LAT-mediated signaling in CD4+CD25+ regulatory T cell development

    Department of Immunology, Duke University Medical Center, Durham, NC 27710

    Engagement of the T cell receptor for antigen (TCR) induces formation of signaling complexes mediated through the transmembrane adaptor protein, the linker for activation of T cells (LAT). LAT plays an important role in T cell development, activation, and homeostasis. A knock-in mutation at Tyr136, which is the phospholipase C (PLC)-1–binding site in LAT, leads to a severe autoimmune disease in mice. In this study, we show that CD4+CD25+ T reg cells that expressed Foxp3 transcription factor were nearly absent in both thymus and peripheral lymphoid organs of LATY136F mice. This defect was not a result of the autoimmune environment as LATY136F T reg cells also failed to develop in healthy LAT–/– mice that received mixed wild-type and LATY136F bone marrow cells. Moreover, adoptive transfer of normal CD4+CD25+ T reg cells protected neonatal LATY136F mice from developing this disease. These T reg cells effectively controlled expansion of CD4+ T cells in LATY136F mice likely via granzymes and/or TGF-–mediated suppression. Furthermore, ectopic expression of Foxp3 conferred a suppressive function in LATY136F T cells. Our data indicate that the LAT–PLC-1 interaction plays a critical role in Foxp3 expression and the development of CD4+CD25+ T reg cells

    Abbreviations used: GFP, green fluorescence protein; GITR, glucocorticoid-induced TNF receptor; LAT, linker for activation of T cells; PLC, phospholipase C.

    Regulatory T (T reg) cells are subsets of CD4+ T cells that play crucial roles in immunological tolerance, transplantation, and autoimmunity (1–3). Among different types of T reg cells, naturally arising CD4+CD25+ T reg cells are the best characterized and studied. These cells comprise 5–10% of CD4+ T cells in peripheral lymphoid organs and represent a unique T cell lineage that undergoes thymic selection and migrates to the periphery (4–8). Natural T reg cells are characterized by cell surface expression of CD25 (the IL-2 receptor -chain), CD62L (L-selectin), cytotoxic T lymphocyte antigen 4, and glucocorticoid-induced TNF receptor (GITR). The role of T reg cells in control of autoimmune diseases was clearly demonstrated in the experimental models in which elimination of CD25+ T cells results in acceleration of disease progression including inflammatory bowel disease, experimental autoimmune encephalomyelitis, and autoimmune diabetes (9).

    Multiple signaling pathways govern the development, function, and homeostasis of natural T reg cells including the IL-2 (10–15) and CD28 signaling pathways (16–18). In addition, the forkhead transcription factor, Foxp3, has recently been shown to be an essential regulator of CD4+CD25+ T reg cell development (19–21). Studies using Foxp3 knock-out mice (Foxp3–/–) have shown that ablation of Foxp3 expression results in mice lacking CD4+CD25+ T reg cells and the development of the lethal lymphoproliferative autoimmune disease characterized by the uncontrolled proliferation of activated T cells. This disease can be prevented by transfer of CD4+CD25+ T reg cells. Moreover, overexpression of Foxp3 in CD4+CD25– T cells converts these lymphocytes into CD4+CD25+ T cells with suppressive biological function (21). These findings suggest an obligatory role of Foxp3 in the development of natural T reg cells.

    Engagement via the TCR is required for CD4+CD25+ T cell development (22–26). TCR engagement activates tyrosine kinases that further phosphorylate many cellular proteins, leading to activation of signaling events such as Ras–MAPK activation and calcium flux. A key protein that couples the signaling events to TCR engagement is the linker for activation of T cells (LAT) (27). LAT is a transmembrane adaptor protein that binds Grb2, Gads, and PLC-1. It is essential during T cell activation and thymocyte development (28). In addition, LAT also plays an important role in immune homeostasis. Mice with a knock-in mutation of LAT at the PLC-1 binding site (Y136) show a severe autoimmune disease (29, 30). Although TCR-mediated phosphorylation of LAT and PLC-1 and Ca2+ mobilization in LATY136F T cells are markedly reduced, T cells in these mice are hyperactivated and produce large amounts of Th2 cytokines, which promotes B cell maturation and isotype switching. Spleens of these mice are enlarged enormously and lymphocytes infiltrate into different organs. It is not clear what causes the severe autoimmune phenotype in these mice. Defects in TCR signaling in LATY136F T cells might affect negative selection as demonstrated recently (31), leading to production of autoreactive T cells in these mice. In addition, the signaling defects might also affect development or survival of CD4+CD25+ T reg cells that are capable of controlling the autoimmunity. In this study, we demonstrated that LATY136F mice lacked CD4+CD25+ T cells expressing Foxp3 in peripheral lymphoid organs. Moreover, transfer of normal CD4+CD25+ T reg cells in LATY136F mice prevented the lymphoproliferative syndrome. Ectopic expression of Foxp3 in LATY136F T cells conferred suppressive function to abrogate lymphoproliferative disease when transferred into LATY136F mice. Our findings provide the evidence that the proximal signaling pathways downstream of the TCR, specifically the LAT–PLC-1 interaction, control T cell homeostasis by regulating Foxp3 expression and development of T reg cells.

    RESULTS

    The absence of CD4+CD25+ T cells in LATY136F mice

    The interaction of the TCR and self-peptides presented by MHC class II plays a critical role in development of CD4+CD25+ T reg cells in the thymus and function in the periphery (6, 32), suggesting that TCR-mediated signaling might also be important in the development of T reg cells. We asked whether the severe autoimmune phenotype of the LATY136F mice might be caused by a defect in development or function of T reg cells. We first examined whether CD4+CD25+ T reg cells were present in LATY136F mice. Cells from thymuses and spleens of LATY136F and WT mice were analyzed for expression of CD4, CD8, and CD25 by FACS. As previously reported (29, 30), LATY136F mice had a partial block in thymocyte development with accumulation of CD4–CD8– cells (Fig. 1 A). Splenomegaly and enlarged lymph nodes became obvious when they were 4–6 wk old. In the periphery, the CD4+ to CD8+ ratio was skewed toward CD4+ cells. Although discreet populations of CD4+CD25+ and CD4+CD25– SP thymocytes could be observed in WT mice, most of CD4+ SP thymocytes in LATY136F mice expressed low levels of CD25 (Fig. 1 A). The aberrant expression of CD25 in LATY136F thymocytes is likely caused by the partial block of thymocyte development in the DN3 (double negative) stage (29, 30), in which thymocytes are CD25+CD44–. Interestingly, double positive thymocytes in LATY136F mice also expressed CD25 (not depicted). Thymocyte development in LATY136F mice might proceed without down-regulation of CD25 expression.

    Because thymocytes from LATY136F mice expressed CD25, we next examined whether CD4+CD25+ T cells are present in the periphery. CD4+CD25+ T cells were clearly missing in the periphery of these mice (Fig. 1 A), although CD4+ T cells from LATY136F mice expressed high levels of GITR (not depicted). We further determined expression of Foxp3, an important transcription factor that is specifically expressed in CD4+CD25+ T reg cells, in LATY136F T cells. CD4+ SP thymocytes and CD4+ splenocytes cells were sorted from both WT and LATY136F mice. Foxp3 expression was determined by RT-PCR. The level of Foxp3 was decreased dramatically in CD4+ SP LATY136F thymocytes compared with that in CD4+ SP WT thymocytes. Decrease in the level of Foxp3 mRNA was also observed in CD4+ T cells from the spleen of LATY136F mice (not depicted). Further quantitation of Foxp3 mRNA by real-time PCR showed that Foxp3 mRNA level in CD4+ SP thymocytes from LATY136F mice was 10-fold less than in WT thymocytes, and Foxp3 RNA level in CD4+ splenic T cells from LATY136F mice was 30-fold less (Fig. 1 B). To exclude the possibility that decreased Foxp3 expression in CD4+ T cells from the mutant mice reflected dilution of Foxp3+ cells because of the expansion of CD4+ T cells, Foxp3 expression in thymocytes and splenocytes from WT and LATY136F mice was examined by intracellular staining with an anti-Foxp3 antibody followed by flow cytometry. As shown in Fig. 1 C, only CD4+ SP thymocytes and CD4+ splenocytes derived from WT, not those from LATY136F mice, expressed Foxp3. Thus, LATY136F mice lack CD4+CD25+ T reg cells.

    To exclude the possibility that the severe autoimmune conditions cause disappearance of T reg cells, we also examined development of T reg cells in younger mutant mice in which the autoimmune disease was not severe. As a result of a partial block in thymocyte development, very few CD4+ or CD8+ T cells were found in the peripheral lymphoid organs of 17-d-old mice (not depicted); however, they appeared in the spleens from 24-d-old mice (Fig. 2 A). Interestingly, a high percentage of CD4+ SP thymocytes expressed CD25 (23.6%). There was also a higher percentage (1.23%; Fig. 2 A) of CD4+CD25+ splenocytes compared with 6-wk-old mice (0.1%; Fig. 1 A). However, these CD4+ cells from the mutant mice did not express Foxp3 as indicated by intracellular staining (Fig. 2A). We also performed a mixed bone marrow chimeras experiment. Lethally irradiated LAT–/– mice were transferred with mixed bone marrow cells from WT Thy1.1+ and LATY136F Thy1.2+ mice. 6 wk after transfer, these mice showed no apparent signs of lymphoproliferative disease, whereas LAT–/– mice received bone marrow cells from LATY136F mice developed the autoimmune disease like LATY136F mice (not depicted). Because of the partial block in thymocyte development in LATY136F mice, fewer Thy1.2+ T cells than Thy1.1+ T cells were detected even though more bone marrow cells from LATY136F Thy1.2+ mice were transferred. As expected, WT Thy1.1+ bone marrow cells gave normal population of CD4+CD25+ T cells. A small population of Thy1.1+ T cells expressed Foxp3, and these Foxp3+ cells also expressed CD25 (Fig. 2 B). In contrast, very few Thy1.2+ cells in thymuses and spleens, if any, expressed Foxp3. Interestingly, different from the 6-wk-old LATY136F mice with the autoimmune disease, a large percentage of Thy1.2+ CD4+ splenocytes expressed CD25. It is possible that CD25 expression is down-regulated with progression of the disease. Because these Thy1.2+LATY136F T cells did not express Foxp3, they likely represented activated T cells. Collectively, these results indicate that LATY136F mice, which express a LAT mutant that does not bind PLC-1, have a defect in Foxp3 expression and development of CD4+CD25+ T reg cells.

    Adoptive transfer of CD4+CD25+ T reg cells

    To investigate whether the absence of CD4+CD25+ T reg cells is indeed one of the underlying mechanisms responsible for the lymphoproliferative syndrome in LATY136F mice, we purified CD4+CD25+ or CD4+CD25– T cells from congenic Thy1.1+ mice by FACS sorting. 2–3 x 105 CD4+CD25+ or CD4+CD25– Thy1.1+ T cells were transferred into 3-d-old Thy1.2+ LATY136F neonatal mice by i.p. injection. These mice were analyzed at 7 wk after injection. Untreated LATY136F mice clearly developed a pathological lymphoproliferative syndrome at 7 wk of age as shown before (29, 30) (Fig. 3 A). Compared with untreated LATY136Fmice, LATY136F mice that received Thy1.1+CD4+CD25+ T cells had a normal size spleen and lymph nodes. In contrast, LATY136F mice injected with CD4+CD25– T cells developed a lymphoproliferative syndrome similar to untreated LATY136F mice (Fig. 3 A).

    We further examined donor cell engraftment and expansion of adoptively transferred cells in LATY136F recipient mice. In normal mice, CD4+CD25+ T cells comprise 5–10% of the peripheral CD4+ T cells or 1–2% of total cells in lymph node and spleen. FACS analysis 7–9 wk after adoptive transfer revealed that 1–2% of the total cells in the lymph node and spleen of LATY136F mice adoptively transferred with CD4+CD25+ T cells were of donor origin, whereas very few donor cells were detected in the thymus (not depicted). This level of engraftment corresponded to 4–9-fold expansion of the initial donor cell inoculum of 2 x 105 cells. A similar degree of donor CD4+CD25+ T cell expansion was observed after injection of CD4+CD25+ T cells into Foxp3sf neonates (21). Likewise, CD4+CD25– injected into LATY136F neonates also underwent a considerable degree of expansion as the number of Thy1.1+CD4+CD25– T cells increased at least 20-fold (not depicted). In contrast, only very few donor-derived CD4+CD25+ and CD4+CD25– cells were detectable when they were transferred into age-matched WT recipients, which had normal T cell compartment. This observation reconciles with the possibility proposed by Fontenot et al. that donor CD4+CD25+ T cells can expand to fill the available homeostatic niche despite a skewed T cell compartment in these mice (21). In addition, adoptive transfer of WT CD4+CD25+ and CD4+CD25– T cells into 3-wk-old LATY136F mice that have not fully developed lymphoproliferative disease also resulted in expansion of both populations of donor-derived T cells (not depicted). However, only the transfer of CD4+CD25+ T cells was able to partially suppress lymphoproliferative disorder in 3-wk-old LATY136F recipients (not depicted). Neonatal adoptive transfer might provide donor-derived CD4+CD25+ T cell population with sufficient time to proliferate and suppress LATY136F T cells to fully suppress lymphoproliferative disorder. FACS analysis showed that in both groups of treated LATY136F mice >99% of the engrafted donor cells were still CD4+ T lymphocytes as expected. At least 75% of the donor CD4+ T cells expressed high levels of CD25 in the LATY136F mice transferred with CD4+CD25+ T cells, whereas the absolute majority of the donor CD4+ T cells were still CD25– in mice that received CD4+CD25– T cells. Adoptive transfer of either population of donor T cells had no effect on expression of CD25 on the host T cells (Fig. 3 B).

    No effect of donor T cells on the phenotypes of recipient T cells

    To evaluate whether adoptive transfer of normal CD4+CD25+ T cells could change activated phenotypes or other intrinsic properties in the recipient T cells leading to suppression of the lymphoproliferative disorder, thymocytes and splenocytes from LATY136F mice that received the adoptive transfer were analyzed and compared with those of untreated WT and LATY136F mice. Thymocyte development in LATY136F mice that received CD4+CD25+ T cells was similar to that in untreated LATY136F mice (Fig. 4 A). In fact, no substantial number of donor T cells was detected in the thymus of these mice (not depicted). However, the LATY136F mice transferred with CD4+CD25+ T cells had a significantly lower percentage of CD4+ T cells in the spleen (3%) compared with untreated WT (27%) and LATY136F (56%) mice. Despite a substantial reduction in the number of CD4+ T cells in LATY136F mice, donor CD4+CD25+ T cells did not alter the activated phenotypes of LATY136F T cells as T cells from both treated and untreated LATY136F mice were identical (CD25–TCRlowCD62LlowCD44high; Fig. 4 B). In addition, T cells from treated LATY136F mice failed to mobilize Ca2+-like T cells from untreated LATY136F mice (not depicted). These results indicate that donor CD4+CD25+ T cells did not change the activated phenotypes of LATY136F T cells based on these parameters we tested. Instead, CD4+CD25+ T reg cells transferred into LATY136F mice likely suppress expansion of CD4+ LATY136F T cells.

    Rescue of the autoimmune phenotype in LATY136F mice

    To determine if transfer of CD4+CD25+ T cells into LATY136F mice could correct the autoimmune disease, we performed a histological analysis of spleen, liver, and kidney from those mice. Histological sections from untreated LATY136F mice showed disorganized B and T cell zones in the spleen, lymphocyte infiltration in the majority of portal veins and liver sinusoids, and immune complex deposition in the glomeruli of the kidneys (Fig. 5 A). In contrast, tissue sections from LATY136F mice treated with CD4+CD25+ T cells appeared relatively normal, although many Thy1.2+ T cells did appear in the B cell zone of the spleen (Fig. 5 A). Histological analysis of the spleen, liver, and kidney of LATY136F mice that received CD4+CD25– T cells exhibited lymphoproliferative diseases similar to untreated LATY136F mice (not depicted).

    Examination of serum antinuclear (not depicted) and anti–double stranded DNA antibodies revealed that LATY136F mice treated with CD4+CD25+ T reg cells had comparable levels to WT controls (Fig. 5 B). These data were in agreement with a reduction in the number of hyperactivated B cells (MHC class IIhi or IgM–B220+) in LATY136F mice treated with CD4+CD25+ T cells (Fig. 5 C). In addition, the concentration of IgG1 (Fig. 5 D) or IgE (not depicted), two antibody isotypes dramatically elevated in LATY136F mice as a result of increased B cell maturation (29), was significantly reduced in mice treated with CD4+CD25+ T cells. Collectively, our data indicate that adoptive transfer of CD4+CD25+ T cells into LATY136F neonates can protect LATY136F mice from further developing lymphoproliferative syndrome.

    Up-regulation of granzymes in transferred CD4+CD25+ T cells

    CD4+CD25+ T reg cells are capable of suppressing autoimmune disease; however, how these cells exert their suppressive function is still not clear. Inhibitory cytokines such as IL-10 and TGF- are considered to be the key molecules involved in T reg cell–mediated immunosuppression (33). Recently, it was reported that CD4+CD25+ T cells up-regulate granzyme B in vitro upon stimulation with anti-CD3 and IL-2 (34). Whether this happens in vivo has not been demonstrated. We asked whether CD4+CD25+ T reg cells up-regulate granzymes (A and B) or inhibitory cytokines when they are transferred into LATY136F mice. At 7 wk after adoptive transfer of CD4+CD25+ or CD4+CD25– Thy1.1+ T cells into LATY136F mice, donor cells were reisolated from these mice by FACS sorting. Because these Thy1.1+CD4+CD25+ T cells were placed in the autoimmune environment and capable of correcting the disease, they must be activated to exert their suppressive function. They were labeled as "activated" in Fig. 5 E. We also isolated Thy1.2+CD4+CD25+ and CD4+CD25– T cells from WT mice as "resting" T reg cells and Thy1.2+CD4+CD25– T cells as a negative control. Total RNAs were prepared from these cells and used in RT-PCR.

    Donor-derived Thy1.1+CD4+CD25+ T cells expressed a high level of Foxp3 similar to Thy1.2+CD4+CD25+ cells directly isolated from WT mice. Thy1.1+ or Thy1.2+ CD4+CD25– T cells did not have a substantial level of Foxp3 expression (Fig. 5 E). Interestingly, Thy1.1+CD4+CD25+ T cells from neonatally injected LATY136F mice expressed high levels of granzyme A, granzyme B, and TGF- RNAs compared with Thy1.2+CD4+CD25+ T cells (Fig. 5 E), whereas their IL-10 expression was comparable to that of resting T reg cells (not depicted). This result is consistent with recent findings in which a granzyme B–dependent mechanism was identified as contact-mediated suppression by CD4+CD25+ T cells in vitro (34). Increased TGF- expression could also function to suppress the expansion of CD4+ T cells from LATY136F mice. It is possible that transferred Thy1.1+CD4+CD25+ T cells use both cytokine-mediated and contact-mediated mechanisms to suppress hyperproliferative host CD4+ T cells in vivo. Although we have not demonstrated that increased granzymes or TGF- indeed mediates suppression in LATY136F mice, our results show that T reg cells indeed up-regulate these proteins in the autoimmune environment.

    Ectopic expression of Foxp3 in LATY136F T cells

    Ectopic expression of Foxp3 was previously shown to be sufficient to activate a program of immunosuppression in CD4+CD25– T cells (21). Because Foxp3 expression was significantly decreased in LATY136F T cells (Fig. 1, B and C), we asked whether reexpression of Foxp3 in LATY136F T cells could confer the regulatory function that normal CD4+CD25+ T cells have. Because LATY136F T cells express low levels of TCRs on their surface, they were difficult to be activated via the TCR. Instead, LATY136F T cells were stimulated with PMA and ionomycin and cultured in the presence of IL-2 for 48 h before transduction with retroviruses expressing Foxp3 and green fluorescence protein (GFP) (pHSpG-Foxp3) or GFP alone (pHSpG-Empty) (Fig. 6 A). After culture for an additional 48 h postretroviral transduction, GFP+CD4+ T cells were FACS sorted, and 2–3 x 105 purified GFP+CD4+ LATY136F T cells were i.p. injected into 3-d-old LATY136F neonates. Expression levels of TCR- and the GITR on transduced LATY136F CD4+ T cells with or without Foxp3 were similar, whereas those of CD25 were slightly higher on pHSpG-Foxp3–transduced cells (Fig. 6 A). Expression of high levels of CD25 in these cells is likely a consequence of stimulation by PMA and ionomycin. 6 wk posttransfer, spleens and lymph nodes were harvested and examined. Mice receiving purified LATY136F T cells expressing only GFP developed a severe lymphoproliferative syndrome like untreated LATY136F mice (Fig. 6 B). In contrast, mice that received LATY136F T cells expressing Foxp3 and GFP exhibited no sign of lymphoproliferative disease as judged by the gross appearance of secondary lymphoid organs (Fig. 6 B). Analysis of GFP– T cells from mice received CD4+ LATY136F T cells expressing Foxp3 and GFP revealed a dramatic decrease in CD4+ T cells similar to that observed in LATY136F mice that received normal CD4+CD25+ T cells. On the other hand, GFP– host T cells from LATY136F mice treated with CD4+ LATY136F T cells expressing GFP alone were predominantly CD4+ similar to those in LATY136F mice (Fig. 6 C). These data indicate that reconstitution of Foxp3 expression in LATY136F T cells is sufficient to induce suppressive function and protect LATY136F mice from lymphoproliferative disease.

    DISCUSSION

    In this study, we demonstrated an essential role of the adaptor protein LAT in Foxp3 expression and CD4+CD25+ T reg cell development. In LATY136F mice with a severe lymphoproliferative disease, CD4+CD25+ cells were nearly absent in peripheral lymphoid organs. Foxp3 expression was also dramatically decreased in the LATY136F T cells. Interestingly, in young LATY136F mice CD4+CD25+ cells could be found; however, they did not express Foxp3. Similar results were also seen in LAT–/– mice that received mixed WT and LATY136F bone marrow chimeras. These data indicate that the LAT–PLC-1 interaction is required for Foxp3 expression and T reg cell development. Severe lymphoproliferative disease in LATY136F knock-in mice could be prevented by transfer of normal CD4+CD25+ T reg cells but not CD4+CD25– T cells. Our results indicate that the lymphoproliferative disease associated with the LATY136F mutation is not only caused by abrogation of central tolerance (31), but also by a breakdown in peripheral tolerance caused by a severe block of CD4+CD25+ T reg cell development.

    Analysis of donor cell engraftment indicates that CD4+CD25+ and CD4+CD25– T cells are able to proliferate and expand in LATY136F mice. Two studies using IL-2R– and Foxp3-deficient mice have previously demonstrated a similar expansion after the transfer of CD4+CD25+ T reg cells (12, 21). A rich Th2 cytokine environment, a consequence of hyperactivated LATY136F CD4+ T cells, may provide an ideal environment to support the expansion of transferred CD4+CD25+ and CD4+CD25– T cells. In LATY136F mice, at 2–3 wk after birth both CD4+ and CD8+ T cells begin to fill in peripheral lymphoid organs as a result of a partial block in thymocyte development. Nonselective expansion of transferred T cells might be caused by the lymphopenic environment in neonatal LATY136F mice. Adoptive transfer of CD4+CD25+ and CD4+CD25– T cells into 3-wk-old LATY136F recipient mice also allowed engraftment and expansion of both T cell populations. Thus, it is also possible that this expansion of transferred T reg cells may result from a proliferative response to fill a homeostatic niche for CD4+CD25+ T cells (10, 35). Although donor-derived CD4+CD25– T cells expanded upon transfer into LATY136F neonates, only transfer of CD4+CD25+ T cells into LATY136F mice rescued the lymphoproliferative disease. Even though CD4+CD25– T cells could convert into CD4+CD25+ T cells upon homeostatic proliferation (36), CD4+CD25– T cells injected into neonatal or 3-wk-old LATY136F mice neither converted into a considerable number of CD4+CD25+ T cells nor rescued the lymphoproliferative disease.

    The appearance of CD4+CD25+ LATY136F T cells in peripheral lymphoid organs of mixed bone marrow chimeras or young mutant mice was unexpected because CD25 expression was almost absent on CD4+ T cells from LATY136F mice older than 6 wk. However, in mice that received treatment of CD4+CD25+ at the neonatal stage CD25 expression on LATY136F CD4+ T cells was still missing. Regardless of CD25 expression in LATY136F T cells from different mice, Foxp3 expression was not detected in these cells, suggesting that these cells are likely activated T cells, not real T reg cells. It is possible that IL-2 produced by WT T cells might induce or maintain a high expression level of CD25 on LATY136F T cells or the autoimmune environment in LATY136F mice may cause T cells to gradually lose CD25 expression. Neonatally injected T reg cells, which do not secret IL-2 (4, 22, 37), failed to restore CD25 expression on LATY136F T cells in these mice and provided only protective function against lymphoproliferative disease. Previous studies show that Foxp3 is up-regulated upon activation of CD4+CD25+ T reg cells, and that ectopic Foxp3 expression confers suppressor function upon peripheral CD4+CD25– T cells (21). Likewise, ectopic expression of Foxp3 conferred LATY136F CD4+ T cells a regulatory function that prevents autoimmunity in these knock-in mice. Although ectopic expression of Foxp3 had no effect on the levels of both TCR- and GITR, CD25 was slightly up-regulated on transduced CD4+ T cells of LATY136F mice. It is difficult to conclude the effect of Foxp3 on CD25 in our experiments because we had to activate LATY136F T cells with PMA and ionomycin, which up-regulate CD25 in vitro, before transducing these cells with retroviruses. Nevertheless, our results suggest that Foxp3-mediated suppression does not require normal function of LAT, perhaps independent of the TCR. Our data strongly support the notion that Foxp3 is a master regulator gene that controls suppressor function in CD4+CD25+ T cells.

    The LAT–PLC-1 interaction is important in TCR-mediated Ca2+ flux and MAPK activation (38, 39). LATY136F T cells have abrogated Ca2+ flux although TCR-mediated MAPK activation is normal (30). As the influx of extracellular Ca2+ after TCR engagement has been implicated in influencing the outcome of both positive and negative selection (40–42), it is also possible that TCR-mediated Ca2+ mobilization and further NFAT activation might be required for induction of Foxp3 expression. Interestingly, the phenotype of LATY136F mice resembles that of mice lacking NFATc2 and NFATc3 (43), which suggests that the autoimmune disease in these mice may be attributed, at least in part, to a decrease in NFAT activation. However, it has been demonstrated that combined NFATc2/c3 deficiency has no effect on development and function of CD4+CD25– T cells but renders CD4+CD25– T cells unresponsive to suppression (44). Based on these findings, we speculate that the LAT–PLC-1 interaction provides signals other than NFAT activation to induce Foxp3 expression. Because LATY136F CD4+ T cells can be suppressed in vivo, NFATc2/c3, which are required for CD4+CD25– T cells to be suppressed, might be activated independent of the TCR. Although our results indicated that the LATY136F mutation affected Foxp3 expression and T reg cell development, we cannot rule out the possibility that the LATY136F T reg cells can develop, but they might not be able to survive in these mice. In addition, because T cell development is partially blocked in the LATY136F mice, it is possible that the defect in Foxp3 expression and T reg cell development might be indirect consequences of the block in thymocyte development. The signaling pathways that link the LAT–PLC-1 association and Foxp3 expression or T reg cell development remain to be explored in the future.

    A dramatic decrease in peripheral CD4+ T cells in treated LATY136F mice strongly indicates that these autoreactive CD4+ T cells are the targets of CD4+CD25+ T cell–mediated suppression. Our data show that donor-derived CD4+CD25+ T cells from treated LATY136F mice expressed high levels of granzyme A, granzyme B, and TGF- compared with CD4+CD25+ T cells isolated directly from WT mice (Fig. 5 E). In normal mice, maintenance of central tolerance mechanisms by negative selection leaves only a scanty number of these potentially harmful T cells in the periphery. The majority of T reg cells may never encounter these self-reactive T cells and thus remain at the resting status. Once placed or exposed to the autoimmune conditions, CD4+CD25+ T reg cells may trigger both contact-dependent and cytokine-mediated mechanisms by secretion of granzymes and TGF-. Whether these proteins indeed function to kill or suppress CD4+ LATY136F T cells in vivo remains to be determined in the future. In conclusion, our study indicates that the proximal signaling pathways downstream of the TCR mediated by the LAT–PLC-1 interaction play an important role in CD4+CD25+ T cell development and opens up the question of what might be the missing link between TCR and Foxp3 induction.

    MATERIALS AND METHODS

    Mice.

    All mice were used in accordance with the National Institutes of Health guidelines. The experiments described in this study were reviewed and approved by the Duke University Institutional Animal Care and Use Committee. Mice were housed in specific pathogen-free conditions at the Duke University Animal Care facility.

    Antibodies.

    Streptavidin-conjugated Texas red and PE-Cy7 and biotinylated FITC, PE, and PE-Cy5, APC-conjugated antibodies to TCR-, CD4, CD8, CD25, B220, CD62L, CD44, GITR, Thy1.1, Thy1.2, IgM, IAb, and mouse Ig were purchased from BD Biosciences. The anti-Foxp3 antibody was from eBioscience.

    Cell purification.

    Cells were maintained in complete RPMI 1640 medium with 10% FCS. To isolate CD4+CD25+ and CD4+CD25– T cells, splenocytes and lymph node cells were isolated. CD8+ T and B cells were depleted by magnetic beads using biotin-conjugated anti-CD8 and anti-B220. The enriched CD4+ lymphocytes were stained with FITC–anti-CD4, PE–anti-CD25, and 7AAD. CD4+CD25+ and CD4+CD25– T cells were purified by cell sorting using a FACSVantage SE flow cytometer (BD Biosciences).

    Neonatal transfer of CD4+ T cells.

    2–3 x 105 WT Thy1.1+-marked CD4+CD25+ or CD4+CD25– T cells were injected i.p. into 3-d-old (Thy1.2+) LATY136F pups or 3-wk-old LATY136F mice. These LATY136F pups were derived from breeding LAT+/Y136F females with LAT–/– males. Injected pups were analyzed at 7–9 wk after the adoptive transfer. Donor cell recovery was calculated based on the total number of lymphocytes multiplied by the percentage of Thy1.1+ cells as determined by FACS analysis.

    Mixed bone marrow transfer.

    T cell–depleted bone marrow cells from Thy1.2+ LATY136F mice (3.0 x 106 cells) were mixed with Thy1.1+ congenic mice (1.5 x 106 cells) and were then injected i.v. into irradiated LAT–/– mice (900 rads). 6 wk after bone marrow reconstitution, thymuses and spleens were harvested and analyzed by FACS.

    Retroviral transduction of LATY136F CD4+CD25– T cells.

    pHSpG and pHSpG/Foxp3 retroviral vectors were used to transfect the Phoenix-ecotropic virus packaging cell line using the calcium phosphate method to produce recombinant retroviruses. To transduce T cells from LATY136F mice, splenocytes from LATY136F mice were first activated using 40 ng ml–1 PMA and 0.5 μg ml–1 ionomycin and recombinant mouse IL-2 (100 ng ml–1) for 36 h. Activated lymphocytes were then transduced by mixing with the retroviral supernatant in the presence of 8 μg ml–1 polybrene and recombinant mouse IL-2 (100 ng ml–1). Cells were then centrifuged at 1,300 g for 2 h at 22°C. After culturing those cells at 37°C for 24 h, the transduction procedure was repeated. At 48 h after viral transduction, GFP+CD4+ T cells were sorted by FACS and 2–3 x 105 GFP+CD4+ T cells were injected i.p. into LATY136F neonates. At 6 wk after injection, lymph node and spleen cells were isolated and analyzed.

    Real-time quantitative PCR and RT-PCR.

    Total RNAs were extracted with the Trizol reagent (Invitrogen) and reverse transcribed using Superscript II reverse transcriptase (Invitrogen). cDNAs were then used as templates in PCR amplification with Taq polymerase. The Foxp3 mRNA level was quantified using the LightCycler system (Roche). The primer pairs used in real-time PCR were the following: -actin, 5'-ACTCCTATGTGGGTGACGAG-3', 5'-CAGGTC-CAGACGCAGGATGGC-3'; Foxp3, 5'-CCCAGGAAAGACAGCAACCTT-3', 5'-TTCTCA-CAACCAGGCCACTTG-3'. The primer pairs used in RT-PCR were the following: Foxp3, 5'-CAGCTGCCTACAGTGCCCCTAG-3', 5'-CATTTGCCAGCAGTGGGTAG-3'; granzyme A, 5'-CTCAAGACCGTATATGGCTCT-3', 5'-CCTGCACAAATCATGTTTAGT-3'; granzyme B, 5'-ACTTTCGATCAAGGATCAGCA-3', 5'-ACTGTCAGCTCAACCTCTTGT-3'; TGF-1, 5'-TGCTGCTTTCTCCCTCAACCT-3', 5'-CACTGCTTCCCGAATGTCTGA-3'.

    Immunohistochemistry.

    Whole spleens, livers, and kidneys were embedded in Tissue-Tek (Sankura Torrance) and sliced into 5-μm-thick section. Sections were applied to poly lysine–coated slides and fixed in acetone. Spleen and liver sections were then stained with FITC-conjugated anti-B220 or biotin-conjugated anti-Thy1.2 followed by alkaline phosphatase-conjugated anti-FITC and horseradish peroxidase–conjugated streptavidin (Sigma-Aldrich). Fast Blue BB and 3-aminoethylcarbazole (Sigma-Aldrich) solution were added for color development. Kidney sections were stained with FITC–anti–mouse IgG (BD Biosciences).

    Autoantibody detection.

    Anti–double-stranded DNA antibodies were detected using ELISA. 96-well plates were coated with 2.5 μg ml–1 calf thymus DNA in Reacti-bind DNA coating solution (Pierce Chemical Co.). Anti-nuclear antibodies were detected using slides of Hep-2 cells adhered to slides from Antibodies Inc.

    Acknowledgments

    We thank Drs. Lawrence Samelson and Paul Love for providing LATY136F mice, Marcella Sarzotti-Kelsoe for help with neonatal injection, Lishan Su for pHSpG and pHSpG/Foxp3 constructs, and Duke University Cancer Center Flow Cytometry facility for FACS analysis.

    This work is supported by National Institutes of Health grant AI048674 and AI056156. W. Zhang is a Leukemia and Lymphoma Society Scholar.

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