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Following solid organ transplantation, small precursor populations of polyclonal CD8+ T cells specific for any graft-expressed antigen preferentially expand their high-affinity clones. This phenomenon, termed "avidity maturation," results in a larger population of CD8+ T cells with increased sensitivity to alloantigen, posing a greater risk for graft rejection. Using a mouse model of minor-mismatched skin transplantation, coupled with the tracking of 2 skin graft-reactive CD8+ T cell receptor-transgenic tracer populations with high and low affinity for the same peptide-major histocompatibility complex, we explored the conventional paradigm that CD8+ T cell avidity maturation occurs through T cell receptor affinity-based competition for cognate antigen. Our data revealed "interclonal CD8-CD8 help," whereby lower/intermediate affinity clones help drive the preferential expansion of their higher affinity counterparts in an interleukin-2/CD25-dependent manner. Consequently, the CD8-helped high-affinity clones exhibit greater expansion and develop augmented effector functions in the presence of their low-affinity counterparts, correlating with more severe graft damage. Finally, interclonal CD8-CD8 help was suppressed by costimulation blockade treatment. Thus, high-affinity CD8+ T cells can leverage help from low-affinity CD8+ T cells of identical specificity to promote graft rejection. Suppressing provision of interclonal CD8-CD8 help may be important to improve transplant outcomes.

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How individual T cells compete for and respond to IL-2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high-affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high-affinity CD8 T cells. As part of an incoherent feed-forward loop, ZFP36L1 has a nonredundant role in suppressing multiple negative regulators of cytokine signaling and mediating a selection mechanism based on competition for IL-2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes the dominance of high-affinity T cells by installing a hierarchical response to IL-2.

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The contribution of low-affinity T cells to autoimmunity in the context of polyclonal T-cell responses is understudied due to the limitations in their capture by tetrameric reagents and low level of activation in response to antigenic stimulation. As a result, low-affinity T cells are often disregarded as nonantigen-specific cells irrelevant to the immune response. Our study aimed to assess how the level of self-antigen reactivity shapes T-cell lineage and effector responses in the context of spontaneous tissue-specific autoimmunity observed in NOD mice. Using multicolor flow cytometry in combination with Nur77GFP reporter of TCR signaling, we identified a dormant population of T cells that infiltrated the pancreatic islets of prediabetic NOD mice, which exhibited reduced levels of self-tissue reactivity based on expression of CD5 and Nur77GFP . We showed that these CD5low T cells had a unique TCR repertoire and exhibited low activation and minimal effector function; however, induced rapid diabetes upon transfer. The CD4+ CD5low T-cell population displayed transcriptional signature of central memory T cells, consistent with the ability to acquire effector function post-transfer. Transcriptional profile of CD5low T cells was similar to T cells expressing a low-affinity TCR, indicating TCR affinity to be an important factor in shaping CD5low T-cell phenotype and function at the tissue site. Overall, our study suggests that autoimmune tissue can maintain a reservoir of undifferentiated central memory-like autoreactive T cells with pathogenic effector potential that might be an important source for effector T cells during long-term chronic autoimmunity.

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The functional avidity (FA) of cytotoxic CD8 T cells impacts strongly on their functional capabilities and correlates with protection from infection and cancer. FA depends on TCR affinity, downstream signaling strength, and TCR affinity-independent parameters of the immune synapse, such as costimulatory and inhibitory receptors. The functional impact of coreceptors on FA remains to be fully elucidated. Despite its importance, FA is infrequently assessed and incompletely understood. There is currently no consensus as to whether FA can be enhanced by optimized vaccine dose or boosting schedule. Recent findings suggest that FA is remarkably stable in vivo, possibly due to continued signaling modulation of critical receptors in the immune synapse. In this review, we provide an overview of the current knowledge and hypothesize that in vivo, codominant T cells constantly "equalize" their FA for similar function. We present a new model of constant FA regulation, and discuss practical implications for T-cell-based cancer immunotherapy.

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Over the past decades, adoptive transfer of T cells has revolutionized cancer immunotherapy. In particular, T-cell receptor (TCR) engineering of T cells has marked important milestones in developing more precise and personalized cancer immunotherapies. However, to get the most benefit out of this approach, understanding the role that TCR affinity, avidity, and functional avidity play on how TCRs and T cells function in the context of tumor-associated antigen (TAA) recognition is vital to keep generating improved adoptive T-cell therapies. Aside from TCR-related parameters, other critical factors that govern T-cell activation are the effect of TCR co-receptors on TCR-peptide-major histocompatibility complex (pMHC) stabilization and TCR signaling, tumor epitope density, and TCR expression levels in TCR-engineered T cells. In this review, we describe the key aspects governing TCR specificity, T-cell activation, and how these concepts can be applied to cancer-specific TCR redirection of T cells.

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T cells are critical in orchestrating protective immune responses to cancer and an array of pathogens. The interaction between a peptide MHC (pMHC) complex on antigen presenting cells (APCs) and T cell receptors (TCRs) on T cells initiates T cell activation, division, and clonal expansion in secondary lymphoid organs. T cells must also integrate multiple T cell-intrinsic and extrinsic signals to acquire the effector functions essential for the defense against invading microbes. In the case of T helper cell differentiation, while innate cytokines have been demonstrated to shape effector CD4+ T lymphocyte function, the contribution of TCR signaling strength to T helper cell differentiation is less understood. In this review, we summarize the signaling cascades regulated by the strength of TCR stimulation. Various mechanisms in which TCR signal strength controls T helper cell expansion and differentiation are also discussed.

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The identity of CD45 isoforms on the T cell surface changes following the activation of naive T cells and impacts intracellular signaling. In this study, we find that the anti-viral memory CD8+ T pool is unexpectedly comprised of both CD45RBhi and CD45RBlo populations. Relative to CD45RBlo memory T cells, CD45RBhi memory T cells have lower affinity and display greater clonal diversity, as well as a persistent CD27hi phenotype. The CD45RBhi memory population displays a homeostatic survival advantage in vivo relative to CD45RBlo memory, and long-lived high-affinity cells that persisted long term convert from CD45RBlo to CD45RBhi. Human CD45RO+ memory is comprised of both CD45RBhi and CD45RBlo populations with distinct phenotypes, and antigen-specific memory to two viruses is predominantly CD45RBhi. These data demonstrate that CD45RB status is distinct from the conventional central/effector T cell memory classification and has potential utility for monitoring and characterizing pathogen-specific CD8+ T cell responses.

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BACKGROUND: Affinity-optimized T cell receptor (TCR)-engineered lymphocytes targeting tumor antigens can mediate potent antitumor responses in cancer patients, but also bear substantial risks for off-target toxicities. Most preclinical studies have focused on T cell responses to antigen-specific stimulation. In contrast, little is known on the regulation of T cell responsiveness through continuous TCR triggering and consequent tonic signaling. Here, we addressed the question whether increasing the TCR affinity can lead to chronic interactions occurring directly between TCRs and MHC-(self) molecules, which may modulate the overall functional potency of tumor-redirected CD8 T cells. For this purpose, we developed two complementary human CD8 T cell models (i.e. HLA-A2 knock-in and knock-out) engineered with incremental-affinity TCRs to the HLA-A2/NY-ESO-1 tumor antigen. METHODS: The impact of HLA-A2 recognition, depending on TCR affinity, was assessed at the levels of the TCR/CD3 complex, regulatory receptors, and signaling, under steady-state conditions and in kinetic studies. The quality of CD8 T cell responses was further evaluated by gene expression and multiplex cytokine profiling, as well as real-time quantitative cell killing, combined with co-culture assays. RESULTS: We found that HLA-A2 per se (in absence of cognate peptide) can trigger chronic activation followed by a tolerance-like state of tumor-redirected CD8 T cells with increased-affinity TCRs. HLA-A2pos but not HLA-A2neg T cells displayed an activation phenotype, associated with enhanced upregulation of c-CBL and multiple inhibitory receptors. T cell activation preceded TCR/CD3 downmodulation, impaired TCR signaling and functional hyporesponsiveness. This stepwise activation-to-hyporesponsive state was dependent on TCR affinity and already detectable at the upper end of the physiological affinity range (KD ≤ 1 µM). Similar findings were made when affinity-increased HLA-A2neg CD8 T cells were chronically exposed to HLA-A2pos-expressing target cells. CONCLUSIONS: Our observations indicate that sustained interactions between affinity-increased TCR and self-MHC can directly adjust the functional potential of T cells, even in the absence of antigen-specific stimulation. The observed tolerance-like state depends on TCR affinity and has therefore potential implications for the design of affinity-improved TCRs for adoptive T cell therapy, as several engineered TCRs currently used in clinical trials share similar affinity properties.

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Adoptive cell transfer (ACT) using T cell receptor (TCR) gene-modified T cells is an exciting and rapidly evolving field. Numerous preclinical and clinical studies have demonstrated various levels of feasibility, safety, and efficacy using TCR-engineered T cells to treat cancer and viral infections. Although evidence suggests their use can be effective, to what extent and how to improve these therapeutics are still matters of investigation. As TCR affinity has been generally accepted as the central role in defining T cell specificity and sensitivity, selection for and generation of high affinity TCRs has remained a fundamental approach to design more potent T cells. However, traditional methods for affinity-enhancement by random mutagenesis can induce undesirable cross-reactivity causing on- and off-target adverse events, generate exhausted effectors by overstimulation, and ignore other kinetic and cellular parameters that have been shown to impact antigen specificity. In this Focussed Research Review, we comment on the preclinical and clinical potential of TCR gene-modified T cells, summarize our contributions challenging the role TCR affinity plays in antigen recognition, and explore how structure-guided design can be used to manipulate antigen specificity and TCR cross-reactivity to improve the safety and efficacy of TCR gene-modified T cells used in ACT.

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The screening and analysis of T cells functional avidity for specific tumor-associated antigens is crucial for the development of personalized immunotherapies against cancer. The affinity and kinetics of a T cell receptor (TCR) binding to the peptide-major histocompatibility complex (pMHC), expressed on tumor or antigen-presenting cells, have shown major implications in T cell activation and effector functions. We introduce an innovative methodology for the two-dimensional affinity analysis of TCR-pMHC in a label-free configuration by employing a multiparametric Surface Plasmon Resonance biosensor (MP-SPR) functionalized with artificial cell membranes. The biomimetic scaffold created with planar lipid bilayers is able to efficiently capture the specific and intact tumor-specific T cells and monitor the formation of the immunological synapse in situ. We have achieved excellent limits of detection for in-flow cell capturing, up to 2 orders of magnitude below the current state-of-the-art for plasmonic sensing. We demonstrate the accuracy and selectivity of our sensor for the analysis of CD8+ T cells bioengineered with TCR of incremental affinities specific for the HLA-A0201/NY-ESO-I157-165 pMHC complex. The study confirmed the significance of providing a biomimetic microenvironment, compared to the traditional molecular analysis, and showed fine agreement with previous results employing flow cytometry. Our methodology is reliable and versatile; thus, it can be applied to more sophisticated photonic and nanoplasmonic technologies for the screening of multiple cell types and boost the development of novel treatments for cancer.

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CD4+ T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4+ T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4+ T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4+ cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4+ and CD8+ T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4+ T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4+ T cells than CD8+ T cells. These results indicate that the CD4+ T cell component of current adoptive therapies using TCRs optimized for CD8+ T cells is below par and that there is room for substantial improvement.

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Cytotoxic T cells recognize, via their T cell receptors (TCRs), small antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells and infected or malignant cells. The efficiency of T cell triggering critically depends on TCR binding to cognate pMHC, i.e., the TCR-pMHC structural avidity. The binding and kinetic attributes of this interaction are key parameters for protective T cell-mediated immunity, with stronger TCR-pMHC interactions conferring superior T cell activation and responsiveness than weaker ones. However, high-avidity TCRs are not always available, particularly among self/tumor antigen-specific T cells, most of which are eliminated by central and peripheral deletion mechanisms. Consequently, systematic assessment of T cell avidity can greatly help distinguishing protective from non-protective T cells. Here, we review novel strategies to assess TCR-pMHC interaction kinetics, enabling the identification of the functionally most-relevant T cells. We also discuss the significance of these technologies in determining which cells within a naturally occurring polyclonal tumor-specific T cell response would offer the best clinical benefit for use in adoptive therapies, with or without T cell engineering.

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Kinetic and biophysical parameters of T cell receptor (TCR) and peptide:MHC (pMHC) interaction define intrinsic factors required for T cell activation and differentiation. Although receptor ligand kinetics are somewhat cumbersome to assess experimentally, TCR:pMHC affinity has been shown to predict peripheral T cell functionality and potential for forming memory. Multimeric forms of pMHC monomers have often been used to provide an indirect readout of higher affinity T cells due to their availability and ease of use while allowing simultaneous definition of other functional and phenotypic characteristics. However, multimeric pMHC reagents have introduced a bias that underestimates the lower affinity components contained in the highly diverse TCR repertoires of all polyclonal T cell responses. Advances in the identification of lower affinity cells have led to the examination of these cells and their contribution to the immune response. In this review, we discuss the identification of high- vs. low-affinity T cells as well as their attributed signaling and functional differences. Lastly, mechanisms are discussed that maintain a diverse range of low- and high-affinity T cells.

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T cell immunity relies on the generation and maintenance of a diverse repertoire of T cell antigen receptors (TCRs). The strength of signaling emanating from the TCR dictates the fate of T cells during development, as well as during the immune response. Whereas development of new T cells in the thymus increases the available TCR repertoire, clonal selection during the immune response narrows TCR diversity through the outgrowth of clonotypes with the fittest TCR. To ensure maintenance of TCR diversity in the antigen-selected repertoire, specific mechanisms can be envisaged that facilitate the participation of T cell clonotypes with less than best fit TCRs. Here, we summarize the evidence for the existence of such mechanisms that can prevent the loss of diversity. A number of T cell-autonomous or extrinsic factors can reverse clonotypic hierarchies set by TCR affinity for given antigen. Although not yet complete, understanding of these factors and their mechanism of action will be critical in interventional attempts to mold the antigen-selected TCR repertoire.

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A repertoire of antigen recognition specificities in mature T cell pool is formed by the selection during T cell development in the thymus. Positive selection is an essential process for the development of functionally competent T cells and is dependent on the interaction between T cell antigen receptors (TCRs) that newly generated thymocytes express and self-peptide-associated major histocompatibility complex (pMHC) molecules that cortical thymic epithelial cells (cTECs) express. Characterization of positive-selection-inducing peptides has revealed that the low-affinity TCR engagement by the positive-selection-inducing pMHC complexes initiates intracellular signals that induce the survival of immature thymocytes and their differentiation into mature T cells. Recent studies suggest unique mechanisms of antigen processing in cTECs for the production of positively selecting MHC-bound self-peptides.

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Maintenance of immune tolerance critically depends upon regulatory T cells that express the transcription factor forkhead box P3 (Foxp3). These CD4(+) T cells can be generated in the thymus, termed thymus-derived regulatory T cells (tTregs), but their developmental pathway remains incompletely understood. tTreg development has been shown to be delayed compared with that of CD4(+) single positive (SP) thymocytes, with tTregs being detected only in neonatal thymi by day 3 after birth. Here, we outline the reasons for this delayed emergence of Foxp3(+) tTregs and demonstrate that thymocyte apoptosis is intrinsically tied to tTreg development. We show that thymic apoptosis leads to the production of TGFß intrathymically from thymic macrophages, dendritic cells, and epithelial cells. This TGFß then induces foxp3 expression and drives tTreg generation. Thymocyte apoptosis has previously been shown to accelerate after birth, which drives increases in TGFß in the neonatal thymus. We highlight a paucity of TGFß in the neonatal thymus, accounting for the delayed development of tTregs compared with CD4(+) SP thymocytes. Importantly, we show that enhanced levels of apoptosis in the thymus result in an augmented tTreg population and, moreover, that decreasing thymic apoptosis results in reduced tTregs. In addition to this, we also show that T-cell receptor (TCR) signals of different affinity were all capable of driving tTreg development; however, to achieve this TGFß signals must also be received concomitant with the TCR signal. Collectively, our results indicate that thymic apoptosis is a key event in tTreg generation and reveal a previously unrecognized apoptosis-TGFß-Foxp3 axis that mediates the development of tTregs.

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Clonal population expansion of T cells during an immune response is dependent on the affinity of the T cell receptor (TCR) for its antigen [1]. However, there is little understanding of how this process is controlled transcriptionally. We found that the transcription factor IRF4 was induced in a manner dependent on TCR-affinity and was critical for the clonal expansion and maintenance of effector function of antigen-specific CD8(+) T cells. We performed a genome-wide expression profiling experiment using RNA sequencing technology (RNA-seq) to interrogate global expression changes when IRF4 was deleted in CD8(+) T cells activated with either a low or high affinity peptide ligand. This allowed us not only to determine IRF4-dependent transcriptional changes but also to identify transcripts dependent on TCR-affinity [2]. Here we describe in detail the analyses of the RNA-seq data, including quality control, read mapping, quantification, normalization and assessment of differential gene expression. The RNA-seq data can be accessed from Gene Expression Omnibus database (accession number GSE49929).

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Adoptive transfer of T cells gene-engineered with antigen-specific T cell receptors (TCRs) has proven its feasibility and therapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCR gene therapy, it is necessary to target immunogenic epitopes that are related to oncogenesis and selectively expressed by tumor tissue, and implement strategies that result in optimal T cell fitness. In addition, in particular for the treatment of solid tumors, it is equally necessary to include strategies that counteract the immune-suppressive nature of the tumor micro-environment. Here, we will provide an overview of the current status of TCR gene therapy, and redefine the following three challenges of improvement: "choice of target antigen"; "fitness of T cells"; and "sensitization of tumor milieu." We will categorize and discuss potential strategies to address each of these challenges, and argue that advancement of clinical TCR gene therapy critically depends on developments toward each of the three challenges.

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Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional αß T cell receptor (TCR) against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR) consisting of a single-chain antibody as an Fv fragment linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the αß TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher-affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher-affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.

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Cytotoxic CD8 T cells mediate immunity to pathogens and they are able to eliminate malignant cells. Immunity to viruses and bacteria primarily involves CD8 T cells bearing high affinity T cell receptors (TCRs), which are specific to pathogen-derived (non-self) antigens. Given the thorough elimination of high affinity self/tumor-antigen reactive T cells by central and peripheral tolerance mechanisms, anti-cancer immunity mostly depends on TCRs with intermediate-to-low affinity for self-antigens. Because of this, a promising novel therapeutic approach to increase the efficacy of tumor-reactive T cells is to engineer their TCRs, with the aim to enhance their binding kinetics to pMHC complexes, or to directly manipulate the TCR-signaling cascades. Such manipulations require a detailed knowledge on how pMHC-TCR and co-receptors binding kinetics impact the T cell response. In this review, we present the current knowledge in this field. We discuss future challenges in identifying and targeting the molecular mechanisms to enhance the function of natural or TCR-affinity optimized T cells, and we provide perspectives for the development of protective anti-tumor T cell responses.