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T cell engaged bispecific antibody (TCB) is an effective immunotherapy for cancer treatment. Through co-targeting CD3 and tumor-associated antigen (TAA), TCB can redirect CD3+ T cells to eliminate tumor cells regardless of the specificity of T cell receptor. Tissue factor (TF) is a TAA that involved in tumor progression. Here, we designed and characterized a novel TCB targeting TF (TF-TCB) for the treatment of TF-positive tumors. In vitro, robust T cell activation, tumor cell lysis and T cell proliferation were induced by TF-TCB. The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB, and was related to TF expression level of tumor cells. In vivo, in both tumor cell/human peripheral blood mononuclear cells (PBMC) co-grafting model and established tumor models with poor T cell infiltration, tumor growth was strongly inhibited by TF-TCB. T cell infiltration into tumors was induced during the treatment. Furthermore, efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors. For the first time, our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.

Atopic dermatitis (AD) and psoriasis represent two of the most common inflammatory skin diseases in developed countries. A hallmark of both diseases is T cell infiltration into the skin. However, it is still not clarified to what extent these infiltrating T cells are antigen-specific skin-homing T cells or unspecific heterogeneous bystander cells. To elucidate this, T cells from lesional skin and from blood of 10 AD and 11 psoriasis patients were compared by receptor (TCR) sequencing. Therefore, peripheral blood mononuclear cells (PBMC) were cell-sorted according to expression of the cutaneous leukocyte antigen (CLA) into skin-homing (CLA+) and non-skin-homing (CLA-) subfractions. Aeroallergen-specific T cell lines were grown from AD patients’ s PBMC in parallel. Intra-individual comparison of TCRB CDR3 regions revealed that clonally expanded T cells in skin lesions of both AD and psoriasis patients corresponded to skin-homing circulating T cells. However, in psoriasis patients, these T cell clones were also detectable to a larger extent among CLA- circulating T cells. Up to 28% of infiltrating cells were identified as allergen-specific by overlapping TCR sequences. Our data shows that in line with the systemic nature of psoriasis, T cells infiltrating psoriatic skin lesions do not exclusively home to the skin and are therefore not specific to antigens that are exclusively encountered at the skin. T cells driving AD skin inflammation appear to home nearly exclusively to the skin and are, to a certain extent, specific to aeroallergens. One Sentence Summary In contrast to psoriasis, T cells driving atopic dermatitis are predominantly skin-homing and are to a certain extent allergen-specific Key Message / Significance Statement T cells that encounter their cognate antigen proliferate clonally and share the same T cell receptor (TCR). Here we describe that clonally expanded T cells from lesional skin in patients with psoriasis and atopic dermatitis (AD) can also be detected within the circulation. While in AD these are mostly homing to the skin, psoriasis-driving T cells do not appear to be exclusively skin-directed. The fact that several co-morbidities are associated with psoriasis matches this observation. In contrast, AD is commonly accompanied by allergic sensitizations. Here we demonstrate that allergen-specific T cells do indeed infiltrate lesional skin and allow a concrete estimate of their frequency.

T cells must be activated and become effectors first before executing allograft rejection, a process that is regulated by diverse signals and transcription factors. In the present study, we studied the basic leucine zipper ATF-like transcription factor (BATF) family members in regulating T cell activities in a heart transplant model and found that mice deficient for both BATF and BATF3 (Batf(−/−)Batf3(−/−) mice) spontaneously accept the heart allografts long-term without tolerizing therapies. Similarly, adoptive transfer of wild type T cells into Rag1(−/−) hosts induced prompt rejection of heart and skin allografts, whereas the Batf(−/−)Batf3(−/−) T cells failed to do so. Analyses of graft-infiltrating cells showed that Batf(−/−)Batf3(−/−) T cells infiltrate the graft but fail to acquire an effector phenotype (CD44(high)KLRG1(+)). Co-transfer experiments in a TCR transgenic TEa model revealed that the Batf(−/−)Batf3(−/−) T cells fail to expand in vivo, retain a quiescent phenotype (CD62L(+)CD127(+)), and unable to produce effector cytokines to alloantigen stimulation, which contrasted sharply to that of wild type T cells. Together, our data demonstrate that the BATF and BATF3 are critical regulators of T effector functions, thus making them attractive targets for therapeutic interventions in transplant settings.

Engineered T cell therapies, mainly chimeric antigen receptor (CAR)-T and T cell receptor (TCR)-T, have become the new frontier of cancer treatment. CAR-T and TCR-T therapies differ in many aspects, including cell persistence and toxicity, leading to different therapeutic outcomes. Both TCR and CAR recognize antigens and trigger T cell mediated antitumor response, but they have distinct molecular structures and signaling properties. TCR represents one of the most complex receptors, while CAR is a single-chain chimera integrating modules from multiple immune receptors. Understanding the mechanisms underlying the strengths and limitations of both systems can pave the way for the development of next-generation T cell therapy. This review synthesizes recent findings on TCR and CAR signaling and highlights the potential strategies of T cell engineering by signaling refinement.

T cells engineered to express transgenes such as chimeric antigen receptors (CAR) or modified T cell receptors (TCR) represent a new pillar of cancer therapy. Use of CRISPR/Cas gene-editing tools now allows even stronger and more precise control over the fate and function of engineered T cell therapies, including multiplex genome editing to facilitate use of off-the-shelf allogeneic T cells and novel approaches which have the potential to overcome some of the limitations of canonical Cas9-mediated DNA cleavage. This review summarizes the CRISPR/Cas techniques that have been used in preclinical research and outlines those that currently being tested in clinical trials.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-β, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-β. Truncating the intracellular signaling domain from TGF-β receptor (TGFβR) II produces a dominant-negative receptor (dnTGFβRII) that dimerizes with endogenous TGFβRI to form a receptor that can bind TGF-β but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02–restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157–165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254–262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-β inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFβRII (e.g., GSK3845097). TGF-β isoforms and a panel of TGF-β–associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-β–positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non–small cell lung cancer setting. Coexpression of dnTGFβRII may therefore improve the efficacy of TCR-transduced T cells.

Contains fulltext : 248576.pdf (Publisher’s version ) (Open Access) Single-cell antigen-receptor gene amplification and sequencing platforms have been used to characterize T cell receptor (TCR) repertoires but typically fail to generate paired full-length gene products for direct expression cloning and do not enable linking this data to cell phenotype information. To overcome these limitations, we established a high-throughput platform for the quantitative and qualitative analysis of human TCR repertoires that provides insights into the clonal and functional composition of human CD4(+) and CD8(+) αβ T cells at the molecular and cellular level. The strategy is a powerful tool to qualitatively assess differences between antigen receptors of phenotypically defined αβ T cell subsets, e.g. in immune responses to cancer, vaccination, or infection, and in autoimmune diseases.

Objectives Antinuclear antibody (ANA) positive Juvenile Idiopathic Arthritis (JIA) is characterized by synovial B cell hyperactivity, but the precise role of CD4+ T cells in promoting local B cell activation is unknown. The objective of this study is to unravel the phenotype and function of synovial CD4+ T cells that promote the aberrant B cell activation in JIA. Methods Flow cytometric analysis was performed to compare the phenotype and cytokine pattern of synovial fluid (SF) PD-1hi CD4+ T cells with tonsil TFH cells. TCRVB next generation sequencing was applied to analyze T cell subsets for signs of clonal expansion. The functional impact of these T cell subsets on B cells was dissected in in vitro co-cultures. Results Multidimensional flow-cytometric analysis revealed the expansion of IL-21 and IFN-γ co-expressing PD-1hi CXCR5- HLA-DR+ CD4+ T cells that accumulate in the joints of ANA+ JIA patients. These T cells exhibited signs of clonal expansion with restricted TCR clonotypes. Phenotypically they resembled peripheral T helper (TPH ) cells with an extrafollicular chemokine receptor pattern and high T-bet and Blimp-1 but low Bcl-6 expression. SF TPH cells particularly skewed B cell differentiation towards a CD21lo/- CD11c+ phenotype by provision of IL-21 and IFN-γ in vitro and correlated with the appearance of SF CD21lo/- CD11c+ CD27- IgM- double-negative B cells (BDN ) in situ. Conclusion Clonally expanded CD4+ TPH cells accumulate in the joints of ANA+ JIA patients and particularly promote CD21lo/- CD11c+ BDN cell differentiation The expansion of TPH and BDN cells might reflect the autoimmune response present in the joints of ANA+ JIA patients.

Mature CD4(+) and CD8(+) T cells constitutively experience weak T cell receptor (TCR) stimulation in response to self-antigens, termed tonic (or basal) signaling. How tonic TCR signal strength impacts T cell responses to foreign antigens is an active area of investigation. Such studies rely on surrogate markers of tonic signal strength, including CD5, Ly6C, and transgenic reporters of Nr4a genes. Recent research indicates that strong tonic TCR signal strength influences basal T cell metabolism, effector differentiation, and TCR signal transduction. T cells that experience the strongest tonic TCR signaling exhibit features of T cell activation and negative regulation. These data suggest a model whereby adaptation to tonic signaling has lasting effects that alter T cell activation and differentiation.

In the past decades, immunotherapies against cancers made impressive progress. Immunotherapy includes a broad range of interventions that can be separated into two major groups: cell-based immunotherapies, such as adoptive T cell therapies and stem cell therapies, and immunomodulatory molecular therapies such as checkpoint inhibitors and cytokine therapies. Genetic engineering techniques that transduce T cells with a cancer-antigen-specific T cell receptor or chimeric antigen receptor have expanded to other cell types, and further modulation of the cells to enhance cancer targeting properties has been explored. Because cell-based immunotherapies rely on cells migrating to target organs or tissues, there is a growing interest in imaging technologies that non-invasively monitor transferred cells in vivo. Here, we review whole-body imaging methods to assess cell-based immunotherapy using a variety of examples. Following a review of preclinically used cell tracking technologies, we consider the status of their clinical translation.

Purpose: Gliomas are intrinsic brain tumors with a high degree of constitutive and acquired resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Glioma subtypes are recognized by characteristic mutations. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy. Experimental Design: Using peptide-based ELISpot assays, we screened for potential recurrent glioma neoepitopes in MHC-humanized mice. Following vaccination, droplet-based single-cell T-cell receptor (TCR) sequencing from established T-cell lines was applied for neoepitope-specific TCR discovery. Efficacy of intraventricular TCR-transgenic T-cell therapy was assessed in a newly developed glioma model in MHC-humanized mice induced by CRISPR-based delivery of tumor suppressor–targeting guide RNAs. Results: We identify recurrent capicua transcriptional repressor (CIC) inactivating hotspot mutations at position 215 CICR215W/Q as immunogenic MHC class II (MHCII)-restricted neoepitopes. Vaccination of MHC-humanized mice resulted in the generation of robust MHCII-restricted mutation-specific T-cell responses against CICR215W/Q. Adoptive intraventricular transfer of CICR215W-specific TCR-transgenic T cells exert antitumor responses against CICR215W-expressing syngeneic gliomas. Conclusions: The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of human leukocyte antigen (HLA)-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T-cell adoptive therapy.

Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4+ T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4+ T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4+ T cell population is unknown. We sought to characterize the relationship between activated CD4+ T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4+ T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4+ T cell reconstitution and in the change in activated CD4+ TCR repertoire diversity following ART. Decreases in activated CD4+ TCR repertoire diversity following ART were predictive of the degree of CD4+ T cell reconstitution. The association of decreased activated CD4+ TCR repertoire diversity and improved CD4+ T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4+ clones. These results provide insight into the dynamic relationship between activated CD4+ TCR diversity and CD4+ T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

Key Points Diagnosis of TCL often relies on PCR-based TCR clonality testing, which displays low specificity and requires high-quality DNA.Targeted NGS and T-cell clonality assessment are complementary techniques that should be applied in hard front-line diagnosis., Immunomorphological diagnosis of T-cell lymphoma (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays detect only 80% of TCL, and clonal lymphocyte populations may also appear in nonneoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques’ performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 nonneoplastic T-cell infiltrates were divided into 2 cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7%-45.5%), whereas no differences were observed in terms of sensitivity (95.1%-97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

Circulating CD4+ T cells are dysfunctional in Alzheimer's disease (AD), however, the underlying molecular mechanisms are not clear. In this study, we demonstrate that CD4+ T cells from AD patients and 5xFAD transgenic mice exhibit elevated levels of β-secretase 1 (BACE1). Overexpression of BACE1 in CD4+ T cells potentiated CD4+ T-cell activation and T-cell-dependent immune responses. Mechanistically, BACE1 modulates prostaglandin E2 (PGE2) synthetase-microsomal prostaglandin E synthase 2 (mPGES2)-to promote mPGES2 maturation and PGE2 production, which increases T-cell receptor (TCR) signalling. Moreover, administration of peripheral PGE2 signalling antagonists partially ameliorates CD4+ T cell overactivation and AD pathology in 5xFAD mice. Overall, our results reveal a potential role for BACE1 in mediating CD4+ T-cell dysfunction in AD.

The use of immunotherapy in cancer treatment over the past decade has resulted in significant advances and improvements in cancer patients survival with the use of checkpoint inhibitors. Nevertheless, only a fraction of solid tumors responds to this immunotherapy modality. Another modality of immunotherapy consists of employing cell-based therapy as an adoptive therapeutic modality. That involves distinct modalities of cellular therapies such as CAR T cells (chimeric antigen receptor T cell), TILs (tumor-infiltrating lymphocytes), and TCR T cells. Those treatments have proven effective in hematologic tumors and could have an impact in tumors that do not respond to checkpoint inhibitors. This review aims to outline the rationale, operation, clinical applicability, and results of adoptive cell therapy for patients with solid tumors.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.

CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αβ sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.

Advancement in our understanding of immune cell recognition and emerging cellular engineering technologies during the last decades made active manipulation of the T cell response possible. Synthetic immunology is providing us with an expanding set of composite receptor molecules capable to reprogram immune cell function in a predefined fashion. Since the first prototypes in the late 1980s, the design of chimeric antigen receptors (CARs; T-bodies, immunoreceptors), has followed a clear line of stepwise improvements from antigen-redirected targeting to designed "living factories" delivering transgenic products on demand. Building on basic research and creative clinical exploration, CAR T cell therapy has been achieving spectacular success in the treatment of hematologic malignancies, now beginning to improve the outcome of cancer patients. In this study, we briefly review the history of CARs and outline how the progress in the basic understanding of T cell recognition and of cell engineering technologies made novel therapies possible.

Protein arginine methyltransferase (PRMT) 5 is the type 2 methyltransferase catalyzing symmetric dimethylation of arginine. PRMT5 inhibition or deletion in CD4 Th cells reduces TCR engagement-induced IL-2 production and Th cell expansion and confers protection against experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. However, the mechanisms by which PRMT5 modulates Th cell proliferation are still not completely understood, and neither are the methylation targets in T cells. In this manuscript, we uncover the role of PRMT5 on alternative splicing in activated mouse T cells and identify several targets of PRMT5 symmetric dimethylation involved in splicing. In addition, we find a possible link between PRMT5-mediated alternative splicing of transient receptor potential cation channel subfamily M member 4 (Trpm4) and TCR/NFAT signaling/IL-2 production. This understanding may guide development of drugs targeting these processes to benefit patients with T cell–mediated diseases.

Though TCRs have been subject to limited engineering in the context of therapeutic design and optimization, they are used largely as found in nature. On the other hand, CARs are artificial, composed of different segments of proteins that function in the immune system. This characteristic raises the possibility of altered response to immune regulatory stimuli. Here we describe a large-scale, systematic comparison of CARs and TCRs across 5 different pMHC targets, with a total of 19 constructs examined in vitro. These functional measurements include CAR- and TCR-mediated activation, proliferation, and cytotoxicity in both acute and chronic settings. Surprisingly, we find no consistent difference between CARs and TCRs as receptor classes with respect to their relative sensitivity to major regulators of T cell activation: PD-L1, CD80/86 and IL-2. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved and compared head to head in functional tests, CARs appear remarkably similar to TCRs with respect to immune modulation.

BACKGROUND/PURPOSE Accurate assessment of malignant T-cell clones in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) is crucial for diagnosis, treatment, and monitoring disease. Although multiple approaches to quantitate malignant T-cell clones have been reported, a cost-effective assay with broad coverage is not available. We report a NanoString-nCounter-Technology-based direct TCR expression assay (DTEA) that was previously developed to quantify both TCR-Vα and TCR-Vβ usages after adoptive immunotherapy. This study was performed to test the effectiveness of DTEA in assessing malignant T-cell clones in L-CTCL patients. METHODS Total RNAs extracted from peripheral blood mononuclear cells of patients before starting extracorporeal photopheresis (ECP) (n = 15) and during therapy at 3 months and 6 months (n = 12) were used for DTEA, with customized probes for 45 TCR-Vα and 46 TCR-Vβ family members. RESULTS At baseline, DTEA detected TCR-Vβ clones in all 15 patients (100%) compared to flow cytometry that detected TCR-Vβ clones in 9 of 13 patients (69.2%). In addition to predominant TCR-Vβ clones, DTEA also detected additional TCR-Vβ clones in 8 of 15 patients (53.3%). Furthermore, DTEA simultaneously identified clonal TCR-Vα usages, which allowed us to pair TCR-Vα and TCRVβ usages by malignant T-cells and identify diversified clonotypes. Changes in the relative frequencies of clonal TCR-Vβ and TCRVα usages over therapy were consistent with patients' clinical responses. CONCLUSIONS Our results indicate that DTEA can effectively assess malignant T-cell clones by detecting clonal TCR-Vα and TCR-Vβ usages. By providing a global view of TCR repertoires, DTEA may also help us understand the origin(s) of malignant T-cells and pathogenesis of CTCL.

Neoantigens play a key role in the recognition of tumour cells by T cells; however, only a small proportion of neoantigens truly elicit T-cell responses, and few clues exist as to which neoantigens are recognized by which T-cell receptors (TCRs). We built a transfer learning-based model named the pMHC–TCR binding prediction network (pMTnet) to predict TCR binding specificities of the neoantigens—and T cell antigens in general—presented by class I major histocompatibility complexes. pMTnet was comprehensively validated by a series of analyses and exhibited great advances over previous works. By applying pMTnet to human tumour genomics data, we discovered that neoantigens were generally more immunogenic than self-antigens, but human endogenous retrovirus E (a special type of self-antigen that is reactivated in kidney cancer) is more immunogenic than neoantigens. We further discovered that patients with more clonally expanded T cells that exhibit better affinity against truncal rather than subclonal neoantigens had more favourable prognosis and treatment response to immunotherapy in melanoma and lung cancer but not in kidney cancer. Predicting TCR–neoantigen/antigen pairing is one of the most daunting challenges in modern immunology; however, we achieved an accurate prediction of the pairing using only the TCR sequence (CDR3β), antigen sequence and class I major histocompatibility complex allele, and our work revealed unique insights into the interactions between TCRs and major histocompatibility complexes in human tumours, using pMTnet as a discovery tool. T-cell immunity is driven by the interaction between peptides presented by major histocompatibility complexes (pMHCs) and T-cell receptors (TCRs). Only a small proportion of neoantigens elicit T-cell responses, and it is not clear which neoantigens are recognized by which TCRs. The authors develop a transfer learning model to predict TCR binding specificity to class-I pMHCs.

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential. H9T, an engineered IL-2 partial agonist, promotes the expansion of T cells while maintaining a stem-cell-like state, leading to improved efficacy of adoptive cell therapy in mouse models of melanoma and acute lymphoblastic leukaemia.

Both B cells and T cells are involved in an effective immune response to SARS‐CoV‐2, the disease‐causing virus of COVID‐19. While B cells—with the indispensable help of CD4+ T cells—are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti‐SARS‐CoV‐2 immunity. In this report, we provide insights into the characteristics of individual HLA‐A*02:01‐ and HLA‐A*24:02‐restricted SARS‐CoV‐2‐reactive TCRs, isolated from convalescent COVID‐19 patients. We observed that SARS‐CoV‐2‐reactive T‐cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re‐expression. The SARS‐CoV‐2‐reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS‐CoV‐2‐reactive TCRs conferred powerful T‐cell effector function to primary CD8+ T cells as evident by a robust anti‐SARS‐CoV‐2 IFN‐γ response and in vitro cytotoxicity. We also provide an example of a long‐lasting anti‐SARS‐CoV‐2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti‐SARS‐CoV‐2 T‐cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS‐CoV‐2., T cell receptors (TCRs) directed against previously identified SARS‐CoV‐2 target epitopes were isolated from convalescent COVID‐19 patients by single‐cell sequencing. Upon ectopic re‐expression, these SARS‐CoV‐2 TCRs conferred potent effector functions such as cytotoxicity and Interferon‐gamma production.

Background aims T cells engineered with synthetic receptors have delivered powerful therapeutic results for patients with relapsed/refractory hematologic malignancies. The authors have recently described the T-cell antigen coupler (TAC) receptor, which co-opts the endogenous T-cell receptor (TCR) and activates engineered T cells in an HLA-independent manner. Here the authors describe the evolution of a next-generation TAC receptor with a focus on developing a TAC-engineered T cell for multiple myeloma. Methods To optimize the TAC scaffold, the authors employed a bona fide antigen-binding domain derived from the B-cell maturation antigen-specific monoclonal antibody C11D5.3, which has been used successfully in the clinic. The authors first tested humanized versions of the UCHT1 domain, which is used by the TAC to co-opt the TCR. The authors further discovered that the signal peptide affected surface expression of the TAC receptor. Higher density of the TAC receptor enhanced target binding in vitro, which translated into higher levels of Lck at the immunological synapse and stronger proliferation when only receptor–ligand interactions were present. Results The authors observed that the humanized UCHT1 improved surface expression and in vivo efficacy. Using TAC T cells derived from both healthy donors and multiple myeloma patients, the authors determined that despite the influence of receptor density on early activation events and effector function, receptor density did not impact late effector functions in vitro, nor did the receptor density affect in vivo efficacy. Conclusions The modifications to the TAC scaffold described herein represent an important step in the evolution of this technology, which tolerates a range of expression levels without impacting therapeutic efficacy.

Activation and expansion of drug reactive T cells are key features in drug hypersensitivity reactions. Drugs may interact directly with immune receptors such as the human leukocyte antigens (HLA) or the T-cell receptors (TCR) itself, the pharmacological interaction [p-i] concept. To analyze whether the drug sulfamethoxazole (SMX) interacts directly with the TCR and thereby contributing to signaling and T cell activation, we analyze two SMX specific T cell clones (TCC “1.3”and “H13”). Proliferation to SMX and 11 related sulfanilamides, Ca++ influx in drug stimulated T-cells and the inhibitory effect of non-reactive sulfanilamides on SMX stimulation were analyzed. In silico docking of SMX and related sulfanilamide to the TCR were used to identify possible drug binding sites, and correlated to in vitro data to find the correct docking. In Ca++ influx assays, reactions occurred as early as 14 sec after adding SMX to TCC and APC. The broadly reactive clone (“H13”) was stimulated by 5 additional sulfanilamide, while one TCC (“1.3”) was reactive exclusively with SMX but not other sulfanilamides. Competition experiments with sulfanilamide inhibited SMX induced Ca++ influx and proliferation of the TCC1.3 ina dose dependent way. Docking experiments with SMX and related sulfanilamides confirmed and explained the in vitro data as docking localized binding sites for SMX and the 5 stimulating sulfanilamides on the CDR2β domain of the clone H13, while the 6 non-stimulatory SA failed to bind. In TCC 1.3, SMX could be docked on the CDR3α of the TCR. The other, non-stimulatory but inhibitory SA could also be docked to the same site. The combined analysis of in vitro and in silico data show that sulfanilamide can bind directly to TCRs. It shows that TCR, like other receptors, appear to be reamenable to manipulations by small molecules.

The mechanism of antigen-specific regulatory T cell (Treg) induction is not yet fully understood. Curcumin has an immune regulatory function. This study aims to induce antigen-specific Tregs by employing extracellular vesicles (EVs) that carry two types of T cell activators. Two types of T cell activators, ovalbumin (OVA)/major histocompatibility complex-II (MHC-II) and tetramethylcurcumin (FLLL31) (a curcumin analog) were carried by dendritic cell-derived extracellular vesicles, designated OFexo. A murine model of allergic rhinitis (AR) was developed with OVA as the specific antigen. AR mice were treated with a nasal instillation containing OFexo. We observed that OFexo recognized antigen-specific T cell receptors (TCR) on CD4+ T cells and enhanced Il10 gene transcription in CD4+ T cells. Administration of the OFexo-containing nasal instillation induced antigen-specific type 1 Tregs (Tr1 cells) in the mouse airway tissues. OFexo-induced Tr1 cells showed immune suppressive functions on CD4+ T cell proliferation. Administration of OFexo efficiently alleviated experimental AR in mice. In conclusion, OFexo can induce antigen-specific Tr1 cells that can efficiently alleviate experimental AR. The results suggest that OFexo has the translational potential to be employed for the treatment of AR or other allergic disorders.

The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.

The role of the microbiota in the development and function of γδ T cells-a T cell subset characterized by a T cell receptor composed of one γ-chain and one δ-chain-has been investigated in multiple organs in mice and humans. Interactions between the microbiota and γδ T cells affect both tissue homeostasis and disease pathologies. Notably, microbiota-induced interleukin-17 (IL-17)-producing-γδ T cells can mediate a range of immunological processes, from metabolic disorders to neuroinflammation via the gut-brain axis. However, the bidirectional interactions between γδ T cells and the microbiota have not been fully determined. In this Perspective, we dissect the roles of microbiota in modulating γδ T cell development and function, and evaluate the evidence for γδ T cell selection of commensal communities. We also discuss the potential implications of these cells in health and disease and the major open questions and research avenues in the field.

A missense mutation (R620W) of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), which encodes lymphoid-tyrosine phosphatase (LYP), confers genetic risk for multiple autoimmune diseases including type 1 diabetes. LYP has been putatively demonstrated to attenuate proximal T and BCR signaling. However, limited data exist regarding PTPN22 expression within primary T cell subsets and the impact of the type 1 diabetes risk variant on human T cell activity. In this study, we demonstrate endogenous PTPN22 is differentially expressed and dynamically controlled following activation. From control subjects homozygous for the nonrisk allele, we observed 2.1- (p < 0.05) and 3.6-fold (p < 0.001) more PTPN22 transcripts in resting CD4+ memory and regulatory T cells (Tregs), respectively, over naive CD4+ T cells, with expression peaking 24 h postactivation. When LYP was overexpressed in conventional CD4+ T cells, TCR signaling and activation were blunted by LYP-620R (p < 0.001) but only modestly affected by the LYP-620W risk variant versus mock-transfected control, with similar results observed in Tregs. LYP overexpression only impacted proliferation following activation by APCs but not anti-CD3– and anti-CD28–coated microbeads, suggesting LYP modulation of pathways other than TCR. Notably, proliferation was significantly lower with LYP-620R than with LYP-620W overexpression in conventional CD4+ T cells but was similar in Treg. These data indicate that the LYP-620W variant is hypomorphic in the context of human CD4+ T cell activation and may have important implications for therapies seeking to restore immunological tolerance in autoimmune disorders.

Gamma-delta (γδ) T cells express T cell receptors (TCR) that are preconfigured to recognize signs of pathogen infection. In primates, γδ T cells expressing the Vγ9Vδ2 TCR innately recognize (E)-4-hydroxy-3-methyl-but- 2-enyl pyrophosphate (HMBPP), a product of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway in bacteria that is presented in infected cells via interaction with members of the B7 family of costimulatory molecules butyrophilin (BTN) 3A1 and BTN2A1. In humans, Listeria monocytogenes (Lm) vaccine platforms have the potential to generate potent Vγ9Vδ2 T cell recognition. To evaluate the activation of Vγ9Vδ2 T cells by Lm-infected human monocyte-derived dendritic cells (Mo-DC) we engineered Lm strains that lack components of the MEP pathway. Direct infection of Mo-DC with these bacteria were unchanged in their ability to activate CD107a expression in Vγ9Vδ2 T cells despite an inability to synthesize HMBPP. Importantly, functional BTN3A1 was essential for this activation. Unexpectedly, we found that cytoplasmic entry of Lm into human dendritic cells resulted in upregulation of cholesterol metabolism in these cells, and the effect of pathway regulatory drugs suggest this occurs via increased synthesis of the alternative endogenous Vγ9Vδ2 ligand isoprenyl pyrophosphate (IPP) and/or its isomer dimethylallyl pyrophosphate (DMAPP). Thus, following direct infection, host pathways regulated by cytoplasmic entry of Lm can trigger Vγ9Vδ2 T cell recognition of infected cells without production of the unique bacterial ligand HMBPP.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1–3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide–human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire. The authors use single-cell profiling and T cell receptor specificity screening to show how tumour antigen recognition shapes the phenotypes of CD8+ T cells and antitumour immune responses.

The unconventional T cell compartment encompasses a variety of cell subsets that straddle the line between innate and adaptive immunity, often reside at mucosal surfaces and can recognize a wide range of non-polymorphic ligands. Recent advances have highlighted the role of unconventional T cells in tissue homeostasis and disease. In this Review, we recast unconventional T cell subsets according to the class of ligand that they recognize; their expression of semi-invariant or diverse T cell receptors; the structural features that underlie ligand recognition; their acquisition of effector functions in the thymus or periphery; and their distinct functional properties. Unconventional T cells follow specific selection rules and are poised to recognize self or evolutionarily conserved microbial antigens. We discuss these features from an evolutionary perspective to provide insights into the development and function of unconventional T cells. Finally, we elaborate on the functional redundancy of unconventional T cells and their relationship to subsets of innate and adaptive lymphoid cells, and propose that the unconventional T cell compartment has a critical role in our survival by expanding and complementing the role of the conventional T cell compartment in protective immunity, tissue healing and barrier function.

Transforming growth factor-beta (TGF-β) family proteins mediate many vital biological functions in growth, development and regulation of the immune system. TGF-β itself controls immune homeostasis and inflammation, including conversion of naive CD4+ T cells into Foxp3+ regulatory T cells (Tregs) in the presence of IL-2 and T cell receptor ligands. The helminth parasite Heligmosomoides polygyrus exploits this pathway through a structurally novel TGF-β mimic (Hp-TGM), which binds to mammalian TGF-β receptors and induces Tregs. Here, we performed detailed comparisons of Hp-TGM with mammalian TGF-β. Compared to TGF-β, Hp-TGM induced greater numbers of Foxp3+ Tregs (iTregs), with more intense Foxp3 expression. Both ligands upregulated Treg functional markers CD73, CD103 and PD-L1, but Hp-TGM induced significantly higher CD39 expression than did TGF-β. Interestingly, in contrast to canonical TGF-β signalling through Smad2/3, Hp-TGM stimulation was slower and more sustained. Gene expression profiles induced by TGF-β and Hp-TGM were remarkably similar, and both types of iTregs suppressed T cell responses in vitro and EAE-driven inflammation in vivo. In vitro, both types of iTregs were equally stable under inflammatory conditions, but Hp-TGM-induced iTregs were more stable in vivo during DSS-induced colitis, with greater retention of Foxp3 expression and lower conversion to a ROR-γt+ phenotype. Altogether, results from this study suggest that the parasite cytokine mimic, Hp-TGM, may deliver a qualitatively different signal to CD4+ T cells with downstream consequences for the long-term stability of iTregs. These data highlight the potential of Hp-TGM as a new modulator of T cell responses in vitro and in vivo.

Selenium (Se) is an essential micronutrient that plays a key role in regulating the immune system. T cells are of particular interest due to their important role in promoting adaptive immunity against pathogens and cancer as well as regulating tolerance, all of which are influenced by dietary Se levels. The biological effects of Se are mainly exerted through the actions of the proteins into which it is inserted, i.e. selenoproteins. Thus, the roles that selenoproteins play in regulating T cell biology and molecular mechanisms involved have emerged as important areas of research for understanding how selenium affects immunity. Members of this diverse family of proteins exhibit a wide variety of functions within T cells that include regulating calcium flux induced by T cell receptor (TCR) engagement, shaping the redox tone of T cells before, during, and after activation, and linking TCR-induced activation to metabolic reprogramming required for T cell proliferation and differentiation. This review summarizes recent insights into the roles that selenoproteins play in these processes and their implications in understanding how Se may influence immunity.

Although there are exceptions and outliers, T cell functional responses generally correlate with the affinity of a TCR for a peptide/MHC complex. In one recently described outlier case, the most promising clinical candidate in a series of TCRs specific for the gp100(209) melanoma antigen bound with the weakest solution affinity and produced the least amount of cytokine in vitro. Hypotheses for this outlier behavior included unusual cytokine expression patterns arising from an atypical TCR binding geometry. Studying this instance in more detail, we found here that outlier behavior is attributable not to unusual cytokine patterns or TCR binding, but the use of a position 2 anchor-modified peptide variant in in vitro experiments instead of the wild type antigen that is present in vivo. Although the anchor-modified variant has been widely used in basic and clinical immunology as a surrogate for the wild type peptide, prior work has shown that TCRs can clearly distinguish between the two. We show that when this differential recognition is accounted for, the functional properties of gp100(209)-specific TCRs track with their affinity towards the peptide/MHC complex. Beyond demonstrating the correlates with T cell function for a clinically relevant TCR, our results provide important considerations for selection of TCRs for immunotherapy and the use of modified peptides in immunology.

The adaptor protein TNFR-associated factor 3 (TRAF3) is required for in vivo T cell effector functions and for normal TCR/CD28 signaling. TRAF3-mediated enhancement of TCR function requires engagement of both CD3 and CD28, but the molecular mechanisms underlying how TRAF3 interacts with and impacts TCR/CD28-mediated complexes to enhance their signaling remains an important knowledge gap. We investigated how TRAF3 is recruited to, and regulates, CD28 as a TCR costimulator. Direct association with known signaling motifs in CD28 was dispensable for TRAF3 recruitment; rather, TRAF3 associated with the CD28-interacting protein linker of activated T cells (LAT) in human and mouse T cells. TRAF3–LAT association required the TRAF3 TRAF-C domain and a newly identified TRAF2/3 binding motif in LAT. TRAF3 inhibited function of the LAT-associated negative regulatory protein Dok1, which is phosphorylated at an inhibitory tyrosine residue by the tyrosine kinase breast tumor kinase (Brk/PTK6). TRAF3 regulated Brk activation in T cells, limiting the association of protein tyrosine phosphatase 1B (PTP1B) with the LAT complex. In TRAF3-deficient cells, LAT complex–associated PTP1B was associated with dephosphorylation of Brk at an activating tyrosine residue, potentially reducing its ability to inhibit Dok1. Consistent with these findings, inhibiting PTP1B activity in TRAF3-deficient T cells rescued basal and TCR/CD28-mediated activation of Src family kinases. These results reveal a new mechanism for promotion of TCR/CD28-mediated signaling through restraint of negative regulation of LAT by TRAF3, enhancing the understanding of regulation of the TCR complex.

The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.

International audience; Protein patterning has emerged as a powerful means to interrogate adhering cells. However, the tools to apply a sub-micrometer periodic stimulus and the analysis of the response are still being standardized. We propose a technique combining electron beam lithography and surface functionalization to fabricate nanopatterns compatible with advanced imaging. The repetitive pattern enables a deep-learning algorithm to reveal that T cells organize their membrane and actin network differently depending upon whether the ligands are clustered or homogeneously distributed, an effect invisible to the unassisted human eye even after extensive image analysis. This fabrication and analysis toolbox should be useful, both together and separately, for exploring general correlation between a spatially structured subcellular stimulation and a subtle cellular response.

With the widespread use of programmed death receptor‐1 (PD‐1) blockade therapy, sensitive and specific predictive biomarkers that guide patient selection are urgently needed. T‐cell receptor (TCR) repertoire, which reflects antitumor T‐cell responses based on antigen specificity, is expected as a novel biomarker for PD‐1 blockade therapy. In the present study, the TCR repertoire of eight patients with gastrointestinal cancer treated with anti‐PD‐1 antibody (nivolumab) was analyzed. To analyze the tumor‐associated T‐cell clones in the blood and their mobilization into the tumor, we focused on T‐cell clones that presented in both blood and tumor (blood‐tumor overlapping clones). Responders to PD‐1 blockade tended to exhibit a higher number of overlapping clones in the tumor and a higher total frequency in the blood. Moreover, a higher total frequency of overlapping clones in blood CD8+ T cells before treatment was associated with a favorable clinical response. Collectively, these results suggest the possibility of blood‐tumor TCR repertoire overlap to predict clinical response to PD‐1 blockade and guide patient selection before the treatment., The T‐cell receptor repertoire of eight patients with gastrointestinal cancer treated with anti‐PD‐1 antibody (nivolumab) was analyzed. We focused on T‐cell clones that presented in both blood and tumor to analyze the tumor‐associated T‐cell clones in the blood. We found that responders to PD‐1 blockade tended to exhibit a higher total frequency of overlapping clones in the blood.

Background:. Endotoxin tolerance (ET) is a protective phenomenon in which pre-treatment with a tolerance dose of lipopolysaccharide (LPS) leads to dramatically elevated survival. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what happens to T cell development in the thymus under ET conditions remains unclear. The purpose of this study was to analyze the alterations in thymocyte populations (double-positive [DP] and single-positive [SP] cells) under ET conditions. Methods:. Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of phosphate-buffered saline was used as a control (control group). Changes in thymus weight, cell counts, and morphology were detected in the three groups. Moreover, surface molecules such as CD4, CD8, CD44, CD69, and CD62L were analyzed using flow cytometry. Furthermore, proliferation, apoptosis, cytokine production, and extracellular signal-regulated kinase (ERK) pathway signaling were analyzed in thymocyte populations. The polymorphism and length of the T-cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) were analyzed using capillary electrophoresis DNA laser scanning analysis (ABI 3730). Results:. Thymus weight and cell counts were decreased in the early stage but recovered by the late stage in a murine model of LPS-induced ET. Moreover, the proportions of DP cells (control: 72.130 ± 4.074, 72-h: 10.600 ± 3.517, 8-day: 84.770 ± 2.228), CD4+ SP cells (control: 15.770 ± 4.419, 72-h: 44.670 ± 3.089, 8-day: 6.367 ± 0.513), and CD8+ SP cells (control: 7.000 ± 1.916, 72-h: 34.030 ± 3.850, 8-day: 5.133 ± 0.647) were obviously different at different stages of ET. The polymorphism and length of TCR β chain CDR3 also changed obviously, indicating the occurrence of TCR rearrangement and thymocyte diversification. Further analysis showed that the expression of surface molecules, including CD44, CD69, and CD62L, on thymocyte populations (DP and SP cells) were changed to different degrees. Finally, the proliferation, apoptosis, cytokine production, and ERK pathway signaling of thymocyte populations were changed significantly. Conclusion:. These data reveal that alterations in thymocyte populations might contribute to the establishment of ET.

Summary Renal cell carcinoma (RCC) is comprised of clear-cell (ccRCC) and non-clear-cell (nccRCC) tumors. Despite definitive surgical resection in localized disease, recurrence often occurs. A commercial method based on a multiplex polymerase chain reaction (PCR) assay exclusively targets rearranged T cell receptor (TCR) genes to generate high-throughput sequencing-based data, allowing characterization of the immune repertoire within tumors. In this study we performed a retrospective analysis on archived tumor samples from patients with recurring versus non-recurring T3 ccRCC and on samples from early nccRCC versus ccRCC. Following genomic DNA extraction and multiplex PCR, the fraction of T cells within tumors, the number of unique receptors (‘richness’) and their relative abundances (‘clonality’) were calculated. Statistical significance and correlations were calculated using Student's t-test and Spearman's rho, respectively. Average fraction and clonality of T cells in tumors from non-recurring patients was 2.5- and 4.3-fold higher than in recurring patients (P = 0.025 and P = 0.043, respectively). A significant positive correlation was found between T cell fraction and clonality (Spearman's rho = 0.78, P = 0.008). The average fraction of T cells in ccRCC tumors was 2.8-fold higher than in nccRCC tumors (P = 0.015). Clonality and estimated richness were similar between ccRCC and nccRCC tumors. In summary, recurrence of ccRCC is associated with a lower fraction and clonality of T cells within tumors; nccRCC tumors are more ‘deserted’ than ccRCC, but similar in their ability to generate a clonal T cell repertoire. Our work suggests associations between the characteristics of T cell infiltrate, histology and tumor recurrence.

Clinical use of various forms of immunotherapeutic drugs in glioblastoma (GBM), has highlighted severe T cell dysfunction such as exhaustion in GBM patients. However, reversing T cell exhaustion using immune checkpoint inhibitors in GBM clinical trials has not shown significant overall survival benefit. Phenotypically, CD8+ T cells with downregulated CD28 coreceptors, low CD27 expression, increased CD57 expression, and telomere shortening are classified as senescent T cells. These senescent T cells are normally seen as part of aging and also in many forms of solid cancers. Absence of CD28 on T cells leads to several functional irregularities including reduced TCR diversity, incomplete activation of T cells, and defects in Ag-induced proliferation. In the context of GBM, presence and/or function of these CD8+CD28– T cells is unknown. In this clinical correlative study, we investigated the effect of aging as well as tumor microenvironment on CD8+ T cell phenotype as an indicator of its function in GBM patients. We systematically analyzed and describe a large population of CD8+CD28– T cells in both the blood and tumor-infiltrating lymphocytes of GBM patients. We found that phenotypically these CD8+CD28– T cells represent a distinct population compared with exhausted T cells. Comparative transcriptomic and pathway analysis of CD8+CD28– T cell populations in GBM patients revealed that tumor microenvironment might be influencing several immune related pathways and thus further exaggerating the age associated immune dysfunction in this patient population.