pMHC Affinity Research Articles

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts.

Reactivation of low avidity tumor-specific CD8+ T cells associates with immunotherapeutic efficacy of anti-PD-1

BackgroundCD8+ T cells are a highly diverse population of cells with distinct phenotypic functions that can influence immunotherapy outcomes. Further insights on the roles of CD8+ specificities and TCR avidity...

PD-1 preferentially inhibits the activation of low-affinity T cells

Anti-PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide-MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1-deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells.

Genomic profiling of solid tumors harboring BRD4-NUT and response to immune checkpoint inhibitors

BackgroundThe translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment. Case presentationThirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLCNOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0–4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases – including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis. ConclusionsCGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.

Improved peptide-MHC class II interaction prediction through integration of eluted ligand and peptide affinity data.

Major histocompatibility complex (MHC) class II antigen presentation is a key component in eliciting a CD4+ T cell response. Precise prediction of peptide-MHC (pMHC) interactions has thus become a cornerstone in defining epitope candidates for rational vaccine design. Current pMHC prediction tools have, so far, primarily focused on inference from in vitro binding affinity. In the current study, we collate a large set of MHC class II eluted ligands generated by mass spectrometry to guide the prediction of MHC class II antigen presentation. We demonstrate that models developed on eluted ligands outperform those developed on pMHC binding affinity data. The predictive performance can be further enhanced by combining the eluted ligand and pMHC affinity data in a single prediction model. Furthermore, by including ligand data, the peptide length preference of MHC class II can be accurately learned by the prediction model. Finally, we demonstrate that our model significantly outperforms the current state-of-the-art prediction method, NetMHCIIpan, on an external dataset of eluted ligands and appears superior in identifying CD4+ T cell epitopes.

Beyond the TCR, Antigen Discrimination in T Cells Continues in the LAT:GRB2:SOS Protein Condensate

T cells activate in response to a stochastic series of single molecule binding events between T cell receptors (TCRs) and peptide ligands presented on major histocompatibility complexes (pMHCs) accumulated at a cell-cell interface. Discrimination between ligands with subtly different affinities ensures selective and robust immunological responses upon TCR triggering. Formation of protein condensates based on the scaffolding protein Linker for Activation of T cells (LAT) is a key intermediate step in TCR signaling. LAT is multi-phosphorylated on tyrosine residues by the kinase, ZAP70, which is activated by triggered TCRs. The phosphotyrosines recruit Grb2, which, via interactions with the multivalent GEF, SOS, forms an extended LAT:Grb2:SOS assembly that resembles a phase transition. We mapped single pMHC:TCR binding events in T cells expressing fluorescent protein-tagged LAT and show that LAT assemblies are discrete and have characteristic features that are independent of the originating binding event. Furthermore, LAT assemblies can become spatially uncoupled from the pMHC:TCR complex. These individual assemblies are capable of deforming the plasma membrane and creating local domains that may participate in protein segregation or control reaction kinetics. The probability of LAT assembly is a function of the pMHC affinity, suggesting a second layer of kinetic proofreading that exists beyond the triggered TCR. We recently introduced a molecular impulse-response assay which maps pMHC:TCR binding events to early T cell activation. Results here argue that LAT assemblies may confer a molecular memory that connects a series of spatiotemporally correlated binding events to cellular decision-making. By extending the impulse-response assay, we show that activating cells experience a characteristic, fast rate of LAT assembly. Taken together, we present new molecular dimensions to T cell activation that could provide actionable therapeutic insights.

Abstract A217: TCR affinity determines the fate of T-cells in tumors

Abstract T-cell-mediated immune responses are triggered by T-cell receptor (TCR) binding to peptide-major histocompatibility complex (pMHC). In acute infections, affinity of TCR:pMHC interaction is a critical determinant of T-cell expansion and effector function. However, little is known about how tumor (neo) antigen affinity impacts T-cell differentiation and dysfunction in tumors. To investigate the functional and molecular programs determined by affinity, we generated an in vivo tumor model expressing altered peptide ligands (APL) derived from a native tumor neoantigen recognized by antigen-specific CD8 T-cells with varying functional avidity. While affinity did not impact T-cell activation in tumor draining lymph nodes, it drove distinct functional and molecular pathways at the tumor site: key transcription factors and effector molecules were regulated by signal strength, preserving a cell-intrinsic functional program in T-cells with lower-affinity interactions, while certain hallmarks of T-cell dysfunction, including the expression of some inhibitory receptors, were affinity-independent. RNAseq and ATACseq analyses revealed distinct affinity-dependent transcriptional and epigenetic programs in low- vs. high-affinity T-cells that drive their functional differences. Together these results reveal that TCR:pMHC affinity plays a critical role in defining the epigenetic and transcriptional states and ultimately fate of tumor-specific T-cells. Citation Format: Mojdeh Shakiba. TCR affinity determines the fate of T-cells in tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A217.

Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

Impact of antigen affinity on T cell dysfunction in solid tumors

Abstract Although tumor-specific T cells (TST) are found in human solid tumors, cancers progress, indicating that these T cells are dysfunctional. TSTs express high levels of inhibitory receptors and fail to execute effector functions; however, the regulatory mechanisms underlying TST dysfunction remain poorly defined. T cell mediated immune response is triggered by T cell receptor (TCR) binding to peptide-major histocompatibility (pMHC) complex on the surface of cells. The affinity of TCR:pMHC interaction is a critical determinant of T cell expansion and effector function in acute infections, where T cells with high affinity interactions generally show superior function. However, little is known about how TCR:pMHC affinity impacts T cells activation, induction of dysfunction and susceptibility to immunotherapy in progressing tumors. To elucidate this, we generated tumor cell lines expressing altered peptide ligands derived from SV40 large T antigen epitope I (Tag) and recognized by Tag-specific transgenic CD8 T cells (TCRTag) with varying functional avidity. We found that tumor-infiltrating TCRTag encountering low and high affinity tumor antigens were equally activated and expressed similar levels of inhibitory receptors, suggesting that even very weak TCR ligations can induce a typical “exhaustion” phenotype. Strikingly, while high affinity TCR:pMHC interactions led to complete loss of cytokine production, T cells with low affinity interactions remained functional, suggesting that TSTs with high affinity TCR:pMHC interactions enter a profound state of dysfunction. Future experiments will test the impact of tumor antigen affinity on the efficacy of therapeutic reprogramming, with important implications for immunotherapy.

Abstract A093: Impact of antigen affinity on T cell dysfunction in solid tumors

Abstract Although tumor-specific CD8 T cells are found in human solid tumors, cancers progress, indicating that these T cells are dysfunctional. When naïve CD8 T cells encounter antigen in an inflammatory context (e.g. acute infection), a cell-intrinsic program is initiated that drives T cells to expand, produce effector cytokines, and differentiate into effector and memory T cells. In contrast, dysfunctional CD8 T cells in solid tumors express high levels of inhibitory receptors and fail to execute effector functions; however, the regulatory mechanisms underlying tumor-specific T cell dysfunction remain poorly defined. T cell-mediated immune responses are triggered by T cell receptor (TCR) binding to peptide-major histocompatibility complex (pMHC) on the surface of antigen presenting cells. The affinity of TCR:pMHC interaction is a critical determinant of CD8 T cell expansion and effector function in acute infections; while both low- and high-affinity antigens can activate naïve antigen-specific CD8 T cells, T cells with high-affinity TCR:pMHC interactions generally show superior effector function. However, little is known about how tumor antigen affinity impacts the activation of T cells, the induction of T cell dysfunction in progressing tumors, and T cell susceptibility to immunotherapeutic reprogramming. To investigate the role of TCR:pMHC affinity in T cell differentiation in progressing tumors, we generated tumor cell lines expressing altered peptide ligands (APL) derived from SV40 large T antigen epitope I (SV40-I). These APLs are recognized by SV40-I-specific transgenic CD8 T cells (TCRSV40-I) with varying functional avidity. We found that tumor-infiltrating TCRSV40-I encountering low- and high-affinity tumor antigens were equally activated and displayed similar expression levels of inhibitory receptors, such as PD1 and Lag3, suggesting that even very weak TCR ligations can induce a typical “exhaustion” phenotype. Strikingly, while high-affinity TCR:pMHC interactions led to complete loss of interferon gamma and tumor necrosis factor alpha production, T cells with low-affinity interactions remained functional and capable of producing high levels of these effector cytokines suggesting that contrary to our expectations, tumor-specific T cells with high-affinity TCR:pMHC interactions enter a profound state of dysfunction. Future experiments will test the impact of tumor antigen affinity on the efficacy of therapeutic reprogramming including checkpoint blockade, which will have important implications for immunotherapy. Citation Format: Mojdeh Shakiba, Mary Philip, Andrea Schietinger. Impact of antigen affinity on T cell dysfunction in solid tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A093.

PMHC affinity controls duration of CD8+ T cell-DC interactions and imprints timing of effector differentiation versus expansion.

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity-primed T cells acquired cytotoxic activity earlier than high affinity-primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity-stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment.

Architecture of a minimal signaling pathway explains the T-cell response to a 1 million-fold variation in antigen affinity and dose

T cells must respond differently to antigens of varying affinity presented at different doses. Previous attempts to map peptide MHC (pMHC) affinity onto T-cell responses have produced inconsistent patterns of responses, preventing formulations of canonical models of T-cell signaling. Here, a systematic analysis of T-cell responses to 1 million-fold variations in both pMHC affinity and dose produced bell-shaped dose-response curves and different optimal pMHC affinities at different pMHC doses. Using sequential model rejection/identification algorithms, we identified a unique, minimal model of cellular signaling incorporating kinetic proofreading with limited signaling coupled to an incoherent feed-forward loop (KPL-IFF) that reproduces these observations. We show that the KPL-IFF model correctly predicts the T-cell response to antigen copresentation. Our work offers a general approach for studying cellular signaling that does not require full details of biochemical pathways.

Preventing tumor escape by targeting a post-proteasomal trimming independent epitope.

Adoptive T cell therapy (ATT) can achieve regression of large tumors in mice and humans; however, tumors frequently recur. High target peptide-major histocompatibility complex-I (pMHC) affinity and T cell receptor (TCR)-pMHC affinity are thought to be critical to preventing relapse. Here, we show that targeting two epitopes of the same antigen in the same cancer cells via monospecific T cells, which have similar pMHC and pMHC-TCR affinity, results in eradication of large, established tumors when targeting the apparently subdominant but not the dominant epitope. Only the escape but not the rejection epitope required postproteasomal trimming, which was regulated by IFN-γ, allowing IFN-γ-unresponsive cancer variants to evade. The data describe a novel immune escape mechanism and better define suitable target epitopes for ATT.

Lower Affinity T Cells are Critical Components and Active Participants of the Immune Response.

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.

Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs.

Fluorochrome-conjugated peptide–MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II–restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR–pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.

Low Affinity CD8+ CD45RBhigh TNF-Producing TCM Are Potently Alloreactive Against High Affinity Antigen in a Model of T Cell Receptor Cross-Reactive Heterologous Immunity.

Memory T cells primed against microbial antigen can cross-react with allogeneic antigen and mediate graft rejection, a process termed heterologous immunity. This scenario requires the T cell receptor (TCR) to recognize both the priming microbial peptide:MHC (pMHC) and an allogeneic pMHC complex, distinct binding interactions that likely have different magnitudes of TCR:pMHC affinity. However, the role that TCR affinity plays during heterologous immunity is unknown. To investigate the impact of TCR affinity during heterologous immune responses, we utilized a model in which transgenic CD8+ T cells are primed via pathogen infection with either high affinity or low affinity antigen, and are subsequently challenged with a skin allograft that constitutively expresses high affinity antigen. Our results demonstrate that infections with high and low affinity antigen generated a similar frequency of KLRG-1lowCD127high memory precursor cells. However, compared to high affinity memory cells, a greater proportion of low affinity memory cells became CD44highCD62Lhigh TCM. Furthermore, the low affinity memory population contained significantly more TNF+ and TNF+IFN-g+ double producer cells when rechallenged with high affinity antigen. Surprisingly, low affinity primed memory CD8+ T cells rejected skin allografts with faster kinetics than high affinity memory CD8+ T cells and were resistant to a costimulation/integrin blockade regimen. We next sought to understand the mechanistic basis underlying this increased potency of low affinity memory CD8+ T cells. High and low affinity memory cells expressed similar levels of TCR, CD8, and CD5; however, low affinity memory cells were predominantly CD45RBhigh, suggesting that CD45RB expression may tune low affinity memory CD8+ T cells to optimally respond to antigen with a broad range of affinity. These studies reveal an important role for low affinity memory CD8+ T cells during graft rejection in the context of heterologous immunity, and identify the phenotypic and functional characteristics associated with their increased potency in vivo.

TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.

Going Pro to enhance T‐cell immunogenicity: Easy as π?

MHC class I molecules bind intracellular oligopeptides and present them on the cell surface for CD8(+) T-cell activation and recognition. Strong peptide/MHC class I (pMHC) interactions typically induce the best CD8(+) T-cell responses; however, many immunotherapeutic tumor-specific peptides bind MHC with low affinity. To overcome this, immunologists can carefully alter peptides for enhanced MHC affinity but often at the cost of decreased T-cell recognition. A new report published in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43:3051-3060] shows that the substitution of proline at the third residue (p3P) of a common tumor peptide increases pMHC affinity and complex stability while enhancing T-cell receptor recognition. X-ray crystallography indicates that stability is generated through newly introduced CH-π bonding between p3P and a conserved residue (Y159) in the MHC heavy chain. This finding highlights a previously unappreciated role for CH-π bonding in MHC peptide binding, and importantly, arms immunologists with a novel and possibly general approach for increasing pMHC stability without compromising T-cell recognition.