Public T-cell Receptor Research Articles

Invariant VD and DJ Motifs Define a Novel Class of Human Antibodies and TCRs Prototyped by antigen receptors of Dual-Expresser Lymphocytes

ABSTRACT Introduction Dual-expressing lymphocytes (DEs) are unique immune cells that express both B cell receptors (BCRs, surface antibody) and T cell receptors (TCRs). In type 1 diabetes, DE antibodies are predominated by one antibody (x-mAb), an IgM monoclonal antibody with a germline-encoded CDR3 that recognizes self-reactive TCRs. We explored if x-mAb and its interacting TCRs have distinct structural features. Methods Using bioinformatics, we compared x-mAb and its most common interacting TCRαβ to billions of antigen receptor sequences to determine if they were unique or randomly generated. Results X-mAb represents a unique class of human antibodies with a conserved CDR3 sequence (CARx1-4DTAMVYYFYDW), consisting of a fixed DJH motif (DTAMVYYFDYW) paired with various VH genes. A public TCRβ clonotype (CASSPGTEAFF) associated with x-mAb on DEs features two invariant segments, VβD (CASSPGT) and DJβ (PGTEAFF), key to two large families of public TCRβ clonotypes—CASSPGT-Jβx and CASSPGT-Jβx—formed by recombining the VβD motif with Jβ genes and the DJβ motif with Vβ genes. B cells also use CASSPGT as a VHD motif for public IGH clonotypes (CASSPGT—Jβx). Discussion DEs, unlike conventional T and B cells, use invariant motifs to create public antibodies and TCRs, a trait previously seen only in cartilaginous fish.

Abstract 1170: An off-the-shelf vaccine activates mutant KRAS-specific T cells in patients with resected pancreatic cancer

Abstract Background: Oncogenic mutations in KRAS (mKRAS) are expressed in up to 90% of pancreatic ductal adenocarcinomas (PDAC) therefore representing a promising immunotherapeutic target. We developed a pooled mKRAS peptide vaccine targeting the 6 most common mutations in PDAC: G12V, G12A, G12C, G12R, G12D, or G13D (NCT04117087). In a phase I study, we evaluated mKRAS-specific T cell responses raised by the vaccine when given in combination with immune checkpoint inhibitors to PDAC patients following resection and adjuvant chemotherapy. Materials and Methods: Patients received the pooled mKRAS peptide vaccine (0.3mg/peptide and 0.5mg poly-ICLC weekly for 4 doses in combination with ipilimumab and nivolumab followed by boosters every 8 weeks with nivolumab. To detect vaccine-induced mKRAS-specific T cells, pre- and post-vaccine peripheral blood mononuclear cells (PBMCs) were restimulated with control or individual mKRAS peptides and IFNγ release was measured by ELISPOT. mKRAS-specific T cell activation, proliferation, memory, and exhaustion profiles were assessed by CyTOF and cytokine secretion was determined by ELISA. For a subset of patients, mKRAS-specific T cells were expanded in vitro and T cell receptor (TCR) β chain sequencing and scRNA/TCRseq were used to identify the KRAS mutation-specific TCR repertoires and define their phenotypes in circulation. Results: mKRAS-specific T cells were detected by IFNγ ELISPOT post-vaccination for all patients, although magnitude and mutation-specific responses varied between patients. CyTOF analysis identified mKRAS-specific Th1 CD4+ central memory (cm, CCR7+IFNγ+) and effector memory (em, CCR7-IFNγ +IL2+ TNFɑ+) responses as well as CD8+ effector (eff, CD137+GZMBhiKi67hi) and effector memory (CCR7-CD137+GZMB+) responses. mKRAS-specific T cells were also detectable in the extended booster phase of treatment (>1 year post initial vaccination). In vitro expanded mKRAS-specific T cells mapped to CD4+ Tcm/em and CD8+ Tem populations within single cell datasets of unstimulated PBMCs collected post-vaccine. 5-25% of the mKRAS-specific T cells significantly expanded to more than one mKRAS antigen suggesting potential cross-reactive T cell clonotypes. In addition, shared public mKRAS-specific T cell clonotypes were identified across patients. Conclusions: This study demonstrates mKRAS vaccine induction of de novo, high quality mKRAS-specific polyfunctional CD4+ and CD8+ T cells. mKRAS-specific TCR analysis identified shared public TCRs across vaccinated patients that have important implications for future “off the shelf” adoptive therapy approaches. Ongoing studies are aimed to validate antigen specificity and map HLA-restriction of mKRAS-specific T cells. Overall, this study demonstrates a positive immunogenic signal of this pooled mutant KRAS vaccine that may be amenable for interception vaccination strategies against PDAC. Citation Format: Amanda L. Huff, Alex Girgis, Saurav D. Haldar, Emily Davis-Marcisak, Thatcher Heumann, Gabriella Longway, Lalitya Andaloori, Alexei Hernandez, Maximilian F. Konig, Brian Mog, Ludmila Danilova, Luciane T. Kagohara, Julie M. Nauroth, Amy M. Thomas, Elana J. Fertig, Won Jin Ho, Elizabeth M. Jaffee, Nilofer Azad, Neeha Zaidi. An off-the-shelf vaccine activates mutant KRAS-specific T cells in patients with resected pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1170.

Colonic mucosal associated invariant T cells in Crohn's disease have a diverse and non-public T cell receptor beta chain repertoire.

Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD). The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin α4β7 in the colon but not in the blood, where they were enriched for α4β7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4β7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.

Single Cell Sequencing Suggests Mucosal Origins of T Cells in HLA-B27-associated Inflammation

Abstract HLA-B27 was one of the first Major Histocompatibility Complex alleles associated with an autoimmune disease, i.e., Ankylosing Spondylitis (AS) and acute anterior uveitis (B27AU), which cause joint and eye inflammation, respectively. As HLA-B27 presents antigens to CD8 T cells, an antigen-specific CD8 T cell response may be central to disease pathogenesis. Consistent with this hypothesis, our recent work identified clonal expansion of public T cell receptors (TCRs) shared across patients with AS and B27AU and enriched in the joint or eye over the blood. These TCRs recognized both bacterial and self-peptides, suggesting a role for molecular mimicry in multiple organs afflicted by HLA-B27-associated inflammation. The main bacterial peptide recognized by these public TCRs is derived from yeiH, a gene present in multiple enteric microbiota. It remains unclear, however, whether these T cells have had a prior antigenic encounter within the gastrointestinal tract. We therefore performed single-cell RNA sequencing (scRNAseq) and T cell receptor sequencing (scTCRseq) on immune cells from the eye and blood during B27AU (n=5) and non-B27AU (n=14). In this analysis, we found that the expanded TCRs in B27AU were associated with CD161 expression and enriched for a transcriptional program from intestinal CD8 T cells. Moreover, we were able to identify an additional expanded TCR sequence that also recognized YEIH. Finally, expanded T cell clones contained higher expression of TNFand IFNG, suggestive of a pro-inflammatory transcriptional program. Collectively, our data suggest that, in HLA-B27-associated inflammation, the early antigen exposure and differentiation of putative pathogenic CD8 T cells may have occurred in enteric organs. This study received support from NIH 5R01AI103867 (KCG), U19AI057229 (KCG), the Howard Hughes Medical Institute (KCG), the Rheumatology Research Foundation (MAP), Arthritis National Research Foundation (MAP), the Rheumatic Diseases Research Resource-based Center at Washington University (NIH P30-AR073752, MAP, WMY), the Bursky Center for Human Immunology and Immunotherapy Programs (WMY), and the Barnes-Jewish Hospital Foundation (WMY).

CD4+ T cell-mediated recognition of a conserved cholesterol-dependent cytolysin epitope generates broad antibacterial immunity

CD4+ Tcell-mediated immunity against Streptococcus pneumoniae (pneumococcus) can protect against recurrent bacterial colonization and invasive pneumococcal diseases (IPDs). Although such immune responses are common, the pertinent antigens have remained elusive. We identified an immunodominant CD4+ Tcell epitope derived from pneumolysin (Ply), a member of the bacterial cholesterol-dependent cytolysins (CDCs). This epitope was broadly immunogenic as a consequence of presentation by the pervasive human leukocyte antigen (HLA) allotypes DPB1∗02 and DPB1∗04 and recognition via architecturally diverse Tcell receptors (TCRs). Moreover, the immunogenicity of Ply427-444 was underpinned by core residues in the conserved undecapeptide region (ECTGLAWEWWR), enabling cross-recognition of heterologous bacterial pathogens expressing CDCs. Molecular studies further showed that HLA-DP4-Ply427-441 was engaged similarly by private and public TCRs. Collectively, these findings reveal the mechanistic determinants of near-global immune focusing on a trans-phyla bacterial epitope, which could inform ancillary strategies to combat various life-threatening infectious diseases, including IPDs.

Comprehensive analysis of T-cell receptor repertoires reveals antigen-driven T-cell clusters in patients with Behçet's syndrome.

T lymphocytes are the major components of adaptive immunity in Behçet's syndrome (BS) pathology. However, the precise mechanism of T-cell-induced inflammatory condition remains to be determined. We applied bulk sequencing of the T-cell receptor (TCR) β chain in peripheral blood samples from 45 patients with BS and 10 healthy donors as controls. TCR repertoires in BS patients displayed more clonality and less diversity than in healthy donors. Male patients exhibited lower diversity metrics of TCR and had a larger proportion in the top 10 clones than females (p = 0.016). There were no TCR clonality differences in other clinical features, such as age, disease duration, organ involvement, disease severity, and activity. By "Grouping of Lymphocyte Interactions by Paratope Hotspots" (GLIPH2) for antigen prediction, we found distinct 2477 clusters of TCR-β sequences that potentially recognize similar antigens shared between BS patients. We observed clonal T-cell expansion in BS patients. Sexual differences in TCR clonal expansion and public TCR groups deserve further study to reveal the underline T-cell-mediated immunity in BS.

SARS-CoV-2-specific CD4+ and CD8+ T cell responses can originate from cross-reactive CMV-specific T cells.

Detection of SARS-coronavirus-2 (SARS-CoV-2) specific CD4+ and CD8+ T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relative high frequency and prevalence of cross-reactive T cells, we hypothesized CMV may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved PBMCs with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+ and spike-specific CD8+ T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+ T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+ T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2.

Sars-Cov-2-Specific CD4+ and CD8+ T Cell Responses Can Originate from Cross-Reactive CMV-Specific T Cells

Introduction: SARS-CoV-2-specific CD4+ and CD8+ T cells play an important role in COVID-19 pathogenesis. In addition to de novo SARS-CoV-2-specific T cell responses in infected individuals, SARS-CoV-2-specific T cells have also been identified in samples frozen down before the pandemic. So far, pre-existing SARS-CoV-2 specific T cell responses has mostly been ascribed to cross-reactive T cells that were initially primed by other coronaviruses. The presence of these cross-reactive T cells is not surprising considering these human coronaviruses share a relatively high sequence homology. However, T cells are also able to cross-react towards dissimilar pathogens, called heterologous immunity. Given the high prevalence and magnitudes of pre-pandemic SARS-CoV-2-specific T cells, we hypothesized that pre-pandemic SARS-CoV-2 T cell immunity could be explained by cross-reactive cytomegalovirus (CMV)-specific T cells. Methods & Results: PBMCs from CMV seropositive and seronegative donors that were cryopreserved before the pandemic were incubated with SARS-CoV-2 peptide pools. To detect SARS-CoV-2-specific T cells using flow cytometry, CD4+ T cell responses were identified by upregulation of CD154 and CD137 whereas CD8+ T cell responses were identified by upregulation of CD137 and IFN-γ production. In the CMV seropositive group, clear CD4+ T cell responses against SARS-CoV-2 membrane and CD8+ T cell responses against SARS-CoV-2 spike were detected whilst this was not observed in CMV seronegative donors. To investigate whether these T cells were reactive against both SARS-CoV-2 and CMV, the SARS-CoV-2 reactive T cells were single cell sorted, clonally expanded, and tested in co-culture assays in which stimulator cells expressing the relevant human leukocyte antigen (HLA) alleles were pulsed with overlapping peptide pools, peptide libraries or single peptides. The results demonstrated that the pre-pandemic SARS-CoV-2 membrane-specific CD4+ T cells recognized both SARS-CoV-2 membrane and CMV-pp65 peptide pools in HLA-DRB3*02:02, and specifically recognized AGILRNLVPM peptide of CMV-pp65. The pre-pandemic SARS-CoV-2 spike-specific CD8+ T cells recognized spike peptide FVSNGTHWF (FVS) and, interestingly, dissimilar CMV-pp65 peptide IPSINVHHY (IPS) both presented by HLA-B*35:01. Importantly, these FVS/IPS-cross-reactive T cells displayed a higher avidity for CMV compared to SARS-CoV-2, which illustrates that these cells were originally primed against CMV since repeated antigen exposure results in a selection for high avidity T cell clonotypes. This selection cannot have occurred in response to SARS-CoV-2-specific peptides in SARS-CoV-2-unexposed individuals. PBMCs from pre-pandemic CMV-seropositive donors that express HLA-B*35:01 were screened for the presence of these dual IPS/FVS-specific CD8+ T cells by peptide-HLA tetramer technology. In multiple donors these cross-reactive T cells were detected, and isolation followed by direct T cell receptor (TCR) sequencing of the IPS/FVS-tetramer positive cells showed that these T cells express an amino acid-identical TCR, illustrating that this SARS-CoV-2/CMV cross-reactivity of the IPS/FVS T cells is caused by a public TCR. To investigate the potential of the IPS/FVS-specific T cells to kill virus-infected cells, and thereby reducing spreading of SARS-CoV-2, the T cells were incubated with SARS-CoV-2-infected airway epithelial Calu-3 cells. Intracellular SARS-CoV-2 RNA was reduced at low virus titers in IPS/FVS-specific T cell co-cultures, indicating that the cross-reactive T cells are killing SARS-CoV-2-infected cells and thereby reduce spreading of SARS-CoV-2 during initial infection. Circulating IPS/FVS-specific T cells were not phenotypically activated in two patients with advanced COVID-19, as measured by flow cytometry analysis of CD38 and HLA-DR expression, indicating that cross-reactive T cells may more likely play a role during early stage of infection. Conclusion: CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-pandemic immunity against SARS-CoV-2. Pre-print link: https://www.biorxiv.org/content/10.1101/2022.07.31.502203v1

Exploration of shared features of B cell receptor and T cell receptor repertoires reveals distinct clonotype clusters.

Although B cells and T cells are integral players of the adaptive immune system and act in co-dependent ways to orchestrate immune responses, existing methods to study the immune repertoire have largely focused on separate analyses of B cell receptor (BCR) and T cell receptor (TCR) repertoires. Based on our hypothesis that the shared history of immune exposures and the shared cellular machinery for recombination result in similarities between BCR and TCR repertoires in an individual, we examine any commonalities and interrelationships between BCR and TCR repertoires. We find that the BCR and TCR repertoires have covarying clonal architecture and diversity, and that the pattern of correlations appears to be altered in immune-mediated diseases. Furthermore, hierarchical clustering of public B and T cell clonotypes in both health and disease based on correlation of clonal proportion revealed distinct clusters of B and T cell clonotypes that exhibit increased sequence similarity, share motifs, and have distinct amino acid characteristics. Our findings point to common principles governing memory formation, recombination, and clonal expansion to antigens in B and T cells within an individual. A significant proportion of public BCR and TCR repertoire can be clustered into nonoverlapping and correlated clusters, suggesting a novel way of grouping B and T cell clonotypes.

Amino acids at position 5 in the peptide/MHC binding region of a public virus-specific TCR are completely inter-changeable without loss of function.

Anti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus-specific T-cell populations in different individuals are known as public TCRs, but also TCRs highly-similar to these public TCRs, with only minor variations in amino acids on specific positions in the Complementary Determining Regions (CDRs), are frequently found. However, the degree of freedom at these positions was not clear. In this study, we used the HLA-A*02:01-restricted EBV-LMP2FLY -specific public TCR as model and modified the highly-variable position 5 of the CDR3β sequence with all 20 amino acids. Our results demonstrate that amino acids at this particular position in the CDR3β region of this TCR are completely inter-changeable, without loss of TCR function. We show that the inability to find certain variants in individuals is explained by their lower recombination probability rather than by steric hindrance.

Public T-Cell Receptors (TCRs) Revisited by Analysis of the Magnitude of Identical and Highly-Similar TCRs in Virus-Specific T-Cell Repertoires of Healthy Individuals

Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 epitopes of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation.

Proteomic Profiling and T Cell Receptor Usage of Abacavir Susceptible Subjects

Type B adverse drug reactions (ADRs) represent a significant threat as their occurrence arises unpredictable and despite proper application of the drug. The severe immune reaction Abacavir Hypersensitivity Syndrome (AHS) that arises in HIV+ patients treated with the antiretroviral drug Abacavir (ABC) strongly correlates to the presence of the human leukocyte antigen (HLA) genotype HLA-B*57:01 and discriminates HLA-B*57:01+ HIV+ patients from ABC treatment. However, not all HLA-B*57:01+ HIV+ patients are affected by AHS, implying the involvement of further patient-specific factors in the development of AHS. The establishment of a reliable assay to classify HLA-B*57:01 carriers as ABC sensitive or ABC tolerant allowed to investigate the T cell receptor (TCR) Vβ chain repertoire of effector cells and revealed Vβ6 and Vβ24 as potential public TCRs in ABC sensitive HLA-B*57:01 carriers. Furthermore, distinct effects of ABC on the cellular proteome of ABC sensitive and tolerant volunteers were observed and suggest enhanced activation and maturation of dentritic cells (DC) in ABC sensitive volunteers. Analysis of ABC-naïve cellular proteomes identified the T cell immune regulator 1 (TCIRG1) as a potential prognostic biomarker for ABC susceptibility and the involvement of significantly upregulated proteins, particularly in peptide processing, antigen presentation, interferon (IFN), and cytokine regulation.

DOP47 Identification and characterization of T-cell receptor sequences associated with Crohn’s Disease

Abstract Background T cells, as part of the adaptive immune system, are a significant driver of inflammation in Crohn’s disease (CD), yet specific T-cell targets are largely unknown. Genetic factors contribute to a small portion of CD risk including several HLA alleles, such as DRB1*07:01 and HLA-DRB1*01:03, associated with CD. The involvement of HLAs suggests that studying specific T cells could lead to new insights into CD development and progression. In this study, we use established immunoSEQ® technology to profile T-cell receptors (TCRs) and identify TCRs associated with CD and CD characteristics. Methods We analyzed TCRs from blood of 1,738 CD cases and 4,970 healthy controls. TCRs that were statistically enriched in cases, but not healthy controls (p <0.001), were termed Enhanced Sequences (ES) associated with CD. An independent cohort of 434 CD cases was used for validation. We inferred associations between the ES and 145 common HLA alleles using data from a separate, HLA-typed dataset. We defined ES clusters by correlating TCRs with single amino acid substitutions. Results We identified 1,121 CD-associated ES in the exploratory cohort. These were also enriched in CD cases in our validation cohort (Fig 1A). Using intestinal tissue samples from a subset of cases, we found that a median of 14% ES from individual cases were shared between blood and tissue samples (Fig. 1B). ES breadth (ES diversity relative to total TCR diversity) was significantly associated with history of CD-related surgery (Fig 1C, p < 1x10-15), with stricturing or fistulizing phenotypes (Fig 1D, p < 1x10-5 for B1 versus B2 or B3), and with ileal or ileocolonic location (Fig 1E, p < 1x10-7 for L2 versus L1 or L3). We found that 202 ES formed clusters of similar sequences consisting of 2–23 members (Fig. 2A). We confidently (p < 0.0001) associated 398 ES to a specific HLA allele (Fig 2B), including 134 of the ES assigned to clusters (Fig 2A). Some clusters, including the largest, had no members that could be assigned to an HLA allele, raising the possibility that these ES clusters bind non-canonical HLAs. Conclusion Our discovery set of public TCRs associated with CD indicates that the immune system of CD patients responds to a consistent set of antigens. Importantly, CD ES were present in both tissue and blood, demonstrating that evaluating TCRs in blood may be a surrogate of TCRs in tissue. The HLA allele associations of these ES potentially point to new risk factors and disease insights, such as the involvement of DP and DQ alleles. The association of ES frequency with CD characteristics strongly suggested that further examination of these TCRs may impact CD patient care and advance understanding of the pathophysiology of the disease.

126. Magnitude and Dynamics of the T-Cell Response to SARS-CoV-2 Infection and Vaccination

BackgroundT cells are central to the early identification and clearance of viral infections and support antibody generation by B cells, making them desirable for assessing the immune response to SARS-CoV-2 infection and vaccines. We combined 2 high-throughput immune profiling methods to create a quantitative picture of the SARS-CoV-2 T-cell response that is highly sensitive, durable, diagnostic, and discriminatory between natural infection and vaccination.MethodsWe deeply characterized 116 convalescent COVID-19 subjects by experimentally mapping CD8 and CD4 T-cell responses via antigen stimulation to 545 Human Leukocyte Antigen (HLA) class I and 284 class II viral peptides. We also performed T-cell receptor (TCR) repertoire sequencing on 1815 samples from 1521 PCR-confirmed SARS-CoV-2 cases and 3500 controls to identify shared public TCRs from SARS-CoV-2-associated CD8 and CD4 T cells. Combining these approaches with additional samples from vaccinated individuals, we characterized the response to natural infection as well as vaccination by separating responses to spike protein from other viral targets.ResultsWe find that T-cell responses are often driven by a few immunodominant, HLA-restricted epitopes. As expected, the SARS-CoV-2 T-cell response peaks about 1-2 weeks after infection and is detectable at least several months after recovery. Applying these data, we trained a classifier to diagnose past SARS-CoV-2 infection based solely on TCR sequencing from blood samples and observed, at 99.8% specificity, high sensitivity soon after diagnosis (Day 3–7 = 85.1%; Day 8–14 = 94.8%) that persists after recovery (Day 29+/convalescent = 95.4%). Finally, by evaluating TCRs binding epitopes targeting all non-spike SARS-CoV-2 proteins, we were able to separate natural infection from vaccination with > 99% specificity.ConclusionTCR repertoire sequencing from whole blood reliably measures the adaptive immune response to SARS-CoV-2 soon after viral antigenic exposure (before antibodies are typically detectable) as well as at later time points, and distinguishes post-infection vs. vaccine immune responses with high specificity. This approach to characterizing the cellular immune response has applications in clinical diagnostics as well as vaccine development and monitoring.Disclosures Thomas M. Snyder, PhD, Adaptive Biotechnologies (Employee, Shareholder) Rachel M. Gittelman, PhD, Adaptive Biotechnologies (Employee, Shareholder) Mark Klinger, PhD, Adaptive Biotechnologies (Employee, Shareholder) Damon H. May, PhD, Adaptive Biotechnologies (Employee, Shareholder) Edward J. Osborne, PhD, Adaptive Biotechnologies (Employee, Shareholder) Ruth Taniguchi, PhD, Adaptive Biotechnologies (Employee, Shareholder) H. Jabran Zahid, PhD, Microsoft Research (Employee, Shareholder) Rebecca Elyanow, PhD, Adaptive Biotechnologies (Employee, Shareholder) Sudeb C. Dalai, MD, PhD, Adaptive Biotechnologies (Employee, Shareholder) Ian M. Kaplan, PhD, Adaptive Biotechnologies (Employee, Shareholder) Jennifer N. Dines, MD, Adaptive Biotechnologies (Employee, Shareholder) Matthew T. Noakes, PhD, Adaptive Biotechnologies (Employee, Shareholder) Ravi Pandya, PhD, Microsoft Research (Employee, Shareholder) Lance Baldo, MD, Adaptive Biotechnologies (Employee, Shareholder, Leadership Interest) James R. Heath, PhD, Merck (Research Grant or Support, Funding (from BARDA) for the ISB INCOV project, but had no role in planning the research or in writing the paper.) Joaquin Martinez-Lopez, MD, PhD, Adaptive Biotechnologies (Consultant) Jonathan M. Carlson, PhD, Microsoft Research (Employee, Shareholder) Harlan S. Robins, PhD, Adaptive Biotechnologies (Board Member, Employee, Shareholder)

Abstract LBA006: Identification of novel, tumor specific pHLA targets recognized by TILs from CPI responders in a high-throughput, high-diversity pHLA library screening platform (3T-TRACE)

Abstract Background The ideal cancer target antigen is selectively expressed on neoplastic cells, absent on normal tissues, and contributes to the survival of cancer cells. Recent clinical trial reports identified T cell receptors (TCRs) recognizing intracellular targets presented as peptides in the context of HLA (pHLA) molecules as the critical molecular mediators of deep and long-lasting anti-tumor responses in a few solid tumor patients with complete responses to checkpoint inhibitor (CPI) treatment. The pHLA targets recognized by these TCRs may thus represent the long sought-after, ideal targets for solid tumor targeting with high-potency therapeutic modalities. Here we describe an immune-response guided target identification platform (3T-TRACE) and its utility to identify novel, intracellular targets, and their corresponding T cell receptors (TCRs) for the treatment of solid tumors. We provide an overview of the frequency and expression levels of the most promising, novel cancer testis antigens (CTAs) and tumor associated antigens (TAAs) identified. Methods By employing single cell sequencing methods, we identified TCRs that are uniquely associated with CPI responses and generated recombinant TCRs for screening against yeast display libraries expressing synthetic peptides in the context of the matching HLA complex, reviewed in (Figure 1). We analyzed over 140,000 CD8/CD4+ TILs from over 90 patient tumor biopsies and/or peripheral blood samples, including colorectal, lung, breast, ovarian, melanoma, and renal cancer (Figure 2). High value TCRs were selected based on the following criteria: high TCR clonality, immunophenotype indicative of T cell activation, shared alpha and/or beta TCR chains across patient samples (Public TCRs) and TCRs with anti-tumor activities. Statistical- and machine learning algorithms were developed to predict human peptides from the library selection data. Predicted target peptides were tested in in vitro T cell co-cultures for TCR activation. Antigen processing assays or mass spec analysis was employed to validate immunological processing of the epitope to identify bona fide targets. Results Among the first 100 targets identified by 3T-TRACE, we found 15 novel cancer testis antigens (CTAs) and 42 tumor associated antigens (TAAs) that were not previously described as targets in CPI treated solid tumor patients (Figure 3). Conclusions By combining TCR repertoire profiling with a library-based, antigen identification approach we found a strong enrichment of CTA- and TAA targets recognized by TCRs on TILs from CPI responders (Figure 4). Our findings are consistent with previous reports, demonstrating long-lasting responses induced by epitope spreading and CD8 T cell responses towards self-antigens, including CTAs and TAAs. Citation Format: Hans-Peter Gerber, Marvin Gee, Leah Sibener. Identification of novel, tumor specific pHLA targets recognized by TILs from CPI responders in a high-throughput, high-diversity pHLA library screening platform (3T-TRACE) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA006.

TCR meta-clonotypes for biomarker discovery with tcrdist3 enabled identification of public, HLA-restricted clusters of SARS-CoV-2 TCRs.

T-cell receptors (TCRs) encode clinically valuable information that reflects prior antigen exposure and potential future response. However, despite advances in deep repertoire sequencing, enormous TCR diversity complicates the use of TCR clonotypes as clinical biomarkers. We propose a new framework that leverages experimentally inferred antigen-associated TCRs to form meta-clonotypes - groups of biochemically similar TCRs - that can be used to robustly quantify functionally similar TCRs in bulk repertoires across individuals. We apply the framework to TCR data from COVID-19 patients, generating 1831 public TCR meta-clonotypes from the SARS-CoV-2 antigen-associated TCRs that have strong evidence of restriction to patients with a specific human leukocyte antigen (HLA) genotype. Applied to independent cohorts, meta-clonotypes targeting these specific epitopes were more frequently detected in bulk repertoires compared to exact amino acid matches, and 59.7% (1093/1831) were more abundant among COVID-19 patients that expressed the putative restricting HLA allele (false discovery rate [FDR]<0.01), demonstrating the potential utility of meta-clonotypes as antigen-specific features for biomarker development. To enable further applications, we developed an open-source software package, tcrdist3, that implements this framework and facilitates flexible workflows for distance-based TCR repertoire analysis.

Autoreactive T cell receptors with shared germline-like α chains in type 1 diabetes.

Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals (“public” chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαβ sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells (“expanded”), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or “private,” TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRβ junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these “innate-like” TCRs differ from previously described immunodominant TCRβ chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.

Characterization of Adaptive-like γδ T Cells in Ugandan Infants during Primary Cytomegalovirus Infection.

Gamma-delta (γδ) T cells are unconventional T cells that help control cytomegalovirus (CMV) infection in adults. γδ T cells develop early in gestation, and a fetal public γδ T cell receptor (TCR) clonotype is detected in congenital CMV infections. However, age-dependent γδ T cell responses to primary CMV infection are not well-understood. Flow cytometry and TCR sequencing was used to comprehensively characterize γδ T cell responses to CMV infection in a cohort of 32 infants followed prospectively from birth. Peripheral blood γδ T cell frequencies increased during infancy, and were higher among CMV-infected infants relative to uninfected. Clustering analyses revealed associations between CMV infection and activation marker expression on adaptive-like Vδ1 and Vδ3, but not innate-like Vγ9Vδ2 γδ T cell subsets. Frequencies of NKG2C+CD57+ γδ T cells were temporally associated with the quantity of CMV shed in saliva by infants with primary infection. The public γδ TCR clonotype was only detected in CMV-infected infants <120 days old and at lower frequencies than previously described in fetal infections. Our findings support the notion that CMV infection drives age-dependent expansions of specific γδ T cell populations, and provide insight for novel strategies to prevent CMV transmission and disease.

Analysis of T cell receptor clonotypes in tumor microenvironment identifies shared cancer-type-specific signatures.

Despite the conventional view that a truly random V(D)J recombination process should generate a highly diverse immune repertoire, emerging reports suggest that there is a certain bias toward the generation of shared/public immune receptor chains. These studies were performed in viral diseases where public T cell receptors (TCR) appear to confer better protective responses. Selective pressures generating common TCR clonotypes are currently not well understood, but it is believed that they confer a growth advantage. As very little is known about public TCR clonotypes in cancer, here we set out to determine the extent of shared TCR clonotypes in the intra-tumor microenvironments of virus- and non-virus-driven head and neck cancers using TCR sequencing. We report that tumor-infiltrating T cell clonotypes were indeed shared across individuals with the same cancer type, where the majority of shared sequences were specific to the cancer type (i.e., viral versus non-viral). These shared clonotypes were not particularly enriched in EBV-associated nasopharynx cancer but, in both cancers, exhibited distinct characteristics, namely shorter CDR3 lengths, restricted V- and J-gene usages, and also demonstrated convergent V(D)J recombination. Many of these shared TCRs were expressed in patients with a shared HLA background. Pattern recognition of CDR3 amino acid sequences revealed strong convergence to specific pattern motifs, and these motifs were uniquely found to each cancer type. This suggests that they may be enriched for specificity to common antigens found in the tumor microenvironment of different cancers. The identification of shared TCRs in infiltrating tumor T cells not only adds to our understanding of the tumor-adaptive immune recognition but could also serve as disease-specific biomarkers and guide the development of future immunotherapies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-021-03047-7.

Profiling of T Cell Repertoire in SARS-CoV-2-Infected COVID-19 Patients Between Mild Disease and Pneumonia.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a public health emergency. The most common symptoms of COVID-19 are fever, cough, and fatigue. While most patients with COVID-19 present with mild illness, some patients develop pneumonia, an important risk factor for mortality, at early stage of viral infection, putting these patients at increased risk of death. So far, little has been known about differences in the T cell repertoires between COVID-19 patients with and without pneumonia during SARS-CoV-2 infection. Herein, we aimed to investigate T cell receptor (TCR) repertoire profiles and patient-specific SARS-CoV-2-associated TCR clusters between COVID-19 patients with mild disease (no sign of pneumonia) and pneumonia. The TCR sequencing was conducted to characterize the peripheral TCR repertoire profile and diversity. The TCR clustering and CDR3 annotation were exploited to further discover groups of patient-specific TCR clonotypes with potential SARS-CoV-2 antigen specificities. Our study indicated a slight decrease in the TCR repertoire diversity and a skewed CDR3 length usage in patients with pneumonia compared to those with mild disease. The SARS-CoV-2-associated TCR clusters enriched in patients with mild disease exhibited significantly higher TCR generation probabilities and most of which were highly shared among patients, compared with those from pneumonia patients. Importantly, using similarity network-based clustering followed by the sequence conservation analysis, we found different patterns of CDR3 sequence motifs between mild disease- and pneumonia-specific SARS-CoV-2-associated public TCR clusters. Our results showed that characteristics of overall TCR repertoire and SARS-CoV-2-associated TCR clusters/clonotypes were divergent between COVID-19 patients with mild disease and patients with pneumonia. These findings provide important insights into the correlation between the TCR repertoire and disease severity in COVID-19 patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10875-021-01045-z.