CD4+CD25+ regulatory T cells (Treg) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on Treg has been shown to be a key player of regulating Treg-mediated suppression. A recent study reports that NF-κB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of Treg but not for its suppression capacity. In this report, we have shown that Treg from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive Treg proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type Treg. Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62Llow Treg both in thymus and periphery than their wild-type littermates. This CD62low subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.

Naturally-arising CD4+CD25+ regulatory T cells (Treg)3 play a central role in maintaining immunologic self-tolerance (1, 2, 3). These cells constitute 5–15% of the peripheral CD4+ T cell compartment. Their sentinel role in tolerance is illustrated by the fact that depletion of this population in vivo results in the development of autoimmunity (4, 5). Furthermore, these cells have also been shown to exert an important immunoregulatory role in transplantation tolerance, tumor immunity, and microbial infection (6, 7, 8). The mechanisms of immunoregulation by Treg are only partially resolved. In vitro, Treg can suppress the proliferative response of CD4+CD25 effector T cells (Teff) through a contact-dependent mechanism, in part mediated by granzymes A and/or B (9, 10). In vivo, a number of cytokines have been implicated, including IL-10 and TGFβ (11).

Glucocorticoid-induced TNFR family-related gene (GITR), a member of the TNFR superfamily, was identified in 1997 (12). Mice deficient of this molecule showed no significant developmental or immunological defects (13). GITR is expressed on activated T cells and constitutively expressed at high levels on Treg. Initially, it was reported that the engagement of GITR on Treg extinguished Treg activities (14, 15). Subsequent studies suggested that GITR signaling resulted in not only counteracting Treg suppressive activities but also providing costimulatory signals to both effector and Treg populations (16, 17, 18).

NF-κB-inducing kinase (NIK), a serine-threonine kinase, which phosphorylates both IκB kinase (IKK) α and IKKβ, plays an integral part of the NF-κB signaling pathway (19, 20). NIK-deficient mice, as well as alymphoplasia (aly) mice, a natural strain with a mutated NIK, have abnormalities in the structural development of lymphoid organs and humoral immunity. Despite the disturbed thymic structure in these mice, the T cell compartment remains relatively normal. The CD4-CD8 T cell ratio and absolute cell numbers showed no significant alterations in both thymus and spleen (21, 22). Although predisposed to immunodeficiency, these mice develop autoimmune syndromes such as chronic inflammation in liver, pancreas, lung, salivary gland, and lacrimal gland by an unknown mechanism (23). Interestingly, a recent study has shown that the expression of mutant NIK in thymic stroma resulted in a reduced number of Treg. Although reduced in number, the remaining Treg had normal suppressive capacity suggesting a role of NIK in the establishment of central tolerance through control of Treg production (24).

The analysis presented herein of Treg subsets in NIK-deficient mice reveals impairment in Treg differentiation. We show that Treg from NIK-deficient mice display a hyperproliferative response upon GITR stimulation. This hyperproliferative response is not mediated by IL-2. Additionally, culture conditions that generally boost Treg proliferation, such as high concentrations of IL-2, anti-CD28 stimulation, or use of GITR ligand (GITRL)-transduced bone marrow-derived dendritic cells (BMDC) as APC, could not ablate this altered proliferative activity. Finally, we show an increased proportion of CD4+CD25+CD62Llow cells in NIK-deficient mice, and it is this Treg subset that is hyperproliferative to GITR signaling. The significance of this findings and their association to the autoimmunity in NIK-deficient mice is discussed.

NIK−/− mice on a 129Sv/Ev background were kindly provided by Dr. R. D. Schreiber (Washington University, St. Louis, MO) (25). In all the experiments, NIK+/− mice were used as control. All mice were maintained by heterozygote mating in a pathogen-free facility.

mAb DTA-1 to mouse GITR was kindly provided by S. Sakaguchi (Kyoto University, Kyoto, Japan). mAb YGL386 to mouse GITRL was kindly provided by H. Waldmann (University of Oxford, Oxford, U.K.). FITC-conjugated anti-CD4 and anti-CD8, Cychrome-conjugated anti-CD4, PE-conjugated anti-CD25, allophycocyanin-conjugated anti-CD62L, PerCP-conjugated streptavidin were purchased from BD Pharmingen. Biotinylated anti-CD28, anti-CD44, anti-CD45RB, anti-CD69, anti-CD103, anti-CD152, anti-CD154, and FITC-conjugated anti-Foxp3 were purchased from ebioscience. CFSE was obtained from Molecular Probes. Recombinant murine GM-CSF, IL-2, and IL-4 was purchased from PeproTech.

Single-cell suspensions were prepared from 8- to 10-wk-old mice and CD4+CD25 and CD4+CD25+ T cell subsets were purified by magnetic separation according to the manufacturer’s instructions (Miltenyi Biotec). Enriched cell populations and purified cells were phenotypically analyzed by FACS. The purities of CD4+CD25 and CD4+CD25+ T cells were >90–95% respectively. For [3H]TdR incorporation assays, 5 × 104 CD4+CD25 and/or the indicated number of CD4+CD25+ T cells were cocultured with irradiated T cell-depleted splenocytes (15 × 104) as APCs and 5 μg/ml anti-CD3 (2C11) in 96-well U-bottom plates treated with anti-GITR Abs (10 μg/ml) or rat Ig as a control for 72 h at 37°C. Cultures were pulsed with [3H]TdR for the last 8 h of culture. For some experiments, plates precoated with 1 μg/ml anti-CD3 and 10 μg/ml anti-CD28 for 1 h at 37°C were used. For ex vivo cytoplasmic dye dilution assay, purified CD4+CD25+ T cells were CFSE-labeled (5 μM) and cultured as above. After 72 h incubation, cells were harvested and analyzed.

Murine GITRL (isolated from the RAW 264.7 macrophage cell line) was cloned into lentiviral transfer vector pWPT (generously provided by the Trono Laboratory, University of Geneva, Geneva, Switzerland) using BamHI and SalI restriction sites (26). For lentivirus production, we cotransfected pWPT vector, and third-generation packaging vectors (encoding gag/pol, VSV-G, and rev) into 293FT cells (Invitrogen Life Technologies) and collected culture supernatants after 48 and 72 h of incubation at 37°C, 5% CO2. We recovered virus by ultracentrifugation (1.5 h at 25,000 rpm in a Beckman SW28 rotor) and resuspended the virus pellet in 25 μl of Opti-MEM media (Invitrogen Life Technologies). Viral titers were determined by serial dilution of concentrated lentivirus on 293FT cells followed by flow cytometry analysis after 48 h. Typical viral preparations yielded 5 × 108 transducing units (TU)/ml. We determined GFP or GITRL expression in transducted cells by flow cytometry 48 h posttransduction. Biotinylated anti-GITRL was used at a 1/200 dilution and then counterstained with streptavidin-allophycocyanin at 1/400.

For BMDC transduction, single-cell suspensions of bone marrow aspirates were ammonia chloride Tris buffer treated. Cells were then incubated for 7 days in complete RPMI 1640 supplemented with 10 ng/ml GM-CSF and 20 ng/ml IL-4. Media were changed with fresh cytokines at days 3 and 5. On day 5, cells received either control (GFP) or GITRL lentivirus at a multiplicity of infection (MOI) of 20 and allowed to incubate overnight. Polybrene was used to facilitate lentiviral transduction (8 μg/ml; Sigma-Aldrich). On day 7, cells were harvested, washed, counted, and applied to FACS to confirm gene expression. Indicated cell numbers were cultured and used as APCs in proliferation and suppression assays as previously described (27).

ELISA was performed to assess the secretion of IL-2 under the same culture conditions used for the proliferation assays described above. Supernatants were collected at the indicated times. IL-2 was quantified according to the manufacturer’s instructions (BD Pharmingen).

CD4+ T cells from NIK-deficient mice were first purified by MACS columns and then labeled with FITC-conjugated anti-CD4, PE-conjugated anti-CD25, and allophycocyanin-conjugated anti-CD62L. Enriched CD4+ T cells were then used for cell sorting. The purities of postsorted CD4+CD25+CD62Lhigh and CD4+CD25+CD62Llow populations were >95%. These cells were used for [3H]TdR incorporation assay as described.

Analysis of proliferation assays between the various treatments were analyzed by the two-tailed, paired Student t test. Values of p < 0.05 were considered significant.

A recent paper has shown that there is a reduced frequency of Treg in the alymphoplasia mouse (Aly), a strain of mouse with a natural mutation of the NIK gene. While reduced numbers of Treg were observed, the Aly/Aly Treg retained their normal suppressive capacity in vitro on a per cell basis (24). Because NIK defect results in the absence of lymph node, we have determined the percentage of Treg in thymus and in spleen. Consistent with this finding, Fig. 1,A illustrates that mice in which NIK has been deleted (NIK−/−) have a 50–75% reduction in the ratio of CD4+CD25+ cells to total CD4 single-positive thymocytes or CD4+ T cells from spleen. Phenotypic analysis of Treg from NIK+/− or NIK−/− mice show similar CD28, CD44, CD103, and GITR expression patterns in either a resting or an activated state (Fig. 1,B). However, the expression of CD45RB, CD62L, and CD69 were distinctive in NIK−/− Treg when compared with the expression pattern in NIK+/−. In contrast to the Treg from NIK−/− mice, the majority of NIK+/− Treg were CD45RBhigh, CD62Lhigh but CD69low. We then asked whether the quantitative and phenotypic differences between Treg from NIK+/− or NIK−/− would change their regulatory function. As shown in Fig. 1 C, the suppressive activity of Treg isolated from either NIK+/− or NIK−/− mice are indistinguishable, as measured by a standard in vitro suppressor assay with different ratios of CD4+CD25/CD4+CD25+ T cells. Taken together, NIK-deficient mice, like NIK mutant mice, have a reduced number of Treg with comparable suppression activities.

FIGURE 1.

Phenotypic and functional comparison of Treg from NIK-deficient mice and their WT littermates. A, CD4 single-positive thymocytes and CD4+ splenocytes from either NIK+/− or NIK−/− have been enriched by MACS columns and then assessed by FACS analysis. The percentages of CD4+CD25+ T cells in total CD4 T cell population have been shown in the figures. B, Freshly isolated or preactivated (48 h with plate-bound anti-CD3 plus IL-2) CD4+CD25+ T cells from NIK+/− or NIK−/− were analyzed by FACS for different cell surface molecules expression. C, Different numbers of isolated CD4+CD25+ T cells from NIK+/− or NIK−/− were cocultured with CD4+CD25 T cells and irradiated T-depleted APC from NIK+/− in a standard suppression assay. Briefly, cells at different CD4+CD25/CD4+CD25+ ratios were stimulated with 5 μg/ml anti-CD3 for 72 h and were pulsed with 1uCi/well [3H]thymidine for the last 8 h of culture. The level of proliferation was measured by [3H]thymidine incorporation.

FIGURE 1.

Phenotypic and functional comparison of Treg from NIK-deficient mice and their WT littermates. A, CD4 single-positive thymocytes and CD4+ splenocytes from either NIK+/− or NIK−/− have been enriched by MACS columns and then assessed by FACS analysis. The percentages of CD4+CD25+ T cells in total CD4 T cell population have been shown in the figures. B, Freshly isolated or preactivated (48 h with plate-bound anti-CD3 plus IL-2) CD4+CD25+ T cells from NIK+/− or NIK−/− were analyzed by FACS for different cell surface molecules expression. C, Different numbers of isolated CD4+CD25+ T cells from NIK+/− or NIK−/− were cocultured with CD4+CD25 T cells and irradiated T-depleted APC from NIK+/− in a standard suppression assay. Briefly, cells at different CD4+CD25/CD4+CD25+ ratios were stimulated with 5 μg/ml anti-CD3 for 72 h and were pulsed with 1uCi/well [3H]thymidine for the last 8 h of culture. The level of proliferation was measured by [3H]thymidine incorporation.

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Previous studies have shown that GITR signaling not only ablates Treg-mediated suppression, but also provides costimulatory signals to both effector and Treg populations (16, 17, 18). Because NIK plays a central role in the signaling of many TNFR family members, the role of NIK deficiency in GITR-induced Treg costimulation and suppressive activities was evaluated.

NIK function is not involved in GITR expression as both Treg from NIK+/− and NIK−/− mice express similar levels of GITR (Fig. 1,B). Furthermore, anti-CD3 induction of GITR on CD4+CD25 T cells was identical in both strains of mice (data not shown). To investigate the potential functional involvement of NIK in GITR signaling, anti-GITR-induced ablation of Treg suppression was evaluated. As shown in Fig. 2,A, Teff proliferation was suppressed by both NIK+/− and NIK−/− Treg in a standard suppression assay. Treg from NIK−/− mice retained normal suppressive activity and suppression was extinguished upon GITR stimulation. Treg from NIK+/− mice were anergized or hypoproliferative to GITR activation in vitro as has been previously shown for wild-type (WT) Treg. In contrast, Treg from NIK−/− mice proliferated in response to GITR stimulation (Fig. 2,A) (14). To confirm Treg proliferation, we purified and CFSE labeled Treg from either NIK+/− or NIK−/− mice and then cocultured with T cell-depleted splenocytes (APCs) for three days with or without GITR stimulation. Only Treg from NIK−/− mice proliferated vigorously in response to GITR activation (Fig. 2,B). Moreover, these GITR-mediated hyperproliferative activities were consistent even when the lower concentrations of anti-GITR were used (Fig. 2 C). These results demonstrate that NIK−/− Treg have distinctive characteristics from NIK+/− Treg, in that they display a hyperproliferative response upon GITR stimulation while their suppressive capacities remain intact.

FIGURE 2.

Treg from NIK-deficient mice displayed a hyperproliferative response upon GITR stimulation. A, CD4+CD25+ T cells isolated from NIK+/− or NIK−/− were cocultured with same number of CD4+CD25 T cells and irradiated T-depleted APC from NIK+/− in the presence of 5 μg/ml anti-CD3 plus 10 μg/ml anti-GITR or RatIg as control in a standard suppression assay as previously described. B, CD4+CD25+ T cells isolated from NIK+/− or NIK−/− were CFSE labeled and cocultured with irradiated T-depleted APC for 72 h in the presence of 5 μg/ml anti-CD3 plus 10 μg/ml anti-GITR or RatIg as control. Cell proliferation was accessed by CFSE dye dilution and FACS analysis. C, CD4+CD25+ T cells isolated from NIK+/− or NIK−/− were cocultured with irradiated T-depleted APC for 72 h in the presence of 5 μg/ml anti-CD3 plus different concentrations of anti-GITR and were pulsed with 1 μCi/well [3H]thymidine for the last 8 h of culture. The level of proliferation was measured by [3H]thymidine incorporation.

FIGURE 2.

Treg from NIK-deficient mice displayed a hyperproliferative response upon GITR stimulation. A, CD4+CD25+ T cells isolated from NIK+/− or NIK−/− were cocultured with same number of CD4+CD25 T cells and irradiated T-depleted APC from NIK+/− in the presence of 5 μg/ml anti-CD3 plus 10 μg/ml anti-GITR or RatIg as control in a standard suppression assay as previously described. B, CD4+CD25+ T cells isolated from NIK+/− or NIK−/− were CFSE labeled and cocultured with irradiated T-depleted APC for 72 h in the presence of 5 μg/ml anti-CD3 plus 10 μg/ml anti-GITR or RatIg as control. Cell proliferation was accessed by CFSE dye dilution and FACS analysis. C, CD4+CD25+ T cells isolated from NIK+/− or NIK−/− were cocultured with irradiated T-depleted APC for 72 h in the presence of 5 μg/ml anti-CD3 plus different concentrations of anti-GITR and were pulsed with 1 μCi/well [3H]thymidine for the last 8 h of culture. The level of proliferation was measured by [3H]thymidine incorporation.

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Given that exogenous IL-2 can break Treg anergy in vitro (28, 29), it is possible that Treg from NIK-deficient mice produce high levels of IL-2 upon GITR activation to facilitate Treg proliferation. IL-2 production from anti-CD3 activated, NIK+/− and NIK−/− CD4+CD25 and CD4+CD25+ T cells was measured. As reported in previous studies using Aly mice (21), CD4+CD25 Teff cells have reduced capacity to produce IL-2 in NIK−/− mice in comparison to NIK+/− cells upon anti-CD3 activation. Interestingly, signaling through GITR further increased the IL-2 secretion in both groups (Fig. 3,A). As for the CD4+CD25+ population, results were also consistent with previous studies that demonstrate undetectable levels of IL-2 production in both NIK−/− and control cultures (Fig. 3 A) (28, 29).

FIGURE 3.

GITR-induced hyperproliferation is not IL-2 dependent. A, CD4+CD25 and CD4+CD25+ T cells from NIK+/− or NIK−/− were cocultured with APC in the presence of 5 μg/ml anti-CD3 plus 10 μg/ml anti-GITR or RatIg as control. Supernatants were collected at the indicated times and IL-2 was quantified by ELISA. (N.D., not detected) B, IL-2 (100 U) was added to the standard suppression assay previously described. Proliferation was accessed by [3H]thymidine incorporation.

FIGURE 3.

GITR-induced hyperproliferation is not IL-2 dependent. A, CD4+CD25 and CD4+CD25+ T cells from NIK+/− or NIK−/− were cocultured with APC in the presence of 5 μg/ml anti-CD3 plus 10 μg/ml anti-GITR or RatIg as control. Supernatants were collected at the indicated times and IL-2 was quantified by ELISA. (N.D., not detected) B, IL-2 (100 U) was added to the standard suppression assay previously described. Proliferation was accessed by [3H]thymidine incorporation.

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Next, we examined the effect of exogenous IL-2 on T cells from NIK-deficient mice. Although exogenous IL-2 broke the unresponsiveness of Treg from both groups as previously shown (28, 29), we have found that both CD4+CD25 and CD4+CD25+ populations from NIK−/− but not from NIK+/− mice, proliferated more extensively in response to GITR stimulation. (Fig. 3 B). Taken together, these data suggest that the GITR-mediated hyperproliferative activity of Treg in NIK−/− mice is not IL-2 dependent.

To confirm that engagement of GITR on NIK−/− Treg results in enhanced proliferation, GITRL was overexpressed on DCs and the proliferation of Treg from WT and NIK−/− mice were evaluated. A lentiviral construct containing a full length GITRL sequence was used to transduce BMDC. The expression levels of GITRL were confirmed by FACS (data not shown). These transduced BMDC were then used as APCs. It is known that in the presence of anti-CD3, DCs can induce the proliferation of Treg in the absence of added cytokines. This phenomenon is believed to be dependent upon B7 costimulation by DCs (30). Consistent with this finding, BMDCs transduced with GFP control virus induced Treg proliferation from both groups (Fig. 4,A). However, overexpression of GITRL resulted in enhanced proliferation of Treg from NIK−/− mice but not Treg from WT mice. Although GITRL expression was significantly higher after transduction, to rule out the interference of endogenous GITRL expressed by BMDCs, similar results were obtained by using an APC-free system with plate-bound anti-CD3/anti-CD28 plus or minus anti-GITR stimulation (Fig. 4 B). These results agree with previous data showing the enhanced proliferation of NIK−/− Treg compared with WT Treg upon GITR activation.

FIGURE 4.

Overexpression of GITRL on BMDC results in heightened proliferation of NIK−/−, but not WT Treg. A, BM cells were grown in complete RPMI 1640 with GM-CSF and IL-4. On day 5, GFP or GITRL lentivirus (MOI of 20) was added into the culture respectively. On day 7, cells were collected, washed, and counted. Some cells were taken to confirm the transduction efficiency; others have been used as APC in a standard suppression assay with a 1:1 ratio of CD4+CD25/CD4+CD25+ T cells as previously described. B, The same amount of CD4+CD25 and CD4+CD25+ T cells from NIK+/− or NIK−/− were cultured in 96-well U-bottom plate precoated with 1 μg/ml anti-CD3/10 μg/ml anti-CD28 plus 10 μg/ml anti-GITR or RatIg as control. (∗, p < 0.03).

FIGURE 4.

Overexpression of GITRL on BMDC results in heightened proliferation of NIK−/−, but not WT Treg. A, BM cells were grown in complete RPMI 1640 with GM-CSF and IL-4. On day 5, GFP or GITRL lentivirus (MOI of 20) was added into the culture respectively. On day 7, cells were collected, washed, and counted. Some cells were taken to confirm the transduction efficiency; others have been used as APC in a standard suppression assay with a 1:1 ratio of CD4+CD25/CD4+CD25+ T cells as previously described. B, The same amount of CD4+CD25 and CD4+CD25+ T cells from NIK+/− or NIK−/− were cultured in 96-well U-bottom plate precoated with 1 μg/ml anti-CD3/10 μg/ml anti-CD28 plus 10 μg/ml anti-GITR or RatIg as control. (∗, p < 0.03).

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CD62L (L-selectin) has been used to discriminate different subsets of Treg (31, 32, 33). Both CD4+CD25+CD62Lhigh and CD4+CD25+CD62Llow T cells have shown to be suppressive in vitro (32, 34). However, in vivo studies have indicated that only the CD62Lhigh Treg subset and not CD62Llow Treg is suppressive (31, 33). As mentioned earlier, analysis of the Treg populations in NIK−/− vs NIK+/− revealed a marked reduction of the CD62Lhigh population in NIK−/− mice. In thymus, ∼50% of the CD4 single-positive and CD25-positive thymocytes from NIK+/− mice were CD62Lhigh, whereas only 36% of the Treg from NIK−/− mice expressed that phenotype. In spleen, over 75% of the Treg from NIK+/− mice were CD62Lhigh, whereas only 50% of the Treg from NIK−/− mice shared that phenotype (Fig. 5,A). To evaluate whether both the CD62Lhigh and CD62Llow subsets from the NIK−/− were hyperproliferative in response to GITR engagement, the CD62Lhigh and CD62Llow populations were sorted and cultured with anti-CD3 and either anti-GITR or rat Ig as control (Fig. 5,A). Interestingly, we found that only the CD62Llow but not CD62Lhigh subset from NIK−/− mice was hyperresponsive upon GITR stimulation (Fig. 5,B). Although there was a marked difference in the ability of GITR engagement to induce proliferation in the two subsets, both populations were suppressive and Foxp3 positive, in agreement with previous studies (Fig. 5, C and D).

FIGURE 5.

NIK-deficient mice have higher frequency of CD4+CD25+ CD62Llow Treg, which accounts for the hyperproliferative response. A, CD4 single-positive, CD25+ thymocytes, and CD4+CD25+ splenocytes from NIK+/− or NIK−/− were stained with CD62L to determine the relative frequency of CD62L high and low cells. The numbers shown in the figures are percent CD62Lhigh positive. CD4+CD25+ T cells from NIK−/− spleen were then sorted into CD62Lhigh and CD62Llow populations. B, CD62Lhigh and CD62Llow Treg subsets from NIK−/− were tested their proliferative capacity in response to anti-CD3 ± anti-GITR. Those cells were used for [3H]TdR incorporation assay as mentioned above. C, The suppressive capacities of CD62Lhigh and CD62Llow Treg subsets were tested in vitro, respectively, using culture conditions noted in Fig. 2. D, CD62Lhigh and CD62Llow Treg subsets from NIK−/− were stained for Foxp3 expression according to manufacturer’s instruction.

FIGURE 5.

NIK-deficient mice have higher frequency of CD4+CD25+ CD62Llow Treg, which accounts for the hyperproliferative response. A, CD4 single-positive, CD25+ thymocytes, and CD4+CD25+ splenocytes from NIK+/− or NIK−/− were stained with CD62L to determine the relative frequency of CD62L high and low cells. The numbers shown in the figures are percent CD62Lhigh positive. CD4+CD25+ T cells from NIK−/− spleen were then sorted into CD62Lhigh and CD62Llow populations. B, CD62Lhigh and CD62Llow Treg subsets from NIK−/− were tested their proliferative capacity in response to anti-CD3 ± anti-GITR. Those cells were used for [3H]TdR incorporation assay as mentioned above. C, The suppressive capacities of CD62Lhigh and CD62Llow Treg subsets were tested in vitro, respectively, using culture conditions noted in Fig. 2. D, CD62Lhigh and CD62Llow Treg subsets from NIK−/− were stained for Foxp3 expression according to manufacturer’s instruction.

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The data presented show that NIK plays a pivotal role in regulating the development and expansion of Treg. NIK deficiency results in a diminished representation of the CD62Lhigh Treg subset, and the hyperproliferative response of the CD62Llow subset following GITR signaling. NIK deficiency does not, however, alter the suppression mediated by Treg on a per cell basis.

It is known that Treg are anergic in vitro, but can proliferate upon Ag immunization in vivo (35, 36). In addition to signaling via the TCR, activation through a number of costimulatory molecules such as CD28/B7, 4-1BB/4-1BBL, OX40/OX40L, and GITR/GITRL also regulate Treg anergy and proliferation (18, 37, 38, 39). In this study, we show that Treg from NIK−/− mice were hyperproliferative to GITR triggering. Like other TNFR family members, GITR recruits several different TNFR-associated factors to its cytoplasmic tail after engagement of its ligand. GITR signals via these adaptor proteins to mediate the activation of multiple signaling pathways including NIK/NFκB and ERK, which in turn phosphorylate and activate downstream transcription factors (17, 40, 41). It appears that NIK tempers the signaling via GITR, such that in its absence, signaling through GITR induces greater levels of proliferation. These observations may be explained by a recent study by Wallach et al. (42) in studies of CD27 signaling (42, 43). These investigators suggested a possible functional role of interaction between NIK and Siva, a proapoptotic protein, which binds to the CD27 cytoplasmic tail after CD70 engagement (42, 43). Interestingly, GITR, like CD27, recruits Siva upon stimulation and it has been shown that GITR and Siva interaction induced apoptosis (44). It is possible that in the absence of NIK, Siva may not be recruited to the GITR tail, and may result in heightened T cell proliferation.

Both IL-2 and CD28 trigger Treg expansion and function. Previous studies have shown that either by adding IL-2 or providing costimulatory signals through anti-CD28 or mature BMDCs, the anergic state of Treg in vitro could be broken (28, 29, 30, 32). It could be argued that heightened IL-2 production by NIK−/− Treg is the basis for GITR-induced hyperproliferation. However, the difference in proliferative activity between NIK−/− and WT Treg was not altered upon the addition IL-2. Moreover, Treg from NIK−/− mice produced little or undetectable IL-2 when triggered through GITR. These data are consistent with the hypothesis that there is an intrinsic defect in NIK−/− Treg that results in heightened signaling via GITR.

The involvement of NIK in a wide spectrum of signaling pathways in many different cell types has made it extremely difficult to clearly delineate the basic mechanisms responsible for autoimmunity in NIK-deficient mice. Among the potential consequences of NIK deficiency, dysregulation of AIRE (autoimmune regulator) in NIK−/− mice may play an important role. AIRE has been shown to be a key mediator in the establishment of central tolerance by regulating ectopic expression of tissue-specific Ags in thymus (45). Lymphotoxin-β receptor deficiency or the mutation of its downstream signaling component, NIK, results in the failure of AIRE induction in the thymus that would ultimately lead to impaired clonal deletion (24, 46). Moreover, although there was no qualitative or quantitative alteration of Treg in AIRE-deficient mice (47), the development of autoimmunity in NIK-deficient/mutant mice may result from the impaired development of Treg in these mice, Kajiura et al. (24) have shown that abnormal NIK expression in the thymic environment resulted in the poor production of Treg. Furthermore, the autoimmune symptoms from mice grafted with a NIK-deficient thymus could be rescued simply by adoptive transfer of more Treg. In our experiments, we have also noted a lower frequency of Treg in NIK-deficient mice.

In addition to the reduced overall numbers of Treg in NIK-deficient mice, we have also shown a disruption in the development of Treg subsets. Treg have been shown to be functionally and phenotypically heterogenous, based in part on the expression of CD62L (31, 33, 48). Previous studies have shown in both a diabetes model and a graft vs host disease model, only the CD62Lhigh but not CD62Llow subset could delay disease or prevent graft rejection (31, 33). This phenomenon has been attributed to the different abilities of Treg to home to lymph nodes to suppress early events in alloantigen-specific T cell activation (31). In other models, such as autoimmune gastritis and colitis, when the suppression of early T cell priming was not required, both CD62Lhigh and CD62Llow subsets functioned equally in the prevention of autoimmunity (48). In NIK-deficient mice, we have found a substantial reduction in the percentage of CD62Lhigh Treg subset in both thymus and spleen compared with their WT littermates. Additional experiments have shown that CD62Llow Treg in NIK-deficient mice was the population that exhibited the hyperproliferative activity in response to GITR stimulation. Taken together, the enhanced discrepancy in population size of CD62Lhigh and CD62Llow Treg in thymus (50% vs 36% CD62Lhigh) and in spleen (75 vs 50% CD62Lhigh) between NIK+/− and NIK−/− mice, respectively, implicate NIK involvement in early Treg subset differentiation in thymus and a continuing skewing in the periphery. Moreover, the pre-existing higher percentage of the CD62Llow population in thymus of NIK−/− mice is in agreement with our in vitro suppression assay results, suggesting these cells belong to a specific Treg lineage, and do not simply represent preactivated conventional T cells.

The involvement of NIK in the development of autoimmunity is complex, impacting different cell types and different signaling pathways. Taken together, our studies and those published illustrate that NIK deficiency results in the escape of autoreactive T cells to the periphery due to impaired AIRE expression. To compound the impact of heightened peripheral autoreactive T cell activities, NIK deficiency results in the reduced development of Treg, and a skewing of the Treg population from CD62Lhigh to CD62low resulting in a less efficient and vigorous regulatory T cell response. Moreover, GITR signaling in the periphery would further aggravate the imbalance of CD62Lhigh and CD62Llow subsets leading to autoimmunity (Fig. 6).

FIGURE 6.

Proposed model for NIK−/− autoimmunity. The role of NIK in the establishment of self-tolerance in WT and NIK-deficient mice is depicted in the left and right panels, respectively.

FIGURE 6.

Proposed model for NIK−/− autoimmunity. The role of NIK in the establishment of self-tolerance in WT and NIK-deficient mice is depicted in the left and right panels, respectively.

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We thank Kathy Bennett for great care of mice. We also thank Gary Ward and Alice Givan for cell sorting. The Trono Laboratory (University of Geneva, Geneva, Switzerland) kindly provided the pWPT-GFP lentivirus construct.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants CA91436-01.

3

Abbreviations used in this paper: Treg, regulatory T cell; Teff, effector T cell; NIK, NF-κB-inducing kinase; GITR, glucocorticoid-induced TNFR family-related gene; IKK, IκB kinase; BMDC, bone marrow-derived dendritic cell; GITRL, GITR ligand; TU, transducing unit; MOI, multiplicity of infection; WT, wild type; AIRE, autoimmune regulator.

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