- Email: [email protected]
CD45RA+ and CD45RO+ T cells differ in susceptibility to cyclosporin A mediated inhibition of interleukin-2 production
Transplant Immunology 1996: 4 : 6 1 - 6 3
C D 4 5 R A + and C D 4 5 R O + T cells differ in susceptibility to cyclosporin A mediated inhibition of interleukin-2 production Reinhard Schwinzer and Renate Siefken Transplantation Laboratory, Clinic for Abdominal and Transplantation Surge©', Medical Highschool Hannover, Hannover
Abstract: Lymphocytes in different states of activation use different intracellular signalling pathways and
may therefore differ in their susceptibility to immunosuppressive agents. In this study we examined the proliferation and production of interleukin-2 (IL-2) by unprimed/naive CD4~CD45RA - T cells and previously activated/memory CD4+CD45RO + T cells from human peripheral blood when stimulated in vilro in the presence of cyclosporin A (CsA). Further, the dependency of the IL-2 response on calcium (Ca>l ions was analysed by the addition of the chelating agent EGTA. The CD4+CD45RO ~ memory T cells were shown to be less susceptible to CsA and less dependent on the level of Ca + ions than the naive CD4+CD45RA + T cells. The subcellular mechanisnls involved in this difference and the potential clinical implications arc discussed.
Introduction
Objective
Despite the broad inhibitory effects of cyclosporin A (CsA), a 30 60% incidence of acute graft rejection still occurs in allografted patients, suggesting the existence of CsA-resistant pathways of T cell activation. The in vitro induction of interleukin-2 (IL-2) production by CD28 mAb (monoclonal antibody) in combination with phorbol ester (PMA) has been demonstrated to be resistant to CsA-mediated inhibition. ~ In contrast, IL-2 production initiated through the T cell receptor (TCR) can usually be effectively inhibited by CsA. The molecular mechanism of CsA-mediated inhibition of T cell activation is the capacity of the drug to interfere with TCR-associated signalling pathways. 2 Recent data suggest that T cells of different activation and/or differentiation states use different signalling pathways for the induction of cytokine gene transcription.3 5 Thus, it is conceivable that different T cell subsets may differ in susceptibility to CsA-mediated immunosuppression.
We studied this question of differential susceptibility to CsA by analysing the effects of CsA on IL-2 synthesis in CD4+CD45RA + (unprimed/naive) and CD4+CD45RO + {previously activated/memory) T cells. The results show that CD4+CD45RO + T cells are less susceptible to CsA-mediated inhibition of IL-2 synthesis compared to the CD4+CD45RA + T cell subset.
Address for correspondence: Reinhard Schwinzer, Transplantationslabor K25, Klinik ftir Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, D-30623 Hannover. Germany. © Arnold 1996
Materials and methods Cells PBMC (peripheral blood mononuclear cells) were isolated from heparinized peripheral human blood by Ficoll gradient centrifugation. Adherent mononuclear cells were removed by plastic adherence overnight at 37°C. Small resting T cells were prepared from nonadherent cells by E-rosetting. CD4+CD45RA + and CD4+CD45RO + T cells were isolated from E + cells by negative selection using the panning technique. Cells were incubated for 30 min on ice with mAb AICD8.1 (CDS, provided by Dr B Schraven, Heidelberg, Germany) plus M E M 56 (CD45RA, provided by Dr V Horejsi, Prague, Czech Republic) or AICD8.1 plus UCHL1 (CD45RO, donated by Dr PCL Beverley, London, UK), washed and lay-
62
R Schwinzer and R Siefken
ered on petri dishes coated with 101xg/ml goat anti-mouse IgG + IgM. After 2 h at 4°C, n o n a d h e r e n t cells were carefully collected, w a s h e d twice and reanalysed. Usually less than 6% of cells from the depleted subset were detected in these preparations as determined by flow cytometry analysis.
T cell activation protocols Purified CD4+CD45RA + and CD4+CD45RO ÷ T cells were stimulated with 1.5 I-~g/ml B M A 031 (anti-(~[3TCR mAb, provided by Dr R Kurrle, Behringwerke, Marburg, G e r m a n y ) in c o m b i n a t i o n with P M A (1 ng/ml) in R P M I 1640 m e d i u m supplemented with 10% FCS (fetal calf serum), 50 U/ml penicillin, 50 Ixg/ml streptomycin, and 4 m M L-glutamine. To study the effect of CsA, cells were incubated with C s A 1 h before stimulation and during culture. C s A was diluted in ethanol and titrated into the culture system ( 2 5 - 2 0 0 ng/ml). The final ethanol concentration was less than 0.5% (v/v) and had no effect on the induction of IL-2 synthesis and proliferation as determined in control experiments. To study the role of calcium (Ca2+), cells were stimulated in the presence of various concentrations of the Ca 2+ chelator E G T A ( 0 . 3 - 0 . 6 m M ) . M e a s u r e m e n t of IL-2 in the supernatant of cell cultures was performed using the IL-2-dependent rat T cell line G2 as described. 6 Results are expressed either as IL-2 units/ml or as proliferative response (cpm) induced in G2 cells.
Results Stimulation of C D 4 + C D 4 5 R A + and CD4+CD45RO + T cells with m A b B M A 031 (anti-c~[3TCR) plus P M A induced similar amounts of IL-2 in the two subsets (Table 1). Treatment of C D 4 + C D 4 5 R A + cells with C s A 50 ng/ml resulted in complete inhibition of IL-2 production. In contrast, significant amounts of IL-2 ( 2 0 - 3 0 U/ml) were detected in the supernatant of CD4+CD45RO + cells stimulated in the presence of C s A 100 ng/ml. C s A is k n o w n to inhibit calcium-dependent signalling pathways by interfering with the serine/threonine phosphatase calcineurin. 7 The differential susceptibility of CD4+CD45RA + and CD4+CD45RO + T cells to C s A could therefore indicate that the two subsets differ in the requirement for calcium to
induce IL-2 gene transcription. To examine this possibility we stimulated CD4+CD45RA + and CD4+CD45RO + cells in the presence of increasing concentrations of the Ca2+-chelator EGTA. The IL-2 activity found in the supernatant of C D 4 5 R A + cells decreased with increasing concentrations of E G T A and was completely absent in cultures stimulated in the presence of 0.5 m M E G T A (Figure 1). In contrast, low concentrations of E G T A (0.3-0.4 m M ) did not inhibit IL-2 production in C D 4 5 R O + cells. A n inhibitory effect was first observed when cells were stimulated in the presence of 0.45 m M EGTA, but even in the presence of 0.6 m M E G T A C D 4 5 R O + cells still produced detectable amounts of IL-2. This pattern of reactivity suggests that the induction of IL-2 production in C D 4 5 R O + cells is less dependent on Ca 2+ c o m p a r e d to C D 4 5 R A + cells.
Discussion T C R - m e d i a t e d elevation of the cytoplasmic calcium concentration [Ca2+]~ is regarded to be an essential step in the induction of IL-2 gene transcription and subsequent IL-2 production, s A consequence of elevated levels of [Ca2+]i is the activation of calcineurin. This serine/threonine phosphatase regulates NF-AT, an important transcription factor involved in the induction of IL-2 gene transcription. 9 The molecular m e c h a n i s m of action of CsA is the inhibition of calcineurin, 7 thereby preventing the induction of IL-2 gene transcription. The data presented here suggest that TCR-associated signalling pathways leading to the induction of IL-2 synthesis are less dependent on Ca 2+ in CD4+CD45RO + m e m o r y T cells
60000
50000E (._ 4 0 0 0 0 -
.o 0
30000-
O (3
._ Table 1 Effect of CsA on IL-2 synthesis in TCR-stimulated CD4+ T cell subsets Stimulus
Subset
20000-
CsA (ng/ml)
10000-
BMA 031 plus PMA
CD45RA + CD45RO +
0
25
50
100
200
75 82
22 62
3 52
<1 26
<1 <1
CD4+CD45RA + and CD4+CD45RO + T cells were stimulated with the antiaI3TCR mAb BMA 031 in combination with PMA. Stimulation was performed in the absence of CsA and in the presence of increasing concentrations of the immunosuppressive drug. After 24 h the culture supernatant was harvested and titrated on the IL-2-dependent cell line G2. Defined serial dilutions of recombinant IL-2 were included in each experiment for control. Proliferation of G2 cells induced by the supernatants was then compared with the IL-2 titration curve to calculate U/ml. Stimulation with either the mAb or PMA alone did not trigger IL-2 synthesis in CD45RA + and CD45RO + cells.
Transplant Immunology 1996; 4 : 6 1 - 6 3
0 0
0.3 0.35 0.4 0.45 0.5
0.6
EGTA conc. (mM) Figure 1 Effect of the Ca2+-chelator EGTA on IL-2 production. CD+CD45RA + and CD4+CD45RO + T cells were negatively isolated and stimulated with the anti-c~[3TCR mAb BMA 031 in combination with PMA. Cells were cultured in the absence or in the presence of increasing concentrations of EGTA. Supernatants from stimulated CD4+CD45RA + (O) and CD4+CD45RO + (11) T cells were harvested after 24 h and titrated on the IL-2-dependent rat T cell line G2. Results are expressed as proliferation of G2 cells in the presence of 25% cell culture supernatant.
Differential susceptibility o f T cell subsets to C s A - m e d i a t e d i m m u n o s u p p r e s s i o n
compared to the CD4+CD45RA + naive T cell subset. The two cell types therefore differ in susceptibility to CsA-mediated inhibition of IL-2 production. The differential susceptibility of C D 4 5 R A + and C D 4 5 R O + cells to CsA-mediated inhibition could either be explained by quantitative or qualitative differences in signalling pathways. Thus, C D 4 5 R O + cells might contain higher basic levels of [Ca-'+]~ or respond to TCR signalling with a stronger increase in [Ca 2~ I,. In both cases, effective inhibition of the signalling pathway would require more CsA compared to C D 4 5 R A + cells. However, the analysis of ICa2~]. in resting cells and in cells stimulated by C D 3 / T C R m A b did not reveal significant differences between the two subsets (data not shown). In addition, by immunoprecipitation experiments we were able to show that C D 4 5 R A + and C D 4 5 R O + cells contain similar amounts of the CsA target molecule calcineurin (data not shown). Together these lindings suggest that the differential susceptibility of the two subsets to CsA is due to qualitative differences in signalling pathways rather than to quantitative differences. The induction of 1L-2 gene transcription is regarded to require two major signalling pathways'~: a Ca'+-dependent pathway leading to calcineurin-mediated dephosphorylation and nuclear translocation of the cytoplasmic c o m p o n e n t of NF-AT and a cascade of k i n a s e s - - i n c l u d i n g protein kinase C (PKC) resulting in the activation of the nuclear c o m p o n e n t of NF-AT. Since the induction of IL-2 production did not require optimal amounts of Ca > in CD45RO* cells (Figure 1 ), one might assume thai IL-2 gene transcription in C D 4 5 R O + cells can be induced by sole activation of PKC. However, activation of PKC by phorbol ester alone did not trigger IL-2 synthesis in C D 4 5 R O - cells, suggesting that in addition to PKC actiwttion a second signal is also required in these cells. If the second signal is mediated via calcineurin, the activation of calcineurin in C D 4 5 R O + cells might be less dependent on Ca ~+ compared to C D 4 5 R A ' cells. Alternatively, it could be possible that dephosphorylation and nuclear translocation of the cytoplasmic c o m p o n e n t of N F - A T is not controlled by calcineurin in C D 4 5 R O ' cells. Significant differences in the regulation of IL-2 gene transcription between C D 4 5 R A - and C D 4 5 R O ' cells are also suggested by the recent demonstration of a silencer of the 1L-2 gene in C D 4 5 R A - but not in C D 4 5 R O + T cells. '° On the basis of the presented data it is likely that the subset composition of the immune system determines the in vivo effects of CsA. Thus, immunosuppression by standard CsA protocols might fail when an immune response is preferentially mediated by C D 4 5 R O ~ cells or when this subset had been
7Yansp/anl lmmuno/ogy 1996;
4:61-63
63
expanded by previous antigenic stimulation. An increased fiequency of C D 4 5 R O + cells can often be detected in the peripheral blood of heart-, liver- and kidney-grafted patients undergoing acute rejection. ~ It remains to be determined whether an expansion of the C D 4 5 R O + memory subset could be the cellular basis for CsA-resistant rejection episodes in presensitized patients.
Acknowledgements We thank A Jeremias for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 265).
References I June CH, Lcdbelter JA. Gillespie MM, Lindsten T, Thompson CB.
2 3
4
5
6
7
T cell proliferation invoh'ing the CD28 palh,,~,ays is associated with cyclosporinc-resistant interleukin 2 gcnc expression. Mol Cell Biol 1987: 7: 4472-81. Liu J. FKS06 and cyclosporin, molecular probes for studying intracellular signal transduclion, lmmtmol Today 1993; 14:290 95. Tamura T. Yanagida T. Nariuchi H. Difference in signal transduc tion i)athway Ikw IL-2 and Ig-4 production in T helper I and T helper 2 cell chines in response to ami-CD3. ,/lmmmlol 1993: 151: 6051 61. Lederer JA, Lion JS, Todd MD, Glimcher LH, Lichtman AH. Regulation of cytokinc gone expression in T helper cell subsets. J lmmunol 1994: 152: 77-86. Barve SS, Cohen DA, DeBenedcni A, Rhoads RE, Kaplan AM. Mechanisms of differential regulation of IL 2 in murine Thl alld Th2 T cell subsets. 1. Induction of IL-2 transcription in Th2 cells by up-regulation of transcription factors with the protein synthesis initiation factor 4E. ,/ lnlnumol 1994; 152:1171 81. Schwinzer R. Schlitt HJ, Wonigeit K. Monochmal antibodies u) common epitopes of the human c,[3 T cell receptor preferentially activate CD45RA + T cells. Cell lmmunol 1992: 140:31-41. Fruman DA, Klee CB, Bierer B, Burakoff SJ. Calcineurin phosphatase activity in T lymphocytes is inhibited by, FK 506 and cyclosporin A. Proc Nat/Acad 5'ci USA 1992; 8 9 : 3 6 8 6 90. Gardner P. Calciunl and T lymphocyte acti\ation. Cell 1989: 59:
15- 20. Rao A. NF-ATp: a transcription l'actor required for the co-ordinate indnclion of several cytokine genes, hmmmo/ Today 1994: 15: 274 81. ll) Mouzaki A, Rungger D. Tucci A. Doucet A, Zubler RH. Occurrcncc of a silencer of the interleukin-2 gene in naive but not memory resting T helper lymplaocytes. Et~r J lmmtmo/ 1993; 23: 1469-74. I1 Winkler M, Schx~.inzer R, Wonigeit K, Ringe B. Pichhnayr R. Analysis of CD45RA CD45RO ~ "memory" T cells in patients after kidney, heart, and lixer tl-ansl)larllalion. T/'a/LSl:,/anl Proc 1992; 24: 2532 34.
C D 4 5 R A + and C D 4 5 R O + T cells differ in susceptibility to cyclosporin A mediated inhibition of interleukin-2 production Reinhard Schwinzer and Renate Siefken Transplantation Laboratory, Clinic for Abdominal and Transplantation Surge©', Medical Highschool Hannover, Hannover
Abstract: Lymphocytes in different states of activation use different intracellular signalling pathways and
may therefore differ in their susceptibility to immunosuppressive agents. In this study we examined the proliferation and production of interleukin-2 (IL-2) by unprimed/naive CD4~CD45RA - T cells and previously activated/memory CD4+CD45RO + T cells from human peripheral blood when stimulated in vilro in the presence of cyclosporin A (CsA). Further, the dependency of the IL-2 response on calcium (Ca>l ions was analysed by the addition of the chelating agent EGTA. The CD4+CD45RO ~ memory T cells were shown to be less susceptible to CsA and less dependent on the level of Ca + ions than the naive CD4+CD45RA + T cells. The subcellular mechanisnls involved in this difference and the potential clinical implications arc discussed.
Introduction
Objective
Despite the broad inhibitory effects of cyclosporin A (CsA), a 30 60% incidence of acute graft rejection still occurs in allografted patients, suggesting the existence of CsA-resistant pathways of T cell activation. The in vitro induction of interleukin-2 (IL-2) production by CD28 mAb (monoclonal antibody) in combination with phorbol ester (PMA) has been demonstrated to be resistant to CsA-mediated inhibition. ~ In contrast, IL-2 production initiated through the T cell receptor (TCR) can usually be effectively inhibited by CsA. The molecular mechanism of CsA-mediated inhibition of T cell activation is the capacity of the drug to interfere with TCR-associated signalling pathways. 2 Recent data suggest that T cells of different activation and/or differentiation states use different signalling pathways for the induction of cytokine gene transcription.3 5 Thus, it is conceivable that different T cell subsets may differ in susceptibility to CsA-mediated immunosuppression.
We studied this question of differential susceptibility to CsA by analysing the effects of CsA on IL-2 synthesis in CD4+CD45RA + (unprimed/naive) and CD4+CD45RO + {previously activated/memory) T cells. The results show that CD4+CD45RO + T cells are less susceptible to CsA-mediated inhibition of IL-2 synthesis compared to the CD4+CD45RA + T cell subset.
Address for correspondence: Reinhard Schwinzer, Transplantationslabor K25, Klinik ftir Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, D-30623 Hannover. Germany. © Arnold 1996
Materials and methods Cells PBMC (peripheral blood mononuclear cells) were isolated from heparinized peripheral human blood by Ficoll gradient centrifugation. Adherent mononuclear cells were removed by plastic adherence overnight at 37°C. Small resting T cells were prepared from nonadherent cells by E-rosetting. CD4+CD45RA + and CD4+CD45RO + T cells were isolated from E + cells by negative selection using the panning technique. Cells were incubated for 30 min on ice with mAb AICD8.1 (CDS, provided by Dr B Schraven, Heidelberg, Germany) plus M E M 56 (CD45RA, provided by Dr V Horejsi, Prague, Czech Republic) or AICD8.1 plus UCHL1 (CD45RO, donated by Dr PCL Beverley, London, UK), washed and lay-
62
R Schwinzer and R Siefken
ered on petri dishes coated with 101xg/ml goat anti-mouse IgG + IgM. After 2 h at 4°C, n o n a d h e r e n t cells were carefully collected, w a s h e d twice and reanalysed. Usually less than 6% of cells from the depleted subset were detected in these preparations as determined by flow cytometry analysis.
T cell activation protocols Purified CD4+CD45RA + and CD4+CD45RO ÷ T cells were stimulated with 1.5 I-~g/ml B M A 031 (anti-(~[3TCR mAb, provided by Dr R Kurrle, Behringwerke, Marburg, G e r m a n y ) in c o m b i n a t i o n with P M A (1 ng/ml) in R P M I 1640 m e d i u m supplemented with 10% FCS (fetal calf serum), 50 U/ml penicillin, 50 Ixg/ml streptomycin, and 4 m M L-glutamine. To study the effect of CsA, cells were incubated with C s A 1 h before stimulation and during culture. C s A was diluted in ethanol and titrated into the culture system ( 2 5 - 2 0 0 ng/ml). The final ethanol concentration was less than 0.5% (v/v) and had no effect on the induction of IL-2 synthesis and proliferation as determined in control experiments. To study the role of calcium (Ca2+), cells were stimulated in the presence of various concentrations of the Ca 2+ chelator E G T A ( 0 . 3 - 0 . 6 m M ) . M e a s u r e m e n t of IL-2 in the supernatant of cell cultures was performed using the IL-2-dependent rat T cell line G2 as described. 6 Results are expressed either as IL-2 units/ml or as proliferative response (cpm) induced in G2 cells.
Results Stimulation of C D 4 + C D 4 5 R A + and CD4+CD45RO + T cells with m A b B M A 031 (anti-c~[3TCR) plus P M A induced similar amounts of IL-2 in the two subsets (Table 1). Treatment of C D 4 + C D 4 5 R A + cells with C s A 50 ng/ml resulted in complete inhibition of IL-2 production. In contrast, significant amounts of IL-2 ( 2 0 - 3 0 U/ml) were detected in the supernatant of CD4+CD45RO + cells stimulated in the presence of C s A 100 ng/ml. C s A is k n o w n to inhibit calcium-dependent signalling pathways by interfering with the serine/threonine phosphatase calcineurin. 7 The differential susceptibility of CD4+CD45RA + and CD4+CD45RO + T cells to C s A could therefore indicate that the two subsets differ in the requirement for calcium to
induce IL-2 gene transcription. To examine this possibility we stimulated CD4+CD45RA + and CD4+CD45RO + cells in the presence of increasing concentrations of the Ca2+-chelator EGTA. The IL-2 activity found in the supernatant of C D 4 5 R A + cells decreased with increasing concentrations of E G T A and was completely absent in cultures stimulated in the presence of 0.5 m M E G T A (Figure 1). In contrast, low concentrations of E G T A (0.3-0.4 m M ) did not inhibit IL-2 production in C D 4 5 R O + cells. A n inhibitory effect was first observed when cells were stimulated in the presence of 0.45 m M EGTA, but even in the presence of 0.6 m M E G T A C D 4 5 R O + cells still produced detectable amounts of IL-2. This pattern of reactivity suggests that the induction of IL-2 production in C D 4 5 R O + cells is less dependent on Ca 2+ c o m p a r e d to C D 4 5 R A + cells.
Discussion T C R - m e d i a t e d elevation of the cytoplasmic calcium concentration [Ca2+]~ is regarded to be an essential step in the induction of IL-2 gene transcription and subsequent IL-2 production, s A consequence of elevated levels of [Ca2+]i is the activation of calcineurin. This serine/threonine phosphatase regulates NF-AT, an important transcription factor involved in the induction of IL-2 gene transcription. 9 The molecular m e c h a n i s m of action of CsA is the inhibition of calcineurin, 7 thereby preventing the induction of IL-2 gene transcription. The data presented here suggest that TCR-associated signalling pathways leading to the induction of IL-2 synthesis are less dependent on Ca 2+ in CD4+CD45RO + m e m o r y T cells
60000
50000E (._ 4 0 0 0 0 -
.o 0
30000-
O (3
._ Table 1 Effect of CsA on IL-2 synthesis in TCR-stimulated CD4+ T cell subsets Stimulus
Subset
20000-
CsA (ng/ml)
10000-
BMA 031 plus PMA
CD45RA + CD45RO +
0
25
50
100
200
75 82
22 62
3 52
<1 26
<1 <1
CD4+CD45RA + and CD4+CD45RO + T cells were stimulated with the antiaI3TCR mAb BMA 031 in combination with PMA. Stimulation was performed in the absence of CsA and in the presence of increasing concentrations of the immunosuppressive drug. After 24 h the culture supernatant was harvested and titrated on the IL-2-dependent cell line G2. Defined serial dilutions of recombinant IL-2 were included in each experiment for control. Proliferation of G2 cells induced by the supernatants was then compared with the IL-2 titration curve to calculate U/ml. Stimulation with either the mAb or PMA alone did not trigger IL-2 synthesis in CD45RA + and CD45RO + cells.
Transplant Immunology 1996; 4 : 6 1 - 6 3
0 0
0.3 0.35 0.4 0.45 0.5
0.6
EGTA conc. (mM) Figure 1 Effect of the Ca2+-chelator EGTA on IL-2 production. CD+CD45RA + and CD4+CD45RO + T cells were negatively isolated and stimulated with the anti-c~[3TCR mAb BMA 031 in combination with PMA. Cells were cultured in the absence or in the presence of increasing concentrations of EGTA. Supernatants from stimulated CD4+CD45RA + (O) and CD4+CD45RO + (11) T cells were harvested after 24 h and titrated on the IL-2-dependent rat T cell line G2. Results are expressed as proliferation of G2 cells in the presence of 25% cell culture supernatant.
Differential susceptibility o f T cell subsets to C s A - m e d i a t e d i m m u n o s u p p r e s s i o n
compared to the CD4+CD45RA + naive T cell subset. The two cell types therefore differ in susceptibility to CsA-mediated inhibition of IL-2 production. The differential susceptibility of C D 4 5 R A + and C D 4 5 R O + cells to CsA-mediated inhibition could either be explained by quantitative or qualitative differences in signalling pathways. Thus, C D 4 5 R O + cells might contain higher basic levels of [Ca-'+]~ or respond to TCR signalling with a stronger increase in [Ca 2~ I,. In both cases, effective inhibition of the signalling pathway would require more CsA compared to C D 4 5 R A + cells. However, the analysis of ICa2~]. in resting cells and in cells stimulated by C D 3 / T C R m A b did not reveal significant differences between the two subsets (data not shown). In addition, by immunoprecipitation experiments we were able to show that C D 4 5 R A + and C D 4 5 R O + cells contain similar amounts of the CsA target molecule calcineurin (data not shown). Together these lindings suggest that the differential susceptibility of the two subsets to CsA is due to qualitative differences in signalling pathways rather than to quantitative differences. The induction of 1L-2 gene transcription is regarded to require two major signalling pathways'~: a Ca'+-dependent pathway leading to calcineurin-mediated dephosphorylation and nuclear translocation of the cytoplasmic c o m p o n e n t of NF-AT and a cascade of k i n a s e s - - i n c l u d i n g protein kinase C (PKC) resulting in the activation of the nuclear c o m p o n e n t of NF-AT. Since the induction of IL-2 production did not require optimal amounts of Ca > in CD45RO* cells (Figure 1 ), one might assume thai IL-2 gene transcription in C D 4 5 R O + cells can be induced by sole activation of PKC. However, activation of PKC by phorbol ester alone did not trigger IL-2 synthesis in C D 4 5 R O - cells, suggesting that in addition to PKC actiwttion a second signal is also required in these cells. If the second signal is mediated via calcineurin, the activation of calcineurin in C D 4 5 R O + cells might be less dependent on Ca ~+ compared to C D 4 5 R A ' cells. Alternatively, it could be possible that dephosphorylation and nuclear translocation of the cytoplasmic c o m p o n e n t of N F - A T is not controlled by calcineurin in C D 4 5 R O ' cells. Significant differences in the regulation of IL-2 gene transcription between C D 4 5 R A - and C D 4 5 R O ' cells are also suggested by the recent demonstration of a silencer of the 1L-2 gene in C D 4 5 R A - but not in C D 4 5 R O + T cells. '° On the basis of the presented data it is likely that the subset composition of the immune system determines the in vivo effects of CsA. Thus, immunosuppression by standard CsA protocols might fail when an immune response is preferentially mediated by C D 4 5 R O ~ cells or when this subset had been
7Yansp/anl lmmuno/ogy 1996;
4:61-63
63
expanded by previous antigenic stimulation. An increased fiequency of C D 4 5 R O + cells can often be detected in the peripheral blood of heart-, liver- and kidney-grafted patients undergoing acute rejection. ~ It remains to be determined whether an expansion of the C D 4 5 R O + memory subset could be the cellular basis for CsA-resistant rejection episodes in presensitized patients.
Acknowledgements We thank A Jeremias for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 265).
References I June CH, Lcdbelter JA. Gillespie MM, Lindsten T, Thompson CB.
2 3
4
5
6
7
T cell proliferation invoh'ing the CD28 palh,,~,ays is associated with cyclosporinc-resistant interleukin 2 gcnc expression. Mol Cell Biol 1987: 7: 4472-81. Liu J. FKS06 and cyclosporin, molecular probes for studying intracellular signal transduclion, lmmtmol Today 1993; 14:290 95. Tamura T. Yanagida T. Nariuchi H. Difference in signal transduc tion i)athway Ikw IL-2 and Ig-4 production in T helper I and T helper 2 cell chines in response to ami-CD3. ,/lmmmlol 1993: 151: 6051 61. Lederer JA, Lion JS, Todd MD, Glimcher LH, Lichtman AH. Regulation of cytokinc gone expression in T helper cell subsets. J lmmunol 1994: 152: 77-86. Barve SS, Cohen DA, DeBenedcni A, Rhoads RE, Kaplan AM. Mechanisms of differential regulation of IL 2 in murine Thl alld Th2 T cell subsets. 1. Induction of IL-2 transcription in Th2 cells by up-regulation of transcription factors with the protein synthesis initiation factor 4E. ,/ lnlnumol 1994; 152:1171 81. Schwinzer R. Schlitt HJ, Wonigeit K. Monochmal antibodies u) common epitopes of the human c,[3 T cell receptor preferentially activate CD45RA + T cells. Cell lmmunol 1992: 140:31-41. Fruman DA, Klee CB, Bierer B, Burakoff SJ. Calcineurin phosphatase activity in T lymphocytes is inhibited by, FK 506 and cyclosporin A. Proc Nat/Acad 5'ci USA 1992; 8 9 : 3 6 8 6 90. Gardner P. Calciunl and T lymphocyte acti\ation. Cell 1989: 59:
15- 20. Rao A. NF-ATp: a transcription l'actor required for the co-ordinate indnclion of several cytokine genes, hmmmo/ Today 1994: 15: 274 81. ll) Mouzaki A, Rungger D. Tucci A. Doucet A, Zubler RH. Occurrcncc of a silencer of the interleukin-2 gene in naive but not memory resting T helper lymplaocytes. Et~r J lmmtmo/ 1993; 23: 1469-74. I1 Winkler M, Schx~.inzer R, Wonigeit K, Ringe B. Pichhnayr R. Analysis of CD45RA CD45RO ~ "memory" T cells in patients after kidney, heart, and lixer tl-ansl)larllalion. T/'a/LSl:,/anl Proc 1992; 24: 2532 34.