Cell Receptor Repertoire Analysis Research Articles

CD4+ T cell heterogeneity in gestational age and preeclampsia using single-cell RNA sequencing.

A balance between pro-inflammatory decidual CD4+ T cells and FOXP3+ regulatory T cells (FOXP3+ Tregs) is important for maintaining fetomaternal tolerance. Using single-cell RNA-sequencing and T cell receptor repertoire analysis, we determined that diversity and clonality of decidual CD4+ T cell subsets depend on gestational age. Th1/Th2 intermediate and Th1 subsets of CD4+ T cells were clonally expanded in both early and late gestation, whereas FOXP3+ Tregs were clonally expanded in late gestation. Th1/Th2 intermediate and FOXP3+ Treg subsets showed altered gene expression in preeclampsia (PE) compared to healthy late gestation. The Th1/Th2 intermediate subset exhibited elevated levels of cytotoxicity-related gene expression in PE. Moreover, increased Treg exhaustion was observed in the PE group, and FOXP3+ Treg subcluster analysis revealed that the effector Treg like subset drove the Treg exhaustion signatures in PE. The Th1/Th2 intermediate and effector Treg like subsets are possible inflammation-driving subsets in PE.

Enhancing comparative T cell receptor repertoire analysis in small biological samples through pooling homologous cell samples from multiple mice

Accurate characterization and comparison of Tcell receptor (TCR) repertoires from small biological samples present significant challenges. The main challenge is the low material input, which compromises the quality of bulk sequencing and hinders the recovery of sufficient TCR sequences for robust analyses. We aimed to address this limitation by implementing a strategic approach to pool homologous biological samples. Our findings demonstrate that such pooling indeed enhances the TCR repertoire coverage, particularly for cell subsets of constrained sizes, and enables accurate comparisons of TCR repertoires at different levels of complexity across Tcell subsets with different sizes. This methodology holds promise for advancing our understanding of Tcell repertoires in scenarios where sample size constraints are a prevailing concern.

Abstract 2295: Circulating T cell receptor repertoire analysis improves cancer early detection

Abstract Background: Cancer screening by liquid biopsy promises to detect cancer early when it can be cured. However, current liquid biopsy approaches based on circulating tumor DNA (ctDNA) detect only a minority of early-stage cancers, because these tumors shed little ctDNA and plasma sample volume is limited. For liquid biopsy to realize its potential for the early detection of cancer, sensitivity for the earliest stage disease must be improved. Methods: We set out to improve sensitivity of liquid biopsy for cancer early detection by exploiting tumor recognition by T cells through sequencing of the circulating T cell receptor repertoire. Using gDNA extracted from blood buffy coats, we studied a cohort of 451 lung cancer patients (86% with stage I disease) and 525 subjects without cancer, enriched for older individuals with a history of smoking. We performed genomic DNA TCR beta chain sequencing to yield a median of 81,088 TCR clonotypes in cancer patients and 112,588 clonotypes in non-cancer controls. We built a TCR sequence similarity nearest neighbor graph to cluster >68M distinct TCR clonotypes into TCR repertoire functional units (RFUs). The TCR frequencies of RFUs were tested for association with cancer status and significantly associated RFUs were combined using a support vector machine model into a TCR RFU cancer score. This model was evaluated by 10-fold cross-validation (CV) and compared to a ctDNA panel of 237 mutation hotspots in 154 lung cancer driver genes (IDT™) and to a panel of 17 lung cancer related protein biomarkers (Olink®) in 87 subjects. Results: We identified 177 cancer-associated TCR RFUs at FDR≤0.10, including 83 RFUs that were enriched in cancer samples with a TCR count log2-fold change between 0.05 and 1.31, and 94 that were enriched in controls with a log2-fold change between 0.07 and 0.40. We observed 56 of the 83 cancer-enriched RFUs had decreasing TCR counts with increasing age across the cohort, coupled with higher counts in cancer patients relative to age-matched controls. The TCR RFU cancer score achieved a cross-validated ROC AUC of 0.69 for cancer status prediction, with the AUC for stage I cancers similar to that of stage II-IV (0.69 vs. 0.70). 44% of stage I cancers (test samples of each CV fold) were detected by the TCR model at a specificity of 80%. We saw a substantial gain in sensitivity for stage I cancer when TCR RFUs were added to ctDNA and proteins, with a ~15-20%-point increase seen at the 90% target specificity level typical for cancer screening tests. Likewise, sensitivity increased at the 99% specificity level used in multi-cancer early detection, with 35% of stage I lung cancers detected, which could not be achieved by any analyte alone. Conclusion: We demonstrate detection of a significant fraction of early lung cancer cases from blood by analyzing the circulating TCR repertoire and show that this signal is complementary to established analytes such as proteins and ctDNA. Citation Format: Yilong Li, Michelle Nahas, Dennis Stephens, Kate Froburg, Emma Hintz, Devin Champagne, Amaneet Lochab, Markus Brown, Jesse Abhyankar, Jasper Braun, Michelle Dinh, María Antonia Fortuño, María-del-Mar Ocon, Andrea Pasquier, Inés María Luque, Christopher W. Seder, Jeffrey A. Borgia, Luis Miguel Seijo, Luis M. Montuenga, Roman Yelensky. Circulating T cell receptor repertoire analysis improves cancer early detection [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 2295.

B cell receptor repertoire analysis in primary Sjogren’s syndrome salivary glands identifies repertoire features associated with clinical activity

BackgroundPrimary Sjogren’s syndrome (pSS) is a complex autoimmune disease featuring damage to salivary and lacrimal glands, with the possibility of manifestations across multiple organs. Antibody-producing B cells have long been appreciated to play a significant role in pSS pathogenesis, with a number of autoreactive antibody species having been identified to be elevated in pSS patients. While several studies have attempted to characterize the BCR repertoires of peripheral blood B cells in pSS patients, much remains unknown about the repertoire characteristics of gland-infiltrating B cells.MethodsThrough paired scRNAseq and scBCRseq, we profiled the BCR repertoires of both infiltrating and circulating B cells in a small cohort of patients. We further utilize receptor reconstruction analyses to further investigate repertoire characteristics in a wider cohort of pSS patients previously profiled through RNAseq.ResultsVia integrated BCR and transcriptome analysis of B cell clones, we generate a trajectory progression pattern for infiltrated memory B cells in pSS. We observe significant differences in BCR repertoires between the peripheral blood and labial gland B cells of pSS patients in terms of relative expansion, isotype usage, and BCR clustering. We further observe significant decreases in IgA2 isotype usage among pSS patient labial and parotid gland B cells these analyses relative to controls as well as a positive correlation between kappa/lambda light chain usage and clinical disease activity.ConclusionsThrough BCR repertoire analysis of pSS patient salivary glands, we identify a number of novel repertoire characteristics that may serve as useful indicators of clinical disease and disease activity. By collecting these BCR repertoires into an accessible database, we hope to also enable comparative analysis of patient repertoires in pSS and potentially other autoimmune disorders.

SARS-CoV2 mRNA-vaccination-induced immunological memory in human non-lymphoid and lymphoid tissues.

Tissue-resident lymphocytes provide organ-adapted protection against invading pathogens. Whereas their biology has been examined in great detail in various infection models, their generation and functionality in response to vaccination has not been comprehensively analyzed in humans. We therefore studied SARS-CoV2 mRNA-vaccine-specific T cells in surgery specimens of kidney, liver, lung, bone marrow and spleen in comparison to paired blood samples from largely virus-naïve individuals. As opposed to lymphoid tissues, non-lymphoid organs harbored significantly elevated frequencies of Spike-specific CD4+ T cells compared to blood showing hallmarks of tissue residency and an expanded memory pool. Organ-derived CD4+ T cells further exhibited increased polyfunctionality over those detected in blood. Single-cell RNA sequencing together with T cell receptor repertoire analysis indicated that the clonotype rather than organ origin is a major determinant of transcriptomic state in vaccine-specific CD4+ T cells. In summary, our data demonstrate that SARS-CoV2 vaccination entails acquisition of tissue memory and residency features in organs distant from the inoculation site, thereby contributing to our understanding of how local tissue protection might be accomplished.

Antigen-driven colonic inflammation is associated with development of dysplasia in primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is an immune-mediated disease of the bile ducts that co-occurs with inflammatory bowel disease (IBD) in almost 90% of cases. Colorectal cancer is a major complication of patients with PSC and IBD, and these patients are at a much greater risk compared to patients with IBD without concomitant PSC. Combining flow cytometry, bulk and single-cell transcriptomics, and T and B cell receptor repertoire analysis of right colon tissue from 65 patients with PSC, 108 patients with IBD and 48 healthy individuals we identified a unique adaptive inflammatory transcriptional signature associated with greater risk and shorter time to dysplasia in patients with PSC. This inflammatory signature is characterized by antigen-driven interleukin-17A (IL-17A)+ forkhead box P3 (FOXP3)+ CD4 T cells that express a pathogenic IL-17 signature, as well as an expansion of IgG-secreting plasma cells. These results suggest that the mechanisms that drive the emergence of dysplasia in PSC and IBD are distinct and provide molecular insights that could guide prevention of colorectal cancer in individuals with PSC.

Abstract 5180: T cell receptor repertoire analysis revealed tissue tropism of tumor-reactive T-cell clones in cell cycle reporter mice

Abstract Tumor-reactive T cells are composed of clones with various TCRs, and each clone has different in vivo kinetics. By analyzing TCR repertoire of tumor and tumor-draining lymph node (dLN), we have demonstrated that Tumor-reactive CD8+ T cells can be classified into “dLN Major”, “Tumor Major”, and “Double Major” clones, which exhibited high frequency in the dLN, tumor, or both tissues. To investigate whether this classification was related to the tissue tropism in the proliferation of each clone, we here employed tumor-bearing Fucci transgenic mice expressing a fluorescent cell-cycle indicator to identify the proliferation of T-cell clones in each tissue. We purified proliferating- and resting-CD8+ T cells from the tumors and dLN and analyzed their TCR repertoire in an LLC subcutaneous tumor model. All Tumor Major clones were proliferated in the tumor, while nearly 0% for dLN Major, indicated their different proliferative capacity in the tumor. The percentage of proliferating clones in the dLN was 20% for Tumor Major and 15% for dLN Major, indicating that these clones had equivalent proliferative capacity in the dLN. These proliferating dLN major clones overlapped more with the tumor than the non-proliferating dLN major clones, suggesting that proliferating dLN Major clones had higher tumor migration capacity. Furthermore, these proliferating dLN major clones were more proliferative in the tumor. These results suggested that dLN Major responded to antigen presentation in the dLN but not in the tumor, whereas Tumor Major responded to those in the dLN and tumor. In addition, there are two types of dLN major, “clones that are proliferating in dLN” and “clones that are not proliferating in dLN” indicating that the former may contribute to the anti-tumor response. Immune checkpoint inhibitor (ICI) treatment not only reactivates clones in the tumor but also activates tumor-reactive clones in the dLN. In conformity with these previous studies, the percentage of proliferating clones among dLN Major increased to about 50% and 80% in the dLN in mice with anti-PD-L1 and anti-CD4 treatment, respectively. This result suggests ICI treatment enhances anti-tumor responses mainly by promoting the activation and proliferation of dLN Major clones. This study shows new findings that tumor-reactive T cells differ in the tissue tropism of their proliferation of each clone. In the future, a quantitative understanding of the contribution of each class of clones to the anti-tumor response will hopefully lead to the development of new combined immunotherapies that optimize the anti-tumor T-cell response. Citation Format: Mikiya Tsunoda, Hiroyasu Aoki, Munetomo Takahashi, Haruka Shimizu, Haru Ogiwara, Shigeyuki Shichino, Kouji Matsushima, Satoshi Ueha. T cell receptor repertoire analysis revealed tissue tropism of tumor-reactive T-cell clones in cell cycle reporter mice. [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 5180.

B cell receptor repertoire analysis from autopsy samples of COVID-19 patients.

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being developed world over. We investigated the possibility of producing artificial antibodies from the formalin fixation and paraffin-embedding (FFPE) lung lobes of a patient who died by coronavirus disease 2019 (COVID-19). The B-cell receptors repertoire in the lung tissue where SARS-CoV-2 was detected were considered to have highly sensitive virus-neutralizing activity, and artificial antibodies were produced by combining the most frequently detected heavy and light chains. Some neutralizing effects against the SARS-CoV-2 were observed, and mixing two different artificial antibodies had a higher tendency to suppress the virus. The neutralizing effects were similar to the immunoglobulin G obtained from healthy donors who had received a COVID-19 mRNA vaccine. Therefore, the use of FFPE lung tissue, which preserves the condition of direct virus sensitization, to generate artificial antibodies may be useful against future unknown infectious diseases.

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.

T cell receptor repertoire analysis in HTLV-1-associated diseases.

Human T lymphotropic virus 1 (HTLV-1) is a human retrovirus identified as the causative agent in adult T-cell leukemia/lymphoma (ATL) and chronic-progressive neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 is estimated to infect between 5-20 million people worldwide, although most infected individuals remain asymptomatic. HTLV-1 infected persons carry an estimated lifetime risk of approximately 5% of developing ATL, and between 0.25% and 1.8% of developing HAM/TSP. Most HTLV-1 infection is detected in CD4+ T cells in vivo which causes the aggressive malignancy in ATL. In HAM/TSP, the increase of HTLV-1 provirus induces immune dysregulation to alter inflammatory milieu, such as expansion of HTLV-1-specific CD8+ T cells, in the central nervous system of the infected subjects, which have been suggested to underlie the pathogenesis of HAM/TSP. Factors contributing to the conversion from asymptomatic carrier to disease state remain poorly understood. As such, the identification and tracking of HTLV-1-specific T cell biomarkers that may be used to monitor the progression from primary infection to immune dysfunction and disease are of great interest. T cell receptor (TCR) repertoires have been extensively investigated as a mechanism of monitoring adaptive T cell immune response to viruses and tumors. Breakthrough technologies such as single-cell RNA sequencing have increased the specificity with which T cell clones may be characterized and continue to improve our understanding of TCR signatures in viral infection, cancer, and associated treatments. In HTLV-1-associated disease, sequencing of TCR repertoires has been used to reveal repertoire patterns, diversity, and clonal expansions of HTLV-1-specific T cells capable of immune evasion and dysregulation in ATL as well as in HAM/TSP. Conserved sequence analysis has further been used to identify CDR3 motif sequences and exploit disease- or patient-specificity and commonality in HTLV-1-associated disease. In this article we review current research on TCR repertoires and HTLV-1-specific clonotypes in HTLV-1-associated diseases ATL and HAM/TSP and discuss the implications of TCR clonal expansions on HTLV-1-associated disease course and treatments.

Suppression of high mobility group box 1 in B16F10 tumor does not inhibit the induction of neoantigen-specific T cells.

Accumulated clinical data of immune checkpoint blockades have suggested the importance of neoantigens in cancer immunity. Tumor antigens are released from dead cancer cells together with cellular components, such as damage-associated molecular patterns (DAMPs), into the tumor microenvironment. We recently reported that high mobility group box 1 (HMGB1), a representative DAMP molecule, showed a negative impact on anti-tumor immunity. However, a positive role of HMGB1 in the initiation of innate and subsequent adaptive immunity has also been demonstrated; thus, the effects of HMGB1 on anti-tumor immunity have not been well understood. In this study, we identified nine immunogenic neoantigen epitopes of B16F10 murine melanoma cells and subsequently investigated the effects of suppression of HMGB1 on the induction of neoantigen-specific immunity using HMGB1-knockout tumors. Neoantigen-reactive T cells were expanded in B16F10 tumor-bearing mice, and T cell receptor repertoire analysis suggested that neoantigen-reactive T cells were oligo-clonally increased in B16F10 tumor bearers. An increase of neoantigen-reactive T cells and oligoclonal expansion of the T cells were similarly detected in HMGB1-knockout tumor-bearing mice. The induction of neoantigen-specific immunity under the suppression of HMGB1 in the tumor microenvironment shown in this study supports further development of combination therapy of HMGB1 suppression with neoantigen-targeted cancer immunotherapies, including immune checkpoint blockade therapy.

Machine Learning Approaches to TCR Repertoire Analysis

Sparked by the development of genome sequencing technology, the quantity and quality of data handled in immunological research have been changing dramatically. Various data and database platforms are now driving the rapid progress of machine learning for immunological data analysis. Of various topics in immunology, T cell receptor repertoire analysis is one of the most important targets of machine learning for assessing the state and abnormalities of immune systems. In this paper, we review recent repertoire analysis methods based on machine learning and deep learning and discuss their prospects.

Longitudinal analysis of anti-drug antibody development in multiple sclerosis patients treated with interferon beta-1a (Rebif™) using B cell receptor repertoire analysis

A significant proportion of multiple sclerosis (MS) patients treated with interferon beta-1a (Rebif™) develop anti-drug antibodies (ADA) with a negative impact on treatment efficacy. We hypothesized that high-throughput B-cell receptor (BCR) repertoire analysis could be used to predict and monitor ADA development. To study this we analyzed 228 peripheral blood samples from 68 longitudinally followed patients starting on interferon beta-1a. Our results show that whole blood BCR analysis does not reflect, and does not predict ADA development in MS patients treated with interferon beta-1a. We propose that BCR analysis of phenotypically selected cell subsets or tissues might be more informative.

Effect of neoadjuvant chemotherapy on the immune microenvironment in gastric cancer as determined by multiplex immunofluorescence and T cell receptor repertoire analysis

BackgroundThe combination of immune checkpoint blockade and chemotherapy has revolutionized the treatment of advanced gastric cancer (GC). It is crucial to unravel chemotherapy-induced tumor microenvironment (TME) modulation and identify which...

Heterogeneous origin of IgE in atopic dermatitis and psoriasis revealed by B cell receptor repertoire analysis.

Epicutaneous sensitization is an important route for the production of IgE, and skin inflammation-induced IgE has recently been reported having features of natural antibody. Atopic dermatitis (AD) and psoriasis have differentially increased level of serum IgE; however, the production mechanism of IgE in these inflammatory skin diseases remains unknown. To explore the origin of IgE in AD and psoriasis by analyzing the B cell receptor repertoire. mRNA was prepared from peripheral blood mononuclear cells of AD and psoriasis patients that had elevated serum levels of IgE, and immunoglobulin heavy chain (IGH) repertoires were sequenced after reverse transcription. Clonal lineages of B cells containing members expressing IgE were identified, and somatic hypermutations in IGH inherited from common ancestors within the clonal lineage were used to infer the relationships between B cells. The proportions of IGHE from AD and psoriasis were higher than that of normal control, which were positively correlated with the levels of serum total IgE. The somatic hypermutation value of IGHE variable region was lower than that of IGHG and IGHA, but higher than IGHM and IGHD, indicating a mixed natural and adaptive origins of IgE; and psoriasis demonstrated lower level of hypermutation than AD. The Shannon indexes of CDR3 in IGHE of AD and psoriasis were higher than that of normal control, also supporting the natural origin. The VH usage of IgE was weakly biased in AD and psoriasis patients with high level of house dust mite-specific IgE. Comparison of the number of shared mutations in multi-isotype lineages containing IgE showed that isotype-switching from IgG-expressing B cells might be the major source of IgE in AD and psoriasis. IgE has heterogeneous origin in AD and psoriasis, and skin inflammation may contribute to the increased production of natural IgE.

G-CSF/Plerixafor Dual-Mobilized Donor Derived CD33CAR T-Cells As Potent and Effective AML Therapy in Pre-Clinical Models

There are currently no known acute myeloid leukemia (AML) specific antigens. Genetic ablation of CD33 using CRISPR/Cas9 engineering of the hematopoietic stem cell (HSC) transplant (VOR33) represents a synthetic biology approach to generating a leukemia-specific antigen in the transplant recipient. VOR33 enables anti-CD33 CAR-T cell killing of AML cells while sparing normal myeloid lineage development and function, thereby potentially avoiding myelosuppression and increasing the therapeutic index of anti-CD33 CAR-T therapy. Mobilized leukapheresis product represents an attractive starting material for the generation of both a CD33 null HSC transplant and a complementary CD33CAR T-cell product. In this study, we sought to determine the impact of dual mobilization with Granulocyte-Colony Stimulating Factor (G-CSF) and plerixafor (mozobil) on immune cell composition, T cell phenotype, and the functionality of these T cells to control AML tumor growth upon chimeric antigen receptor (CAR) transduction.Mobilized (mob) peripheral blood mononuclear cells (PBMCs) were collected from healthy donors injected with G-CSF (10µg/kg/day, 5 consecutive days) and plerixafor (240µg/kg, on day 4 and 5). Non-mobilized (non-mob) PBMCs, collected from the same donors, were used as controls. Cells were analyzed by flow cytometry for immunophenotyping and T cell characterization including differentiation and bone marrow homing markers, as well as responses to T cell activation with anti-CD3 (OKT3) and IL-2. Non-/mob PBMC populations were also analyzed by single-cell next generation sequencing (CITEseq) using 127 immune cell phenotypic markers in combination with extensive transcriptome and T cell receptor repertoire analysis. In addition, lentiviral transduction of anti-CD33 CAR constructs enabled functional comparisons of mob- and non-mob-CAR T-cells in AML cell co-cultures as well as AML mouse models.Ex vivo immunophenotyping of PBMC from a total of over 30 healthy donor samples showed that mobilization decreases the overall percentage of CD3 + T cells but increases that of naïve T cells (CD45RA +/CCR7 +), at the expense of T effector-memory (CD45RA -/CCR7 -) and central-memory (CD45RA -/CCR7 +) populations. Bone marrow homing factors (e.g.: CXCR4) were increased in mob compared to non-mob T cell samples. As expected, higher percentages of monocytes (CD14 +) were detected in mob compared to non-mob donor samples, but this difference disappeared after culture under T cell activation conditions. T cell activation also led to similar increases in CD25, CD69 and CD137 expression, and a decrease in CD62L expression. Single cell sequencing analyses confirmed mobilization-induced increases in naïve T cells as well as changes in monocytes/macrophages, CD4 + T cells and NK cells percentages. Notably, functional in vitro cytotoxic assays demonstrated that mob-CD33-CAR T-cells are as effective as non-mob-CD33-CAR T-cells in killing CD33 + AML cells, with reduced ‘bystander’ activation of non-transduced T cells. Furthermore, results from in vivo AML mouse models indicate that mob-CD33-CAR T-cells are equally effective in clearing CD33 + tumors as non-mob-CD33-CAR T-cells.Our analysis showed phenotypical ex vivo differences between mob and non-mob PBMCs, which disappeared upon activation, indicating similar responses to T cell-specific stimulation. These findings are corroborated by similar in vitro cytotoxicity profiles of non-/mob-CAR T-cells. Non-transduced T cells in the mob-CAR T-cell population showed limited ‘bystander’ activation, indicating a potentially favorable clinical toxicity profile. Additional in vivo assessment of mob-CAR T-cell function shows effective tumor clearance, which supports further efforts towards their clinical use in combination with engineered HSCs for the treatment of AML patients. DisclosuresCanesin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hoyt: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Williams: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Silva: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chng: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ung: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Qiu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Shin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hu: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Scherer: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Kassim: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

Diversity of draining lymph node-tumor overlapping T cell receptor repertoire reflects the anti-tumor effect of cancer immunotherapies

Abstract CD8+ tumor-infiltrating lymphocytes, the main effector cells of anti-tumor immune responses, are composed of various T cell clones. Numerous studies have demonstrated the presence of tumor-reactive T cell clones with high frequency in tumors. However, it remains unclear as to which of the two mechanisms, the expansion of limited tumor-specific clones or the induction of diverse tumor-reactive ones, is more beneficial for the action of immune checkpoint inhibitors (ICIs). To address this question, we performed a T cell receptor repertoire analysis on B16F10 and on Lewis lung carcinoma tumor-bearing mice treated with immunotherapy including anti-PD-L1 monoclonal antibody (mAb) and anti-CD4 depletion mAb, which enhance the expansion of tumor-reactive CD8+ T cells. We focused on the T cell clones that overlapped between the tumor and draining lymph node (dLN) to analyze the T cell clones that were mobilized into the anti-tumor response after treatment. In anti-PD-L1 and anti-CD4 treated mice, the total frequency of CD8+ dLN-tumor overlapping clones in the tumor was increased. Surprisingly, although the expansion of the top10 overlapping clones did not differ, ICI treatment increased the total frequency of the 11th or lower overlapping clones, indicating the broadening of the tumor-reactive T cell repertoire. Moreover, the administration of FTY720 to tumor-bearing mice, which inhibits egress of T cells from the LNs to circulation, reduced the total frequency of the 11th or lower overlapping clones in the tumor and reduced the long-term anti-tumor effect of anti-PD-L1 treatment. These results suggest that mobilization of diverse tumor-reactive clones is essential for enhancing and persistent anti-tumor effect of ICIs.

The energy sensor AMPK orchestrates metabolic and translational adaptation in expanding T helper cells.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in‐depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK‐deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo.

HLA-A*24:02 is associated with metronidazole-induced cutaneous adverse drug reactions in Han Chinese individuals: A pilot case-control study with both HLA gene and T cell receptor repertoire analysis.

Metronidazole, a widely used drug for the treatment of infections with anaerobic and facultative anaerobic bacteria and protozoa, can frequently cause metronidazole-induced cutaneous adverse reactions (McADRs). The aim of the present study was to investigate the association between human leucocyte antigen (HLA) alleles and McADRs in a Chinese Han population. The frequency of HLA-B*24:02 carriers among the McADR patients was 73.3%, which was significantly higher than that of the population controls (32.16%, OR=5.80, 95% CI=[1.80-18.72], Pc=0.004) and of the metronidazole-tolerant patients (26.67%, OR=7.56, 95% CI=[2.02-28.35], Pc=0.004). Molecular docking showed that metronidazole and one of its major metabolites had the potential to bind in the HLA groove and that there was a relatively stable binding state of the HLA-B*24:02-metronidazole/the metabolite complex. The CDR3 repertoires of both T cell receptor (TCR)Vα and Vβ of the patients showed a significantly skewed or an oligoclonal distribution. The TCRVβ CDR3 of the patients shared a similar motif, "CASSxxxxxxQxF." The current study demonstrated that both the HLA-A*24:02 allele and TCR are involved in the pathogenesis of McADRs.

Autoreactive B Cell Differentiation in Diffuse Ectopic Lymphoid-Like Structures of Inflamed Pemphigus Lesions

Pemphigus is an organ-specific autoimmune disease that targets skin and/or mucous membranes. Our previous study showed that infiltrating lymphocytes in pemphigus vulgaris (PV) lesions produce anti-desmoglein (Dsg) 1/3 antibodies after invitro culture. In this study, we found diffuse ectopic lymphoid-like structures (ELSs) commonly present in the lesions of both PV and pemphigus foliaceus. Notably, pemphigus lesions contained centroblasts, plasmablasts, and plasma cells, which recapitulated the different stages of B cell differentiation. Elevated mRNA expression levels of the differentiation-related transcription factors BLIMP-1, IRF4, and BCL-6 were observed in pemphigus lesions. Moreover, B cell receptor repertoire analysis revealed the clonal expansion of the lesional B cells. Lesional B cells might recirculate among lesions, lymph nodes, and peripheral blood. Increased mRNA expression levels of multiple chemokines in pemphigus lesions and elevated expression levels of chemokine receptors on lesional B cells were also observed. Collectively, these results show that the ELSs in pemphigus lesions might act as a niche, supporting in situ B cell differentiation and clonal expansion.