TCRα Chain Research Articles

ThymoSpheres culture: A model to study human polyclonal unconventional T cells.

In vitro cultures remain crucial for studying the fundamental mechanisms of human T-cell development. Here, we introduce a novel in vitro cultivation system based on ThymoSpheres (TS): dense spheroids consisting of DLL4-expressing stromal cells and human hematopoietic precursor cells, in the absence of thymic epithelial cells. These spheroids are subsequently cultured at the air-liquid interphase. TS generate large numbers of mature T cells, are easy to manipulate, scalable, and can be repeatably sampled to monitor T-cell differentiation. The mature T cells generated from primary human hematopoietic precursor cells were extensively characterized using single-cell RNA and combined T-cell receptor (TCR) sequencing. These predominantly CD8α T cells exhibit transcriptional and TCR CDR3 characteristics similar to the recently described human polyclonal αβ unconventional T cell (UTC) lineage. This includes the expression of hallmark genes associated with agonist selection, such as IKZF2 (Helios), and the expression of various natural killer receptors. The TCR repertoire of these UTCs is polyclonal and enriched for CDR3-associated autoreactive features and early rearrangements of the TCR-α chain. In conclusion, TS cultures offer an intriguing platform to study the development of this human polyclonal UTC lineage and its inducing selection mechanisms.

Germline-like TCR-α chains shared between autoreactive T cells in blood and pancreas

Human type 1 diabetes (T1D) is caused by autoimmune attack on the insulin-producing pancreatic beta cells by islet antigen-reactive T cells. How human islet antigen-reactive (IAR) CD4+ memory T cells from peripheral blood affect T1D progression in the pancreas is poorly understood. Here, we aim to determine if IAR T cells in blood could be detected in pancreas. We identify paired αβ (TRA/TRB) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new-onset, and established T1D donors, and measured sequence overlap with TCRs in pancreata from healthy, at risk and T1D organ donors. We report extensive TRA junction sharing between IAR T cells and pancreas-infiltrating T cells (PIT), with perfect-match or single-mismatch TRA junction amino acid sequences comprising ~29% total unique IAR TRA junctions (942/3,264). PIT-matched TRA junctions were largely public and enriched for TRAV41 usage, showing significant nucleotide sequence convergence, increased use of germline-encoded versus non-templated residues in epitope engagement, and a potential for cross-reactivity. Our findings thus link T cells with distinctive germline-like TRA chains in the peripheral blood with T cells in the pancreas.

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

BackgroundHematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due...

Integration of ζ-deficient CARs into the CD3ζ gene conveys potent cytotoxicity in T and NK cells

AbstractChimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR’s activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

Exploration of B and T Cell Receptor Repertoires Reveals Distinct Mechanisms in Pure Red Cell Aplasia, Autoimmune Hemolytic Anemia, and Aplastic Anemia

Background: Pure red cell aplasia (PRCA), autoimmune hemolytic anemia (AIHA) and aplastic anemia (AA) are immune-mediated diseases that mainly attack erythrocyte or erythroid progenitor cells. This study aimed to investigate changes related to autoimmunity in the B cell receptor (BCR) and T cell receptor (TCR) repertoires in these diseases. Methods: Patients with newly diagnosed primary PRCA, AIHA, and AA and healthy individuals with matched age and sex (normal controls, NC) were recruited. Peripheral blood was collected, and BCR and TCR repertoires were sequenced by next-generation immunosequencing. The diversity and clonal expansion of the repertoires, gene and gene combination usage, somatic hypermutation (SHM) of BCR, and the sequence length, hydrophobicity, motifs of the most diverse epitope encoded (complementary-determining region 3, CDR3), T cell clustering, and BCR light chain and TCRα chain were analyzed. Results: 10 PRCA, 10 AIHA, 10 AA, and 7 NC were finally enrolled. For the broad repertoire metrics analysis, only the TCR repertoire of the AA group was more diverse compared with NC (p＜0.05). For BCR and TCR repertoires, PRCA, AIHA, and AA showed uniform characteristics in gene usage. The preferential gene usage of PRCA immunoglobulin heavy (IGH) chains such as IGHV3-23, IGHV3-74 were correlated with memory cells and red blood cell antigen recognition; the higher usage of IGHV4-31 in the AIHA group was associated with secretion of auto-antibodies; whereas AA patients had more gene usage of neutralizing antibodies (p＜0.05). In the gene usage in T cell receptor β (TRB) chains, PRCA patients had skewed usage of genes associated with T cell dysregulation and hyperinflammation such as TRBV1 and TRBV11-2; the increased gene usage in AIHA and AA were also found in other autoimmune diseases, which correlated with abnormal antigen recognition. PRCA and AA also had specific TRBV-J gene combination usage. Only PRCA had higher BCR SHM compared with NC (p＜0.05). The length of CDR3 was similar but the hydrophobicity was different among different disease groups. 12, 28, and 22 motifs were found respectively in BCR of PRCA, AIHA, and AA; and 13, 6, and 6 motifs were found in TCR. Compared with NC, PRCA, AIHA, and AA showed a considerable lack of physiology T cell clusters, and 147, 98, and 141 disease-specific T cell clusters in each disease was found, respectively. For light chain of BCR, PRCA and AIHA had the tendency of more usage of κ chains, whereas AA had more usage of λ chains. For TCRα chains, the three diseases used more genes related to hypersensitivity reactions (p＜0.05). Conclusion: PRCA, AIHA, and AA had different BCR and TCR repertoire characteristics in gene and gene combination usage, hydrophobicity and motifs of CDR3, and T cell clustering, which might contribute to autoimmune antigen recognition. The abnormalities were mainly T cell-related for PRCA, B cell-related for AIHA and both for AA. Keywords: pure red cell aplasia; autoimmune hemolytic anemia; aplastic anemia; BCR; TCR

Mucosal-associated invariant T cells display both pathogenic and protective roles in patients with inflammatory bowel diseases.

An important subtype of the innate-like T lymphocytes is mucosal-associated invariant T (MAIT) cells expressing a semi-invariant T cell receptor α (TCR-α) chain. MAIT cells could be activated mainly by TCR engagement or cytokines. They have been found to have essential roles in various immune mediated. There have been growing preclinical and clinical findings that show an association between MAIT cells and the physiopathology of inflammatory bowel diseases (IBD). Of note, published reports demonstrate contradictory findings regarding the role of MAIT cells in IBD patients. A number of reports suggests a protective effect, whereas others show a pathogenic impact. The present review article aimed to explore and discuss the findings of experimental and clinical investigations evaluating the effects of MAIT cells in IBD subjects and animal models. Findings indicate that MAIT cells could exert opposite effects in the course of IBD, including an anti-inflammatory protective effect of blood circulating MAIT cells and an effector pathogenic effect of colonic MAIT cells. Another important finding is that blood levels of MAIT cells can be considered as a potential biomarker in IBD patients.

Mini-TCRs: Truncated T cell receptors to generate T cells from induced pluripotent stem cells

Allogeneic Tcell platforms utilizing induced pluripotent stemcell (iPSC) technology exhibit significant promise for the facilitation of adoptive immunotherapies. While mature Tcell receptor (TCR) signaling plays a crucial role in generating Tcells from iPSCs, the introduction of exogenous mature TCR genes carries a potential risk of causing graft-versus-host disease (GvHD). In this study, we present the development of truncated TCRα and TCRβ chains, termed mini-TCRs, which lack variable domains responsible for recognizing human leukocyte antigen (HLA)-peptide complexes. We successfully induced cytotoxic T lymphocytes (CTLs) from iPSCs by employing mini-TCRs. Combinations of TCRα and TCRβ fragments were screened from mini-TCR libraries based on the surface localization of CD3 proteins and their ability to transduce Tcell signaling. Consequently, mini-TCR-expressing iPSCs underwent physiological Tcell development, progressing from the CD4 and CD8 double-positive stage to the CD8 single-positive stage. The resulting iPSC-derived CTLs exhibited comparable cytokine production and cytotoxicity in comparison to that of full-length TCR-expressing T lymphocytes when chimeric antigen receptors (CARs) were expressed. These findings demonstrate the potential of mini-TCR-carrying iPSCs as a versatile platform for CAR Tcell therapy, offering a promising avenue for advancing adoptive immunotherapies.

Large-scale template-based structural modeling of T-cell receptors with known antigen specificity reveals complementarity features.

T-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. Identification 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. Using 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. Our results provide a methodology for creating high-quality TCR-peptide-MHC models for antigens of interest that can be utilized to predict TCR specificity.

Tnpo3 controls splicing of the pre-mRNA encoding the canonical TCR α chain of iNKT cells

Unconventional T cells, such as innate natural killer T cells (iNKT) cells, are an important part of vertebrate immune defences. iNKT recognise glycolipids through a T cell receptor (TCR) that is composed of a semi-invariant TCR α chain, paired with a restricted set of TCR β chains. Here, we show that splicing of the cognate Trav11-Traj18-Trac pre-mRNA encoding the characteristic Vα14Jα18 variable region of this semi-invariant TCR depends on the presence of Tnpo3. The Tnpo3 gene encodes a nuclear transporter of the β-karyopherin family whose cargo includes various splice regulators. The block of iNKT cell development in the absence of Tnpo3 can be overcome by transgenic provision of a rearranged Trav11-Traj18-Trac cDNA, indicating that Tnpo3 deficiency does not interfere with the development of iNKT cells per se. Our study thus identifies a role for Tnpo3 in regulating the splicing of the pre-mRNA encoding the cognate TCRα chain of iNKT cells.

Gene expression and TCR amino acid sequences selected by HLA-A02:01-restricted CTLs specific to HTLV-1 in ATL patients.

Adult T-cell leukaemia/lymphoma (ATL) is an aggressive malignancy of peripheral T cells caused by human T-cell lymphotropic virus type-1 (HTLV-1). Tax is the most important regulatory protein for HTLV-1. We aimed to reveal a unique amino acid sequence (AA) of complementarity-determining region 3 (CDR3) of the T-cell receptor (TCR)β and TCRα chains of HLA-A*02:01-restricted Tax11-19 -specific cytotoxic T cells (Tax-CTLs). The gene expression profiles (GEP) of Tax-CTLs were assessed by the next-generation sequence (NGS) method with SMARTer technology. Tax-CTLs seemed to be oligoclonal, and their gene compositions were skewed. The unique motifs of 'DSWGK' in TCRα and 'LAG' in TCRβ at CDR3 were observed in almost all patients. Tax-CTL clones harbouring the 'LAG' motif with BV28 had a higher binding score than those without either of them, besides a higher binding score associated with longer survival. Tax-CTLs established from a single cell showed killing activities against Tax-peptide-pulsed HLA-A2+ T2 cell lines. GEP of Tax-CTLs revealed that genes associated with immune response activity were well preserved in long-term survivors with stable status. These methods and results can help us better understand immunity against ATL, and should contribute to future studies on the clinical application of adoptive T-cell therapies.

Modeling cell-mediated immunity in human type 1 diabetes by engineering autoreactive CD8+ T cells.

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing β-cells is thought to be mediated primarily by antigen-specific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human β-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells.

Analysis of thymic generation of shared T-cell receptor α repertoire associated with recognition of tumor antigens shows no preference for neoantigens over wild-type antigens.

The number of mutations in cancer cells is an important predictor of a positive response to cancer immunotherapy. It has been suggested that the neoantigens produced by these mutations are more immunogenic than nonmutated tumor antigens, which are likely to be protected by immunological tolerance. However, the mechanisms of tolerance as regards tumor antigens are incompletely understood. Here, we have analyzed the impact of thymic negative selection on shared T-cell receptor (TCR) repertoire associated with the recognition of either mutated or nonmutated tumor antigens by comparing previously known TCR-antigen-pairs to TCR repertoires of 21 immunologically healthy individuals. Our results show that TCRα chains associated with either type of tumor antigens are readily generated in the thymus, at a frequency similar to TCRα chains associated with nonself. In the peripheral repertoire, the relative clone size of nonself-associated chains is higher than that of the tumor antigens, but importantly, there is no difference between TCRα chains associated with mutated or nonmutated tumor antigens. This suggests that the tolerance mechanisms protecting nonmutated tumor antigens are non-deletional and therefore potentially reversible. As unmutated antigens are, unlike mutations, shared by a large number of patients, they may offer advantages in designing immunological approaches to cancer treatment.

Single-cell profiling identifies a novel human polyclonal unconventional T cell lineage.

In the human thymus, a CD10+ PD-1+ TCRαβ+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double-positive stage. Here, we identify the progeny of this unconventional lineage in antigen-inexperienced blood. These unconventional T cells (UTCs) in thymus and blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e., ZNF683 and IKZF2), and a polyclonal TCR repertoire with autoreactive features, exhibiting a bias toward early TCRα chain rearrangements. Single-cell RNA sequencing confirms a common developmental trajectory between the thymic and blood UTCs and clearly delineates this unconventional lineage in blood. Besides MME+ recent thymic emigrants, effector-like clusters are identified in this heterogeneous lineage. Expression of Helios and KIR and a decreased CD8β expression are characteristics of this lineage. This UTC lineage could be identified in adult blood and intestinal tissues. In summary, our data provide a comprehensive characterization of the polyclonal unconventional lineage in antigen-inexperienced blood and identify the adult progeny.

T-cell engineering strategies for tumors with low antigen density, and T-cell survival in the immunosuppressive tumor microenvironment of relapsed/refractory diffuse large B-cell lymphoma

Refractory and/or relapsed (r/r) diffuse large B-cell lymphomas after treatment with two lines of systemic chemoimmunotherapy exhibit diversity in genetics, tissue biology, and pathology, as well as poor prognosis. Patient TCRαβ cells engineered with a CD19-specific chimeric antigen receptor (CAR) have shown promising clinical outcomes in r/r diffuse large B-cell lymphoma. The ZUMA-1 study, the JULIET study, and the TRANSCEND NHL 001 study of three prototype 19CAR-T cells have indicated an overall response rate of 52–82%, a complete response rate of 40–58%, and a 12-month progression-free survival of 33.2%–46.6%, with clinically manageable treatment related toxicity. At the 5-year follow-up, relapse was observed in approximately 57% of patients within 1 year. Understanding of the risk factors for non-response remains insufficient. In addition to intrinsic tumor resistance, such as aberrant apoptotic signaling, downregulation or loss of tumor-associated antigens (TAA), an immunosuppressive tumor microenvironment, and CAR-T cell exhaustion in vivo have been suggested to be important risk factors. Mechanisms underlying 19CAR-T cell exhaustion under chronic TAA exposure, and limited 19CAR-T cell trafficking and infiltration into the tumor mass have been reported. Moreover, tumor escape in the presence of low TAA density remains a challenge in 1928ζ CAR-T cell treatment. In this review, we provide an overview of modified modular CAR elements and their synergistic effects in controlling T-cell function. We then briefly discuss novel strategies against tumors with low TAA density, such as bispecific tandem or loop CAR recognition domains, the development of human leukocyte antigen-independent synthetic TCRαβ double-chain receptors integrated into the constant region of the TCRα chain, and armored CAR-T cells targeting the tumor microenvironment.

Preclinical Activity of Allogeneic CS1-Specific CAR T-Cells (UCARTCS1) in Multiple Myeloma

Introduction: The substantial activity of chimeric antigen receptor (CAR) T-cells in multiple myeloma (MM) has recently been shown by the success of BCMA-CAR T-cell therapy. Nevertheless the difficulties in (timely) generation of CART cells for each patient, the limited availability of manufacturing slots, and the frequent BCMAlow MM relapses after therapy urge the investigators to search for i) alternative, "off the shelf", available sources of CAR T-cells and ii) alternative target antigens than BCMA. UCARTCS1 cells are "off the shelf" allogeneic CAR T-cells derived from healthy donors targeting CS1 (SLAMF7), which is a highly attractive target, due to its overexpression on MM cells. Furthermore, the genes coding for the TCRα chain of the TCRab receptor and for CS1 are disrupted in UCARTCS1 cells using Cellectis' TALEN® gene-editing technology. These modifications aim to minimize the potential risk for allo-TCR-mediated graft-versus-host disease (GvHD) upon infusion in patients and diminish CS1-mediated fratricide. In this study, we evaluated the preclinical activity of UCARTCS1 in MM cell lines, in bone marrow (BM) samples obtained from MM patients and in a MM mouse model. We also investigated the potential impact of previous therapy and tumor characteristics on the in vitro efficacy of UCARTCS1. Methods: Luciferase-transduced MM cell lines with different expression levels of CS1 were incubated with UCARTCS1 cells or control (non-transduced CS1/TCRαβ double knock-out) T-cells at different E:T ratios for 24 hours. MM cell lysis was assessed by bioluminescence. Anti-MM activity of UCARTCS1 was also evaluated in 25 BM samples obtained from newly diagnosed (n=10; NDMM), daratumumab-naïve relapsed/refractory patients (n=10; RRMM; median of 3 prior therapies) and from daratumumab-refractory patients (n=5; DRMM; median of 3 prior therapies) in 24-hour flow cytometry-based cytotoxicity assays. The results were correlated to CS1 expression levels of MM cells, Treg frequency, and several other patient characteristics such as treatment history. Results: UCARTCS1 induced strong CS1 CAR-mediated and dose-dependent lysis of different MM cell lines expressing variable levels of CS1 (MM1s, L363, UM9, U266). UCARTCS1 cells also effectively lysed primary MM cells in a dose-dependent and CAR-mediated manner, with a mean maximal lysis of 96.9% (Figure 1). In these BM samples, UCARTCS1 cells also targeted CS1+ non-malignant hematopoietic cells, such as CD4+ T-cells, CD8+ T-cells, B cells, and NK cells, while sparing the CS1low fraction in each cell subset. However, the lysis of normal cells was less efficient than MM cell lysis, which could be explained by substantially lower CS1 expression on normal cells compared to MM cells (Figure 2). CS1 expression on MM cells and UCARTCS1 cytotoxic activity were both comparable in the different patient subgroups. In addition, baseline tumor and immune characteristics, such as the expression level of CS1 on MM cells, the tumor load (MM cell percentage in BM), or frequency of Treg cells in the BM did not affect CAR T-cell mediated cytotoxicity. Finally, UCARTCS1 exhibited durable dose-dependent anti-MM activity in mouse xenograft models after i.v. injection of CAR T-cells compared to mice treated with CAR- control T-cells. Conclusion: UCARTCS1 has potent anti-MM activity against MM cell lines and primary MM cells, as well as in a MM xenograft model. There was no difference in activity between heavily pretreated and newly diagnosed patients, indicating that mechanisms of resistance to prior therapies do not result in reduced susceptibility to UCARTCS1-mediated lysis in vitro. UCARTCS1 was also effective in samples from patients with relatively low CS1 expression, with high tumor burden, or with high Treg numbers. These data support the ongoing phase 1 clinical trial with UCARTCS1 in heavily pretreated MM patients (NCT04142619). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adipocyte CD1d Gene Transfer Induces T Cell Expansion and Adipocyte Inflammation in CD1d Knockout Mice.

CD1d, a lipid Ag-presenting molecule for invariant NKT (iNKT) cells, is abundantly expressed on adipocytes and regulates adipose homeostasis through iNKT cells. CD1d gene expression was restored in visceral adipose tissue adipocytes of CD1d knockout (KO) mice to investigate the interactions between adipocytes and immune cells within adipose tissue. We developed an adipocyte-specific targeting recombinant adeno-associated viral vector, with minimal off-target transgene expression in the liver, to rescue CD1d gene expression in visceral adipose tissue adipocytes of CD1d KO mice, followed by assessment of immune cell alternations in adipose tissue and elucidation of the underlying mechanisms of alteration. We report that adeno-associated virus-mediated gene transfer of CD1d to adipocytes in CD1d KO mice fails to rescue iNKT cells but leads to massive and selective expansion of T cells within adipose tissue, particularly CD8+ T effector cells, that is associated with adipocyte NLRP3 inflammasome activation, dysregulation of adipocyte functional genes, and upregulation of apoptotic pathway proteins. An NLRP3 inhibitor has no effect on T cell phenotypes whereas depletion of CD8+ T cells significantly attenuates inflammasome activation and abolishes the dysregulation of adipocyte functional genes induced by adipocyte CD1d. In contrast, adipocyte overexpression of CD1d fails to induce T cell activation in wild-type mice or in invariant TCR α-chain Jα18 KO mice that have a normal lymphocyte repertoire except for iNKT cells. Our studies uncover an adipocyte CD1d → CD8+ T cell → adipocyte inflammasome cascade, in which CD8+ T cells function as a key mediator of adipocyte inflammation likely induced by an allogeneic response against the CD1d molecule.

A novel mutation in TRAC in a patient with abnormal newborn screening for severe combined immunodeficiency

Background: The T cell receptor (TCR)-α chain plays a key role in TCR structure and function. Biallelic mutations in TRAC, encoding the constant region of the TCR-α chain, obliterates TCR expression and results in immunodeficiency. TCR-α chain deficiency presents at infancy or childhood with repeated viral and bacterial infections, enlarged liver, spleen, and lymph nodes as well as autoimmune features and lymphoma (OMIM #615387). Aim: To broaden the genotypic and phenotypic spectrum of TCR-α chain deficiency. Methods: We present a case report of a patient with severe combined immunodeficiency (SCID) due to a novel autosomal recessive mutation in TRAC. Results: Our patient was identified at 13 days of life due to abnormal T cell receptor excision circle levels detected by newborn screening (NBS). Immune evaluation revealed profound lymphopenia, depressed responses to the mitogen PHA and a skewed T cell repertoire, all consistent with SCID. The patient was found to carry a novel homozygous mutation in the TRAC gene. Conclusion: A novel homozygous mutation in the TRAC gene caused profound T cell lymphopenia and aberrant in vitro mitogenic response, the hallmarks of SCID. Statement of Novelty: TCR-α chain deficiency is a rare and relatively new condition and not very well defined. We herein report a novel mutation in TRAC resulting in SCID.

Moringa oleifera polysaccharide regulates colonic microbiota and immune repertoire in C57BL/6 mice.

This study investigated the effects of Moringa oleifera polysaccharide (MOP) on serum immune indices, immune organ indices, colonic microflora and immune repertoire of mice. Forty male SPF C57BL/6 mice were randomly divided into four groups and subjected to gavage of 0, 20, 40 and 60mg/kg MOP for 28days. Mice were sacrificed on the last day of the experiment and their thymus, spleen, blood and colon contents were collected for further detection. Our findings suggested that MOP could significantly increase the thymus index (P<0.01) and spleen index (P<0.05), and significantly decrease the levels of interleukin-6 and tumour necrosis factor-α in mice (P<0.05). And MOP could regulate the proportion of colonic microflora of mice, significantly increase the abundance of Muribaculaceae and significantly decrease the abundance values of Proteobacteria, Helicobacter, Stenotrophomonas, etc (P<0.05). In addition, MOP could regulate the usage frequencies of TRBV15 (P=0.06) and TRBV9 (P=0.10) on the TCRα chain and 9 V-J pairs were found to have remarkable usage frequency changes. These results implied that MOP exerted positive effects on the immune performance and intestinal health of mice.

Autoreactive T cell receptors with shared germline-like α chains in type 1 diabetes.

Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals (“public” chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαβ sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells (“expanded”), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or “private,” TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRβ junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these “innate-like” TCRs differ from previously described immunodominant TCRβ chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.

64 A cloning and expression system of the neoantigen-specific TCRs from tumor-infiltrating lymphocytes by single-cell sequencing of paired TCRα and TCRβ chains

BackgroundT-cells that target tumor neoantigens arising from cancer mutations are the primary mediators of cancer immunotherapies. Identifying neoantigens and T-cells that recognize them is essential for T-cell-based immunotherapy. However, neoantigen-reactive Tumor-infiltrating lymphocytes (TILs) are highly differentiated or exhausted with a limited proliferative capacity; it is challenging to expand them for a sufficient number to probe their specificity. Therefore, we developed a novel cloning and expression system to examine TCRs discovered by single-cell sequencing of TILs for their neoantigen-specificity.MethodsTILs of lung cancer and sarcoma were analyzed. Surgically removed tumors were divided into several pieces. They were enzymatically digested to prepare fresh tumor digest (FTD) and cryopreserved. They were used to generate TIL cultures and perform WES and RNA-Seq to identify tumor-specific mutations. MHCflurry was used to predict the binding affinity of potential epitopes arising from these mutations to HLA class I. Peptides that were predicted to bind to patients‘ own MHC class I molecules strongly were then synthesized. Single TILs isolated with the ICELL8® cx system (Takara Bio) were dispensed into a nanowell TCR chip containing preprinted barcodes. Barcoded cDNAs were PCR-amplified in-chip, pooled off-chip, and used as a template in the TCR-specific PCR or for the whole transcriptome library generation of 5’ ends of all transcripts. Based on single-cell transcriptome data and TCR profiles of TILs, we predict and prioritize neoantigen-specific TCRs and cloned them into siTCR® retrovirus vectors. These TCRs were transduced into SUP-T1-based reporter cells in which ZsGreen fluorescent protein expression is controlled by AP-1 and NFAT binding sites. TCR-expressing reporter cells were cocultured with patient autologous APCs pulsed with a pool of candidate neoantigen peptides. ZsGreen expression indicates that TCRs match their cognate neoantigens.ResultsIn a lung cancer patient, we set up 18 TIL cultures and obtained 12 TILs. TILs were cocultured with FTD; IFN-γ production was measured by ELISA to evaluate their reactivity to the autologous tumor. NGS identified 197 somatic mutations, 4 fusion genes, and 8 highly expressed cancer-testis antigens. Among them, 339 candidate peptides were synthesized and screened. In addition, we cloned 3 pairs of TCRαβ chains from most expanded TIL cultures and 4 TCRs from ex vivo TILs with exhausted phenotype. Two reporter cells that express TCRs from exhausted TILs responded to the same neoantigen peptide.ConclusionsGenerating TCR expressing cell lines facilitated the identifying neoantigens and their cognate TCR sequences from patients.Ethics ApprovalG3545