Antigen presentation is a specialized function of the immune system where peptides generated from cellular or internalized proteins are presented on Major Histocompatibility Complex molecules on the surface of antigen-presenting cells for recognition by T cells. The binding of these MHC-peptide complexes to hypervariable receptors on the surface of a T cell leads to its activation. T cell activation induces increases in cell-surface activation markers such as CD69, which can be tracked by flow cytometry. Here we describe a method that uses flow cytometry and a recombinant T cell to track the presentation of an MHC-II HLA-DR1 restricted epitope generated from the influenza hemagglutinin protein.

BackgroundThe role of CD4 T cells in the control of viral infections beyond their traditional helper activity has been increasingly recognized, and CD4 T cells with cytotoxic capacity have been reported for the nearly ubiquitous betaherpesviruses HCMV (human cytomegalovirus) and HHV-6B (human herpesvirus 6B).ObjectiveWe sought to investigate the functional landscape of cytotoxic CD4 T cells responding to HHV-6B and HCMV epitopes presented by DRB1*03:01 and to identify public T-cell receptors (TCRs) (i.e., shared by multiple subjects).ApproachWe tetramer-sorted epitope-specific CD4 T cells from healthy donors and performed RNA and TCR sequencing to assess functional profiles and identify TCR clonotypes. We evaluated the publicity of the repertoire and tested the functionality, epitope specificity, and sensitivity of selected public clonotypes.ResultsDifferential gene expression analysis comparing T cells expanded with HHV-6B and HCMV epitopes showed differences in their functional profiles, with the HCMV-expanded T cells displaying a more robust cytotoxic gene expression signature. Tens to hundreds of TCR clonotypes responding to HHV-6B or HCMV were identified in each subject. The TCR repertoires were dominated by private clonotypes in all subjects, but 3 public TCRα/β, along with 41 public TCRα and TCRβ clonotypes were identified. Some of these clonotypes and closely related variants were found in a substantial fraction of DRB1*03:01 subjects in datasets of total peripheral blood TCR repertoires. TCRs associated with two HHV-6B epitopes (U11.306–323 and U85.88-104) and one HCMV epitope (pp65.509-523) were cloned for validation and biochemical characterization. Using an in vitro activation assay, the epitope specificity was confirmed for each selected TCRα/β, with half-maximal activation observed at 5–50 nM peptide concentration. With one exception, all TCRs bound tightly to the corresponding peptide-major histocompatibility complex (pMHC) tetramer. Finally, minimal peptide mapping combined with structural modeling of pMHCs identified potential sites of TCR interaction.ConclusionsCD4 T cells recognizing HHV-6B or HCMV exhibit cytotoxic signatures and can lyse antigen-pulsed target cells, with the HCMV-specific population exhibiting greater activity. The TCR repertoires of CD4 T cells recognizing HHV-6B or HCMV epitopes presented by DRB1*03:01 are broad but include public TCR clonotypes. These TCRs may be useful to monitor infection, reactivation under immunosuppressive conditions, and response to therapy.

Major histocompatibility (MHC) class II molecules can exist in two distinct conformational states based on alternative pairing of transmembrane domain GxxxG dimerization motifs (i.e., M1- and M2-paired MHC class II). M1- and M2-paired MHC class II molecules drive different levels of T cell activation and B cell signaling; consequently, differential peptide loading would impact the level of immune response elicited by various antigens/epitopes. In previous studies of a single model antigen, we show that while peptide from BCR-bound antigen is selectively loaded onto M1-paired I-Ak class II, peptide from fluid phase processing of the same antigen is loaded onto both M1- and M2-paired I-Ak. To expand this analysis, we determined the immunnopeptidomes of M1-paired vs. total I-Ak class II molecules isolated from murine B cells. By comparing the two immunopeptidomes as well as the source proteins (antigens), a picture emerges highlighting the unique access each class II conformer has to antigens from different subcellular compartments. Sequence analysis of the two immunopeptidomes suggests a high degree of similarity between the peptide binding grooves of the two class II conformers. Analysis of class II-associated invariant chain (Ii)-derived peptides reveals the robust presence of a nested set of non-CLIP peptides that associate primarily with M1-paired class II, likely outside of the canonical peptide binding groove. In total, these results further highlight the differential peptide loading of M1- vs. M2-paired MHC class II molecules and support the idea that differential peptide loading could impact overall immune responsiveness.

Type 1 diabetes (T1D) arises from T cell-mediated destruction of insulin-secreting pancreatic β cells. Inflammatory triggers have been hypothesized to induce presentation of new epitopes for pathogenic T cells, but the naturally processed MHC-bound peptides presented by primary human islet β cells are largely unknown. We used mass spectrometry to identify native and post-translationally modified self-peptides presented by MHC proteins from human cadaveric islet samples treated in vitro with cytokines to identify epitopes in an inflamed pancreas. Of >4,300 islet peptides presented by 60 different MHC molecules, we identified 28 autoimmune epitopes targeted by T cells from patients with T1D, 31 additional epitopes from previously identified autoantigens, and 100 additional candidate autoantigens. The epitopes derive from inflammation, unfolded protein response, and secretory hormone processing pathways. These results identify naturally processed islet peptides targeted by autoimmune T cells in T1D and provide a resource for investigating T1D etiology and progression.

This proceedings article summarizes the inaugural "T Cells in the Brain" symposium held at Columbia University. Experts gathered to explore the role of T cells in neurodegenerative diseases. Key topics included characterization of antigen-specific immune responses, T cell receptor (TCR) repertoire, microbial etiology in Alzheimer's disease (AD), and microglia-T cell crosstalk, with a focus on how T cells affect neuroinflammation and AD biomarkers like amyloid beta and tau. The symposium also examined immunotherapies for AD, including the Valacyclovir Treatment of Alzheimer's Disease (VALAD) trial, and two clinical trials leveraging regulatory T cell approaches for multiple sclerosis and amyotrophic lateral sclerosis therapy. Additionally, single-cell RNA/TCR sequencing of T cells and other immune cells provided insights into immune dynamics in neurodegenerative diseases. This article highlights key findings from the symposium and outlines future research directions to further understand the role of T cells in neurodegeneration, offering innovative therapeutic approaches for AD and other neurodegenerative diseases. HIGHLIGHTS: Researchers gathered to discuss approaches to study T cells in brain disorders. New technologies allow high-throughput screening of antigen-specific T cells. Microbial infections can precede several serious and chronic neurological diseases. Central and peripheral T cell responses shape neurological disease pathology. Immunotherapy can induce regulatory T cell responses in neuroinflammatory disorders.

Abstract Molecular mimicry is the immunological mechanism by which pathogen-derived peptides can modulate immune responses due to sequence similarity with self-peptides. Herein, we report molecular mimicry associated with pathogen immune evasion. A quantitative analysis of the immunopeptidome presented by in vivo mycobacteria tuberculosis (MTB)-matured dendritic cells in HLA-DR0101 (DR1) transgenic mice identified an MTB-processed peptide which presented immunosuppressive capability and shared a DR1 core sequence with a human and mouse self-protein. The TB peptide could expand nTreg with generation of immunosuppressive cytokines in DR1 mice and DR1 subjects diagnosed with active or latent TB. In TB-infected patients the tolerogenic TB peptide reduced the inflammatory response generated to a series of immunogenic TB peptides as quantified by the QuantiFERON test. Additionally, the tolerogenic TB peptide presented an MHC-binding core sequence shared by a multitude of pathogens. Our analysis characterizes a pathogen-derived peptide which, through molecular mimicry facilitate immune evasion by activating/inducing nTreg.

IntroductionHuman Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined.MethodsWe set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches.ResultsWe demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 – ULBP1 complex indicates some similarities to the m152-RAE1γ complex.

The lymphatic fluid is the conduit by which part of the tissue “omics” is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics, herein we investigate the lymph proteomic composition, uncovering that its composition varies according to the tissue of origin. Tissue specificity is also reflected in the dendritic cell-major histocompatibility complex class II-eluted immunopeptidome harvested from the cervical and mesenteric nodes. Following inflammatory disruption of the gut barrier, the lymph antigenic and inflammatory loads are analyzed in both mice and subjects with inflammatory bowel diseases. Gastrointestinal tissue damage reflects the lymph inflammatory and damage-associated molecular pattern signatures, microbiome-derived by-products, and immunomodulatory molecules, including metabolites of the gut-brain axis, mapped in the afferent mesenteric lymph. Our data point to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance.

The ability of mammals to mount adaptive immune responses culminating with the establishment of immunological memory is predicated on the ability of the mature T cell repertoire to recognize antigenic peptides presented by syngeneic MHC class I and II molecules. Although it is widely believed that mature T cells are highly skewed towards the recognition of antigenic peptides originating from genetically diverse (for example, foreign or mutated) protein-coding regions, preclinical and clinical data rather demonstrate that novel antigenic determinants efficiently recognized by mature T cells can emerge from a variety of non-mutational mechanisms. In this Review, we describe various mechanisms that underlie the formation of bona fide non-mutational neoantigens, such as epitope mimicry, upregulation of cryptic epitopes, usage of non-canonical initiation codons, alternative RNA splicing, and defective ribosomal RNA processing, as well as both enzymatic and non-enzymatic post-translational protein modifications. Moreover, we discuss the implications of the immune recognition of non-mutational neoantigens for human disease.

Major histocompatibility (MHC) bound viral peptides are the immune signatures to initiate the selective and cross-reactive T cell responses. Identifying the antigenic targets of adaptive immunity to SARS-CoV-2 is a key towards understanding the pathogenesis of Sars-CoV-2 and the rapidly evolving mutants. We isolated the immunopeptidomes to identify naturally processed and presented MHC-II (HLA-DR, HLA-DP) and MHC-I (HLA-ABC) bound canonical and out-of-frame viral peptides. We identified 5 HLA-DR canonical epitopes (13 peptides) from spike protein, 2 HLA-DP canonical epitopes (4 peptides) from spike and membrane proteins, 1 canonical MHC-Class I peptides and 5 out-of-frame MHC-I peptides from different ORF proteins. The viral peptides were presented in lower abundances as compared to host peptides obscuring the identification of the MHC bound viral peptides. Some of these peptides were shown to recall T cell responses in COVID-19 donors in other reported studies. We observed the downregulation of surface and total HLA-ABC, HLA-DR and HLA-DP expression upon Sars-CoV-2 infection possibly restricting the presentation of viral peptides and therefore delaying the immune response. The mechanisms by which SARS-CoV-2 evades adaptive immune responses mediated by MHC-I are studied in detail by different groups. Hence, we further investigating the modulators of the MHC-II pathway using proteomics-based approach. We observed significant downregulation of CD74 protein, CIITA protein and cathepsins in infected cells affecting the processing and presentation of viral peptides. These results may help in understanding significantly altered antigen presentation in Sars-CoV-2 infected cells.

Seasonal “common-cold” human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but the T-cell response to seasonal coronaviruses remains largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal coronavirus OC43. We identified MHC-bound peptides derived from each of the viral structural proteins (spike, nucleoprotein, hemagglutinin-esterase, membrane, and envelope) as well as non-structural proteins nsp3, nsp5, nsp6, and nsp12. Eighty MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. Fewer and less abundant MHC-I bound OC43-derived peptides were observed, possibly due to MHC-I downregulation induced by OC43 infection. The MHC-II peptides elicited low-abundance recall T-cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T-cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T-cell lines. Among the validated epitopes, spike protein S 903-917 presented by DPA1*01:03/DPB1*04:01 and S 1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. Nucleoprotein N 54-68 and hemagglutinin-esterase HE 128-142 presented by DRB1*15:01 and HE 259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow CD4 T-cell cross-reactivity after infection or vaccination, and to guide selection of epitopes for inclusion in pan-coronavirus vaccines.

The ability to understand and characterize phosphorylation is important to the study of cell signaling and to synthetic biology approaches. Current methods for characterizing kinase-substrate interactions are limited by their inherently low throughput and the heterogeneity of samples analyzed. Recent advances in yeast surface display techniques provide new opportunities for studying individual kinase-substrate interactions in a stimulus-independent fashion. Here, we describe techniques for building substrate libraries into full-length domains of interest that, when co-localized intracellularly with individual kinases, result in the display of phosphorylated domains on the yeast surface, as well as fluorescence-activated cell sorting and magnetic bead selection techniques for enriching from these libraries based on phosphorylation state.

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumor microenvironments. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies, which are being harnessed to improve solid tumor CAR T cell therapies. Here, we describe fully optimized CAR T cells targeting tumor-associated glycoprotein-72 (TAG72) for the treatment of solid tumors, identifying the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. These findings have culminated into a phase 1 trial evaluating safety, feasibility, and bioactivity of TAG72-CAR T cells for the treatment of patients with advanced ovarian cancer ( NCT05225363 ). Preclinically, we found that CAR T cell-mediated IFNγ production facilitated by IL-12 signaling was required for tumor cell killing, which was recapitulated by expressing an optimized membrane-bound IL-12 (mbIL12) molecule on CAR T cells. Critically, mbIL12 cell surface expression and downstream signaling was induced and sustained only following CAR T cell activation. CAR T cells with mbIL12 demonstrated improved antigen-dependent T cell proliferation and potent cytotoxicity in recursive tumor cell killing assays in vitro and showed robust in vivo anti-tumor efficacy in human xenograft models of ovarian cancer peritoneal metastasis. Further, locoregional administration of TAG72-CAR T cells with antigen-dependent IL-12 signaling promoted durable anti-tumor responses against both regional and systemic disease in mice and was associated with improved systemic T cell persistence. Our study features a clinically-applicable strategy to improve the overall efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting both regional and systemic disease.

Background Chimeric antigen receptor (CAR) T cell therapy has demonstrated marked success in the control of hematological malignancies. However, CAR T cell therapy has been less successful when applied to solid tumors. This has been attributed to multiple factors including tumor infiltration, antigen heterogenicity, and the immunosuppressive tumor microenvironment. The hostile milieu and chronic exposure to antigen that CAR T cells face in the tumor microenvironment induces T cell exhaustion. Characterized by an increased expression of inhibitory receptors, reduced proliferation, and a loss of cytotoxic function, T cell exhaustion is associated with an overall loss of therapeutic efficacy. Methods Strategies for overcoming T cell exhaustion are being developed to improve CAR T cell efficacy in the treatment of solid tumors. One strategy focuses on incorporating the expression of survival-promoting proteins, such as cytokines, in CAR T cells to produce a local immune supportive environment. Interleukin-2 (IL-2) is a cytokine that acts as a potent growth factor in T cells; however, therapeutic application of systemic IL-2 has been associated with severe toxicities and T cell exhaustion. To address this, IL-2 partial agonists have been developed and shown to have reduced STAT5 signaling and has been shown to promote the maintenance of a stem-like phenotype in effector T cells.1 We have bound IL-2 and IL-2 partial agonists to the cell surface membrane of CAR T cells with a variety of tethers. As IL-2 is a secreted protein that has known cis and trans signaling capabilities, tethering the cytokines to the surface of the CAR T cells has the potential to maximize autocrine signaling and prevent side-effects. Results Here, we evaluate the influence of partial agonist and tether composition on the proliferation, exhaustion state, and cytotoxic function of CAR T cells. CAR T cells expressing the membrane tethered IL-2 and IL-2 partial agonists had reduced reliance on exogenously delivered IL-2 and CAR T cells expressing the membrane tethered IL-2 partial agonist demonstrated improved cytotoxic function. Conclusions Exhaustion of antigen-specific T cells is a challenge in multiple cancer immunotherapy modalities, and the proposed membrane tethered IL2 partial agonists have the potential to revolutionize immunotherapy for solid tumors as an effective and widely applicable solution. Reference Mo F, Yu Z, Li P, Oh J, Spolski R, Zhao L, Glassman CR, Yamamoto TN, Chen Y, Golebiowski FM, Hermans D, Majri-Morrison S, Picton LK, Liao W, Ren M, Zhuang X, Mitra S, Lin JX, Gattinoni L, Powell JD, Restifo NP, Garcia KC, Leonard WJ. An engineered IL-2 partial agonist promotes CD8+ T cell stemness. Nature. 2021 Sep;597(7877):544–548. doi: 10.1038/s41586–021-03861–0. Epub 2021 Sep 15. PMID: 34526724; PMCID: PMC9172917.