Increased sensitivity to antigen in high avidity CD8(+) T cells results from augmented membrane proximal T-cell receptor signal transduction

The functional avidity of a cytotoxic T lymphocyte (CTL) is known to be a critical determinant of the efficacy with which it clears pathogens. High avidity cells, which are by definition highly sensitive to peptide antigen, are superior for elimination of viruses and tumours. Our studies have established the ability of T cells to undergo avidity modulation as a result of antigen encounter. High and low avidity cells established in this manner exhibit significant differences in the amount of peptide required to elicit effector function. However, how signalling is regulated in these cells as it relates to the control of peptide sensitivity remains to be defined. To address this question, we compared T-cell receptor (TCR) signal transduction events in high and low avidity CTL generated from OT-I(rag2-) TCR transgenic mice. Our data suggest that divergent signalling is initiated at the TCR-associated CD3ζ, with low avidity CTL requiring higher amounts of pMHC to achieve threshold levels of phosphorylated CD3ζ compared with high avidity CTL. Further, this difference is transduced further downstream to mitogen-activated protein kinase and Ca(2+) signalling pathways. These results suggest that regulated control of the initiation of TCR signalling in high versus low avidity cells determines the amount of peptide required for T-cell activation.     

Low-dose antigen-experienced CD4+ T cells display reduced clonal expansion but facilitate an effective memory pool in response to secondary exposure

The strength and duration of an antigenic signal at the time of initial stimulation were assumed to affect the development and response of effectors and memory cells to secondary stimulation with the same antigen. To test this assumption, we used T-cell receptor (TCR)-transgenic CD4⁺ T cells that were stimulated in vitro with various antigen doses. The primary effector CD4⁺ T cells generated in response to low-dose antigen in vitro exhibited reduced clonal expansion upon secondary antigenic exposure after adoptive transfer to hosts. However, the magnitude of their contraction was much smaller than both those generated by high-dose antigen stimulation and by naïve CD4⁺ T cells, resulting in higher numbers of antigen-specific CD4⁺ T cells remaining until the memory stage. Moreover, secondary effectors and memory cells developed by secondary antigen exposure were not functionally impaired. In hosts given the low-dose antigen-experienced CD4⁺ T cells, we also observed accelerated recall responses upon injection of antigen-bearing antigen-presenting cells. These results suggest that primary TCR stimulation is important for developing optimal effectors during initial antigen exposure to confer long-lasting memory CD4⁺ T cells in response to secondary exposure.     

The avidity of cross-reactive virus-specific T cells for their viral and allogeneic epitopes is variable and depends on epitope expression

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through cross-reactivity of their T-cell receptor (TCR). In a transplantation setting, such allo-HLA cross-reactivity may contribute to harmful immune responses towards the allograft, provided that the cross-reactive T cells get sufficiently activated upon recognition of the allo-HLA. An important determinant of T-cell activation is TCR avidity, which to date, has remained largely unexplored for allo-HLA-cross-reactive virus-specific T cells.For this purpose, cold target inhibition assays were performed using allo-HLA-cross-reactive virus-specific memory CD8⁺ T-cell clones as responders, and syngeneic cells loaded with viral peptide and allogeneic cells as hot (radioactively-labeled) and cold (non-radioactively-labeled) targets. CD8 dependency of the T-cell responses was assessed using interferon γ (IFNγ) enzyme-linked immunosorbent assay (ELISA) in the presence and absence of CD8-blocking antibodies.At high viral-peptide loading concentrations, T-cell clones consistently demonstrated lower avidity for allogeneic versus viral epitopes, but at suboptimal concentrations the opposite was observed. In line, anti-viral reactivity was CD8 independent at high, but not at suboptimal viral-peptide-loading concentrations.The avidity of allo-HLA-cross-reactive virus-specific memory CD8⁺ T cells is therefore highly dependent on epitope expression, and as a consequence, can be both higher and lower for allogeneic versus viral targets under different (patho)physiological conditions.     

A low antigen dose selectively promotes expansion of high-avidity autoreactive T cells with distinct phenotypic characteristics: A study of human autoreactive CD4+T cells specific for GAD65

T cells specific for pancreatic islet proteins can be detected in type 1 diabetes patients and at-risk individuals, suggesting a failure of the central tolerance and negative selection. We addressed the question, how antigen dose shapes the diversity of CD4+ autoreactive T cells specific for glutamate decarboxylase 65 (GAD65) in a healthy HLA-DR*0404+ individual, with a persistent GAD65-specific T-cell response. CD4+T cells from this subject were stimulated with decreasing concentrations of the GAD65 555-567 (557I) peptide, and T-cell clones were derived from the tetramer-binding cell population. Functional and structural avidity, TcR-Vβ usage, and cytokine profiles were investigated at a clonal level. T-cell clones established with a low antigen dose (0.1 and 1 μg/ml) displayed higher avidity in contrast to the clones established with the highest antigen dose (10 μg/ml; Mann–Whitney U test, p = 0.003 and 0.006, respectively). The T-cell clones stimulated with the lowest peptide dose also had a higher tetramer-binding affinity than clones stimulated with the highest dose (p = 0.026). The majority (60.0%) of the high-avidity clones expressed TcR-Vβ5.1 chain whereas only one (12.5%) low-avidity clone did. All clones displayed Th0/Th2 cytokine profiles, but intermediate and high-avidity clones produced more IL-10 than low-avidity clones (p = 0.032). The results demonstrate an important role of the antigen dose in the determination of characteristics of the responding T-cell repertoire. High IL-13 and IL-10 production by GAD65-reactive T cells suggests a more anti-inflammatory profile of this healthy individual underlying protection from T1D.     

Avidity optimization of a MAGE-A1-specific TCR with somatic hypermutation

A T-cell receptor (TCR) with optimal avidity to a tumor antigen can be used to redirect T cells to eradicate cancer cells via adoptive cell transfer. Cancer testis antigens (CTAs) are attractive targets because they are expressed in the testis, which is immune-privileged, and in the tumor. However, CTAs are self-antigens and natural TCRs to CTAs have low affinity/avidity due to central tolerance. We previously described a method of directed evolution of TCR avidity using somatic hypermutation. In this study, we made several improvements to this method and enhanced the avidity of the hT27 TCR, which is specific for the cancer testis antigen HLA-A2-MAGE-A1_278-286. We identified eight point mutations with varying degrees of improved avidity. Human T cells transduced with TCRs containing these mutations displayed enhanced tetramer binding, IFN-γ and IL2 production, and cytotoxicity. Most of the mutations have retained specificity, except for one mutant with extremely high avidity. We demonstrate that somatic hypermutation is capable of optimizing avidity of clinically relevant TCRs for immunotherapy.     

Dose-dependent modulation of CD8 and functional avidity as a result of peptide encounter

The generation of an optimal CD8(+) cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8(+) cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8alpha and CD8beta. Importantly the ensuing CD8(low) and CD8(high) CTL populations were not the result of the selective outgrowth of naive CD8(+) T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to 'fine tune' the sensitivity of responding CTL to a defined concentration of antigen.     

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification

We set out to clone Bax-specific CD8⁺ T cells from peripheral blood samples of patients with primary chronic lymphocytic leukaemia. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-amino-acid sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (> 95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild-type sequence to generate functionally active peptides. Computer modelling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However, these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the T-cell receptor. This modified product formed < 1% of the original P603 crude peptide preparation and < 0·05% of the original 23-peptide mixture used for T-cell stimulation. The data presented here, illustrate the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlight the exquisite capacity of T-cell receptors to discriminate between structurally similar peptide sequences. Furthermore, this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses.     

DAPK1 (death associated protein kinase 1) mediates mTORC1 activation and antiviral activities in CD8+ T cells

Mechanistic target of rapamycin complex 1 (mTORC1) regulates CD8⁺ T-cell differentiation and function. Despite the links between PI3K-AKT and mTORC1 activation in CD8⁺ T cells, the molecular mechanism underlying mTORC1 activation remains unclear. Here, we show that both the kinase activity and the death domain of DAPK1 are required for maximal mTOR activation and CD8⁺ T-cell function. We found that TCR-induced activation of calcineurin activates DAPK1, which subsequently interacts with TSC2 via its death domain and phosphorylates TSC2 to mediate mTORC1 activation. Furthermore, both the kinase domain and death domain of DAPK1 are required for CD8⁺ T-cell antiviral responses in an LCMV infection model. Together, our data reveal a novel mechanism of mTORC1 activation that mediates optimal CD8⁺ T-cell function and antiviral activity.     

Generation of functionally active HIV-1 specific CD8+ CTL in intestinal mucosa following mucosal, systemic or mixed prime-boost immunization

Gastrointestinal and vaginal mucosa are major sites of entry in natural HIV infection and therefore the preferred sites to elicit high-avidity CD8⁺ CTL by vaccination. We directly compare systemic and mucosal immunization in mice after DNA priming and boosting with rgp160 env expressed either in MVA or Ad for their ability to induce mucosal as well as systemic HIV-specific CTL. The optimal CTL response in the gut mucosa was observed after priming with the HIV-1 gp160 env DNA vaccine and boosting with rMVA or rAd encoding the same envelope gene all administered intrarectally (IR). Maximum levels of high-avidity CD8⁺ T cells were seen in intestinal lamina propria following this regimen. When the prime and boost routes were distinct, the delivery site of the boost had a greater impact than the DNA priming. IM DNA prime and IR rMVA boost were more effective than IR DNA prime and IM rMVA boost for eliciting mucosal CD8⁺ T-cell avidity. A systemic DNA-prime-followed by systemic rMVA boost induced high levels of high-avidity CD8⁺ T cells systemically, but responses were undetectable in mucosal sites. A single systemic immunization with rMVA was sufficient to induce high-avidity IFN-γ secreting CD8⁺ T cells in systemic organs, whereas a single mucosal immunization with rMVA was not sufficient to elicit high-avidity CD8⁺ T cells in mucosa. Thus, a heterologous mucosal DNA prime-viral vectored boost strategy was needed. The requirement for a heterologous DNA prime-recombinant viral boost strategy for generation of high-avidity CD8⁺ T cells in mucosal sites in mice may be more stringent than for the induction of high-avidity CD8⁺ T cells in systemic compartments.     

Murine CD8 T-cell functional avidity is stable in vivo but not in vitro: Independence from homologous prime/boost time interval and antigen density

It is known that for achieving high affinity antibody responses, vaccines must be optimized for antigen dose/density, and the prime/boost interval should be at least 4 weeks. Similar knowledge is lacking for generating high avidity T-cell responses. The functional avidity (FA) of T cells, describing responsiveness to peptide, is associated with the quality of effector function and the protective capacity in vivo. Despite its importance, the FA is rarely determined in T-cell vaccination studies. We addressed the question whether different time intervals for short-term homologous vaccinations impact the FA of CD8 T-cell responses. Four-week instead of 2-week intervals between priming and boosting with potent subunit vaccines in C57BL/6 mice did not improve FA. Equally, similar FA was observed after vaccination with virus-like particles displaying low versus high antigen densities. Interestingly, FA was stable in vivo but not in vitro, depending on the antigen dose and the time interval since T-cell activation, as observed in murine monoclonal T cells. Our findings suggest dynamic in vivo modulation for equal FA. We conclude that low antigen density vaccines or a minimal 4-week prime/boost interval are not crucial for the T-cell's FA, in contrast to antibody responses.     

Dual immunoregulatory pathways of 4-1BB signaling

It is perhaps rare to encounter among the various immunologically competent receptor–ligand pairs that a single cell surface determinant unleashes both a hidden suppressive function and costimulation. 4-1BB, an activation-induced tumor necrosis factor receptor family member chiefly viewed as a powerful T-cell costimulatory molecule, is one such example. Accumulated evidence in recent years uncovered an unknown facet of in vivo 4-1BB signaling (i.e., “active suppression”). Although in vitro signaling via 4-1BB is shown to support both CD4⁺ and CD8⁺ T-cell responses, the same induces a predominant CD8⁺ T-cell response suppressing CD4⁺ T-cell function when applied in vivo. How, when, and why such dual immunoregulatory effect of anti-4-1BB monoclonal antibody (MAB) comes into play is currently the focus of intense research. Existing data, although not complete, uncover several important aspects of in vivo 4-1BB signaling in the amelioration or exacerbation of various immune disorders. Despite minor disagreements, a majority agree that upregulation of interferon (IFN)-γ is critical to anti-4-1BB MAB therapy in addition to immune modulators such as interleukin 2, transforming growth factor β, and indolamine 2,3-dioxygenase⁵, all of which contribute greatly to the success of anti-4-1BB MAB-based immunotherapy. Anti-4-1BB MAB-mediated expansion of novel CD11c⁺CD8⁺ T cells is additional weaponry that appears critical for its in vivo suppressive function. These CD11c⁺CD8⁺ T cells express high levels of IFN-γ, become effective killers, and mediate selective suppression of CD4⁺ T cells. In this review, we discuss the dual nature (costimulatory and suppressive) of 4-1BB-mediated immune regulation, its current status, future direction, and its impact on the immune system, with special reference to its immunotherapy.     

TCR repertoire evolution during maintenance of CMV-specific T-cell populations

During infections and cancer, the composition of the T-cell receptor (TCR) repertoire of antigen-specific CD8⁺ T cells changes over time. TCR avidity is thought to be a major driver of this process, thereby interacting with several additional regulators of T-cell responses to form a composite immune response architecture. Infections with latent viruses, such as cytomegalovirus (CMV), can lead to large T-cell responses characterized by an oligoclonal TCR repertoire. Here, we review the current status of experimental studies and theoretical models of TCR repertoire evolution during CMV infection. We will particularly discuss the degree to which this process may be determined through structural TCR avidity. As engineered TCR-redirected T cells have moved into the spotlight for providing more effective immunotherapies, it is essential to understand how the key features of a given TCR influence T-cell expansion and maintenance in settings of infection or malignancy. Deeper insights into these mechanisms will improve our basic understanding of T-cell immunology and help to identify optimal TCRs for immunotherapy.     

Hepatic effector CD8+ T-cell dynamics

CD8⁺ T cells play a critical role in hepatitis B virus (HBV) pathogenesis. During acute, self-limited infections, these cells are instrumental to viral clearance; in chronic settings, they sustain repetitive cycles of hepatocellular necrosis that promote hepatocellular carcinoma development. Both CD8⁺ T-cell defensive and destructive functions are mediated by antigen-experienced effector cells and depend on the ability of these cells to migrate to the liver, recognize hepatocellular antigens and perform effector functions. Understanding the signals that modulate the spatiotemporal dynamics of CD8⁺ T cells in the liver, particularly in the context of antigen recognition, is therefore critical to gaining insight into the pathogenesis of acute and chronic HBV infection. Here, we highlight recent data on how effector CD8⁺ T cells traffic within the liver, and we discuss the potential for novel imaging techniques to shed light on this important aspect of HBV pathogenesis.     

HLA-E-restricted recognition of human cytomegalovirus by a subset of cytolytic T lymphocytes

Natural killer (NK)-cytotoxic T lymphocytes (CTL) are a subset of CD8(+) cytolytic T lymphocytes that express human leukocyte antigen (HLA) class I-specific inhibitory receptors. They are detectable as monoclonal expansions in the blood of cytomegalovirus (CMV)-seropositive individuals displaying particular HLA-Cw allotypes. Similar to NK cells, they are capable of killing various allogeneic tumor cell lines, a function referred to as "NK-like activity." The mechanism underlying this unusual functional property has recently been clarified. Via their T-cell receptor, NK-CTL recognize the nonclassical HLA class I molecule HLA-E, which is characterized by a limited polymorphism and by the ability to bind peptides derived from the leader sequence of various HLA class I alleles as well as from few viral proteins. The analysis of the T-cell receptor avidity revealed that NK-CTL recognize with high avidity a CMV UL40-derived peptide. The HLA-E-restricted recognition of CMV by NK-CTL may represent an important immunologic strategy in defenses against this virus. Indeed, unlike conventional CTL, NK-CTL mediated lysis is apparently not affected by the downregulation of major histocompatibility complex class I that occurs during CMV infection. Because the CMV UL40-derived peptide is identical to the one generated from the leader sequence of various HLA-Cw alleles, NK-CTL are also able to display an "HLA-E-dependent alloreactivity" against allogeneic target cells expressing appropriate HLA-Cw alleles. This broad ability to recognize and kill allogeneic cells may pose serious problems in transplantation.     

Regulatory role of leukocyte common antigen‐related molecule (LAR) in thymocyte differentiation

The strength of interaction between the antigenic peptide‐loaded MHC (MHC/p) and the TCR determines T‐cell fate in the thymus. A high avidity interaction between the TCR and the MHC/p induces apoptosis of self‐reactive T cells (negative selection), whereas a moderate avidity interaction rescues thymocytes from apoptosis and permits further differentiation to mature T cells (positive selection). Leukocyte common antigen‐related molecule (LAR), a receptor‐like protein tyrosine phosphatase, is expressed on immature thymocytes, but its role in thymocyte differentiation has not yet been fully elucidated. We analyzed LAR‐deficient mice and demonstrated that LAR deficiency affected the differentiation and expansion of immature thymocytes as well as positive and negative selection. Furthermore, LAR deficiency resulted in a lower Ca²⁺ response. The results indicate that LAR is an important modulator of TCR signaling that controls thymocyte differentiation.     

Antibody-dependent cellular cytotoxicity (ADCC)-mediated destruction of human immunodeficiency virus (HIV)-coated CD4+ T lymphocytes by acquired immunodeficiency syndrome (AIDS) effector cells

The acquired immunodeficiency syndrome (AIDS) is defined in clinical terms by the development of Kaposi's sarcoma and/or severe opportunistic infections in persons without predisposing conditions. A hallmark of the syndrome has been a decrease in the number of CD4⁺ T helper cells. The reduction in the frequency of the CD4⁺ lymphocytes has been postulated to be primarily the result of human immunodeficiency virus (HIV) tropism and cytophathogenicity for the T-cell subset. Yet only a small percentage of cells is actually infected with HIV. Recently, we provided evidence indicating that AIDS patients' natural killer cells can mediate normal levels of antibody-dependent cellular cytotoxicity (ADCC) despite exhibiting a defect in natural killer (NK) effector function (J Immunol 139:55, 1987). This finding prompted us to investigate whether AIDS patients' effector cells could mediate ADCC against circulating CD4⁺ T cells infected with or expressing HIV antigen. The findings reported herein demonstrate that AIDS effector cells can mediate lysis of CEM (CD4⁺ T-cell line) coated with HIV protein in the presence of HIV-specific antibody. Lysis was specific, as non-HIV-coated CEM or the addition of HIV-negative serum resulted in no lysis. We then examined HIV-coated peripheral blood-derived CD4⁺ T lymphocytes as targets in ADCC. We demonstrate that in the presence of HIV-specific antibody, HIV-coated CD4⁺ T lymphocytes serve as targets for ADCC by AIDS effector cells. The lytic activity obtained with AIDS effector cells was comparable to that obtained with normal effector cells. These results demonstrate that AIDS effector cells can mediate ADCC against HIV-coated CD4⁺ T lymphocytes and suggest that ADCC may play a rolein vivo in the pathogenesis of AIDS.     

The P9 peptide sidechain specificity of I-A

The murine MHC class II variant I-A^d confers susceptibility to herpes simplex virus (HSV)-induced keratitis and relative protection against type 1 diabetes mellitus. The association to these autoimmune diseases appears to be largely determined by the peptide sidechain specificity of the P9 pocket, which we therefore have analyzed in detail. Assessment of T-cell responses and I-A^d binding capacity of position 446-substituted analogs of an IgG2a allotype b (IgG2a^b) heavy chain peptide demonstrates that engagement of the P9 pocket is crucial for effective peptide presentation. Sidechain size rather than charge decides the capacity to engage the P9 pocket. Thus, small, uncharged sidechains are accepted, whereas acidic and aromatic amino acids as well as lysine and arginine are disfavored. The specificity of the P9 pocket of I-A^d (serine^β57) is distinct from that of the diabetes-associated I-A^g7 (aspartic acid^β57), supporting the contention that the polymorphism at residue β57 influences diabetes susceptibility via P9-specific effects on the repertoires of self peptides presented to T cells. Furthermore, the data rationalize the susceptibility to HSV-induced keratitis conferred by the a and the protection conferred by the b allotypes of the IgG2a heavy chain. Keratitogenic T cells, which cross-react with the viral UL6 protein and a corneal antigen, are silenced in IgG2a^b mice because of antigenic mimicry with γ2a^b 435–451. Our finding that the lysine P9 residue of the corresponding γ2a^a allopeptide precludes high-affinity binding to I-A^d indicates that the susceptibility of IgG2a^a mice reflects inefficient thymic presentation of autologous IgG2a and thus failure to purge the T-cell repertoire of the pathogenic clones.     

Multifunctional cytomegalovirus (CMV)-specific CD8+ T cells are not restricted by telomere-related senescence in young or old adults

Antigen-specific multifunctional T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α simultaneously after activation are important for the control of many infections. It is unclear if these CD8⁺ T cells are at an early or late stage of differentiation and whether telomere erosion restricts their replicative capacity. We developed a multi-parameter flow cytometric method for investigating the relationship between differentiation (CD45RA and CD27 surface phenotype), function (cytokine production) and replicative capacity (telomere length) in individual cytomegalovirus (CMV) antigen-specific CD8⁺ T cells. This involves surface and intracellular cell staining coupled to fluorescence in situ hybridization to detect telomeres (flow-FISH). The end-stage/senescent CD8⁺ CD45RA⁺ CD27⁻ T-cell subset increases significantly during ageing and this is exaggerated in CMV immune-responsive subjects. However, these end-stage cells do not have the shortest telomeres, implicating additional non-telomere-related mechanisms in inducing their senescence. The telomere lengths in total and CMV (NLV)-specific CD8⁺ T cells in all four subsets defined by CD45RA and CD27 expression were significantly shorter in old compared with young individuals in both a Caucasian and an Asian cohort. Following stimulation by anti-CD3 or NLV peptide, similar proportions of triple-cytokine-producing cells are found in CD8⁺ T cells at all stages of differentiation in both age groups. Furthermore, these multi-functional cells had intermediate telomere lengths compared with cells producing only one or two cytokines after activation. Therefore, global and CMV (NLV)-specific CD8⁺ T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α are at an intermediate stage of differentiation and are not restricted by excessive telomere erosion.     

Opposing peripheral fates of tissue-restricted self antigen-specific conventional and regulatory CD4+ T cells

The development of self antigen-specific T cells is influenced by how the self antigen is expressed. Here, we created a mouse in which a model self antigen is conditionally expressed in different tissue environments. Using peptide:MHCII tetramer-based cell enrichment methods, we examined the development of corresponding endogenous self antigen-specific CD4⁺ T cell populations. While ubiquitous self antigen expression resulted in efficient deletion of self antigen-specific T cells in the thymus, some tissue-restricted expression patterns resulted in partial deletion of the population in peripheral lymphoid organs. Deletion specifically affected Foxp3⁻ conventional T cells (Tconv) with a bias towards high avidity TCR expressing cells in the case of thymic, but not peripheral deletion. In contrast, Foxp3⁺ Treg exhibited elevated frequencies with increased TCR avidity. T cells surviving deletion were functionally impaired, with Tconv cells exhibiting more impairment than Tregs. Collectively, our results illustrate how postthymic recognition of tissue-restricted self antigens results in opposing developmental fates for Tconv and Treg cell subsets.