The impact of SARS-CoV-2 spike mutation on peptide presentation is HLA allomorph-specific.

CD8+ T cells are crucial for viral elimination and recovery from viral infection. Nonetheless, the current understanding of the T cell response to SARS-CoV-2 at the antigen level remains limited. The Spike protein is an external structural protein that is prone to mutations, threatening the efficacy of current vaccines. Therefore, we have characterised the immune response towards the immunogenic Spike-derived peptide (S976-984, VLNDILSRL), restricted to the HLA-A*02:01 molecule, which is mutated in both Alpha (S982A) and Omicron BA.1 (L981F) variants of concern. We determined that the mutation in the Alpha variant (S982A) impacted both the stability and conformation of the peptide, bound to HLA-A*02:01, in comparison to the original S976-984. We identified a longer and overlapping immunogenic peptide (S975-984, SVLNDILSRL) that could be presented by HLA-A*02:01, HLA-A*11:01 and HLA-B*13:01 allomorphs. We showed that S975-specific CD8+ T cells were weakly cross-reactive to the mutant peptides despite their similar conformations when presented by HLA-A*11:01. Altogether, our results show that the impact of SARS-CoV-2 mutations on peptide presentation is HLA allomorph-specific, and that post vaccination there are T cells able to react and cross-react towards the variant of concern peptides.

Enhanced T cell receptor specificity through framework engineering.

Development of T cell receptors (TCRs) as immunotherapeutics is hindered by inherent TCR cross-reactivity. Engineering more specific TCRs has proven challenging, as unlike antibodies, improving TCR affinity does not usually improve specificity. Although various protein design approaches have been explored to surmount this, mutations in TCR binding interfaces risk broadening specificity or introducing new reactivities. Here we explored if TCR specificity could alternatively be tuned through framework mutations distant from the interface. Studying the 868 TCR specific for the HIV SL9 epitope presented by HLA-A2, we used deep mutational scanning to identify a framework mutation above the mobile CDR3ß loop. This glycine to proline mutation had no discernable impact on binding affinity or functional avidity towards the SL9 epitope but weakened recognition of SL9 escape variants and led to fewer responses in a SL9-derived positional scanning library. In contrast, an interfacial mutation near the tip of CDR3α that also did not impact affinity or functional avidity towards SL9 weakened specificity. Simulations indicated that the specificity-enhancing mutation functions by reducing the range of loop motions, limiting the ability of the TCR to adjust to different ligands. Although our results are likely to be TCR dependent, using framework engineering to control TCR loop motions may be a viable strategy for improving the specificity of TCR-based immunotherapies.

Distinct sets of molecular characteristics define tumor-rejecting neoantigens.

Challenges in identifying tumor-rejecting neoantigens limit the efficacy of neoantigen vaccines to treat cancers, including cutaneous squamous cell carcinoma (cSCC). A minority of human cSCC tumors shared neoantigens, supporting the need for personalized vaccines. Using a UV-induced mouse cSCC model which recapitulated the mutational signature and driver mutations found in human disease, we found that CD8 T cells constrain cSCC. Two MHC class I neoantigens were identified that constrained cSCC growth. Compared to the wild-type peptides, one tumor-rejecting neoantigen exhibited improved MHC binding and the other had increased solvent accessibility of the mutated residue. Across known neoantigens that do not impact MHC binding, structural modeling of the peptide/MHC complexes indicated that increased solvent accessibility, which will facilitate TCR recognition of the neoantigen, distinguished tumor-rejecting from non-immunogenic neoantigens. This work reveals characteristics of tumor-rejecting neoantigens that may be of considerable importance in identifying optimal vaccine candidates in cSCC and other cancers.

Accurate modeling of peptide-MHC structures with AlphaFold.

Major histocompatibility complex (MHC) proteins present peptides on the cell surface for T cell surveillance. Reliable in silico prediction of which peptides would be presented and which T cell receptors would recognize them is an important problem in structural immunology. Here, we introduce an AlphaFold-based pipeline for predicting the three-dimensional structures of peptide-MHC complexes for class I and class II MHC molecules. Our method demonstrates high accuracy, outperforming existing tools in class I modeling accuracy and class II peptide register prediction. We validate its performance and utility with new experimental data on a recently described cancer neoantigen/wild-type peptide pair and explore applications toward improving peptide-MHC binding prediction.

Structural and physical features that distinguish tumor-controlling from inactive cancer neoepitopes.

Neoepitopes arising from amino acid substitutions due to single nucleotide polymorphisms are targets of T cell immune responses to cancer and are of significant interest in the development of cancer vaccines. However, understanding the characteristics of rare protective neoepitopes that truly control tumor growth has been a challenge, due to their scarcity as well as the challenge of verifying true, neoepitope-dependent tumor control in humans. Taking advantage of recent work in mouse models that circumvented these challenges, here, we compared the structural and physical properties of neoepitopes that range from fully protective to immunologically inactive. As neoepitopes are derived from self-peptides that can induce immune tolerance, we studied not only how the various neoepitopes differ from each other but also from their wild-type counterparts. We identified multiple features associated with protection, including features that describe how neoepitopes differ from self as well as features associated with recognition by diverse T cell receptor repertoires. We demonstrate both the promise and limitations of neoepitope structural analysis and predictive modeling and illustrate important aspects that can be incorporated into neoepitope prediction pipelines.

Prior viral infection primes cross-reactive CD8+ T cells that respond to mouse heart allografts.

Introduction: Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed. Methods: Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8+ T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens. Results: Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8+ T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer+ CD8+ T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8+ T cells that were GFP+ was <1%. These data show that a significant percentage of viral-specific memory CD8+ T cells expressed T cell receptors that also recognized alloantigens in vivo. Notably, the frequency of cross-reactive CD8+ T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity. Discussion: In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.

CD8+ T Cell-Dependent Antitumor Activity In Vivo of a Mass Spectrometry-Identified Neoepitope despite Undetectable CD8+ Immunogenicity In Vitro.

Identification of neoepitopes that can control tumor growth in vivo remains a challenge even 10 y after the first genomics-defined cancer neoepitopes were identified. In this study, we identify a neoepitope, resulting from a mutation in the junction plakoglobin (Jup) gene (chromosome 11), from the mouse colon cancer line MC38-FABF (C57BL/6). This neoepitope, Jup mutant (JupMUT), was detected during mass spectrometry of MHC class I-eluted peptides from the tumor. JupMUT has a predicted binding affinity of 564 nM for the Kb molecule and a higher predicted affinity of 82 nM for Db. However, whereas structural modeling of JupMUT and its unmutated counterpart Jup wild-type indicates that there are little conformational differences between the two epitopes bound to Db, large structural divergences are predicted between the two epitopes bound to Kb. Together with in vitro binding data with RMA-S cells, these data suggest that Kb rather than Db is the relevant MHC class I molecule of JupMUT. Immunization of naive C57BL/6 mice with JupMUT elicits CD8-dependent tumor control of a MC38-FABF challenge. Despite the CD8 dependence of JupMUT-mediated tumor control in vivo, CD8+ T cells from JupMUT-immunized mice do not produce higher levels of IFN-γ than do naive mice. The structural and immunological characteristics of JupMUT are substantially different from those of many other neoepitopes that have been shown to mediate tumor control.

T cell receptor therapeutics: immunological targeting of the intracellular cancer proteome.

The T cell receptor (TCR) complex is a naturally occurring antigen sensor that detects, amplifies and coordinates cellular immune responses to epitopes derived from cell surface and intracellular proteins. Thus, TCRs enable the targeting of proteins selectively expressed by cancer cells, including neoantigens, cancer germline antigens and viral oncoproteins. As such, TCRs have provided the basis for an emerging class of oncology therapeutics. Herein, we review the current cancer treatment landscape using TCRs and TCR-like molecules. This includes adoptive cell transfer of T cells expressing endogenous or engineered TCRs, TCR bispecific engagers and antibodies specific for human leukocyte antigen (HLA)-bound peptides (TCR mimics). We discuss the unique complexities associated with the clinical development of these therapeutics, such as HLA restriction, TCR retrieval, potency assessment and the potential for cross-reactivity. In addition, we highlight emerging clinical data that establish the antitumour potential of TCR-based therapies, including tumour-infiltrating lymphocytes, for the treatment of diverse human malignancies. Finally, we explore the future of TCR therapeutics, including emerging genome editing methods to safely enhance potency and strategies to streamline patient identification.

The Energetic Landscape of Catch Bonds in TCR Interfaces.

Recognition of peptide/MHC complexes by αß TCRs has traditionally been viewed through the lens of conventional receptor-ligand theory. Recent work, however, has shown that TCR recognition and T cell signaling can be profoundly influenced and tuned by mechanical forces. One outcome of applied force is the catch bond, where TCR dissociation rates decrease (half-lives increase) when limited force is applied. Although catch bond behavior is believed to be widespread in biology, its counterintuitive nature coupled with the difficulties of describing mechanisms at the structural level have resulted in considerable mystique. In this review, we demonstrate that viewing catch bonds through the lens of energy landscapes, barriers, and the ensuing reaction rates can help demystify catch bonding and provide a foundation on which atomic-level TCR catch bond mechanisms can be built.

Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/ß sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/ß cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Single cell transcriptomic analysis of renal allograft rejection reveals novel insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/ß sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8 + T cell clonal expansion (CD8 EXP ), independent of HLA mismatch or IS type. Subcloning of TCRα/ß cDNAs from CD8 EXP into Jurkat76 cells (TCR -/- ) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8 EXP revealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8 EXP , while CD8 EXP were maintained during treatment-refractory rejection. Finally, most rBx-derived CD8 EXP were also observed in matching urine samples. Overall, our data define the clonal CD8 + T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.

A class-mismatched TCR bypasses MHC restriction via an unorthodox but fully functional binding geometry.

MHC restriction, which describes the binding of TCRs from CD4+ T cells to class II MHC proteins and TCRs from CD8+ T cells to class I MHC proteins, is a hallmark of immunology. Seemingly rare TCRs that break this paradigm exist, but mechanistic insight into their behavior is lacking. TIL1383I is a prototypical class-mismatched TCR, cloned from a CD4+ T cell but recognizing the tyrosinase tumor antigen presented by the class I MHC HLA-A2 in a fully functional manner. Here we find that TIL1383I binds this class I target with a highly atypical geometry. Despite unorthodox binding, TCR signaling, antigen specificity, and the ability to use CD8 are maintained. Structurally, a key feature of TIL1383I is an exceptionally long CDR3ß loop that mediates functions that are traditionally performed separately by hypervariable and germline loops in canonical TCR structures. Our findings thus expand the range of known TCR binding geometries compatible with normal function and specificity, provide insight into the determinants of MHC restriction, and may help guide TCR selection and engineering for immunotherapy.

Validation and promise of a TCR mimic antibody for cancer immunotherapy of hepatocellular carcinoma.

Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens, T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However, the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe, and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC), a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm, which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the γ and δ subunits of a TCR, producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTCR + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment, no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably, one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant.

Peptide-dependent tuning of major histocompatibility complex motional properties and the consequences for cellular immunity.

T cell receptors (TCRs) and other receptors of the immune system recognize peptides presented by class I or class II major histocompatibility complex (MHC) proteins. Although we generally distinguish between the MHC protein and its peptide, at an atomic level the two form a structural composite, which allows peptides to influence MHC properties and vice versa. One consequence is the peptide-dependent tuning of MHC structural dynamics, which contributes to protein structural adaptability and influences how receptors identify and bind targets. Peptide-dependent tuning of MHC protein dynamics can impact processes such as antigenicity, TCR cross-reactivity, and T cell repertoire selection. Motional tuning extends beyond the binding groove, influencing peptide selection and exchange, as well as interactions with other immune receptors. Here, we review recent findings showing how peptides can affect the dynamic and adaptable nature of MHC proteins. We highlight consequences for immunity and demonstrate how MHC proteins have evolved to be highly sensitive dynamic reporters, with broad immunological consequences.

Physicochemical Heuristics for Identifying High Fidelity, Near-Native Structural Models of Peptide/MHC Complexes.

There is long-standing interest in accurately modeling the structural features of peptides bound and presented by class I MHC proteins. This interest has grown with the advent of rapid genome sequencing and the prospect of personalized, peptide-based cancer vaccines, as well as the development of molecular and cellular therapeutics based on T cell receptor recognition of peptide-MHC. However, while the speed and accessibility of peptide-MHC modeling has improved substantially over the years, improvements in accuracy have been modest. Accuracy is crucial in peptide-MHC modeling, as T cell receptors are highly sensitive to peptide conformation and capturing fine details is therefore necessary for useful models. Studying nonameric peptides presented by the common class I MHC protein HLA-A*02:01, here we addressed a key question common to modern modeling efforts: from a set of models (or decoys) generated through conformational sampling, which is best? We found that the common strategy of decoy selection by lowest energy can lead to substantial errors in predicted structures. We therefore adopted a data-driven approach and trained functions capable of predicting near native decoys with exceptionally high accuracy. Although our implementation is limited to nonamer/HLA-A*02:01 complexes, our results serve as an important proof of concept from which improvements can be made and, given the significance of HLA-A*02:01 and its preference for nonameric peptides, should have immediate utility in select immunotherapeutic and other efforts for which structural information would be advantageous.

Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA.

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrently mutated driver genes. Here we describe a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant PIK3CA, among the most frequently genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this strategy, we developed a panel of TCRs that recognize an endogenously processed neopeptide encompassing a common PIK3CA hotspot mutation restricted by the prevalent human leukocyte antigen (HLA)-A*03:01 allele. Mechanistically, immunogenicity to this public NeoAg arises from enhanced neopeptide/HLA complex stability caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a comparatively 'featureless' surface dominated by the peptide's backbone. To bind this epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface facilitated by an unusually long CDR3ß loop. In patients with diverse malignancies, we observed NeoAg clonal conservation and spontaneous immunogenicity to the neoepitope. Finally, adoptive transfer of TCR-engineered T cells led to tumor regression in vivo in mice bearing PIK3CA-mutant tumors but not wild-type PIK3CA tumors. Together, these findings establish the immunogenicity and therapeutic potential of a mutant PIK3CA-derived public NeoAg.

Engineering the T cell receptor for fun and profit: Uncovering complex biology, interrogating the immune system, and targeting disease.

T cell receptors (TCRs) orchestrate cellular immunity by recognizing peptide antigens bound and presented by major histocompatibility complex (MHC) proteins. Due to the TCR's central role in immunity and tight connection with human health, there has been significant interest in modulating TCR properties through protein engineering methods. Complicating these efforts is the complexity and vast diversity of TCR-peptide/MHC interfaces, the interdependency between TCR affinity, specificity, and cross-reactivity, and the sophisticated relationships between TCR binding properties and T cell function, many aspects of which are not well understood. Here we review TCR engineering, starting with a brief historical overview followed by discussions of more recent developments, including new efforts and opportunities to engineer TCR affinity, modulate specificity, and develop novel TCR-based constructs.

A previously unappreciated polymorphism in the beta chain of I-As expressed in autoimmunity-prone SJL mice: Combined impact on antibody, CD4 T cell recognition and MHC class II dimer structural stability.

In the process of structure-function studies on the MHC class II molecule expressed in autoimmunity prone SJL mice, I-As, we discovered a disparity from the reported sequence of the MHC class II beta chain. The variant is localized at a highly conserved site of the beta chain, at residue 58. Our studies revealed that this single amino acid substitution of Pro for Ala at this residue, found in I-As, changes the structure of the MHC class II molecule, as evidenced by a loss of recognition by two monoclonal antibodies, and elements of MHC class II conformational stability identified through molecular dynamics simulation. Two other rare polymorphisms in I-As involved in hydrogen bonding potential between the alpha chain and the peptide main chain are located at the same end of the MHC class II binding pocket, studied in parallel may impact the consequences of the ß chain variant. Despite striking changes in MHC class II structure, CD4 T cell recognition of influenza-derived peptides was preserved. These disparate findings were reconciled by discovering, through monoclonal antibody blocking approaches, that CD4 T cell recognition by I-As restricted CD4 T cells focused more on the region of MHC class II at the peptide's amino terminus. These studies argue that the conformational variability or flexibility of the MHC class II molecule in that region of I-As select a CD4 T cell repertoire that deviates from the prototypical docking mode onto MHC class II peptide complexes. Overall, our results are consistent with the view that naturally occurring MHC class II molecules can possess polymorphisms that destabilize prototypical features of the MHC class II molecule but that can maintain T cell recognition of the MHC class II:peptide ligand via alternate docking modes.