Quantification of T-Cell Receptor Excision Circles (TRECs).

Aging is a natural process that compromises the immune system's functionality increasing the risk of infectious, tumors, and autoimmune diseases. The thymus involution is an age-dependent process characterized by decreased cellularity, peripheral lymphocyte infiltration into the perivascular space, and expansion of adipose tissue. All those modifications hamper the functionality of the organ and lead to a decline of naïve T-cell production with a shrinking of the T-cell repertoire. Thymus atrophy is described in several disorders including autoimmune diseases. The quantification of T-cell receptor excision circles (TRECs) in recent thymus emigrants is a standard procedure to investigate the thymic function. In this chapter, we discuss the methodology used to quantify this molecule in peripheral blood mononuclear cells and isolated CD4+ and CD8+ T cells.

[Development of off-the-shelf universal T cell therapies from ES/iPS cells: applications in cancer and viral infection].

Methods in which patient-derived T cells are genetically modified in vitro and administered to patients have been demonstrated effective in the area of cancer immunotherapy. However, these methods have some unresolved issues such as cost, time, and unstable quality. Several groups have developed strategies to overcome these barriers by regenerating T cells from iPSCs. We have been developing a method in which specific TCR genes are introduced into iPSCs and T cells are regenerated from these iPSCs (TCR-iPSC method). We are now using starting iPSCs from the iPSC stock lines provided by CiRA-F, as the iPSC stock cells are less likely to be rejected. A study aimed at application to solid tumors demonstrated the therapeutic effect of regenerated T cells in a patient tissue xenograft model of WT1 antigen-positive renal cell carcinoma. This article will also discuss strategies by other groups to regenerate various types of T cells from iPSCs.

Chemical Complementarity of Blood-Sourced, Breast Cancer-Related TCR CDR3s and the CMV UL29 and IE1 Antigens is Associated with Worse Overall Survival.

Cytomegalovirus (CMV) infection is common and becomes a particular concern in immunocompromised patients. Understanding the potential role CMV plays in breast cancer patients' disease progression is important for providing more patient-specific treatments. In this study, we analyzed whether a breast cancer patient's blood-sourced T-cell receptor (TCR) complementarity determining-3 (CDR3) amino acid (AA) sequences could provide an indication of the impact of a systemic CMV infection. Specifically, we assessed the chemical complementarity of patient TCR CDR3 AAs and CMV antigens to determine whether patients with greater complementarity also represented different survival probabilities. Initially, we examined five distinct CMV antigens, of which two, IE1 and UL29, represented TCR (TRA+ RB)-CDR3-CMV antigen complementarity scores (CSs) whereby cases representing the upper 50th percentile of CSs had a worse overall survival (log-rank p = 5.034E-3, for IE1). Then, an analysis of CSs representing previously identified, TCR IE1 epitopes indicated that greater TRB CDR3-IE1 epitope complementarities represented a worse OS (log-rank p = 0.0111). These results raise the question of whether a systemic, anti-CMV response leads to increased systemic inflammation, which is either directly or indirectly supportive of tumor growth; or are patients succumbing to a direct impact of CMV functions on tumor growth or metastasis?

Insights into cytomegalovirus-associated T cell receptors in recipients following allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation is a serious problem in recipients of allogeneic hematopoietic stem cell transplantation. Long-term latency depends on specific T cell immune reconstitution, which identifies various pathogens by T cell receptors (TCRs). However, the mechanisms underlying the selection of CMV-specific TCRs in recipients after transplantation remain unclear. METHODS: Using high-throughput sequencing and bioinformatics analysis, the T cell immune repertoire of seven CMV reactivated recipients (CRRs) were analyzed and compared to those of seven CMV non-activated recipients (CNRs) at an early stage after transplant. RESULTS: The counts of unique complementarity-determining region 3 (CDR3) were significantly higher in CNRs than in CRRs. The CDR3 clones in the CNRs exhibit higher homogeneity compared to the CRRs. With regard to T cell receptor ß-chain variable region (TRBV) and joint region (TRBJ) genotypes, significant differences were observed in the frequencies of TRBV6, BV23, and BV7-8 between the two groups. In addition to TRBV29-1/BJ1-2, TRBV2/BJ2-2, and TRBV12-4/BJ1-5, 11 V-J combinations had significantly different expression levels between CRRs and CNRs. CONCLUSIONS: The differences in TCR diversity, TRBV segments, and TRBV-BJ combinations observed between CNRs and CRRs might be associated with post-transplant CMV reactivation and could serve as a foundation for further research.

Cis-interaction between CD52 and T cell receptor complex interferes with CD4+ T cell activation in acute decompensation of cirrhosis.

BACKGROUND: Immune dysfunction contributes to a high rate of infection in patients with acute decompensation of cirrhosis. CD52 is a glycoprotein prominently expressed in lymphocytes. Immune regulation by CD52 may be involved in adaptive immune dysfunction in cirrhosis. This study aimed to investigate the function of CD52 on CD4+ T cells on the blood of patients with acute decompensation of cirrhosis. METHODS: The expression of CD52 in the peripheral blood lymphocytes of 49 patients with cirrhosis was investigated using flow cytometry and transcriptomics. Potential cis-membrane ligands of CD52 were discovered via proximity labelling followed by proteomics. The function of CD52 on antigen-specific activation of CD4+ T cells was examined using flow cytometry in CD52 CRISPR-Cas9 knockout primary T cells. FINDINGS: CD52 expression was elevated in CD4+ T cells in acute decompensation of cirrhosis, and this elevation was correlated with increased disease severity and mortality. Components of the T cell receptor complex including TCRß, CD3γ and CD3ε were identified and validated as cis-membrane ligands of CD52. Knockout of CD52 promoted antigen-specific activation, proliferation, and pro-inflammatory cytokine secretion. INTERPRETATION: Membrane bound CD52 demonstrated cis-interaction with the T cell receptor and served as a dynamic regulator of antigen-specific activation of CD4+ T cells. The upregulation of CD52 in the periphery of acute decompensation of cirrhosis hinders the recognition of the T cell receptor by MHC, contributing to impaired T cell function. The development of an alternative anti-CD52 antibody is required to restore T cell function and prevent infections in cirrhosis. FUNDING: This study was supported by the NIHR Imperial Biomedical Research Centre, Institute for Translational Medicine and Therapeutics (P74713), Wellcome Trust (218304/Z/19/Z), and Medical Research Council (MR/X009904/1 and MR/R014019/1).

Conventional PCR-based versus next-generation sequencing-based approach for T-cell receptor γ gene clonality assessment in mature T-cell lymphomas: A phase 3 diagnostic accuracy study.

Background: Clonality assessment is currently the major molecular analysis utilized to support the diagnosis of suspicious lymphoid malignancies. Clonal rearrangements of the V-J segments of T-cell receptor G chain locus (TCRγ or TRG) have been observed in almost all types of T neoplasms, such as T-cell-related non-Hodgkin lymphomas and leukemias. At present, the gold standard for clonality evaluation is multiplex polymerase chain reaction (PCR), plus subsequent capillary electrophoresis/heteroduplex analyses, and/or Sanger sequencing. This approach overcomes the problem with the conventional Southern blot hybridization and is more efficient, simple, fast, and reproducible. In the recent years, the new next-generation sequencing (NGS) technologies provided alternative techniques for the analysis of antigen receptors genes, which presented several advantages, such as increased efficiency, specificity (SP), sensitivity (ST), resolution, and objectivity of the results, leading to a better classification, stratification, and monitoring of lymphoid malignancies. Nonetheless, these technologies are still far from being the new gold standard since further studies are warranted to prove their utility. The present study aimed to assess the diagnostic accuracy of these two methods by comparing a commercial NGS-based assay for the evaluation of TRG locus with the gold standard PCR-based one, to fulfill the requirements of a phase 3 diagnostic accuracy study. Methods: We assessed the TRG gene rearrangements in 72 cases using the conventional and highly-validated PCR-based assay proposed by EuroClonality consortium, an alternative commercial PCR-based assay, namely, IdentiClone® TCR Gamma Gene Rearrangement Assay 2.0, and a commercial NGS-based assay, that is, Invivoscribe LymphoTrack® Dx MiSeq® (both by Invivoscribe Technologies Inc., San Diego, CA, USA), to determine the diagnostic accuracy of the latter, and compare them with reference diagnoses made based on observation of clinical manifestations, cytohistological, and immunohistochemical analyses. Statistical values were calculated using the Oxford CATmaker software package. Results: Using standardized criteria of interpretation, the obtained results showed a diagnostic accuracy of 90.3% (correspondence in 65 out of 72 cases) of the test under investigation, with a ST of 86%, a SP of 95%, a positive predicting value of 94%, and a negative predicting value of 88%, demonstrating that it had high efficiency and reliability in detecting clonal TRG gene rearrangements in T-cell non-Hodgkin lymphomas. Conclusions: This diagnostic accuracy study yielded comparable results using a validated PCR-based approach and a new NGS-based one. Subsequent studies and cost-effectiveness evaluation are needed to put the NGS-based clonality assessment into routine diagnostic practice.

Targeting epigenetic regulation and post-translational modification with 5-Aza-2' deoxycytidine and SUMO E1 inhibition augments T-cell receptor therapy.

BACKGROUND: Cellular immunotherapy using modified T cells offers new avenues for cancer treatment. T-cell receptor (TCR) engineering of CD8 T cells enables these cells to recognize tumor-associated antigens and tumor-specific neoantigens. Improving TCR T-cell therapy through increased potency and in vivo persistence will be critical for clinical success. METHODS: We evaluated a novel drug combination to enhance TCR therapy in mouse models for acute myeloid leukemia (AML) and multiple myeloma (MM). RESULTS: Combining TCR therapy with the SUMO E1 inhibitor TAK981 and the DNA methylation inhibitor 5-Aza-2' deoxycytidine resulted in strong antitumor activity in a persistent manner against two in vivo tumor models of established AML and MM. We uncovered that the drug combination caused strong T-cell proliferation, increased cytokine signaling in T cells, improved persistence of T cells, and reduced differentiation towards exhausted phenotype. Simultaneously the drug combination enhanced immunogenicity of the tumor by increasing HLA and co-stimulation and surprisingly reducing inhibitory ligand expression. CONCLUSION: Combining T-cell therapy with TAK981 and 5-Aza-2' deoxycytidine may be an important step towards improved clinical outcome.

LYP regulates SLP76 and other adaptor proteins in T cells.

BACKGROUND: The LYP tyrosine phosphatase presents a SNP (1858C > T) that increases the risk of developing autoimmune diseases such as type I diabetes and arthritis. It remains unclear how this SNP affects LYP function and promotes the development of these diseases. The scarce information about LYP substrates is in part responsible for the poor understanding of LYP function. RESULTS: In this study, we identify in T lymphocytes several adaptor proteins as potential substrates targeted by LYP, including FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2. We also show that LYP co-localizes with SLP76 in microclusters, upon TCR engagement. CONCLUSIONS: These data indicate that LYP may modulate T cell activation by dephosphorylating several adaptor proteins, such as FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2 upon TCR engagement.

Differential roles of kinetic on- and off-rates in T-cell receptor signal integration revealed with a modified Fab'-DNA ligand.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRß H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.

Protective effects of Ganoderma lucidum spores on estradiol benzoate-induced TEC apoptosis and compromised double-positive thymocyte development.

Backgroud: Thymic atrophy marks the onset of immune aging, precipitating developmental anomalies in T cells. Numerous clinical and preclinical investigations have underscored the regulatory role of Ganoderma lucidum spores (GLS) in T cell development. However, the precise mechanisms underlying this regulation remain elusive. Methods: In this study, a mice model of estradiol benzoate (EB)-induced thymic atrophy was constructed, and the improvement effect of GLS on thymic atrophy was evaluated. Then, we employs multi-omics techniques to elucidate how GLS modulates T cell development amidst EB-induced thymic atrophy in mice. Results: GLS effectively mitigates EB-induced thymic damage by attenuating apoptotic thymic epithelial cells (TECs) and enhancing the output of CD4+ T cells into peripheral blood. During thymic T cell development, sporoderm-removed GLS (RGLS) promotes T cell receptor (TCR) α rearrangement by augmenting V-J fragment rearrangement frequency and efficiency. Notably, biased Vα14-Jα18 rearrangement fosters double-positive (DP) to invariant natural killer T (iNKT) cell differentiation, partially contingent on RGLS-mediated restriction of peptide-major histocompatibility complex I (pMHCⅠ)-CD8 interaction and augmented CD1d expression in DP thymocytes, thereby promoting DP to CD4+ iNKT cell development. Furthermore, RGLS amplifies interaction between a DP subpopulation, termed DPsel-7, and plasmacytoid dendritic cells (pDCs), likely facilitating the subsequent development of double-negative iNKT1 cells. Lastly, RGLS suppresses EB-induced upregulation of Abpob and Apoa4, curbing the clearance of CD4+Abpob+ and CD4+Apoa4+ T cells by mTECs, resulting in enhanced CD4+ T cell output. Discussion: These findings indicate that the RGLS effectively mitigates EB-induced TEC apoptosis and compromised double-positive thymocyte development. These insights into RGLS's immunoregulatory role pave the way for its potential as a T-cell regeneration inducer.