Role of Nuclear Factor of Activated T Cell (NFAT) Transcription Factors in Skin and Vascularized Cardiac Allograft Rejection

The fungal metabolites cyclosporine A and tacrolimus (FK506) are among the most potent immunosuppressive drugs available today (1). As inhibitors of the calcium-dependent serine/threonine phosphatase calcineurin, these drugs inhibit the dephosphorylation of the transcription factor nuclear factor of activated T cells (NFAT), a process that is essential for the nuclear localization of the cytoplasmic components of the NFAT transcription complex. Currently, five different NFAT family members have been identified: NFAT1 (also known as NFATp/NFATc2), NFAT2 (NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx/NFATc3), and NFAT5 (2). Our collaborator has previously demonstrated a preferential T helper 2 (Th2) differentiation and modulated T-cell receptor responsiveness of naive T cells in mice deficient in NFATc2 and NFATc3 (NFATc2/c3 double deficient [DKO] mice: BALB/c (H-2d) background) (3). Moreover, several studies of one or two NFAT family members have attempted to address the role of NFAT in regulation of CD4+ Th1/Th2 differentiation (4–8). Although NFAT family members play crucial roles in the development and function of the immune system, little work seems to have addressed their direct role in transplantation. In this study, we sought to explore the role of these molecules in both skin and cardiac transplantation models using allografts from BALB.B (H-2b) donors. Allograft survival was slightly but significantly prolonged in mice deficient in NFATc2 and NFATc3 (DKO) recipients of skin grafts (18 days vs. 12.5 days, P<0.0001) (Fig. 1b) and in recipients of heart allografts (14 days vs. 8 days, P=0.0004) (Fig. 1a). Our in vitro studies showed that alloantigen proliferative responses are unaffected in DKO recipients by mixed lymphocyte reaction (data not shown). Enzyme-linked immunosorbent spot (ELISPOT) showed that interferon (IFN)-γ production was significantly lower in NFATc2- and NFATc3-deficient mice than in controls (353.6±84.6 vs. 1067±78.4, P<0.0001), and interleukin (IL)-4 generation was significantly enhanced by NFATc2 and NFATc3 deficiency (147.5±8.7 vs. 112.9±7.7, P<0.0076) (Fig. 1c). These data point to enhanced alloantigen-specific Th2 cytokine generation. To further elucidate the role of IL-4 in prolonging allograft survival, we crossed DKO mice with BALB/c IL-4-deficient mice to generate a mouse triply deficient in NFATc2, NFATc3, and IL-4 (NFATc2/c3 and IL-4 triple-deficient [TKO]). Although NFATc2/NFATc3 double deficiency led to allograft prolongation, graft survival in NFATc2/NFATc3/IL-4 TKO recipients was abrogated in both the cardiac (P<0.0001 vs. DKO; Fig. 1d) and skin transplantation models (P=0.0003 vs. DKO; Fig. 1e). In fact, TKO recipient mice rejected both skin (14 days, n=5, P=not significant) and cardiac allografts (7 days, n=8, P=not significant) as promptly as wild-type BALB/c controls (Fig. 1d, e). Rejection was delayed 6 to 7 days in NFATc2 and NFATc3 (DKO) compared to wildtype BALB/c animals due to NFATc2 and c3 deficiency promoting a Th2 milieu (Fig. 1a, b). The Th1/Th2 paradigm proposes that Th2 lymphocytes favor tolerance induction by producing IL-4 and IL-10, whereas Th1 lymphocytes hinder tolerance induction by secreting IL-2 and IFN-γ (9, 10). However, studies using STAT4−/− mice (deficient in the Th1 response) or STAT6−/− mice (deficient in the Th2 response) and IFN-γ-deficient mice as recipients indicate that an absence of Th1 cytokines and/or a Th2 switch protects against chronic transplant rejection (11–13). Our preliminary data showed that IL-4-deficient mice abrogated the graft-prolonging effects of CD28-B7 blockade by CTLA4-Ig (250 μg intraperitoneally on day 2) in a fully major histocompatibility complex-mismatched cardiac transplant model (16 days, n=5 vs. 29.5 days, n=6 in controls; P<0.0008). Thus, delayed allograft rejection in DKO animals results from dysregulated expression of the Th2 cytokine IL-4. Investigation of individual NFAT family members may improve our understanding of allograft tolerance. Our results identify NFATc2 and NFATc3 as critical, though not necessary, components of allograft rejection, at least, in part, through their ability to control IL-4 production. The mechanisms by which these NFAT proteins inhibit Th2 differentiation remain controversial, but proposed mechanisms include competitive repression, perhaps against NFATc1 or GATA3, of IL-4, other Th2 cytokine genes, or the IL-4/IL-5/IL-13 locus as a whole (7). Given the importance of these and other NFAT family members in T-cell effector function (14), further dissection of the contributions of individual NFAT members in the regulation of the immune response should yield insight into their roles as therapeutic targets in transplantation medicine.FIGURE 1.: Allograft survival in DKO recipients; (a and b) Allograft survival was significantly prolonged in DKO recipients of skin grafts (b) and somewhat less prolonged in recipients of heart allografts (a). Th2 cytokine generation was enhanced by NFATc2/c3 deficiency; (c) production of the Th1 cytokine IFN-γ was significantly lower in the NFATc2- and NFATc3-deficient (DKO) mice. DKO mice enhanced their production of IL-4. Data are representative of three independent experiments using at least n=3 mice per group. Allograft survival in TKO recipients; (d and e) TKO mice rejected both skin (e) and cardiac allografts (d) as promptly as wild-type BALB/c controls. Data from the figure (a and b): allograft survival in control and DKO is also used in the figure (d and e). **P less than 0.001; ***P less than 0.0001. DKO, NFATc2/c3 double-deficient; NFAT, nuclear factor of activated T cells; IL, interleukin; TKO, NFATc2/c3 and IL-4 triple-deficient.Takuya Ueno1,2 Akira Yamada1 Toshiro Ito1 Melissa Y. Yeung2 Rostic Gorbatov3 Tetsunosuke Shimizu2 Reza Abdi2 Mohamed H. Sayegh2 Hugh Auchincloss, Jr.1 Nader Najafian2 1 Transplantation Unit Surgical Services Massachusetts General Hospital Harvard Medical School Boston, MA 2 Transplantation Research Center Brigham and Women's Hospital & Children's Hospital Harvard Medical School Boston, MA 3 Center for Systems Biology Massachusetts General Hospital Harvard Medical School Boston, MA ACKNOWLEDGMENT The authors thank Laurie H. Glimcher (Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA) for her generous gift of mice deficient in NFATc2 and NFATc3.