Cell Receptor Repertoire Diversity Research Articles

MicroRNAs targeting TGF-β signaling exacerbate central nervous system autoimmunity by disrupting regulatory T cell development and function.

Transforming growth factor beta (TGF-β) signaling is essential for a balanced immune response by mediating the development and function of regulatory T cells (Tregs) and suppressing autoreactive T cells. Disruption of this balance can result in autoimmune diseases, including multiple sclerosis (MS). MicroRNAs (miRNAs) targeting TGF-β signaling have been shown to be upregulated in naïve CD4 T cells in MS patients, resulting in a limited in vitro generation of human Tregs. Utilizing the murine model experimental autoimmune encephalomyelitis, we show that perinatal administration of miRNAs, which target the TGF-β signaling pathway, enhanced susceptibility to central nervous system (CNS) autoimmunity. Neonatal mice administered with these miRNAs further exhibited reduced Treg frequencies with a loss in T cell receptor repertoire diversity following the induction of experimental autoimmune encephalomyelitis in adulthood. Exacerbated CNS autoimmunity as a result of miRNA overexpression in CD4 T cells was accompanied by enhanced Th1 and Th17 cell frequencies. These findings demonstrate that increased levels of TGF-β-associated miRNAs impede the development of a diverse Treg population, leading to enhanced effector cell activity, and contributing to an increased susceptibility to CNS autoimmunity. Thus, TGF-β-targeting miRNAs could be a risk factor for MS, and recovering optimal TGF-β signaling may restore immune homeostasis in MS patients.

Abstract 5216: Peripheral T cell receptor repertoire diversity is associated with outcome in bladder cancer

Abstract Bladder cancer is the sixth most common cancer overall, and treatment often involves removal of the bladder, which comes with a significant reduction in quality of life. Within this cancer type, an easily applicable prognostic biomarker may have clinical utility, potentially stratifying patients into bladder-sparing treatments. Expansion of T cell populations targeting cancer-specific neoantigens is an early response to malignancy. To investigate if the T cell receptor (TCR) repertoire could serve as a biomarker for cancer progression, we analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer, treated with neoadjuvant chemotherapy before undergoing radical cystectomy. Peripheral and tumor TCR repertoires were produced using amplicon-based sequencing of the TCR beta chain. Additionally, T cell fraction was determined from whole exome sequencing data using TcellExTRECT, and latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. To explore the T cell repertoire subtypes, single-cell sequencing was performed on four fresh blood samples. In pre-treatment peripheral blood, low-diversity TCR repertoires were associated with worse survival (HR=2.3, p=0.024). Particularly poor outcome was observed in a subset of patients with both low TCR diversity and low fraction of circulating T cells (HR=4.7, p=5e-4). Low-diversity repertoires were dominated by expanded T cell clones. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections (p=2e-13), and cytomegalovirus DNA detection was strongly associated with decreased TCR diversity (p=5e-5). Additionally, tumor TCR sequencing revealed that expanded clones were rarely found in the tumor. Single-cell sequencing of peripheral blood found that most expanded clones consisted of cytotoxic T cells, while non-expanded clones mostly consisted of naive T cells. This indicates that low TCR diversity may represent a contraction of the naive T cell compartment, resulting in reduced immune competence. Finally, analysis of patient-matched longitudinal samples revealed that chemotherapy and cystectomy reduced TCR diversity in patients with high pre-treatment diversity (p=0.001), indicating that treatment negatively affected the immune system in the most immune-competent patients. Our results suggest that both low TCR diversity and low T cell fraction represent decreased immune competency. This could be associated with an elevated risk of progression due to a reduced ability to control cancer growth and limit systemic cancer cell dissemination. These findings underline that immune health is an important factor during malignant disease, even when patients are not treated with immunotherapies. Therefore, improving immune health could be a potential future goal for cancer treatment and prevention. Citation Format: Asbjørn Kjær, Nanna Kristjánsdóttir, Iver Nordentoft, Randi Istrup Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen Bjerggaard Jensen, Nicolai J Birkbak, Lars Dyrskjøt. Peripheral T cell receptor repertoire diversity is associated with outcome in bladder 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 5216.

Metabolic and senescence characteristics associated with the immune microenvironment in non-small cell lung cancer: insights from single-cell RNA sequencing.

Non-small lung cancer (NSCLC) has been defined as a highly life-threatening heterogeneous disease, with high mortality and occurrence. Recent research has indicated that tumor-infiltrating lymphocytes play a key determinant role in cancer progression. Emerging single-cell RNA sequencing (also termed scRNA-seq) has been extensively applied to depict the baseline landscape of the cell composition and function phenotype in the tumor environment (TME). Herein, we dissected the cell types in NSCLC samples (including tissue and blood) and identified three types of cell marker genes including cancer cells, T cells, and macrophages by integrating two NSCLC-associated scRNA-seq datasets in GEO. Survival analysis indicated that 17 marker genes were related to tumor prognosis. Function annotation was used to scrutinize the molecular mechanism of these marker genes in different cells. Besides, we investigated the developmental trajectory and T cell receptor repertoire diversity of tumor-infiltrating T cells. Our analysis will help further understand the complexity of cell components and the heterogeneity of TME in NSCLC.

Dietary restriction mitigates the age-associated decline in mouse B cell receptor repertoire diversity

Aging impairs the capacity to respond to novel antigens, reducing immune protection against pathogens and vaccine efficacy. Dietary restriction (DR) extends life- and health span in diverse animals. However, little is known about the capacity of DR to combat the decline in immune function. Here, we study the changes in B cell receptor (BCR) repertoire during aging in DR and control mice. By sequencing the variable region of the BCR heavy chain in the spleen, we show that DR preserves diversity and attenuates the increase in clonal expansions throughout aging. Remarkably, mice starting DR in mid-life have repertoire diversity and clonal expansion rates indistinguishable from chronic DR mice. In contrast, in the intestine, these traits are unaffected by either age or DR. Reduced within-individual B cell repertoire diversity and increased clonal expansions are correlated with higher morbidity, suggesting a potential contribution of B cell repertoire dynamics to health during aging.

Multimodal single-cell analyses of peripheral blood mononuclear cells of COVID-19 patients in Japan

SARS-CoV-2 continues to spread worldwide. Patients with COVID-19 show distinct clinical symptoms. Although many studies have reported various causes for the diversity of symptoms, the underlying mechanisms are not fully understood. Peripheral blood mononuclear cells from COVID-19 patients were collected longitudinally, and single-cell transcriptome and T cell receptor repertoire analysis was performed. Comparison of molecular features and patients’ clinical information revealed that the proportions of cells present, and gene expression profiles differed significantly between mild and severe cases; although even among severe cases, substantial differences were observed among the patients. In one severely-infected elderly patient, an effective antibody response seemed to have failed, which may have caused prolonged viral clearance. Naïve T cell depletion, low T cell receptor repertoire diversity, and aberrant hyperactivation of most immune cell subsets were observed during the acute phase in this patient. Through this study, we provided a better understanding of the diversity of immune landscapes and responses. The information obtained from this study can help medical professionals develop personalized optimal clinical treatment strategies for COVID-19.

A phase I study of a mutant KRAS-targeted long peptide vaccine combined with ipilimumab/nivolumab in resected pancreatic cancer and MMR-proficient metastatic colorectal cancer.

TPS814 Background: Novel strategies are needed to improve immune responses in “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) and mismatch repair-proficient colorectal cancer (MMRp CRC). As a frequent oncogenic driver, mutant KRAS (mKRAS) neoantigens are attractive targets to augment anti-tumor immunity in both diseases. Recently, adoptive transfer of mKRAS G12D-specific T cells has shown durable tumor regressions in patients with metastatic CRC and PDAC (Tran et. al., 2020; Leidner et. al., 2022). Furthermore, our preclinical work has demonstrated that combining a mKRAS neoantigen vaccine with immune-modulating agents prevents progression of premalignant lesions to PDAC in mice (Keenan et. al., 2014). Based on this rationale, our study pairs a pooled synthetic long peptide (SLP) mKRAS vaccine with dual checkpoint blockade to assess safety and immunogenicity in patients with resected PDAC and chemorefractory MMRp CRC. Methods: This is a first-in-human, single-arm, open-label phase I trial evaluating a pooled SLP mKRAS vaccine combined with ipilimumab/nivolumab (ipi/nivo) in patients with resected PDAC (Cohort A, n = 12) and MMRp metastatic CRC (Cohort B, n = 12) The vaccine consists of poly-ICLC adjuvant admixed with SLPs corresponding to six common mKRAS subtypes: G12D, G12R, G12V, G12A, G12C, and G13D. In priming phase, the mKRAS vaccine is given on days 1, 8, 15, and 22 along with ipi/nivo. In boost phase, the mKRAS vaccine is given on weeks 13, 21, 29, 37, and 45 along with nivo alone. Cohort A patients who remain disease-free can continue to receive boost vaccines in a 12-month extended treatment phase. Eligible patients must have molecular tumor testing that demonstrates one of the six KRAS mutations listed above. Cohort A patients must be disease-free following completion of adjuvant chemotherapy within 6 months prior to study entry. Cohort B patients must have confirmed MMRp status, exposure to ≥ 2 prior lines of standard chemotherapy, and measurable disease amenable to biopsies at baseline and week 7. The co-primary endpoints of this study are safety and T cell response. Adverse events will be graded per NCI CTCAE v5.0. T cell response will be determined by the maximal percent change in IFNγ-producing mKRAS-specific T cell density within 16 weeks post-vaccination compared to baseline. Secondary endpoints include disease control and objective response rates at 16 weeks per RECIST v1.1/iRECIST (Cohort B only) as well as disease-free/progression-free and overall survival. Correlative studies will examine treatment-associated changes in T cell receptor (TCR) repertoire diversity by next-generation TCR sequencing of peripheral blood and tumor specimens. Patient accrual began in May 2020 and is completed for Cohort A. Enrollment is currently ongoing for Cohort B. Study drug support provided by Bristol Myers Squibb. Clinical trial information: NCT04117087 .

Amphibians as a model to study the role of immune cell heterogeneity in host and mycobacterial interactions

Mycobacterial infections represent major concerns for aquatic and terrestrial vertebrates including humans. Although our current knowledge is mostly restricted to Mycobacterium tuberculosis and mammalian host interactions, increasing evidence suggests common features in endo- and ectothermic animals infected with non-tuberculous mycobacteria (NTMs) like those described for M. tuberculosis. Importantly, most of the pathogenic and non-pathogenic NTMs detected in amphibians from wild, farmed, and research facilities represent, in addition to the potential economic loss, a rising concern for human health. Upon mycobacterial infection in mammals, the protective immune responses involving the innate and adaptive immune systems are highly complex and therefore not fully understood. This complexity results from the versatility and resilience of mycobacteria to hostile conditions as well as from the immune cell heterogeneity arising from the distinct developmental origins according with the concept of layered immunity. Similar to the differing responses of neonates versus adults during tuberculosis development, the pathogenesis and inflammatory responses are stage-specific in Xenopus laevis during infection by the NTM M. marinum. That is, both in human fetal and neonatal development and in tadpole development, responses are characterized by hypo-responsiveness and a lower capacity to contain mycobacterial infections. Similar to a mammalian fetus and neonates, T cells and myeloid cells in Xenopus tadpoles and axolotls are different from the adult immune cells. Fetal and amphibian larval T cells, which are characterized by a lower T cell receptor (TCR) repertoire diversity, are biased toward regulatory function, and they have distinct progenitor origins from those of the adult immune cells. Some early developing T cells and likely macrophage subpopulations are conserved in adult anurans and mammals, and therefore, they likely play an important role in the host-pathogen interactions from early stages of development to adulthood. Thus, we propose the use of developing amphibians, which have the advantage of being free-living early in their development, as an alternative and complementary model to study the role of immune cell heterogeneity in host-mycobacteria interactions.

The T Cell Receptor Repertoire Diversity Following Hematopoietic Stem Cell Transplantation.

Immune reconstitution after hematopoietic stem cell transplantation (HSCT) with a conditioning regimen has appeared to be a promising treatment for autoimmune diseases and hematologic malignancies. This study aimed to assess the T cell receptor (TCR) repertoire diversity in CD4+ cells of patients with hematological malignancies who received allogeneic or autologous HSCT. The diversity of the TCR repertoire was evaluated in 13 patients with hematologic malignancies before and four months after HSCT. Amino acid changes in the 25 Vβ families were evaluated using Spectratyping and data were presented as Hamming distance (HD). HD more than 20% was considered a change in TCR repertoire after HSCT. The mean HD was significantly changed after transplantation in all Vβ gene families, with most amino acid changes in p4 and p22 families. There was a strong negative correlation between the HD as the index of TCR repertoire and age (r = -0.62,). The results revealed no association between HD mean and parameters such as sex, disease, conditioning regimen, and type of transplantation. Our data revealed that commonly used conditioning regimens in Iran could successfully cause TCR repertoire diversity in patients with hematologic malignancies in the short term. The amount of change in TCR repertoire was inversely correlated with the increasing age of patients.

Abstract LB094: First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery

Abstract T cell receptor repertoire diversity and T cell cytokine production are the important attributes for the assessment of effective T cell immunity against cancer and pathogens, yet existing single-cell genomics platforms cannot interrogate TCR sequences and proteomic cytokines simultaneously. IsoPlexis’ functional proteomics has demonstrated the values in cell therapy and cancer immunology against blood cancers and solid tumors. The ability to connect the T cell receptor diversity to the most potent single cells enables a wide variety of applications for tumor infiltrating lymphocytes, personalized neoantigen TCR’s, cancer immunology in terms of understanding antigen specificity, as well as self-potency. IsoPlexis single-cell TCR Duomic platform provide a unique measurement for connecting T cell receptor sequences to those T cell cytokine profiles, for the first time. Human PBMC-derived T cell line established from healthy donor was stimulated with PMA/ionomycin (2ul/ml, Biolegend) for 6hrs, stained and resuspended in Reverse transcription premix containing reverse transcriptase with buffer and protein phosphatase inhibitor at 1 x 106/ml and then loaded on Duomic chips. Ran on IsoLight overnight, cDNA was collected from the chip followed by cleanup and amplification, proceeded with TCR amplification and sequencing on NextSeq 1000/2000. Proteomic data from single cells was automatically generated and analyzed by IsoSpeak software. The single-cell TCR sequencing data on the IsoPlexis TCR Duomics platform shows that 78.9% cells have TCR alpha chain recombination and 94.4% cells have TCR beta chain recombination. Totally 77.7% cells have combination on both alpha and beta chain. VDJ Usage Plot further displays the top10 most common V (D) J combination flow between T cell receptor alpha chain and beta chain among the single cells, revealing the diversity of TCR repertoire at the single-cell level. In addition, the proteomics data identifies the dominant proteins among the single cells are Granzyme B, GM-CSF, IFN-g and TNF-a. Furthermore, we define 3 distinct cell clusters based on functional proteins and an association of VDJ distribution with these cell clusters, suggesting the occurrence of V(D)J sequences in the most common reconstructed alpha and beta sequence. IsoPlexis single-cell TCR Duomics platform, for the first time, enables simultaneous profiling of functional proteomics and TCR repertoires/TCR clonotypes as well as identification of specific TCR V(D)J combination with functional cell clusters across the same individual T cells. The combination of these two analytes at the single cells provides a key new modality of tracking the most potent and also the antigen specific immune cells together to fight cancer and infectious disease, for the first time. Citation Format: Jennifer Yang, Jing Zhou, Sean Mackay. First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB094.

BCG hydrogel promotes CTSS-mediated antigen processing and presentation, thereby suppressing metastasis and prolonging survival in melanoma

BackgroundThe use of intralesional Mycobacterium bovis BCG (intralesional live BCG) for the treatment of metastatic melanoma resulted in regression of directly injected, and occasionally of distal lesions. However, intralesional-BCG is...

Comprehensive immune profiling unravels evolution of spatial distribution and immune repertoire in tumor microenvironment from primary to metastatic triple-negative breast cancer.

1079 Background: Immune checkpoint inhibitors (ICI) have improved PFS and OS in metastatic triple-negative breast cancer (mTNBC), but benefit is limited to PD-L1 positive tumors. Metastatic tumors are notorious for deficient immune cell infiltration and immunosuppressive features that may limit responses to ICI in mTNBC. However, the underlying mechanisms for the weak immunogenicity of the metastatic tumor immune microenvironment (TIME) and related poor ICI responses are still not well understood. The current study was designed to investigate the evolution of the TIME between paired primary and metastatic TNBCs. Methods: Spatial distribution of 37 key immune regulators using the NanoString digital spatial profiling (DSP) platform was analyzed. 452 regions of interests (ROIs) from 33 primary tumors (PT) and 29 metastatic tumors (MT) including 28 paired specimens, were selected based on CD45+ immune hotspots, and the protein expression levels of the key immune regulators were quantified within pan-cytokeratin (panCK) and CD45 masked regions, respectively. In parallel, we examined the clonality of tumor infiltrating B cell receptors by reconstructing the immune repertoire from bulk RNA-seq data. Results: Using the DSP platform, we confirmed reduced immune infiltration (e.g., CD3 and CD20) in both panCK and CD45 masked regions of MT, while CD8A and CD11c (padj = 2.8×10-7 and 2.1×10-6) expression was only observed in panCK masked regions of MT compared with PT. A significant shift in myeloid composition between PT and MT as evidenced by increased CD68 signal (padj = 5.8×10-4) in CD45 masked regions of MT was identified. Within MT, PD-L1 signal was substantially higher (padj = 0.030) in CD45 masked regions only, while PD1 counts were lower (padj = 0.035) in panCK masked regions. This suggests the limited responses to ICI for MT may stem from relatively low expression of activated and targetable T cell subsets in MT islands. In support of the lower CD20 counts in MT, immune repertoire analysis revealed B cell receptor (BCR) repertoire diversity (represented by Gini index) was substantially lower in MT than PT (p = 0.041) suggesting that the ability of B cells to recognize a wide variety of tumor antigens in MT is greatly reduced in contrast to PT. Conclusions: Through comprehensive analysis of the TIME spatial organization within paired PT and MT, a significant reduction in dendritic cell/macrophage ratios (CD11c/CD68), reduced tumor localized T cell activation (CD8, PD1, PD-L1), and reduced B cell diversity (BCR clonality) are key features of the reduced immunogenicity of the metastatic TIME in TNBC. Further work to understand key mechanistic features driving the evolution of these differences in TIME between primary and metastatic tumors are ongoing.

Single-cell RNA sequencing coupled to TCR profiling of large granular lymphocyte leukemia T cells

T-cell large granular lymphocyte leukemia (T-LGLL) is a lymphoproliferative disease and bone marrow failure syndrome which responds to immunosuppressive therapies. We show single-cell TCR coupled with RNA sequencing of CD3+ T cells from 13 patients, sampled before and after alemtuzumab treatments. Effector memory T cells and loss of T cell receptor (TCR) repertoire diversity are prevalent in T-LGLL. Shared TCRA and TCRB clonotypes are absent. Deregulation of cell survival and apoptosis gene programs, and marked downregulation of apoptosis genes in CD8+ clones, are prominent features of T-LGLL cells. Apoptosis genes are upregulated after alemtuzumab treatment, especially in responders than non-responders; baseline expression levels of apoptosis genes are predictive of hematologic response. Alemtuzumab does not attenuate TCR clonality, and TCR diversity is further skewed after treatment. Inferences made from analysis of single cell data inform understanding of the pathophysiologic mechanisms of clonal expansion and persistence in T-LGLL.

Response and recurrence correlates in individuals treated with neoadjuvant anti-PD-1 therapy for resectable oral cavity squamous cell carcinoma

SummaryNeoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer.

Thymic Aging May Be Associated with COVID-19 Pathophysiology in the Elderly.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the global pandemic of coronavirus disease 2019 (COVID-19) and particularly exhibits severe symptoms and mortality in elderly individuals. Mounting evidence shows that the characteristics of the age-related clinical severity of COVID-19 are attributed to insufficient antiviral immune function and excessive self-damaging immune reaction, involving T cell immunity and associated with pre-existing basal inflammation in the elderly. Age-related changes to T cell immunosenescence is characterized by not only restricted T cell receptor (TCR) repertoire diversity, accumulation of exhausted and/or senescent memory T cells, but also by increased self-reactive T cell- and innate immune cell-induced chronic inflammation, and accumulated and functionally enhanced polyclonal regulatory T (Treg) cells. Many of these changes can be traced back to age-related thymic involution/degeneration. How these changes contribute to differences in COVID-19 disease severity between young and aged patients is an urgent area of investigation. Therefore, we attempt to connect various clues in this field by reviewing and discussing recent research on the role of the thymus and T cells in COVID-19 immunity during aging (a synergistic effect of diminished responses to pathogens and enhanced responses to self) impacting age-related clinical severity of COVID-19. We also address potential combinational strategies to rejuvenate multiple aging-impacted immune system checkpoints by revival of aged thymic function, boosting peripheral T cell responses, and alleviating chronic, basal inflammation to improve the efficiency of anti-SARS-CoV-2 immunity and vaccination in the elderly.

Thymopoiesis, Alterations in Dendritic Cells and Tregs, and Reduced T Cell Activation in Successful Extracorporeal Photopheresis Treatment of GVHD

Acute graft-versus-host disease (aGVHD) is a significant complication of allogeneic hematopoietic stem cell transplant (HSCT) and negatively affects T cell reconstitution. Extracorporeal photopheresis (ECP) reduces aGVHD, but the mechanisms remain incompletely understood. Our objective was to examine the impact of ECP on thymopoiesis in pediatric aGVHD and the mechanisms at a cellular and transcriptional level. Sixteen pediatric HSCT patients were recruited: 6 with ECP-treated aGVHD, 5 without aGVHD, and 5 with aGVHD treated with corticosteroids only. Thymopoiesis was evaluated by measuring naive T cells, TRECs, IL-7, and T cell receptor repertoire diversity. Regulatory T cell (Treg) enumeration and function and dendritic cell (DC) enumeration and phenotype were analyzed using flow cytometry. T cell transcriptome analysis was performed on ECP patients after treatment and responders pre- and post-treatment. Four ECP responders demonstrated thymic-dependent T cell recovery, and superior median naïve T cell numbers at 8 and 12 months post-HSCT compared to the aGVHD corticosteroid group. Increased Tregs and Treg suppressive function, reduced cDC/pDC and DC co-stimulatory marker expression in ECP responders suggest upregulated peripheral tolerance; these findings were not observed in partial responders. Responder post-ECP CD3+ T cell transcriptional profile demonstrated 3333 downregulated and 364 upregulated genes, with significant downregulation of ERRα and GαS pathways, and reduced expression of pro-inflammatory and adhesion proteins.Thymic function improves with successful ECP treatment. ECP reduces T cell activation and impacts peripheral tolerance via DCs and Tregs. Differences in thymic recovery, DC, and Treg cellular patterns and the T cell transcriptome were observed between ECP responders and partial responders and require further validation and investigation in additional patients.

Distinct T cell receptor repertoire diversity of clinically defined high-grade serous ovarian cancer treatment subgroups

SummaryIn patients with high-grade serous ovarian cancer (HGSC), it is unclear which genomic features are related to complete gross resection (R0), which is typically associated with better clinical outcomes, or response to neoadjuvant chemotherapy (NACT). In this study, we evaluated T cell receptor (TCR) repertoire diversity in primary and metastatic tumor samples (n = 90) from clinically well-annotated patients with HGSC who achieved R0 or received NACT with excellent or poor response based on a laparoscopic triage algorithm. TCR sequencing followed by an integrative analysis with comprehensive multi-omics data identified higher TCR diversity (e.g., higher number of unique productive sequences and less clonal relatedness) in the R0 than NACT groups. We found enrichment of specific TCRβ genes usage, distinct mutual exclusiveness and co-occurrence pattern of TCRβ genes among the groups. We also found significantly positive correlations between clonal relatedness and neoantigens, copy number variations, and mutation load in the groups.

Ageing compromises mouse thymus function and remodels epithelial cell differentiation.

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

Identification and Validation of the Immune Subtypes of Lung Adenocarcinoma: Implications for Immunotherapy.

Lung adenocarcinoma (LUAD) is a devastating disease with poor patient survival. Cancer immunotherapy has revolutionized the treatment of LUAD, but only a limited number of patients effectively respond to this treatment. Thus, the work to elucidate the LUAD immune heterogeneity could be crucial in developing new immunotherapeutic strategies with better efficacy. Non-negative matrix factorization-based deconvolution was performed to identify robust clusters of 489 LUAD patients in The Cancer Genome Atlas (TCGA) and verify their reproducibility and stability in an independent LUAD cohort of 439 patients from the Gene Expression Omnibus (GEO). We used the graph learning-based dimensionality reduction to visualize the distribution of individual patients. In this study, four reproducible immune subtypes, Clusters 1–4 (C1–C4) associated with distinct gene module signatures, clinicopathological features, molecular and cellular characteristics were identified and validated. The immune-cold subtype, C3, was associated with the Dead event, the most advanced T stage, N stage, TNM stage and the worst prognosis for LUAD patients. Moreover, C3 exhibited the lowest infiltrating levels of B cells, T cell receptor (TCR) repertoire diversity and the highest level of neoantigen and mutation rate among C1–C4. On the other hand, the immune-hot subtype (C4) exhibited the highest infiltration of six types of infiltrating immune cells as well as the greatest leukocyte fraction, TCR and B cell receptor (BCR) repertoire diversity. C1 and C2 subtypes showed diverse clinicopathological and immunological features. Finally, our investigations discovered a complex immune landscape with a scattered immune subtype profile. This work may help inform immunotherapeutic decision-making and design advanced immunotherapy strategies for the treatment of lung cancer.

Characterizing the TCR repertoire in Neuropsychiatric Lupus

Abstract Approximately 40% of lupus patients experience neurological and/or psychiatric disorders. However, the mechanisms contributing to the wide range of neuropsychiatric manifestations (NPSLE) have not yet been fully elucidated. We have demonstrated that the choroid plexus (CP) in the MRL/lpr NPSLE lupus mouse model is heavily infiltrated with effector T cells. We investigated T cell receptor (TCR) usage and repertoire diversity in CP infiltrating T cells, to enhance our understanding of this T cell population and its possible contribution to neuropsychiatric disease. The CP, salivary glands, and spleens were dissected from female MRL/lpr mice (n=9, n=9, n=4 respectively) and the spleens from female MRL/MpJ (n=5) at 17–22 weeks of age, at which time MRL/lpr mice display prominent neurobehavioral deficits. The TCR repertoire was evaluated by isolating genomic DNA, amplifying the CDR3 sequence by multiplex PCR (Adaptive Technologies), and analyzing the sequences with the Adaptive Technologies immunoSeq Analyzer and the R software, LymphoSeq. MRL/lpr CP tissues had increased numbers of T cell sequences, unique productive sequences, and clonality. The top 10 productive rearrangements were more abundant in MRL/lpr CP when compared to the MRL/lpr and MRL/MpJ splenic tissues (p = 0.0673 and p = 0.0003 respectively). The MRL/lpr CP had an increased frequency of TCRBV02, TCRBV04, TCRBV12, and TCRBV26 genes compared to MRL/lpr splenic and salivary gland tissues. Despite the lymphoproliferation found in this lupus strain, the increased oligoclonal expansion and limited TCR V beta gene usage in the MRL/lpr CP T cell repertoire, as compared to other MRL/lpr tissues, implies a distinct and specific infiltration of T cells into the CP.

Abstract LB-190: Single-cell polyfunctionality of circulating CD4+ T cells correlates with T cell receptor repertoire diversity of PBMCs, indicating potential synergistic activity and peripheral biomarker predictive of efficacy of intratumor injection of the toll-like receptor 4 agonist G100

Abstract Predictive biomarker plays a crucial role in identifying cancer patients who might benefit from a therapy and facilitating a personalized medication. Polyfunctional T cells (co-secretion of 2+ proteins per cell) measured by single-cell IsoCode chip proteomics was demonstrated to predict patient response to CAR-T therapy. Herein, we utilized the IsoCode chip technology to study the efficacy of G100 on eliciting systemic polyfunctional T cells and the association of T cell polyfunctionality with the diversity of T cell receptor (TCR) repertoire in patients with soft tissue sarcomas (STS). PBMC were collected pre- and post-treatment from patients with metastatic STS treated with intratumoral (IT) G100. CD4+ and CD8+ T cells were enriched by microbeads, stimulated with anti-CD3/CD28 at 37°C, 5% CO2 for 24 hours and loaded into an IsoCode chip containing ~12,000 microchambers pre-patterned with a complete copy of a 32-plex antibody array. After 16-hour incubation, protein secretions were analyzed from ~1000 single cells; polyfunctional profile was evaluated across 5 groups: Effector: Granzyme B, TNF-α, IFN-γ, MIP-1α, Perforin, TNF-β; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGF-β1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β. Polyfunctional strength index (PSI) was calculated as the percentage of polyfunctional cells multiplied by the intensities of secreted proteins. TCR deep sequencing of PBMCs was performed to evaluate the TCR repertoire diversity. Both CD4+ and CD8+ T cells had heterogeneous polyfunctional profiles pre- and post-treatment. CD4+ T cells showed a marked increase in polyfunctionality while CD8+ T cells exhibited a polyfunctional decrease after treatment. A greater variety of proteins associated with antitumor immunity was detected in CD4+ T cells post treatment. TNF-α, IL-8, Granzyme B, IL-5, MIP-1β and sCD137 primarily drove the enhanced PSI of CD4+ T cells. More importantly, the pre-treatment CD4 PSI positively correlated with the TCR diversity of PBMCs (P = 0.0185). Interestingly, the functional subgroups of effector, stimulatory and regulatory secretions, but not chemoattractive and inflammatory secretions, contributed to the significant association between pretherapy CD4 PSI and PBMC diversity. Single-cell multiplexed proteomics sensitively identifies circulating polyfunctional T cells induced by IT injection of G100 in patients with STS. The pre-treatment PSI of blood CD4+ T cells correlates with TCR diversity, providing a potential peripheral metric to predict patient response to G100 for the development of more effective therapeutics. Citation Format: Sean Mackay, Jon Chen, Jeremy Patino, Y. David Seo, Seth Pollack, Jing Zhou. Single-cell polyfunctionality of circulating CD4+ T cells correlates with T cell receptor repertoire diversity of PBMCs, indicating potential synergistic activity and peripheral biomarker predictive of efficacy of intratumor injection of the toll-like receptor 4 agonist G100 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-190.