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The ability to identify and track T-cell receptor (TCR) sequences from patient samples is becoming central to the field of cancer research and immunotherapy. Tracking genetically engineered T cells expressing TCRs that target specific tumor antigens is important to determine the persistence of these cells and quantify tumor responses. The available high-throughput method to profile TCR repertoires is generally referred to as TCR sequencing (TCR-Seq). However, the available TCR-Seq data are limited compared with RNA sequencing (RNA-Seq). In this paper, we have benchmarked the ability of RNA-Seq-based methods to profile TCR repertoires by examining 19 bulk RNA-Seq samples across 4 cancer cohorts including both T-cell-rich and T-cell-poor tissue types. We have performed a comprehensive evaluation of the existing RNA-Seq-based repertoire profiling methods using targeted TCR-Seq as the gold standard. We also highlighted scenarios under which the RNA-Seq approach is suitable and can provide comparable accuracy to the TCR-Seq approach. Our results show that RNA-Seq-based methods are able to effectively capture the clonotypes and estimate the diversity of TCR repertoires, as well as provide relative frequencies of clonotypes in T-cell-rich tissues and low-diversity repertoires. However, RNA-Seq-based TCR profiling methods have limited power in T-cell-poor tissues, especially in highly diverse repertoires of T-cell-poor tissues. The results of our benchmarking provide an additional appealing argument to incorporate RNA-Seq into the immune repertoire screening of cancer patients as it offers broader knowledge into the transcriptomic changes that exceed the limited information provided by TCR-Seq. [ABSTRACT FROM AUTHOR]

The specificity of T cells is that each T cell has only one T cell receptor (TCR). A T cell clone represents a collection of T cells with the same TCR sequence. Thus, the number of different T cell clones in an organism reflects the number of different T cell receptors (TCRs) that arise from recombination of the V(D)J gene segments during T cell development in the thymus. TCR diversity and more specifically, the clone abundance distribution, are important factors in immune functions. Specific recombination patterns occur more frequently than others while subsequent interactions between TCRs and self-antigens are known to trigger proliferation and sustain naive T cell survival. These processes are TCR-dependent, leading to clone-dependent thymic export and naive T cell proliferation rates. We describe the heterogeneous steady-state population of naive T cells (those that have not yet been antigenically triggered) by using a mean-field model of a regulated birth-death-immigration process. After accounting for random sampling, we investigate how TCR-dependent heterogeneities in immigration and proliferation rates affect the shape of clone abundance distributions (the number of different clones that are represented by a specific number of cells, or clone counts). By using reasonable physiological parameter values and fitting predicted clone counts to experimentally sampled clone abundances, we show that realistic levels of heterogeneity in immigration rates cause very little change to predicted clone-counts, but that modest heterogeneity in proliferation rates can generate the observed clone abundances. Our analysis provides constraints among physiological parameters that are necessary to yield predictions that qualitatively match the data. Assumptions of the model and potentially other important mechanistic factors are discussed.

Antigen-specific retargeting of cytotoxic lymphocytes against tumorassociated antigens has thus far remained largely dependent on chimeric antigen receptors (CARs) that can be constructed by the fusion of an extracellular targeting domain (classically a single-chain variable fragment from an antibody) fused with intracellular signaling domains to trigger activation of T or natural killer (NK) cells. A major limitation of CAR-based therapies is that this technology only allows for the targeting of antigens that would be located on the surface of target cells while non-surface antigens, which affect approximately three-fourths of all human genes, remain out of reach. The targeting of non-surface antigens is only possible using inherent T cell receptor (TCR) mechanisms. However, introducing a second TCR into T cells via genetic modification is problematic due to the heterodimeric nature of the TCR ligand-binding domain, which is composed of TCR α and β chains. It has been observed that the delivery of a second TCR α/β pair may lead to the mispairing of new TCR chains with the endogenously expressed ones and create mixed TCR dimers, and this has negatively affected the advancement of TCR-based T cell therapies. Recently, NK cells have been put forward as possible effectors for TCR gene therapy. Since NK cells do not endogenously express TCR chains, this seems to be an infallible approach to circumventing the problem of mispairing. Moreover, the similarity of intracellular signaling pathways and mechanisms of cytotoxicity between NK and T cells ensures that the triggering of antigen-specific responses by the TCR/CD3 complex can be used to induce antigen-specific cytotoxicity by TCR-modified NK (TCR-NK) cells. This review provides an overview of the initial studies of TCR-NK cells, identifies open questions in the field, and defines the place of this approach within the spectrum of adoptive immunotherapy techniques that rely on cytotoxic lymphocytes.

Abstract Background Sepsis is a frequent complication in critically ill patients, is highly heterogeneous and is associated with high morbidity and mortality rates, especially in the elderly population. Utilizing RNA sequencing (RNA-Seq) to analyze biological pathways is widely used in clinical and molecular genetic studies, but studies in elderly patients with sepsis are still lacking. Hence, we investigated the mortality-relevant biological features and transcriptomic features in elderly patients who were admitted to the intensive care unit (ICU) for sepsis. Methods We enrolled 37 elderly patients with sepsis from the ICU at Taichung Veterans General Hospital. On day-1 and day-8, clinical and laboratory data, as well as blood samples, were collected for RNA-Seq analysis. We identified the dynamic transcriptome and enriched pathways of differentially expressed genes between day-8 and day-1 through DVID enrichment analysis and Gene Set Enrichment Analysis. Then, the diversity of the T cell repertoire was analyzed with MiXCR. Results Overall, 37 patients had sepsis, and responders and non-responders were grouped through principal component analysis. Significantly higher SOFA scores at day-7, longer ventilator days, ICU lengths of stay and hospital mortality were found in the non-responder group, than in the responder group. On day-8 in elderly ICU patients with sepsis, genes related to innate immunity and inflammation, such as ZDHCC19, ALOX15, FCER1A, HDC, PRSS33, and PCSK9, were upregulated. The differentially expressed genes (DEGs) were enriched in the regulation of transcription, adaptive immune response, immunoglobulin production, negative regulation of transcription, and immune response. Moreover, there was a higher diversity of T-cell receptors on day-8 in the responder group, than on day-1, indicating that they had better regulated recovery from sepsis compared with the non-response patients. Conclusion Sepsis mortality and incidence were both high in elderly individuals. We identified mortality-relevant biological features and transcriptomic features with functional pathway and MiXCR analyses based on RNA-Seq data; and found that the responder group had upregulated innate immunity and increased T cell diversity; compared with the non-responder group. RNA-Seq may be able to offer additional complementary information for the accurate and early prediction of treatment outcome.

Cutaneous delayed drug hypersensitivity reactions (DHRs) are common iatrogenic events with potentially life-threatening consequences. Delayed DHRs encompass diverse phenotypes and can be classified by their distinct T-cell responses to drug antigens. Interaction between the immune receptors, human leukocyte antigen (HLA) and T-cell receptor (TCR), and the complementary antigenic peptide is required for the development of delayed DHRs. These idiosyncratic interactions can be elicited by the formation of antigenic drug-protein adducts (hapten hypothesis) or from direct interactions of drugs with the immune receptors (pharmacological interaction of drugs with immune receptors concept, altered peptide repertoire model, and altered TCR model). In addition, viral infections may play a role by providing co-stimulatory signals or enhancing TCR/HLA expression on T-cells. The associations of HLA allele polymorphisms and DHRs are phenotype and ethnicityspecific. The discovery of genetic polymorphisms associated with DHRs has provided a strategy to prevent and diagnose potentially life-threatening reactions. Recently, advances in next-generation sequencing technologies, such as the incorporation of whole-exome or whole-genome sequencing, enabled the comprehensive detection of susceptibility loci. Several HLA associations have shown clinical utility and cost-effectiveness, such as HLA-B*15:02 (carbamazepine-induced Stevens–Johnson syndrome/toxic epidermal necrolysis in Han Chinese), HLA-B*58:01 (allopurinol-induced severe cutaneous adverse reactions in Han Chinese), HLA-B*57:01 (abacavir hypersensitivity reactions in Caucasians), and HLA-B*13:01 (dapsone-induced drug reaction with eosinophilia and systemic symptoms in Han Chinese). Herein, we summarize the current knowledge of the pathogenesis, antigen presentation models, and HLA associations of cutaneous delayed DHRs.

Abstract α2,6‐sialylation, catalyzed by α2,6‐sialyltransferase (ST6GAL1), plays a pivotal role in immune responses. However, the role of ST6GAL1 in the pathogenesis of ulcerative colitis (UC) remains unknown. ST6GAL1 mRNA is highly expressed in UC tissues compared with the corresponding adjacent normal tissues, and α2,6‐sialylation is significantly increased in the colon tissues of patients with UC. The expression of ST6GAL1 and proinflammatory cytokines, such as interleukin (IL)‐2, IL‐6, IL‐17, and interferon‐gamma, is also increased. The number of CD4+ T cells increases in UC patients. St6gal1 gene knockout (St6gal1−/‐) rats are established by clustered regularly interspaced short palindromic repeats (CRISPR)‐associated gene knockout system. St6gal1 deficiency reduces the levels of pro‐inflammatory cytokines and alleviates colitis symptoms in UC model rats. Ablation of α2,6‐sialylation inhibits the transport of the TCR to lipid rafts and suppresses CD4+ T‐cell activation. The attenuation of TCR signaling downregulates the expression of NF‐κB in ST6GAL1‐/‐ CD4+ T‐cells. Moreover, NF‐κB could bind to the ST6GAL1 promoter to increase its transcription. Ablation of ST6GAL1 downregulates the expression of NF‐κB and reduces the production of proinflammatory cytokines to relieve UC pathogenesis, which is a potential novel target for the clinical treatment of UC.

IntroductionImmunotherapy with checkpoint inhibitors is an efficient treatment for metastatic melanoma. Development of vitiligo upon immunotherapy represents a specific immune-related adverse event (irAE) diagnosed in 15% of patients and associated with a positive clinical response. Therefore, a detailed characterization of immune cells during vitiligo onset in melanoma patients would give insight into the immune mechanisms mediating both the irAE and the anti-tumor response. MethodsTo better understand these aspects, we analyzed T cell subsets from peripheral blood of metastatic melanoma patients undergoing treatment with anti-programmed cell death protein (PD)-1 antibodies. To deeply characterize the antitumoral T cell response concomitant to vitiligo onset, we analyzed T cell content in skin biopsies collected from melanoma patients who developed vitiligo. Moreover, to further characterize T cells in vitiligo skin lesion of melanoma patients, we sequenced T cell receptor (TCR) of cells derived from biopsies of vitiligo and primary melanoma of the same patient.Results and discussionStratification of patients for developing or not developing vitiligo during anti-PD-1 therapy revealed an association between blood reduction of CD8-mucosal associated invariant T (MAIT), T helper (h) 17, natural killer (NK) CD56bright, and T regulatory (T-reg) cells and vitiligo onset. Consistently with the observed blood reduction of Th17 cells in melanoma patients developing vitiligo during immunotherapy, we found high amount of IL-17A expressing cells in the vitiligo skin biopsy, suggesting a possible migration of Th17 cells from the blood into the autoimmune lesion. Interestingly, except for a few cases, we found different TCR sequences between vitiligo and primary melanoma lesions. In contrast, shared TCR sequences were identified between vitiligo and metastatic tissues of the same patient. These data indicate that T cell response against normal melanocytes, which is involved in vitiligo onset, is not typically mediated by reactivation of specific T cell clones infiltrating primary melanoma but may be elicited by T cell clones targeting metastatic tissues. Altogether, our data indicate that anti-PD-1 therapy induces a de novo immune response, stimulated by the presence of metastatic cells, and composed of different T cell subtypes, which may trigger the development of vitiligo and the response against metastatic tumor.

IntroductionT-cell receptor (TCR) recognition of foreign peptides presented by the major histocompatibility complex (MHC) initiates the adaptive immune response against pathogens. While a large number of TCR sequences specific to different antigenic peptides are known to date, the structural data describing the conformation and contacting residues for TCR-peptide-MHC complexes is relatively limited. In the present study we aim to extend and analyze the set of available structures by performing highly accurate template-based modeling of these complexes using TCR sequences with known specificity. MethodsIdentification of CDR3 sequences and their further clustering, based on available spatial structures, V- and J-genes of corresponding T-cell receptors, and epitopes, was performed using the VDJdb database. Modeling of the selected CDR3 loops was conducted using a stepwise introduction of single amino acid substitutions to the template PDB structures, followed by optimization of the TCR-peptide-MHC contacting interface using the Rosetta package applications. Statistical analysis and recursive feature elimination procedures were carried out on computed energy values and properties of contacting amino acid residues between CDR3 loops and peptides, using R.ResultsUsing the set of 29 complex templates (including a template with SARS-CoV-2 antigen) and 732 specificity records, we built a database of 1585 model structures carrying substitutions in either TCRα or TCRβ chains with some models representing the result of different mutation pathways for the same final structure. This database allowed us to analyze features of amino acid contacts in TCR - peptide interfaces that govern antigen recognition preferences and interpret these interactions in terms of physicochemical properties of interacting residues.ConclusionOur results provide a methodology for creating high-quality TCR-peptide-MHC models for antigens of interest that can be utilized to predict TCR specificity.

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with few accepted prognostic factors that limit the efficiency of therapy. The aim of the current study was to assess the clinical and laboratory features of T-cell receptor (TCR) aberrations and early T-cell precursor (ETP) subtype as well as their outcome to therapy. Methods: Sixty-three newly diagnosed pediatric T-ALL patients were assessed for the ETP status using immunophenotyping. Screening of TCRA/D aberrations was done by fluorescent in situ hybridization (FISH). The data were correlated to the patients' clinical features, response to treatment, and survival rates. Results: Seven patients (11%) had ETP-ALL. The ETP-ALL patients were older (P = 0.013), presented with lower white blood cell (WBC) count (P = 0.001) and lower percentage of peripheral blood (PB) blast cells (P = 0.037), more likely to have hyperdiploid karyotype (P = 0.009), and had been associated with TCRA/D gene amplification (P = 0.014) compared to other T-ALL patients. Of note, the same associations had been significantly observed in patients with TCRA/D gene amplification. Patients with TCRA/D amplification frequently coincided with TCRβ aberrations (P = 0.025). TCR-β aberrations were significantly associated with negative MRD at the end of induction compared to TCR-β-negative patients. There was a nonsignificant trend of ETP-positive cases to have lower overall survival (OS) (P = 0.06). Patients with TCR aberrations had no significant differences regarding disease-free survival (DFS) or OS rates compared to those with normal TCR. Conclusion: ETP-ALL patients tend to have increased mortalities. There was no significant impact of TCR aberrations on the survival rates of the patients. [ABSTRACT FROM AUTHOR]

Lichen planus (LP) is a cutaneomucosal chronic inflammatory disease characterized by a CD8+ cytotoxic T‐lymphocytes (CTL) infiltrate. In erosive oral LP, we found HPV16‐specific activated CTL in lesions, supporting a pathogenic contribution of HPV16. Here, we investigated whether a similar scenario occurs in other clinical forms of LP and in lichen sclerosus et atrophicus (LSA), another chronic disease also affecting the mucosa and/or the skin. Blood CTL from LP and LSA patients expressed significant higher levels of granzyme B, perforin and CD107a proteins than healthy donors. Expansions of TCRVß3+ CTL, with presence of TCR clonotypes identical to those previously detected in erosive oral LP, were found both in blood and mucosal/skin lesions of LP, and not of LSA patients. These expansions were enriched with HPV16‐specific CD8+ T‐cells as shown by their recognition of the E711‐20 immunodominant epitope. In LSA patients, the peripheral repertoire of CTL was oligoclonal for TCRVß6+ CTL. Finally, although patients with LP and LSA have developed antibodies against HPV16 capsid L1, antibodies against HPV16 E6 were only observed in patients with LP. Overall, our data collectively suggest an involvement of HPV16‐specific CTL in different clinical forms of LP, not only in erosive oral LP, while a different scenario operates in LSA. [ABSTRACT FROM AUTHOR]

Background T-cell receptor (TCR-T) therapies are based on the expression of an introduced TCR targeting a tumor associated antigen (TAA) which has been studied in several trials in cutaneous melanoma. We conducted a systematic review and meta-analysis aiming to assess the primary efficacy of TCR-based adoptive cell therapy in cutaneous melanoma. Methods We searched through PubMed electronic database from its inception until May 21, 2022. Primary endpoints were pooled objective response rate (ORR) and disease control rate (DCR). We conducted logistic regression analyses to identify potential predictive factors for tumor response. Results From 187 patients, 50 showed an objective response (pooled ORR 28%; 95% CI, 20%-37%) and a pooled DCR of 38% (95% CI, 27%-50%). Median PFS was 2, 9 months (95% CI, 1.4-3.1). A trend toward higher PFS was demonstrated for patients treated with cancer/testis antigens targeting TCR-T cells (HR 0.91 95% CI, 0.64-1.3, P =.61) among whom, patients treated with NYESO-1 targeting TCR-T showed a significantly higher PFS (HR 0.63 95% CI, 0.64-0.98, P =.03). In addition, the number of infused cells was associated with a significantly higher likelihood of tumor response (OR 6.61; 95% CI, 1.68-21.6; P =.007). Conclusion TCR-T therapy shows promising results in terms of antitumor activity and survival similar to those reported for TILs with a significantly higher benefit for cancer/testis antigens targeting cells. Since TCR-based therapy shows advantages of great potential over classic ACT strategies, further research in solid cancers is warranted (PROSPERO ID CRD42022328011). [ABSTRACT FROM AUTHOR]

Acute Respiratory Distress Syndrome (ARDS) is an illness that typically develops in people who are significantly ill or have serious injuries. ARDS is characterized by fluid build-up that occurs in the alveoli. T-cells are implicated as playing a role in the modulation of the aberrant response leading to excessive tissue damage and, eventually, ARDS. Complementarity Determining Region 3 (CDR3) sequences derived from T-cells are key players in the adaptive immune response. This response is governed by an elaborate specificity for distinct molecules and the ability to recognize and vigorously respond to repeated exposures to the same molecules. Most of the diversity in T-cell receptors (TCRs) is contained in the CDR3 regions of the heterodimeric cell-surface receptors. For this study, we employed the novel technology of immune sequencing to assess lung edema fluid. Our goal was to explore the landscape of CDR3 clonal sequences found within these samples. We obtained more than 3615 CDR3 sequences across samples in the study. Our data demonstrate that: (1) CDR3 sequences from lung edema fluid exhibit distinct clonal populations, and (2) CDR3 sequences can be further characterized based on biochemical features. Analysis of these CDR3 sequences offers insight into the CDR3-driven T-cell repertoire of ARDS. These findings represent the first step towards applications of this technology with these types of biological samples in the context of ARDS. [ABSTRACT FROM AUTHOR]

Adoptive T-cell therapy (ACT) is successfully applied in cancer treatment; however, its efficiency can be limited by a low viability, short persistence time, and loss of functional activity of T-cells after adoptive transfer. The search for novel immunomodulators that can improve the viability, expansion, and functions of T-cells after their infusion with the minimal side effects could contribute to the development of more efficient and safe ACT strategies. Recombinant human cyclophilin A (rhCypA) is of particular interest in this respect, as it exhibits pleiotropic immunomodulatory activity and stimulates both innate and adaptive anti-tumor immunity. Here, we evaluated the effect of rhCypA on the efficacy of ACT in the mouse EL4 lymphoma model. Lymphocytes from transgenic 1D1a mice with an inborn pool of EL4-specific T-cells were used as a source of tumor-specific T-cells for ACT. In models of immunocompetent and immunodeficient transgenic mice, the course (3 days) rhCypA administration was shown to significantly stimulate EL4 rejection and prolong the overall survival of tumor-bearing mice after adoptive transfer of lowered doses of transgenic 1D1a cells. Our studies showed that rhCypA significantly improved the efficacy of ACT by enhancing the effector functions of tumor-specific cytotoxic T-cells. These findings open up the prospects for the development of innovative strategies of adoptive T-cell immunotherapy for cancer using rhCypA as an alternative to existing cytokine therapies. [ABSTRACT FROM AUTHOR]

Conventionally T-cell receptors (TCRs) have so far been considered as a T-lymphocyte privilege. However, recent findings also place TCR expression in non-lymphoid cells, namely neutrophils, eosinophils and macrophages. In order to examine the ectopic expression of TCR, this study focused on RAW 264.7 cells, which have been broadly used for their macrophage properties. Immunofluorescence staining detected 70% and 40% of the cells to express TCRαβ and TCRγδ respectively, which was also verified by RT-PCR experiments and confocal microscopy analysis. Interestingly, except from the predicted 292 and 288 bp gene products for the α- and γ-chain, additional products at 220 and 550 bp could be detected, respectively. RAW 264.7 cells also expressed the co-stimulatory CD4 and CD8 markers at a percentage of 61% and 14% respectively, which supported the expression of TCRs. However, only low numbers of cells expressed CD3ε and CD3ζ (9% and 7% respectively). Such observations contradicted the existing knowledge, and indicated that TCRs would be supported by other molecules for reaching the membrane and transducing their signal. Such candidate molecules could be the Fcγ receptors (FcγRs). Indeed, the FcγRII/III receptor was found to be expressed in 75% of the cells, which also expressed at a percentage of 25% major histocompatibility complex (MHC) class II molecules. Engagement of the FcγRII/III receptor by a recombinant IgG2aCH2 fragment, except from stimulating the macrophage-dependent properties of the cells, was shown to reduce expression of TCRαβ and γδ indicating that FcγRII/III was indeed used by TCRs for their transport to the cell membrane. In order to examine the ability of RAW 264.7 cells to simultaneously display antigen presenting- and T-cell properties, functional experiments as to antigen-specific antibody and IL-2 production were performed. In in vitro immunization assays in the presence of naïve B cells, RAW264.7 failed to promote antibody production. However, RAW 264.7 cells could compete with antigen-stimulated macrophages but not T cells when applied to a system of in vivo antigen-sensitized cells followed by an in vitro immunization protocol. Interestingly, simultaneous addition of antigen and the IgG2aCH2 fragment to RAW 264.7 cells could promote IL-2 production from the cells, indicating that FcγRII/III activation could also support TCR stimulation. Extrapolating these findings to cells of the myeloid origin, the above results dictate novel regulatory mechanisms towards the alteration of the immune response. [Display omitted] • RAW 264.7 macrophages express TCRαβ/TCRγδ and CD4/CD8 but insufficient CD3 levels. • Recombinant IgG2aCH2 decreases TCRαβ/TCRγδ indicating that FcγRII/III is involved in TCR membrane transport. • RAW 264.7 cells may compete macrophages but not T cells in an antigen specific antibody production. • Stimulation of RAW 264.7 cells with rIgG2aCH2 and antigen induces IL-2 production. • Novel regulatory pathways by TCR+ myeloid cells towards stimulation of immunity. [ABSTRACT FROM AUTHOR]

T cells use their T‐cell receptors (TCRs) to discriminate between lower‐affinity self and higher‐affinity foreign peptide major‐histocompatibility‐complexes (pMHCs) based on the TCR/pMHC off‐rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off‐rate remains debated. Here, we measured the effect of mechanical force on the off‐rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower‐affinity TCR/pMHCs with faster solution off‐rates were more resistant to mechanical force (weak slip or catch bonds) than higher‐affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best‐characterized catch bond, involving the OT‐I TCR, has a low affinity and an exceptionally fast solution off‐rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA‐1 in force‐shielding the TCR/pMHC interaction. Synopsis: The T‐cell antigen receptor (TCR) needs to discriminate between low‐affinity self‐peptide MHC antigens (pMHCs) and higher affinity foreign pMHCs. This study shows that mechanical force likely impairs TCR discrimination, supporting a role for adhesion receptors in shielding the TCR/pMHC interaction from mechanical forces.A cell‐free assay is used to systematically measure the off‐rate of 13 different TCR/pMHC interactions under different applied forces.In contrast to studies using intact T cells, we found that applied force increased the off‐rate of high‐affinity TCR/pMHC interactions by more than it did for low‐affinity interactions.Structured‐based molecular dynamics simulations confirmed these experimental findings.A kinetic proofreading model incorporating the observed effects of mechanical forces predicts that reducing the force on the TCR/pMHC interaction improves antigen discrimination and sensitivity.The ability of CD2 and LFA‐1 to improve antigen discrimination and sensitivity supports a role for these adhesion receptors in force‐shielding the TCR/pMHC interaction. [ABSTRACT FROM AUTHOR]

Background: Minimal change disease (MCD) is the major cause of childhood idiopathic nephrotic syndrome, which is characterized by massive proteinuria and debilitating edema. Proteinuria in MCD is typically rapidly reversible with corticosteroid therapy, but relapses are common, and children often have many adverse events from the repeated courses of immunosuppressive therapy. The pathobiology of MCD remains poorly understood. Prior clinical observations suggest that abnormal T-cell function may play a central role in MCD pathogenesis. Based on these observations, we hypothesized that T-cell responses to specific exposures or antigens lead to a clonal expansion of T-cell subsets, a restriction in the T-cell repertoire, and an elaboration of specific circulating factors that trigger disease onset and relapses. Methods: To test these hypotheses, we sequenced T-cell receptors in fourteen MCD, four focal segmental glomerulosclerosis (FSGS), and four membranous nephropathy (MN) patients with clinical data and blood samples drawn during active disease and during remission collected by the Nephrotic Syndrome Study Network (NEPTUNE). We calculated several T-cell receptor diversity metrics to assess possible differences between active disease and remission states in paired samples. Results: Median productive clonality did not differ between MCD active disease (0.0083; range: 0.0042, 0.0397) and remission (0.0088; range: 0.0038, 0.0369). We did not identify dominant clonotypes in MCD active disease, and few clonotypes were shared with FSGS and MN patients. Conclusions: While these data do not support an obvious role of the adaptive immune system T-cells in MCD pathogenesis, further study is warranted given the limited sample size. A higher resolution version of the Graphical abstract is available as Supplementary information. [ABSTRACT FROM AUTHOR]

High-grade serous ovarian carcinoma (HGSOC) is a fatal gynecological malignancy. Somatic recombination occurring during T-cell receptor (TCR) development results in TCR diversity, and the TCR repertoire, thus produced, is associated with immune response. This study analyzed the difference in the TCR repertoire and their prognostic significance in 51 patients with HGSOC. The patient's clinical characteristics, gene expression pattern, TCR clonotypes, and degree of tumor-infiltrating leukocytes (TILs) were analyzed, and the patients were divided into groups depending on their recurrence pattern, tumor-infiltrating leukocyte (TIL) score, and homologous recombinant repair pathway deficiency (HRD)-associated mutations. The TCR repertoire was low in patients with recurrence and showed the expansion of eight TCR segments. Interestingly, a few genes correlated with the TCRs also showed a difference in expression according to the prognosis. Among them, seven genes were related to immune responses and KIAA1199 was up-regulated in ovarian cancer. Our study shows that the differences in the TCR repertoire in patients with ovarian cancer and their associated immune pathways could affect the prognosis of HGSOC. [ABSTRACT FROM AUTHOR]

Understanding racial disparities in cancer outcomes continues to be a challenge, with likely many factors at play, including socioeconomic factors and genetic polymorphisms impacting basic cellular and molecular functions. Additionally, it is possible that specific combinations of environment and genetics have specific impacts. T‐cell receptor (TCR) gene segment usage, HLA allele combinations have been associated with autoimmune and infectious disease courses, and more recently, TCR gene segment usage, HLA allele combinations have been associated with distinct survival outcomes in cancer as well. We examined several such, previously reported cancer‐related TCR gene segment usage, HLA allele combinations for evidence of racial disparities, with regard to the prevalence of the combination in different racial groups. Results indicated that TCR gene segment usage, potentially reflecting environmental factors related to previous pathogen exposure, in combination with certain HLA alleles or independently, may represent a novel explanation for racial disparities in cancer outcomes. Overall, at this point, a genetic connection to racial disparities in cancer outcomes is detectable but remains modest, suggesting that other factors, such as socioeconomic factors, remain as important considerations. [ABSTRACT FROM AUTHOR]

T lymphocytes are the major components of adaptive immunity in Behçet's syndrome (BS) pathology. However, the precise mechanism of T‐cell‐induced inflammatory condition remains to be determined. We applied bulk sequencing of the T‐cell receptor (TCR) β chain in peripheral blood samples from 45 patients with BS and 10 healthy donors as controls. TCR repertoires in BS patients displayed more clonality and less diversity than in healthy donors. Male patients exhibited lower diversity metrics of TCR and had a larger proportion in the top 10 clones than females (p = 0.016). There were no TCR clonality differences in other clinical features, such as age, disease duration, organ involvement, disease severity, and activity. By "Grouping of Lymphocyte Interactions by Paratope Hotspots" (GLIPH2) for antigen prediction, we found distinct 2477 clusters of TCR‐β sequences that potentially recognize similar antigens shared between BS patients. We observed clonal T‐cell expansion in BS patients. Sexual differences in TCR clonal expansion and public TCR groups deserve further study to reveal the underline T‐cell‐mediated immunity in BS. [ABSTRACT FROM AUTHOR]

Chronic pancreatitis (CP) is considered an irreversible fibroinflammatory pancreatic disease. Despite numerous animal model studies, questions remain about local immune characteristics in human CP. We profiled pancreatic immune cell characteristics in control organ donors and CP patients including those with hereditary and idiopathic CP undergoing total pancreatectomy with islet autotransplantation. Flow cytometric analysis revealed a significant increase in the frequency of CD68+ macrophages in idiopathic CP. In contrast, hereditary CP samples showed a significant increase in CD3+ T cell frequency, which prompted us to investigate the T cell receptor β (TCRβ) repertoire in the CP and control groups. TCRβ sequencing revealed a significant increase in TCRβ repertoire diversity and reduced clonality in both CP groups versus controls. Interestingly, we observed differences in Vβ-Jβ gene family usage between hereditary and idiopathic CP and a positive correlation of TCRβ rearrangements with disease severity scores. Immunophenotyping analyses in hereditary and idiopathic CP pancreases indicate differences in innate and adaptive immune responses, which highlights differences in immunopathogenic mechanisms of disease among subtypes of CP. TCR repertoire analysis further suggests a role for specific T cell responses in hereditary versus idiopathic CP pathogenesis, providing insights into immune responses associated with human CP.

Abstract Profiling the T-Cell Receptor (TCR) repertoire is establishing as a potent approach to investigate autologous and treatment-induced antitumor immune response. Technical and computational breakthroughs, including high throughput next-generation sequencing (NGS) approaches and spatial transcriptomics, are providing unprecedented insight into the mechanisms underlying antitumor immunity. A precise spatiotemporal variation of T-cell repertoire, which dynamically mirrors the functional state of the evolving host-cancer interaction, allows the tracking of the T-cell populations at play, and may identify the key cells responsible for tumor eradication, the evaluation of minimal residual disease and the identification of biomarkers of response to immunotherapy. In this review we will discuss the relationship between global metrics characterizing the TCR repertoire such as T-cell clonality and diversity and the resultant functional responses. In particular, we will explore how specific TCR repertoires in cancer patients can be predictive of prognosis or response to therapy and in particular how a given TCR re-arrangement, following immunotherapy, can predict a specific clinical outcome. Finally, we will examine current improvements in terms of T-cell sequencing, discussing advantages and challenges of current methodologies.

Abstract Background Clear cell renal cell cancer (ccRCC) is accompanied by T-cell infiltration. In this study, we sought to determine the difference in T-cell infiltration and the T-cell receptor (TCR) immune repertoire between ccRCC and peritumour tissue. Methods T-cell infiltration was examined using immunohistochemistry (IHC) and haematoxylin and eosin (HE) staining. The chi-squared test and Pearson correlation analysis were applied to evaluate the relationship between clinical traits and CD3, CD4, and CD8 expression. Immune repertoire sequencing (IR-Seq) was used to describe the profile of the TCR repertoire. Results The adjacent tissue showed increased expression of CD3, CD4 and CD8 compared with ccRCC tissue (P CD3 = 0.033; P CD4 = 0.014; P CD8 = 0.004). Indicated CD3+ T-cell density in ccRCC tissue was positively correlated with that in peritumour tissue (P = 0.010, r = 0.514), which implied the T cells in peritumour tissue directly infect the number of cells infiltrating in ccRCC tissue. Moreover, there was a positive correlation between Vimentin expression and indicated positive T-cell marker in ccRCC tissue (P CD3 = 0.035; P CD4 = 0.020; P CD8 = 0.027). Advanced stage revealed less CD4+ T-cell infiltration in ccRCC tissue (P CD4 = 0.023). The results from IR-Seq revealed an obvious increase in VJ and VDJ segment usage, as well as higher complementarity-determining region 3 (CDR3) amino acid (aa) clonotypes in ccRCC. The matched antigen recognized by the TCR of ccRCC may be potential targets. Conclusions The current study collectively demonstrates diminished T-cell infiltration and increased CDR3 aa diversity in ccRCC, which may be associated with immunotherapeutic targets for ccRCC patients.

Abstract Background Severe cutaneous adverse drug reactions (SCARs) are a group of serious clinical conditions caused by immune reaction to certain drugs. The allelic variance of human leukocyte antigens of HLA-B*13:01 has been strongly associated with hypersensitivities induced by dapsone (DDS). T-cell receptor mediated activation of cytotoxic T lymphocytes (CTLs) has also been suggested to play an essential role in pathogenesis of SCARs. However, HLA-B*13:01-DDS-TCR immune synapse that plays role in drug-induced hypersensitivity syndrome (DIHS) associated T cells activation remains uncharacterized. Methods To investigate the molecular mechanisms for HLA-B*13:01 in the pathogenesis of Dapsone-induced drug hypersensitivity (DDS-DIHS), we performed crystallization and expanded drug-specific CTLs to analyze the pathological role of DDS-DIHS. Results Results showed the crystal structure of HLA-B*13:01-beta-2-microglobulin (β2M) complex at 1.5 Å resolution and performed mutation assays demonstrating that I118 or I119, and R121 of HLA-B*13:01 were the key residues that mediate the binding of DDS. Subsequent single-cell TCR and RNA sequencing indicated that TCRs composed of paired TRAV12-3/TRBV28 clonotype with shared CDR3 region specifically recognize HLA-B*13:01-DDS complex to trigger inflammatory cytokines associated with DDS-DIHS. Conclusion Our study identified the novel p-i-HLA/TCR as the model of interaction between HLA-B*13:01, DDS and the clonotype-specific TCR in DDS-DIHS. Graphical Abstract

Background: Sepsis is a frequent complication in critically ill patients, is highly heterogeneous and is associated with high morbidity and mortality rates, especially in the elderly population. Utilizing RNA sequencing (RNA-Seq) to analyze biological pathways is widely used in clinical and molecular genetic studies, but studies in elderly patients with sepsis are still lacking. Hence, we investigated the mortality-relevant biological features and transcriptomic features in elderly patients who were admitted to the intensive care unit (ICU) for sepsis. Methods: We enrolled 37 elderly patients with sepsis from the ICU at Taichung Veterans General Hospital. On day-1 and day-8, clinical and laboratory data, as well as blood samples, were collected for RNA-Seq analysis. We identified the dynamic transcriptome and enriched pathways of differentially expressed genes between day-8 and day-1 through DVID enrichment analysis and Gene Set Enrichment Analysis. Then, the diversity of the T cell repertoire was analyzed with MiXCR. Results: Overall, 37 patients had sepsis, and responders and non-responders were grouped through principal component analysis. Significantly higher SOFA scores at day-7, longer ventilator days, ICU lengths of stay and hospital mortality were found in the non-responder group, than in the responder group. On day-8 in elderly ICU patients with sepsis, genes related to innate immunity and inflammation, such as ZDHCC19, ALOX15, FCER1A, HDC, PRSS33, and PCSK9, were upregulated. The differentially expressed genes (DEGs) were enriched in the regulation of transcription, adaptive immune response, immunoglobulin production, negative regulation of transcription, and immune response. Moreover, there was a higher diversity of T-cell receptors on day-8 in the responder group, than on day-1, indicating that they had better regulated recovery from sepsis compared with the non-response patients. Conclusion: Sepsis mortality and incidence were both high in elderly individuals. We identified mortality-relevant biological features and transcriptomic features with functional pathway and MiXCR analyses based on RNA-Seq data; and found that the responder group had upregulated innate immunity and increased T cell diversity; compared with the non-responder group. RNA-Seq may be able to offer additional complementary information for the accurate and early prediction of treatment outcome. [ABSTRACT FROM AUTHOR]

Clonality assessment using the unique rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes in lymphocytes is a widely applied supplementary test for the diagnosis of B-cell and T-cell lymphoma. To enable a more sensitive detection and a more precise comparison of clones compared with conventional clonality analysis based on fragment analysis, the EuroClonality NGS Working Group developed and validated a next-generation sequencing (NGS)-based clonality assay for detection of the IG heavy and kappa light chain and TR gene rearrangements for formalin-fixed and paraffin-embedded tissues. We outline the features and advantages of NGS-based clonality detection and discuss potential applications for NGS-based clonality testing in pathology, including site specific lymphoproliferations, immunodeficiency and autoimmune disease and primary and relapsed lymphomas. Also, we briefly discuss the role of T-cell repertoire of reactive lymphocytic infiltrations in solid tumors and B-lymphoma. [ABSTRACT FROM AUTHOR]

Antigen-specific retargeting of cytotoxic lymphocytes against tumor-associated antigens has thus far remained largely dependent on chimeric antigen receptors (CARs) that can be constructed by the fusion of an extracellular targeting domain (classically a single-chain variable fragment from an antibody) fused with intracellular signaling domains to trigger activation of T or natural killer (NK) cells. A major limitation of CAR-based therapies is that this technology only allows for the targeting of antigens that would be located on the surface of target cells while non-surface antigens, which affect approximately three-fourths of all human genes, remain out of reach. The targeting of non-surface antigens is only possible using inherent T cell receptor (TCR) mechanisms. However, introducing a second TCR into T cells via genetic modification is problematic due to the heterodimeric nature of the TCR ligand-binding domain, which is composed of TCR α and β chains. It has been observed that the delivery of a second TCR α/β pair may lead to the mispairing of new TCR chains with the endogenously expressed ones and create mixed TCR dimers, and this has negatively affected the advancement of TCR-based T cell therapies. Recently, NK cells have been put forward as possible effectors for TCR gene therapy. Since NK cells do not endogenously express TCR chains, this seems to be an infallible approach to circumventing the problem of mispairing. Moreover, the similarity of intracellular signaling pathways and mechanisms of cytotoxicity between NK and T cells ensures that the triggering of antigen-specific responses by the TCR/CD3 complex can be used to induce antigen-specific cytotoxicity by TCR-modified NK (TCR-NK) cells. This review provides an overview of the initial studies of TCR-NK cells, identifies open questions in the field, and defines the place of this approach within the spectrum of adoptive immunotherapy techniques that rely on cytotoxic lymphocytes. [ABSTRACT FROM AUTHOR]

High-throughput RNA-sequencing (RNA-seq) technologies provide an unprecedented opportunity to explore the individual transcriptome. Unmapped reads are a large and often overlooked output of standard RNA-seq analyses. Here, we present Read Origin Protocol (ROP), a tool for discovering the source of all reads originating from complex RNA molecules. We apply ROP to samples across 2630 individuals from 54 diverse human tissues. Our approach can account for 99.9% of 1 trillion reads of various read length. Additionally, we use ROP to investigate the functional mechanisms underlying connections between the immune system, microbiome, and disease. ROP is freely available at https://github.com/smangul1/rop/wiki .

Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.

Clonality assessment using the unique rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes in lymphocytes is a widely applied supplementary test for the diagnosis of B-cell and T-cell lymphoma. To enable a more sensitive detection and a more precise comparison of clones compared with conventional clonality analysis based on fragment analysis, the EuroClonality NGS Working Group developed and validated a next-generation sequencing (NGS)-based clonality assay for detection of the IG heavy and kappa light chain and TR gene rearrangements for formalin-fixed and paraffin-embedded tissues. We outline the features and advantages of NGS-based clonality detection and discuss potential applications for NGS-based clonality testing in pathology, including site specific lymphoproliferations, immunodeficiency and autoimmune disease and primary and relapsed lymphomas. Also, we briefly discuss the role of T-cell repertoire of reactive lymphocytic infiltrations in solid tumors and B-lymphoma.

Profiling the T-Cell Receptor (TCR) repertoire is establishing as a potent approach to investigate autologous and treatment-induced antitumor immune response. Technical and computational breakthroughs, including high throughput next-generation sequencing (NGS) approaches and spatial transcriptomics, are providing unprecedented insight into the mechanisms underlying antitumor immunity. A precise spatiotemporal variation of T-cell repertoire, which dynamically mirrors the functional state of the evolving host-cancer interaction, allows the tracking of the T-cell populations at play, and may identify the key cells responsible for tumor eradication, the evaluation of minimal residual disease and the identification of biomarkers of response to immunotherapy. In this review we will discuss the relationship between global metrics characterizing the TCR repertoire such as T-cell clonality and diversity and the resultant functional responses. In particular, we will explore how specific TCR repertoires in cancer patients can be predictive of prognosis or response to therapy and in particular how a given TCR re-arrangement, following immunotherapy, can predict a specific clinical outcome. Finally, we will examine current improvements in terms of T-cell sequencing, discussing advantages and challenges of current methodologies. [ABSTRACT FROM AUTHOR]

Background While diagnostic, therapeutic, and vaccine development in the coronavirus disease 2019 (COVID-19) pandemic has proceeded at unprecedented speed, critical gaps in our understanding of the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unaddressed by current diagnostic strategies. Methods A statistical classifier for identifying prior SARS-CoV-2 infection was trained using >4000 SARS-CoV-2–associated T-cell receptor (TCR) β sequences identified by comparing 784 cases and 2447 controls from 5 independent cohorts. The T-Detect COVID (Adaptive Biotechnologies) assay applies this classifier to TCR repertoires sequenced from blood samples to yield a binary assessment of past infection. Assay performance was assessed in 2 retrospective (n = 346; n = 69) and 1 prospective cohort (n = 87) to determine positive percent agreement (PPA) and negative percent agreement (NPA). PPA was compared with 2 commercial serology assays, and pathogen cross-reactivity was evaluated. Results T-Detect COVID demonstrated high PPA in individuals with prior reverse transcription–polymerase chain reaction (RT-PCR)–confirmed SARS-CoV-2 infection (97.1% 15+ days from diagnosis; 94.5% 15+ days from symptom onset), high NPA (∼100%) in presumed or confirmed SARS-CoV-2 negative cases, equivalent or higher PPA than 2 commercial serology tests, and no evidence of pathogen cross-reactivity. Conclusions T-Detect COVID is a novel T-cell immunosequencing assay demonstrating high clinical performance for identification of recent or prior SARS-CoV-2 infection from blood samples, with implications for clinical management, risk stratification, surveillance, and understanding of protective immunity and long-term sequelae. [ABSTRACT FROM AUTHOR]

Background: Clear cell renal cell cancer (ccRCC) is accompanied by T-cell infiltration. In this study, we sought to determine the difference in T-cell infiltration and the T-cell receptor (TCR) immune repertoire between ccRCC and peritumour tissue.Methods: T-cell infiltration was examined using immunohistochemistry (IHC) and haematoxylin and eosin (HE) staining. The chi-squared test and Pearson correlation analysis were applied to evaluate the relationship between clinical traits and CD3, CD4, and CD8 expression. Immune repertoire sequencing (IR-Seq) was used to describe the profile of the TCR repertoire.Results: The adjacent tissue showed increased expression of CD3, CD4 and CD8 compared with ccRCC tissue (PCD3 = 0.033; PCD4 = 0.014; PCD8 = 0.004). Indicated CD3+ T-cell density in ccRCC tissue was positively correlated with that in peritumour tissue (P = 0.010, r = 0.514), which implied the T cells in peritumour tissue directly infect the number of cells infiltrating in ccRCC tissue. Moreover, there was a positive correlation between Vimentin expression and indicated positive T-cell marker in ccRCC tissue (PCD3 = 0.035; PCD4 = 0.020; PCD8 = 0.027). Advanced stage revealed less CD4+ T-cell infiltration in ccRCC tissue (PCD4 = 0.023). The results from IR-Seq revealed an obvious increase in VJ and VDJ segment usage, as well as higher complementarity-determining region 3 (CDR3) amino acid (aa) clonotypes in ccRCC. The matched antigen recognized by the TCR of ccRCC may be potential targets.Conclusions: The current study collectively demonstrates diminished T-cell infiltration and increased CDR3 aa diversity in ccRCC, which may be associated with immunotherapeutic targets for ccRCC patients. [ABSTRACT FROM AUTHOR]

The roles and mechanisms of T-cell receptor (TCR)-associated transmembrane adaptor 1 (TRAT1) in lung adenocarcinoma (LAC) have not yet been reported in the relevant literature. Therefore, this study aimed to understand the roles and mechanisms of TRAT1 in LAC using bioinformatics and in vitro experiments. TRAT1 expression levels in LAC samples were analysed using various databases. TRAT1 co-expressed genes were acquired by the correlation analysis of LAC tissues. The functional mechanisms and protein network of TRAT1 co-expressed genes were analysed using bioinformatics analysis. The expression of TRAT1 was activated in LAC cells, and the roles of TRAT1 overexpression in the growth and migration of cancer cells was investigated using flow cytometry, Cell Counting Kit-8 (CCK-8), and migration and invasion assays. The relationship between TRAT1 overexpression, the immune microenvironment, and RNA modification was evaluated using correlation analysis. TRAT1 expression levels were significantly abnormal at multiple mutation sites and were related to the prognosis of LAC. TRAT1 co-expressed genes were involved in cell proliferation, adhesion, and differentiation, and TRAT1 overexpression significantly inhibited cell viability, migration, and invasion and promoted apoptosis of A549 and H1299 cells, which might be related to the TCR, B cell receptor (BCR), MAPK, and other pathways. TRAT1 expression levels were significantly correlated with the ESTIMATE, immune, and stromal scores in the LAC microenvironment. Additionally, TRAT1 expression levels were significantly correlated with the populations of B cells, CD8 T cells, cytotoxic cells, and other immune cells. TRAT1 overexpression was significantly correlated with the expression of immune cell markers (such as PDCD1, CD2, CD3E) and genes involved in RNA modification (such as ALKBH1, ALKBH3, ALKBH5). In conclusions, TRAT1 overexpression inhibited the growth and migration of LAC cells, thereby delaying cancer progression, and was correlated with the LAC microenvironment and RNA modifications. [ABSTRACT FROM AUTHOR]

T‐cell receptor (TCR)‐like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next‐generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR‐like Abs using a rabbit system. We humanized previously generated rabbit‐derived TCR‐like Abs reacting Epstein–Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA‐A24 molecules, produced chimeric antigen receptor (CAR)‐T cells, and evaluated their antitumor effects using in vitro and in vivo tumor models. Humanization of the rabbit‐derived TCR‐like Abs using the complementarity‐determining region grafting technology maintained their specificity and affinity. We prepared a second‐generation CAR using single‐chain variable fragment of the humanized TCR‐like Abs and then transduced them into human T cells. The CAR‐T cells specifically recognized BRLF1p/MHC molecules and lysed the target cells in an antigen‐specific manner in vitro. They also demonstrated antitumor activity in a mouse xenograft model. We report the generation of CAR‐T cells using humanized rabbit‐derived TCR‐like Abs. Together with our established and efficient generation procedure for TCR‐like Abs using rabbits, our platform for the clinical application of humanized rabbit‐derived TCR‐like Abs to CAR‐T cells will help improve next‐generation cancer immunotherapy. [ABSTRACT FROM AUTHOR]

In the diagnostic workup of poorly differentiated tumors, T-cell receptor (TCR) clonality has long been considered as evidence of T-cell lymphoma. MET exon 14 skipping (METex14) is a mutation typically seen in lung adenocarcinoma. Herein, we present the first report of METex14 lung adenocarcinoma with isolated monoclonal TCRγ gene rearrangement. A 69-year-old woman presented to an outside hospital with pleural effusions. A pleural decortication demonstrated malignant cells positive for CD30 and CD138 but negative for BerEP4, KRT5, and EMA. An equivocal HHV8 staining was interpreted as positive, leading to the erroneous outside diagnosis of primary effusion lymphoma. Additional workup at our institution revealed a lack of HHV8 and T-cell markers but the presence of TCRγ clonality, pankeratin, and TTF1 expression. Repeat TCRγ testing on the in-house biopsy was negative for clonality. Next-generation sequencing detected METex14, confirming the diagnosis of lung adenocarcinoma. The potential diagnostic pitfall and prognostic/predictive implications are discussed. [ABSTRACT FROM AUTHOR]

The thymus-derived lymphocytes of jawed vertebrates have four T-cell receptor (TCR) chains that play a significant role in immunity. As chickens have commercial value, their immune systems require a great deal of attention. Local chicken breeds are an essential part of poultry genetic resources in China. Here, we used high-throughput sequencing to analyze the TCRα and TCRβ repertoires and their relative expression levels in the native chicken breeds Baier Buff, Longyou Partridge, Xiaoshan, and Xianju. We found that TCR Vα and TCR Vβ were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vβ1, and Vβ2 subgroups, respectively. V-J pairing was biased; Jα11 was utilized by nearly all Vα segments and was the most commonly used. Breed-specific V segments and V-J pairings were detected as well. The results of the principal coordinate analysis (PCoA) as well as the V-J pairing and CDR3 diversity analyses suggested that the four local chicken breeds did not significantly differ in terms of TCR diversity. Hence, they expressed not significant differentiation, and they are rich genetic resources for the development and utilization of immune-related poultry breeding. • Both Vα subgroups and Vβ subgroups are expressed in chicken. • Various TCR diversity with V segments, V-J pairing, and CDR3 diversity. • No significant differentiation of TCR diversity parameters among local chicken breeds. [ABSTRACT FROM AUTHOR]

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells. Most patients with T-PLL present with lymphocytosis, anemia, thrombocytopenia, and hepatosplenomegaly. Correct identification of T-PLL is essential because treatment for this disease is distinct from that of other T-cell neoplasms. In 2019, the T-PLL International Study Group (TPLL-ISG) established criteria for the diagnosis, staging, and assessment of response to treatment of T-PLL with the goal of harmonizing research efforts and supporting clinical decision-making. T-PLL pathogenesis is commonly driven by T-cell leukemia 1 (TCL1) overexpression and ATM loss, genetic alterations that are incorporated into the TPLL-ISG diagnostic criteria. The cooperativity between TCL1 family members and ATM is seemingly unique to T-PLL across the spectrum of T-cell neoplasms. The role of the T-cell receptor, its downstream kinases, and JAK/STAT signaling are also emerging themes in disease pathogenesis and have obvious therapeutic implications. Despite improved understanding of disease pathogenesis, alemtuzumab remains the frontline therapy in the treatment of naïve patients with indications for treatment given its high response rate. Unfortunately, the responses achieved are rarely durable, and the majority of patients are not candidates for consolidation with hematopoietic stem cell transplantation. Improved understanding of T-PLL pathogenesis has unveiled novel therapeutic vulnerabilities that may change the natural history of this lymphoproliferative neoplasm and will be the focus of this concise review.

Objective: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health emergency. T-cell receptors (TCRs) are crucial mediators of antiviral adaptive immunity. This study sought to comprehensively characterize the TCR repertoire changes in patients with COVID-19. Methods: A large sample size multi-center randomized controlled trial was implemented to study the features of the TCR repertoire and identify COVID-19 disease-related TCR sequences. Results: It was found that some T-cell receptor beta chain (TCRβ) features differed markedly between COVID-19 patients and healthy controls, including decreased repertoire diversity, longer complementarity-determining region 3 (CDR3) length, skewed utilization of the TCRβ variable gene/joining gene (TRBV/J), and a high degree of TCRβ sharing in COVID-19 patients. Moreover, this analysis showed that TCR repertoire diversity declines with aging, which may be a cause of the higher infection and mortality rates in elderly patients. Importantly, a set of TCRβ clones that can distinguish COVID-19 patients from healthy controls with high accuracy was identified. Notably, this diagnostic model demonstrates 100% specificity and 82.68% sensitivity at 0–3 days post diagnosis. Conclusions: This study lays the foundation for immunodiagnosis and the development of medicines and vaccines for COVID-19 patients.

IntroductionT cells are crucial for pathogenesis as well as control for tuberculosis (TB). Although much is known about the signaling pathways which are required for the activation of T cells during acute infection but the way these cells respond during persistent of infection still remained elusive. Therefore, it is rationale to understand T cell activation during tuberculous pleural effusion (TPE), which is similar to bacterial persistency system.MethodsHerein, we will employ T cell receptor (TCR) based approaches for studying events of T cell activation pathways in cells of blood and pleural fluid among patients with TPE. We performed spectrofluorimetric analysis to study effect of M. tuberculosis antigens, ESAT-6 and Ag85A stimulation on intracellular calcium levels, Phosphorylation levels of ZAP-70 (Zeta-chain-associated protein kinase 70), PKC-θ (Protein kinase C theta), Erk1/2 (Extracellular signal-regulated kinase 1 and 2) and p-38 two important members of MAPKs (Mitogen activated Protein kinases) in CD3 and CD28 induced cells of blood and pleural fluid of same patients with TPE by western blotting. Patients with non-TPE were also included as matching disease controls in this study.ResultsWe observed significantly higher intracellular calcium levels, Phosphorylation levels of ZAP-70, Erk1/2 and p-38 in CD3 and CD28 induced cells of pleural fluid as compared to the blood cells of same patients with TPE. Alteration in the activation of these events has also been noted after stimulation of ESAT-6 and Ag85A.DiscussionPresent study demonstrated up-regulated activation of TCR mediated T cell signaling events at local disease site (Pleural fluid) as compared to the blood sample of TB pleurisy patients which could be involved in T-cell dysfunctioning during the progression of the disease and also could be responsible for Th 1 dominance at local disease site in patients with TPE.

Summary: Background: Primary Sjogren's syndrome (SS) is a chronic inflammatory disease with unknown aetiology. Although clonal expansion of autoreactive T cells has been identified in patients with SS, the clinical correlation of T-cell receptor (TCR) variance in SS remains unclear. Methods: TCRβ repertoire sequencing was performed on 260 SS patients with 3-6 months of follow-up in a cohort study to dynamically assess the characteristics of TCR diversity and their clinical significance. Findings: We found that SS patients had lower TCR diversity, but higher frequency of public clones than healthy controls (HCs). Significant differences were identified in the usage of the variable (V) gene, joining (J) gene, and V-J pairing between SS and HCs. Eighteen SS-associated clones were identified, showing a high sensitivity and specificity for disease classification. TCR diversity was correlated with the presence of dental caries, thrombocytopenia, hepatocholangeitis, antinuclear antibody, anti-SSA/SSB, and hypergammaglobulinemia but not with disease course, number of relapses, arthritis, rheumatoid factor, hypocomplementemia or disease activity defined by SSDAI. During follow-up, the TCR abnormalities remained, represented by more altered V/J usage and higher frequencies of SS-associated clones. Among SS patients, the sensitive subgroup had increased TCR diversity after treatment. Eighty-five SS-sensitivity associated TCRs were identified and used for sensitivity classification by cross validation with high specificity and sensitivity. Interpretation: These results demonstrate that the TCR repertoire could provide insights into the disease status and prognosis in SS and other autoimmune diseases. Funding: This study was funded by the National Key Research and Development Program of China (2016YFC0906201), Sichuan Science and Technology Program (2020YJ0223), and the 1·3·5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYGD18015).

T-cell receptors (TCRs) are formed by random recombination of genomic precursor elements, some of which mediate the recognition of cancerassociated antigens. Due to the complicated process of T-cell immune response and limited biological empirical evidence, the practical strategy for identifying TCRs and their recognized peptides is the computational prediction from population and/or individual TCR repertoires. In recent years, several machine/deep learning-based approaches have been proposed for TCRpeptide binding prediction. However, the predictive performances of these methods can be further improved by overcoming several significant flaws in neural network design. The interrelationship between amino acids in TCRs is critical for TCR antigen recognition, which was not properly considered by the existing methods. They also did not pay more attention to the amino acids that play a significant role in antigen-binding specificity. Moreover, complex networks tended to increase the risk of overfitting and computational costs. In this study, we developed a dual-input deep learning framework, named AttnTAP, to improve the TCR-peptide binding prediction. It used the bidirectional long short-term memory model for robust feature extraction of TCR sequences, which considered the interrelationships between amino acids and their precursors and postcursors. We also introduced the attention mechanism to give amino acids different weights and pay more attention to the contributing ones. In addition, we used the multilayer perceptron model instead of complex networks to extract peptide features to reduce overfitting and computational costs. AttnTAP achieved high areas under the curves (AUCs) in TCR-peptide binding prediction on both balanced and unbalanced datasets (higher than 0.838 on McPAS-TCR and 0.908 on VDJdb). Furthermore, it had the highest average AUCs in TPP-I and TPP-II tasks compared with the other five popular models (TPP-I: 0.84 on McPAS-TCR and 0.894 on VDJdb; TPP-II: 0.837 on McPAS-TCR and 0.893 on VDJdb). In conclusion, AttnTAP is a reasonable and practical framework for predicting TCR-peptide binding, which can accelerate identifying neoantigens and activated T cells for immunotherapy to meet urgent clinical needs. [ABSTRACT FROM AUTHOR]

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS-from CD28-mediated costimulation during early Tcell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4+ T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes. [ABSTRACT FROM AUTHOR]

Autoimmune disease (AID) is a condition in which the immune system breaks down and starts to attack the body. Some common AIDs include systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes mellitus and so forth. The changes in T-cell receptor (TCR) repertoire have been found in several autoimmune diseases, and may be responsible for the breakdown of peripheral immune tolerance. In this review, we discussed the processes of TCR revision in peripheral immune environment, the changes in TCR repertoire that occurred in various AIDs, and the specifically expanded T cell clones. We hope our discussion can provide insights for the future studies, helping with the discovery of disease biomarkers and expanding the strategies of immune-targeted therapy. Restricted TCR repertoire and biased TCR-usage are found in a variety of AIDs. TCR repertoire shows tissue specificity in a variety of AID diseases. The relationship between TCR repertoire diversity and disease activity is still controversial in AIDs. Dominant TCR clonotypes may help to discover new disease biomarkers and expand the strategies of immune-targeted therapy. [ABSTRACT FROM AUTHOR]

Objectives: Although adoptive cell therapy with T‐cell receptor‐engineered T cells (TCR‐Ts) has mediated effective antitumor responses in several cancers, senescence of T cells could impair the therapeutic effect of TCR‐Ts. Thus, it is essential to elucidate the characteristics of senescent TCR‐Ts and how to subsequently improve their antitumor effect. Here, we focused on the influence of autophagy on TCR‐Ts, since autophagy is tightly associated with the regulation of T‐cell activation, proliferation and differentiation. Methods: We first evaluated autophagy level of senescent TCR‐Ts, and then the senescent TCR‐Ts were expanded in vitro for 7 days with and without spermidine treatment, respectively. Furthermore, the proliferative potential, phenotypical characteristics and functionality of the propagated senescent TCR‐Ts were analysed in vitro and in vivo after 7‐day ex vivo expansion. Results: We found that autophagic flux of senescent TCR‐T cells was significantly impaired. The restoration of autophagic flux via spermidine treatment reduced the expression of inhibitory immunoreceptors (PD‐1, TIM‐3 or LAG‐3), enhanced proliferation and effector functions and subsequently demonstrated the superior in vitro and in vivo antitumor activity of TCR‐Ts. Conclusion: These data suggest that spermidine treatment presents an opportunity to improve the antitumor effect of TCR‐Ts for the treatment of solid tumors. [ABSTRACT FROM AUTHOR]

The startling diversity in αβ T‐cell receptor (TCR) sequences and structures complicates molecular‐level analyses of the specificity and sensitivity determining T‐cell immunogenicity. A number of three‐dimensional (3D) structures are now available of ternary complexes between TCRs and peptides: major histocompatibility complexes (pMHC). Here, to glean molecular‐level insights we analyze structures of TCRs bound to human class I nonamer peptide–MHC complexes. Residues at peptide positions 4–8 are found to be particularly important for TCR binding. About 90% of the TCRs hydrogen bond with one or both of the peptide residues at positions 4 and 8 presented by MHC allele HLA‐A2, and this number is still ~79% for peptides presented by other MHC alleles. Residue 8, which lies outside the previously‐identified central peptide region, is crucial for TCR recognition of class I MHC‐presented nonamer peptides. The statistics of the interactions also sheds light on the MHC residues important for TCR binding. The present analysis will aid in the structural modeling of TCR:pMHC complexes and has implications for the rational design of peptide‐based vaccines and T‐cell‐based immunotherapies. [ABSTRACT FROM AUTHOR]

T-cell receptors (TCRs) are formed by random recombination of genomic precursor elements, some of which mediate the recognition of cancerassociated antigens. Due to the complicated process of T-cell immune response and limited biological empirical evidence, the practical strategy for identifying TCRs and their recognized peptides is the computational prediction from population and/or individual TCR repertoires. In recent years, several machine/deep learning-based approaches have been proposed for TCRpeptide binding prediction. However, the predictive performances of these methods can be further improved by overcoming several significant flaws in neural network design. The interrelationship between amino acids in TCRs is critical for TCR antigen recognition, which was not properly considered by the existing methods. They also did not pay more attention to the amino acids that play a significant role in antigen-binding specificity. Moreover, complex networks tended to increase the risk of overfitting and computational costs. In this study, we developed a dual-input deep learning framework, named AttnTAP, to improve the TCR-peptide binding prediction. It used the bidirectional long short-term memory model for robust feature extraction of TCR sequences, which considered the interrelationships between amino acids and their precursors and postcursors. We also introduced the attention mechanism to give amino acids different weights and pay more attention to the contributing ones. In addition, we used the multilayer perceptron model instead of complex networks to extract peptide features to reduce overfitting and computational costs. AttnTAP achieved high areas under the curves (AUCs) in TCR-peptide binding prediction on both balanced and unbalanced datasets (higher than 0.838 on McPAS-TCR and 0.908 on VDJdb). Furthermore, it had the highest average AUCs in TPP-I and TPP-II tasks compared with the other five popular models (TPP-I: 0.84 on McPAS-TCR and 0.894 on VDJdb; TPP-II: 0.837 on McPAS-TCR and 0.893 on VDJdb). In conclusion, AttnTAP is a reasonable and practical framework for predicting TCR-peptide binding, which can accelerate identifying neoantigens and activated T cells for immunotherapy to meet urgent clinical needs. [ABSTRACT FROM AUTHOR]

γδ T cells play important roles in immune responses by rapidly producing large quantities of cytokines. Recently, γδ T cells have been found to be involved in tissue homeostatic regulation, playing roles in thermogenesis, bone regeneration and synaptic plasticity. Nonetheless, the mechanisms involved in γδ T-cell development, especially the regulation of TCRδ gene transcription, have not yet been clarified. Previous studies have established that NOTCH1 signaling plays an important role in the Tcrg and Tcrd germline transcriptional regulation induced by enhancer activation, which is mediated through the recruitment of RUNX1 and MYB. In addition, interleukin-7 signaling has been shown to be required for Tcrg germline transcription, VγJγ rearrangement and γδ T-lymphocyte generation as well as for promoting T-cell survival. In this study, we discovered that interleukin-7 is required for the activation of enhancer-dependent Tcrd germline transcription during thymocyte development. These results indicate that the activation of both Tcrg and Tcrd enhancers during γδ T-cell development in the thymus depends on the same NOTCH1- and interleukin-7-mediated signaling pathways. Understanding the regulation of the Tcrd enhancer during thymocyte development might lead to a better understanding of the enhancer-dependent mechanisms involved in the genomic instability and chromosomal translocations that cause leukemia. [ABSTRACT FROM AUTHOR]