T-cell Receptor Engagement Research Articles

Mapping the SLP76 interactome in T cells lacking each of the GRB2-family adaptors reveals molecular plasticity of the TCR signaling pathway.

The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations.

Expression of Non-T Cell Activation Linker (NTAL) in Jurkat Cells Negatively Regulates TCR Signaling: Potential Role in Rheumatoid Arthritis.

T lymphocytes are key players in adaptive immune responses through the recognition of peptide antigens through the T Cell Receptor (TCR). After TCR engagement, a signaling cascade is activated, leading to T cell activation, proliferation, and differentiation into effector cells. Delicate control of activation signals coupled to the TCR is needed to avoid uncontrolled immune responses involving T cells. It has been previously shown that mice deficient in the expression of the adaptor NTAL (Non-T cell activation linker), a molecule structurally and evolutionarily related to the transmembrane adaptor LAT (Linker for the Activation of T cells), develop an autoimmune syndrome characterized by the presence of autoantibodies and enlarged spleens. In the present work we intended to deepen investigation into the negative regulatory functions of the NTAL adaptor in T cells and its potential relationship with autoimmune disorders. For this purpose, in this work we used Jurkat cells as a T cell model, and we lentivirally transfected them to express the NTAL adaptor in order to analyze the effect on intracellular signals associated with the TCR. In addition, we analyzed the expression of NTAL in primary CD4+ T cells from healthy donors and Rheumatoid Arthritis (RA) patients. Our results showed that NTAL expression in Jurkat cells decreased calcium fluxes and PLC-γ1 activation upon stimulation through the TCR complex. Moreover, we showed that NTAL was also expressed in activated human CD4+ T cells, and that the increase of its expression was reduced in CD4+ T cells from RA patients. Our results, together with previous reports, suggest a relevant role for the NTAL adaptor as a negative regulator of early intracellular TCR signaling, with a potential implication in RA.

JAML immunotherapy targets recently activated tumor-infiltrating CD8+ Tcells.

Junctional adhesion molecule-like protein (JAML) serves as a co-stimulatory molecule in γδ Tcells. While it has recently been described as a cancer immunotherapy target in mice, its potential to cause toxicity, specific mode of action with regard to its cellular targets, and whether it can be targeted in humans remain unknown. Here, we show that JAML is induced by Tcell receptor engagement, reveal that this induction is linked to cis-regulatory interactions between the CD3D and JAML gene loci. When compared with other immunotherapy targets plagued by low target specificity and end-organ toxicity, we find JAML to be mostly restricted to and highly expressed by tissue-resident memory CD8+ Tcells in multiple cancer types. By delineating the key cellular targets and functional consequences of agonistic anti-JAML therapy in a murine melanoma model, we show its specific mode of action and the reason for its synergistic effects with anti-PD-1.

Recent insights into the role of Akt in CD4 T-cell activation and differentiation: alternative splicing and beyond.

The activation and differentiation of CD4+ T cells is a complex process that is controlled by many factors. A critical component of the signaling pathway triggered following T-cell receptor (TCR) engagement is the serine threonine kinase Akt. Akt is involved in the control of many cellular processes including proliferation, metabolism, and differentiation of specific TH-cell subsets. Recent work has shown that, depending on the nature or strength of the TCR activation, Akt may activate different sets of substrates which then lead to differential cellular outcomes. Akt plays an important role in controlling the strength of the TCR signal and several recent studies have identified novel mechanisms including control of the expression of negative regulators of TCR signaling, and the influence on regulatory T cells (Treg) and TH17 differentiation. Many of these functions are mediated via control of the FoxO family of transcription factors, that play an important role in metabolism and Th cell differentiation. A theme that is emerging is that Akt does not function in the same way in all T-cell types. We highlight differences between CD4 and CD8 T cells as well as between Treg, TH17, and TFH cells. While Akt activity has been implicated in the control of alternative splicing in tumor cells, recent studies are emerging that indicate that similar functions may exist in CD4 T cells. In this mini review, we highlight some of the recent advances in these areas of Akt function that demonstrate the varied role that Akt plays in the function of CD4 T cells.

CD8+ T Cells from Patients with CLL Show Evidence of Granzyme B and Perforin Cytotoxic Capacity, Which Is Enhanced upon Exposure to Exogenous IL2 and/or IL21

CD8+ T Cells from Patients with CLL Show Evidence of Granzyme B and Perforin Cytotoxic Capacity, Which Is Enhanced upon Exposure to Exogenous IL2 and/or IL21

Autologous blood extracellular vesicles and specific CD4+ T-cell co-activation.

Extracellular vesicles (EVs), which are generated by cell membrane budding in diverse cells, are present in variable numbers in the blood. An immunoregulatory role has been demonstrated principally for heterologous EVs, but the function of the EVs present naturally in blood remains unknown. We hypothesize that these autologous EVs might also modulate the phenotype and function of immune system cells, especially CD4+ T lymphocytes (TLs), as previously described for heterologous EVs. Several membranes and soluble immunoregulatory molecules were studied after the treatment of CD4+ TLs with autologous EVs. No direct activation was detected with autologous EVs, contrasting with the findings for heterologous EVs. However, following treatment with autologous EVs, a soluble form of CD27 (sCD27) was detected. sCD27 is strongly associated with lymphoproliferation. Autologous EVs have been shown to increase TL proliferation only after T-cell receptor (TcR) engagement due to polyclonal or specific-antigen stimulation. Our results therefore suggest that the EVs present in the blood have an immunomodulatory role different from that of heterologous EVs. These findings should be taken into account in future studies, particularly those focusing on infectious diseases, autotransfusion or doping practices.

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

Transcriptional profiling of human Vδ1 Tcells reveals a pathogen-driven adaptive differentiation program.

SUMMARYγδ T cells are generally considered innate-like lymphocytes, however, an ‘‘adaptive-like’’ γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+ BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αβ T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct ‘‘innate-effector’’ transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.

Chemical augmentation of mitochondrial electron transport chains tunes T cell activation threshold in tumors

BackgroundCancer immunotherapy shows insufficient efficacy for low immunogenic tumors. Furthermore, tumors often downregulate antigen and major histocompatibility complex expression to escape recognition by T cells, resulting in insufficient T cell...

Structural Basis for Unusual TCR CDR3β Usage Against an Immunodominant HIV-1 Gag Protein Peptide Restricted to an HLA-B*81:01 Molecule.

In HIV infection, some closely associated human leukocyte antigen (HLA) alleles are correlated with distinct clinical outcomes although presenting the same HIV epitopes. The mechanism that underpins this observation is still unknown, but may be due to the essential features of HLA alleles or T cell receptors (TCR). In this study, we investigate how T18A TCR, which is beneficial for a long-term control of HIV in clinic, recognizes immunodominant Gag epitope TL9 (TPQDLTML180-188) from HIV in the context of the antigen presenting molecule HLA-B*81:01. We found that T18A TCR exhibits differential recognition for TL9 restricted by HLA-B*81:01. Furthermore, via structural and biophysical approaches, we observed that TL9 complexes with HLA-B*81:01 undergoes no conformational change after TCR engagement. Remarkably, the CDR3β in T18A complexes does not contact with TL9 at all but with intensive contacts to HLA-B*81:01. The binding kinetic data of T18A TCR revealed that this TCR can recognize TL9 epitope and several mutant versions, which might explain the correlation of T18A TCR with better clinic outcomes despite the relative high mutation rate of HIV. Collectively, we provided a portrait of how CD8+ T cells engage in HIV-mediated T cell response.

Abstract P035: A promising cancer immunotherapy target: Novel fully human agonist antibodies against the human T-cell costimulatory receptor CD27

Abstract CD27 is a member of the TNF receptor superfamily and plays a critical role in T-cell activation by providing a costimulatory signal. CD27 signaling enhances T-cell proliferation, activation and differentiation of effector and memory T cells and therefore promotes cytotoxic T cell (CTL)-based anti-tumor immunity. Agonistic stimulation of CD27 is a promising cancer immunotherapy approach to boost specific T cell driven anti-tumor responses. In this study, we generated a series of 147 fully human monoclonal anti-CD27 antibodies and tested their agonist properties to stimulate T cell activation. Using a NF-κB reporter Jurkat cell line, we evaluated in vitro the ability of anti-CD27 antibodies to induce CD27 receptor activation. With this assay, five antibodies have been selected for their agonist properties. When combined with suboptimal T cell receptor (TCR) stimulation, agonist antibodies induced CD27 receptor activation with an EC50 of 1-5 ug/mL. We also used human peripheral blood T cells to characterize the CD27-mediated costimulatory effects of agonist antibodies in combination with TCR stimulation. Our anti-CD27 monoclonal antibodies boosted T cell proliferation and induced IL-2 and TNFalpha secretion only in a presence of TCR engagement. Moreover, CD27 agonists induce strong T cell proliferation in a Mixed Lymphocyte Reaction. CD27 antibodies were shown to bind human and cynomolgus monkey CD27 with a KD value of 5-20 nM as determined by BioLayer Interferometry, but do not bind to mouse CD27. In vivo experiments are currently ongoing to demonstrate the efficient anti-tumor activity of the selected CD27 agonist antibodies in different mice tumor models. In conclusion, we have developed and successfully selected efficient fully human immunostimulatory agonist CD27 mAbs as a promising cancer immunotherapy. Citation Format: Yulia Ovechkina, Shaarwari Sridhar, David Jurchen, David Peckham, Eric Tarcha, Shawn Iadonato, Thierry Guillaudeux. A promising cancer immunotherapy target: Novel fully human agonist antibodies against the human T-cell costimulatory receptor CD27 [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P035.

Unconventional modes of peptide-HLA-I presentation change the rules of TCR engagement.

The intracellular proteome of virtually every nucleated cell in the body is continuously presented at the cell surface via the human leukocyte antigen class I (HLA-I) antigen processing pathway. This pathway classically involves proteasomal degradation of intracellular proteins into short peptides that can be presented by HLA-I molecules for interrogation by T-cell receptors (TCRs) expressed on the surface of CD8+ T cells. During the initiation of a T-cell immune response, the TCR acts as the T cell's primary sensor, using flexible loops to mould around the surface of the pHLA-I molecule to identify foreign or dysregulated antigens. Recent findings demonstrate that pHLA-I molecules can also be highly flexible and dynamic, altering their shape according to minor polymorphisms between different HLA-I alleles, or interactions with different peptides. These flexible presentation modes have important biological consequences that can, for example, explain why some HLA-I alleles offer greater protection against HIV, or why some cancer vaccine approaches have been ineffective. This review explores how these recent findings redefine the rules for peptide presentation by HLA-I molecules and extend our understanding of the molecular mechanisms that govern TCR-mediated antigen discrimination.

S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse.

Efficient immune responses require Ca2+ fluxes across ORAI1 channels during engagement of T cell receptors (TCR) at the immune synapse (IS) between T cells and antigen presenting cells. Here, we show that ZDHHC20-mediated S-acylation of the ORAI1 channel at residue Cys143 promotes TCR recruitment and signaling at the IS. Cys143 mutations reduced ORAI1 currents and store-operated Ca2+ entry in HEK-293 cells and nearly abrogated long-lasting Ca2+ elevations, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement in Jurkat T cells. The acylation-deficient channel remained in cholesterol-poor domains upon enforced ZDHHC20 expression and was recruited less efficiently to the IS along with actin and TCR. Our results establish S-acylation as a critical regulator of ORAI1 channel trafficking and function at the IS and reveal that ORAI1 S-acylation enhances TCR recruitment to the synapse.

133P Novel fully human agonist antibodies against the T-cell costimulatory receptor CD27 shape adaptive anti-tumor immunity

133P Novel fully human agonist antibodies against the T-cell costimulatory receptor CD27 shape adaptive anti-tumor immunity

TCR-independent Activation in Presence of a Src-family Kinase Inhibitor Improves CAR-T Cell Product Attributes.

Chimeric antigen receptor expressing T cells (CAR-T cells) have shown remarkable efficacy against some blood cancers and have potential to treat many other human diseases. During CAR-T cell manufacturing, T cells are activated via engagement of the T-cell receptor (TCR); however, persistent TCR engagement can induce unchecked activation, differentiation, and exhaustion, which can negatively affect CAR-T cell product quality and in vivo potency. In addition, T cells may not uniformly respond to TCR-dependent activation (TCRD) contributing to lot-to-lot variability, poor expansion, and manufacturing failures. TCRD also presents challenges during manufacturing of allogeneic CAR-T cells when endogenous TCR is deleted to prevent graft-versus-host disease. Thus, novel strategies to activate T cells may help improve CAR-T cell product attributes and reduce manufacturing failures. In this study, we compared the effect of TCRD and TCR-independent activation (TCRI) on CAR-T cell product attributes. We found that TCRI in presence of a Src-kinase inhibitor significantly improved CAR-T cell expansion and yield without affecting viability and CD4/CD8 ratio. Markers of T-cell activation, exhaustion and differentiation were also reduced in these CAR-T cells compared with CAR-T cells manufactured by TCRD. TCRI did not affect CAR-T cell in vitro potency; however, following co-culture with target cells, CAR-T cells manufactured by TCRI released significantly less inflammatory cytokines compared with CAR-T cells manufactured by TCRD. Together, these data suggest that manufacturing CAR-T cells by TCRI activation in the presence of a Src-kinase inhibitor improves product quality attributes and may help reduce manufacturing failures and improve CAR-T cell safety and efficacy in vivo.

Upregulation of Phosphodiesterase 2A Augments T Cell Activation by Changing cGMP/cAMP Cross-Talk.

3′,5′-cyclic adenosine monophosphate (cAMP) is well-known for its diverse immunomodulatory properties, primarily inhibitory effects during T cell activation, proliferation, and production of pro-inflammatory cytokines. A decrease in cAMP levels, due to the hydrolyzing activity of phosphodiesterases (PDE), is favoring inflammatory responses. This can be prevented by selective PDE inhibitors, which makes PDEs important therapeutic targets for autoimmune disorders. In this study, we investigated the specific roles of PDE2A and PDE3B in the regulation of intracellular cAMP levels in different mouse T cell subsets. Unexpectedly, T cell receptor (TCR) activation led to a selective upregulation of PDE2A at the protein level in conventional T cells (Tcon), whereas no changes were detected in regulatory T cells (Treg). In contrast, protein expression of PDE3B was significantly higher in both non-activated and activated Tcon subsets as compared to Treg, with no changes upon TCR engagement. Live-cell imaging of T cells expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, has enabled cAMP measurements in real time and revealed stronger responses to the PDE2A inhibitors in activated vs non-activated Tcon. Importantly, stimulation of intracellular cGMP levels with natriuretic peptides led to an increase of cAMP in non-activated and a decrease of cAMP in activated Tcon, suggesting that TCR activation changes the PDE3B-dependent positive to PDE2A-dependent negative cGMP/cAMP cross-talk. Functionally, this switch induced higher expression of early activation markers CD25 and CD69. This constitutes a potentially interesting feed-forward mechanism during autoimmune and inflammatory responses that may be exploited therapeutically.

Signaling through polymerization and degradation: Analysis and simulations of T cell activation mediated by Bcl10.

The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR to a key transcriptional regulator, NF-κB. The transmission of this signal involves a highly dynamic process in which helical filaments of Bcl10, a key protein constituent of the TCR signaling cascade, undergo competing processes of polymeric assembly and macroautophagy-dependent degradation. Through computational analysis of three-dimensional, super-resolution optical micrographs, we quantitatively characterize TCR-stimulated Bcl10 filament assembly and length dynamics, and demonstrate that filaments become shorter over time. Additionally, we develop an image-based, bootstrap-like resampling method that demonstrates the preferred association between autophagosomes and both Bcl10-filament ends and punctate-Bcl10 structures, implying that autophagosome-driven macroautophagy is directly responsible for Bcl10 filament shortening. We probe Bcl10 polymerization-depolymerization dynamics with a stochastic Monte-Carlo simulation of nucleation-limited filament assembly and degradation, and we show that high probabilities of filament nucleation in response to TCR engagement could provide the observed robust, homogeneous, and tunable response dynamic. Furthermore, we demonstrate that the speed of filament disassembly preferentially at filament ends provides effective regulatory control. Taken together, these data suggest that Bcl10 filament growth and degradation act as an excitable system that provides a digital response mechanism and the reliable timing critical for T cell activation and regulatory processes.

Gene Edited Human CD4+ T Cells Reveal that Loss of PTPN22 Function Enhances Mildly Autoreactive T Cell Responses

Abstract A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases. PTPN22 is a non-receptor tyrosine phosphatase that negatively regulates proximal T cell receptor (TCR) signaling. PTPN22 deficiency increases TCR signaling in mouse and human T cells. Mouse models of the variant similarly exhibit increased TCR signaling, while primary T cells from human carriers of the variant exhibit decreased signaling. Here, we utilize gene editing in primary human T cells to address these discordant findings and explore how the risk variant impacts TCR signaling. Using Crispr/Cas9 nucleases and DNA repair templates, we established a robust platform for precise homology-directed-repair based gene editing of PTPN22, allowing the generation of PTPN22 risk edited, control edited (silent modification), and knock-out T cells from the same donor. In contrast to control edited cells, PTPN22 risk edited T cells exhibit increased activation in response to TCR engagement with higher expression of activation markers including CD40L and CD25, mimicking PTPN22 KO cells. Further, using lentiviral delivery of transgenic TCRs (derived from autoreactive T cells of T1D patients), we demonstrate that loss of PTPN22 leads to enhanced TCR signaling in weakly autoreactive, but not strongly autoreactive T cells, causing increased phosphorylation of S6 kinase, enhanced proliferation, and greater Th1 skewing. Taken together, these findings suggest that direct effects of the PTPN22 rs2476601 variant represents a loss of function in T cells, increasing TCR signaling in mildly autoreactive T cells, potentially expanding the pool of autoreactive T cells and skewing them toward an inflammatory phenotype.

Decreased Levels of STAT1 and Interferon-γ-Induced STAT1 Phosphorylation in Rheumatoid Arthritis CD4 and CD8 T Cells.

ObjectiveWe investigated whether a previously reported association of IFNGR expression with rheumatoid arthritis (RA) and its radiographic severity reflects differences in proximal interferon‐γ (IFN‐γ) signaling in T cells from patients with RA compared with healthy controls (HC).MethodsUsing phosphoflow cytometry, we compared IFN‐γ–stimulated signal transducer and activator of transcription 1 (STAT1) activation in CD4+ and CD8+ T‐cell populations from patients with RA and HC.ResultsCompared with controls, patients with RA had a higher proportion of CD4+ T cells, associated with expansion of the CD4+ effector memory subset. Several CD4+ T‐cell types exhibited reduced IFN‐γ–induced phosphoSTAT1Y701 (pSTAT1Y701) in patients with RA compared with HC. Engaging the T‐cell receptor (TCR) complex on CD4+ T cells during IFN‐γ stimulation abrogated the reduction in STAT1 activation in patients with RA but had no effect in HC. The phosphorylation of STAT1S727 was similar in CD4+ T cells from patients with RA and HC. In contrast to CD4+ T cells, IFN‐γ–induced pSTAT1Y701 levels in CD8+ T cells were equivalent or higher in patients with RA compared with HC. Total STAT1 levels (phosphorylated + unphosphorylated) were lower in CD4+ and CD8+ T cells from patients with RA compared with HC.ConclusionWe report diminished IFN‐γ–induced pSTAT1Y701 levels in CD4+ T cells in patients with RA, which were restored by TCR engagement. There were lower levels of total STAT1 in patients with RA compared with HC, but this likely does not explain diminished IFN‐γ–induced pSTAT1Y701 levels in CD4+ T cells because activation in CD8+ T cells was higher or equivalent to that seen in HC. The enhanced IFNGR expression in patients with RA reported previously may reflect a compensatory mechanism to overcome deficiency in IFN‐γ responsiveness.

CD5 levels reveal distinct basal T-cell receptor signals in T cells from non-obese diabetic mice.

Type 1 diabetes in non-obese diabetic (NOD) mice occurs when autoreactive T cells eliminate insulin producing pancreatic β cells. While extensively studied in T-cell receptor (TCR) transgenic mice, the contribution of alterations in thymic selection to the polyclonal T-cell pool in NOD mice is not yet resolved. The magnitude of signals downstream of TCR engagement with self-peptide directs the development of a functional T-cell pool, in part by ensuring tolerance to self. TCR interactions with self-peptide are also necessary for T-cell homeostasis in the peripheral lymphoid organs. To identify differences in TCR signal strength that accompany thymic selection and peripheral T-cell maintenance, we compared CD5 levels, a marker of basal TCR signal strength, on immature and mature T cells from autoimmune diabetes-prone NOD and -resistant B6 mice. The data suggest that there is no preferential selection of NOD thymocytes that perceive stronger TCR signals from self-peptide engagement. Instead, NOD mice have an MHC-dependent increase in CD4+ thymocytes and mature T cells that express lower levels of CD5. In contrast, T cell-intrinsic mechanisms lead to higher levels of CD5 on peripheral CD8+ T cells from NOD relative to B6 mice, suggesting that peripheral CD8+ T cells with higher basal TCR signals may have survival advantages in NOD mice. These differences in the T-cell pool in NOD mice may contribute to the development or progression of autoimmune diabetes.