T-cell Receptor Repertoire Research Articles

T-cell receptor determinants of response to chemoradiation in locally-advanced HPV16-driven malignancies.

The effect of chemoradiation on the anti-cancer immune response is being increasingly acknowledged; however, its clinical implications in treatment responses are yet to be fully understood. Human papillomavirus (HPV)-driven malignancies express viral oncogenic proteins which may serve as tumor-specific antigens and represent ideal candidates for monitoring the peripheral T-cell receptor (TCR) changes secondary to chemoradiotherapy (CRT). We performed intra-tumoral and pre- and post-treatment peripheral TCR sequencing in a cohort of patients with locally-advanced HPV16-positive cancers treated with CRT. An in silico computational pipeline was used to cluster TCR repertoire based on epitope-specificity and to predict affinity between these clusters and HPV16-derived epitopes. Intra-tumoral repertoire diversity, intra-tumoral and post-treatment peripheral CDR3β similarity clustering were predictive of response. In responders, CRT triggered an increase peripheral TCR clonality and clonal relatedness. Post-treatment expansion of baseline peripheral dominant TCRs was associated with response. Responders showed more baseline clustered structures of TCRs maintained post-treatment and displayed significantly more maintained clustered structures. When applying clustering by TCR-specificity methods, responders displayed a higher proportion of intra-tumoral TCRs predicted to recognise HPV16 peptides. Baseline TCR characteristics and changes in the peripheral T-cell clones triggered by CRT are associated with treatment outcome. Maintenance and boosting of pre-existing clonotypes are key elements of an effective anti-cancer immune response driven by CRT, supporting a paradigm in which the immune system plays a central role in the success of CRT in current standard-of-care protocols.

Identification of TCR repertoire patterns linked with anti-cancer immunotherapy.

The highly diverse T cell receptor (TCR) repertoire is a crucial component of the adaptive immune system that aids in the protection against a wide variety of pathogens. This TCR repertoire, comprising the collection of all TCRs in an individual, is a valuable source of information on both recent and ongoing T cell activation. Cancer cells, like pathogens, have the ability to trigger an adaptive immune response. However, because cancer cells use a variety of strategies to escape immune responses, this is often insufficient to completely eradicate them. As a result, immunotherapy is a promising treatment option for cancer patients. This treatment is expected to increase T cell activation and subsequently alter the TCR repertoire composition in these patients. Monitoring TCR repertoires before and after immunotherapy can therefore provide additional insight into T cell responses and might identify cancer-associated TCR sequences. Here we present a computational strategy to identify those changes in the TCR repertoire that occur after treatment with immunotherapy. Since this method allows the identification of TCR patterns that might be treatment-associated, it can help future research by revealing those patterns that are related with response. This TCR analysis workflow is illustrated using public data from three different cancer patients who received anti-PD-1 treatment.

The molecular mechanisms of CD8+ T cell responses to SARS-CoV-2 infection mediated by TCR-pMHC interactions.

Cytotoxic CD8+ T lymphocytes (CTLs) have been implicated in the severity of COVID-19. The TCR-pMHC ternary complex, formed by the T cell receptor (TCR) and peptide-MHC (major histocompatibility complex), constitutes the molecular basis of CTL responses against SARS-CoV-2. While numerous studies have been conducted on T cell immunity, the molecular mechanisms underlying CTL-mediated immunity against SARS-CoV-2 infection have not been well elaborated. In this review, we described the association between HLA variants and different immune responses to SARS-CoV-2 infection, which may lead to varying COVID-19 outcomes. We also summarized the specific TCR repertoires triggered by certain SARS-CoV-2 CTL epitopes, which might explain the variations in disease outcomes among different patients. Importantly, we have highlighted the primary strategies used by SARS-CoV-2 variants to evade T-cell killing: disrupting peptide-MHC binding, TCR recognition, and antigen processing. This review provides valuable insights into the molecule mechanism of CTL responses during SARS-CoV-2 infection, aiding efforts to control the pandemic and prepare for future challenges.

Multimodal profiling of chordoma immunity reveals distinct immune contextures

BackgroundChordomas are rare cancers from the axial skeleton which present a challenging clinical management with limited treatment options due to their anatomical location. In recent years, a few clinical trials...

Clinical significance of peripheral T-cell receptor repertoire profiling and individualized nomograms in patients with gastrointestinal cancer treated with anti-programmed death 1 antibody.

Immune checkpoint inhibitors (ICIs) have significant clinical benefit for a subset of patients with gastrointestinal cancers (GICs) including esophageal cancer, gastric cancer and colorectal cancer. However, it is difficult to predict which patients will respond favorably to immune checkpoint blockade therapy. Thus, this study was initiated to determine if peripheral T-cell receptor (TCR) repertoire profiling could predict the clinical efficacy of anti-programmed death 1 (PD-1) treatment. Blood samples from 31 patients with GICs were collected before anti-PD-1 antibody treatment initiation. The clinical significance of the combinatorial diversity evenness of the TCR repertoire [the diversity evenness 50 (DE50), with high values corresponding to less clonality and higher TCR diversity] from peripheral blood mononuclear cells (PBMCs) was evaluated in all the enrolled patients. A highly predictive nomogram was set up based on peripheral TCR repertoire profiling. The performance of the nomogram was assessed by receiver operating characteristic (ROC) curve, concordance index (C-index), and calibration curves, and decision curve analysis (DCA) was used to assess its clinical applicability. Compared to non-responders [progression disease (PD)], the DE50 scores were significantly higher in responders [stable disease (SD) and partial response (PR)] (P=0.018). Patients with a high DE50 score showed better progression-free survival (PFS) than those with a low DE50 score (P=0.0022). The multivariable Cox regression demonstrated that high DE50 and low platelet-lymphocyte ratio (PLR) were significant independent predictors for better PFS when treated with anti-PD-1 antibody. Furthermore, a highly predictive nomogram was set up based on peripheral TCR repertoire profiling. The area under the curves (AUCs) of this system at 3-, 6- and 12-month PFS reached 0.825, 0.802, and 0.954, respectively. The nomogram had a C-index of 0.768 [95% confidence interval (CI): 0.658-0.879]. Meanwhile, the calibration curves also demonstrated the reliability and stability of the model. High DE50 scores were predictive of a favorable response and longer PFS to anti-PD-1 treatment in GIC patients. The nomogram based on TCR repertoire profiling was a reliable and practical tool, which could provide risk assessment and clinical decision-making for individualized treatment of patients.

Perturbations of the T-cell receptor repertoire in response to SARS-CoV-2 in immunocompetent and immunocompromised individuals

Functional T-cell responses are essential for virus clearance and long-term protection after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, whereas certain clinical factors, such as older age and immunocompromise, are associated with worse outcome. We sought to study the breadth and magnitude of T-cell responses in patients with coronavirus disease 2019 (COVID-19) and in individuals with inborn errors of immunity (IEIs) who had received COVID-19 mRNA vaccine. Using high-throughput sequencing and bioinformatics tools to characterize the T-cell receptor β repertoire signatures in 540 individuals after SARS-CoV-2 infection, 31 IEI recipients of COVID-19 mRNA vaccine, and healthy controls, we quantified HLA class I- and class II-restricted SARS-CoV-2-specific responses and also identified several HLA allele-clonotype motif associations in patients with COVID-19, including a subcohort of anti-type 1 interferon (IFN-1)-positive patients. Our analysis revealed that elderly patients with COVID-19 with critical disease manifested lower SARS-CoV-2 T-cell clonotype diversity as well as T-cell responses with reduced magnitude, whereas the SARS-CoV-2-specific clonotypes targeted a broad range of HLA class I- and class II-restricted epitopes across the viral proteome. The presence of anti-IFN-I antibodies was associated with certain HLA alleles. Finally, COVID-19 mRNA immunization induced an increase in the breadth of SARS-CoV-2-specific clonotypes in patients with IEIs, including those who had failed to seroconvert. Elderly individuals have impaired capacity to develop broad and sustained T-cell responses after SARS-CoV-2 infection. Genetic factors may play a role in the production of anti-IFN-1 antibodies. COVID-19 mRNA vaccines are effective in inducing T-cell responses in patients with IEIs.

Alteration of γδ T cell subsets in non-human primates transplanted with GGTA1 gene-deficient porcine blood vessels.

αGal-deficient xenografts are protected from hyperacute rejection during xenotransplantation but are still rejected more rapidly than allografts. Despite studies showing the roles of non-Gal antibodies and αβ T cells in xenograft rejection, the involvement of γδ T cells in xenograft rejection has been limitedly investigated. Six male cynomolgus monkeys were transplanted with porcine vessel xenografts from wild-type (n=3) or GGTA1 knockout (n=3) pigs. We measured the proportions and T cell receptor (TCR) repertoires of blood γδ T cells before and after xenotransplant. Grafted porcine vessel-infiltrating immune cells were visualized at the end of experiments. Blood γδ T cells expanded and infiltrated into the graft vessel adventitia following xenotransplantation of α-Gal-deficient pig blood vessels. Pre- and post-transplant analysis of γδ TCR repertoire revealed a transition in δ chain usage post-transplantation, with the expansion of several clonotypes of δ1, δ3, or δ7 chains. Furthermore, the distinctions between pre- and post-transplant δ chain usages were more prominent than those observed for γ chain usages. γδ TCR repertoire was significantly altered by xenotransplantation, suggesting the role of γδ T cells in sustained xenoreactive immune responses.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency

AbstractRecombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based “universal gene-correction” approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Decoding the diagnostic potential of T cell repertoires in peripheral blood of patients from amnestic mild cognitive impairment to Alzheimer's disease.

Alzheimer's disease (AD) is currently an incurable neurodegenerative disorder and is the most common etiological cause of dementia. Consequently, it has severe burden on its patients and on their caregivers and represents a global health concern. Clinical investigations have indicated that a dysregulation of peripheral T cell immune homeostasis may be involved in the pathogenesis of AD, as well as in the early stages of AD, characterized by mild cognitive impairment (MCI). However, the characteristics and concomitant feasibility of the use of T-cell receptor (TCR) typing for disease diagnosis remains largely unknown. We employed a high-throughput sequencing and multidimensional bioinformatics analyses for the identification of TCR repertoires present in peripheral blood samples of 10 patients with amnestic MCI (aMCI), 10 patients with AD, and 10 healthy controls (HCs). Based on the characteristics of the TCR repertoires in the amount and diversity of combinations of V-J, the spectrum of immune defense, and differentially expressed genes (DEGs), single and specific TCR profiles were observed in the patient samples of aMCI and AD compared to profiles of HCs. In particular, the diversity of TCR clonotypes manifested a pattern of "decreased first and then increased" pattern during the progression from aMCI to AD, a pattern that was not observed in HC samples. Additionally, a total of 46 and 35 amino acid CDR3 sequences with consistent and reverse expressive abundance with diversity of TCR clonotypes were identified, respectively. Taken together, we provide novel and essential preliminary evidence demonstrating the presence of diversity of T cell repertoires from differentially expressed V-J gene segments and amino acid clonotypes using peripheral blood samples from patients with AD, aMCI, and from HC. Such findings have the potential to reveal potential mechanisms through which aMCI progresses to AD and provide a reference for the future development of immune-related diagnoses and therapies for AD.

Glioma-derived ANXA1 suppresses the immune response to TLR3 ligands by promoting an anti-inflammatory tumor microenvironment.

A highly immunosuppressive tumor microenvironment (TME) and the presence of the blood‒brain barrier are the two major obstacles to eliciting an effective immune response in patients with high-grade glioma (HGG). Here, we tried to enhance the local innate immune response in relapsed HGG by intracranially injecting poly(I:C) to establish a robust antitumor immune response in this registered clinical trial (NCT03392545). During the follow-up, 12/27 (44.4%) patients who achieved tumor control concomitant with survival benefit were regarded as responders in our study. We found that the T-cell receptor (TCR) repertoire in the TME was reshaped after poly(I:C) treatment. Based on the RNA-seq analysis of tumor samples, the expression of annexin A1 (ANXA1) was significantly upregulated in the tumor cells of nonresponders, which was further validated at the protein level. In vitro and in vivo experiments showed that ANXA1 could induce the production of M2-like macrophages and microglia via its surface receptor formyl peptide receptor 1 (FPR1) to establish a Treg cell-driven immunosuppressive TME and suppress the antitumor immune response facilitated by poly(I:C). The ANXA1/FPR1 signaling axis can inhibit the innate immune response of glioma patients by promoting an anti-inflammatory and Treg-driven TME. Moreover, ANXA1 could serve as a reliable predictor of response to poly(I:C), with a notable predictive accuracy rate of 92.3%. In light of these notable findings, this study unveils a new perspective of immunotherapy for gliomas.

Abstract A003: High-throughput TCR sequencing demonstrates induction of long-lasting HPV16-specific T cell responses in VB10.16 vaccinated advanced cervical cancer patients

Abstract Background: Assessment of vaccine-induced T cell responses is a primary pharmacodynamic readout in cancer vaccine clinical trials. High-throughput sequencing of T cell receptors (TCRs) is emerging as a rapid and scalable method, suitable for late-stage clinical trials, and offers sensitive and accurate quantification of the full T cell repertoire. Here we performed ELISpot and TCR sequencing on serial peripheral blood mononuclear cell (PBMC) samples from a clinical phase 2 trial investigating the therapeutic HPV16 cancer vaccine VB10.16 in combination with atezolizumab in advanced cervical cancer patients (NCT04405349). Methods: T cell responses were assessed by ex vivo IFN-g ELISpot (n=36). Immunosequencing of the TCRB locus was performed in 10 patients with available PBMCs, representing clinical response, stable disease, and progressive disease as best overall response. Immunosequencing of PBMC-derived gDNA was performed on up to five timepoints per patient, ranging from week 10 to 52. Novel and expanded clones from baseline to on-treatment timepoints were determined by a differential abundance framework, using binomial models in pair-wise comparisons, enabling longitudinal tracking of significantly expanded T cell clones. The sequences were matched and annotated to HPV16-specific TCRs present in a proprietary database of confirmed HLA class I HPV16-specific T cell clones; the database was constructed from querying the T cell repertoires of 92 healthy donors with 146 peptides derived from HPV16 E6 and E7 in MIRA assay. Results: An increased T cell response was significantly associated with disease control assessed by RECIST1.1 (n=24 patients with disease control vs n=12 patients with progressive disease, p=0.0113) and patients with a >2-fold increase measured by ex vivo IFN-g ELISpot showed a numerically improved progression-free survival (8 vs 3.7 months median PFS). The longitudinal tracking of TCRs provided a detailed insight into the dynamics of the overall TCR repertoire during treatment. Expansion of both pre-existing and newly expanded T cell clones was observed from week 10 and persisted until the end of treatment. Despite the non-exhaustive database, at least one verified HPV16-specific CD8 T cell clone was expanded in 8 out of 10 patients, supporting that the clonotypic expansion is caused by VB10.16. In 5 out of 7 patients with disease control, the breadth of HPV16-specific TCRs increased after vaccination, demonstrating induction of clinically relevant T cell responses. Conclusions: We demonstrate induction of strong and long-lasting HPV16-specific T cell responses after treatment with VB10.16 and atezolizumab in advanced cervical cancer patients. Induction of HPV16-specific T cell responses was significantly correlated with clinical efficacy. Immunosequencing and HPV16-specific annotation allowed longitudinal tracking of expanded TCRs and demonstrated a potential clinical relevance of increased repertoire diversity of HPV16-specific CD8 T cells in patients. Citation Format: Kaja C G Berg, Paula Bousquet, Milena Blaga, Thomas Bello, Mohammad Arabpour, Mikkel W Pedersen, Karoline Schjetne. High-throughput TCR sequencing demonstrates induction of long-lasting HPV16-specific T cell responses in VB10.16 vaccinated advanced cervical cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A003.

Abstract C007: Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping 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. A new technology we developed that combines profiling of all human TCR and BCR variable regions with phenotypic characterization of immune cells using the same workflow could be particularly useful. The TCR and BCR immunophenotyping method proposed involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, and subsequently determining the expression levels of the most informative T- and B-cell phenotyping genes. Preliminary 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 from the TCR and BCR clonotypes profile combined with RNA expression profiling of the same samples results in a richer data set that includes the identification of major immune cell subtypes and their activation status. Data from human cancer tissues and whole blood samples will be presented. Citation Format: Alex Chenchik, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Khadija Ghias, Lester Kobzik. Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C007.

Machine learning analysis of the T cell receptor repertoire identifies sequence features of self-reactivity

The Tcell receptor (TCR) determines specificity and affinity for both foreign and self-peptides presented by the major histocompatibility complex (MHC). Although the strength of TCR interactions with self-pMHC impacts Tcell function, it has been challenging to identify TCR sequence features that predict Tcell fate. To discern patterns distinguishing TCRs from naive CD4+ Tcells with low versus high self-reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that identifies population-level differences between TCRβ sequence sets. This approach revealed that weakly self-reactive Tcell populations were enriched for longer CDR3β regions and acidic amino acids. We tested our ML predictions of self-reactivity using retrogenic mice with fixed TCRβ sequences. Extrapolating our analyses to independent datasets, we predicted high self-reactivity for regulatory Tcells and slightly reduced self-reactivity for Tcells responding to chronic infections. Our analyses suggest a potential trade-off between TCR repertoire diversity and self-reactivity. A record of this paper's transparent peer review process is included in the supplemental information.

Exploration of B and T Cell Receptor Repertoires Reveals Distinct Mechanisms in Pure Red Cell Aplasia, Autoimmune Hemolytic Anemia, and Aplastic Anemia

Background: Pure red cell aplasia (PRCA), autoimmune hemolytic anemia (AIHA) and aplastic anemia (AA) are immune-mediated diseases that mainly attack erythrocyte or erythroid progenitor cells. This study aimed to investigate changes related to autoimmunity in the B cell receptor (BCR) and T cell receptor (TCR) repertoires in these diseases. Methods: Patients with newly diagnosed primary PRCA, AIHA, and AA and healthy individuals with matched age and sex (normal controls, NC) were recruited. Peripheral blood was collected, and BCR and TCR repertoires were sequenced by next-generation immunosequencing. The diversity and clonal expansion of the repertoires, gene and gene combination usage, somatic hypermutation (SHM) of BCR, and the sequence length, hydrophobicity, motifs of the most diverse epitope encoded (complementary-determining region 3, CDR3), T cell clustering, and BCR light chain and TCRα chain were analyzed. Results: 10 PRCA, 10 AIHA, 10 AA, and 7 NC were finally enrolled. For the broad repertoire metrics analysis, only the TCR repertoire of the AA group was more diverse compared with NC (p＜0.05). For BCR and TCR repertoires, PRCA, AIHA, and AA showed uniform characteristics in gene usage. The preferential gene usage of PRCA immunoglobulin heavy (IGH) chains such as IGHV3-23, IGHV3-74 were correlated with memory cells and red blood cell antigen recognition; the higher usage of IGHV4-31 in the AIHA group was associated with secretion of auto-antibodies; whereas AA patients had more gene usage of neutralizing antibodies (p＜0.05). In the gene usage in T cell receptor β (TRB) chains, PRCA patients had skewed usage of genes associated with T cell dysregulation and hyperinflammation such as TRBV1 and TRBV11-2; the increased gene usage in AIHA and AA were also found in other autoimmune diseases, which correlated with abnormal antigen recognition. PRCA and AA also had specific TRBV-J gene combination usage. Only PRCA had higher BCR SHM compared with NC (p＜0.05). The length of CDR3 was similar but the hydrophobicity was different among different disease groups. 12, 28, and 22 motifs were found respectively in BCR of PRCA, AIHA, and AA; and 13, 6, and 6 motifs were found in TCR. Compared with NC, PRCA, AIHA, and AA showed a considerable lack of physiology T cell clusters, and 147, 98, and 141 disease-specific T cell clusters in each disease was found, respectively. For light chain of BCR, PRCA and AIHA had the tendency of more usage of κ chains, whereas AA had more usage of λ chains. For TCRα chains, the three diseases used more genes related to hypersensitivity reactions (p＜0.05). Conclusion: PRCA, AIHA, and AA had different BCR and TCR repertoire characteristics in gene and gene combination usage, hydrophobicity and motifs of CDR3, and T cell clustering, which might contribute to autoimmune antigen recognition. The abnormalities were mainly T cell-related for PRCA, B cell-related for AIHA and both for AA. Keywords: pure red cell aplasia; autoimmune hemolytic anemia; aplastic anemia; BCR; TCR

Somatic and Germline HLA Determinants of Immune Surveillance and Escape in Myelodysplastic Syndromes

While the contribution of canonical myeloid genetic drivers to myelodysplastic syndromes/neoplasms (MDS) pathobiology is well established, other genetic events operative under special circumstances, and by itself not constituting intrinsic drivers, may affect the disease course. These include genetic defects conveying resistance to specific therapies, clonally restricted DNA repair alterations favoring the accumulation of additional mutations, and/or lesions facilitating the resistance to inflammation or immune escape. Here, we focus on this latter aspect as it may shed light on the pressure exerted by immune tumor surveillance or acquired resistance to cancer immune therapies, a field that has lagged in myeloid neoplasia as compared to the dramatic changes obtained in solid tumors. We hypothesize that during MDS ontogeny and leukemia evolution a spectrum of immunological mechanisms may shape the oncogenic landscape and facilitate the emergence of clones with diverse immunogenic potential as targets, leading to selective immunotherapeutic liabilities. We accrued a clinically and molecularly well annotated cohort of 101 MDS patients at various stages of the disease spectrum [N=51 low risk (LR)-MDS, N=32 high risk (HR)-MDS, N=18 secondary AML-sAML)]. This cohort included specific clinical sub-entities wherein the contribution of immune forces appears to be a prominent feature i.e., hypocellular MDS (hMDS, N=9) or MDS with co-occurring T large granular lymphocytosis (LGL) (N=5). By combining human leukocyte antigen (HLA) and T cell receptor (TCR) immunosequencing at disease onset, we leveraged our bioinformatics expertise to impute HLA mutations and losses, HLA evolutionary divergence (HED), and characteristics of T cell repertoires. The median age of the whole cohort was 71.2 years (IQR: 63.4-77.7), and median bone marrow (BM) blast count was 3% (1-11%). Cytogenetics revealed normal karyotype in 41 patients (40%), while complex karyotype was observed in 21 (20%) and partial or complete loss of chromosome 7 in 10 cases (10%). Comparisons with a cohort of 784 ethnicity-matched healthy individuals (HC), showed no differences in terms of locus-specific HED configurations. However, when HED scores were analyzed across the disease spectrum, we observed a heterogeneous distribution in MDS patients for the HLA-C locus, with higher values in hypoplastic and LGL-associated MDS as compared to HR diseases (p= 0.0068) and sAML (p= 0.0409). The screening for immune evasion highlighted the presence of both somatic mutations and losses in the HLA region in 14 patients, with both type of alterations found in 1 case. Of those, 6 patients (20% LGL-MDS, 5% LR-MDS and 17% sAML) harbored HLA mutations (in HLA-A, B, C and DPB1 loci), whereas HLA losses were observed in 9 patients (22% hMDS, 5% LR-MDS, 10% HR MDS, 11% sAML) almost exclusively in class II alleles (chiefly DPB1, DRB1 and DPA1). When exploring the degree of immunocompetence by TCR immunosequencing as a proxy, we found a higher degree of diversity (imputed via inverse Simpson index) as compared to healthy controls (p=1.5e-07), possibly in relationship to diverse T cell responses across the disease spectrum. However, no difference between MDS sub-entities was found. Of interest is that the maximal productive frequency (an indicator of the degree of T cell expansion) was higher in patients with hMDS and LGL-associated MDS as compared to HR-MDS (p=0.0079). Finally, inter-sample comparisons of shared clonotypes showed a high degree of overlap with regards to TCR repertoires among MDS as compared to healthy subjects. Our data emphasize the adaptive immune-molecular network underpinning MDS development. HLA constellations and their interactions with TCR repertoires may create selective immune pressure. Somatic HLA aberrations may serve as a marker of ongoing immune pressure and correspond to oligoclonal T-cell expansions and variations in TCR diversity. While virtually no HLA mutations have been previously found in AML/MDS, we show a relatively higher frequency of HLA aberrations in hMDS and MDS-LGL, immunologically stressed scenarios reminiscent of aplastic anemia and post-allogenic transplant AML/MDS cases, whereby respectively 36% 1 and 38% 2 of patients harbored HLA lesions as a mean of escape from cytotoxic lymphocytes or allogeneic Graft-vs-Leukemia reactions.

Single Cell Multi-Omic Dissection of Response and Resistance to Chimeric Antigen Receptor T Cells Against BCMA in Relapsed Multiple Myeloma

Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment of relapsed/refractory multiple myeloma (RRMM). Robust variables that predict long-term response are currently missing. Limited data are especially available on the impact of bridging therapies on manufacturing and outcome. We conducted a longitudinal single-cell multi-omics study to identify factors that predict response to BCMA-directed CAR T cells. Changes in the immune microenvironment associated with response were analyzed as well as the impact of prior bridging therapy with bispecific antibodies on subsequent CAR T cell manufacturing and outcome. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 29 consecutive MM patients treated with commercially available anti-BCMA CAR T cells on the day of leukapheresis as well as days 30 and 100 after CAR T cell infusion. PBMCs were subjected to single cell RNA, T-cell receptor (TCR) and B-cell receptor (BCR) sequencing. A custom panel of 57 oligonucleotide-coupled antibodies was used to study surface proteomics. Downstream analyses were performed with Seurat. Differences in cellular compositions at all three time points were analyzed with scCODA. To analyze CAR T cell functionality, CAR T cells from peripheral blood were isolated 7 days after infusion and subjected to an in vitro cytotoxicity assay after expansion and stimulation. Patients were grouped based on their best response following CAR T cell infusion (CR: n=12, non CR: n=17). Results: In total, 375,338 cells were sequenced (median 7246 cells/sample, range 1,569-10,972 cells) and 354,878 cells (94.5%) passed quality assessment. Quantitative and qualitative differences in the cellular composition of peripheral blood between CR and non CR patients were detected at the time of leukapheresis as well as on days 30 and 100 following infusion. CR patients harbored significantly more CD8+ effector memory T cells (TEM) at leukapheresis and less NK cells on day 30 after therapy compared to non CR patients. Regulatory T cells isolated at the time of leukapheresis from non CR patients exhibited significantly higher surface protein levels of CXCR3, CD40, CD95 and KLRG1 (p<0.015, respectively) that have been associated with T cell senescence and impaired tumor immunity. No significant differences in cell numbers between CR and non CR were detected on day 100 after CAR T cell infusion. However, single cell TCR analysis revealed an increasing diversity in the TCR repertoire over time in patients with CR, while Shannon diversity decreased from leukapheresis over day 30 to day 100 in non CR patients (p=0.004). The prior administration of the bispecific antibody teclistamab had no significant impact on the quantitative cellular composition at the time of leukapheresis. However, termination of manufacturing in the first attempt occurred in all patients with a close proximity of teclistimab administration and apheresis. Differential gene expression analysis showed that the application of teclistamab was associated with impaired T cell activation and exhaustion indicated by upregulation of e.g. CTLA4, TIGIT, LAG3 and GZMK. After discontinuation of teclistamab (median 4 weeks) and successful manufacturing of CAR T cells, we found no significant differences for in vitro cytotoxicity and in vivo expansion of CAR T cells. CAR T cells isolated at day 7 post-infusion from patients in CR, non CR or with prior teclistamab exposure, effectively eliminated MM cells (U-266). Tracking of single CAR T cells over time showed that the majority of CAR+ cells were CD8+ TEMs regardless of remission achievement or prior teclistamab exposure. Conclusion: We demonstrate that differences between MM patients achieving a CR and patients with suboptimal response upon anti-BCMA CAR T cell therapy can already be identified at the time of leukapheresis. Long-term changes associated with CR include a diversification of the TCR repertoire. Successful CAR T cell manufacturing is hampered by exposure to bispecific antibodies but can be successfully achieved by allowing for a wash-out phase of ca. 4 weeks.

Correlation of TCR Repertoire Diversity with Myeloid Gene Mutations in Chronic and Advanced Phase Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are characterized by a chronic phase (CP), with indolent clinical behavior and, in 10-30% of cases, progression to advanced phases (AP), with aggressive behavior and compromised prognosis. Numerous factors have been associated with progression to AP, including the acquisition of mutations in recurrently mutated myeloid genes (1,2). On the other hand, decreased diversity in the T-cell receptor (TCR) repertoire has been associated with worse prognosis in solid tumors. The purpose of this work is to study the correlation between the acquisition of mutations in myeloid genes and the diversity of the TCR repertoire in chronic and advanced myeloproliferative neoplasms. Methods: Patients with chronic CP and AP myeloproliferative neoplasms were selected. The selected patients underwent an NGS panel of 48 myeloid genes. Only pathogenic (P) or likely pathogenic (LP) variants were considered. In parallel, the study of the TCR repertoire by NGS with the panel “AmpliSeq for Illumina Immune Repertoire Plus, TCR beta Panel” (Illumina™) was performed on the available samples. Finally, the obtained data were correlated. The study was approved by the local ethics committee. Results: A total of 22 patients were selected, 10 in CP and 12 in AP. Regarding diagnosis, 8 were polycythemia vera (PV), 4 essential thrombocythemia (ET), 6 secondary myelofibrosis (MFS) (1 to PV and 5 to ET), 2 systemic mastocytosis (MS) and 1 primary myelofibrosis (MFP). Eighty percent were male and the mean age was 68 years. The AP patients were significantly older (74 vs. 62 years of age, p<0,001). Regarding the myeloid NGS panel result, 41 mutations were found in 19 patients. Fourteen percent of the patients had no mutation, 36% had an isolated driver mutation and 50% had additional mutations. Of these, 9% were in CP and 81% were in AP. The most recurrently mutated genes were JAK2 (45.4%), CALR (18.2%), ASXL1, IDH2, SRSF2, IDH2 (all 13.6%), MPL and KIT (both 9.1%). Regarding the results of the TCR repertoire, it was possible to perform the study in 11 patients, 4 in CP and 7 in AP. Only 6/11 had results of sufficient quality to allow analysis. Of these 6 patients, 3 were in CP and 3 in AP. As can be seen in Figure 1, the diversity of the TCR between CP patients and PC patients was different. Thus, while the pattern of diversity in the CP phase shows the expected distribution, the diversity in AP shows a characteristic pattern. Conclusions: APs of MPNs show an abnormal TCR diversity pattern. This pattern may be related to the acquisition of myeloid gene mutations in AP. To our knowledge, this is the first study demonstrating an alteration in TCR diversity in AP and opens the door to future fields of research.

The Overlap of Skin and Blood T-Cell Clones in Early-Stage Mycosis Fungoides

Introduction: Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma that initially starts in the skin but can progress to involve blood with significant mortality in late stages. Even in early stages and in the absence of blood involvement, changes in blood T-cell receptor (TCR) repertoire have been observed in MF patients. While prior work has explored the significance of dominant blood T-cell clones in the prognosis of early MF, the relationship between TCR sequences and T-cell repertoires of blood and skin in early-stage MF patients has not been characterized. Objectives: To determine the relationship of blood and skin T-cell repertories and to reassess the impact of dominant blood T-cell clones on selected outcome endpoints in early-stage MF. Materials and Methods: We used high-throughput sequencing to interrogate TCR sequences in blood and skin of MF patients without blood involvement at the Jefferson Cutaneous Lymphoma Clinic (Adaptive Biotechnologies). ImmunoSEQ Analyzer provided T-cell repertoire overlap metric (Morisita's index) and diversity measure (Simpson's clonality score). Time to systemic treatment (TTST) was calculated as the time from initial diagnosis to initiation of first systemic therapy. Results: 60 MF patients with no blood involvement were enrolled. 28% had a dominant clone in blood; of these, 18% were identical to dominant skin clones and 82% were distinct from the dominant clones identified in skin. We found that MF patients with discordant dominant T-cell clones in blood and skin had a longer TTST when compared to the identical clone cohort (P=0.0057) (Figure 1A). Furthermore, patients with discordant dominant clones had a lower degree of T-cell repertoire overlap between blood and skin (P<0.0001) and higher blood T-cell repertoire diversity scores (P=0.013) when compared to the identical clone group (Figure 1B). In all patients, the degree of T-cell overlap in blood and skin did not significantly change over a period of months to years or among skin biopsies obtained from different anatomical locations. Finally, in a subset of patients with discordant clones, the dominant skin clone did not appear to be detected in blood even at low frequencies. Conclusion: Our data highlight the importance of establishing clonal associations between dominant T-cell clones of the skin and blood in MF. We propose that early-stage MF patients with dominant clones in blood segregate into two cohorts with distinct prognoses and T-cell repertoires based on the identity of their peripheral T-cell clones. In addition, our findings suggest that not all MF patients have reservoirs of malignant T-cell clones readily available in peripheral circulation to seed new lesions in skin. Figure 1: A: Kaplan-Meier analyses of time to systemic treatment (TTST) in skin-limited mycosis fungoides (MF) patients with an identical dominant T-cell clone in blood and skin, a discordant dominant T-cell clone in blood and skin, and no dominant blood clones. NS- not significant (P =0.5274), ** (P =0.0057), *** (P <0.0003). B: Average Morisita's index for blood and skin overlap for patients in indicated groups of I: identical dominant clones in blood and skin, D: discordant dominant clones in blood and skin, and N: no dominant clone in blood. **** (P <0.0001).

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.