T-cell Receptor Vβ Research Articles

Investigation of Long-Term CD4+ T Cell Receptor Repertoire Changes Following SARS-CoV-2 Infection in Patients with Different Severities of Disease.

The difference in the immune response to severe acute respiratory syndrome coro-navirus 2 (SARS-CoV-2) in patients with mild versus severe disease remains poorly understood. Recent scientific advances have recognised the vital role of both B cells and T cells; however, many questions remain unanswered, particularly for T cell responses. T cells are essential for helping the generation of SARS-CoV-2 antibody responses but have also been recognised in their own right as a major factor influencing COVID-19 disease outcomes. The examination of T cell receptor (TCR) family differences over a 12-month period in patients with varying COVID-19 disease severity is crucial for understanding T cell responses to SARS-CoV-2. We applied a machine learning approach to analyse TCR vb family responses in COVID-19 patients (n = 151) across multiple timepoints and disease severities alongside SARS-CoV-2 infection-naïve (healthy control) individ-uals (n = 62). Blood samples from hospital in-patients with moderate, severe, or critical disease could be classified with an accuracy of 94%. Furthermore, we identified significant variances in TCR vb family specificities between disease and control subgroups. Our findings suggest advantageous and disadvantageous TCR repertoire patterns in relation to disease severity. Following validation in larger cohorts, our methodology may be useful in detecting protective immunity and the assessment of long-term outcomes, particularly as we begin to unravel the immunological mechanisms leading to post-COVID complications.

SARS-CoV-2-Specific T-Cell as a Potent Therapeutic Strategy against Immune Evasion of Emerging COVID-19 Variants.

Despite advances in vaccination and therapies for coronavirus disease, challenges remain due to reduced antibody longevity and the emergence of virulent variants like Omicron (BA.1) and its subvariants (BA.1.1, BA.2, BA.3, and BA.5). This study explored the potential of adoptive immunotherapy and harnessing the protective abilities using virus-specific T cells (VSTs). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VSTs were generated by stimulating donor-derived peripheral blood mononuclear cells with spike, nucleocapsid, and membrane protein peptide mixtures. Phenotypic characterization, including T-cell receptor (TCR) vβ and pentamer analyses, was performed on the ex vivo-expanded cells. We infected human leukocyte antigen (HLA)-partially matched human Calu-3 cells with various authentic SARS-CoV-2 strains in a Biosafety Level 3 facility and co-cultured them with VSTs. VSTs exhibited a diverse TCR vβ repertoire, confirming their ability to target a broad range of SARS-CoV-2 antigens from both the ancestral and mutant strains, including Omicron BA.1 and BA.5. These ex vivo-expanded cells exhibited robust cytotoxicity and low alloreactivity against HLA-partially matched SARS-CoV-2-infected cells. Their cytotoxic effects were consistent across variants, targeting conserved spike and nucleocapsid epitopes. Our findings suggest that third-party partial HLA-matching VSTs could counter immune-escape mechanisms posed by emerging variants of concern.

Local chicken breeds exhibit abundant TCR-V segments but similar repertoire diversity

The thymus-derived lymphocytes of jawed vertebrates have four T-cell receptor (TCR) chains that play a significant role in immunity. As chickens have commercial value, their immune systems require a great deal of attention. Local chicken breeds are an essential part of poultry genetic resources in China. Here, we used high-throughput sequencing to analyze the TCRα and TCRβ repertoires and their relative expression levels in the native chicken breeds Baier Buff, Longyou Partridge, Xiaoshan, and Xianju. We found that TCR Vα and TCR Vβ were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vβ1, and Vβ2 subgroups, respectively. V-J pairing was biased; Jα11 was utilized by nearly all Vα segments and was the most commonly used. Breed-specific V segments and V-J pairings were detected as well. The results of the principal coordinate analysis (PCoA) as well as the V-J pairing and CDR3 diversity analyses suggested that the four local chicken breeds did not significantly differ in terms of TCR diversity. Hence, they expressed not significant differentiation, and they are rich genetic resources for the development and utilization of immune-related poultry breeding.

Abstract 1425: Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status

Abstract Background: Epigenetic silencing of MHC-I, and poor T cell infiltration and function in the tumor microenvironment (TME) are prevalent in solid malignancies and associate with lack of response to immune checkpoint blockade (ICB). Novel combination therapies may overcome these barriers, thereby providing clinical benefit across solid tumors. STAR0602 is a first-in-class bifunctional therapeutic molecule, that simultaneously engages a nonclonal mode of T cell receptor (TCR) activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. Here, we examined the tumor-suppressive ability and mode of action of a murine surrogate of STAR0602 (mSTAR1302) in combination with the class I HDAC inhibitor Entinostat, an epigenetic modulator that enhances tumor immune cell recognition, in multiple tumor models, including ICB-refractory. Methods: mSTAR1302 and/or Entinostat were administered to mice bearing ICB-responsive breast EMT6 wild-type (wt) and ICB-refractory EMT6 β2-microglobulin (B2m)-deleted and CT26 (Kras G12Dmut colon) tumors. Anti-tumor efficacy, survival, and protective memory were monitored. Immune cell depletion studies were conducted. Comprehensive proteomic and immune profiling of tumor, tumor-draining lymph node (tdLN), and spleen cell populations was conducted. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: Combination therapy demonstrated significant enhancement in tumor suppression and prolonged survival in CT26 (MHC-I+), EMT6 wt (MHC-I+) and EMT6 β2m-deleted (MHC-Inull) tumors relative to single agent therapy, as well as enhanced tumor-specific protective memory responses. Tumor suppression was independent of CD4+ T cell immunity and associated with sustained activation and expansion of Vβ13 CD8+ T cells. Conclusions: Ongoing mechanistic studies indicate combination therapy to elicit complimentary mechanisms of tumor suppression beyond CD8+ T cells. Ongoing proteomic and single-cell transcriptomic investigation of MHC-I competent and null tumors will provide a deeper insight into the mode of action of this combination therapy. Collectively, these data support the use of STAR0602 in combination with Entinostat for the treatment of solid malignancies, including for patients harboring ICB-refractory tumors devoid of MHC class I. Citation Format: Katherine E. Lothstein, Asma S. Khelifa, Masaya Miyamoto, Christine M. Minnar, Lisa Poppe, Nicholas Roller, Andrew Bayliffe, Ke Liu, Jacques Moisan, Madan Katragadda, Zhen Su, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status [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 1425.

Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy

Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vβ2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vβ chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vβ scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vβ2+ Jurkat cells and Vβ2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vβ2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.

Expanded T lymphocytes in the cerebrospinal fluid of multiple sclerosis patients are specific for Epstein-Barr-virus-infected B cells.

Epstein-Barr virus (EBV) infection has long been associated with multiple sclerosis (MS), but the role of EBV in the pathogenesis of MS is not clear. Our hypothesis is that a major fraction of the expanded clones of T lymphocytes in the cerebrospinal fluid (CSF) are specific for autologous EBV-infected B cells. We obtained blood and CSF samples from eight relapsing-remitting patients in the process of diagnosis. We stimulated cells from the blood with autologous EBV-infected lymphoblastoid cell lines (LCL), EBV, varicella zoster virus, influenza, and candida and sorted the responding cells with flow cytometry after 6 d. We sequenced the RNA for T cell receptors (TCR) from CSF, unselected blood cells, and the antigen-specific cells. We used the TCR Vβ CDR3 sequences from the antigen-specific cells to assign antigen specificity to the sequences from the CSF and blood. LCL-specific cells comprised 13.0 ± 4.3% (mean ± SD) of the total reads present in CSF and 13.3 ± 7.5% of the reads present in blood. The next most abundant antigen specificity was flu, which was 4.7 ± 1.7% of the reads in the CSF and 9.3 ± 6.6% in the blood. The prominence of LCL-specific reads was even more marked in the top 1% most abundant CSF clones with statistically significant 47% mean overlap with LCL. We conclude that LCL-specific sequences form a major portion of the TCR repertoire in both CSF and blood and that expanded clones specific for LCL are present in MS CSF. This has important implications for the pathogenesis of MS.

Durable immunity to EBV after rituximab and third-party LMP-specific T cells: a Children’s Oncology Group study

AbstractPosttransplant lymphoproliferative disease (PTLD) in pediatric solid organ transplant (SOT) recipients is characterized by uncontrolled proliferation of Epstein-Barr virus–infected (EBV+) B cells due to decreased immune function. This study evaluated the feasibility, safety, clinical and immunobiological outcomes in pediatric SOT recipients with PTLD treated with rituximab and third-party latent membrane protein–specific T cells (LMP-TCs). Newly diagnosed (ND) patients without complete response to rituximab and all patients with relapsed/refractory (R/R) disease received LMP-TCs. Suitable LMP-TC products were available for all eligible subjects. Thirteen of 15 patients who received LMP-TCs were treated within the prescribed 14-day time frame. LMP-TC therapy was generally well tolerated. Notable adverse events included 3 episodes of rejection in cardiac transplant recipients during LMP-TC therapy attributed to subtherapeutic immunosuppression and 1 episode of grade 3 cytokine release syndrome. Clinical outcomes were associated with disease severity. Overall response rate (ORR) after LMP-TC cycle 1 was 70% (7/10) for the ND cohort and 20% (1/5) for the R/R cohort. For all cohorts combined, the best ORR for LMP-TC cycles 1 and 2 was 53% and the 2-year overall survival was 70.7%. vβT-cell receptor sequencing showed persistence of adoptively transferred third-party LMP-TCs for up to 8 months in the ND cohort. This study establishes the feasibility of administering novel T-cell therapies in a cooperative group clinical trial and demonstrates the potential for positive outcomes without chemotherapy for ND patients with PTLD. This trial was registered at www.clinicaltrials.gov as #NCT02900976 and at the Children's Oncology Group as ANHL1522.

Clinical Usefulness of T-Cell Receptor Vβ Repertoire Analysis for Differentiating Multisystem Inflammatory Syndrome in Japanese Children From Toxic Shock Syndrome and Kawasaki Disease.

The specific expansion of T-cell receptor β chain variable region (TCR-Vβ21.3 + ) CD4 + and CD8 + T cells was observed in Japanese patients with multisystem inflammatory syndrome in children. In contrast, these findings were not observed in patients with toxic shock syndrome and Kawasaki disease. T-cell receptor β chain variable region repertoire analysis to detect specific expansion of Vβ21.3 + T cells might be useful for differentiating multisystem inflammatory syndrome in children from toxic shock syndrome and Kawasaki disease.

Aplastic Anemia Patients Have Diverse T Cell Repertoires and Flexible T Cell Responses Similar to Healthy Individuals

Acquired aplastic anemia (AA) is a bone marrow failure disease caused by T cell-mediated autoimmune attack on hematopoietic stem and progenitor cells (HSPCs). Identifying autoreactive T cells in AA has been challenging. Advances in T cell receptor (TCR) sequencing and the discovery of Human Leukocyte Antigen Class I (HLA-I) risk alleles linked to autoantigen presentation in AA have rekindled interest in exploring T cell repertoires in AA. We hypothesized that autoreactive CD8 + T cells in AA may have convergent TCR structures allowing them to recognize AA autoantigens presented by specific HLA-I risk alleles. To test our hypothesis, we analyzed TCR repertoires and HLA-restricted CD8 + T cell reactivity in AA patients. Using TCR Vβ (TRB) sequencing, we analyzed 83 patient samples obtained at AA diagnosis or relapse. High-risk alleles HLA-B*14:02 and B*40:02 were present in 26% and 10% of patients, respectively. HLA-DR*15 was present in 24%. Patient samples were largely sorted bone marrow (BM) CD8 + T lymphocytes. Patient TRB repertoires were compared to those in 709 control samples, largely of unsorted peripheral blood (PB). To account for sampling differences, we downsampled sequences to the minimum number of clones in the matching tissue. Normalized clone size distributions were visualized on rank plots (Fig 1). TRB repertoires in both patients and controls were diverse. Highly expanded clones (>5%) were rare in either group. Patients had 1,415,069 unique CDR3 amino acid sequences (CDR3AA), of which 106,446 (7.5%) were shared by ≥2 patients. Among the 106,446 shared CDR3AA, 95.4% were found in controls, and 4.3% TCRs had reported specificity to exogenous (mostly viral) or endogenous antigens in VDJdb. 4,920 (4.6%) of the shared CDR3AA were detected only in patients (median 2 patients (2-25)). Of these, 190 exhibited TCR convergence in ≥1 patient, such that the same CDR3AA was encoded by ≥2 distinct nucleotide sequences. Clustering 4,920 patient-associated CDR3AA using GLIPH (Grouping of lymphocyte interactions by paratope hotspots) identified 43 TCR antigen convergence groups. CDR3AA in each cluster were not significantly expanded in the majority of patients and were not enriched for the high-risk alleles HLA-B*14:02 or B*40:02. Our data suggest that most CDR3AA in AA are private (unique to individual patients). Shared autoreactive CDR3AA may exist in patients but are likely to also be present in healthy individuals. As the next step towards identifying autoreactive T cells, we focused on AA patients with known HLA-I risk alleles. We tested their CD8 + T cells for reactivity with endogenously expressed antigens by stimulating T cells with hematopoietic cell lines engineered to express single HLA-I alleles matching those of the patients (Fig 2). TRB sequencing showed increased clonality and significant expansion of a subset of clonotypes after T cell stimulation. Interferon γ enzyme-linked immunospot assays confirmed the expanded T cells' responsiveness to antigens presented by HLA alleles used for T cell stimulation. Additionally, these expanded T cells demonstrated variable reactivity to cells expressing other HLA alleles from the same patient. Among the 1% of CDR3AA clonotypes most highly expanded with single HLA-I alleles (63-3000-fold), we identified several that were expanded in an HLA-restricted manner. However, several highly expanded CDR3AA were highly expanded with multiple HLA alleles. Potential reasons for expansion of some CDR3AA after stimulation with cells expressing different HLA alleles include 1) binding of the same CDR3AA to different antigens presented by distinct HLA alleles, 2) expansion in response to the same antigens presented by distinct HLA alleles, or 3) expansion in response to non-HLA antigens. In conclusion, our data show diverse and mostly private TRB repertoires in AA patients. Our in vitro findings suggest that much like T cell responses in non-AA individuals, T cell responses in AA are diverse and exhibit flexibility with respect to activation in response to various endogenous antigens in the context of different self HLA-I alleles. Given the diversity of TCR responses observed in AA patients, the tissue specificity of autoimmune attack and established association with risk HLA-I alleles in AA remain a mystery and likely require HSPC-specific autoantigen expression and efficient autoantigen presentation by individual patients' HLA-I alleles.

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

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 (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) 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 g7 MHC 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 manifested by Vβ3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs 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 immune disorders.

Clinical Features and Outcomes in Large Granular Lymphocyte Leukemia-Associated Pure Red Cell Aplasia with STAT3 Mutation

Background Large granular lymphocyte leukemia (LGLL) is a rare disease frequently complicated with pure red cell aplasia (PRCA) [1, 2]. STAT3 mutations have been describled in 30-40% of LGLL patients [3]. The aim of this work was to evaluate whether STAT3 mutations might be associated with specific clinical features and outcomes in LGLL-associated PRCA. Methods and results From January 2016 to July 2023, 81 patients with LGLL-associated PRCA were enrolled in the China Eastern Cooperation Group for Anemia (CECGA) database (ChiCTR2100043485). As an initial step in the STAT3 mutational analysis, we screened all patients with Sanger sequencing or Next-generation sequencing (NGS). The initial dose of CsA was 3-5mg/kg/day, and the serum concentration was adjusted to be 150-200ng/mL based on adverse reactions. After 12 months of maintenance, the dosage was slowly reduced. Cyclophosphamide combined with prednisone (CP) regimen was used as a salvage therapy for patients who failed to CsA. The initial dose of cyclophosphamide and prednisone were 100mg/day and 0.5-1mg/kg/day, respectively. Among the 81 cases, 26% of them were positive for STAT3 mutations. The clinical characteristics of patients with or without STAT3 mutation were summarized in Table 1. Patients with STAT3 mutations had a higher reticulocyte percentage (0.88% vs 0.28%, P=0.039) and red cell distribution width-coefficient of variation (18.8% vs 15.8%, P=0.008) than patients without STAT3 mutations. Y640F mutation were found in 9 of 21 cases. Subgroup analysis showed that patients with Y640F mutation were associated with younger age (44 vs 65 years old, P=0.007) and higer lymphocyte percentage in peripheral blood (63.7% vs 34.4%, P=0.033). Deep sequencing of rearranged T-cell receptor Vβ complementarity-determining region 3 by NGS was conducted in 61 patients. The predominant V-gene of LGLL clonotypes belonged to the TRBV06 family gene was detected in 12 (25%) patients. The expression of TRBV06 family gene was a litter lower in patients with STAT3 mutation than non-mutant groups (8% vs 31%, P=0.189). The complete response rate (CRR) [31% (5/16) vs 33% (19/58), P=0.909] and overall response rate (ORR) [56% (9/16) vs 50% (29/58), P=0.658] of cyclosporine (CsA) treatment were similar in patients with STAT3 mutations or not. In STAT3 mutant group, the CRR [54% (7/13) vs 31% (5/16), P=0.274] and ORR [85% (11/13) vs 56% (9/16), P=0.130] of CP regimen tended to be better than CsA . 6 patients (67%) relapsed among the 9 patients who responded to CsA in STAT3 mutant group. In patients without STAT3 mutation, 19 of 29 (66%) CsA-responders relapsed. There was no siginificant difference in the relapse-free survival between the two group (Figure 1). Discussion/Conclusions In summary, STAT3 mutation was frequently recognized in LGLL-associated PRCA, and the hotspot site was Y640F. Patients with STAT3 mutations responded to CsA as well as those without the mutation. CP regimen could be used as a salvage therapy for patients who failed to CsA. Reference 1. Balasubramanian SK, Sadaps M, Thota S, et al. Rational management approach to pure red cell aplasia. Haematologica. 2018, 103(2): 221-230. 2. Wu X, Cheng L, Liu X, et al. Clinical characteristics and outcomes of 100 adult patients with pure red cell aplasia. Ann Hematol. 2022, 101(7):1493-1498. 3. Barilà G, Teramo A, Calabretto G, et al. Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients. Leukemia. 2020, 34: 1116-1124.

Influence of Radiation Fractionation on Immune Repertoire Diversity in Solid Tumor Patients

Radiation (RT)-induced lymphopenia (RIL) occurs in up to 75% of patients undergoing RT and is associated with worse tumor control and survival across a spectrum of solid tumors. Patients undergoing hypofractionated RT are at lower risk of RIL compared with patients treated with more prolonged RT courses. However, it is unknown whether immune repertoire diversity is similarly affected by fractionation scheme in patients undergoing RT. This prospective study analyzed RT-induced changes in immune repertoire diversity in patients treated with conventionally (CFRT) vs hypofractionated RT (HFRT). RNA-based T and B cell receptor sequencing was performed on peripheral lymphocytes collected prospectively before RT and within 4 weeks of the last RT fraction from 23 patients (18 men, 5 women, median age 67 y) with primary solid tumors undergoing CFRT (≤3 Gy/day x ≥10 days, n = 13) or HFRT (≥5 Gy/day x ≤5 days, n = 10). Absolute lymphocyte counts (ALC; cells/μL) were obtained from clinical laboratory data. The number of unique CDR3 receptors (uCDR3) and Shannon entropy were used to monitor changes in T (TCR Vβ) and B (BCR IgH) receptor diversity. ALC decreased after RT in 90% (20/22) of patients (mean pre-RT ALC 1830 vs 1040 post-RT, p <0.001). Mean % ALC loss was greater in CFRT vs HFRT patients (44.3 vs. 35.2%). After RT, entropy in IgH and Vβ decreased in 18/23 (78%) and 17/23 (74%) patients, respectively; uCDR3 in IgH and Vβ decreased in 14/23 (61%) and 15/23 (65%). Among patients with concordant decreases in ALC and uCDR3, a moderate correlation between magnitude of ALC loss and uCDR3 levels in the T-cell receptor Vβ was observed (r = 0.64, p = 0.02). For both receptor species studied (IgH and Vβ), HFRT patients were more likely to have an increase in either entropy or uCDR3 in the face of decreased ALC (36 vs 15%, X2 p = 0.03). Furthermore, while decreases in entropy were observed among the CFRT patients for both IgH (median entropy 10.4 vs 9.4, p = 0.06) and Vβ (9.7 vs 8.1, p = 0.02), entropy did not significantly change following RT in the HFRT patients (IgH 10.6 vs 10.4, p = 0.74 and Vβ 10.9 vs 10.8, p = 0.24). RT-induced changes in immune repertoire diversity are variably reflected in the peripheral ALC. Both HFRT and CFRT depleted circulating lymphocytes, but patients undergoing HFRT were more likely to experience increases in T and B cell diversity metrics despite lymphopenia. It is therefore possible that relative sparing of repertoire diversity among patients undergoing HFRT could increase the likelihood of tumor antigen recognition by peripheral blood lymphocytes. As immune repertoire diversity is associated with the likelihood of response to immunotherapy, these findings also have implications for RT-immunotherapy combinations. Further study is required to understand the relationship between RT exposure to circulating lymphocyte populations and immune repertoire diversity.

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)

TCRvβ-CART therapy mediates high-precision targeting of malignant T-cell clones.

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoid malignancies associated with poor prognosis due to ineffective treatment options and high rates of relapse. The success of chimeric antigen receptor T-cell (CART) therapy for certain hematologic malignancies makes it an attractive treatment option for PTCLs. However, shared expression of potential target antigens by both malignant and healthy T cells poses a challenge. Current prospective CART approaches cause a high degree of on-target, off-tumor activity, resulting in fratricide during CART expansion, depletion of healthy Tcells invivo, and immune compromise in the patient. To limit off-tumor targeting, we sought to develop a CART platform specific for a given T-cell receptor vβ (TCRvβ) family thatwould endow CAR-modified T cells with the ability to mediate lysis of the clonal malignant population while preserving the majority of healthy T cells. Here, CAR constructs specific for multiple TCRvβ family members were designed and validated. Our results demonstrate that TCRvβ-family-specific CARTs (TCRvβ-CARTs) recognize and kill TCRvβ-expressing target cells. This includes specific self-depletion of the targeted cell subpopulation in the CART product and lysis of cell lines engineered to express a target TCRvβ family. Furthermore, TCRvβ-CARTs eliminated the dominant malignant TCRvβ clonein 2 patient samples. Finally, in immunodeficient mice, TCRvβ-CARTs eradicated malignant cells in a TCRvβ-dependent manner. Importantly, the nontargeted TCRvβ families were spared in all cases. Thus, TCRvβ-CART therapy provides a potential option for high-precision treatment of PTCL with limited healthy T-cell depletion.

Abstract LB338: A novel class of T cell-activators targeting germline encoded TCRβ chains promotes antitumor activity in PD1-refractory models through expansion of a clonally enriched effector memory T cell subset

Abstract Background: Here we describe a new class of selective T cell activator molecule that combines antibody targeting of the variable (V) β regions of the T cell receptor (TCR) with a costimulatory cytokine. STAR0602, a molecule that selectively targets T cells expressing Vβ6 and Vβ10 TCRs and delivers IL-2R co-stimulation, activates and expands human Vβ6 and Vβ10 T cells that are enriched in tumor infiltrating lymphocytes (TIL) relative to other Vβ subsets. Using this molecular design, STAR0602 and molecules targeting other Vβ T cell subsets simultaneously engage a novel, non-clonal mode of TCR activation with cytokine co-stimulation. Methods: The effects of STAR0602 on activation and expansion of primary human T cells were assessed in vitro by flow cytometry, homogeneous time-resolved fluorescence, TCRseq, and NanoString. Several murine surrogate molecules targeting different Vβ T cell subsets with a range of baseline frequencies were tested in murine syngeneic tumor models. Tumors were excised for phenotyping of TILs including tetramer staining of antigen specific T cells using flow cytometry, and scRNAseq/TCRseq. Results: In vitro, STAR0602 induced TCR signaling and IL-2R pathway activation in human T cells that preceded expansion of Vβ6/Vβ10 T cells that acquired an atypical central memory T cell (TCM) phenotype. Across PD1-refractory syngeneic murine tumor models, monotherapy with fusion molecules targeting different Vβ TCR and delivering IL-2R co-stimulation induced potent anti-tumor activity that was associated with expansion and accumulation of targeted Vβ T cell subsets in blood and tumors. In vivo anti-tumor activity was dependent on the accumulation of effector memory T or TCM Vβ T cells expressing a novel effector gene signature. Additional analysis of TILs showed increased clonality in de novo expanded Vβ T cells compared to controls in addition to increased numbers of tumor-specific T cells. Conclusions: To our knowledge, the use of therapeutic antibodies to selectively target and expand Vβ T cell subsets and thereby modulate the germline TCR repertoire for therapeutic benefit is novel. Here we show that STAR0602 selectively binds and activates human Vβ6/Vβ10 T cells and promotes a novel TCM phenotype. Murine surrogate molecules targeting different Vβ T cell subsets demonstrated potent and durable single-agent anti-tumor activity even when targeting low frequency Vβ subsets. These expanded Vβ T cells appear to drive enhanced anti-tumor activity due to their increased effector potentials, novel memory phenotype, and a striking enrichment in clonal diversity. In summary, STAR0602 and similar Vβ TCR-targeting constructs have the potential to remodel the adaptive immune response to solid tumors that are refractory to anti-PD1 therapy. A Phase 1/2 clinical trial of STAR0602 is ongoing in patients with advanced metastatic cancers. Citation Format: Jacques Moisan, Madan Katraggada, Jonathan Hsu, Jessica Lowry, Wei Huang, Jian Tang, Roya Servattalab, Gurkan Guntas, Raj Chopra, Zhen Su, Andrew Bayliffe. A novel class of T cell-activators targeting germline encoded TCRβ chains promotes antitumor activity in PD1-refractory models through expansion of a clonally enriched effector memory T cell subset [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB338.

Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy

BackgroundThe wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons...

Clonal composition and persistence of antigen-specific circulating T follicular helper cells.

T follicular helper (TFH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH ) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non-cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non-cTFH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu-specific cTFH and non-cTFH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1+ cTFH cells had a "GC TFH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cTFH and non-cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non-cTFH cells and on the heterogeneity and persistence of antigen-specific human cTFH cells.

Rapid evaluation of T cell clonality in the diagnostic work-up of mature T cell neoplasms: TRBC1-based flow cytometric assay experience

The identification of mature T cell neoplasms by flow cytometry is often challenging, due to overlapping features with reactive T cells and limitations of currently available T cell clonality assays. The description of an antibody specific for one of two mutually exclusive T cell receptor (TCR) β-chain constant regions (TRBC1) provides an opportunity to facilitate the detection of clonal TCRαβ+ T cells based on TRBC-restriction. Here we prospectively analyzed 14 healthy controls and 63 patients with the flow cytometry protocol currently used for suspected T cell neoplasm implemented with immunostaining targeting TRBC1. Specimens were firstly classified in 3 groups based on clinical records data, laboratory findings and immunophenotypic features. T cell clonality was assessed by TCR Vβ repertoire analysis and the new rapid TRBC1 assay. Results showed that TRBC1 unimodal expression was unequivocally associated with samples presenting with immunophenotypic aberrancies. Moreover, we demonstrated that the use of TRBC1 is useful in solving uncertain cases and confirmed the high sensitivity of the method in identifying small T cell clones of uncertain significance (T-CUS). Finally, we found a high degree of concordance (97%) comparing the currently available clonality assessment methods with the proposed new method. In conclusion, our results provided real-life evidence of the utility of TRBC1 introduction in the flow cytometric clonality evaluation for the routine diagnostic work-up of T cell neoplasms.

T-cell receptor variable region usage in Chagas disease: A systematic review of experimental and human studies.

T cells recognize their ligand, the peptide major histocompatibility complex (MHC), via the T-cell receptor (TCR), which is composed of covalently linked α and β or γ and δ chains. This recognition is critical for T-cell ontogeny and controls the selection, activation, and function of T lymphocytes. Specific TCR αβ variable regions have been associated with immunopathogenesis of Chagas disease. Here, we present a systematic review that compiles experimental in vivo and human data regarding the preferential expression of variable alpha (Vα) and variable beta (Vβ) chain regions in Trypanosoma cruzi infection. The original studies indexed in PubMed/Medline, Scopus, and Web of Science databases were screened according to the PRISMA strategy. The analysis showed that expression of TCR Vα subfamilies were evaluated in one human study, and, unlike TCR Vβ, TCR Vα presented a more restricted usage. Despite the great variability in the usage of TCR Vβ regions in human Chagas disease, a down-regulation of TCR Vβ5 expression by T cells from patients in the acute phase of the disease was shown. Opposingly, this TCR region was found overly expressed in CD4+ T cells from chronic Chagas patients. It was also demonstrated that murine Vβ9+ T cells derived from nonlymphoid organs of T. cruzi-infected animals had a modulatory profile, while splenic Vβ9+ T cells produced inflammatory cytokines, indicating that although they display the same TCR Vβ region usage, these cells are functionally distinct. Despite the limitations of few papers and year of publication of the studies, compiling the data derived from them reveals that further investigation of TCR usage will point to their potential role in protective or pathogenic responses, as biomarkers of disease progression, and in the search for dominant peptides potentially useful for the development of vaccines or therapies.

Oligoclonal T-cell expansion in a patient with bone marrow failure after CD19 CAR-T therapy for Richter-transformed DLBCL

The advent of chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape of refractory B-cell malignancies. Real-world evidence has highlighted the high incidence of hematological toxicity, including prolonged and profound cytopenia. Here, we present a case of bone marrow (BM) failure in a 57-year old man with DLBCL-type Richter transformation receiving tisagenlecleucel in a standard-of-care setting. His clinical course was notable for underlying BM infiltration, severe cytokine release syndrome, and multiple infectious complications in the setting of prolonged, profound neutropenia. Cytokine profiling revealed a signature consistent with acquired aplastic anemia - including downregulation of CD40-L and EGF. Flow cytometric analyses demonstrated migration of CAR T-cells to the BM, as well as peripheral blood expansion of both CAR and non-CAR bearing CD8+CD57+ T-cells, an immunophenotype linked to oligoclonality in aplastic anemia. From single-cell level transcriptomics, this cytotoxic T-cell population was characterized by T-cell receptor Vβ oligoclonal expansion and post-CAR clonal drift. Gene expression profiling revealed upregulation of exhaustion markers on (CAR) T-cells and decreased expression of genes involved in STAT signalling and inflammatory response. In conclusion, this case highlights the complex nature of CAR-T-related hematological toxicity and introduces oligoclonal (CAR) T-cell expansion as a potential contributing pathophysiologic mechanism.