Type B thymomas in patients with myasthenia gravis display a distinctive pattern of αß TCR and IL-7 receptor α expression on CD4+CD8+ thymocytes.

Thymoma is closely associated with myasthenia gravis (MG). However, due to the heterogeneity of thymoma and the intricate pathogenesis of MG, it remains unclear why some patients with thymoma develop MG and others do not. In this study, we conducted a comparative phenotype analysis of thymocytes in type B thymomas in patients with MG (MG (+) thymomas) and without MG (MG (-) thymomas) via fluorescence-activated cell sorting (FACS). Our results show that the developmental stages defined by the expression of CD3, CD4, and CD8 were largely maintained in both MG (+) and MG (-) thymomas, with CD4+CD8+ cells constituting the majority of thymocytes in type B thymoma, and no significant difference between this cell population was observed in MG (+) and MG (-) thymomas.We discovered that CD4+CD8+ thymocytes in MG (+) thymomas expressed low levels of αß TCR and high levels of IL-7 receptor α (IL-7Rα), whereas in MG (-) thymomas, CD4+CD8+ thymocytes exhibited the opposite pattern of αß TCR and IL-7Rα expression. These results suggest that the positive and negative selection processes of CD4+CD8+ thymocytes might differ between MG (+) thymomas and MG (-) thymomas. The expression of the Helios transcription factor is induced during negative selection and marks a group of T cells that have undergone negative selection and are likely to be deleted due to strong TCR binding with self-peptides/MHC ligands. We observed that the percentage of Helios-positive CD4SP T cells was greater in MG (-) than in MG (+) thymomas. Thus, the differentially regulated selection process of CD4+CD8+ thymocytes, which involves TCR and IL-7/IL-7Rα signaling, is associated with the presence of MG in type B thymomas.

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vß21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

Local chicken breeds exhibit abundant TCR-V segments but similar repertoire diversity.

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 TCRß 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 Vß were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vß1, and Vß2 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.

Identification of apolipoprotein B-reactive CDR3 motifs allows tracking of atherosclerosis-related memory CD4+T cells in multiple donors.

Introduction: Atherosclerosis is a major pathological condition that underlies many cardiovascular diseases (CVDs). Its etiology involves breach of tolerance to self, leading to clonal expansion of autoreactive apolipoprotein B (APOB)-reactive CD4+T cells that correlates with clinical CVD. The T-cell receptor (TCR) sequences that mediate activation of APOB-specific CD4+T cells are unknown. Methods: In a previous study, we had profiled the hypervariable complementarity determining region 3 (CDR3) of CD4+T cells that respond to six immunodominant APOB epitopes in most donors. Here, we comprehensively analyze this dataset of 149,065 APOB-reactive and 199,211 non-reactive control CDR3s from six human leukocyte antigen-typed donors. Results: We identified 672 highly expanded (frequency threshold > 1.39E-03) clones that were significantly enriched in the APOB-reactive group as compared to the controls (log10 odds ratio ≥1, Fisher's test p < 0.01). Analysis of 114,755 naïve, 91,001 central memory (TCM) and 29,839 effector memory (TEM) CDR3 sequences from the same donors revealed that APOB+ clones can be traced to the complex repertoire of unenriched blood T cells. The fraction of APOB+ clones that overlapped with memory CDR3s ranged from 2.2% to 46% (average 16.4%). This was significantly higher than their overlap with the naïve pool, which ranged from 0.7% to 2% (average 1.36%). CDR3 motif analysis with the machine learning-based in-silico tool, GLIPHs (grouping of lymphocyte interactions by paratope hotspots), identified 532 APOB+ motifs. Analysis of naïve and memory CDR3 sequences with GLIPH revealed that ~40% (209 of 532) of these APOB+ motifs were enriched in the memory pool. Network analysis with Cytoscape revealed extensive sharing of the memory-affiliated APOB+ motifs across multiple donors. We identified six motifs that were present in TCM and TEM CDR3 sequences from >80% of the donors and were highly enriched in the APOB-reactive TCR repertoire. Discussion: The identified APOB-reactive expanded CD4+T cell clones and conserved motifs can be used to annotate and track human atherosclerosis-related autoreactive CD4+T cells and measure their clonal expansion.

Selection, engineering, and in vivo testing of a human leukocyte antigen-independent T-cell receptor recognizing human mesothelin.

BACKGROUND: Canonical α/ß T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/ß TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. METHODS: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. RESULTS: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. CONCLUSION: HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.

Immune checkpoint therapy responders display early clonal expansion of tumor infiltrating lymphocytes.

Immune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRß) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRß repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRß repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRß clonotypes was observed in responding tumours. Machine learning identified TCRß CDR3 signatures unique to each tumour model, and signatures associated with ICT response at various timepoints before or during ICT. Clonally expanded CD8+ T cells in responding tumours post ICT displayed effector T cell gene signatures and phenotype. An early burst of clonal expansion during ICT is associated with response, and we report unique dynamics in TCRß signatures associated with ICT response.

Structure-Based Rational and General Strategy for Stabilizing Single-Chain T-Cell Receptors to Enhance Affinity.

The T-cell receptor (TCR) is a crucial molecule in cellular immunity. The single-chain T-cell receptor (scTCR) is a potential format in TCR therapeutics because it eliminates the possibility of αß-TCR mispairing. However, its poor stability and solubility impede the in vitro study and manufacturing of therapeutic applications. In this study, some conserved structural motifs are identified in variable domains regardless of germlines and species. Theoretical analysis helps to identify those unfavored factors and leads to a general strategy for stabilizing scTCRs by substituting residues at exact IMGT positions with beneficial propensities on the consensus sequence of germlines. Several representative scTCRs are displayed to achieve stability optimization and retain comparable binding affinities with the corresponding αß-TCRs in the range of µM to pM. These results demonstrate that our strategies for scTCR engineering are capable of providing the affinity-enhanced and specificity-retained format, which are of great value in facilitating the development of TCR-related therapeutics.

Myeloid Cells and Sphingosine-1-Phosphate Are Required for TCRαß Intraepithelial Lymphocyte Recruitment to the Colon Epithelium.

Intraepithelial lymphocytes (IELs) are T cells important for the maintenance of barrier integrity in the intestine. Colon IELs are significantly reduced in both MyD88-deficient mice and those lacking an intact microbiota, suggesting that MyD88-mediated detection of bacterial products is important for the recruitment and/or retention of these cells. Here, using conditionally deficient MyD88 mice, we show that myeloid cells are the key mediators of TCRαß+ IEL recruitment to the colon. Upon exposure to luminal bacteria, myeloid cells produce sphingosine-1-phosphate (S1P) in a MyD88-dependent fashion. TCRαß+ IEL recruitment may be blocked using the S1P receptor antagonist FTY720, confirming the importance of S1P in the recruitment of TCRαß+ IELs to the colon epithelium. Finally, using the TNFΔARE/+ model of Crohn's-like bowel inflammation, we show that disruption of colon IEL recruitment through myeloid-specific MyD88 deficiency results in reduced pathology. Our results illustrate one mechanism for recruitment of a subset of IELs to the colon.

TCRαß-depleted hematopoietic stem cell transplant and third-party CD45RA+ depleted adoptive cell therapy for treatment of post-transplant parvovirus B19 aplastic crisis.

This is a case report of a 6-year-old girl with relapsed B cell acute lymphoblastic leukemia in which adoptive cell therapy was applied successfully to treat refractory human parvovirus (HPV) B19 infection. Allogenic chimeric antigen receptor (CAR) T-cell therapy (bispecific CD19/CD22) was bridged to hematopoietic stem cell transplantation (HSCT) using a haploidentical paternal donor. However, HPV B19 DNAemia progressed and transfusion-related graft versus host disease occurred. After finding a third-party related donor with a better HLA match, haploidentical HPV B19-seropositive CD45RA+ depleted cells (16.5 × 106/kg) were administered and paternal TCRαß+ depleted stem cell were retransplanted. The HPV B19 DNAemia became negative within 1 week and the reticulocyte, neutrophil, hemoglobin, and platelet counts gradually normalized. The patient remained stable during the 1-year outpatient follow-up period. Thus, our case report highlights that persistent B19 infection can lead to pancytopenia, aplastic crisis, and graft rejection and TCRαß+ depleted haplo-HSCT is an effective means of hematopoiesis recovery. CD45RO memory T-cell therapy is the key to treating and preventing the development of refractory severe HPV B19 infection.

A comparative analysis of TCR immune repertoire in COVID-19 patients.

The coronavirus disease 2019 (COVID-19) has merged as a global health threat since its outbreak in December 2019. Despite widespread recognition, there has been a paucity of studies focusing on the T cell receptor (TCR) bias in adaptive immunity induced by SARS-CoV-2. This research conducted a comparative analysis of the TCR immune repertoire to identify notable αß TCR bias sequences associated with the SARS-CoV-2 virus antigen. The present study encompassed 73 symptomatic COVID-19 patients, categorized as moderate/mild or severe/critical, along with 9 healthy controls. Our findings revealed specific TCR chains prominently utilized by moderate and severe patients, identified as TRAV30-J34-TRBV3-1-J2-7 and TRAV12-3-J6-TRBV28-J1-1, respectively. Additionally, our research explored critical TCR preferences in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients at various disease stages. Indeed, monitoring the dynamics of immune repertoire changes in COVID-19 patients could serve as a crucial biomarker for predicting disease progression and recovery. Furthermore, the study explored TCR bias in both peripheral blood mononuclear cells (PBMCs) and BALF. The most common αß VJ pair observed in BALF was TRAV12-3-J18-TRBV7-6-J2-7. In addition, a comparative analysis with the VDJdb database indicated that the HLA-A*02:01 allele exhibited the widest distribution and highest frequency in COVID-19 patients across different periods. This comprehensive examination provided a global characterization of the TCR immune repertoire in COVID-19 patients, contributing significantly to our understanding of TCR bias induced by SARS-CoV-2.

TCR CDR3-CMV Antigen Chemical Complementaries Are Associated With a Worse Outcome for Renal Cell Carcinoma.

BACKGROUND/AIM: Due to still unresolved questions regarding viruses as either a primary cause or a comorbidity in cancer, we examined a potential immune response to cytomegalovirus (CMV) in the renal cell carcinoma (RCC) setting using genomics and bioinformatics approaches. MATERIALS AND METHODS: Specifically, we assessed chemical complementarity scores (CSs) for solid tissue normal resident, T-cell receptor (TCR) complementarity determining region 3 (CDR3s) and CMV antigens and determined whether higher or lower CS groups were associated with a higher or lower survival probability. RESULTS: This was indeed the case, with all such analyses consistently indicating a lower overall and progression-free survival for the cases representing the higher TCR CDR3-CMV antigen chemical CSs. This basic result was obtained for two separate RCC datasets and multiple CMV antigens. CONCLUSION: The results raise the question, to what extent a systemic CMV infection may represent an important co-morbidity for RCC.

RORγt up-regulates RAG gene expression in DP thymocytes to expand the Tcra repertoire.

Recombination activating gene (RAG) expression increases as thymocytes transition from the CD4-CD8- double-negative (DN) to the CD4+CD8+ double-positive (DP) stage, but the physiological importance and mechanism of transcriptional up-regulation are unknown. Here, we show that a DP-specific component of the recombination activating genes antisilencer (DPASE) provokes elevated RAG expression in DP thymocytes. Mouse DP thymocytes lacking the DPASE display RAG expression equivalent to that in DN thymocytes, but this supports only a partial Tcra repertoire due to inefficient secondary Vα-Jα rearrangement. These data indicate that RAG up-regulation is required for a replete Tcra repertoire and that RAG expression is fine-tuned during lymphocyte development to meet the requirements of distinct antigen receptor loci. We further show that transcription factor RORγt directs RAG up-regulation in DP thymocytes by binding to the DPASE and that RORγt influences the Tcra repertoire by binding to the Tcra enhancer. These data, together with prior work showing RORγt to control Tcra rearrangement by regulating DP thymocyte proliferation and survival, reveal RORγt to orchestrate multiple pathways that support formation of the Tcra repertoire.

Unraveling the chicken T cell repertoire with enhanced genome annotation.

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in ß and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/ß/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the ß and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

Identification, expression and molecular polymorphism of T-cell receptors α and ß from the glacial relict Hucho bleekeri.

The T-cell receptor (TCR) is a specific molecule on the surface of all T cells that mediates cellular adaptive immune responses to antigens. Hucho bleekeri is a critically endangered species and is regarded as a glacial relict that has the lowest-latitude distribution compared with any Eurasian salmonid. In the present study, two TCR genes, namely, TCR α and ß, were identified and characterized in H. bleekeri. Both TCR α and TCR ß have typical TCR structures, including the IgV domain, IgC domain, connecting peptide, transmembrane and cytoplasmic domains. The two TCR genes were constitutionally expressed in various tissues, with the highest expression found in the spleen for TCR α and in the trunk kidney for TCR ß. Challenge of H. bleekeri with LPS or poly(I:C) resulted in significant upregulation of both TCR α and ß expression in headkidney and spleen primary cells, indicating their potential roles in the immune response. Molecular polymorphism analysis of the whole ORF regions of TCR α and ß in different individuals revealed high diversity of IgV domains of these two genes, especially in complementarity-determining region (CDR) 3. The ratio of nonsynonymous substitution occurred at a significantly higher frequency than synonymous substitution in the CDR of TCR α and ß, demonstrating the existence of positive selection. The results obtained in the present study enhance our understanding of TCR roles in regulating immune mechanisms and provide new information for the study of TCR lineage diversity in fish.

TRBC1-targeting antibody-drug conjugates for the treatment of T cell cancers.

Antibody and chimeric antigen receptor (CAR) T cell-mediated targeted therapies have improved survival in patients with solid and haematologic malignancies1-9. Adults with T cell leukaemias and lymphomas, collectively called T cell cancers, have short survival10,11 and lack such targeted therapies. Thus, T cell cancers particularly warrant the development of CAR T cells and antibodies to improve patient outcomes. Preclinical studies showed that targeting T cell receptor ß-chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity12, making TRBC1 an attractive target to treat T cell cancers. However, the first-in-human clinical trial of anti-TRBC1 CAR T cells reported a low response rate and unexplained loss of anti-TRBC1 CAR T cells13,14. Here we demonstrate that CAR T cells are lost due to killing by the patient's normal T cells, reducing their efficacy. To circumvent this issue, we developed an antibody-drug conjugate that could kill TRBC1+ cancer cells in vitro and cure human T cell cancers in mouse models. The anti-TRBC1 antibody-drug conjugate may provide an optimal format for TRBC1 targeting and produce superior responses in patients with T cell cancers.

Homozygous HLA-DQB1*06:02 combined with T-cell receptor alpha polymorphism results in narcolepsy onset - A familial case report.

Narcolepsy is a life-long neurological disorder with well-established genetic risk factors. Human leukocyte antigen-DQB1*06:02 remains the strongest genetic predeterminant; however, polymorphisms in genes encoding the T-cell receptor alpha chain are also strongly linked. This case report shows the inheritance pathway of these genetic markers contributing to narcolepsy onset in a 17-year-old female.

Pathways and mechanisms of CD4+CD8αα+ intraepithelial T cell development.

The mammalian small intestine epithelium harbors a peculiar population of CD4+CD8αα+ T cells that are derived from mature CD4+ T cells through reprogramming of lineage-specific transcription factors. CD4+CD8αα+ T cells occupy a unique niche in T cell biology because they exhibit mixed phenotypes and functional characteristics of both CD4+ helper and CD8+ cytotoxic T cells. The molecular pathways driving their generation are not fully mapped. However, recent studies demonstrate the unique role of the commensal gut microbiota as well as distinct cytokine and chemokine requirements in the differentiation and survival of these cells. We review the established and newly identified factors involved in the generation of CD4+CD8αα+ intraepithelial lymphocytes (IELs) and place them in the context of the molecular machinery that drives their phenotypic and functional differentiation.

Virus-Associated CD8+ T-Cells Are Not Activated Through Antigen-Mediated Interaction Inside Atherosclerotic Lesions.

INTRODUCTION: Viral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8+ T-cells in the atherosclerotic lesion. METHODS: The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8+ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-ß sequencing and single-cell T-cell receptor (α and ß) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8+ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined. RESULTS: Virus-associated CD8+ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P=0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8+ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8+ T-cells were phenotypically highly similar to other CD8+ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8+ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion. CONCLUSIONS: This study suggests that virus-specific CD8+ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms.

The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αß T cell counts at birth persisted over time, with normal memory αß and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αß T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αß T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αß T cells, autoimmune conditions were more frequent in these patients compared with the general population.