T-cell Receptor Repertoire Research Articles

A differential analysis of TCR repertoire between patients with EGFR-mutant and KRAS-mutant non–small-cell lung cancer.

e21012 Background: T-cell receptor (TCR) repertoire could partially represent tumor micro-environment. EGFR and KRAS are common driver genes mutated and often mutually exclusive in non-small cell lung cancer (NSCLC). There’s a hypothesis that the immune system responds differently according to different mutations. Here we investigated the TCR features, including diversity, clonality, evenness and sequence similarity in EGFR and KRAS mutated NSCLC patients, to better understand the immune response in carcinogenesis. Methods: A total of 71 EGFR-mutant and 31 KRAS-mutant NSCLC tissue samples underwent multiplex PCR based TCR-β sequencing. TCR-β cdr3 amino acid sequences were obtained from the sequencing data using MiXCR and VDJtools. To compare TCR repertoires in EGFR-mutant and KRAS-mutant groups, each assemble CDR3 amino acid sequence was taken as a word, and each repertoire was taken as a document. Each repertoire was compared with every other one as a sample pair. TF-IDF (term frequency-inverse document frequency) document vectors was built for each sample and cosine similarity scores were computed for sample pairs. Results: The total cosine similarity score within EGFR-mutant group is 11.07 and average cosine similarity score is 0.0044±0.01, while in the KRAS-group, the total cosine similarity score is 13.41 and average cosine similarity score is 0.029±0.04. Between these two groups, the total cosine similarity score is 8.76 and average cosine similarity score is 0.0019±0.0009. See table for summary. The intergroup similarity score is significantly less than that within each group respectively (p < 1E10). Conclusions: Our results demonstrated that the TCR repertoire was clearly associated with cancer mutational profile. The mechanism of how mutations affect the immune system and shape the TCR repertoire is yet to be investigated. [Table: see text]

Exploration of biomarkers in potentially resectable stage IIIB NSCLC patients receiving neoadjuvant chemoimmunotherapy.

e20591 Background: Non–small cell lung cancer (NSCLC) is one of the main leading causes of cancer mortality in the world. NSCLC is incurable in most patients with potentially resectable locally advanced stage IIIB disease. Numerous studies suggest that advantageous chemoimmunotherapy is a promising treatment for resectable NSCLC. The determination of PD-L1, T-cell receptor (TCR) repertoire and tumor mutational burden (TMB) have been reported to predict the response to immunotherapy. However, the value of pathologic response determination in the context of neoadjuvant chemoimmunotherapy in resectable stage IIIB NSCLC is limited. Therefore, it is necessary to identify biomarkers for the prediction of the benefit from neoadjuvant chemoimmunotherapy in patients with potentially resectable stage IIIB NSCLC. Methods: A total of 15 patients with potentially resectable clinical stage IIIB NSCLC were enrolled into the study receiving neoadjuvant sintilimab (200mg IV Q3W) plus chemotherapy therapies for 4-6 weeks followed by surgery. All peripheral blood and tissue samples were collected and detected by using TCR repertoire sequencing before and after chemoimmunotherapy. The patients were divided into MPR group (n=5) and non-MPR group (n=10) according to MPR (<10% viable tumour at resection) as an index. Furthermore, TMB spectra of 1021-gene panels and PD-L1 immunohistochemical staining were performed in paraffin-embedded human NSCLC tissue before neoadjuvant chemoimmunotherapy. The Pearson correlation coefficient method was used to analyze correlation. The ROC analysis were performed using “pROC” R package. Results: In baseline tissue samples, the levels of TMB and NAD were higher in MPR compared with non-MPR groups (P=0.0328, P=0.015, respectively). The negative correlation was existed between pretreatment tissue TCR richness and TMB in non-MPR group (P=0.028, R=-0.66). In MPR group, TCR clones and evenness showed significant difference (P=0.00237, P=0.04658, respectively). In non-MPR group, the TCR repertoire of tissue in posttreatment showed significant difference compared with those of pretreatment, including clones (P=1.04x10-6), shannon’s diversity (P=0.00142), evenness (P=0.00433), richness (P=0.00591). The clinical data showed that MPR patients with DDR mutation had longer DFS after sintilimab plus chemotherapy therapies (18.4±5.41 months, X-squared=9.8, P=0.02034). DDR mutation, TMB and TCR richness revealed better sensitivity and specificity to classify patients achieving MPR after neoadjuvant chemoimmunotherapy (AUC=1.000, AUC=0.796, AUC=0.773, respectively). Conclusions: This study confirmed the superiority of neoadjuvant chemoimmunotherapy in patients with potentially resectable stage IIIB NSCLC in terms of MPR. TCR repertoire, TMB and DDR mutation were associated with pathologic response to neoadjuvant chemoimmunotherapy as biomarkers for effective anti-tumor immunity. Clinical trial information: ChiCTR2000040673 .

The peripheral TCR repertoire diversity indexes can be used as an independent indicator for long term survival rate of ovarian cancer patients.

e17558 Background: According to GLOBOCAN 2020 data, ovarian cancer (OC) ranks 19th in incidence and 15th in mortality with a rate of 1.6% and 2.1%, respectively, High-grade serous OC is the most common sub-type of malignant ovarian tumors, remains the leading cause of cancer-related death among all gynecological cancers in the developed parts of the world. In the United States in 2016, 5-year survival rate was only 10-25% in stage III or IV OC patients, and there was considerable variability in survival in an advanced stage. At present, there is no reliable molecular marker to identify patients with better or worse prognosis. As is known to all, the adaptive immune system defends the body against different foreign antigens, using two main types of antigen receptors: T-cell receptors (TCRs) and antibodies. Now we use high-throughput sequencing approach to analyze the expressed TCRβ repertoires in the peripheral blood of OC patients and healthy women donors. Methods: The experimental design and participant recruitment were approved by Medical Ethics Committee of West China Second University Hospital (Ethics Approval for Research: 2022). Total RNA was extracted from PBMC using TRIZOL reagent. High through put sequencing of the TCRβ gene, assignment of the Vβ, Dβ, and Jβ germline gene, and TCR diversity indexes analyses were conducted essentially following our previously published protocols. Statistical analysis and graph drawing were performed using R language (R 4.0.3). Results: Comparison of the TCR repertoires between the 232 OC patients (OC group) and the 250 healthy women donors (HD group) revealed significant differences. For examples, the VJ combinations of the OC group were distinct from those of the HD group. In addition, the TCR repertoire diversity indexes of the OC group were significantly lower than those of the HD group ( p < 0.001), including D50s, Simpson indexes, the number of highly expressed clones (HECs), and the frequencies of the largest TCR clones. As reported before, the TCR repertoire diversity indexes decreased gradually with increasing age in the HD group (all p < 0.001). Moreover, the TCR repertoire diversities in the survival group were significantly higher than those in the death group (D50: p = 0.0342; the number of HECs: p = 0.0414). Using as the cut off value, the OC patients with D50 < 0.032 had significantly higher mortality rates compared to the OC patients with D50 > 0.032 ( p = 0.0019). Similarly, using the numbers of HECs or the frequencies of the largest TCR clones can also distinguish OC patients in long term survival. Conclusions: Analyzing the peripheral TCR repertoires in the OC and HD revealed significant differences, and the peripheral TCR repertoire diversity indexes may be used as a potential predictor for long term survival for ovarian cancer.

Multi-omics analysis to reveal the change of peripheral blood TCR repertoire and tumor burden during neoadjuvant chemotherapy combined with sequential PD-1 blockade for patients with non-small cell lung cancers: A single-center, single-arm phase 2 trial.

e20582 Background: Preoperative neoadjuvant chemotherapy combined with immunotherapy has been applied to the treatment of non-small cell lung cancer (NSCLC). However, the optimal dosage of chemotherapy and sequencing for chemotherapy and immunotherapy remains controversial. Methods: In this single-arm phase 2 trial, patients with stage IB to IIIA resectable NSCLC were enrolled and received 2 cycles of dose-limiting platinum-based chemotherapy (albumin-bound paclitaxel or pemetrexed plus cisplatin or carboplatin) on day 1, combined with sintilimab (200mg) on day 5 before undergoing radical surgery. Combination postoperative adjuvant therapy with the same regimen was administered. The primary endpoints were objective response rate (ORR) and major pathological response (MPR). Biopsies and plasma at different timepoint were collected and performed with RNA sequencing, circulating tumor DNA sequencing and T cell receptor (TCR) sequencing. Results: 17 patients were enrolled. After neoadjuvant treatment, patients with completed response, partial responses and stable diseases were 11.8% (2/17), 70.6% (12/17) and 17.6% (3/17), respectively, yielding an ORR of 82.4%. Radiological downstaging was achieved in 82.4% (14/17) of patients. All patients (17/17, 100%) experienced the R0 resection. Pathologic complete response and MPR were identified in 35.2% (6/17) patients and 23.5% (4/17) patients, respectively. Only 1 of the 17 patients (5.9%) experienced grade 3 or greater adverse event. Multi-omics analysis revealed chemotherapy combined with sequential PD-1 blockade results in the increase of dominated TCR clones and the decrease of the tumor burden. Especially during chemotherapy of cycle 1, the dominated TCR clones were significantly increased, and the maxVAF of ctDNA was significantly decreased. The change between the dominated TCR clones and maxVAF of ctDNA was negatively correlated. Conclusions: In patients with stage IB to IIIA resectable NSCLC, the neoadjuvant therapy with dose-limiting platinum-based chemotherapy combined with sequential sintilimab was safer and resulted in significant antitumor activity in patients with resectable NSCLC. The changes in TCR repertoire and ctDNA during treatment were analyzed, which provided evidence for the rationality of neoadjuvant chemotherapy combined with sequential PD-1 blockade. Clinical trial information: ChiCTR2000033092 .

Single-cell RNA and T cell receptor (TCR) sequencing in patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) after induction CTLA4 and PD-1 immune checkpoint blockade (ICB).

6056 Background: Combination CTLA-4 and PD-1 ICB results in durable responses in patients with recurrent and metastatic HNSCC and therefore justifies further investigation in the induction or neoadjuvant setting. Methods: We investigated features of the tumor immune microenvironment (TIME) associated with MHR after induction CTLA-4 and PD-1 ICB in patients with newly diagnosed HPV-positive HNSCC. Tumor samples were collected at baseline and on treatment from 31 patients enrolled in a phase 2 trial of a 6-week cycle of CTLA-4 and PD-1 ICB induction followed by dose/volume-adapted IMRT (50-66Gy) concurrent with cycle 2. Tumors were evaluated for histological response and single-cell RNA sequencing (scRNA-seq) and scTCR-seq were performed on the 10X genomics platform. After stringent clustering and annotation of scRNA-seq data, 76,319 CD8+ and 78,622 CD4+ T cells with paired TCR sequence data were evaluable for transcriptomic, TCR repertoire and clonotype differentiation analysis. Results: Percent reduction in tumor viability on histology correlated significantly with reduction in tumor cell proportion in scRNA-seq data (R2= 0.7). Thirteen (48%) of 27 patients with paired samples (missing data due to COVID) had MHR, defined as ≤10% residual tumor viability on ICB treatment. Through TCR tracing, over 100 T cell clonotypes that expanded significantly (p < 0.05, Fisher’s exact) post-ICB were identified. Highly expanded clonotypes resided mainly in exhausted CD8+ T cell clusters and exhibited a tissue resident memory (TRM) phenotype. Examination of their functional states revealed a variety of T cell modulatory trajectories, including reverse transition from exhausted to less exhausted states and from memory to functional states. When compared to patients without MHR, those with MHR had significantly higher ICB-induced TCR clonotype expansion (p = 0.025, Wilcoxon), reductions of activated Tregs (p = 0.003, Wilcoxon) and terminally exhausted CD8+ T cells (p = 0.003, Wilcoxon), and increase of polyfunctional CD8+ T cells (p = 0.014, Wilcoxon). A lasso regression model associated T cell expansion, basal 7-gene TRM and 5-gene effector scores, and on-treatment proliferation score and effector cell proportion with percent reduction in tumor viability (R2 = 0.66). Conclusions: In HPV-HNSCC, induction CTLA4 and PD1 ICB lead to extensive reinvigoration of CD8+ T cell clonotypes with both an exhausted and TRM phenotype, revealing a high degree of plasticity of distinct T cell clones. Paired RNA and TCR profiling facilitated identification of potentially tumor reactive and ICB-responsive T cells and clonotypes and identified several TIME features associated with tumor cell death. Clinical trial information: NCT03799445 .

Association of host immune signatures with response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC).

4550 Background: Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with immunotherapy-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods: We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. Bulk RNA-sequencing (RNA-seq) and T-cell receptor sequencing (TCR-seq) were performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-seq was additionally performed on available pre-treatment primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results: In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts. Following quality-control, TCR-seq and RNA-seq data were available for 367 and 134 patient samples, respectively. Responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Conclusions: We were successfully able to characterize T-cell receptor and IgH repertoires in a large cohort of patients with mRCC and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development.

Trial START-001: A phase 1/2 study of STAR0602, a first-in-class, selective T cell receptor (TCR)-targeting, bifunctional antibody-fusion molecule, as monotherapy in patients with antigen-rich tumors post checkpoint inhibitor (CPI) treatment.

TPS2671 Background: Many patients do not respond to CPI-based therapies and most responders eventually develop resistance. Thus, the development of more effective therapies for CPI treatment resistance is a significant unmet medical need. STAR0602, a first-in-class, TCR β chain-targeting bifunctional antibody fused to a costimulatory molecule, binds the germline Vβ6/Vβ10 TCRs of human alpha/beta (aβ) T cells, and promotes the selective activation and expansion of CD8+ and CD4+ Vβ6/Vβ10 effector memory T cells by simultaneously engaging direct TCR activation with IL-2 receptor binding and activation in cis. Preclinical studies with STAR0602 and its murine surrogate showed potent single-agent anti-tumor activity in multiple CPI-refractory human organoid and murine syngeneic tumor models, and acceptable tolerability and limited cytokine release in monkeys and mice. Durable anti-tumor responses, with long-term protection from tumor re-challenge, was associated with de novo expansion and tumor infiltration of targeted Vβ T cell subsets with a novel effector memory gene signature and striking increase in TCR repertoire diversity. These data provide strong scientific rationale to investigate the safety and efficacy of STAR0602 in patients with CPI-resistant cancers. Methods: Using a 3+3 trial design, Phase 1 of START-001 is a dose escalation through eight provisional dose levels of STAR0602, administered via an intravenous infusion once every two weeks, to determine a recommended Phase 2 dose (RP2D), based on safety and tolerability, pharmacokinetics, pharmacodynamics, and preliminary anti-tumor activity. Subjects must have one of the following, histologically confirmed solid tumors that are unresectable, locally advanced or metastatic and for which standard curative therapies do not exist or are no longer effective: 1) high mutational burden solid tumors (TMB-H); 2) MSI-H/mismatch repair-deficient (dMMR) cancers; 3) Virally associated tumors including Merkel cell carcinoma, cervical, oropharyngeal, anal, penile, vaginal, and vulval cancers. After an RP2D is selected, Phase 2 (dose expansion at this RP2D) will begin to further investigate the safety and preliminary anti-tumor activity of STAR0602 in 10 provisional cohorts of patients who have the following solid tumors: TMB-H, CPI experienced; TMB-H, no prior CPI experience; MSI-H/dMMR; virally associated tumors; K-RAS wild-type and MSI-L/MMR proficient colorectal cancer (CRC); K-Ras mutant CRC; metastatic triple-negative breast cancer; relapsed and refractory epithelial ovarian cancer; metastatic castration-resistant prostate cancer; primary stage IV or recurrent non-small cell lung cancer. Phase 1 enrollment has begun since January 2023. Clinical trial information: NCT05592626 .

Leveraging T-cell receptor - epitope recognition models to disentangle unique and cross-reactive T-cell response to SARS-CoV-2 during COVID-19 progression/resolution.

Despite the general agreement on the significance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain. Understanding this aspect could provide insights for adjusting vaccines and maintaining robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to SARS-CoV-2 epitopes unique to the virus (SC2-unique) or shared with other coronaviruses (CoV-common), we trained a large number of T-cell receptor (TCR) - epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. These models were then applied to longitudinal CD8+ TCR repertoires from critical and non-critical COVID-19 patients. In spite of comparable initial CoV-common TCR repertoire depth and CD8+ T-cell depletion, the temporal dynamics of SC2-unique TCRs differed depending on the disease severity. Specifically, while non-critical patients demonstrated a large and diverse SC2-unique TCR repertoire by the second week of the disease, critical patients did not. Furthermore, only non-critical patients exhibited redundancy in the CD8+ T-cell response to both groups of epitopes, SC2-unique and CoV-common. These findings indicate a valuable contribution of the SC2-unique CD8+ TCR repertoires. Therefore, a combination of specific and cross-reactive CD8+ T-cell responses may offer a stronger clinical advantage. Besides tracking the specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire, our analytical framework can be expanded to more epitopes and assist in the assessment and monitoring of CD8+ T-cell response to other infections.

Antigen-specific and cross-reactive T cells in protection and disease.

Human T cells have a diverse T-cell receptor (TCR) repertoire that endows them with the ability to identify and defend against a broad spectrum of antigens. The universe of possible antigens that T cells may encounter, however, is even larger. To effectively surveil such a vast universe, the T-cell repertoire must adopt a high degree of cross-reactivity. Likewise, antigen-specific and cross-reactive T-cell responses play pivotal roles in both protective and pathological immune responses in numerous diseases. In this review, we explore the implications of these antigen-driven T-cell responses, with a particular focus on CD8+ T cells, using infection, neurodegeneration, and cancer as examples. We also summarize recent technological advances that facilitate high-throughput profiling of antigen-specific and cross-reactive T-cell responses experimentally, as well as computational biology approaches that predict these interactions.

Detection of mutant antigen-specific T cell receptors against multiple myeloma for T cell engineering

Multiple myeloma (MM) remains an incurable hematological neoplasm. Neoantigen-specific Tcell receptor (TCR)-engineered T(TCR-T) cell therapy is a potential alternative treatment. Particularly, TCRs derived from a third-party donor may cover broad ranges of neoantigens, whereas TCRs in patients suffering from immune disorders are limited. However, the efficacy and feasibility of treating MM have not been evaluated thoroughly. In this study, we established a system for identifying immunogenic mutant antigens on MM cells and their corresponding TCRs using healthy donor-derived peripheral blood mononuclear cells (PBMCs). Initially, the immune responses to 35 candidate peptides predicted by the immunogenomic analysis were investigated. Peptide-reactive T lymphocytes were enriched, and subsequently, TCR repertoires were determined by single-cell TCR sequencing. Eleven reconstituted TCRs showed mutation-specific responses against 4 peptides. Particularly, we verified the HLA-A∗24:02-binding QYSPVQATF peptide derived from COASY S55Y as the naturally processed epitope across MM cells, making it a promising immune target. Corresponding TCRs specifically recognized COASY S55Y+HLA-A∗24:02+ MM cells and augmented tumoricidal activity. Finally, adoptive cell transfer of TCR-T cells showed objective responses in the xenograft model. We initiatively proposed the utility of tumor mutated antigen-specific TCR genes to suppress MM. Our unique strategy will facilitate further identification of neoantigen-specific TCRs.

Individual Epitope-Specific CD8+ T Cell Immune Responses Are Shaped Differently during Chronic Viral Infection.

A hallmark in chronic viral infections are exhausted antigen-specific CD8+ T cell responses and the inability of the immune system to eliminate the virus. Currently, there is limited information on the variability of epitope-specific T cell exhaustion within one immune response and the relevance to the T cell receptor (TCR) repertoire. The aim of this study was a comprehensive analysis and comparison of three lymphocytic choriomeningitis virus (LCMV) epitope-specific CD8+ T cell responses (NP396, GP33 and NP205) in a chronic setting with immune intervention, e.g., immune checkpoint inhibitor (ICI) therapy, in regard to the TCR repertoire. These responses, though measured within the same mice, were individual and independent from each other. The massively exhausted NP396-specific CD8+ T cells revealed a significantly reduced TCR repertoire diversity, whereas less-exhausted GP33-specific CD8+ T cell responses were rather unaffected by chronicity in regard to their TCR repertoire diversity. NP205-specific CD8+ T cell responses showed a very special TCR repertoire with a prominent public motif of TCR clonotypes that was present in all NP205-specific responses, which separated this from NP396- and GP33-specific responses. Additionally, we showed that TCR repertoire shifts induced by ICI therapy are heterogeneous on the epitope level, by revealing profound effects in NP396-, less severe and opposed effects in NP205-, and minor effects in GP33-specific responses. Overall, our data revealed individual epitope-specific responses within one viral response that are differently affected by exhaustion and ICI therapy. These individual shapings of epitope-specific T cell responses and their TCR repertoires in an LCMV mouse model indicates important implications for focusing on epitope-specific responses in future evaluations for therapeutic approaches, e.g., for chronic hepatitis virus infections in humans.

Recruitment of epitope-specific T cell clones with a low-avidity threshold supports efficacy against mutational escape upon re-infection

Repetitive pathogen exposure leads to the dominant outgrowth of Tcell clones with high Tcell receptor (TCR) affinity to the relevant pathogen-associated antigens. However, low-affinity clones are also known to expand and form immunological memory. While these low-affinity clones contribute less immunity to the original pathogen, their role in protection against pathogens harboring immune escape mutations remains unclear. Based on identification of the TCR repertoire and functionality landscape of naive epitope-specific CD8+ Tcells, we reconstructed defined repertoires that could be followed as polyclonal populations during immune responses invivo. We found that selective clonal expansion is governed by clear TCR avidity thresholds. Simultaneously, initial recruitment of broad TCR repertoires provided a polyclonal niche from which flexible secondary responses to mutant epitopes could be recalled. Elucidating how Tcell responses develop "from scratch" is informative for the development of enhanced immunotherapies and vaccines.

Quantitative annotations of T-Cell repertoire specificity.

The specificity of a T-cell receptor (TCR) repertoire determines personalized immune capacity. Existing methods have modeled the qualitative aspects of TCR specificity, while the quantitative aspects remained unaddressed. We developed a package, TCRanno, to quantify the specificity of TCR repertoires. We created deep-learning-based, epitope-aware vector embeddings to infer individual TCR specificity. Then we aggregated clonotype frequencies of TCRs to obtain a quantitative profile of repertoire specificity at epitope, antigen and organism levels. Applying TCRanno to 4195 TCR repertoires revealed quantitative changes in repertoire specificity upon infections, autoimmunity and cancers. Specifically, TCRanno found cytomegalovirus-specific TCRs in seronegative healthy individuals, supporting the possibility of abortive infections. TCRanno discovered age-accumulated fraction of severe acute respiratory syndrome coronavirus 2 specific TCRs in pre-pandemic samples, which may explain the aggressive symptoms and age-related severity of coronavirus disease 2019. TCRanno also identified the encounter of Hepatitis B antigens as a potential trigger of systemic lupus erythematosus. TCRanno annotations showed capability in distinguishing TCR repertoires of healthy and cancers including melanoma, lung and breast cancers. TCRanno also demonstrated usefulness to single-cell TCRseq+gene expression data analyses by isolating T-cells with the specificity of interest.

Coexpression of natural killer cell antigens by T-cell large granular lymphocytes in hydroa vacciniforme lymphoproliferative disorder and the involvement of Vδ1 + epithelial-type γδT cells

Hydroa vacciniforme lymphoproliferative disorder (HV-LPD) is a cutaneous variant of chronic active Epstein–Barr virus disease. We examined the coexpression of T- and natural killer (NK)-cell antigens in five patients with classic HV (cHV) and five with systemic HV (sHV). T-cell receptor (TCR) repertoire analysis was performed with high‑throughput sequencing. All five cHV patients had increased γδT cells (> 5%), whereas five sHV patients showed γδT- and αβT-cell dominance in two patients each, and a mixture of abnormal γδT and αβT cells in one. Circulating CD3 + T cells expressed CD16/CD56 at 7.8–42.3% and 1.1–9.7% in sHV and cHV, respectively. The percentage of CD16/CD56 + T cells was higher in the large granular lymphocyte or atypical T-cell fractions in sHV, but no TCR Vα24 invariant chain characteristic of NKT cells was detected. Considerable numbers of CD3 + cells expressing CD56 were observed in sHV skin infiltrates. Of the circulating γδT cells tested, TCR Vδ1 + cells characteristic of the epithelial type of γδT cells were dominant in two sHV cases. Thus, atypical αβT and γδT cells in HV-LPD can express NK-cell antigens, such as CD16 and CD56, and Vδ1 + epithelial-type γδT cells are a major cell type in some HV-LPD cases.

The T cell receptor repertoire reflects the early dynamics of the immune response to vaccination

Abstract Early ascertainment of the immune response to vaccinations is important. The T cell receptor (TCR) is a promising target to achieve this, with the complete TCR repertoire in PBMCs reflecting the T cells present in an individual. To this end, we developed Tseek, an unbiased and accurate method for profiling the TCR repertoire by sequencing the TCR α and β chains and developing a suite of tools for repertoire analysis. We explored the T cell response to both the COVID-19 mRNA vaccine (n=9) and the seasonal inactivated Influenza vaccine (n=5) at several time points. Neutralizing antibody titers were also measured in the covid vaccine samples. The COVID-19 vaccine elicited a broad T cell response involving multiple expanded clones, whereas the Influenza vaccine elicited a narrower response involving fewer clones. Distinct T cell clones responded at different time points over a month, providing temporal details lacking in the antibody measurements. The physical symptoms following administration of the vaccinations were not indicative of the TCR/antibody responses while the TCR responses broadly presaged the antibody responses. Additionally, the TCR repertoire is akin to a fingerprint: donors of blood samples taken years apart could be identified from the TCR repertoire. TCR repertoire profiling shows great promise as an early and sensitive measure of the adaptive immune response to vaccination, which can help improve immunogen selection and optimize vaccine dosage and spacing between doses.

Reshaping the anti-tumor CD4 T-cell repertoire contributes to oncolytic virus therapeutic efficacy in malignant glioma

Abstract Malignant gliomas are deadly brain tumors with no cure. We engineered herpes simplex virus (HSV)-1 and developed oncolytic HSV (oHSV) to treat brain tumors, given its potential to selectively kill tumor cells while sparing normal brain cells, and to boost anti-tumor immunity. The therapy shows great promise based on our Phase I trial results. However, the precise mechanisms for its enhanced anti-tumor immune responses remain poorly understood. Using an oHSV secreting murine IL-12 (M002), murine intracranial glioma models and adoptive transfer approaches, we observed that M002 treatment induced unique CD4 +T-cell populations to acquire cytolytic effector phenotypes, promoting anti-glioma responses in an MHCII-dependent manner. Notably, single-cell RNA sequencing and T-cell receptor (TCR) repertoire analysis revealed that M002 treatment shifted the TCR dominance while reducing the clonal diversity. These results suggest that the capacity of oncolytic virus therapy to reshape the anti-tumor CD4 +T-cell repertoire contributes to its improved efficacy. Insights obtained from this study can facilitate the development of better therapeutic strategies for brain tumor patients. Supported by DoD W81XH-18-1-0315 and UAB Faculty Start-up Funds (to JWL)

T-cell and B-cell receptor repertoire profiling for biomarker discovery

Abstract T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B-cells. We developed a new technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells in the same workflow. The TCR and BCR immunophenotyping method involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, as well as determining the expression levels of the most informative T- and B-cell phenotyping genes. Results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data allows profiling of the TCR and BCR clonotypes and identifcation of major immune cell subtypes and their activation status. Preliminary data from rheumatoid arthritis blood samples and others will be presented.

Deletion of Vβ3 +CD4 +T-cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8 +T-cells

Abstract In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T-cells. Interactions between both helper CD4 +and cytotoxic CD8 +T-cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T-cells arise from deleterious interactions between relatively common genes which regulate aspects of T-cell activation/effector function (Ctla-4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T-cell receptor signaling (Ptpn22). In this study, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded Mtv3 provirus as a genetic element affecting CD4 +/CD8 +T-cell interactions through an additional mechanism, altering the T-cell receptor (TCR) repertoire. Mtv3 encodes a superantigen that deletes the majority of Vβ3 +thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3 +T-cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8 +T-cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifest by Vβ3 +CD4 +T-cells, which unless deleted by Mtv3, accumulate in insulitic lesions and trigger in B6 background mice the pathogenic activation of diabetogenic CD8 +T-cells. Our findings provide evidence that endogenous Mtv superantigens can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human disorders. Supported by grants from the American Diabetes Association (1-14-BS-051) and NIH (AI130656)

Integrated analysis of single-cell and bulk transcriptome identifies a signature based on NK cell marker genes to predict prognosis and therapeutic response in clear cell renal cell carcinoma.

Accumulating evidence has highlighted the effects of natural killer (NK) cells on shaping anti-tumor immunity. This study aimed to construct an NK cell marker gene signature (NKMS) to predict prognosis and therapeutic response of clear cell renal cell carcinoma (ccRCC) patients. Publicly available single-cell and bulk RNA profiles with matched clinical information of ccRCC patients were collected from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), ArrayExpress, and International Cancer Genome Consortium (ICGC) databases. A novel NKMS was constructed, and its prognostic value, associated immunogenomic features and predictive capability to immune checkpoint inhibitors (ICIs) and anti-angiogenic therapies were evaluated in ccRCC patients. We identified 52 NK cell marker genes by single-cell RNA-sequencing (scRNA-seq) analysis in GSE152938 and GSE159115. After least absolute shrinkage and selection operator (LASSO) and Cox regression, the most prognostic 7 genes (CLEC2B, PLAC8, CD7, SH3BGRL3, CALM1, KLRF1, and JAK1) composed NKMS using bulk transcriptome from TCGA. Survival and time-dependent receiver operating characteristic (ROC) analysis exhibited exceptional predictive capability of the signature in the training set and two independent validation cohorts (E-MTAB-1980 and RECA-EU cohorts). The seven-gene signature was able to identify patients within high Fuhrman grade (G3-G4) and American Joint Committee on Cancer (AJCC) stage (III-IV). Multivariate analysis confirmed the independent prognostic value of the signature, and a nomogram was built for clinical utility. The high-risk group was characterized by a higher tumor mutation burden (TMB) and greater infiltration of immunocytes, particularly CD8+ T cells, regulatory T (Treg) cells and follicular helper T (Tfh) cells, in parallel with higher expression of genes negatively regulating anti-tumor immunity. Moreover, high-risk tumors exhibited higher richness and diversity of T-cell receptor (TCR) repertoire. In two therapy cohorts of ccRCC patients (PMID32472114 and E-MTAB-3267), we demonstrated that high-risk group showed greater sensitivity to ICIs, whereas the low-risk group was more likely to benefit from anti-angiogenic therapy. We identified a novel signature that can be utilized as an independent predictive biomarker and a tool for selecting the individualized treatment for ccRCC patients.

Universal, open MHC-I for rapid ligand loading and enhanced complex stability across classical and non-classical allotypes.

Abstract The instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents an important biochemical challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs) for autologous therapy development. Here, we leverage the coupled binding between ligand and the light chain (beta-2 microglobulin, β 2m) to the MHC-I heavy chain with the aim of developing conformationally stable molecules by engineering a disulfide bond to bridge the interface between the heavy and light chain. The resulting MHC-I molecules are stabilized in a ligand-receptive/open state, exhibit favorable ligand exchange properties, and are readily loaded with moderate affinity ligands to afford properly conformed protein complexes of enhanced stability compared to the wild type. We demonstrate the generality of the structural design using polymorphic HLA representatives covering multiple HLA-A and B supertypes, the nonclassical and oligomorphic HLA-Ib molecules, as well as MHC-like molecules, MR1 and CD1. Our platform allows spontaneous and rapid ligand exchange, which enables a range of approaches for ligand screening and the functional screening and characterization of polyclonal TCR repertoires. Supported by grants from NIH (R01AI143997, R35GM125034, U01DK112217).