Essential roles of NF-κB–inducing kinase (NIK) for the development of medullary thymic epithelial cells (mTECs) and regulatory T cells have been highlighted by studies using a strain of mouse bearing a natural mutation of the NIK gene (aly mice). However, the exact mechanisms underlying the defect in thymic cross-talk leading to the breakdown of self-tolerance in aly mice remain elusive. In this study, we demonstrated that production of regulatory T cells and the final maturation process of positively selected conventional αβ T cells are impaired in aly mice, partly because of a lack of mature mTECs. Of note, numbers of thymic dendritic cells and their expression of costimulatory molecules were also affected in aly mice in a thymic stroma–dependent manner. The results suggest a pivotal role of NIK in the thymic stroma in establishing self-tolerance by orchestrating cross-talk between mTECs and dendritic cells as well as thymocytes. In addition, we showed that negative selection was impaired in aly mice as a result of the stromal defect, which accounts for the development of organ-specific autoimmunity through a lack of normal NIK.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery (1). Although the stromal elements that control these processes still remain elusive, much attention has recently been focused on the epithelial cell component of the stroma (i.e., thymic epithelial cells [TECs]). In particular, medullary TECs (mTECs) are considered to play a pivotal role in eliminating pathogenic autoreactive T cells by negative selection through expressing a large set of tissue-restricted self-Ags (TRAs) for parenchymal organs (2, 3). TRA gene expression in mTECs has also been implicated in the production of CD4+Foxp3+ regulatory T cells (Tregs) in the thymus (4, 5), cells that are involved in preventing autoimmunity.

NF-κB–inducing kinase (NIK), encoded by the Map3k14 gene, is a pivotal upstream kinase acting primarily in the noncanonical NF-κB activation pathway (6, 7). We and others have previously demonstrated that a strain of mouse bearing a natural mutation of the NIK gene (NIK-mutant aly mice) (8, 9) shows defective development of mTECs associated with dramatically reduced expression of TRA genes (10, 11). When embryonic thymi from aly mice were grafted into nude mice, the recipients developed organ-specific autoimmune disease, suggesting a pathogenic role of structurally disorganized mTECs in the autoimmunity of this strain (10). Although previous studies have also demonstrated impaired production of Tregs in aly mice (10, 12), the processes and cell types responsible for the defective NIK-dependent production of Tregs remain unclear. Furthermore, the relevance of negative selection to the breakdown of central tolerance in aly mice has not been formally examined.

In addition to mTECs, thymic dendritic cells (DCs) constitute an important component that present self-Ags for the induction of negative selection as well as for the production of Tregs within the thymic microenvironment (13). Although cross-presentation of mTEC-derived self-Ags by thymic DCs has been recognized as an important interplay between mTECs and DCs (14), little is known about how the thymic stroma affects the development and function of thymic DCs. The issue becomes more complex when considering the role of NIK in this context because NIK has been implicated in the intrinsic function of hematopoietic cells including T cells (1520) as well as DCs (12, 21). In this regard, it is particularly important to note that the cell-autonomous role of NIK in T cells has recently been debated. One study has suggested that NIK is dispensable in T cells and that it is NIK in DCs that is responsible for priming the effector function of T cells (21). Given that thymic DCs play an important role in the production of Tregs in an NIK-dependent manner, we further wanted to examine how the function of DCs may be affected by the interaction with thymic stroma that also expresses NIK. Therefore, we considered that thymic cross-talk may not be confined to the interplay between developing thymocytes and mTECs and that cross-talk between DCs and mTECs should also be taken into account.

To investigate the characteristics of thymic cross-talk required for the establishment of self-tolerance, we investigated the thymic stroma-dependent development of thymocytes, including Tregs, together with the development and function of thymic DCs in NIK-mutant mice. In this study, we show that the phenotypes of thymic DCs are strongly influenced by their interaction with mTECs expressing NIK, as is the case for developing thymocytes. Specifically, numbers of thymic DCs and their expression of costimulatory molecules are affected by NIK in a thymic stroma-dependent manner. In light of the fact that, in turn, thymic DCs can affect the function of thymocytes in an NIK-dependent manner (21), we suggest that thymic cross-talk can be viewed as an integrated circuit involving a broader range of cell types, including mTECs, thymocytes (including both Treg, and non-Tregs), and DCs, as revealed by the pleiotropic role of NIK.

aly mice and B6 mice were purchased from CLEA Japan. Genotyping of aly mice was performed as described previously (22). TCR-transgenic (Tg) mice specific for male H-Y Ag on a Rag2-deficient background (23) were purchased from Taconic Farms. Aire-deficient mice on a C57BL/6 (B6) background (24), Aire/GFP-knockin (KI) mice (25), and Rag1/GFP-KI mice (26) were generated as described previously. B6 (CD45.1) mice and OT-I and OT-II Tg mice (27, 28) were from The Jackson Laboratory. Tg mice expressing OVA under control of the rat insulin promoter (RIP-OVA Tg) were kindly provided by Dr. Michael J. Bevan (Department of Immunology, Howard Hughes Medical Institute, University of Washington, Seattle, WA) (14). The mice were maintained under pathogen-free conditions. The protocols used in this study were in accordance with the Guidelines for Animal Experimentation of Tokushima University School of Medicine.

Preparation of TECs and flow cytometric analysis with an FACScalibur (BD Biosciences), an FACSAria II (BD Biosciences), and a Gallios (Beckman Coulter) were performed as described previously (25, 29). The mAbs used were anti-CD4, anti-CD11c, anti-CD24, anti-CD25, anti-CD45, anti-CD45.1, anti-CD45.2, anti-CD62L, anti-CD69, anti-CD80, anti-CD86, anti–I-A/I-E, anti–Qa-2, anti-Foxp3, anti-γδ, and anti-NK1.1, all purchased from eBioscience. Anti-Sirpα mAb, anti-Vα2 mAb, and anti-Vβ5 mAb were from BD Biosciences. Anti-CD8α mAb, anti-CD40 mAb, anti–CTLA-4 mAb, anti-Helios mAb, B220 mAb, and and plasmacytoid DC (pDC) Ag-1 mAb were from BioLegend. Ulex europaeus agglutinin 1 (UEA-1) was from Vector Laboratories. Rabbit polyclonal anti–sphingosine 1-phosphate receptor type 1 (S1P1) Ab was kindly provided by Dr. J. G. Cyster (Department of Microbiology and Immunology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA).

Bone marrow (BM) transfer and thymus grafting were performed as previously described (10, 24). In brief, thymic lobes were isolated from embryos at 14.5 d postcoitus. The lobes were then cultured for 4 d on Nucleopore filters (Whatman) placed on culture media containing 1.35 mM 2′-deoxyguanosine (Sigma-Aldrich) to eliminate BM-derived cells, including DCs, within the thymus prior to the graft. The recipient mice were used for analyses 6–10 and 5 wk after BM transfer and thymus grafting, respectively.

Immunohistochemical analysis of the thymus was performed as described previously (29, 30). Anti-CD70 mAb was from eBioscience.

One microgram PE-labeled CD4 mAb (clone GK1.5: eBioscience) was i.v. injected. Thymi were harvested 5 min after the injection, and CD4PE-labeled cells were counted by flow cytometry, as previously described (31). For the inhibition of thymic egress, FTY720 (Cayman Chemical) was injected 12 h before CD4PE administration.

RNA was extracted from total thymus with RNeasy Mini Kits (Qiagen) and made into cDNA with SuperScript III RT Kits (Invitrogen) in accordance with the manufacturer’s instructions. Real-time PCR for quantification of the OVA, insulin 2, C-reactive protein (CRP), and Hprt gene was performed as described previously (25, 32).

Sorted thymic DC subsets were subjected to real-time PCR for the measurement of the expression of chemokines and chemokine receptors. Primers and probes for the detection were employed following the methods described in previous reports (21, 33).

All results are expressed as mean ± SEM. Statistical analysis was performed using the Student two-tailed unpaired t test for comparisons between two groups. Differences were considered significant if p values were ≤0.05.

We previously demonstrated that aly (aly/aly) mice have impaired production of Tregs in the thymus, which was associated with disorganization of the thymic medulla. The medullary region identified by staining with ER-TR5 mAb was sparse, and Aire-expressing mTECs were absent (10). In addition, flow cytometric analysis demonstrated dramatically reduced percentages of UEA-1+MHC class II (MHC-II)high cells, as well as UEA-1+CD80high and UEA-1+CD86high cells, in comparison with control aly/+ mice (Y. Mouri and M. Matsumoto, unpublished observations). Although this finding implied that NIK in the thymic stroma was required for the de novo production of Tregs, other mechanisms controlling the total numbers of Tregs in the thymus were not formally excluded. For example, a reduced number of resident Tregs [Tregs that remain resident in the thymus for long periods (34) but eventually migrate into the periphery], but not a reduced number of newly developed Tregs, could account for the reduction in the total numbers of thymic Tregs in aly mice. In addition, resident Tregs include recirculating mature Tregs that have migrated from the periphery. One study has suggested that resident Tregs account for up to 60% of the total number of Tregs in the thymus, if Foxp3 expression is employed only for the identity of CD4+ Tregs (35). Therefore, the processes and cell types responsible for the defective production of Tregs in an NIK-dependent manner need to be studied by considering the heterogeneity of Tregs within the thymus.

To focus on the ability to produce Tregs de novo within the thymic microenvironment of aly mice, we discriminated newly developed Tregs from resident Tregs by using Rag1/GFP-KI mice (26). Newly developed Foxp3+ Tregs in the thymus retain GFP expression, whereas resident Foxp3+ Tregs (including recirculating Tregs in the thymus) have already lost their GFP expression (34, 35). The use of this system to discriminate between newly developed Tregs and resident Tregs in the thymus was first validated by evaluating the GFP+Foxp3+ or GFPFoxp3+ Tregs in the thymus at different ages. At day 3, when the production of thymic Tregs has just started (4), although newly developed GFP+ Tregs were present, GFP resident Tregs were scarcely detected in the thymus (Supplemental Fig. 1A). In contrast, thymi taken from mice at the age of 1 and 4 wk contained not only GFP+ (newly developed) Tregs but also GFP (resident) Tregs. These newly developed and resident Tregs equally expressed Treg markers such as CD25, CTLA-4, and Helios (4) (Supplemental Fig. 1B). Similarly, only GFP+ (newly developed) Tregs were present at day 3 in the spleen (Supplemental Fig. 1C), whereas we observed both GFP+ (newly developed) and GFP (resident) Tregs at later time points (i.e., at the age of 1 and 4 wk), and these cells also expressed CD25, CTLA-4, and Helios (Supplemental Fig. 1D).

Within this experimental setting, we transferred BM from Rag1/GFP-KI into aly/+ or aly/aly recipient mice and evaluated the percentages and numbers of GFP+Foxp3+ and GFPFoxp3+ Tregs. It is important to note that reconstitution by the transferred BM cells assessed by the congenic markers (e.g., CD45.1 versus CD45.2) showed >99% of the replacement in our BM transfer system (Y. Mouri and M. Matsumoto, unpublished observations). Furthermore, total numbers of CD4+ single-positive (SP) cells from BM chimeras were indistinguishable between aly/+ and aly/aly recipient mice (data not shown). GFP+Foxp3+ Tregs but not GFPFoxp3+ Tregs were reduced in the thymi of aly/aly recipient mice compared with thymi from aly/+ recipient mice (Fig. 1A, top panel), suggesting that NIK in the thymic stroma controls the process of de novo thymic Treg production. In the spleen, GFP cells were the major population of Foxp3+ Tregs, and the numbers of GFP cells were indistinguishable between aly/+ and aly/aly recipient mice (Fig. 1A, bottom panel). Although these results were obtained 10 wk after BM transfer, we also observed lower numbers of GFP+Foxp3+ Tregs in aly/aly mice at earlier time points (i.e., 6 wk after BM transfer; Y. Mouri and M. Matsumoto, unpublished observations). This suggests that the lower numbers of GFP+Foxp3+ Tregs in aly/aly mice is not a result of impaired long-term maintenance of the BM graft.

FIGURE 1.

NIK mutation affects de novo production of Tregs in the thymus. (A) Percentages and numbers of newly developed GFP+Foxp3+ Tregs and resident GFPFoxp3+ Tregs in the thymus (top panel) and spleen (bottom panel) were evaluated in aly/+ or aly/aly recipient mice (Rc) after transfer with BM from Rag1/GFP-KI. In aly/aly recipient mice (black columns: n = 4), newly developed GFP+Foxp3+ Tregs, but not resident GFPFoxp3+ Tregs, were reduced in the thymus (top panel) but not in the spleen (bottom panel), unlike those from aly/+ recipient mice (white columns: n = 4). (B) aly/aly recipient mice (black columns: n = 4) were defective in the production of both CD25+Foxp3 Treg precursors and mature CD25+Foxp3+ Tregs in the thymus. Wild-type CD45.1 congenic mouse BM cells were transferred into recipient mice (CD45.2), and the data were analyzed after gating for CD45.1+ cells. White columns are for control animals: n = 4 for aly/+ recipient mice. Data were accumulated from a total of three experiments. *p < 0.05, **p < 0.01.

FIGURE 1.

NIK mutation affects de novo production of Tregs in the thymus. (A) Percentages and numbers of newly developed GFP+Foxp3+ Tregs and resident GFPFoxp3+ Tregs in the thymus (top panel) and spleen (bottom panel) were evaluated in aly/+ or aly/aly recipient mice (Rc) after transfer with BM from Rag1/GFP-KI. In aly/aly recipient mice (black columns: n = 4), newly developed GFP+Foxp3+ Tregs, but not resident GFPFoxp3+ Tregs, were reduced in the thymus (top panel) but not in the spleen (bottom panel), unlike those from aly/+ recipient mice (white columns: n = 4). (B) aly/aly recipient mice (black columns: n = 4) were defective in the production of both CD25+Foxp3 Treg precursors and mature CD25+Foxp3+ Tregs in the thymus. Wild-type CD45.1 congenic mouse BM cells were transferred into recipient mice (CD45.2), and the data were analyzed after gating for CD45.1+ cells. White columns are for control animals: n = 4 for aly/+ recipient mice. Data were accumulated from a total of three experiments. *p < 0.05, **p < 0.01.

Close modal

Immature CD4SP T cells develop into CD25+Foxp3 Treg precursors through TCR signaling together with costimulatory receptors such as CD28. CD25+Foxp3 Treg precursors further develop into mature CD25+Foxp3+ Tregs in the thymus through TCR-independent signals such as IL-2 and IL-15 (4, 36). Because aly mice had reduced numbers of newly developed Tregs in the thymus, we examined whether the TCR-dependent or TCR-independent step for de novo production of Tregs was impaired by NIK mutation. We found that both CD25+Foxp3 and CD25+Foxp3+ CD4SP T cells in the thymus were reduced in aly/aly recipient mice relative to aly/+ recipient mice (both are CD45.2+) when the BM cells from NIK-sufficient mice (CD45.1+) were transferred (Fig. 1B). These results suggest that a BM-extrinsic factor affects Treg differentiation in NIK-mutant mice.

To obtain more direct evidence for the link between the reduction in CD80high mTECs and the impairment of Treg production in aly mice, we conducted thymus grafting experiments. Thymi obtained from aly/+ embryos and aly/aly embryos were simultaneously grafted into a single nude mouse, under the left and right renal capsule, respectively. Five weeks later, Treg production together with the development of CD80high mTECs in the stroma were analyzed for each lobe grafted. In this experimental system, grafted thymi on both sides were repopulated by the hematopoietic cells derived from the same recipient mouse. Thymic lobes from aly/aly embryos contained fewer Foxp3+ CD4SP Tregs than those from aly/+ embryos (Fig. 2A, 2B, top panels), associated with lower percentages of UEA-1+CD80high stromal cells in aly/aly lobes compared with those from aly/+ lobes in all the individual recipients examined (Fig. 2A, 2B, bottompanels). Taken together, these results suggest that impaired Treg production in aly mice is at least partly stroma dependent and associated with the defect in the production of mature CD80high mTECs.

FIGURE 2.

Impaired production of Tregs in the thymus from aly mice is associated with reduced CD80high mTECs. (A) Thymic chimera experiments indicating that impaired Treg production in the thymus of aly mice is associated with lack of CD80high mTECs. Single nude mice were simultaneously engrafted with embryonic thymi from aly/+ mice and aly/aly mice under the left and right renal capsule, respectively. Five weeks after thymus grafting, production of Tregs and CD80high mTECs within each lobe was analyzed in the individual recipients. Representative flow cytometric analysis of Tregs (top panel) and CD80high mTECs (bottom panel) in one recipient mouse from a total of four is shown. (B) Percentages of Foxp3+ CD4SP Tregs and UEA-1+CD80+ mTECs (gated for CD45 cells) from simultaneously grafted aly/+ and aly/aly lobes from individual recipients were plotted using the same symbols. Bars represent the mean values. (C) Reciprocal BM transfer experiments demonstrating that hematopoietic cells from aly mice also contribute to the impaired Treg production in the thymus. Flow cytometric analysis of Foxp3+CD4SP Tregs from lethally irradiated wild-type CD45.1 congenic mice reconstituted with aly/+ mice or aly/aly mouse BM (n = 6 for each; top panel), and from untreated aly/+ mice and aly/aly mice (n = 5 for each; bottom panel). Production of Foxp3+CD4SP Tregs was assessed after gating for CD45.2+ thymocytes in the toppanels. *p < 0.05, **p < 0.01. FT, fetal thymus.

FIGURE 2.

Impaired production of Tregs in the thymus from aly mice is associated with reduced CD80high mTECs. (A) Thymic chimera experiments indicating that impaired Treg production in the thymus of aly mice is associated with lack of CD80high mTECs. Single nude mice were simultaneously engrafted with embryonic thymi from aly/+ mice and aly/aly mice under the left and right renal capsule, respectively. Five weeks after thymus grafting, production of Tregs and CD80high mTECs within each lobe was analyzed in the individual recipients. Representative flow cytometric analysis of Tregs (top panel) and CD80high mTECs (bottom panel) in one recipient mouse from a total of four is shown. (B) Percentages of Foxp3+ CD4SP Tregs and UEA-1+CD80+ mTECs (gated for CD45 cells) from simultaneously grafted aly/+ and aly/aly lobes from individual recipients were plotted using the same symbols. Bars represent the mean values. (C) Reciprocal BM transfer experiments demonstrating that hematopoietic cells from aly mice also contribute to the impaired Treg production in the thymus. Flow cytometric analysis of Foxp3+CD4SP Tregs from lethally irradiated wild-type CD45.1 congenic mice reconstituted with aly/+ mice or aly/aly mouse BM (n = 6 for each; top panel), and from untreated aly/+ mice and aly/aly mice (n = 5 for each; bottom panel). Production of Foxp3+CD4SP Tregs was assessed after gating for CD45.2+ thymocytes in the toppanels. *p < 0.05, **p < 0.01. FT, fetal thymus.

Close modal

Although the data presented above suggested that impaired de novo production of Tregs in aly mice is thymic stroma dependent, we and others previously demonstrated that NIK mutation also affects T cell reactivity in a cell-autonomous manner (1520) that can affect Treg lineage commitment (4). Therefore, we examined whether impaired de novo production of Tregs also depends on NIK in hematopoietic cells by generating reciprocal BM chimeras. When aly/aly BMs (CD45.2+) were transferred into wild-type recipient mice (CD45.1+), the percentages of Tregs (CD45.2+) were reduced in comparison with the chimeras in which aly/+ BM cells were transferred into wild-type recipient mice (CD45.1+) (Fig. 2C, top panels). This demonstrates that NIK mutation in hematopoietic cells also affected Treg production. However, in this experimental setting, we cannot determine whether this is because of the T cell–intrinsic role of NIK or secondary to the NIK-deficient DCs (21) because our BM transfer experiment does not allow us to examine the requirement of NIK in T cells and/or DCs (see 17Discussion).

Recently, it was demonstrated that a thymic medulla expressing normal RelB was required for the generation of Tregs, whereas continued maturation of positively selected conventional αβ T cells was not dependent on RelB expression from mTECs (37). Although both NIK and RelB are involved in the noncanonical NF-κB activation pathway (6, 38), NIK has been suggested to be involved in the canonical NF-κB activation pathway downstream of CD27 (39), indicating a broader impact of NIK on immune regulation than RelB. Therefore, we investigated the developmental process of mature SP T cells from aly mice. Compared with aly/+ mice, aly/aly mice showed increased percentages of terminally differentiated CD62LhighCD24low CD4SP T cells (Fig. 3A, top panels) and CD62LhighCD24low CD8SP T cells (Fig. 3B, top panels), although the levels of Qa-2 expression on both CD4SP and CD8SP T cells were not significantly different (Fig. 3A, 3B, bottom panels). This result is consistent with the notion that the aly mouse thymus contains increased numbers of mature SP T cells with phenotypes of recent thymic emigrants and may show disturbed thymocyte export (11). The reciprocal BM transfer experiment indicated that increased percentages of terminally differentiated CD62LhighCD24low CD4SP T cells were stroma dependent (data not shown). We observed that the levels of S1P1 expression on mature CD62Lhigh CD4SP T cells were slightly but significantly higher in aly/aly mice than in aly/+ mice (Fig. 3C, left panel); however, levels of S1P1 expression on semimature CD62Llow CD4SP T cells were indistinguishable between aly/+ mice and aly/aly mice (Fig. 3C, right panel). These results suggest that the process of development of mature conventional αβ T cells beyond Treg production is affected by the NIK-mutant medullary thymic microenvironment, suggesting a broader impact of NIK than RelB in mTECs.

FIGURE 3.

Alteration of the developmental process of mature SP thymocytes in aly mice. CD4SP T cells (A) and CD8SP T cells (B) with mature phenotypes defined by CD62LhighCD24low expression were increased in aly/aly mice (n = 7) compared with those in aly/+ mice (n = 8) (top panel). Qa-2 expression from CD4SP T cells (A) and that from CD8SP T cells (B) were indistinguishable between aly/+ and aly/aly mice (bottom panel). (C) Flow cytometric analysis showed higher expression of S1P1 from mature CD62Lhigh CD4SP T cells (left panel), but not from semimature CD62Llow CD4SP T cells (right panel) in aly/aly mice compared with those in aly/+ mice. Red and blue lines indicate aly/+ mice and aly/aly mice, respectively. Gray lines represent staining with control serum. One representative experiment from a total of three repeats is shown. (D and E) Levels of GFP expression (i.e., MFI of GFP) in the final maturation stage of the CD62LhighCD69low population were significantly lower in aly/aly recipient mice transferred with BM from Rag1/GFP-KI than in those of aly/+ recipient mice. The GFP signal from CD4SP T cells at each maturation stage is defined by the expression of CD62L and CD69 from Rag1/GFP-KI (D). MFI of GFP from aly/+ recipient mice (white columns) in each population was defined as 1 and compared with that from aly/aly recipient mice (black columns) (n = 8 for each) (E). Representative profile of GFP expression from CD62LhighCD69low CD4SP T cells is shown in the rightpanel. (F) Flow cytometric detection of intravascularly CD4PE-labeled SP cells in the thymus of aly/+ recipient mice (Rc) (middle panel) or aly/aly Rc (right panel) transferred with wild-type BM and their absence after 12 h of FTY720 treatment (left panel). Data were accumulated from a total of three experiments using six aly/+ recipient mice (white circles) and seven aly/aly recipient mice (black circles) in the right panel. Black lines represent mean values. *p < 0.05, **p < 0.01.

FIGURE 3.

Alteration of the developmental process of mature SP thymocytes in aly mice. CD4SP T cells (A) and CD8SP T cells (B) with mature phenotypes defined by CD62LhighCD24low expression were increased in aly/aly mice (n = 7) compared with those in aly/+ mice (n = 8) (top panel). Qa-2 expression from CD4SP T cells (A) and that from CD8SP T cells (B) were indistinguishable between aly/+ and aly/aly mice (bottom panel). (C) Flow cytometric analysis showed higher expression of S1P1 from mature CD62Lhigh CD4SP T cells (left panel), but not from semimature CD62Llow CD4SP T cells (right panel) in aly/aly mice compared with those in aly/+ mice. Red and blue lines indicate aly/+ mice and aly/aly mice, respectively. Gray lines represent staining with control serum. One representative experiment from a total of three repeats is shown. (D and E) Levels of GFP expression (i.e., MFI of GFP) in the final maturation stage of the CD62LhighCD69low population were significantly lower in aly/aly recipient mice transferred with BM from Rag1/GFP-KI than in those of aly/+ recipient mice. The GFP signal from CD4SP T cells at each maturation stage is defined by the expression of CD62L and CD69 from Rag1/GFP-KI (D). MFI of GFP from aly/+ recipient mice (white columns) in each population was defined as 1 and compared with that from aly/aly recipient mice (black columns) (n = 8 for each) (E). Representative profile of GFP expression from CD62LhighCD69low CD4SP T cells is shown in the rightpanel. (F) Flow cytometric detection of intravascularly CD4PE-labeled SP cells in the thymus of aly/+ recipient mice (Rc) (middle panel) or aly/aly Rc (right panel) transferred with wild-type BM and their absence after 12 h of FTY720 treatment (left panel). Data were accumulated from a total of three experiments using six aly/+ recipient mice (white circles) and seven aly/aly recipient mice (black circles) in the right panel. Black lines represent mean values. *p < 0.05, **p < 0.01.

Close modal

We hypothesized that one reason for the increased proportion of terminally differentiated mature SP T cells in aly mice may be the impaired exit of mature SP T cells from the NIK-mutant medullary thymic microenvironment. Therefore, we evaluated the exit process of mature SP T cells using the Rag1/GFP-KI system [i.e., a molecular timer (34)]. CD4SP T cells were divided into CD62LlowCD69high, CD62LintermediateCD69high, CD62LhighCD69high, and CD62LhighCD69low populations as they matured, and GFP expression was monitored for each population (Fig. 3D). Although levels of GFP expression (i.e., mean fluorescence intensity [MFI] of GFP) in the CD62LlowCD69high, CD62LintermediateCD69high, and CD62LhighCD69high populations were indistinguishable between T cells from aly/+ recipient mice and aly/aly recipient mice transferred with BM from Rag1/GFP-KI, GFP expression was significantly lower in the final maturation stage of the CD62LhighCD69low population from aly/aly recipient mice (Fig. 3E). This suggests that terminally differentiated mature SP T cells in aly mice stay longer in the thymus.

In addition, we examined the defective thymic egress in aly mice in a thymic stroma-dependent manner, by taking advantage of an in vivo labeling technique, in which i.v. injection of PE-conjugated CD4 Ab (CD4PE) labels thymocytes that have just emigrated into the vascular system but have not yet left the thymus (31). When FTY720, which inhibits thymic egress by disturbing S1P activity, had been injected before CD4PE administration, the number of CD4PE-labeled SP cells in the thymus was reduced, as expected (Fig. 3F, left panel) (31). Because we wanted to determine stroma-dependent thymic egress events, we injected CD4PE into aly/+ or aly/aly mice transferred with wild-type BM. We observed reduced percentages of CD4PE-labeled SP thymocytes from aly/aly recipient mice compared with those from aly/+ recipient mice transferred with wild-type BM (Fig. 3F). This suggests a slower thymic output because of the stromal defect in aly mice. Taken together, these results indicate that the exit of terminally differentiated CD4SP T cells from the thymus was inhibited in the NIK-mutant medullary thymic microenvironment, accounting for an increased proportion of terminally differentiated mature SP T cells in aly mice.

In the reciprocal BM chimeric mice described above, chimeric mice reconstituted with aly/aly mouse BM produced reduced numbers of Tregs compared with mice reconstituted with aly/+ mouse BM (Fig. 2C), suggesting that NIK mutation in hematopoietic cells also plays a role in Treg production. This finding supports a T cell–intrinsic role of NIK (1520). However, one study has suggested that the phenotypes of T cells from aly mice are attributable to abnormal function of DCs lacking normal NIK (21). BM cells from mice expressing wild-type NIK in DCs but lacking NIK in other hematopoietic cells (including T cells) were transferred into wild-type recipient mice, and the authors found that Treg production in the thymus was restored in this setting, suggesting a cell-intrinsic role of NIK in DCs but not in T cells. In addition, we considered that phenotypes of thymic DCs may be affected by the interaction with disorganized thymic stroma lacking normal NIK, as was the case for the development of thymocytes described above (i.e., the production of Tregs and final maturation process of SP conventional αβ T cells). Therefore, we examined the thymic stroma-dependent phenotypes of thymic DCs by preparing reciprocal BM chimeras. We transferred NIK-wild-type BM (CD45.1+) into either aly/+ or aly/aly recipient mice (CD45.2+), and after gating for the congenic marker for NIK-wild-type BM (CD45.1), we examined three different types of thymic DCs [i.e., Sirpα+ conventional DCs (cDCs), Sirpα cDCs, and pDCs] (13) (Fig. 4A, Supplemental Fig. 2A). In the NIK-mutant thymic microenvironment, all three types of DCs were reduced compared with the NIK-sufficient microenvironment (Fig. 4A, top panel), as was the case for the untreated aly/aly mice (Fig. 4A, bottom panel). This was a result of the overall reduction in CD11c+ cells in thymi from aly/aly recipient mice compared with thymi from aly/+ recipient mice (Supplemental Fig. 2B).

FIGURE 4.

Impaired development and function of thymic DCs from aly mice is associated with reduced CD80high mTECs. Reciprocal BM transfer experiments demonstrating that the reduction in both the numbers and function of thymic DCs is due to a stromal defect with NIK mutation. (A) Flow cytometric analysis of Sirpα+ cDCs, Sirpα cDCs and pDCs in thymi and spleens from lethally irradiated aly/+ or aly/aly recipient mice (Rc) (CD45.2) reconstituted with wild-type CD45.1 congenic mouse BM (n = 5 for each; top panel), from lethally irradiated wild-type CD45.1 congenic mice reconstituted with aly/+ or aly/aly mouse BM (n = 4 for each; middle panel) and from untreated aly/+ mice (n = 5) and aly/aly mice (n = 3) (bottom panel). Production of each DC type was assessed after gating for CD45.1+ and CD45.2+ cells in the top and middlepanels, respectively. Total numbers of Sirpα+ cDCs, Sirpα cDCs, and pDCs in the thymus and spleen from each group are also indicated: white columns and black columns are for aly/+ mice and aly/aly mice, respectively. (B) Expression of CD80 and CD86 on thymic DCs from BM chimeric mice transferred with wild-type (WT) CD45.1 congenic mouse BM shown in (A), top panel, BM chimeric mice transferred with aly/+ or aly/aly mouse BM shown in (A), middlepanel, and untreated aly/+ mice and aly/aly mice shown in (A), bottompanel. Profiles with red and blue lines are those for the aly/+ mice and aly/aly mice, respectively (top and bottom panels). Profiles with orange and green lines are those using BM from aly/+ mice and aly/aly mice, respectively (middle panel). Gray lines represent staining with isotype control. (C) Expression of CD80 and CD86 on splenic DCs from BM chimeric mice are shown in the same way as in (B). *p < 0.05, **p < 0.01.

FIGURE 4.

Impaired development and function of thymic DCs from aly mice is associated with reduced CD80high mTECs. Reciprocal BM transfer experiments demonstrating that the reduction in both the numbers and function of thymic DCs is due to a stromal defect with NIK mutation. (A) Flow cytometric analysis of Sirpα+ cDCs, Sirpα cDCs and pDCs in thymi and spleens from lethally irradiated aly/+ or aly/aly recipient mice (Rc) (CD45.2) reconstituted with wild-type CD45.1 congenic mouse BM (n = 5 for each; top panel), from lethally irradiated wild-type CD45.1 congenic mice reconstituted with aly/+ or aly/aly mouse BM (n = 4 for each; middle panel) and from untreated aly/+ mice (n = 5) and aly/aly mice (n = 3) (bottom panel). Production of each DC type was assessed after gating for CD45.1+ and CD45.2+ cells in the top and middlepanels, respectively. Total numbers of Sirpα+ cDCs, Sirpα cDCs, and pDCs in the thymus and spleen from each group are also indicated: white columns and black columns are for aly/+ mice and aly/aly mice, respectively. (B) Expression of CD80 and CD86 on thymic DCs from BM chimeric mice transferred with wild-type (WT) CD45.1 congenic mouse BM shown in (A), top panel, BM chimeric mice transferred with aly/+ or aly/aly mouse BM shown in (A), middlepanel, and untreated aly/+ mice and aly/aly mice shown in (A), bottompanel. Profiles with red and blue lines are those for the aly/+ mice and aly/aly mice, respectively (top and bottom panels). Profiles with orange and green lines are those using BM from aly/+ mice and aly/aly mice, respectively (middle panel). Gray lines represent staining with isotype control. (C) Expression of CD80 and CD86 on splenic DCs from BM chimeric mice are shown in the same way as in (B). *p < 0.05, **p < 0.01.

Close modal

Furthermore, we examined the expression of costimulatory molecules (i.e., CD80 and CD86) from thymic DCs that had developed in either an NIK-sufficient or an NIK-mutant thymic microenvironment because expression of these costimulatory molecules critically affects APC function of DCs (13). In the NIK-mutant thymic microenvironment, levels of CD80 and CD86 expression were significantly reduced in Sirpα+ cDCs and Sirpα cDCs (Fig. 4B, top panel, Supplemental Fig. 4A, top panel), although levels of CD40 expression were similar (data not shown). Except for small changes in CD86 expression (Fig. 4C, top panel, Supplemental Fig. 4B, top panel), these phenotypes were not obvious in splenic DCs (Fig. 4A, top panel for the numbers), indicating that changes in the phenotypes of DCs in aly mice were thymus specific.

Reverse experiments in which NIK-mutant BM was transferred into wild-type recipient mice demonstrated no changes in the number of thymic DCs (Fig. 4A, middle panel). In addition, changes in the expression of costimulatory molecules of thymic DCs were minimal (i.e., CD80 expression on Sirpα cDCs) (Fig. 4B, middle panel, Supplemental Fig. 4A, bottom panel). In contrast, splenic DCs showed more significant changes in the number (Fig. 4A, middle panel) or expression of costimulatory molecules (Fig. 4C, middle panel, Supplemental Fig. 4B, bottom panel) when NIK-mutant BM was transferred into wild-type recipient mice.

Thymus graft experiments indicated more directly that altered expression of CD80 and CD86 on thymic DCs was thymic stroma dependent. We used MHC-II–deficient mice as recipients in this experiment to reduce the influence of interactions between thymocytes and DCs (both are derived from recipient mice). DCs colonized in the grafted thymi were derived from MHC-II–deficient DCs, as expected (Supplemental Fig. 2C). Those MHC-II–deficient DCs are considered to have less chance of being influenced by the interaction with thymocytes, thereby allowing a more direct assessment of the cross-talk between thymic stroma and DCs by minimizing the effect of thymic cross-talk between thymocytes and DCs. Thymi obtained from aly/+ embryos and aly/aly embryos were simultaneously grafted into a single MHC-II–deficient mouse under the left and right renal capsule, respectively. We observed lower expression levels of CD80 and CD86 on both Sirpα+ cDCs (Fig. 5, top panel) and Sirpα cDCs (Fig. 5, bottom panel) from aly/aly thymic lobes compared with aly/+ thymic lobes, and the effect was greater in Sirpα cDCs, a population that is produced intrathymically (33). These data suggest that the development and function of thymic DCs are under the control of an organized mTEC compartment expressing normal NIK.

FIGURE 5.

mTECs expressing normal NIK are required for the expression of costimulatory molecules of thymic DCs. Thymi obtained from aly/+ embryos and aly/aly embryos were simultaneously grafted into a single MHC-II–deficient mouse under the left and right renal capsule, respectively. Expression of CD80 and CD86 on both Sirpα+ cDC (top panel) and Sirpα cDC (bottom panel) from aly/aly thymic lobes were compared with those from aly/+ thymic lobes (n = 4 for each). Percentages of CD80+CD86+ cells are shown. **p < 0.01.

FIGURE 5.

mTECs expressing normal NIK are required for the expression of costimulatory molecules of thymic DCs. Thymi obtained from aly/+ embryos and aly/aly embryos were simultaneously grafted into a single MHC-II–deficient mouse under the left and right renal capsule, respectively. Expression of CD80 and CD86 on both Sirpα+ cDC (top panel) and Sirpα cDC (bottom panel) from aly/aly thymic lobes were compared with those from aly/+ thymic lobes (n = 4 for each). Percentages of CD80+CD86+ cells are shown. **p < 0.01.

Close modal

A previous study has reported that the expression of many chemokines/chemokine receptors from thymic DCs was reduced and implicated in the altered function of thymic DCs in aly mice (21). Therefore, we also examined the expression of chemokines/chemokine receptors from thymic DCs isolated from BM chimeras. Consistent with the previous report (21), we observed reduced expression of several chemokines/chemokine receptors from thymic DCs isolated from untreated aly/aly mice compared with those isolated from untreated aly/+ mice (Supplemental Fig. 3). However, the overall expression of chemokines/chemokine receptors from thymic DCs was not affected in aly/aly recipient mice compared with aly/+ recipient mice transferred with wild-type mouse BM except for CXCR2 expression from Sirpα+ cDC. Similarly, in the reciprocal BM chimeric mice, thymic DCs isolated from wild-type mice reconstituted with aly/+ mouse BM or aly/aly mouse BM showed indistinguishable levels of chemokine/chemokine receptor expression except for CCR9 from pDCs (Supplemental Fig. 3), suggesting that reduced expression of chemokines/chemokine receptors in aly mice was not determined by either cell-intrinsic (in DCs) or cell-extrinsic NIK (in thymic stroma) alone. Instead, this phenotype was dependent on both thymic stroma and BM-derived cells lacking normal NIK activity, further strengthening the importance of cross-talk between thymic stroma and DCs. This is in contrast to CD80/86 expression levels from thymic DCs, in which normal NIK activity from thymic stroma is a predominant determinant of this action (Figs. 4, 5, Supplemental Fig. 4). Therefore, the contribution of NIK in thymic stroma appears to differ depending on the functional properties of the thymic DCs being investigated.

Although the defect in the production of Tregs in the thymus can partly account for the development of autoimmunity in aly mice, negative selection in the disorganized thymic medulla in this strain has not been formally assessed. Therefore, we examined whether NIK in the thymic stroma is required for the negative selection process by using TCR Tg mouse models. Using H-Y TCR-Tg mice specific for male Ag (23), we found that NIK was dispensable for the clonal deletion of the T cells specific for ubiquitously expressed self-Ag in males (Y. Mouri and M. Matsumoto, unpublished observations), consistent with a previous study that had employed a similar H-Y TCR-Tg system (40). Furthermore, we employed another TCR Tg model for clonal deletion of T cells directed against organ-specific self-Ag from the thymic stroma (14, 27, 28). When BM cells from OT-I Tg or OT-II Tg were transferred into aly/aly mice crossed with RIP-OVA Tg, deletion of the clonotypic CD8SP (Fig. 6A) and CD4SP thymocytes (Fig. 6B), respectively, was impaired in comparison with transfer into aly/+ mice crossed with RIP-OVA Tg.

FIGURE 6.

Impaired negative selection in aly mice assessed with the Tg model for expression of organ-specific self-Ag from the thymic stroma. aly/+ mice or aly/aly mice crossed with RIP-OVA Tg were transferred with BM from OT-I (A) and OT-II Tg mice (B). After gating for CD8SP (for OT-I) (A) or CD4SP (for OT-II) T cells (B), clonotypic T cells were assessed with anti-Vα2 and anti-Vβ5 mAbs. Percentages together with absolute numbers of clonotypic T cells are shown in the rightpanels: non-Tg recipient mice are shown as clear circles, whereas solid circles represent RIP-OVA Tg recipient mice. One circle represents one mouse analyzed, and each experimental group contains three to six mice. Bars represent the mean values. (C) Expression of OVA, insulin 2 (Ins 2), and CRP was examined using RNAs extracted from total thymi of non-Tg aly/+ mice (clear columns), aly/+ mice crossed with RIP-OVA Tg (gray columns), and aly/aly mice crossed with RIP-OVA Tg (black columns) by real-time PCR. The expression level of Hprt was used as an internal control for RNAs, and the abundance of each gene was normalized relative to Hprt expression. Thymus from aly/aly RIP-OVA Tg showed levels of OVA indistinguishable from those of aly/+ RIP-OVA Tg, whereas expression of the insulin 2 (Ins 2) and CRP genes was dramatically reduced in the thymus from aly/aly RIP-OVA Tg. Results are expressed as the mean ± SEM for triplicate wells of one representative experiment from a total of two repeat experiments. U.D., under the limit of detection. **p < 0.01.

FIGURE 6.

Impaired negative selection in aly mice assessed with the Tg model for expression of organ-specific self-Ag from the thymic stroma. aly/+ mice or aly/aly mice crossed with RIP-OVA Tg were transferred with BM from OT-I (A) and OT-II Tg mice (B). After gating for CD8SP (for OT-I) (A) or CD4SP (for OT-II) T cells (B), clonotypic T cells were assessed with anti-Vα2 and anti-Vβ5 mAbs. Percentages together with absolute numbers of clonotypic T cells are shown in the rightpanels: non-Tg recipient mice are shown as clear circles, whereas solid circles represent RIP-OVA Tg recipient mice. One circle represents one mouse analyzed, and each experimental group contains three to six mice. Bars represent the mean values. (C) Expression of OVA, insulin 2 (Ins 2), and CRP was examined using RNAs extracted from total thymi of non-Tg aly/+ mice (clear columns), aly/+ mice crossed with RIP-OVA Tg (gray columns), and aly/aly mice crossed with RIP-OVA Tg (black columns) by real-time PCR. The expression level of Hprt was used as an internal control for RNAs, and the abundance of each gene was normalized relative to Hprt expression. Thymus from aly/aly RIP-OVA Tg showed levels of OVA indistinguishable from those of aly/+ RIP-OVA Tg, whereas expression of the insulin 2 (Ins 2) and CRP genes was dramatically reduced in the thymus from aly/aly RIP-OVA Tg. Results are expressed as the mean ± SEM for triplicate wells of one representative experiment from a total of two repeat experiments. U.D., under the limit of detection. **p < 0.01.

Close modal

Furthermore, we investigated whether impaired negative selection of OVA-specific T cells in NIK-mutant thymus may be because of the defective expression of OVA. Because the numbers of mTECs were very low to allow isolation from aly mice, we measured the OVA transcript from total thymi. We found that total thymic expression of OVA from aly/aly mice was not reduced compared with aly/+ mice (Fig. 6C), despite the reduced numbers of CD80highMHC-IIhigh mTECs in aly/aly mice. In contrast, RNA transcripts for insulin 2 and CRP were almost absent from aly/aly mouse thymi. These results suggest that, at least in this Tg model, NIK in the thymic stroma controls deletional tolerance of T cells specific for organ-specific self-Ag, not by control of self-Ag expression at the transcriptional level but by the creation of a negative selection niche through some other means, such as processing and/or presentation of OVA peptides from existing mTECs and/or thymic DCs for which development and function are NIK dependent, as demonstrated in the current study.

Finally, we examined the expression of costimulatory receptors on mature SP thymocytes from aly mice as a reflection of altered provision of costimulatory signals from thymic APCs because costimulatory signals provided by thymic APCs, such as mTECs and thymic DCs, are critical determinant factors for the production of thymic Tregs (4, 36) as well as for the clonal deletion of autoreactive thymocytes (14). BM transfer experiments showed that CD28 expression on both CD4SP and CD8SP thymocytes (gated for CD45.1+ NIK-sufficient donor cells) was upregulated from aly/aly recipient mice (Fig. 7A, top panel), most likely reflecting the paucity of CD80high mTECs together with the reduced CD80/86 expression from thymic DCs in aly/aly recipient mice. Similarly, CD27 expression on both CD4SP and CD8SP thymocytes was upregulated from aly/aly recipient mice (Fig. 7A, bottom panel). Consistent with the upregulation of CD27 on SP thymocytes, immunohistochemical analysis showed that compared with thymi from aly/+ recipient mice, thymi from aly/aly recipient mice had significantly weaker expression of CD70 from the medullary region that included both CD11c+ DCs and mTECs (Fig. 7B). Taking all the results together, we suggest an NIK-dependent thymic cross-talk in the establishment of self-tolerance, as illustrated in Fig. 8.

FIGURE 7.

Altered expression of costimulatory receptors on mature SP thymocytes from aly mice. (A) Expression of CD28 (top panel) and CD27 (bottom panel) on CD4SP (left panel) or CD8SP thymocytes (right panel) from lethally irradiated aly/+ or aly/aly recipient mice (CD45.2) reconstituted with wild-type(WT) CD45.1 congenic mouse BM was examined by flow cytometric analysis after gating for CD45.1+ thymocytes. Profiles with red and blue lines are those for aly/+ mouse SP T cells and aly/aly mouse SP T cells, respectively. Gray lines represent staining with the isotype control. (B) Immunohistochemical analysis of thymi showed weaker staining for CD70 (in red) from the medulla in lethally irradiated aly/aly recipient mice (Rc) reconstituted with wild-type CD45.1 congenic mouse BM compared with that from aly/+ recipient mice. CD11c+ cells (in green) were located predominantly in the medulla. One representative experiment from a total of two repeats is shown. Scale bar, 100 μm.

FIGURE 7.

Altered expression of costimulatory receptors on mature SP thymocytes from aly mice. (A) Expression of CD28 (top panel) and CD27 (bottom panel) on CD4SP (left panel) or CD8SP thymocytes (right panel) from lethally irradiated aly/+ or aly/aly recipient mice (CD45.2) reconstituted with wild-type(WT) CD45.1 congenic mouse BM was examined by flow cytometric analysis after gating for CD45.1+ thymocytes. Profiles with red and blue lines are those for aly/+ mouse SP T cells and aly/aly mouse SP T cells, respectively. Gray lines represent staining with the isotype control. (B) Immunohistochemical analysis of thymi showed weaker staining for CD70 (in red) from the medulla in lethally irradiated aly/aly recipient mice (Rc) reconstituted with wild-type CD45.1 congenic mouse BM compared with that from aly/+ recipient mice. CD11c+ cells (in green) were located predominantly in the medulla. One representative experiment from a total of two repeats is shown. Scale bar, 100 μm.

Close modal
FIGURE 8.

Pleiotropic role of NIK in thymic cross-talk. Conventional thymic cross-talk has been recognized as a bidirectional and interdependent dialogue between developing thymocytes and mTECs (left side). In the current study, we have demonstrated that NIK in mTECs controls Treg production and terminal differentiation of conventional αβ SP thymocytes in this context (black arrow at left). Many reported studies have also demonstrated the effect of thymocytes in the development and function of mTECs, as drawn by a gray arrow (1, 3, 38, 46). We further demonstrated that the development and function of thymic DCs are strongly influenced by mTECs in a NIK-dependent manner (black arrow at right). Given that thymic DCs can affect the function of thymocytes in turn (21) (gray arrow), we further suggest that thymic cross-talk can be viewed as an integrated circuit involving at least three components: mTECs, thymocytes, and thymic DCs. Possible actions indicated by clear arrows need to be further investigated. mTECs with nuclear dot protein at right represent Aire+ mTECs.

FIGURE 8.

Pleiotropic role of NIK in thymic cross-talk. Conventional thymic cross-talk has been recognized as a bidirectional and interdependent dialogue between developing thymocytes and mTECs (left side). In the current study, we have demonstrated that NIK in mTECs controls Treg production and terminal differentiation of conventional αβ SP thymocytes in this context (black arrow at left). Many reported studies have also demonstrated the effect of thymocytes in the development and function of mTECs, as drawn by a gray arrow (1, 3, 38, 46). We further demonstrated that the development and function of thymic DCs are strongly influenced by mTECs in a NIK-dependent manner (black arrow at right). Given that thymic DCs can affect the function of thymocytes in turn (21) (gray arrow), we further suggest that thymic cross-talk can be viewed as an integrated circuit involving at least three components: mTECs, thymocytes, and thymic DCs. Possible actions indicated by clear arrows need to be further investigated. mTECs with nuclear dot protein at right represent Aire+ mTECs.

Close modal

Conventional thymic cross-talk has been recognized as a dialogue primarily between thymocytes and mTECs, contributing to shaping of the self-tolerant T cell repertoire (1). In the current study, we demonstrated that the development and function of thymic DCs is also under the control of NIK-expressing mTECs, and we propose that DCs are also critically involved in thymic cross-talk. Therefore, the thymus can now be viewed as a platform where two essential types of interplay (i.e., between thymocytes and mTECs and between DCs and mTECs) further cross. Given that thymic DCs control some aspects of thymocyte function in an NIK-dependent manner (21), our present study has revealed a pleiotropic role of NIK for thymic cross-talk in the establishment of self-tolerance, as illustrated in Fig. 8.

The production of Tregs can be divided into two steps: the production of CD25+Foxp3 Treg precursors and their maturation into CD25+Foxp3+ Tregs (4, 36). The fact that the production of CD25+Foxp3 Treg precursors was impaired in aly mice is consistent with our notion that NIK in the thymic stroma is required for the de novo production of Tregs in the thymus. Impaired production of CD25+Foxp3 Treg precursors in aly mice suggests an impairment of either the expression of agonistic self-Ags from APCs (i.e., role of NIK in mTECs and/or thymic DCs) or of signals downstream from TCR coupled with costimulatory activities required for the positive selection of Tregs (i.e., the role of NIK in T cells), or both, as a result of NIK mutation. BM transfer experiments suggested the involvement of both the thymic stroma (i.e., mTECs) and hematopoietic cells (i.e., DCs and/or T cells) in this defect. Although several studies including ours have demonstrated the cell-intrinsic roles of NIK in T cell reactivity (1520), one study has suggested that NIK does not play a major role in T cells because BMs from mice expressing wild-type NIK in DCs but lacking NIK in other hematopoietic cells (including T cells) were able to restore Treg production in the thymus when transferred to wild-type recipient mice (21). This suggested that NIK in DCs could explain many of the T cell phenotypes from aly mice, thereby emphasizing that T cell phenotypes are controlled by DCs in an NIK-dependent manner. However, the issue has proved to be more complex because, in the current study, we have demonstrated that phenotypes of DC were also under the control of the thymic stroma and dependent on NIK. From this viewpoint, our present findings do not exclude a cell-autonomous role of NIK in nonstromal cells (i.e., T cells or DCs) in immune regulation or indicate the cell types, T cells or DCs, in which NIK plays a predominant role in thymic cross-talk. Instead, our results suggest a more complex interplay between mTECs, T cells, and DCs, as further discussed below.

We have demonstrated that fewer Tregs were developed when aly/aly mice received wild-type BM (Fig. 1). This could be a result of the following scenarios: 1) NIK-mutant mTECs were unable to support Treg generation (we assume that this primarily occurs through the cross-talk between thymic stroma and thymocytes); 2) NIK-mutant mTECs altered the development and function of thymic DCs (Figs. 4, 5), thereby affecting the Treg generation; or 3) a combination of both. These possibilities are not contradictory to the notion that Treg generation is dependent on the function of the cell-intrinsic role of NIK in thymic DCs (21). Of note, the study reported by Hofmann et al. (21) highlighted the significance of the cross-talk between thymic DCs and thymocytes because they transferred NIK-rescued DCs into an NIK-sufficient thymic stromal microenvironment. In contrast, we have investigated the effect of NIK-deficient thymic stroma on thymocytes (possibility 1) and/or DCs (possibility 2). Given that DCs are the critical determinant for the production of Tregs in an NIK-dependent manner (21), we support the idea that altered phenotypes of NIK-sufficient DCs interacting with NIK-deficient thymic stroma has an impact on Treg generation (possibility 2). At this point, it remains unclear which types of factors in the thymic stroma control the DC phenotypes that we observed within the thymic microenvironment. However, cytokines and chemokines are most likely involved. Signals mediated via the lymphotoxin β receptor, which requires NIK activity for its role in lymphoid organogenesis (8, 41), induce a wide variety of cytokines and chemokines from stromal cells (42). The possible presence of a reverse effect in thymic cross-talk, from DCs toward mTECs (Fig. 8), also needs to be investigated in the future.

Conversely, when aly/aly mouse BM was transferred into wild-type mice, thymic DC differentiation (in terms of the numbers and costimulatory expression) was apparently normal in these BM chimeras (Fig. 4). This result appears to be in contrast to the fact that NIK mutation is responsible for the decreased production of splenic DCs in a cell-autonomous manner (12). There has been an interesting precedent for this phenomenon. Proietto et al. (43) compared the phenotype and function of DC subsets in both the thymus and spleen by focusing on the expression of genes encoding chemokines/chemokine receptors and TLRs. They observed significant differences in chemokine production between splenic and thymic DC subsets. The results suggested that the functional differences are intrinsic to DCs reflecting a different developmental pathway, or they may be a result of a microenvironmental factor that influences the phenotype and functional properties of the resident DCs or both. Although NIK-deficient thymic DCs apparently developed normally in an NIK-sufficient microenvironment, they may remain responsible for the reduced Tregs in this experimental setting. It is still possible that other aspects of NIK-deficient DCs, beyond the numbers and costimulatory molecule expression, could contribute to the altered production of thymic Tregs.

We found that CD27 expression on both CD4SP and CD8SP thymocytes was upregulated in aly recipient mice (Fig. 7A). Although we were unsuccessful in quantitatively monitoring the levels of CD70 expression in mTECs or thymic DCs by flow cytometric analysis (Y. Mouri and M. Matsumoto, unpublished observations), immunohistochemistry consistently demonstrated weaker staining of CD70 in the thymic medulla of aly/aly mice relative to the medulla of aly/+ mice (Fig. 7B), possibly accounting for the upregulation of CD27 on CD4SP and CD8SP thymocytes in aly/aly mice. In this context, a recent report suggesting that mTECs, together with thymic DCs, promote Treg production via the CD27–CD70 pathway merits attention (44). The authors suggested that the CD27–CD70 pathway rescues developing Tregs from apoptosis. It is possible that weaker activation of the CD27–CD70 pathway induced by the interaction between Treg precursors and mTECs and/or thymic DCs in aly mice may account for the impaired production of Treg precursors in this strain. Even if this were the case, it would be difficult to determine whether upregulation of CD27 in T cells [a result of the cell-intrinsic role of NIK downstream of CD27 (39)] or downregulation of CD70 from mTECs and/or thymic DCs occurs first in the context of the complex thymic cross-talk we suggest.

Finally, although aly mice developed autoimmune pathology that was dependent on the thymic stroma in a polyclonal setting (10), it was unclear whether the development of autoimmunity in this strain involved any defect of negative selection beyond that in the production of Tregs. Although NIK was dispensable for clonal deletion of T cells specific for the ubiquitously expressed self-Ag, H-Y male Ag (23), we demonstrated that there was impairment of negative selection of CD4SP (OT-II) and CD8SP (OT-I) T cells specific for transgenically expressed TRA (i.e., OVA) in aly mice. However, crossing of OT-I Tg with RIP-OVA Tg on an aly/aly background did not lead to the development of overt diabetes. However, upon transfer of splenocytes from OT-I Tg crossed with RIP-OVA Tg on an aly/aly background into RIP-OVA Tg, we observed the development of diabetes in the recipient mice in some cases (Y. Mouri and M. Matsumoto, unpublished observations). One reason why OT-I Tg crossed with RIP-OVA Tg on aly/aly background did not develop diabetes, despite the defect in negative selection, may have been a defect in the development of secondary lymphoid organs (i.e., lack of peripheral lymph nodes and Peyer’s patches together with a disorganized splenic architecture) (8, 9, 41). In addition, the dispensable role of NIK in the H-Y TCR-Tg system (23) may have been a result of clonal deletion of autoreactive T cells within cortical TECs (40), for which development was NIK independent. An alternative possibility is that H-Y male Ag was so abundant that the defect in Ag presentation by mTECs and/or DCs to the clonotypic T cells in aly mice may have been easily overcome and indiscernible.

OVA transcripts from total thymi of aly mice were not reduced, despite the dramatically reduced numbers of CD80highMHC-IIhigh mTECs. One possible explanation for the defect in negative selection with preserved OVA transcripts from the thymic stroma may have been that existing mTECs with NIK mutation may have had a defect in the processing and/or presentation of the self-Ag required for deletional tolerance. Similar findings have been reported for Aire-deficient mice. Aire-deficient thymi showed a defect in the negative selection of OVA-specific T cells in the same RIP-OVA Tg mouse system, although the abundance of OVA transcripts remained unchanged in mTECs lacking Aire (45). Because deletion of OT-II cells in RIP-OVA Tg requires the participation of BM-APCs (14), it is also possible that mTECs from aly mice show defective transfer of self-Ags to thymic DCs. Alternatively, thymic DCs from aly mice may have defective elimination of autoreactive T cells because of the reduction in their numbers, together with the impaired APC function (i.e., altered expression of costimulatory molecules from thymic DCs) that we have demonstrated in the current study. To address these issues, it will be necessary to precisely identify the cell types present among mTECs presenting self-Ags (i.e., OVA in this case) and to elucidate the unique tolerogenic functions of mTECs.

We thank Drs. J.G. Cyster and M.J. Bevan for provision of anti-S1P1 Ab and RIP-OVA Tg, respectively.

This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Core Research for Evolutionary Science and Technology grant from the Japan Science Technology Agency (to M.M.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

BM

bone marrow

cDC

conventional DC

CRP

C-reactive protein

DC

dendritic cell

KI

knockin mouse

MHC-II

MHC class II

mTEC

medullary thymic epithelial cell

NIK

NF-κB–inducing kinase

pDC

plasmacytoid DC

RIP-OVA Tg

transgenic mice expressing OVA under the control of rat insulin promoter

SP

single-positive

S1P

sphingosine 1-phosphate

S1P1

sphingosine 1-phosphate receptor type 1

TEC

thymic epithelial cell

Tg

transgenic mice

TRA

tissue-restricted self-Ag

Treg

regulatory T cell

UEA-1

Ulex europaeus agglutinin 1.

1
van Ewijk
W.
,
Shores
E. W.
,
Singer
A.
.
1994
.
Crosstalk in the mouse thymus.
Immunol. Today
15
:
214
217
.
2
Hogquist
K. A.
,
Baldwin
T. A.
,
Jameson
S. C.
.
2005
.
Central tolerance: learning self-control in the thymus.
Nat. Rev. Immunol.
5
:
772
782
.
3
Kyewski
B.
,
Klein
L.
.
2006
.
A central role for central tolerance.
Annu. Rev. Immunol.
24
:
571
606
.
4
Sakaguchi
S.
2004
.
Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses.
Annu. Rev. Immunol.
22
:
531
562
.
5
Aschenbrenner
K.
,
D’Cruz
L. M.
,
Vollmann
E. H.
,
Hinterberger
M.
,
Emmerich
J.
,
Swee
L. K.
,
Rolink
A.
,
Klein
L.
.
2007
.
Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells.
Nat. Immunol.
8
:
351
358
.
6
Malinin
N. L.
,
Boldin
M. P.
,
Kovalenko
A. V.
,
Wallach
D.
.
1997
.
MAP3K-related kinase involved in NF-κB induction by TNF, CD95 and IL-1.
Nature
385
:
540
544
.
7
Sun
S. C.
2012
.
The noncanonical NF-κB pathway.
Immunol. Rev.
246
:
125
140
.
8
Shinkura
R.
,
Kitada
K.
,
Matsuda
F.
,
Tashiro
K.
,
Ikuta
K.
,
Suzuki
M.
,
Kogishi
K.
,
Serikawa
T.
,
Honjo
T.
.
1999
.
Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-κ b-inducing kinase.
Nat. Genet.
22
:
74
77
.
9
Miyawaki
S.
,
Nakamura
Y.
,
Suzuka
H.
,
Koba
M.
,
Yasumizu
R.
,
Ikehara
S.
,
Shibata
Y.
.
1994
.
A new mutation, aly, that induces a generalized lack of lymph nodes accompanied by immunodeficiency in mice.
Eur. J. Immunol.
24
:
429
434
.
10
Kajiura
F.
,
Sun
S.
,
Nomura
T.
,
Izumi
K.
,
Ueno
T.
,
Bando
Y.
,
Kuroda
N.
,
Han
H.
,
Li
Y.
,
Matsushima
A.
, et al
.
2004
.
NF-κ B-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner.
J. Immunol.
172
:
2067
2075
.
11
Boehm
T.
,
Scheu
S.
,
Pfeffer
K.
,
Bleul
C. C.
.
2003
.
Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho-epithelial cross talk via LTβR.
J. Exp. Med.
198
:
757
769
.
12
Tamura
C.
,
Nakazawa
M.
,
Kasahara
M.
,
Hotta
C.
,
Yoshinari
M.
,
Sato
F.
,
Minami
M.
.
2006
.
Impaired function of dendritic cells in alymphoplasia (aly/aly) mice for expansion of CD25+CD4+ regulatory T cells.
Autoimmunity
39
:
445
453
.
13
Dresch
C.
,
Leverrier
Y.
,
Marvel
J.
,
Shortman
K.
.
2012
.
Development of antigen cross-presentation capacity in dendritic cells.
Trends Immunol.
33
:
381
388
.
14
Gallegos
A. M.
,
Bevan
M. J.
.
2004
.
Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation.
J. Exp. Med.
200
:
1039
1049
.
15
Matsumoto
M.
,
Yamada
T.
,
Yoshinaga
S. K.
,
Boone
T.
,
Horan
T.
,
Fujita
S.
,
Li
Y.
,
Mitani
T.
.
2002
.
Essential role of NF-κ B-inducing kinase in T cell activation through the TCR/CD3 pathway.
J. Immunol.
169
:
1151
1158
.
16
Ishimaru
N.
,
Kishimoto
H.
,
Hayashi
Y.
,
Sprent
J.
.
2006
.
Regulation of naive T cell function by the NF-κB2 pathway.
Nat. Immunol.
7
:
763
772
.
17
Yamada
T.
,
Mitani
T.
,
Yorita
K.
,
Uchida
D.
,
Matsushima
A.
,
Iwamasa
K.
,
Fujita
S.
,
Matsumoto
M.
.
2000
.
Abnormal immune function of hemopoietic cells from alymphoplasia (aly) mice, a natural strain with mutant NF-κ B-inducing kinase.
J. Immunol.
165
:
804
812
.
18
Jin
W.
,
Zhou
X. F.
,
Yu
J.
,
Cheng
X.
,
Sun
S. C.
.
2009
.
Regulation of Th17 cell differentiation and EAE induction by MAP3K NIK.
Blood
113
:
6603
6610
.
19
Sánchez-Valdepeñas
C.
,
Martín
A. G.
,
Ramakrishnan
P.
,
Wallach
D.
,
Fresno
M.
.
2006
.
NF-κB-inducing kinase is involved in the activation of the CD28 responsive element through phosphorylation of c-Rel and regulation of its transactivating activity.
J. Immunol.
176
:
4666
4674
.
20
Murray
S. E.
,
Polesso
F.
,
Rowe
A. M.
,
Basak
S.
,
Koguchi
Y.
,
Toren
K. G.
,
Hoffmann
A.
,
Parker
D. C.
.
2011
.
NF-κB–inducing kinase plays an essential T cell–intrinsic role in graft-versus-host disease and lethal autoimmunity in mice.
J. Clin. Invest.
121
:
4775
4786
.
21
Hofmann
J.
,
Mair
F.
,
Greter
M.
,
Schmidt-Supprian
M.
,
Becher
B.
.
2011
.
NIK signaling in dendritic cells but not in T cells is required for the development of effector T cells and cell-mediated immune responses.
J. Exp. Med.
208
:
1917
1929
.
22
Macpherson
A. J.
,
Uhr
T.
.
2003
.
The donor splice site mutation in NFκB-inducing kinase of alymphoplasia (aly/aly) mice.
Immunogenetics
54
:
693
698
.
23
Kisielow
P.
,
Blüthmann
H.
,
Staerz
U. D.
,
Steinmetz
M.
,
von Boehmer
H.
.
1988
.
Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes.
Nature
333
:
742
746
.
24
Kuroda
N.
,
Mitani
T.
,
Takeda
N.
,
Ishimaru
N.
,
Arakaki
R.
,
Hayashi
Y.
,
Bando
Y.
,
Izumi
K.
,
Takahashi
T.
,
Nomura
T.
, et al
.
2005
.
Development of autoimmunity against transcriptionally unrepressed target antigen in the thymus of Aire-deficient mice.
J. Immunol.
174
:
1862
1870
.
25
Yano
M.
,
Kuroda
N.
,
Han
H.
,
Meguro-Horike
M.
,
Nishikawa
Y.
,
Kiyonari
H.
,
Maemura
K.
,
Yanagawa
Y.
,
Obata
K.
,
Takahashi
S.
, et al
.
2008
.
Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance.
J. Exp. Med.
205
:
2827
2838
.
26
Kuwata
N.
,
Igarashi
H.
,
Ohmura
T.
,
Aizawa
S.
,
Sakaguchi
N.
.
1999
.
Cutting edge: absence of expression of RAG1 in peritoneal B-1 cells detected by knocking into RAG1 locus with green fluorescent protein gene.
J. Immunol.
163
:
6355
6359
.
27
Kurts
C.
,
Heath
W. R.
,
Carbone
F. R.
,
Allison
J.
,
Miller
J. F.
,
Kosaka
H.
.
1996
.
Constitutive class I-restricted exogenous presentation of self antigens in vivo.
J. Exp. Med.
184
:
923
930
.
28
Barnden
M. J.
,
Allison
J.
,
Heath
W. R.
,
Carbone
F. R.
.
1998
.
Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements.
Immunol. Cell Biol.
76
:
34
40
.
29
Nishikawa
Y.
,
Hirota
F.
,
Yano
M.
,
Kitajima
H.
,
Miyazaki
J.
,
Kawamoto
H.
,
Mouri
Y.
,
Matsumoto
M.
.
2010
.
Biphasic Aire expression in early embryos and in medullary thymic epithelial cells before end-stage terminal differentiation.
J. Exp. Med.
207
:
963
971
.
30
Mouri
Y.
,
Yano
M.
,
Shinzawa
M.
,
Shimo
Y.
,
Hirota
F.
,
Nishikawa
Y.
,
Nii
T.
,
Kiyonari
H.
,
Abe
T.
,
Uehara
H.
, et al
.
2011
.
Lymphotoxin signal promotes thymic organogenesis by eliciting RANK expression in the embryonic thymic stroma.
J. Immunol.
186
:
5047
5057
.
31
Zachariah
M. A.
,
Cyster
J. G.
.
2010
.
Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction.
Science
328
:
1129
1135
.
32
Niki
S.
,
Oshikawa
K.
,
Mouri
Y.
,
Hirota
F.
,
Matsushima
A.
,
Yano
M.
,
Han
H.
,
Bando
Y.
,
Izumi
K.
,
Matsumoto
M.
, et al
.
2006
.
Alteration of intra-pancreatic target-organ specificity by abrogation of Aire in NOD mice.
J. Clin. Invest.
116
:
1292
1301
.
33
Proietto
A. I.
,
van Dommelen
S.
,
Wu
L.
.
2009
.
The impact of circulating dendritic cells on the development and differentiation of thymocytes.
Immunol. Cell Biol.
87
:
39
45
.
34
McCaughtry
T. M.
,
Wilken
M. S.
,
Hogquist
K. A.
.
2007
.
Thymic emigration revisited.
J. Exp. Med.
204
:
2513
2520
.
35
Cuss
S. M.
,
Green
E. A.
.
2012
.
Abrogation of CD40-CD154 signaling impedes the homeostasis of thymic resident regulatory T cells by altering the levels of IL-2, but does not affect regulatory T cell development.
J. Immunol.
189
:
1717
1725
.
36
Hsieh
C. S.
,
Rudensky
A. Y.
.
2005
.
The role of TCR specificity in naturally arising CD25+ CD4+ regulatory T cell biology.
Curr. Top. Microbiol. Immunol.
293
:
25
42
.
37
Cowan
J. E.
,
Parnell
S. M.
,
Nakamura
K.
,
Caamano
J. H.
,
Lane
P. J.
,
Jenkinson
E. J.
,
Jenkinson
W. E.
,
Anderson
G.
.
2013
.
The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development.
J. Exp. Med.
210
:
675
681
.
38
Matsumoto
M.
2007
.
Transcriptional regulation in thymic epithelial cells for the establishment of self tolerance.
Arch. Immunol. Ther. Exp. (Warsz.)
55
:
27
34
.
39
Ramakrishnan
P.
,
Wang
W.
,
Wallach
D.
.
2004
.
Receptor-specific signaling for both the alternative and the canonical NF-κB activation pathways by NF-κB-inducing kinase.
Immunity
21
:
477
489
.
40
McCaughtry
T. M.
,
Baldwin
T. A.
,
Wilken
M. S.
,
Hogquist
K. A.
.
2008
.
Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.
J. Exp. Med.
205
:
2575
2584
.
41
Matsumoto
M.
,
Iwamasa
K.
,
Rennert
P. D.
,
Yamada
T.
,
Suzuki
R.
,
Matsushima
A.
,
Okabe
M.
,
Fujita
S.
,
Yokoyama
M.
.
1999
.
Involvement of distinct cellular compartments in the abnormal lymphoid organogenesis in lymphotoxin-α-deficient mice and alymphoplasia (aly) mice defined by the chimeric analysis.
J. Immunol.
163
:
1584
1591
.
42
Dejardin
E.
,
Droin
N. M.
,
Delhase
M.
,
Haas
E.
,
Cao
Y.
,
Makris
C.
,
Li
Z. W.
,
Karin
M.
,
Ware
C. F.
,
Green
D. R.
.
2002
.
The lymphotoxin-β receptor induces different patterns of gene expression via two NF-κB pathways.
Immunity
17
:
525
535
.
43
Proietto
A. I.
,
Lahoud
M. H.
,
Wu
L.
.
2008
.
Distinct functional capacities of mouse thymic and splenic dendritic cell populations.
Immunol. Cell Biol.
86
:
700
708
.
44
Coquet
J. M.
,
Ribot
J. C.
,
Bąbała
N.
,
Middendorp
S.
,
van der Horst
G.
,
Xiao
Y.
,
Neves
J. F.
,
Fonseca-Pereira
D.
,
Jacobs
H.
,
Pennington
D. J.
, et al
.
2013
.
Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway.
J. Exp. Med.
210
:
715
728
.
45
Anderson
M. S.
,
Venanzi
E. S.
,
Chen
Z.
,
Berzins
S. P.
,
Benoist
C.
,
Mathis
D.
.
2005
.
The cellular mechanism of Aire control of T cell tolerance.
Immunity
23
:
227
239
.
46
Anderson
G.
,
Lane
P. J.
,
Jenkinson
E. J.
.
2007
.
Generating intrathymic microenvironments to establish T-cell tolerance.
Nat. Rev. Immunol.
7
:
954
963
.

The authors have no financial conflicts of interest.

Supplementary data