Repertoire Analysis Research Articles

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

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

SARS-CoV-2 mRNA vaccination-induced immunological memory in human nonlymphoid 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 have not been comprehensively analyzed in humans. We therefore studied SARS-CoV-2 mRNA vaccine-specific T cells in surgery specimens of kidney, liver, lung, bone marrow, and spleen compared with paired blood samples from largely virus-naive individuals. As opposed to lymphoid tissues, nonlymphoid organs harbored significantly elevated frequencies of spike-specific CD4+ T cells compared with 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-Seq 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-CoV-2 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.

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

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

Composition and diversity analysis of the TCR CDR3 repertoire in patients with idiopathic orbital inflammation using high-throughput sequencing

BackgroundIdiopathic orbital inflammation (IOI) is a nonspecific orbital inflammatory disease with the third highest prevalence among orbital diseases, and its pathogenesis is associated with T-cell-mediated immune responses. This study aimed to investigate the differences in T-cell receptor (TCR) expression between IOI patients and healthy subjects by high-throughput sequencing and to characterize TCR expression in patients with IOI and with respect to glucocorticoid response.MethodsA total of 19 subjects were enrolled in this study and were divided into the idiopathic orbital inflammation group (IOI group, n = 13) and the healthy control group (HC group, n = 6), and within the IOI group were further divided into the glucocorticoid therapy sensitive group (IOI(EF) group, n = 6) and the glucocorticoid therapy ineffective group (IOI(IN) group, n = 7) based on the degree of effectiveness to glucocorticoid therapy. High-throughput TCR sequencing was performed on peripheral blood mononuclear cells of IOI patients and healthy control individuals using 5’ RACE technology combined with Unique Identifier (UID) digital tag correction technology. The TCR CDR3 region diversity, sharing patterns, and differential sequences between the IOI and HC groups, and between the IOI(EF) and IOI(IN) groups were analyzed.ResultsIt was found that the diversity of TCR CDR3 in the IOI group was significantly lower than that in the HC group, and the frequency of V gene use was significantly different between groups. The diversity of TCR CDR3 in patients in the IOI(EF) group was significantly lower than that in patients in the IOI(IN) group, and the frequency of V and J gene use was significantly different between the IOI(EF) group and the IOI(IN) group. Additionally, we found 133 nucleotide sequences shared in all IOI samples and screened two sequences with higher expression from them.ConclusionsOur results suggested that abnormal clonal expansion of specific T-cells exists in IOI patients and that TCR diversity may had an impact on the prognosis of glucocorticoid-treated IOI. This study may contribute to a better understanding of the immune status of IOI and provide new insights for T-cell -associated IOI pathogenesis, diagnosis and treatment prediction.

Altered Cellular Dynamics and Surface Expression Promote Long-Lived Plasma Cell Survival

Serological memory following vaccination decline with aging, indicating reduced survival of new plasma cells (PCs). We report that with age, bone marrow (BM) PC pool is enriched in long-lived plasma cells (LLPCs), leading to reduced PC turnover, suggesting increased competition. Using in vivo imaging, we find that LLPCs are highly organized into April-dependent clusters. Based on bulk RNA sequencing, and flow-based surface phenotyping, LLPCs exhibit unique transcriptome and proteome, fine tuning expression of key cell surface molecules, including CD93, CD81, CXCR4, CD326, CD44 and CD48, important for adhesion and homing. To test their functional role, Cxcr4 was conditionally deleted in PCs following immunization, which led to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and accelerated decay of antibody titer. Analysis of endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting non-random selection into the LLPC pool that is age-dependent.

Comparative analysis of behavioural repertoires for Mahogany glider and Brushtail possum using accelerometer loggers and machine learning algorithms

AbstractGliding has evolved independently as an isolated adaptive event within many vertebrate taxa. Yet, the underlying selection forces that led to these innovative adaptations remain ambiguous, especially in species that preclude direct observation. Our study utilized accelerometry and machine learning algorithms to compare the behavioural repertoires of two sympatric species, the Mahogany glider (Petaurus gracilis) and brushtail possum (Trichosaurus vulpecula), as to explore previously proposed selection pressures such as energy expenditure (VeBA), canopy use and ground avoidance measured by activity budgets. We found that mahogany gliders on average expend more activity‐related energy than brushtail possums but at different stages throughout the day. Canopy use was observed to be greater amongst mahogany gliders than brushtail possums, and we observed frequent ground use in brushtail possums yet none in mahogany gliders. The study found strong evidence to support ground avoidance as a potential driver for gliding evolution. The implications of these findings are important when considering the lack of knowledge surrounding evolved gliding behaviours in marsupials. Furthermore, the use of accelerometers and machine learning algorithms in behavioural studies has proven to be a robust and informative method and should be incorporated into future studies to understand the evolution of gliding behaviour.

Single-Cell Characterization of TP53-Mutated AML Patients Treated with Frontline Azacitidine, Venetoclax, and Magrolimab Reveals Mechanisms of Response and Resistance

Background: TP53 mutations, found in around 20% of AML cases, often lead to therapy resistance and unfavorable outcomes. Recent studies hint at the potential efficacy of CD47, a ‘don't eat me’ signal, in treating TP53-mutated AML. The ongoing clinical trial (NCT04435691) is assessing the efficacy of a triple therapy combo-magrolimab (anti-CD47 mAb), azacitidine, and venetoclax (AVM)-with promising initial results in TP53-mutated AML patients. However, understanding resistance and relapse remains crucial. Methods: We performed paired 5' scRNA and scTCR profiling on 3 healthy donors and 27 samples from 11 treatment-naive TP53-mutated AML patients (median age 63, range 59-73 years; 8 responders (CR/CRi); 3 non-responders (NR)), both pre-AVM (n=11) and post-AVM-point of remission (n=11), relapse (n=2), or primary resistance (n=3). Results: 42,910 AML cells and 137,169 bone marrow microenvironment cells passed quality checks. AML cell identification was confirmed via correlation with clinical flow cytometry, inferred copy number alterations, and immunohistochemistry markers. We leveraged Symphony to phenotypically infer AML cell types based on their transcriptional similarity to normal hematopoietic cells. Overall, samples displayed heterogeneous distribution of cell types at time of diagnosis. However, the 3 samples from the 3 primary resistant patients demonstrated a marked and significant expansion of AML cell proportion exhibiting an erythroid differentiation phenotype post-AVM (Fig. A). Also, the 2 patients who relapsed 4.8- and 7.1-months following AVM demonstrated expansion of erythroid differentiation proportion (Fig. A) and higher LSC17 at time of relapse. This suggests a selection favoring erythroid clones as a mechanism of primary and acquired resistance following AVM therapy. We conducted a pathway enrichment analysis using Hallmark gene sets to identify significantly altered AML-intrinsic biological pathways between responders and non-responders. CR/CRi patient-derived AML cells exhibited an enriched inflammatory response, specifically in TNFα and IFNγ signaling pathways-known CD47 expression regulators-and increased CD47 expression (Fig. B). In contrast, AML cells from non-responsive patients showed an enrichment of oxidative phosphorylation (Fig. B). Of note, phagocytic activity of myeloid cells was high in non-responders suggesting that despite the likely activity of magrolimab, other mechanisms of primary resistance were at play. As anti-CD47-mediated phagocytosis can stimulate antigen presentation and trigger T cell activation, we delved into the T cell subtypes and their corresponding cellular responses. There were not significant differences in the distribution of CD4 and CD8 pretreatment in CR/CRi versus NR patients. However, CD8 dysfunction score was significantly higher in non-responders at baseline. Following therapy, the CD8 dysfunction score was also significantly higher at time of relapse among responders. Further, effector CD8 T cells of NR patients had enrichment for cytotoxicity and TCR signaling pathways, and higher expression of exhaustion-related genes. Conclusions: Through single cell transcriptomic profiling of TP53-mutated AML patients treated with the triplet combination of AVM on protocol we identified erythroid differentiation and oxidative phosphorylation as possible intrinsic resistance mechanisms. Additionally, non-responders exhibited a dysfunctional T cell state prior to treatment, while relapses were associated with development of CD8 dysfunction. Overall, this data provides valuable insight into the mechanisms of response and resistance to magrolimab-based therapy. TCR repertoire and AML-immune cell interaction analyses are ongoing.

Distinct Minor Neoplastic Follicular T-Cell Subsets Dictate Lymphoma Fates through Functional Specialization

Background: The presence and role of follicular T-cell populations other than T follicular helper (Tfh) cells, such as T follicular regulatory (Tfr) and cytotoxic (Tfc) cells, are gaining increasing attention in certain pathological states. However, the ecosystem of follicular T cells in the tumor microenvironment (TME) has not been fully elucidated. In particular, the significance of minor follicular T-cell subsets in the neoplastic follicular environment remains elusive. Here, we aimed to reveal the landscape of follicular T-cell alterations in various cancers, with a particular emphasis on the follicular lymphoma (FL) TME. Methods: We analyzed single-cell RNA/TCR sequencing data of >500,000 human T cells from FL (obtained from four cohorts) and 25 other cancer types, as well as homeostatic and reactive lymph nodes (LNs), to construct a comprehensive single-T-cell atlas. We investigated differentially expressed genes, RNA velocity, and TCR clonality using this atlas. To determine the functions of neoplastic follicular regulatory (Tnfr) and cytotoxic (Tnfc) T cells, we performed in vitro cytokineproduction and co-culture assays, in combination with cell activation/suppression, cell division, and apoptosis assays, using human FL samples. With the PhenoCycler-Fusion system, we conducted multiplex digital spatial profiling (DSP) of 169 FL samples from two independent cohorts (now being extended to 242 FL samples from three cohorts) for >25 antibodies. We also performed single-cell spatial and protein expression profiling and prognostic analysis. Results: In FL, distinct minor neoplastic follicular T-cell subsets-Tnfr and CD4 (Tnfc4) and CD8 (Tnfc8) Tnfc cells-increased relative to homeostatic LNs. The TCR repertoire analysis revealed that Tnfr cells shared clonotypes with conventional effector regulatory T (Trg) and Tfh cells, whereas Tnfc4 and Tnfc8 cells shared clonotypes with Tfh cells and effector and exhausted (Tcex) cytotoxic CD8 T cells, respectively. In line with these findings, the RNA velocity survey suggested that Tnfr, Tnfc4, and Tnfc8 cells originated from Trg, Tfh, and naïve-like CD8 T cells, respectively. Tnfr and Tnfc cells expressed higher levels of effector genes, including those involved in cytokine release, chemokine response, migration, and PD-1 signaling, than their reactive LN counterparts. The pan-cancer survey revealed that Tfr and CD4 Tfc cells were exclusive to FL, whereas the prevalence and gene expression profiles of CD8 Tfc cells varied across cancers. Tnfr cells were marked by abundant expression of IL10 and IL21, whereas Tnfc cells displayed a unique phenotype, as they concomitantly expressed markers of effector Tfh (e.g., CXCL13, CXCR5, and PDCD1), naïve/stem (e.g., CCR7 and TCF7), central memory (e.g., CD27, CD28, and SELL), and tissue-resident memory (e.g., ITGAE) cells. Hierarchical clustering demonstrated that Tnfc8 cells had transcriptional profiles similar to those of melanoma TCF1 +PD-1 +CD8 + stem-like T cells. DSP of FL detected Tnfr and Tnfc cells frequently localized within and around neoplastic follicles, forming a cellular neighborhood that allowed them to interact closely. Tnfr cells were distributed predominantly near Tfh cells. The functional co-culture assays demonstrated that Tnfr cells suppressed Tfh-cell activation and division, thereby inhibiting Tfh-mediated malignant B-cell activation and survival. Tnfc8 cells showed a higher cell division capability than that of Tcex cells, suggesting that Tnfc8 cells function as a pool of CD8 T cells in neoplastic follicles. The prognostic analysis revealed that Tnfr and Tnfc cell proportions correlated with early disease relapse (i.e., POD24) and predicted a significantly longer time-to-relapse ( P <0.05 for Tnfr and <0.001 for Tnfc cells) in FL. In the multivariate analysis, the prognostic impact of these two cell subsets was independent of the FLIPI. The prognostic analysis findings were confirmed using a validation cohort. Conclusions: Our multi-omics approach identified the expansion of minor neoplastic follicular T-cell subsets that carry unique transcriptional and functional profiles and robust prognostic impacts. These findings deepen our understanding of the biological and immunological roles of non-Tfh follicular T cells in the lymphoma TME and highlights their clinical potential for patient risk stratification and future therapeutic interventions.

Alterations in Intratumoral Immune Response before and during Early-On Nivolumab Treatment for Unresectable Advanced or Recurrent Gastric Cancer

We investigated the tumor immune response in gastric cancer patients receiving third-line nivolumab monotherapy to identify immune-related biomarkers for better patient selection. Nineteen patients (10 males, median age 67 years) who received nivolumab as a third- or later-line therapy were enrolled. We analyzed the tumor immune response in durable clinical benefit (DCB) and non-DCB patients. Pre-treatment and early-on-treatment tumor transcriptomes were examined, and gene expression profiles, immunograms, and T cell receptor (TCR) repertoire were analyzed. DCB was observed in 15.8% of patients, with comparable secondary endpoints (ORR; objective response rate, OS; overall survival, PFS; progression-free survival) to previous trials. The immunograms of individual subjects displayed no significant changes before or early in the treatment, except for the regulatory T cell (Treg) score. Moreover, there were no consistent alterations observed among cases experiencing DCB. The intratumoral immune response was suppressed by previous treatments in most third- or later-line nivolumab recipients. TCR repertoire analysis revealed newly emerged clonotypes in early-on-treatment tumors, but clonal replacement did not impact efficacy. High T cell/Treg ratios and a low UV-radiation-response gene signature were linked to DCB and treatment response. This study emphasizes the tumor immune response’s importance in nivolumab efficacy for gastric cancer. High T cell/Treg ratios and specific gene expression signatures show promise as potential biomarkers for treatment response. The tumor-infiltrating immune response was compromised by prior treatments in third-line therapy, implying that, to enhance immunotherapeutic outcomes, commencing treatment at an earlier stage might be preferable. Larger cohort validation is crucial to optimize immune-checkpoint inhibitors in gastric cancer treatment.

IGH repertoire analysis at scale: deciphering the complexity of B cell infiltration and migration in esophageal squamous cell carcinoma.

Tumor-infiltrating B-lineage cells have become predictors of prognosis and immunotherapy responses in various cancers. However, limited knowledge about their infiltration and migration patterns has hindered the understanding of their anti-tumor functions. Here, we examined the immunoglobulin heavy chain (IGH) repertoires in 496 multi-regional tumor, 107 normal tissue, and 48 metastatic lymph node samples obtained from 107 patients with esophageal squamous cell carcinoma (ESCC). Our study revealed higher IgG-type B-lineage cells infiltration in tumors than in healthy tissue, which was associated with improved patient outcomes. Genes such as ACTN1, COL6A5, and pathways like focal adhesion, which shapes the physical structure of tumors, could affect B-lineage cell infiltration. Notably, the IGH sequence was used as an identity-tag to monitor B cell migration, and their infiltration schema within the tumor were depicted based on our multi-regional tumor specimens. This analysis revealed an escalation in B cell clones overlapped between metastatic lymph nodes and tumors. Therefore, the Lymph Node Activation Index was defined, which could predict the outcomes of patients with lymph node metastasis. This research introduces a novel framework for probing B cell infiltration and migration within the tumor microenvironment using large-scale transcriptome data, while simultaneously providing fresh perspectives on B cell immunology within ESCC.

Application and challenges of TCR and BCR sequencing to investigate T- and B-cell clonality in elastase-induced experimental murine abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays autoimmune disease characteristics. Particularly, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial immune response. Single-cell RNA (scRNA) T cell receptor (TCR) and B cell receptor (BCR) sequencing is a powerful tool for investigating clonality. However, difficulties such as limited numbers of isolated cells must be considered during implementation and data analysis, making biological interpretation challenging. Here, we perform a representative single-cell immune repertoire analysis in experimental murine AAA and show a reliable bioinformatic processing pipeline highlighting opportunities and limitations of this approach. We performed scRNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aorta of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham-operated mice at days 3 and 28 and non-operated mice served as controls. Comparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed neither differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified several clones in 11 of 16 AAA samples and one of eight control samples. Immune receptor repertoire comparison indicated that only a few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2 + TRBV13-2. We found no clonal expansion of B cells but evidence for clonal expansion of T cells in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive number of lymphocytes to prevent undersampling and potentially detect rare clones. Thus, further experiments are necessary to confirm our findings. In summary, this paper examines TCR and BCR sequencing results, identifies limitations and pitfalls, and offers guidance for future studies.

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

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

CTIM-16. DEVELOPING PATIENT SPECIFIC TUMOR INFILTRATING LYMPHOCYTES (TILS) WITH INCREASED TUMOR SPECIFICITY AS A THERAPY FOR GLIOBLASTOMA

Abstract Immunotherapy failure in glioblastoma results from three main hurdles; limited numbers of T cells within the tumor, T cell exhaustion and significant intra-tumoral and intra-patient heterogeneity leading to lack of T cell tumor recognition. Our group has developed an expansion protocol to isolate and expand less exhausted tumor infiltrating lymphocytes (TILs) from the tumor periphery, selecting for TILs that recognize tumor antigen ex vivo. This process will increase the number of T-cells specific for an individual patient’s diverse tumor neoantigens. We have successfully expanded glioblastoma associated TILs from 66% of the novel Lukens trap substrate, which collects TILs and tumor from the tumor periphery. Cells are expanded in two stages, preREP and REP, through novel methods based on a protocol with clinical success in lung cancer and modified to increase the percent of tumor specific T cells. Through co-culture with patient matched glioma neurospheres we have identified activated T cells from each stage of culture (initial product, preREP, REP) based on CD137+ expression and tumor specific cytokine release (IFNy+TNFa+IL2+IL17a+) by flow cytometry. Through 5’scRNA sequencing paired with TCR repertoire analysis we can identify activation specific TCRs. Overrepresented TCR alpha and beta chains will be linked by a P2A site, cloned into a lentiviral vector, and transduced into TCR negative Jurkat cells to confirm tumor reactivity. This strategy will allow us to enrich and assess tumor specific TCR activity compared to bystander activation in our TIL product. Following additional preclinical assessment of cytotoxicity against autologous tumor, we will evaluate safety and feasibility of by administering our novel TIL product intrathecally in patients with glioblastoma.

Cold-blooded vertebrates evolved organized germinal center-like structures.

Germinal centers (GCs) or analogous secondary lymphoid microstructures (SLMs) are thought to have evolved in endothermic species. However, living representatives of their ectothermic ancestors can mount potent secondary antibody responses upon infection or immunization, despite the apparent lack of SLMs in these cold-blooded vertebrates. How and where adaptive immune responses are induced in ectothermic species in the absence of GCs or analogous SLMs remain poorly understood. Here, we infected a teleost fish (trout) with the parasite Ichthyophthirius multifiliis (Ich) and identified the formation of large aggregates of highly proliferating IgM+ B cells and CD4+ T cells, contiguous to splenic melanomacrophage centers (MMCs). Most of these MMC-associated lymphoid aggregates (M-LAs) contained numerous antigen (Ag)-specific B cells. Analysis of the IgM heavy chain CDR3 repertoire of microdissected splenic M-LAs and non-M-LA areas revealed that the most frequent B cell clones induced after Ich infection were highly shared only within the M-LAs of infected animals. These M-LAs represented highly polyclonal SLMs in which Ag-specific B cell clonal expansion occurred. M-LA-associated B cells expressed high levels of activation-induced cytidine deaminase and underwent significant apoptosis, and somatic hypermutation of Igμ genes occurred prevalently in these cells. Our findings demonstrate that ectotherms evolved organized SLMs with GC-like roles. Moreover, our results also point to primordially conserved mechanisms by which M-LAs and mammalian polyclonal GCs develop and function.

Application of Machine Learning for Repertoire Analysis in Antibody-Mediated Rejection (ABMR) of Kidney Transplantation

Application of Machine Learning for Repertoire Analysis in Antibody-Mediated Rejection (ABMR) of Kidney Transplantation

T-cell receptor repertoire analysis of CD4-positive T cells from blood and an affected organ in an autoimmune mouse model.

One hallmark of some autoimmune diseases is the variability of symptoms among individuals. Organs affected by the disease differ between patients, posing a challenge in diagnosing the affected organs. Although numerous studies have investigated the correlation between T cell antigen receptor (TCR) repertoires and the development of infectious and immune diseases, the correlation between TCR repertoires and variations in disease symptoms among individuals remains unclear. This study aimed to investigate the correlation of TCRα and β repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and β of CD4+ CD44high CD62Llow T cells in the blood and stomachs of mice deficient in autoimmune regulator (Aire) (AIRE KO), a mouse model of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Data analysis revealed that the degree of similarity in TCR sequences between the blood and stomach varied among individual AIRE KO mice and reflected the extent of T cell infiltration in the stomach. We identified a set of TCR sequences whose frequencies in blood might correlate with extent of the stomach manifestations. Our results propose a potential of using TCR repertoires not only for diagnosing disease development but also for diagnosing affected organs in autoimmune diseases.

Yclon: Ultrafast clustering of B cell clones from high-throughput immunoglobulin repertoire sequencing data

MotivationThe next-generation sequencing technologies have transformed our understanding of immunoglobulin (Ig) profiles in various immune states. Clonotyping, which groups Ig sequences into B cell clones, is crucial in investigating the diversity of repertoires and changes in antigen exposure. Despite its importance, there is no widely accepted method for clonotyping, and existing methods are computationally intensive for large sequencing datasets. ResultsTo address this challenge, we introduce YClon, a fast and efficient approach for clonotyping Ig repertoire data. YClon uses a hierarchical clustering approach, similar to other methods, to group Ig sequences into B cell clones in a highly sensitive and specific manner. Notably, our approach outperforms other methods by being more than 30 to 5000 times faster in processing the repertoires analyzed. Astonishingly, YClon can effortlessly handle up to 2 million Ig sequences on a standard laptop computer. This enables in-depth analysis of large and numerous antibody repertoires. Availability and implementationYClon was implemented in Python3 and is freely available on GitHub.

Tidytcells: standardizer for TR/MH nomenclature.

T cell receptors (TR) underpin the diversity and specificity of T cell activity. As such, TR repertoire data is valuable both as an adaptive immune biomarker, and as a way to identify candidate therapeutic TR. Analysis of TR repertoires relies heavily on computational analysis, and therefore it is of vital importance that the data is standardized and computer-readable. However in practice, the usage of different abbreviations and non-standard nomenclature in different datasets makes this data pre-processing non-trivial. tidytcells is a lightweight, platform-independent Python package that provides easy-to-use standardization tools specifically designed for TR nomenclature. The software is open-sourced under the MIT license and is available to install from the Python Package Index (PyPI). At the time of publishing, tidytcells is on version 2.0.0.

Lenvatinib activates anti-tumor immunity by suppressing immunoinhibitory infiltrates in the tumor microenvironment of advanced hepatocellular carcinoma

BackgroundLenvatinib, a multiple receptor tyrosine kinase inhibitor, might exert antitumor effects via tumor immune modulation. However, changes in the tumor immune microenvironment induced by lenvatinib are poorly understood. We investigated the effect of lenvatinib on immune features in clinical samples from patients with hepatocellular carcinoma.MethodsFifty-one patients with advanced hepatocellular carcinoma who received lenvatinib monotherapy as first-line treatment were enrolled. We collected blood sample (n = 51) and tumor tissue (n, baseline/four weeks after treatment initiation/post-progression = 50/8/12). DNA, RNA, and proteins extracted from the tissues were subjected to multi-omics analysis, and patients were classified into two groups according to baseline immune status. Each group was investigated in terms of the dynamics of tumor signaling. We also longitudinally analyzed circulating immune proteins and chemokines in peripheral blood.ResultsHere we show that lenvatinib has similar anti-tumor efficacy with objective response rate and progression-free survival in both Immune-Hot and Immune-Cold subtypes. Immune signatures associated with T-cell functions and interferon responses are enriched in the early phase of treatment, while signatures associated with immunoinhibitory cells are downregulated along with efficient vascular endothelial growth factor receptor and fibroblast growth factor receptor blockades. These findings are supported by imaging mass cytometry, T-cell receptor repertoire analysis and kinetics of circulating proteins. We also identify interleukin-8 and angiopoietin-2 as possible targets of intervention to overcome resistance to existing immunotherapies.ConclusionsOur findings show the ability of lenvatinib to modulate tumor immunity in clinical samples of hepatocellular carcinoma.

Immune cell abundance and T cell receptor landscapes suggest new patient stratification strategies in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a molecularly and spatially heterogeneous disease frequently characterized by impairment of immunosurveillance mechanisms. Despite recent success with immunotherapy treatment, disease progression still occurs quickly after treatment in the majority of cases, suggesting the need to improve patient selection strategies. In the quest for biomarkers that may help inform response to checkpoint blockade, we characterized the tumour microenvironment (TME) of 162 HNSCC primary tumours of diverse aetiological and spatial origin, through gene expression and immunohistochemistry (IHC) profiling of relevant immune proteins, T cell receptor (TCR) repertoire analysis and whole-exome sequencing. We identified five HNSCC TME categories based on immune/stromal composition: (1) cytotoxic, (2) plasma cell rich, (3) dendritic cell rich, (4) macrophage rich and (5) immune-excluded. Remarkably, the cytotoxic and plasma cell rich subgroups exhibited a phenotype similar to tertiary lymphoid structures (TLS), which have been previously linked to immunotherapy response. We also found an increased richness of the TCR repertoire in these two subgroups and in never smokers. Mutational patterns evidencing APOBEC activity were enriched in the plasma cell high subgroup. Furthermore, specific signal propagation patterns within the Ras/ERK and PI3K/AKT pathways associated with distinct immune phenotypes. While traditionally CD8/CD3 T cell infiltration and immune checkpoint expression (e.g. PD-L1) have been used in the patient selection process for checkpoint blockade treatment, we suggest that additional biomarkers, such as TCR productive clonality, smoking history and TLS index, may have the ability to pull out potential responders to benefit from immunotherapeutic agents.