T-cell Receptor Repertoire Diversity Research Articles

Functional and biological implications of clonotypic diversity among human donor-unrestricted T cells.

Tcells express a T-cell receptor (TCR) heterodimer that is the product of germline rearrangement and junctional editing resulting in immense clonotypic diversity. The generation of diverse TCR repertoires enables the recognition of pathogen-derived peptide antigens presented by polymorphic major histocompatibility complex (MHC) molecules. However, Tcells also recognize nonpeptide antigens through nearly monomorphic antigen-presenting systems, such as cluster of differentiation 1 (CD1), MHC-related protein 1 (MR1) and butyrophilins (BTNs). This potential for shared immune responses across genetically diverse populations led to their designation as donor-unrestricted Tcells (DURTs). As might be expected, some CD1-, MR1- and BTN-restricted Tcells express a TCR that is conserved across unrelated individuals. However, several recent studies have reported unexpected diversity among DURT TCRs, and increasing evidence suggests that this diversity has functional consequences. Recent reports also challenge the dogma that immune cells are either innate or adaptive and suggest that DURT TCRs may act in both capacities. Here, we review this evidence and propose an expanded view of the role for clonotypic diversity among DURTs in humans, including new perspectives on how DURT TCRs may integrate their adaptive and innate immune functions.

Increase in HIV reservoir and T cell immune response after CoronaVac vaccination in people living with HIV

IntroductionCoronaVac, an inactivated vaccine developed by Sinovac Life Sciences, has been widely used for protection against Coronavirus Disease 2019 (COVID-19). This study investigates its effect on the HIV reservoir and T cell repertoires in people living with HIV (PLWHs). MethodsBlood samples were collected from fifteen PLWHs who were administered at least two doses of CoronaVac between April 2021 and February 2022. The levels of cell-associated HIV RNA (CA HIV RNA) and HIV DNA, as well as the T cell receptor (TCR) repertoire profiles, TCR clustering and TCRβ annotation, were studied. ResultsA significant increase was observed in CA HIV RNA at 2 weeks (431.5 ± 164.2 copies/106 cells, P = 0.039) and 12 weeks (330.2 ± 105.9 copies/106 cells, P = 0.019) after the second dose, when compared to the baseline (0 weeks) (73.6 ± 23.7 copies/106 cells). Various diversity indices of the TCRβ repertoire, including Shannon index, Pielou's evenness index, and Hvj Index, revealed a slight increase (P < 0.05) following CoronaVac vaccination. The proportion of overlapping TCRβ clonotypes increased from baseline (31.9 %) to 2 weeks (32.5 %) and 12 weeks (40.4 %) after the second dose. We also found that the breadth and depth of COVID-19-specific T cells increased from baseline (0.003 and 0.0035) to 12 weeks (0.0066 and 0.0058) post the second dose. ConclusionsOur study demonstrated an initial increase in HIV reservoir and TCR repertoire diversity, as well as an expansion in the depth and breadth of COVID-19-specific T-cell clones among CoronaVac-vaccinated PLWHs. These findings provide important insights into the effects of COVID-19 vaccination in PLWHs.

Abstract 70: Unlocking the potential of the T-cell receptor in prostate cancer

Abstract Introduction: Prostate cancer (PC) is the most commonly diagnosed cancer among western men, with its diagnosis and subsequent monitoring still heavily dependent on the prostate specific antigen (PSA) test. However, the prognostic accuracy remains poor, leading to overtreatment of patients with non-aggressive cancer and undertreatment of those with aggressive cancer in need of intervention. Malignant cells are subject to constant monitoring and elimination by T-cell lymphocytes recognizing cancer-specific antigens through the T-cell receptor (TCR). Consequently, to establish itself and progress, cancer must evade or modulate the surveillance mechanisms of the immune system. As cancer growth accelerates, immune evasion becomes more difficult, resulting in a more pronounced impact on the immune system. Recently, our research group documented the clinical value of tumor-infiltrating T-cells as markers of aggressive PC. Here, we hypothesize that this signal can also be found in circulating T-cells isolated from the blood and used as proxy for the antitumoral immune response in tissue. Methods: This study utilizes a diverse and well-characterized cohort, encompassing 220 castration resistant PC patients (CRPC) with blood samples taken at the time of CRPC diagnosis, 30 newly diagnosed hormone-naïve PC patients, and 30 healthy male donors. For a deeper understanding of the mechanisms underlying the antitumoral immune response and its impact on disease progression, a subset of CRPC patients (n = 25) are TCR sequenced throughout their disease course and uniquely, the TCR repertoire of their metastatic biopsies is characterized. Results: In an initial comparison of 8 CRPC patients and 8 age-matched healthy males, we observed that cancer patients had a higher proportion of hyper-expanded T-cell clones (P = 0.003; Fisher’s exact test) and a less diverse TCR repertoire in blood. Furthermore, 86.5 % of hyper-expanded clones found in peripheral blood samples could also be found in malignant prostate tissue from matched CRPC patients, while overlap between patients was virtually non-existent Conclusion: Our pilot study detected differences in the TCR repertoire between cancer patients and matched controls. The clones contributing most to this difference were concurrently found in malignant tissue indicating that the antitumoral immune response can indeed be detected in the periphery. Given these promising results, we are currently running the larger study described above to obtain a better understanding of the TCR repertoire and establish its prognostic potential in PC. While the findings showcased in this abstract exclusively stem from the pilot study, results for the full cohort will be presented at the conference. Citation Format: Eske Nøhr Glud, Jacob H. Fredsøe, Iver K. Nordentoft, Asbjørn Kjær, Maria Rusan, Britt E. Laursen, Bodil G. Pedersen, Nicolai J. Birkbark, Lars Dyrskjøt, Michael Borre, Karina Dalsgaard Sørensen. Unlocking the potential of the T-cell receptor in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 70.

Abstract 1515: Tertiary lymphoid structures restrain cancer evolution by supporting stem-like CD8+ T cells for sustained anti-tumor immunity

Abstract Currently, our understanding of the mechanism underlying the immune response on extending the survival of patients with aggressive tumors remains limited. In this study, we generated multi-regional genomic and transcriptional data coupled with digital pathology from 28 long-term survivors (LTSs) and 34 stage-matched short-term survivors, with intrahepatic cholangiocarcinoma (iCCA). Our investigation unveiled that mature tertiary lymphoid structures (TLSs) within the tumors of LTSs exerted significant immune pressure on the tumor, leading to the restriction in generating clonal mutations and increased subclonal diversity. Employing single-cell profiling, we identified significant enrichment of IgM+ naïve B cells and TCF7+ stem-like CD8+ T cells in TLS-positive tumors. Spatial profiling further highlighted that TCF7+ stem-like CD8+ T cells preferentially localized in TLS, facilitating reduced vulnerability to unfavorable environmental factors. Functionally, TCF7+ stem-like CD8+ T cells displayed strong capacity to differentiate into effector subsets while maintaining a diverse T-cell receptor repertoire. Mechanistically, we identified that naïve B cells promote activation and differentiation of stem-like CD8+ T cells within the TLS niche through IgM-FcμR axis, which primed these T cells for enhanced anti-tumor immunity. In summary, our findings underscore that TLS plays a pivotal role in orchestrating enduring and effective anti-tumor immunity, which yield strong immune stress on cancer evolution and ultimately contributing to better prognosis of patients. Citation Format: Pengxiang Wang, Chi Zhou, Zhiting Wei, Yue Wang, Yuli Gao, Fangliangzi Meng, Jian Zhou, Jia Fan, Qi Liu, Yunfan Sun. Tertiary lymphoid structures restrain cancer evolution by supporting stem-like CD8+ T cells for sustained anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1515.

Mechanism study of ubiquitination in T cell development and autoimmune disease.

T cells play critical role in multiple immune processes including antigen response, tumor immunity, inflammation, self-tolerance maintenance and autoimmune diseases et. Fetal liver or bone marrow-derived thymus-seeding progenitors (TSPs) settle in thymus and undergo T cell-lineage commitment, proliferation, T cell receptor (TCR) rearrangement, and thymic selections driven by microenvironment composed of thymic epithelial cells (TEC), dendritic cells (DC), macrophage and B cells, thus generating T cells with diverse TCR repertoire immunocompetent but not self-reactive. Additionally, some self-reactive thymocytes give rise to Treg with the help of TEC and DC, serving for immune tolerance. The sequential proliferation, cell fate decision, and selection during T cell development and self-tolerance establishment are tightly regulated to ensure the proper immune response without autoimmune reaction. There are remarkable progresses in understanding of the regulatory mechanisms regarding ubiquitination in T cell development and the establishment of self-tolerance in the past few years, which holds great potential for further therapeutic interventions in immune-related diseases.

Intratumoral T-cell receptor repertoire composition predicts overall survival in patients with pancreatic ductal adenocarcinoma

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune checkpoint inhibitor therapy. However, intratumoral T-cell infiltration correlates with improved overall survival (OS). Herein, we characterized the diversity and antigen specificity of the PDAC T-cell receptor (TCR) repertoire to identify novel immune-relevant biomarkers. Demographic, clinical, and TCR-beta sequencing data were collated from 353 patients across three cohorts that underwent surgical resection for PDAC. TCR diversity was calculated using Shannon Wiener index, Inverse Simpson index, and “True entropy.” Patients were clustered by shared repertoire specificity. TCRs predictive of OS were identified and their associated transcriptional states were characterized by single-cell RNAseq. In multivariate Cox regression models controlling for relevant covariates, high intratumoral TCR diversity predicted OS across multiple cohorts. Conversely, in peripheral blood, high abundance of T-cells, but not high diversity, predicted OS. Clustering patients based on TCR specificity revealed a subset of TCRs that predicts OS. Interestingly, these TCR sequences were more likely to encode CD8+ effector memory and CD4+ T-regulatory (Tregs) T-cells, all with the capacity to recognize beta islet-derived autoantigens. As opposed to T-cell abundance, intratumoral TCR diversity was predictive of OS in multiple PDAC cohorts, and a subset of TCRs enriched in high-diversity patients independently correlated with OS. These findings emphasize the importance of evaluating peripheral and intratumoral TCR repertoires as distinct and relevant biomarkers in PDAC.

Characterization of T cell receptor repertoire in penile cancer

Tumor-infiltrating lymphocytes (TILs) play a key role in regulating the host immune response and shaping tumor microenvironment. It has been previously shown that T cell infiltration in penile tumors was associated with clinical outcomes. However, few studies have reported the T cell receptor (TCR) repertoire in patients with penile cancer. In the present study, we evaluated the TCR repertoires in tumor and adjacent normal tissues from 22 patients with penile squamous cell carcinoma (PSCC). Analysis of the T cell receptor beta-variable (TRBV) and joining (TRBJ) genes usage and analysis of complementarity determining region 3 (CDR3) length distribution did not show significant differences between tumor and matched normal tissues. Moreover, analysis of the median Jaccard index indicated a limited overlap of TCR repertoire between these groups. Compared with normal tissues, a significantly lower diversity and higher clonality of TCR repertoire was observed in tumor samples, which was associated with clinical characteristics. Further analysis of transcriptional profiles demonstrated that tumor samples with high clonality showed increased expression of genes associated with CD8 + T cells. In addition, we analyzed the TCR repertoire of CD4 + T cells and CD8 + T cells isolated from tumor tissues. We identified that expanded clonotypes were predominantly in the CD8 + T cell compartment, which presented with an exhausted phenotype. Overall, we comprehensively compared TCR repertoire between penile tumor and normal tissues and demonstrated the presence of distinct T cell immune microenvironments in patients with PSCC.

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.

Large-Scale Post-Transplant TCR Deep Sequencing Reveals a Major T Cell Diversity Bottleneck with Post-Transplant Cyclophosphamide with Implications for Both Efficacy and Toxicity: Results of the BMT CTN 1801 Study

Background: BMT CTN 1703 was a Phase 3 trial comparing Tac/MTX (n = 217) to post-transplant cyclophosphamide (PT-Cy)/Tac/MMF (n = 214) GVHD prophylaxis after reduced intensity conditioning unrelated donor hematopoietic cell transplant (HCT). PT-Cy significantly improved GVHD-free Relapse-Free Survival (Bolanos-Meade et al NEJM 2023). However overall survival was not significantly different and the # of Grade 2 infections was higher with PT-Cy. To investigate its biologic underpinnings 1703 was linked to a mechanistic study, BMT CTN 1801, which co-enrolled 324 pts (159 CNI/MTX 165 PT-Cy). The resulting dataset enabled an analysis of post-HCT T cell reconstitution at an unprecedented level of detail. Methods: Samples were collected from the HCT infusion and on D+7, 14, 28, 63, 98, 180, 270, 1- and 2-yr. We extracted DNA (2,225 samples) and performed Adaptive Biotech β-strand T Cell Receptor (TCR) Immunosequencing (TCR-Seq). We profiled 44,077 (median) TCRs/sample resulting in 215,155,719 T cells analyzed. We evaluated multiple TCR diversity measures longitudinally and before/after cGVHD and infections. Each measure evaluates a specific aspect of TCR diversity, including Simpsons Clonality (focuses on the most expanded clones) Diversity Slope (mid-range clones) Richness (both mid-range and less-expanded clones) and Singletons (the TCR-Seq equivalent of naïve T cells). This analysis was robust to the full range (1000-50,000) of down-sampled TCRs sequenced. Results: We identified major differences in TCR reconstitution in Tac/MTX vs PT-Cy that begin early and persist through 2 yr post-HCT. These resulted in a significantly less diverse TCR repertoire with PT-Cy with substantial implications for its efficacy and toxicity. The most salient differences are: (1) PT-Cy caused significant in-vivo T cell depletion early post-HCT (% T cells/total WBC at D+28: 8.2% (Tac/MTX) vs 2.2% (PT-Cy) p=2.2x10 -23). (2) The remaining donor T cells in PT-Cy undergo substantial clonal expansion ( Fig 1) resulting in significantly constrained TCR diversity measured by Clonality, Slope, Richness, Singletons (not shown) and Singletons/Richness ( Fig 2) which began early (D+14) and persisted for 2 yr. (3) Linked T cell depletion and clonal expansion resulted in a TCR diversity bottleneck after PT-Cy ( Fig 1). Due to the bottleneck, while the fraction of T cells originating from the transplant infusion at =/&amp;gt; 1 yr was similar between arms (18% vs 24%, p=0.7) there were significantly fewer Singleton TCRs in PT-Cy, suggesting a reduction in naïve T cells ( Fig 2), an outcome that has critical consequences for both cGVHD and infection. Thus while cGVHD onset with Tac/MTX was linked to a significant drop in TCR diversity due to singleton clonal expansion (median log-2 fold change in singletons with Tac/MTX = -0.25 p = 0.01) this drop was not observed with PT-Cy (log-2 fold change = -0.05, p = 0.1). Along with the significantly decreased # singletons at all time-points in PT-Cy ( Fig 2) this suggests that while cGVHD after Tac/MTX is linked to substantial naïve T cell expansion, the same is not true with PT-Cy, identifying a novel mechanism for cGVHD control with PT-Cy. The substantially fewer singletons in PT-Cy vs Tac/MTX may also increase the risk of Grade 2+ infections. T cell responses to new infections rely on antigen recognition/effector maturation of naïve (singleton TCR) T cells. After infection, the TCR repertoire became less diverse for both Tac/MTX and PT-Cy (log-2-fold singleton change -0.17 and -1.2 p = 0.0006 and 0.01) indicating that singletons in both cohorts were capable of expanding to pathogens. However the markedly decreased # singletons in PT-Cy at all timepoints was associated with significantly fewer singletons prior to infection (mean 3979 at 5K read-depth for Tac/MTX vs 2820 for PT-Cy p&amp;lt;0.001). This singleton deficit would place PT-Cy patients at higher risk of missing the critical mass of TCRs required to respond effectively to infections, consistent with their increased rate of Gr 2 infections. Conclusions: We performed longitudinal TCR-Seq on &amp;gt;200 million T cells from 324 HCT patients, representing the largest post-HCT TCR-Seq analysis in the history of the field, and enabling novel insights into TCR dynamics. These data identify a previously unappreciated global TCR diversity bottleneck with PT-Cy that simultaneously protects patients from cGVHD and places them at higher risk of infectious complications.

Efficacy and Safety of BCMA-Specific CAR T Cell-Based Therapy in Relapsed/Refractory Multiple Myeloma Patients with Extramedullary Disease

Background Extramedullary disease (EMD) is an aggressive subentity of multiple myeloma (MM), characterized by the ability of a subclone to grow independent of the bone marrow microenvironment, resulting in a high-risk state associated with increased proliferation, evasion of immune surveillance and treatment resistance. Relapsed/refractory multiple myeloma (R/R MM) patients with EMD have unfavorable prognoses and no effective treatment options. B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies have shown promising efficacy in R/R MM; however, data on patients with EMD remain limited. We aimed to assess the efficacy and safety of CAR T-cell therapy in R/R MM patients with EMD. Methods We conducted a retrospective multi-institutional study of 55 R/R MM patients confirmed with EMD at referral. The definition of EMD included (1) soft tissue masses in extraosseous locations resulting from hematogenous spread (EM-E) and (2) bone-related plasmacytomas that extend via disruption of cortical bones into contiguous soft tissues (EM-B). The overall response, long-term outcomes, and safety were assessed. We also characterized differential response between medullary and extramedullary disease, analyzed unique attributes of compartmental toxicity, and explored patterns of relapse in the setting of EMD. Gene expression profiling was performed using nCounter Human CAR-T Characterization Panel (NanoString, #XT-CSO-HCART1-12) on 40 biopsy specimens from patients enrolled in this study, including EMD and bone marrow biopsy specimens at pretreatment or/and at relapse. Results were analyzed based on outcome (no durable response [progression within 6 months, NDR] vs durable response [progression beyond 6 months, DR]). Multiplex immunofluorescence (mIF) was performed for detection of populations and phenotyping of infiltrated CD8 + T cells (Panel A: CD8/PD-1/LAG-3/TIM-3) and macrophages (Panel B: CD68/CD86/CD163) with EMD biopsies. Results The infusion resulted in an overall response rate of 90.9% (95% confidence interval [CI], 80.4-96.1) in medullary disease and 70.9% (95% CI, 57.9-81.2) in EMD ( P = 0.008). Discrepant outcomes between medullary and extramedullary response were observed, with suboptimal and delayed overall response and shortened duration of response achieved in EMD. With a median follow-up of 27.3 months, the median progression-free survival was 8.7 months (95% CI, 3.7-18.8), and the median overall survival was 16.0 months (95% CI, 13.5-20.1). Landmark analysis was conducted to compare OS in patients who progressed versus remained progression-free at the 6-month landmark time point. We demonstrated that progression before 6 months post-infusion is strongly associated with an increased risk of death (HR = 11.15 [95% CI, 3.49 to 35.60]; P &amp;lt; 0.001). Local cytokine release syndrome (CRS) was depicted in 21.8% patients and was moderately associated with the occurrence of systemic CRS (r = 0.322; P = 0.017). 78.2% patients experienced post-infusion progression in EMD, and BCMA + progression constituted the main pattern of EMD progression. At the time of EMD progression, peripheral CAR + cells were detectable, and 50% patients had ongoing documented B cell aplasia. We next interrogated NanoString gene expression data of tumor microenvironment (TME) genes for possible association with clinical response and toxicity. Pretreatment TME enriched for immune checkpoint, and type I interferon signaling were associated negatively with clinical outcomes. We found that the infiltration of type 2 macrophages (CD68 +CD163 +) in pretreatment TME is associated with the density of activated or exhausted CD8 + T cell ( PD-1 +LAG-3 +/-), and is associated with no durable response in EMD. We interrogated TME dynamic changes of patients developing EMD progression between cohorts of DR and NDR. An increase in immune counter-regulatory marker and lower diversity of T-cell receptor (TCR) repertoire, was observed at relapse in cohort of NDR. Discussion Our study shows that anti-BCMA CAR-T therapy has provided apparent therapeutic advantage over the existing drugs in response rate and long-term survival. Compared with medullary disease, BCMA-specific CAR T cell-based therapy showed limited and discrepant efficacy in EMD, perhaps due to the the properties of EMD-specific microenvironment.

Large-Scale Characterization and Functional Assessment of Adaptive Immunity in Patients with MGUS and SMM Reveals Significant Defects

Introduction Patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Multiple Myeloma (SMM) exhibit signs of immune dysregulation despite their early stage of oncogenesis. The SARS-CoV-2 pandemic and subsequent broad vaccination of the population presented an opportunity to compare the real-life immune response of patients and healthy individuals following exposure to a common antigen. Here, we have used SARS-CoV-2 vaccination as a model to study the adaptive immune response and have thus comprehensively assessed the adaptive immune response of patients with MGUS/SMM to SARS-CoV-2 vaccination compared to healthy donors (HD). Methods We performed (i) paired 5' single-cell RNA and T cell receptor (TCR) sequencing on 224 peripheral blood mononuclear cell (PBMC) samples drawn serially before and after two doses of SARS-CoV-2 vaccination (HD, n=26; MGUS, n=20; SMM, n=48; Multiple Myeloma (MM), n=24), (ii) ELISA for anti-Spike IgG antibodies post-vaccination (post-vx), (HD, n=237; MGUS, n=550; SMM, n=947) and MMR antibodies at baseline (HD, n=20; SMM, n=20), (iii) bulk TCR sequencing pre- and post-vx (HD, n=2; SMM, n=2) (Adaptive Biotechnologies), and (iv) IFN gamma ELISpot assay following in vitro stimulation with Spike peptide (HD, n=20; SMM, n=20). All post-vx samples were drawn after 2 doses of vaccination. Results Patients and HD showed comparable anti-Spike antibody titers within 14-50 days post-vx, however, we observed faster waning in patients with MGUS/SMM, who had significantly lower titers 50-100 days post-vx (p=0.014 &amp; p=0.007, respectively), suggesting a potential defect in long-lived plasma cell maintenance. Indeed, patients with SMM showed significantly lower titers of antibodies against Mumps and Rubella, antigens encountered in childhood, compared to age-matched HD (p=0.002 &amp; p=0.03, respectively), showing that this defect may also lead to loss of plasma cell memory. Next, we performed single-cell RNA/TCR-seq to assess the patients' T cell response to vaccination. Patients with SMM showed a significant increase in TCR repertoire diversity post-vx (paired t-test, p=0.046), suggesting at least partial preservation of T cell responses with recruitment of rare clones. Indeed, using bulk TCR-seq, we observed Spike-specific T cells in patients with MGUS/SMM. Interestingly, however, a significant enrichment in Spike-specific clones by single-cell TCR-seq was only observed in HD post-vx (paired t-test, p=0.01), suggesting patient T cell responses may be suboptimal. Functional assessment of T cell activation following stimulation with the Spike peptide showed significantly lower activation in patients with SMM compared to HD (t-test, p=0.002), confirming the suspected defect. Notably, a larger proportion of Spike-specific clones in patients was of the Treg phenotype compared to HD (empirical p=0.002), which may partly explain the observed defect. Conclusions This study demonstrates that despite the absence of symptoms, patients with MGUS/SMM may demonstrate defective humoral and cellular immune responses, including loss of plasma cell memory. Specific interventions, such as changes in the vaccination schedule, may be warranted for this population, and the morbidity associated with these defects should be studied further.

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&amp;lt;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.

TCR Sequencing in Mouse Models of Allorecognition Unveils the Features of Directly and Indirectly Activated Clonotypes.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

Factors regulating the differences in frequency of infiltration of Th17 and Treg of the blood-brain barrier.

Controlling CD4+ immune cell infiltration of the brain is a leading aim in designing therapeutic strategies for a range of neuropathological disorders such as multiple sclerosis, Alzheimer's disease, and depression. CD4+ T cells are a highly heterogeneous and reprogrammable family, which includes various distinctive cell types such as Th17, Th1, and Treg cells. Interestingly Th17 and Treg cells share a related transcriptomic profile, where the TGFβ-SMADS pathway plays a fundamental role in regulating the differentiation of both of these cell types. However, Th17 could be highly pathogenic and was shown to promote inflammation in various neuropathological disorders. Conversely, Treg is anti-inflammatory and is known to inhibit Th17. It could be noticed that Th17 frequencies of infiltration of the blood-brain barrier in various neurological disorders are significantly upregulated. However, Treg infiltration numbers are significantly low. The reasons behind these contradicting observations are still unknown. In this perspective, we propose that the difference in the T-cell receptor repertoire diversity, diapedesis pathways, chemokine expression, and mechanical properties of these two cell types could be contributing to answering this intriguing question.

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

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

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

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

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

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

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

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

Abstract 796: Altered immune response to vaccination in patients with plasma cell premalignancy

Abstract Introduction Patients with hematological malignancies, including multiple myeloma (MM), experience sub-optimal responses to SARS-CoV-2 vaccination. Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM) are precursors to MM and exhibit altered immune cell composition and function. The SARS-CoV-2 pandemic and the subsequent population-wide vaccination represent an opportunity to study the real-life immune response to a common antigen. Here, we present updated results from the IMPACT study, a study we launched in November 2020 to characterize the effect of plasma cell premalignancy on response to SARS-CoV-2 vaccination (vx). Methods We performed: (i) ELISA for SARS-CoV-2-specific antibodies on 1,887 peripheral blood (PB) samples (237 healthy donors (HD), and 550 MGUS, 947 SMM, and 153 MM patients) drawn pre- and post-vx; (ii) single-cell RNA, T cell receptor (TCR), and B cell receptor (BCR) sequencing (10x Genomics) on 224 PB samples (26 HD, and 20 MGUS, 48 SMM, and 24 MM patients) drawn pre- and post-vx; (iii) plasma cytokine profiling (Olink) on 106 PB samples (32 HD, and 38 MGUS and 36 SMM patients) drawn pre- and post-vx; and (iv) bulk TCR sequencing (Adaptive Biotechnologies) on 8 PB samples from 4 patients (2 MGUS, 2 SMM) drawn pre- and post-vx. Results Patients with MGUS and SMM achieved comparable antibody titers to HD two months post-vx. However, patient titers waned significantly faster, and 4 months post-vx we observed significantly lower titers in both MGUS (Wilcoxon rank-sum, p=0.030) and SMM (p=0.010). These results indicate impaired humoral immune response in patients with MGUS and SMM.At baseline, the TCR repertoire was significantly less diverse in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.039), while no significant difference was observed in the BCR repertoire (p=0.095). Interestingly, a significant increase in TCR repertoire diversity was observed post-vx in patients with SMM (paired t-test, p=0.014), indicating rare T cell clone recruitment in response to vaccination. In both HD and patients, recruited clones showed upregulation of genes associated with CD4+ naïve and memory T cells, suggesting preservation of the T cell response in SMM, which was confirmed by bulk TCR-sequencing in 4 patients.Lastly, by cytokine profiling, we observed a defect in IL-1β and IL-18 induction post-vx in patients with SMM compared to HD (Wilcoxon rank-sum, p=0.047 and p=0.015, respectively), two key monocyte-derived mediators of acute inflammation, suggesting an altered innate immune response as well. Conclusion Taken together, our findings highlight that despite the absence of clinical manifestations, plasma cell premalignancy is associated with defects in both innate and adaptive immune responses. Therefore, patients with plasma cell premalignancy may require adjusted vaccination strategies for optimal immunization. Citation Format: Yoshinobu Konishi, Romanos Sklavenitis-Pistofidis, Michelle P. Aranha, Ting Wu, Hong Yue, Elizabeth D. Lightbody, Mahshid Rahmat, Michael Timonian, Shohreh Varmeh, Daniel Heilpern-Mallory, Michael P. Agius, Nang K. Su, Jacqueline Perry, Erica Horowitz, Maya I. Davis, Anna V. Justis, Radosław P. Nowak, Mark Hamilton, Daniel Auclair, Catherine R. Marinac, Eric S. Fischer, Gad Getz, Irene M. Ghobrial. Altered immune response to vaccination in patients with plasma cell premalignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 796.

Abstract 1719: VHLloss enables immune checkpoint blockade therapy by boosting interferon response

Abstract Background: The von-Hippel Lindau (VHL) as a part of E3 ubiquitin ligase is a key regulator of hypoxia induced factors (HIFs). It is also characterized as a tumor suppressor gene mutated in up to 80% clear cell renal cell carcinoma (ccRCC) cases. Immune checkpoint blockade (ICB) therapy has shown improved ccRCC treatment outcomes. However, the biological roles of the VHL gene in ICB therapy are to be explored. Methods: The association between VHL gene mutation and overall survival of patients receiving ICB treatments was determined by integrating multiple human datasets. CRISPR/Cas9 was used to generate VHL knockout (KO) for in vivo tumor growth and in vitro mechanistic studies. The anti-tumor responses of genetic VHL loss in combination with anti-PD1 treatment were evaluated in three syngeneic murine tumor models. Tumor infiltrated lymphocytes and intratumoral T-cell receptor (TCR) diversity were analyzed by flow cytometry and TCR sequencing, respectively. Mitochondrial defects resulted from VHL loss were tested by JC-1 mitochondrial membrane potential assay, immunofluorescence microscopy, and qRT-PCR. VHL loss-mediated downstream signaling were further examined by western blot and bulk RNA sequencing. Results: The substantial overall survival benefits among ICB-treated patients withVHL mutations across different cancers, including RCC, in multiple human cohorts. Genetic knockout of VHL enhanced anti-PD1 therapy in three murine tumor models, including those of non-RCC origins. Moreover, VHL loss caused significant infiltration of immune effector cells and increased diversity of intratumoral TCR repertoire. VHL deficiency resulted in HIFα accumulation, increased expression of pro-mitophagy factor BNIP3, and impaired mitochondrial membrane potential that led to the cytoplasmic leakage of mitochondrial DNA (mtDNA), triggering cGAS-STING activation and type I interferon production. HIFα overexpression showed similar results as demonstrated in VHL-KO cells. In contrast, double knockdown of VHL and HIFα reversed VHL-loss induced BNIP3 upregulation, rescued mtDNA leakage to the cytoplasm, and disrupted constitutive activation of cGAS-STING signaling. Furthermore, blocking type I interferon signaling abrogated VHL-deficiency induced tumor suppression. Conclusion: Our study uncovered a novel role for VHL loss in creating an immunogenic tumor microenvironment through the activation of mtDNA-cGAS-STING pathway and type I interferon signaling in tumor cells via VHL-HIFα-BNIP3 axis, making them susceptible to ICB therapy. This finding provides a mechanistic explanation for ccRCC responsiveness to ICB therapy despite a moderate tumor mutation burden and suggests potential therapeutic strategies targeting ccRCC and other VHL-deficient tumors based on constitutive cGAS-STING and type I interferon activation. Citation Format: Meng Jiao, Xuhui Bao, Mengjie Hu, Dong Pan, Jonathan Kim, Xinjian Liu, Fang Li, Chuan-Yuan Li. VHLloss enables immune checkpoint blockade therapy by boosting interferon response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1719.