Cell Repertoire Research Articles

14P Integrated modelling of T cell repertoires to identify clonotype signatures of ICI response

14P Integrated modelling of T cell repertoires to identify clonotype signatures of ICI response

Single cell transcriptomic heterogeneity between sporadic early and late‐onset of Alzheimer’s disease

AbstractBackgroundRegulatory T‐cells (Tregs) are key modulators of immune responses, and may play a key role in the pathophysiology of various neurodegenerative disorders, including Alzheimer’s disease (AD). However, the impact of Tregs modulation of immune responses during disease progression is poorly understood and still controversial. Thus, the main objective of our ongoing study is to depict a high‐resolution transcriptomic landscape of blood immune cells during spontaneous disease progression, which will facilitate a better understanding of the protective and/or pathogenic immune responses at different stages of the disease1‐3.MethodIn 2019 a Multi‐partner consortium to expand dementia research in Latin America (ReDLat) was created. The Consortium aims to combine genomic, neuroimaging and behavioral (clinical, cognitive, socioeconomic status –SES‐) data to improve dementia characterization and identify novel inroads to treat neurodegeneration in diverse populations. ReDLat initiative supports a basic platform anchored in Argentina, Brazil, Colombia, Peru, Mexico, Chile and USA. allowing us to expand our research and combine genomic, neuroimaging and behavioral (clinical, cognitive, socioeconomic status) data to improve dementia characterization.To investigate changes in individual T cell subsets across progressive clinical conditions we included 4 healthy controls (CTLs), 4 early‐onset sporadic AD (EOAD), 4 late onset AD (LOAD) and 4 Parkinson disease patients (PD) from ReDLat. Peripheral blood mononuclear cells (PBMCs) were isolated and a 5' single‐cell transcriptome and immune repertoire sequencing was performed.ResultsWe identified transcriptionally distinct subpopulations, heterogeneity of the immune system, T cell repertoire diversity and antigen specificity among groups. Interestingly, CD4+CD25+Foxp3+ T‐regs gene expression heterogeneity was observed between sporadic EOAD and LOAD, mainly related to cytotoxicity and inflammation. Our next step is to perform multiple association studies incorporating imaging, sex, cognition and socio‐economics status (SES).ConclusionAlthough full analysis is still in progress, we identified significant changes in individual T‐cell subsets among individuals across the four proposed groups, providing a unique opportunity to identify new risk factors and therapeutics targets for AD.1‐ Fu J (2021) Front. Aging Neurosci.2‐ Cira Dansokho (2016). Brain.3‐Baek H (2016) Oncotarget.

A bispecific Clec9A-PD-L1 targeted type I interferon profoundly reshapes the tumor microenvironment towards an antitumor state

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2. Moreover, we report empowered diversity in the CD8+ T cell repertoire and induction of a shift from naive, dysfunctional CD8+ T cells towards effector, plastic cytotoxic T lymphocytes together with increased presence of NK and NKT cells as well as decreased regulatory T cell levels. These dynamic changes were associated with potent antitumor activity. Tumor clearance and immunological memory, therapeutic immunity on large established tumors and blunted tumor growth at distant sites were obtained upon co-administration of a non-curative dose of chemotherapy.Overall, this study illuminates further application of type I interferon as a safe and efficient way to reshape the suppressive tumor microenvironment and induce potent antitumor immunity; features which are of major importance in overcoming the development of metastases and tumor cell resistance to immune attack. The strategy described here has potential for application across to a broad range of cancer types.

A T cell receptor β chain-directed antibody fusion molecule activates and expands subsets of T cells to promote antitumor activity.

Despite the success of programmed cell death-1 (PD-1) and PD-1 ligand (PD-L1) inhibitors in treating solid tumors, only a proportion of patients respond. Here, we describe a first-in-class bifunctional therapeutic molecule, STAR0602, that comprises an antibody targeting germline Vβ6 and Vβ10 T cell receptors (TCRs) fused to human interleukin-2 (IL-2) and simultaneously engages a nonclonal mode of TCR activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. In solution, STAR0602 binds IL-2 receptors in cis with Vβ6/Vβ10 TCRs on the same T cell, promoting expansion of human Vβ6 and Vβ10 CD4+ and CD8+ T cells that acquire an atypical central memory phenotype. Monotherapy with a mouse surrogate molecule induced durable tumor regression across six murine solid tumor models, including several refractory to anti-PD-1. Analysis of murine tumor-infiltrating lymphocyte (TIL) transcriptomes revealed that expanded Vβ T cells acquired a distinct effector memory phenotype with suppression of genes associated with T cell exhaustion and TCR signaling repression. Sequencing of TIL TCRs also revealed an increased T cell repertoire diversity within targeted Vβ T cell subsets, suggesting clonal revival of tumor T cell responses. These immunological and antitumor effects in mice were recapitulated in studies of STAR0602 in nonhuman primates and human ex vivo models, wherein STAR0602 boosted human antigen-specific T cell responses and killing of tumor organoids. Thus, STAR0602 represents a distinct class of T cell-activating molecules with the potential to deliver enhanced antitumor activity in checkpoint inhibitor-refractory settings.

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.

Immune Dynamics Associated with Patient Outcomes Identified By Multimodal Single-Cell Analysis in Multiple Myeloma Patients Receiving Talquetamab Monotherapy

Background: Talquetamab (Tal) is a CD3xGPRC5D bispecific antibody (BiAb) that redirects endogenous T cells to mediate killing of GPRC5D-expressing multiple myeloma (MM) cells. In the recent phase I/II MonumenTAL-1 trial (NCT03399799), Tal showed ≥ 70% response rate when given at the recommended phase 2 doses (RP2D) in heavily pretreated MM patients (Chari A et al., NEJM 2022). However, there is still a need to identify predictive biomarkers and better understand the immune dynamics associated with treatment response and durability with this novel agent. Methods:We identified 30 patients who received Tal monotherapy and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and PB collected at cycle 3 day 1 (C3D1). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed to understand transcriptional changes and cell surface proteomics. Total cells were isolated by Ficoll separation from PB and BM samples and were then analyzed separately and only samples of good quality were included in the analysis. Samples were filtered for good quality based on total cell count: at least 1000 cells with more than 500 unique molecular identifiers (UMIs) or genes and less than 4-5 standard deviations above median for UMI, genes and mitochondrial transcripts. A total of 60,851 BM cells (15 pts at baseline) and 299,996 PB cells (23 pts at baseline and 17 pts on C3D1) were analyzed. Downstream analysis was performed using the Seurat R package. All patients included in this study had signed consent for an IRB-approved institutional sub-study (GCO # 18-00456). Results: The 30 patients had a median age of 66.2 years and 57% were female. They received a median of 6 prior lines of therapy, with 83% being triple-class refractory and 50% penta-drug refractory. Only 2 patients were previously treated with anti-BCMA CAR-T cell therapy and none were treated with other BiAbs. 43% of patients had high-risk cytogenetics (including t(4;14), t(14;16) and 17p deletion) and 13% had extramedullary disease at time of initiating therapy. Ten patients (30%) received the recommended phase 2 dose (RP2D) of Tal (0.4 mg/kg weekly or 0.8 mg/kg every other week). Patients were followed for a median of 32.5 months and had a median progression-free survival (PFS) of 6.7 months. 22/30 patients (73%) achieved a partial response (PR) or better, and 14 patients had a short PFS of <150 days. There was no association between the prevalence of different immune cell subsets and depth of response, but rather with PFS. In the baseline BM, a longer PFS was associated with a higher percentage of CD8+ cytotoxic T cells (central memory p=0.00031, effector memory p=0.0022, EMRA p=0.014) and a trend towards higher CD4+ cytotoxic T cells (p=0.072). Of note, we did not find the same associations in the baseline PB samples, which may indicate that the PB may not always reflect the tumor microenvironment in the BM. When we next looked at immune changes in the PB of patients with longer PFS, we found that they exhibited higher percentage of NK cells (p=0.0018 for CD56 low, p=0.04 for CD56 high) and Gamma Delta T cells (p=0.04) at C3D1. Gamma Delta T cells are novel players that warrant further investigation, as they only constitute 1-5% of the T cell repertoire yet are involved in early immune response and do not require major histocompatibility complex (MHC) antigen presentation to function. Profiling of activation and exhaustion markers of these immune subsets is underway. Conclusion: Our findings highlight that the baseline BM immune repertoire and the PB immune changes during Tal treatment may predict long term outcomes. Further in vitro mechanistic studies of T cell function and fitness and validation of these results in independent cohorts will be critical to develop these into predictive biomarkers of long-term response to Tal.

Personal and Shared Tumor-Antigen Prioritization in Diffuse Large B Cell Lymphoma Patients Undergoing CD19 CAR T Cell Treatment

Backgroun d: Durable response to Axi-cel CD19 CAR T cell therapy is associated with activation and clonotypic expansion of the host T cell repertoire (Cheloni et al Blood, ASH, 2022). The characterization of the tumor microenvironment (TME) and the identification of tumor antigens associated with therapeutic efficacy is a critical area of investigation. Aim: We sought to determine the association between the host immune activation in response to tumor-associated and tumor-specific antigens and the clinical response to CAR-T cell treatment in diffuse large B cell lymphoma patients. Methods: RNAseq was performed on FFPE lymphoma samples collected before CAR T cell infusion (N=16). Patients were divided into 3 distinct cohorts: stable progressive disease at 1 month post-CAR T (non-responders, n=2), disease relapse within 6 months post-CAR T (relapsed, n=3), and on-going response lasting greater than 1 year (long-term responders, n=11). For neoantigens, mutations were retrieved following the GATK best practices for RNAseq data and annotated with Variant Effect Predictor (VEP). Tumor-specific antigens and tumor-associated antigens were detected by prioritizing 9-11mer amino acid sequences, preferentially presented by class I molecules, observed solely or primarily in tumor samples, respectively, as compared to a pool of healthy controls derived from 250 RNAseq samples, corresponding to 29 tissues (GTEx Portal). Antigen prioritization was performed following weighted scoring considering RNA expression, translation potential, peptide-MHC binding affinity, predicted immunogenicity, physicochemical properties, and clonality, in the case of neoantigens. Using deconvolution methods and canonical marker expression, we characterized the major immune cell types within the TME. Results: Higher cell proportions of lymphocytes and innate immune cells, including NK, and activated dendritic cells were observed in long-term responders, suggesting that a higher infiltration of these cell types in the tumor bed is associated with a better response to CAR T cell therapy. Notably, a positive correlation between the mutational burden and the a/b clonotypic expansion of peripheral blood cytotoxic T cell populations, detected from single-cell immunoprofiling (Cheloni et al Blood, ASH, 2022) was observed in eight long-term responders, consistent with prior studies highlighting the significance of neoantigens in eliciting robust and durable immune response against cancer cells. A high occurrence of neoantigens originating from the Titin ( TTN) gene in two long-term responders with the highest mutational burden was observed, and from ACTB and ACTG1, mutated genes associated with lymphoid cancers. A substantial enrichment of shared tumor antigens originating from retroelements was observed in long-term responders, suggesting the presence of common epitopes that drive an antigen-specific response. Conclusion: These findings shed light on the potential immunogenicity of specific neoantigens and shared antigens derived from repetitive elements, which could serve as promising targets for immunotherapeutic strategies in lymphoma patients. Understanding the tumor immune landscape and antigenic features associated with long-term response to CAR T cell therapy may pave the way for the development of personalized and effective combinatorial treatments for lymphoid cancers.

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.

B cell MHC haplotype affects follicular inclusion, germinal center participation and plasma cell differentiation in a mouse model of lupus.

MHC class II molecules are essential for appropriate immune responses against pathogens but are also implicated in pathological responses in autoimmune diseases and transplant rejection. Previous studies have shed light on the systemic contributions of MHC haplotypes to the development and severity of autoimmune diseases. In this study, we addressed the B cell intrinsic MHC haplotype impact on follicular inclusion, germinal center (GC) participation and plasma cell (PC) differentiation in the context of systemic lupus erythematosus (SLE). We leveraged the 564Igi mouse model which harbors a B cell receptor knock-in from an autoreactive B cell clone recognizing ribonuclear components, including double-stranded DNA (dsDNA). This model recapitulates the central hallmarks of the early stages of SLE. We compared 564Igi heterozygous offspring on either H2b/b, H2b/d, or H2d/d background. This revealed significantly higher germinal center (GC) B cell levels in the spleens of H2b/b and H2b/d as compared to H2d/d (p<0.0001) mice. In agreement with this, anti-dsDNA-antibody levels were higher in H2b/b and H2b/d than in H2d/d (p<0.0001), with H2b/b also being higher compared to H2b/d (p<0.01). Specifically, these differences held true both for autoantibodies derived from the knock-in clone and from wild-type (WT) derived clones. In mixed chimeras where 564Igi H2b/b, H2b/d and H2d/d cells competed head-to-head in the same environment, we observed a significantly higher inclusion of H2b/b cells in GC and PC compartments relative to their representation in the B cell repertoire, compared to H2b/d and H2d/d cells. Furthermore, in mixed chimeras in which WT H2b/b and WT H2d/d cells competed for inclusion in GCs associated with an epitope spreading process, H2b/b cells participated to a greater extent and contributed more robustly to the PC compartment. Finally, immature WT H2b/b cells had a higher baseline of BCRs with an autoreactive idiotype and were subject to more stringent negative selection at the transitional stage. Taken together, our findings demonstrate that B cell intrinsic MHC haplotype governs their capacity for participation in the autoreactive response at multiple levels: follicular inclusion, GC participation, and PC output. These findings pinpoint B cells as central contributors to precipitation of autoimmunity.

Characterization of the TCRβ repertoire of peripheral MR1-restricted MAIT cells in psoriasis vulgaris patients

Psoriasis vulgaris (PV) is an inflammatory skin disease largely driven by aberrant αβT cells. Mucosal-associated invariant T (MAIT) cells, which constitute the largest circulating innate-like αβT cell community in human adults, are characterized by a semi-invariant TCRVα7.2 receptor and MR1-restricted affinity toward microbial metabolites. Limited MAIT TCRα diversity is complemented by a more variable TCRβ repertoire, but its footprint in the MAIT repertoire of PV patients has never been tested. Here, we used bulk TCRSeq, MiXCR, VDJTools, and Immunarch pipelines to decipher and compare TCRβ clonotypes from flow-sorted, peripheral TCRVα7.2+MR1-5-OP-RU-tet+MAIT cells from 10 PV patients and 10 healthy, matched controls. The resulting TCRβ collections were highly private and individually unique, with small public clonotype content and high CDR3β amino acid length variability in both groups. The age-related increase in the ‘hyperexpanded’ clonotype compartment was observed in PV, but not in healthy MAIT repertoires. The TCRβ repertoires of PV patients were also marked by skewed TRBV/TRBJ pairing, and the emergence of PV-specific, public CDR3β peptide sequences closely matching the published CDR3β record from psoriatic skin. Overall, our study provides preliminary insight into the peripheral MAIT TCRβ repertoire in psoriasis and warrants further evaluation of its diagnostic and clinical significance.

A Public Neoantigen Produced By the SF3B1 K700E Mutation Is a Bona Fide T Cell Target in AML/MDS

Myelodysplastic syndrome (MDS) is a life-threatening disease in which malignant bone marrow cells fail to differentiate, resulting in anemia, cytopenia, and increased risk of progression to acute myeloid leukemia (AML). T cell-based therapies have great potential in treating AML/MDS, given that allogeneic stem cell transplantation (aSCT) can be curative in such settings. aSCT is associated with the graft-vs-leukemia (GvL) effect, in which T cells within the donor graft recognize and target antigens expressed by leukemia cells. Unfortunately, aSCT is also associated with considerable morbidity and mortality, as donor T cells may also recognize antigens expressed by non-malignant tissues and kill them, leading to graft-vs-host disease (GvHD). A T cell-based therapy that targets an AML/MDS-exclusive antigen has the potential to eliminate leukemic cells while sparing healthy tissues. Most known antigens expressed on AML/MDS are also expressed by healthy myeloid cells. An ideal T cell target would be 1.) only expressed by leukemia cells, including leukemic stem cells (LSCs), 2.) expressed by all leukemia cells, and 3.) required for continued leukemia persistence. Somatic mutations in genes encoding spliceosomal machinery are common in MDS and have the potential to generate such neoantigens. In particular, a conserved missense mutation encoding a K700E substitution in SF3B1 is present in about 20% of patients with MDS, and approximately 80% of patients with MDS with ringed sideroblasts (MDS-RS). Moreover, SF3B1 K700E-mutant MDS has been proposed to be a distinct disease entity in which the SF3B1 mutation is a necessary, disease-initiating event. Further, SF3B1 K700E mutations are broadly detected across the spectrum of myeloid neoplasms from clonal hematopoiesis (CH) to relapsed/refractory AML and are associated with worse overall survival (OS) in the latter. We demonstrate that the SF3B1 K700E mutant protein produces a leukemia-restricted T cell target. NetMHCpan4.1 binding algorithm predicted that a nonameric SF3B1 K700E-derived peptide could bind HLA-A*02:01, the most common HLA-A allele. A similar peptide derived from the wild type (WT) SF3B1 sequence was also predicted to bind HLA-A*02:01 with similar affinity. We identify potent TCRs from the T cell repertoires of healthy HLA-A*02:01 positive individuals, which effectively discriminate between WT and K700E peptides. We further show that the SF3B1 K700E: HLA-A*02:01 is a bona fide T cell antigen, as it is naturally processed by the proteasome and presented on the surface of SF3B1 K700E mutant cell lines and primary patient samples. Primary human CD8 T cells that were retrovirally engineered to express an SF3B1 K700E:HLA-A2 specific TCR are activated by, and kill SF3B1 K700E mutant cancer cells, while sparing SF3B1 WT cells. Antigen dose response experiments demonstrate that this TCR has an approximately 4 order of magnitude specificity for the K700E peptide over the WT peptide, and amino acid substitution scanning experiments tiling the mutant peptide suggested no other naturally occurring HLA-A*02:01 restricted peptides from the human proteome that could activate the TCR. The EC 50 of the K700E:A0201-specific TCR in peptide pulsing experiments was &amp;lt;1nM, which is comparable to the potency observed in viral antigen-specific TCRs. In summary, we report that the SF3B1 K700E mutation yields an HLA-A*02:01 restricted AML/MDS-specific neoantigen which can be effectively targeted with TCR-T cells. We identified a highly potent TCR that eliminates cancer cells while sparing mutation negative cells. These data support further development of this TCR as a potential therapeutic for MDS and AML.

High-Throughput Identification of Graft-Vs-Leukemia TCRs in AML Patient Samples after Allogeneic Stem Cell Transplantation

For patients with intermediate and high-risk AML/MDS, allogeneic stem cell transplantation (aSCT) is a potentially curative therapy. Efficacy is driven by the graft-versus-leukemia (GvL) response, in which donor-derived T cells recognize peptide:MHC antigens presented by leukemia cells, and subsequently kill them. The antigens that drive GvL responses are attractive therapeutic targets for engineered cell therapies and/or biologics. However, only a small number of GvL antigens have been identified, limiting the therapeutic potential of targeted immunotherapies. Deep interrogation of the T cell repertoires of patients who have responded favorably to aSCT presents an opportunity to identify bona fide GvL antigens, but high-throughput methods need to be developed to do this effectively. A popular method to identify antigen-specific T cells is fluorescent peptide:MHC tetramer staining, whereby T cells whose T cell receptors (TCR) bind a particular peptide:MHC antigen become fluorescent and identifiable by flow cytometry. However, a major challenge to identifying GvL T cells using tetramers is that the prediction of immunogenic antigens - that is, those that induce a T cell response in vivo - is poor; studies of solid tumors suggest that only ~1% of predicted MHC-binding neoantigens are immunogenic in patients. In AML/MDS aSCT patients, there are thousands of peptides that are predicted to bind MHCI and represent potential GvL antigens, but due to practical limitations of patient blood and bone marrow sample quantities, only a small subset can be screened by flow cytometry. Other higher throughput methods using heavy-metal tagged peptide:MHC tetramers followed by mass cytometry, or DNA-barcoded tetramers followed by bulk sequencing have been employed to increase antigen screening scale, but these methods are limited in sensitivity and destroy the T cell without providing TCR sequences. To overcome these limitations and rapidly identify GvL T cells in patient samples, we developed a single cell sequencing based method that enables the screening of thousands of peptide:MHC antigens simultaneously, using a single patient sample. We generated peptide: MHCI monomers using UV-mediated peptide exchange and generated large panels of combinatorial DNA-barcoded peptide:MHCI tetramers at microscale. We integrated our methodology into the 10X Genomics Single Cell Immune Profiling workflow to enable simultaneous recovery of TCR antigen-specificity, paired TCR alpha beta sequences, and single cell RNA-Seq data. These methods were used to screen post-transplant AML patient blood and bone marrow, and we identified expanded GvL T cell clonotypes that target novel minor histocompatibility antigens, overexpressed “aberrant self” proteins, and other leukemia associated antigens. We generated primary CD8 T cells that retrovirally express these TCRs and demonstrate that they are potent and have the expected antigen specificities. The methods described are highly sensitive, specific, efficient, and scalable, and enable high confidence identification of antigen specific clonotypes in patient samples. They markedly expand the throughput compared to existing methods, and simultaneously yield paired alpha beta TCR sequences, facilitating a full characterization of the antigen-TCR drivers of GvL. Furthermore, these methods have broad utility beyond SCT, as they are applicable to studying T cell responses to other immune settings, including anti-cancer, autoimmune, and anti-microbial responses.

Overall avidity declines in TCR repertoires during latent CMV but not EBV infection.

The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. To address these issues, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCRαβ clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. The parameters involved during the long-term persistence of virus-specific T cell clonotypes were further evaluated by gene expression profiling, phenotype and functional analyses. Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. These findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data further suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.

Peripheral T cell activation, not thymic selection, expands the T follicular helper repertoire in a lupus-prone murine model.

Many autoimmune diseases are characterized by the activation of autoreactive T cells. The T cell repertoire is established in the thymus; it remains uncertain whether the presence of disease-associated autoreactive T cells reflects abnormal T cell selection in the thymus or aberrant T cell activation in the periphery. Here, we describe T cell selection, activation, and T cell repertoire diversity in female mice deficient for B lymphocyte-induced maturation protein (BLIMP)-1 in dendritic cells (DCs) (Prdm1 CKO). These mice exhibit a lupus-like phenotype with an expanded population of T follicular helper (Tfh) cells having a more diverse T cell receptor (TCR) repertoire than wild-type mice and, in turn, develop a lupus-like pathology. To understand the origin of the aberrant Tfh population, we analyzed the TCR repertoire of thymocytes and naive CD4 T cells from Prdm1 CKO mice. We show that early development and selection of T cells in the thymus are not affected. Importantly, however, we observed increased TCR signal strength and increased proliferation of naive T cells cultured in vitro with antigen and BLIMP1-deficient DCs compared to control DCs. Moreover, there was increased diversity in the TCR repertoire in naive CD4+ T cells stimulated in vitro with BLIMP1-deficient DCs. Collectively, our data indicate that lowering the threshold for peripheral T cell activation without altering thymic selection and naive T cell TCR repertoire leads to an expanded repertoire of antigen-activated T cells and impairs peripheral T cell tolerance.

Single-cell transcriptomes provide insights into expansion of glial cells in Bombyx mori.

The diversity of cell types in the brain and how these change during different developmental stages, remains largely unknown. The life cycle of insects is short and goes through 4 distinct stages including embryonic, larval, pupal, and adult stages. During postembryonic life, the larval brain transforms into a mature adult version after metamorphosis. The silkworm, Bombyx mori, is a lepidopteran model insect. Here, we characterized the brain cell repertoire of larval and adult B. mori by obtaining 50708 single-cell transcriptomes. Seventeen and 12 cell clusters from larval and adult brains were assigned based on marker genes, respectively. Identified cell types include Kenyon cells, optic lobe cells, monoaminergic neurons, surface glia, and astrocyte glia. We further assessed the cell type compositions of larval and adult brains. We found that the transition from larva to adult resulted in great expansion of glial cells. The glial cell accounted for 49.8% of adult midbrain cells. Compared to flies and ants, the mushroom body kenyon cell is insufficient in B. mori, which accounts for 5.4% and 3.6% in larval and adult brains, respectively. Analysis of neuropeptide expression showed that the abundance and specificity of expression varied among individual neuropeptides. Intriguingly, we found that ion transport peptide was specifically expressed in glial cells of larval and adult brains. The cell atlas dataset provides an important resource to explore cell diversity, neural circuits and genetic profiles.

High-sensitive spatially resolved T cell receptor sequencing with SPTCR-seq

Spatial resolution of the T cell repertoire is essential for deciphering cancer-associated immune dysfunction. Current spatially resolved transcriptomic technologies are unable to directly annotate T cell receptors (TCR). We present spatially resolved T cell receptor sequencing (SPTCR-seq), which integrates optimized target enrichment and long-read sequencing for highly sensitive TCR sequencing. The SPTCR computational pipeline achieves yield and coverage per TCR comparable to alternative single-cell TCR technologies. Our comparison of PCR-based and SPTCR-seq methods underscores SPTCR-seq’s superior ability to reconstruct the entire TCR architecture, including V, D, J regions and the complementarity-determining region 3 (CDR3). Employing SPTCR-seq, we assess local T cell diversity and clonal expansion across spatially discrete niches. Exploration of the reciprocal interaction of the tumor microenvironmental and T cells discloses the critical involvement of NK and B cells in T cell exhaustion. Integrating spatially resolved omics and TCR sequencing provides as a robust tool for exploring T cell dysfunction in cancers and beyond.

Plasma Cell Differentiation, Antibody Quality, and Initial Germinal Center B Cell Population Depend on Glucose Influx Rate.

Serum Ab concentrations, selection for higher affinity BCRs, and generation of higher Ab affinities are important elements of immune response optimization and functions of germinal center (GC) reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by mouse GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether glucose uptake or glycolysis increases in GC B cells compared with their naive precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that reduction of the glucose transporter GLUT1 in mice after establishment of a preimmune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and plasma cell outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway, whose activity was important in controlling reactive oxygen species (ROS) and Ab-secreting cell production. In modeling how glucose usage by B cells promotes the Ab response, the control of ROS appeared insufficient. Surprisingly, the combination of galactose, which mitigated ROS, with provision of mannose, an efficient precursor to glycosylation, supported robust production of and normal Ab secretion by Ab-secreting cells under glucose-free conditions. Collectively, the findings indicate that GCs depend on normal glucose influx, especially in plasma cell production, but reveal an unexpected metabolic flexibility in hexose requirements.

Unraveling the molecular mechanisms of nitrite-induced physiological disruptions in largemouth bass

Largemouth bass (LMB; Micropterus salmoides) is an economically consequential piscine species spanning North America, Europe, and China. Elevated nitrite concentrations in aquatic environments cause significant physiological changes in fish, including stunted growth, appetite changes, reduced feed intake, and impaired oxygen transport, collectively affecting overall fish health. This study embarked on uncovering the molecular mechanisms underscoring the influence of various nitrite concentrations (0 mg/L, 400 mg/L, 600 mg/L, and 600 mg/L followed by recovery in nitrite-free water) on LMB, leveraging transcriptomics and metabolomics. Our findings revealed that a nitrite exposure of 400/600 mg/L may incite temporary, recoverable damage to the spleen, heart, and gills, but potentially enduring, irreversible hepatic pathologies. The spleen emerged as the most vulnerable organ to nitrite-induced deleterious impacts, causing acute, reversible damage and widespread apoptosis. Nitrite reprogramed splenocyte gene expressions, and prompted apoptosis, autophagy, and changes in metabolic pathways. It also triggered cell-type transitions and promoted adhesion-related gene expressions, as well as inhibited nucleotide metabolism and cell cycle progression. Notably, the immune cell repertoire within the spleen was affected by different nitrite concentrations, possibly linking to nitrite-induced spleen tissue impairment. Following the construction of a LMB protein–protein interaction (PPI) network, several pivotal genes uniquely expressed in distinct nitrite concentrations were identified, such as hdac4, hdac5, and brg1 in the N2 group (400 mg/L), hdac1, hells, and nccrp1 in the N3 group (600 mg/L), and Actr1, brd4, atf7a, and kat2b in the N4 group (600 mg/L recovery). The LMB spleen transcriptome was most profoundly influenced in the N3 group. Applying the MCODE algorithm for network module segmentation, we discerned eight MCODE subnetworks. Detailed analyses of these subnetworks shed light on their core functional pathways and pivotal genes. In particular, the MCODE1 core network implicated mainly Notch-HLH transcription, histone deacetylation, and protein deacetylation pathways, with pivotal genes such as ESRRGB, ATF7A, JDP2A, BRD4, and THRB. Metabolomic analysis results paralleled those of transcriptomic examination, corroborating analogous metabolic pathway alterations. This study holds substantial implications for comprehending the toxicological impact of nitrite on economically important fish species like LMB, and provides insights for refining aquaculture practices to minimize this toxicity.

CTIM-21. T-CELL RECEPTOR REPERTOIRE ANALYSIS IN PBMCS FROM PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT), which involves transferring tumor-specific lymphocytes, has shown promising clinical efficacy across various cancer types. However, accurately tracking these transferred cells has been challenging. To overcome this limitation, we utilized T-cell receptor (TCR) sequencing to investigate the TCR repertoire in peripheral blood mononuclear cells (PBMCs). Specifically, we examined PBMCs stimulated ex vivo using autologous dendritic cells (DCs) loaded with total tumor RNA, which we applied for infusion in pediatric patients. We also collected blood samples prior to and following immunotherapy to compare the TCR repertoire dynamics. METHODS We isolated total RNA from patient PBMCs obtained before the initiation of adoptive cellular therapy (ACT) and continued collection on a weekly basis for one month, followed by monthly collections after immunotherapy treatment. To generate cDNA, we used RACE technology with the addition of a common adapter at the 5' end. The raw sequencing data obtained was then preprocessed using MiXCR software. Subsequent TCR repertoire analysis was performed using the Immunarch R-package and VDJtools software. RESULTS&amp; CONCLUSIONS The analysis of T-cell receptor (TCR) diversity across ex vivo extended T-cell samples showed a range of 593 to 34,000 unique CDR3 clones among 28 patients with recurrent medulloblastoma. The observed range suggests that different patients exhibit distinct levels of TCR diversity. Our results revealed significant instability in the TCR repertoire within the peripheral blood during the initial 28 days after ACT for all patients. Subsequently, the regeneration was mainly driven by de novo thymic production of naïve T-cells. TCR sequencing of PBMCs stimulated with autologous DCs loaded with total tumor RNA allowed us to gain a deeper understanding of the TCR repertoire in the context of immunotherapy. This approach enabled us to track and analyze the tumor-specific T-cells in the repertoire, providing valuable insights into their behavior and response to the treatment.

Changes in the immune cell landscape in the infarcted mouse heart

Abstract Background Myocardial Infarction (MI) and Ischemic Heart Failure represent the leading causes of death globally. Myocardial remodeling after MI is accompanied and driven by a strong immune response with an accumulation of several, partially opposing immune cell types. The temporal and spatial dynamics of this cardiac specific immune cell infiltration, however, have not been systematically evaluated. Purpose We characterized the dynamics of the immune cell repertoire in murine hearts following myocardial infarction by multi-color flow cytometry using a novel 12-marker panel allowing us to characterize major principal leukocyte lineages with a total of 10 distinct leukocyte populations. In parallel, we studied the immune response after MI in secondary lymph organs such as heart´s draining lymph nodes and the spleen. In addition, we performed transcriptional analysis of the healthy and infarcted hearts by bulk RNA at selected time points. Methods Permanent ligation of the left descending coronary artery (LAD), ischemic/reperfusion injury (I/R) and sham surgery was performed on C57BL6/J, 10 weeks old male mice. Leukocytes dynamics and T cell responses were screened in infarcted and remote areas of the heart, as well as in blood, spleen, and heat´s draining lymph nodes after 1, 3, 7, 14 and 28 days by multi-color flow cytometry. Also, immune activation and exhaustion transcription analysis by bulk RNA of healthy and infarcted hearts was performed at day 3,7 and 28 days after MI. Results We describe a sequential shift in the resident heart leukocyte population, a change in T cell activation states and identified potential new targets such as NK and γ/δ T cells. While macrophages area the most frequent leukocytes under steady state, making up to 75% of the total relative leukocyte population, at day 28 Lymphocytes represent 50%, among which B cells represent the most of them, followed by T helper cells. At early stages, CD4+ T cells secret pro inflammatory cytokines in the heart and in the heart´s draining lymph nodes, however, as stable scar in the heart is formed at day 7 immunomodulatory cytokines secretion such as IL-10 was observed. This is consistent with the build-up of a relevant adaptive immune response involving antigen presenting DCs and effector T and B cells in the healing heart. Conclusions Our findings contribute to identify novel cellular and transcriptional targets in the fight against ischemic heart failure.