tissue sections, we demonstrate that CsA specifically inhibits the maturation of thymocytes in organ culture. Furthermore, we find that this inhibition is not due to the absence of certain lymphokines important for Tcell growth and maturation, nor to the loss of MHC class I1 expression in the thymic medullary epithelium.
2 Materials and methods 2.1 Fetal thymic organ cultures C57BL/6Ca mice were obtained from the National Cancer Institute breeding facility and were bred in our animal colony. The appearance of the vaginal plug was counted as day 0 of gestation. Organ culture was performed basically as described by others [13].Three to six thymic lobes were removed from fetuses at the indicated days of gestation and placed on HAWP 0.5 pm filters (Millipore Corp. Bedford, MA) which had been boiled in distilled water for 30 min and sterilized prior t o use. Filters were placed on gelatin sponges (Gelfoam: Upjohn Co., Kalamazoo, MI) which were immersed in 3 ml of culture medium (RPMI 1640 supplemented with 10% FCS, 10 pg/ml penicillin and streptomycin, 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 m~ nonessential amino acids and 5 X lop52-ME) with or without addition of other agents. Cultures were maintained in a 37 "C,5% COz environment and the medium and additives were changed every 3-4 days 0014-2980/90/0404-0753$02 solo
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of culture. At the end of the culture period, thymocytes were harvested by suspending the filters in 0.1% collagenase (Sigma Co., St. Louis, MO) solution at 37 "C for 30 min with agitation and vigorous pipetting. The collagenase was neutralized by washing the resulting cell suspension in an equal volume of FCS.
2.2 CsA and lymphokines CsA powder was the generous gift of Dr. B. Ryffel, Sandoz, Co.,Geneva, Swizerland. CsA was dissolved in DMSO at a concentration of 1 m g / d and then diluted into culture medium to a final concentration of 100 pg/ml for storage. Control cultures received the amount of DMSO equivalent to the highest dose of CsA used in each experiment. Murine rIL 2 was a generous gift of the Ajinomoto Co,Yokahama, Japan. D10.G4 SN was harvested from bulk cultures of the T H clone ~ D10.G4 stimulated for 3 days with conalbumin and irradiated AKR spleen cells. SN were tested for lymphokine activity by their ability to stimulate growth of the HT2 IL 2/IL 4-dependent cell line in the presence of appropriate blocking antibodies. The concentration of IL 4 in the D10.G4 SN used in these experiments was found to be = 16 U/ml.
Eur. J. Immunol. 1990. 20: 753-757
3 Results 3.1 T cells in day16 organ cultures sequentially aquire differentiation markers We chose to begin cultures at day 16of gestation as this was shown t o be the stage of development immediately preceding surface expression of the TcR a / p heterodimer [151. As defined by double staining with CD4 and CD8 at day 16 of gestation (Fig. l A ) , all populations were present except C D 4 T D 8 - cells, which are the last to appear during ontogeny. Only a small percentage of day-16 thymocytes were CD3+ (Fig. lB), and these cells probably expressed the TcR $6, as they did not stain with a mAb directed against theTcR a / p framework region [16]. After 8 days of organ culture (Fig. lC), large numbers of CD4+CD8- and 4,
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2.3 Immunofluorescence analysis of frozen tissue sections Thymus lobes were snap-frozen in OCT embedding compound and 10-pm cryostat sections were made.The sections were fixed in acetone for 20 min, blocked with 5% normal mouse serum (Pel-Freeze; Brown Deer,WI), and incubated with primary and secondary antibodies for 45 min each with the 3-min washes in between each step. The sections were mounted in 9 : 1glycerol :DABCO (Sigma Co.)in PBS, pH 8.6 to prevent quenching and were photographed with a Nikon photomicroscope. For FCM analysis, cells were resuspendedinFCM buffer (PBS with0.5% BSAandO.l% NaN3) at a concentration of = 1 x lo7 cells/ml and incubated with primary and secondary reagents for 20 min at 4 "C,with 2 washes between each step. After staining, the cells were fixed with 0.5% formaldehyde overnight and then returned to FCM buffer for analysis. FCM was performed on a modified Becton Dickinson (Sunnyvale, CA) FCM IV analyzer. Gates on the two-color analyses were determined by comparison to negative controls. For one-color CD3 staining, the percentage of CD3bn@"cells was determined as described by White [14]by reflecting the peak of the positively stained cell population around its axis after subtracting background fluorescence. The mAb used in these experiments were biotin-conjugated anti-CD8 and R-PE-conjugated anti-CD4 (Becton Dickinson) anti I-Ab (M5/114.15.2), anti-a/p framework region (H57-597), and anti-CD3~(500A2, a gift from Dr. Jim Allison, U.C. Berkeley). For two-color immunofluorescence, biotinylated antibodies were followed by streptavidin-FITC or streptavidin-R-PE (Tag0 Immunochemicals, Burlingame, CA). For double staining with CD4 or CD8 vs. CD3, cells were incubated with anti-CD3 antibody followed by FITCcoupled goat anti-hamster antibody, CD4-PE or CD8biotin was then added with a tenfold excess of rat IgG to prevent cross-reaction of the anti-hamster secondary antibody. CD8 staining was visualized by incubating the cells with streptavidin-PE.
11.1
3
e.d. 16+8 3pgfmlCsA
2 1 0
I
4-1
I
I Adult Thymus
3 2 1 0
0
1
2
3 CD8
4
5
,
.
.
.
.
.
0
1
2
3
4
5
CD3
Figure I. Representative FCM profiles showing the effects of CsA on day-16 thymic lobes cultured for 8 days. Adult and fresh day-16 thymocytes are shown as controls. In the two-color profiles (A, C, E, G) staining was with anti-CD8-biotin followed by streptavidinFITC and anti-CD4-PE.The percentage of cells positive for control staining with streptavidin-FITC alone was subtracted from experimental values. Single-colorstaining (B, D, F, H) was performed by incubation with 1 pg/ml hamster Ig (dotted lines) or SN from the anti-CD3 hybridoma 500A2 (solid lines). Primary staining was followed by goat anti-hamster FITC.
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Table 1. Effccts o f CsA on thymocytc development in organ culture 500 ng/ml CsA. Although doublestaining with CD4 vs. CD8 showed little effect of CsA on the CD8+CD4- single-positive subset, this subset is known to contain both mature CD3+ TcR a / p and y/6 Tcells as well as an immature CD3- subset. Maturation of Tcells within
6.24 f 0.38 2.26 f 0.03 2.06 f 0.40 0.82 f 0.23 0.89 f 0.14 0.99 f 0.35 36.48 f 2.24 32.57 k 4.28 13.30 f 4.02
14.33 f 1.35 15.10 f 3.98
2.10
1.on I1.d.
a) Thymic lobes were removed from day-16 C57BL/6 mouse embryos and cultured for 8 days.Values are the mean f SEM, for experiments which were performed more than once. Most values represent the results of at least three separate experiments. Boldface entries indicate values which differ from controls with a significance of p < 0.05 in an unpaired separate variance t-test [26].
this population was affected by CsA, as double-staining with CD8 vs. CD3 showed a twofold decrease in the percentage of CD3+ cells within the CD8+ population in CsA-treated cultures compared with controls (Table 1). FCM analysis of reactivity with anti-CD3 mAb revealed a large decrease in numbers of CD3bnghtcells in CsA-treated cultures when compared to mock-treated controls (Fig. 1D and F). The percentage of cells expressing CD3 in CsAtreated cultures was similar to the adult thymus. In contrast to the adult thymus, however, most of the CD3+ cells developing in the presence of CsA lacked expression of the TcR a/p framework determinant, showing that CsA specifically affects development of these cells (Table 1). One explanation of these effects could be a toxic effect of CsA on matureTcells. However, this possibility was ruled out by the observation that addition of CsA for 3 days to day 16 fetal thymic lobes already cultured for 5 days did not reduce the levels of T cells with mature phenotype (data not shown). Taken together, this data demonstrates a specific and almost complete block in development of phenotypically mature TcR a/p Tcells imposed by CsA in the isolated thymus.
3.3 IL 18, IL 2 or IL 4 cannot reverse the CsA-induced differentiation block Since inhibition of IL 2 production is a major effect of CsA on peripheral Tcells, CsA may be blocking Tcell development in this system by inhibiting secretion of this or other lymphokines essential to Tcell development. To determine if this was the case, we added various sources of lymphokines thought to be important inTcell development to our culture system in the presence and absence of CsA.Without CsA, D10.G4 SN (a rich source of IL4), and rIL2 decreased the total yield of cells but did not affect significantly expression of CD4 or CD8 markers. IL l p increased total cell numbers, particularly those of mature phenotype, indicating that physiological quantities of the lymphokine were reaching the developing thymocytes in this system. However, none of the lymphokines tested,
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Table 2. UK lp, IL 2 and IL 4 fail to reverse the effects of CsAa)
Conditions
Cell number per lobe: Cells x 10-5 3 pglml CsA and 1 pglml CsA and 10 Ulml IL 1p IL 2 100 Ulml 20% D10 SN
Celldobe X cD4-CD8CD4+CD8+ CD4+CD8CD8+CD4~~3bmi
5.60 (- 0.2%) 5.60 (- 1.4%) 3.08 (-5.0%) 3.58(+31.7%) 0.79 (+ 15.2%) 0.69 (-28.5%) 0.34 (- 15.5%) 0.52 (- 0.25%) 1.26 (-2.9%) 0.67 (- 1.27%) 0.63(+5.8%) 1.22(+ 2.74%)
7.33 (- 2.67%) 2.01 (+3.79%) 2.97 (- 7.08%) 0.97 (+ 3.53%) 1.21 (+0.61%)
1.07(+4.04%)
including rIL l p and IL 2, D10.G4 SN, Con A SN, or the SN of the IL 2-secreting MLA cell line, could diminish the effects of CsA (Table 2). Thus a simple block in the secretion of any one of these lymphokines cannot account for the inhibition of T cell development by CsA.
a) Day-16 fetal lobes were cultured for 8 days in the presence of CsA with or without the indicated lymphokines. Thymocytes were harvested and analyzed by FCM.The numbers given in parenthesesfor each subset indicate the percent change in cell numbers compared to a parallel culture treated with CsA alone.
anti-IAb mAb showed no loss of class I1 expression in the medullary areas of CsA-treated organ-cultured lobes (Fig. 2).
4 Discussion 3.4 Class II-expressing cells are preserved in the medullae of CsA-treated thymic lobes
Medullary atrophy and a decrease in class I1 expression by epithelial cells in the medulla has been suggested to be a major effect of in vivo CsA treatment on the mouse and rat thymus [19]. However,whether or not medullary atrophy is a cause of the T cell maturation defect or a result of long-term treatment with CsA is not known.To resolve this question, we were interested in the effect of CsA on the distribution and density of class I1 expression in CsAtreated thymic lobes in organ culture. Histological analysis of frozen sections of CsA vs. mock-treated lobes did show somewhat smaller medullary areas in CsA-treated lobes. This could be a result of the smaller numbers of thymocytes filling the medullary compartment after CsA treatment, since FCM analysis of CsA-treated lobes revealed lower percentages of thymocytes with a medullary phenotype. More significantly, indirect immunofluorescence with an
The results of these experiments show that within the controlled and isolated environment of fetal thymic organ cultures, CsA specifically blocks the appearance of phenotypically mature CD4 and CD8 Tcells. As observed in the in vivo experiments with CsA,we found a selective effect onT cells expressing the TcR a@. However, sinceTcells 6/y may have already been present at the beginning of our culture period, we cannot make any direct conclusions about the effect of CsA on the development of these cells in organ culture.The fact that CsA blocksTcell development in vitro rules out any indirect effects such as the release of soluble mediators from other organs which may have been playing a role in the in vivo experiments. Our results correspond with a recently published report describing the effects of CsA in FTOC [20]. However, we observe an opposite effect on CD4+CD8+ immature thymocytes, seeing an overall increase in numbers as opposed to a decrease. This is probably because we began our cultures 1day later in gestation, when these cells were already present in the fetal
Figure 2. Immunostaining of frozen sections of cultured fetal thymic lobes. (A) and (C) were from day-16 lobes cultured for 8 days with diluent alone and (B) and (D) were from parallel cultures treated with 3 pglml CsA. (A) and (B) were incubated with 1:50 dilution of anti-LAb (M5/114) ascites and (C) and (D) were incubated with 1 pg/ml of rat IgG as a nonspecific control. Binding of the primar y antibodies was visualized by a second incubation with FITC-conjugated mouse
anti-rat IgG.
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thymus. In addition, our data on lymphokine addition and MHC class I1 expression in thymus sections yield novel insights into the possible mechanisms of CsA action in this system.
publication). These mice will enable us to define the conditions required for clonal deletion of these T cells in
The observations that class I1 expression was preserved in CsA-treated thymic lobes strongly argues against a reduction in the density of class I1 expression being a primary cause for the CsA-induced T cell maturation defect. Medullary epithelial cells may be more sensitive to the effects of long-term in v i v o treatment with CsA. Additionally, experimental down-modulation of class I1 expression has been shown to affect development of CD4 T cells only, while we have demonstrated decreases in both CD4 and CD8 subsets of mature T cell with CsA treatment. An earlier report [21] claimed that differentiation of CDVCD4- cells was unaffected by CsA. While this is superficially correct, our results show that the CD8 singlepositive cells which remain after CsA treatment are actually CD3- or TcR y/&expressing cells, neither of which are end products of the classical T cell differentiation pathway. We have not been able to reverse the effects of CsA with preparations containing large amounts of IL 1, 2, or 4, which have been implicated in the growth and differentiation of various immature thymocyte subsets [22,23]. Thus CsA does not appear to exert is effects through inhibiting the secretion of any one of these lymphokines. It is still possible however, that CsA works by inhibiting the secretion of other lymphokines alone or in combination. Experiments are in progress to investigate these possibilities.
We would like to thank Dr. Richard Schmidt for helpful advice, Dr. Allan Pickard for performing the FCM analyses; Drs. Michael R Cancro and Ali Naji for use of equipment; Sara Kinsman, Jennifer Punt, Dr. Lilly Goumnerova, and Dr. Yasu Hashimoto, for critical reading of this manuscript.
Another explanation for the dramatic effects of CsA on T cell development, and the one that is most consistent with our results, stems from recent advances in our knowledge about the effects of this agent on events inTcell activation. CsA specifically interferes with genetic events triggered by occupation of the TcR without affecting the immediate biochemical consequences of TcR cross-linking such as Ca2+ influx [24, 251. It is possible that CsA blocks the genetic events controlling differentiation and clonal deletion which are thought to result from TcR-mediated signaling in these cells. If CsA is acting solely on developing thymocytes in this system, in principle its effects should be reversible as is the inhibition of mature Tcell activation in vitro. In experiments designed to test this possibility, CsA was added to organ cultures and then removed by extensive washing with fresh medium. Remarkably, as little as 3 h of incubation with CsA was enough to reproduce the effects of CsA treatment for the whole culture period. In these experiments however, the possibility of retention of the drug by the organ lobes or filters after removal from the culture medium cannot be ruled out. Because of the relatively small numbers of cells generated in fetal thymic organ cultures it is difficult to examine the effect of CsA and other agents on clonal deletion of Tcells bearing specific Vg segments. However, we have recently created a transgenic mouse line in which almost 100% of T cells express a single Vg gene which confers reactivity against the Mlsa antigen (K. Yui et al., submitted for
vitro.
Received December 9, 1989.
5 References 1 Strominger, J. L., Science 1989. 244: 943. 2 Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M. and Von Boehmer, H., Nature 1988. 333: 742. 3 Kappler, J. W., Roehm, N. and Marrack, P., Cell. 1987. 49: 273. 4 MacDonald, H. R., Schneider, R., Lees, R. K., Howe, R . C., Acha-Orbea, H., Festenstein, H., Zinkernagel, R. M. and Hengartner, H . , Nature 1988. 332: 40. 5 Kisielow, P., Sia Heh, H., Bluthmann, H . and Von Boehmer, H., Nature 1988. 335: 730. 6 Sha, W. C., Nelson, C. A., Newberry, R. D., Kranz, D. M., Russell, J. H. and Loh, D.Y., Nature 1988. 336: 73. 7 Yui, K.,Wadsworth, S.,Yellen, A., Hashimoto,Y., Kokai,Y. and Greene, M. I., Immunol. Rev. 1988. 104: 120. 8 Kosugi, A . , Sharrow, S. 0. and Shearer, G. M., J. Immunol. 1989. 142: 3026: 9 Glazier, A., Tutchka, P.J., Farmer, E. R. and Santos, G.W., J. Exp. Med. 1983. 158: 1. 10 Sakaguchi, S. and Sakaguchi, N., J. Immunol. 1989. 142: 471. 11 Gao, E. K., Lo, D., Cheney, R . , Kanagawa, 0. and Sprent, J., Nature 1988. 336: 176. 12 Jenkins, M. K., Schwartz, R. H. and Pardoll, D. M., Science 1988. 241: 1655. 13 Shipman, €? M., Schmidt, R. R. and Chepenik, K. P., J. Immunol. 1988. 140: 2714. 14 White, J. A., Herman, A . , Pullen, A . M., Kubo, R., Kappler,J. W. and Marrack, P., Cell 1989. 56: 27. 15 Havran,W. L. and Allison, J. P., Nature 1988. 335: 443. 16 Kubo, R. T., Born,W., Kappler, J. W., Marrack, P. and Pigeon, M., J. Immunol. 1989. 142: 2736. 17 Scollay, R. and Shortman, K., in Watson, J., Marbrook, J. (Eds.), Recognition and Regulation in Cell-Mediated Immunity, Marcel Dekker, Inc., New York and Basel 1985, p. 3. 18 Kisielow, I?, Leiserson,W. and Von Boehmer, H., J. Immunol. 1984. 133: 1117. 19 Cheney, R. T. and Sprent, J., Transplant. Proc. 1985. XVII: 528. 20 Kosugi, A . , Zuniga-Pflucker, J. C., Sharrow, S., Kruisbeek, A. and Shearer, G . , J. Immunol. 1989. 143: 3134. 21 Takeuchi,Y., Habu, S., Okumura, K. and Suzuki, G . , Immunology 1989. 66: 362. 22 Raulet, D. H., Nature 1985. 314: 101. 23 Zlotnik, A . , Ransom, J., Frank, G., Fischer, M. and Howard, M., Proc. Natl. Acad. Sci. USA 1987. 84: 3856. 24 Wiskocil, R., Weiss, A . , Imboden, J., Kamin-Lewis, R. and Stobo, J., J. Immunol. 1985. 134: 1599. 25 Crabtree, G . R., Science 1989. 243: 355. 26 Ott, L., An Introduction to Statistical Methods and Data Analysis. 3rd edit., PWS-Kent, Boston 1988, p. 175.
