Differential expression of CLIP: MHC class II and conventional endogenous peptide: MHC class II complexes by thymic epithelial cells and peripheral antigen-presenting cells

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide : MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of Eα52–68: I-A^b and class II-associated invariant chain peptide (CLIP): I-A^b complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of Eα52–68:I-A^b complexes in thymic epithelial cells is approximately 5–10 % of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A^b complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.     

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus

TCRαβ⁺ CD8α⁺CD8β^–intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely β2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86^neg/low and MHCI⁺. However, selective depletion of β2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.     

Vβ11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in mice bearing H-2q versus H-2b haplotypes

We have recently demonstrated that toxic shock syndrome toxin-1 (TSST-1) expanded Vβ11⁺ T lymphocytes contribute to Staphylococcus aureus arthritis and sepsis-induced mortality. Interestingly, Vβ11⁺ T-cell mediated joint pathology varies in different mouse strains. In this study, we characterized the in vitro pattern of Vβ11⁺ T-cell expansion by TSST-1 in mice with various genetic backgrounds. Mice expressing major histocompatibility complex (MHC) class II I-E molecules did not expand Vβ11⁺ T cells upon stimulation with TSST-1. Using B10 congeneic I-E negative mouse strains, we found that the TSST-1-expanded Vβ11⁺ T cells in B10Q (H-2^q) and B10M (H-2^f) mice but not in B10B (H-2^b) mice. Antigen-presenting cells (APC) from B10Q mice, L cells and lymphoma cell line transfected with a q gene did not restore the deficient Vβ11⁺ T-cell expansion by TSST-1 in purified T cells from B10B mice. In contrast, I-A^b APC were able to stimulate Vβ11⁺ T cells from H-2^q mice. Furthermore, Vβ11⁺ T cells in H-2^b mice did expand when exposed to staphylococcal enterotoxin A (SEA). These findings suggest that the T-cell repertoire, skewed by clonal deletion and inactivation of self-reactive T cells, accounts for the different magnitude of Vβ11⁺ T-cell expansion among the different mouse strains.     

Gene complementation in the T lymphocyte proliferative response to poly(Glu56Lys35Phe9)nFunctional evidence for a restriction element coded for by both the I-A and I-E subregions

The T lymphocyte proliferative response to poly(Glu⁵⁶Lys³⁵Phe⁹)_n (GLΦ) is under the control of two immune response genes, Ir-GLΦ-β and Ir-GLΦ-α, mapping in the I-A and I-E/C subregions of the major histocompatibility complex, respectively. Previous studies have demonstrated that in order to generate a response to GLΦ, both gene products must be expressed in the antigen-presenting cell (APC) but that neither responder allele has to be present in the responding T lymphocyte, provided that the T cell has matured in a responder environment. These results suggested that both gene products function as restricting elements in GLΦ presentation by APC. In this report, we provide further evidence for this model from experiments designed to test histocompatibility restrictions in antigen presentation at the I-E/C subregion. Genetic identity at the I-A subregion between T cells and APC was required for GLΦ presentation. To assess the requirements at I-E/C, B10. A(5R) T cells (I-A^b, I-E^k) primed to GLΦ were stimulated in vitro with GLΦ-pulsed spleen cells from F₁ hybrids between C57BL/10 (B10: I-A^b, I-E^b) which made the cells compatible at I-A, and a variety of B10 congenics bearing other H-2 haplotypes. Although none of the parental spleen cells could present GLΦ to B10.A(5R) T cells, spleen cells from F₁ hybrids between B10 and strains possessing H-2I of k, d, p and r presented GLΦ, whereas hybrids with strains possessing H-2I of f, q and s failed to present. This pattern of complementation for GLΦ presentation could not be explained on the basis of the responder status of the I-E/C donating parental haplotypes nor by invoking inhibitory stimuli from mixed lymphocyte reactions induced by the F_l APC. Rather, the pattern correlated with the presence of the serologic marker Ia.7 coded for by the I-E subregion of the complementing parental haplotype and the possession of an I-E-encoded a chain which has been shown by peptide mapping to be very similar in strains bearing the k, d, p and r haplotypes. These results suggest that the restriction element involved in the presentation of GLΦ to B10.A(5R) T cells is composed of a β chain encoded in I-A^b and an a chain encoded in I-E for which the allelic products of the k, d, p and r haplotypes are functionally equivalent. This correlation between structure and function represents the strongest evidence so far that Ia antigen-bearing molecules are the Ir gene products.     

An endogenously processed self peptide and the corresponding exogenous peptide bound to the same MHC class II molecule could be distinct ligands for TCR with different kinetic stability

Immunization with self peptides often elicits activation of CD4⁺ T cells in vivo. Although such peptides have been suggested to be derived from minor self determinants or self antigens sequestered from the immune system, we found that immunization with Eα peptide (Eα52 – 68), a major self determinant bound to I-A^b molecules, elicits an immune response in Eα-transgenic C57BL/6 (Eα-B6) mice where Eα52 – 68 is endogenously processed and presented by I-A^b molecules in the thymus and periphery. To better understand this response, a panel of T cell hybridomas raised against exogenous Eα52 – 68 were analyzed for their reactivity to spleen cells from Eα-B6 mice. Some hybridomas were stimulated with Eα-B6 spleen cells in the absence of exogenous Eα52 – 68, whereas others were not stimulated with them. The Eα52 – 68/I-A^b complex recognized by the TCR that is expressed on the hybridoma with reactivity to Eα-B6 spleen cells was found to be quite stable, whereas the complex recognized by the TCR on the hybridoma specific for the exogenous Eα52 – 68 lost the stimulation activity by incubation the complex at 37 °C for 10 min. Stimulation experiments using extensively substituted Eα analogue peptides suggested that amino acid residues at positions 57, 58, 60 and 62 of Eα52 – 68 are involved in the interaction with TCR recognizing the Eα52 – 68/I-A^b complex expressed on Eα-B6 spleen cells. While amino acid substitutions at positions 60 and 62 also affected the recognition of TCR specific for exogenous Eα52 – 68, all or many amino acid substitutions were allowed at position 58 or 57, respectively, without impairing the TCR recognition. Taken together, these results suggest that endogenously processed self peptide and the corresponding exogenous peptide bound to the same MHC class II molecule could be distinct TCR ligands with different kinetic stability and probably with different configuration.     

Both positive and negative effects on immune responses by expression of a second class II MHC molecule

It is perplexing why vertebrates express a limited number of major histocompatibility complex (MHC) molecules when theoretically, having a greater repertoire of MHC molecules would increase the number of epitopes presented, thereby enhancing thymic selection and T cell response to pathogens. It is possible that any positive effects would either be neutralized or outweighed by negative selection restricting the T cell repertoire. We hypothesize that the limit on MHC number is due to negative consequences arising from expressing additional MHC. We compared T cell responses between B6 mice (I-A⁺) and B6.E⁺ mice (I-A⁺, I-E⁺), the latter expressing a second class II MHC molecule, I-E^b, due to a monomorphic Eα^k transgene that pairs with the endogenous I-Eβ^b chain. First, the naive T cell Vβ repertoire was altered in B6.E⁺ thymi and spleens, potentially mediating different outcomes in T cell reactivity. Although the B6 and B6.E⁺ responses to hen egg-white lysozyme (HEL) protein immunization remained similar, other immune models yielded differences. For viral infection, the quality of the T cell response was subtly altered, with diminished production of certain cytokines by B6.E⁺ CD4⁺ T cells. In alloreactivity, the B6.E⁺ T cell response was significantly dampened. Finally, we observed markedly enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE) in B6.E⁺ mice. This correlated with decreased percentages of nTreg cells, supporting the concept of Tregs exhibiting differential susceptibility to negative selection. Altogether, our data suggest that expressing an additional class II MHC can produce diverse effects, with more severe autoimmunity providing a compelling explanation for limiting the expression of MHC molecules.     

Prevention of murine lupus by an I-E α chain transgene: Protective role of I-E α chain-derived peptides with a high affinity to I-Ab molecules

The expression of a transgene encoding the I-E α chain prevents a lupus-like autoimmune syndrome in BXSB mice. However, it had not been elucidated whether the Eα^d transgene-mediated protective effect results from I-E expression or from the generation of I-E α chain-derived peptides (Eα peptide) displaying high affinity for the I-A^b molecule. To address this question, two different BXSB lines expressing the transgene at low or high levels were crossed with lupus-prone MRL mice; this resulted in three types of (MRL × BXSB)F₁ mice, differing in the expression levels of I-E molecules and of Eα peptides presented by I-A^b molecules. Comparative analysis of these three (MRL × BXSB)F₁ mice as well as several BXSB transgenic lines showed that the Eα^d transgene-mediated protection paralleled the expression levels of Eα peptide presented by I-A^b molecules, but not of I-E molecules on B cells. In addition, use of transgenic and nontransgenic double bone marrow chimeras showed a selective activation of nontransgenic B cells during I-A^b-restricted T cell-dependent immune responses, while both transgenic and nontransgenic B cells were comparably activated during T cell-independent responses. These results favor a model of autoimmunity prevention based on competition for antigen presentation, in which excessive generation of Eα peptides prevents, because of their high affinity to the I-A molecules, activation of potential autoreactive T and B cells.     

Comparative Sequence Analysis of cDNA Clones Encoding I-A Molecules of the CH12 B Cell Lymphoma: Nucleotide Differences do not Account for their ‘Defective’ Function in B Cell Stimulation

The CH12 B cell lymphoma can be stimulated to secrete antibody by helper T cells that interact with I-E^K but not I-A^K molecules expressed on its membrane. Both molecules present antigen to the appropriate T cells. We have analyzed the mRNA by Northern blot analysis and obtained partial sequences of cDNA clones encoding A_α and A_β of the I-A molecule to determine if deletions or mutations in the cytoplasmic or transmembrane domains account for the “defect” in triggering following interaction with I-A restricted helper T cells. The results provide no evidence for structural alterations in either A_α or A_β which could account for these observations. The implications of these findings on the role of class II molecules in B cell activation is discussed.     

Quantitative Analysis of the Efficiency of Clonal Deletion in the Thymus

One of the major mechanisms for establishing self-tolerance is the clonal deletion of self-reactive T cells during their development in the thymus. Using a TCR transgenic mouse model, we have established a quantitative ex vivo assay for examining the sensitivity and specificity of negative selection. Thymic organ cultures established from mice of varying MHC haplotypes were incubated with antigen, and the efficiency of clonal deletion assessed. We show here that clonal deletion of CD4⁺8⁺ thymocytes is sensitive to both the gene dosage and the allelic variation of MHC class II molecules expressed on thymic antigen-presenting cells. We also find that when epithelial cells in the thymic cortex are the only antigen-presenting cells expressing the appropriate MHC class II molecules, negative selection of CD4⁺8⁺ cells is as efficient as when antigen is presented on all thymic antigen-presenting cells. These studies demonstrate that the induction of self-tolerance via clonal deletion in the thymus is a function not only of antigen concentration, but also of MHC class II cell-surface density. In addition, together with the reports of others, these results confirm that cortical epithelial cells can mediate negative selection, and demonstrate that they do so in the intact thymic microenvironment.     

Inhibition of I-Ad-, but not Db-restricted peptide-induced thymic apoptosis by glucocorticoid receptor antagonist RU486 in T cell receptor transgenic mice

Thymocytes differentiate by positive and negative selection of immature CD4⁺ CD8⁺ T cells. Negative selection occurs by default or by high-affinity recognition of peptides bound to proteins encoded by the major histocompatibility complex (MHC). MHC class I molecules are expressed on many different cell types, although at different levels, whereas MHC class II molecules are selectively expressed on thymic epithelial cells (TEC) and dendritic cells (DC). We investigated the role of the glucocorticoid receptor (GR) in thymic negative selection using the receptor antagonist RU486. Glucocorticoids (GC) are known to be potent inducers of apoptosis in CD4⁺ CD8⁺ thymocytes, and we have earlier shown that anti-CD3-induced thymic apoptosis can be blocked by RU486 in vivo. We now show that anti-CD3 induces thymic apoptosis in mice that have been adrenalectomized (ADX), and that RU486 inhibits anti-CD3 antibody-mediated thymocyte killing in newborn thymic organ cultures. Thymocyte apoptosis induced by ovalbumin peptide OVA323–339 treatment of mice transgenic for the DO11.10T cell receptor (TCR), which recognizes this peptide in the context of I-A^d, was found to be inhibited by RU486. These mice responded to peptide treatment by an extensive activation of the peripheral immune system, which became lethal in 60% of the mice when accompanied by simultaneous RU486 treatment. In contrast, RU486 had no effect on thymic apoptosis induced by the influenza A nucleoprotein NP366–374 peptide, recognized in context of D^b, in F5 TCR transgenic mice. We interpret the results to demonstrate that different deletion systems operate in the thymus. We propose that endogenous GC may be important for negative selection by default and by high-affinity recognition of endogenous MHC-presented peptides on TEC.     

Effect of the expression of DRαEβNOD molecule on the development of insulitis and diabetes in the non-obese diabetic (NOD) mouse

Previous studies have shown that a transgenic I-Eα gene, the mouse homologue of human DRα gene, prevents the development of insulitis and hence of diabetes in NOD mice. To investigate the mechanism of this prevention, we generated two strains of NOD mice expressing DRαEβ molecule: DRα-24-NOD expressing DRαEβ molecule on thymic epithelial cells (TEC) and bone marrow-derived cells (BDC), and DRα-30-NOD expressing DRαEβ molecule only on the TEC, and these mice were monitored for disease development. Because the DRαEβ molecule reconstituted I-E controlled immune regulation, it would become clear which cell type, TEC or BDC, was responsible for the I-E-mediated disease protection. To our surprise, however, DRα-24-NOD developed insulitis and diabetes comparably to non-transgenic littermates. This suggested that the difference in structure between DRα and Eα molecules contributed to the difference in preventive effect on the development of insulitis and diabetes between DRα-24-NOD and Eα-NOD. In an analysis of the T cell proliferative responses to glutamic acid decarboxylase (GAD) 65-derived peptides which were known to be diabetogenic autoantigens, it was shown that DRα-24-NOD and NOD acquired comparable level of T cell response to GAD 509–528 but 5–10-fold higher response was observed in Eα-NOD. This suggested that I-A^NOD and EαEβ^NOD molecules could present GAD 509–528 peptide to T cells, while DRαEβ^NOD could not. Furthermore, T cells from DRα transgenic mice showed proliferative response to antigen-presenting cells from Eα transgenic mice in primary mixed lymphocyte reaction. This also suggested that the EαEβ molecule does differ in structure and peptide binding from the DRαEβ molecule. Present data suggested a possibility that the T cell repertoire selection, or the T cell response to GAD 65 and/or other unknown antigens specifically mediated by I-E molecule, may contribute to the prevention of disease development in Eα-NOD.     

Thymic heterotypic cellular complexes in gene-targeted mice with defined blocks in T cell development and adhesion molecule expression

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1^−/−, TCRβ^−/−, and p56^lck−/− mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRβ and p56^lck gene mutations but partially decreased in RAG-1^−/− mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRβ chain. Genetic blocks in CD8 lineage commitment (CD8^−/− and IFN regulatory factor-1^−/− mice) and positive and negative T cell selection (CD45^−/−, TCRα^−/−, and CD30^−/− mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4^−/− mice, but not MHC class II^−/− mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4^−/− block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.     

A peptide binding weakly to the major histocompatibility molecule augments T cell responses

An I-A^d-derived peptide PB1 was found to enhance the reactivity of I-A^d-restricted T cells. The augmentative effect was not due to the cross-reactivity of PB1 peptide with antigens. PB1 had no effect on T cells specific for I-A^b and I-E^k, nor did PB1 increase the T cell responses to concanavalin A and staphyloccocal enterotoxin B. The strict I-A^d specificity suggests that PB1 enhances the recognition of antigen-I-A^d complex by T cell receptor. PB1 bound to I-A^d weakly. The augmentative effect could be found on other I-A^d-binding peptides in appropriate conditions; however, PB1 was distinct in its prominently augmentative effect on all the I-A^d-restricted T cells analyzed. A similar enhancing activity was demonstrated on a synthetic transferrin receptor peptide with minimum affinity for I-A^d. The unusual enhancing activity of PB1 may thus be attributed to the low I-A^d binding affinity. It was postulated that the binding of low-affinity PB1 would not only stabilize I-A^d structure, but also enhance the binding of other peptides. This was supported by the increased binding of OVA 323-339 and cI 84–98 to I-A^d in the presence of PB1. The inclusion of PB1 in the immunization mixture also enhanced T cell responses in vivo, suggesting the possibility of using low-affinity peptide to promote specific immunity.     

Major histocompatibility complex regulation of T helper functions mapped to a peptide C terminus that controls ligand density

The functional status (Th1- versus Th2-like) of CD4 T cells primed against human collagen type IV (hCol IV) or a single 30mer peptide from the α2 chain of this molecule is predicted by the major histocompatibility complex (MHC) class II (I-A) genotype of the responding mice. H-2^s mice elicit Th1-like cell-mediated responses to these antigens, whereas Th2-like humoral responses are primed in H-2^b,d,k mice. We now report that the ability of MHC to dictate T helper function in this system depends upon a single amino acid of the minimal α2(IV) peptide. The C terminus of this minimal (12mer) peptide is -G-G-P-K, which is predicted to form a β-turn. The present data demonstrate that the terminal lysine (K) stabilizes the immunogens full biological effects necessary for exclusive cellmediated responses in H-2^s mice. The lysine-truncated (11mer) peptide with otherwise identical sequence effectively primes T helper function in both H-2^b and H-2^s genotypes. Most importantly, our direct analysis of these peptides' presentation by live antigen-presenting cells (APC) reveals that the 12mer is bound at a log higher density on H-2^s APC than on H-2^b APC, and that the 11mer is presented at an equally low relative density on APC from both genotypes. In vitro analyses of 12mer/11mer cross-reactive Th clones demonstrate that I-A^s restricted clones require about 1–2 log lower doses of 12mer peptide than 11mer peptide to stimulate equivalent thymidine incorporation and cytokine release. By contrast, I-A^b-restricted (12mer/11mer cross-reactive) Th clones show no preference for the 12mer and require relatively high peptide doses similar to those required to stimulate the I-A^s clones with the 11mer peptide. Thus, the peptide dose requirements of Th clones reflect the high density of presentation associated with the 12mer: I-A^s ligand. Taken together, the results directly support the role of ligand density as an important control point in the functional decision of CD4 T cells.     

Mechanisms by which the I-ABM12 Mutation Influences Susceptibility to Experimental Myasthenia Gravis: a Study in Homozygous and Heterozygous Mice

The I-A^bm12 mutation in C57B1/6 (B6) mice yields the B6. C-H-2^bm12 (bm12) strain, which is resistant to Experimental Myasthenia Gravis (EMG) induced by immunization with Torpedo acetylcholine receptor (TAChR), while the parental B6 strain is highly susceptible to EMG. CD4⁺ cells from bm12 mice immunized with TAChR do not recognize three sequence regions of the TAChR Q subunit which dominate the CD4⁺ cell sensitization in B6 mice. We immunized with TAChR bm12, B6 and (bm12B6)Fl mice. B6 and F1 mice developed EMG with comparable frequency. Their CD4⁺ cells recognized the same TAChR α subunit peptide sequences (Tα150–169, Tα181–200 and Tα360–378). CD4⁺ cells from TAChR-sensitized Fl mice were challenged with TAChR and α subunit epitope peptides, using F1, B6 or bml 2 APC. B6 and F1 APC presented all these Ag efficiently, while bm 12 APC presented TAChR and peptide Tα150–169 poorly and erratically. Anti-TAChR and anti-α subunit epitope CD4⁺ lines propagated from Fl and B6 mice had similar TcR Vβ usage. All lines but those specific for the sequence Tα150–169 had unrestricted Vβ usage. Anti-Tα150–169 lines from both B6 and Fl mice had a strong preferential usage of Vβ6. Anti-Tα150–169 lines from Fl mice had also a slightly higher Vβ14 usage. B6, bm12 and Fl mice developed similar anti-TAChR Ab titres, and had Ab bound to muscle AChR in comparable amounts. Therefore EMG resistance of bm12 mice must be due to a subtle shift in the anti-AChR Ab repertoire, and absence of special Ab able to cause destruction and/or dysfunction of muscle AChR. This is probably related to the absence of CD4⁺ cells sensitized to epitopes within the sequence Tα 150–160, consequent to the inability of the I-A^bm12 molecule to present this sequence.     

Spatial (Tbata) expression in mature medullary thymic epithelial cells

The Spatial gene is expressed in highly polarized cell types such as testis germ cells, brain neurons and thymic epithelial cells (TEC). Its expression was documented in testis and brain but poorly characterized in thymus. Here, we characterize for the first time Spatial‐expressing TEC throughout ontogeny and adult mouse thymus. Spatial is expressed in thymic‐fated domain by embryonic day E10.5 and persists in subcapsular, cortical, medullary epithelial cells and in MTS24⁺ progenitor TEC. Using mouse strains in which thymocyte development is blocked at various stages, we show that Spatial expression is independent of thymocyte‐derived signals during thymus organogenesis. Analyses on purified thymic cell subsets show that Spatial short isoforms are expressed in cortical TEC (cTEC) and mature medullary TEC (mTEC). Spatial long isoforms were detected in the same TEC population. Spatial presents a nuclear distribution specific to mature mTEC expressing UEA1 and Aire. Aire‐ and RANKL‐deficient mice revealed that Spatial expression is drastically reduced in the thymus of these mutants. These findings reveal a critical function of Aire in regulating Spatial expression, which is compatible with promiscuous Spatial gene expression.