Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

SASH3 variants cause a novel form of X-linked combined immunodeficiency with immune dysregulation.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA-corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1-/- and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival.

Poor T-cell receptor ß repertoire diversity early posttransplant for severe combined immunodeficiency predicts failure of immune reconstitution.

BACKGROUND: Development of a diverse T-cell receptor ß (TRB) repertoire is associated with immune recovery following hematopoietic cell transplantation (HCT) for severe combined immunodeficiency (SCID). High-throughput sequencing of the TRB repertoire allows evaluation of clonotype dynamics during immune reconstitution. OBJECTIVES: We investigated whether longitudinal analysis of the TRB repertoire would accurately describe T-cell receptor diversity and illustrate the quality of T-cell reconstitution following HCT or gene therapy for SCID. METHODS: We used high-throughput sequencing to study composition and diversity of the TRB repertoire in 27 infants with SCID at 3, 6, and 12 months and yearly posttreatment(s). Total RNA from peripheral blood was used as template to amplify TRB rearrangements. RESULTS: TRB sequence analysis showed poor diversity at 3 months, followed by significant improvement by 6 months after cellular therapies. Kinetics of development of TRB diversity were similar in patients with a range of underlying gene defects. However, in patients with RAG and DCLRE1C defects, HCT with no conditioning or immune suppression only resulted in lower diversity than did HCT with conditioning. HCT from a matched donor correlated with higher diversity than did HCT from a mismatched donor. Naive CD4+ T-cell count at 6 months post-HCT correlated with higher TRB diversity. A Shannon index of diversity of 5.2 or lower 3 months after HCT predicted a need for a second intervention. CONCLUSIONS: TRB repertoire after hematopoietic cell therapies for SCID provides a quantitative and qualitative measure of diversity of T-cell reconstitution and permits early identification of patients who may require a second intervention.

c-Rel employs multiple mechanisms to promote the thymic development and peripheral function of regulatory T cells in mice.

The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel-/- ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel-/- mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel-/- tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.

SATB1 ensures appropriate transcriptional programs within naïve CD8+ T cells.

Special AT-binding protein 1 (SATB1) is a chromatin-binding protein that has been shown to be a key regulator of T-cell development and CD4+ T-cell fate decisions and function. The underlying function for SATB1 in peripheral CD8+ T-cell differentiation processes is largely unknown. To address this, we examined SATB1-binding patterns in naïve and effector CD8+ T cells demonstrating that SATB1 binds to noncoding regulatory elements linked to T-cell lineage-specific gene programs, particularly in naïve CD8+ T cells. We then assessed SATB1 function using N-ethyl-N-nitrosourea-mutant mice that exhibit a point mutation in the SATB1 DNA-binding domain (termed Satb1m1Anu/m1Anu ). Satb1m1Anu/m1Anu mice exhibit diminished SATB1-binding, naïve, Satb1m1Anu/m1Anu CD8+ T cells exhibiting transcriptional and phenotypic characteristics reminiscent of effector T cells. Upon activation, the transcriptional signatures of Satb1m1Anu/m1Anu and wild-type effector CD8+ T cells converged. While there were no overt differences, primary respiratory infection of Satb1m1Anu/m1Anu mice with influenza A virus (IAV) resulted in a decreased proportion and number of IAV-specific CD8+ effector T cells recruited to the infected lung when compared with wild-type mice. Together, these data suggest that SATB1 has a major role in an appropriate transcriptional state within naïve CD8+ T cells and ensures appropriate CD8+ T-cell effector gene expression upon activation.

The Impact of MHC Class I Dose on Development and Maintenance of the Polyclonal Naive CD8+ T Cell Repertoire.

Naive CD8+ T cell survival in the periphery is critically dependent on tonic TCR signaling through peptide + MHC class I (MHCI) recognition; however, little is known about how natural variation in MHCI levels impacts the naive CD8+ T cell repertoire. Using mice that are hemizygous or homozygous for a single MHCI allele, we showed that despite a reduction in peripheral CD8+ T cell numbers of ∼50% in MHCI hemizygous mice, MHCI levels had no notable impact on the rate of thymic generation or emigration of CD8 single-positive T cells. Moreover, the peripheral T cell repertoire in hemizygous mice showed selective retention of T cell clonotypes with a greater competitive advantage as evidenced by increased expression of CD5 and IL-7Rα. The qualitative superiority of CD8+ T cells retained in hemizygous mice was also seen during influenza A virus infection, in which epitope-specific CD8+ T cells from hemizygous mice had a higher avidity for pMHCI and increased cytokine polyfunctionality, despite a reduced response magnitude. Collectively, this study suggests that natural variation in MHCI expression levels has a notable and biologically relevant impact on the maintenance, but not generation, of the naive CD8+ T cell repertoire.

Cell death and thymic tolerance.

The differentiation of hematopoietic precursors into the many functionally distinct T-cell types produced by the thymus is a complex process. It proceeds through a series of stages orchestrated by a variety of thymic microenvironments that shape the T-cell developmental processes. Numerous cytokine and cell surface receptors direct thymocyte differentiation but the primary determinant of cell fate is the engagement of the T-cell antigen receptor (TCR). The strength of the TCR signal and the maturation stage of the thymocyte receiving it can direct the various differentiation programs or, alternatively, end the process by inducing cell death. The regulation of thymocyte death is critical for the efficiency of thymic T-cell differentiation and the preservation of immune tolerance. A detailed knowledge of mechanisms that eliminate thymocytes from the T-cell repertoire is essential to understand the "logic" of T-cell selection in the thymus. This review focuses on the central role of the BCL-2 family of proteins in the apoptotic checkpoints that punctuate thymocyte differentiation and the consequences of defects in these processes.

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3+ regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes.

αß T-cell receptors with a central CDR3 cysteine are enriched in CD8αα intraepithelial lymphocytes and their thymic precursors.

The thymus plays a crucial role in immune tolerance by exposing developing T cells (thymocytes) to a myriad of self-antigens. Strong T-cell receptor (TCR) engagement induces tolerance in self-reactive thymocytes by stimulating apoptosis or selection into specialized T-cell lineages, including intestinal TCRαß+ CD8αα+ intraepithelial lymphocytes (IEL). TCR-intrinsic amino acid motifs that can be used to predict whether a TCR will be strongly self-reactive remain elusive. Here, a novel TCR sequence alignment approach revealed that T-cell lineages in C57BL/6 mice had divergent usage of cysteine within two positions of the amino acid at the apex of the complementarity-determining region 3 (CDR3) of the TCRα or TCRß chain. Compared to pre-selection thymocytes, central CDR3 cysteine usage was increased in IEL and Type A IEL precursors (IELp) and markedly decreased in Foxp3+ regulatory T cells (T-reg) and naïve T cells. These findings reveal a TCR-intrinsic motif that distinguishes Type A IELp and IEL from T-reg and naïve T cells.

Quantitative reduction of the TCR adapter protein SLP-76 unbalances immunity and immune regulation.

Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies.

Ndfip1 mediates peripheral tolerance to self and exogenous antigen by inducing cell cycle exit in responding CD4+ T cells.

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains--an Ndfip1-YFP reporter and an Ndfip1-deficient strain--to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4(+) T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4(+) cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity.

A timeline demarcating two waves of clonal deletion and Foxp3 upregulation during thymocyte development.

Thymocytes that bind strongly to self-antigens are prevented from becoming naive T cells by several mechanisms. They undergo clonal deletion at two stages of development; wave 1 in immature thymocytes lacking the medulla-homing chemokine receptor, CCR7, or wave 2 in more mature CCR7(+) thymocytes. Alternatively, self-reactive thymocytes upregulate Foxp3 to become T-regulatory cells. Here, we describe the differential timing of the two waves of deletion and Foxp3 upregulation relative to the immature proliferating stage. Proliferating thymocytes were pulse-labeled in normal C57BL/6 mice with 5-ethynyl-2'-deoxyuridine (EdU). Thymocytes progressed into wave 1 (CCR7(-)) and wave 2 (CCR7(+)) of clonal deletion ~2 and 5 days after proliferation, respectively. Foxp3 upregulation occurred between 4 and 8 days after proliferation, predominantly in thymocytes with a Helios(+) CCR7(+) phenotype. These findings establish a timeline that suggests that wave 1 of clonal deletion occurs in the thymic cortex, whereas wave 2 and Foxp3 upregulation both occur in the thymic medulla.

A role for intrathymic B cells in the generation of natural regulatory T cells.

B cells inhabit the normal human thymus, suggesting a role in T cell selection. In this study, we report that B cells can modulate thymic production of CD4+ Foxp3+ T cells (regulatory T cells [Tregs]). Mice with transgenic expression of BAFF (BAFF-Tg) harbor increased numbers of Helios+ Foxp3+ thymic Tregs and, similar to some human autoimmune conditions, also exhibit increased numbers of B cells colonizing the thymus. Distinct intrathymic B cell subpopulations were identified, namely B220+, IgM+, CD23(hi), CD21(int) cells; B220+, IgM+, CD23(lo), CD21(lo) cells; and a population of B220+, IgM+, CD23(lo), CD21(hi) cells. Anatomically, CD19+ B cells accumulated in the thymic medulla region juxtaposed to Foxp3+ T cells. These intrathymic B cells engender Tregs. Indeed, thymic Treg development was diminished in both B cell-deficient BAFF-Tg chimeras, but also B cell-deficient wild-type chimeras. B cell Ag capture and presentation are critical in vivo events for Treg development. In the absence of B cell surface MHC class II expression, thymic expansion of BAFF-Tg Tregs was lost. Further to this, expansion of Tregs did not occur in BAFF-Tg/Ig hen egg lysozyme BCR chimeras, demonstrating a requirement for Ag specificity. Thus, we present a mechanism whereby intrathymic B cells, through the provision of cognate help, contribute to the shaping of the Treg repertoire.

Program Evaluation of the radKIDS® Youth Personal Empowerment Safety Education Program.

Developing sound evidence of program effectiveness can be difficult for many programs initiated by schools and communities, and impedes many beneficial programs from broader dissemination. This paper shares results of an evaluation approach used with a bullying and victimization prevention program with elementary school children called the radKIDS® Personal Empowerment and Safety Education Program. The purpose of this study was to examine indicators of initial effectiveness of the radKIDS® program for elementary school child safety skill development and instructor training to reduce child victimization and associated trauma and empower healthy psychosocial child development. The study involved 330 active radKIDS® instructors surveyed during two separate two-week periods, resulting in 148 completed questionnaires (45%). Instructors rated their perceptions of what children effectively learned in radKIDS®, the effectiveness of instructor training, and on Social Emotional Learning (SEL) competencies addressed in the program. Evaluation findings confirmed the theoretical model of the program, and that the developmental safety domains impacting children in radKIDS® differs from those in other bullying prevention interventions focused on SEL and other competencies. Recommended areas of improvement for the program included making training less time consuming and more flexible in delivery, provide more practice opportunities and time on skill acquisition during training, and increase supervision and guidance during program implementation. Supplementary Information: The online version contains supplementary material available at 10.1007/s40653-024-00618-5.

The NF-κB transcription factor c-Rel is required for Th17 effector cell development in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease involving effector Th subsets such as Th1 and Th17. In this study, we demonstrate that mice lacking the NF-κB transcription factor family member c-Rel (rel(-/-)), which are known to be resistant to EAE, show impaired Th17 development. Mixed bone marrow chimeras and EAE adoptive transfer experiments show that the deficiency of effector Th17 cells in rel(-/-) mice is T cell intrinsic. Consistent with this finding, c-Rel was activated in response to TCR signaling in the early stages of Th17 development and controlled the expression of Rorc, which encodes the Th17 transcription factor retinoic acid-related orphan receptor γt. CD28, but not IL-2, repression of Th17 development was dependent on c-Rel, implicating a dual role for c-Rel in modulating Th17 development. Adoptive transfer experiments also suggested that c-Rel control of regulatory T cell differentiation and homeostasis influences EAE development and severity by influencing the balance between Th17 and regulatory T cells. Collectively, our findings indicate that in addition to promoting Th1 differentiation, c-Rel regulates the development and severity of EAE via multiple mechanisms that impact on the generation of Th17 cells.

T-cell regulation by casitas B-lineage lymphoma (Cblb) is a critical failsafe against autoimmune disease due to autoimmune regulator (Aire) deficiency.

Autoimmune polyendocrinopathy syndrome type 1 (APS1) results from homozygous Aire mutations that cripple thymic deletion of organ-specific T cells. The clinical course in man and mouse is characterized by high variability both in the latent period before onset of autoimmune disease and in the specific organs affected, but the reasons for this are unknown. Here we test the hypothesis that the latent period reflects the failsafe action of discrete postthymic mechanisms for imposing self-tolerance in peripheral T cells. Aire-deficient mice were crossed with mice of a uniform major histocompatibility complex (MHC) haplotype and genetic background carrying specific genetic defects in one of four distinct peripheral tolerance mechanisms: activation-induced cell death (Fasl(gld/gld)), anergy and requirement for CD28 costimulation (Cblb(-/-)), inhibition of ICOS and T(FH) cells (Rc3h1(san/san)), or decreased numbers of Foxp3(+) T regulatory cells (Card11(unm/unm)). Cblb-deficiency was unique among these four in precipitating rapid clinical autoimmune disease when combined with Aire-deficiency, resulting in autoimmune exocrine pancreatitis with median age of survival of only 25 d. Massive lymphocytic infiltration selectively destroyed most of the exocrine acinar cells of the pancreas and submandibular salivary gland, and CD4(+) and CD8(+) subsets were necessary and sufficient to transfer the disease. Intrinsic regulation of peripheral T cells by CBL-B thus serves a uniquely critical role as a failsafe against clinical onset of autoimmune disease in AIRE deficiency, and multiple peripheral tolerance mechanisms may need to fail before onset of clinical autoimmunity to many organs.

Regulation of the IL-21 gene by the NF-κB transcription factor c-Rel.

IL-21 is a member of the common gamma-chain-dependent cytokine family and is a key modulator of lymphocyte development, proliferation, and differentiation. IL-21 is highly expressed in activated CD4(+) T cells and plays a critical role in the expansion and differentiation of the Th cell subsets, Th17 and follicular helper T (T(FH)) cells. Because of its potent activity in both myeloid and lymphoid cell immune responses, it has been implicated in a number of autoimmune diseases and has also been used as a therapeutic agent in the treatment of some cancers. In this study, we demonstrate that c-Rel, a member of the NF-kappaB family of transcription factors, is required for IL-21 gene expression in T lymphocytes. IL-21 mRNA and protein levels are reduced in the CD4(+) cells of rel(-/-) mice when compared with rel(+/+) mice in both in vitro and in vivo models. A c-Rel binding site identified in the proximal promoter of il21 is confirmed to bind c-Rel in vitro and in vivo and to regulate expression from the il21 promoter in T cells. Downstream of IL-21 expression, Th17, T(FH), and germinal center B cell development are also impaired in rel(-/-) mice. The administration of IL-21 protein rescued the development of T(FH) cells but not germinal center B cells. Taken together, c-Rel plays an important role in the expression of IL-21 in T cells and subsequently in IL-21-dependent T(FH) cell development.

How Thymocyte Deletion in the Cortex May Curtail Antigen-Specific T-Regulatory Cell Development in the Medulla.

CD4+ T cell responses to self-antigens are pivotal for immunological self-tolerance. Activation of Foxp3- T-conventional (T-conv) cells can precipitate autoimmune disease, whereas activation of Foxp3+ T-regulatory (T-reg) cells is essential to prevent autoimmune disease. This distinction indicates the importance of the thymus in controlling the differentiation of self-reactive CD4+ T cells. Thymocytes and thymic antigen-presenting cells (APC) depend on each other for normal maturation and differentiation. In this Hypothesis and Theory article, we propose this mutual dependence dictates which self-antigens induce T-reg cell development in the thymic medulla. We postulate self-reactive CD4+ CD8- thymocytes deliver signals that stabilize and amplify the presentation of their cognate self-antigen by APC in the thymic medulla, thereby seeding a niche for the development of T-reg cells specific for the same self-antigen. By limiting the number of antigen-specific CD4+ thymocytes in the medulla, thymocyte deletion in the cortex may impede the formation of medullary T-reg niches containing certain self-antigens. Susceptibility to autoimmune disease may arise from cortical deletion creating a "hole" in the self-antigen repertoire recognized by T-reg cells.