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Understanding racial disparities in cancer outcomes continues to be a challenge, with likely many factors at play, including socioeconomic factors and genetic polymorphisms impacting basic cellular and molecular functions. Additionally, it is possible that specific combinations of environment and genetics have specific impacts. T-cell receptor (TCR) gene segment usage, HLA allele combinations have been associated with autoimmune and infectious disease courses, and more recently, TCR gene segment usage, HLA allele combinations have been associated with distinct survival outcomes in cancer as well. We examined several such, previously reported cancer-related TCR gene segment usage, HLA allele combinations for evidence of racial disparities, with regard to the prevalence of the combination in different racial groups. Results indicated that TCR gene segment usage, potentially reflecting environmental factors related to previous pathogen exposure, in combination with certain HLA alleles or independently, may represent a novel explanation for racial disparities in cancer outcomes. Overall, at this point, a genetic connection to racial disparities in cancer outcomes is detectable but remains modest, suggesting that other factors, such as socioeconomic factors, remain as important considerations., (© 2022 John Wiley & Sons Ltd.)

T-cell repertoire characterization is a methodology that enables the identification of T-cell receptor (TCR) gene sequences in a T-cell population. TCR genes are composed of modular gene segments V (D) J that undergo somatic recombination, resulting in unique and unpredictable sequences that can be utilized to identify each T-cell clone. The analysis of the TCR composition in a T-cell population can give information on the biological phenomenon such as antigen-driven expansion and heterogeneity of T-cell responses. Bulk TCR analysis can give useful information on the clonality and can help track a specific clonotype over time or in different compartments, although the information about pairing cannot be provided. Single-cell TCR sequencing, on the other hand, can provide pairing information that are necessary to reconstruct the TCR and confirm antigen specificity.Here we describe common methods to characterize T-cell repertoires based on both bulk and single-cell next-generation sequencing., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The complete germline repertoires of the channel catfish, Ictalurus punctatus , T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish form eight subgroups based on members sharing 75% nucleotide identity. However, the 149 TRAD genes in zebrafish form 53 subgroups. This difference in subgroup numbers between catfish and zebrafish is best explained by expansions of catfish TRADV subgroups, which likely occurred through multiple, relatively recent gene duplications. Similarly, 112 catfish TRBV genes form 30 subgroups, while the 51 zebrafish TRBV genes are placed into 36 subgroups. Notably, several catfish and zebrafish TRB subgroups share ancestor nodes. In addition, the complete catfish TR gene annotation was used to compile a TR gene segment database, which was applied in clonotype analysis of an available gynogenetic channel catfish transcriptome. Combined, the TR annotation and clonotype analysis suggested that the expressed TRA, TRB, and TRD repertoires were generated by different mechanisms. The diversity of the TRB repertoire depends on the number of TRBV subgroups and TRBJ genes, while TRA diversity relies on the many different TRAJ genes, which appear to be only minimally trimmed. In contrast, TRD diversity relies on nucleotide additions and the utilization of up to four TRDD segments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Crider, Quiniou, Felch, Showmaker, Bengtén and Wilson.)

Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy., Competing Interests: D.H.B. is co-founder of STAGE Cell Therapeutics GmbH (now Juno Therapeutics, a Bristol-Myers Squibb Company) and T Cell Factory B.V. (now Kite, a Gilead Company). D.H.B. has a consulting contract with and receives sponsored research support from Juno Therapeutics., (© 2021 The Authors.)

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes., (© 2021. The Author(s).)

Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice was achieved in 2 of 14 SS cases. Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading. These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection often causes severe complications and even death. However, asymptomatic infection has also been reported, highlighting the difference in immune responses among individuals. Here we performed single-cell chromatin accessibility and T cell-receptor analyses of peripheral blood mononuclear cells collected from individuals convalescing from COVID-19 and healthy donors. Chromatin remodelling was observed in both innate and adaptive immune cells in the individuals convalescing from COVID-19. Compared with healthy donors, recovered individuals contained abundant TBET-enriched CD16 + and IRF1-enriched CD14 + monocytes with sequential trained and activated epigenomic states. The B-cell lineage in recovered individuals exhibited an accelerated developmental programme from immature B cells to antibody-producing plasma cells. Finally, an integrated analysis of single-cell T cell-receptor clonality with the chromatin accessibility landscape revealed the expansion of putative SARS-CoV-2-specific CD8 + T cells with epigenomic profiles that promote the differentiation of effector or memory cells. Overall, our data suggest that immune cells of individuals convalescing from COVID-19 exhibit global remodelling of the chromatin accessibility landscape, indicative of the establishment of immunological memory.

Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRβ) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRβ signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients' repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients., (Copyright © 2020 Kelkka, Savola, Bhattacharya, Huuhtanen, Lönnberg, Kankainen, Paalanen, Tyster, Lepistö, Ellonen, Smolander, Eldfors, Yadav, Khan, Koivuniemi, Sjöwall, Elo, Lähdesmäki, Maeda, Nishikawa, Leirisalo-Repo, Sokka-Isler and Mustjoki.)

Objective: To investigate the clinical features of AIHA/ES patients with clonal Ig/TCR gene rearrangement., Methods: Ig/TCR gene rearrangements were measured by BIOMED-2 PCR. 44 primary AIHA/ES patients were enrolled in the study. Clinical characteristics were analyzed and compared between patients with and without clonal Ig/TCR gene rearrangement., Results: Clonal Ig/TCR rearrangements were identified in 34.09% (15/44) patients including 18.18% (8/44) clonal Ig rearrangement and 15.91% (7/44) clonal TCR rearrangement. 11.37% (5/44) patients showed TCR γ rearrangement, and 2.27% (1/44) patient showed β rearrangement. 2.27% (1/44) patient showed both γ and β chain rearrangement. The median ages of patients with Ig/TCR clonality (8 male and 7 female) and without Ig/TCR clonality (10 male and 19 female) were 60 (16 ∼ 81) and 53 (17 ∼ 78) years old, respectively. No significant differences were found in age or gender between the two groups ( p  = .26, p  = .378). Hb and RBC of patients with Ig/TCR clonality [(64.31 ± 5.72) g/L, (1.78 ± 0.22) × 10 12 /L] were significantly lower than those of patients without Ig/TCR clonality ( p  = .0053 and p  = .0189, respectively). The percentage of reticulocytes of Ig/TCR clonality group was obviously higher than that of patients without Ig/TCR clonality ( p  = .0248). No significant differences were found in levels of TBIL, IBIL, LDH, FHb, Hp, IgG, IgA, IgM, IgE, C3, C4, CD5 + CD19 + /CD19 + ratio, and CD3 + CD4 + /CD3 + CD8 + ratio between the two groups. Treatment response of Ig/TCR clonality group occurred significantly later than that of the non-clonality group ( p  = .0016). There were no differences in relapse rate, time to recurrence, and duration of remission between two groups ( p  = .083, p  = .72, and p  = .61, respectively)., Conclusion: AIHA/ES patients with clonal Ig/TCR gene rearrangement presented more severe haemolysis and anaemia. Longer treatment is needed for these patients to obtain remission.

Background: This study aims to investigate the T-cell receptor (TCR) repertoire in patients with acute coronary syndrome (ACS)., Methods: The TCR repertoires of 9 unstable angina patients (UA), 14 acute myocardial infarction patients (AMI) and 9 normal coronary artery (NCA) patients were profiled using high-throughput sequencing (HTS). The clonal diversity of the TCR repertoires in different groups was analyzed, as well as the frequencies of variable (V), diversity (D) and joining(J) gene segments., Results: ACS patients including UA and AMI, showed reduced TCRβ diversity than NCA patients. ACS patients presented higher levels of clonal expansion. The clonotype overlap of complementarity determining region 3(CDR3) was significantly varied between different groups. A total of 10 V genes and 1 J gene were differently utilized between ACS and NCA patients. We identified some shared CDR3 amino acid sequences that were presented in ACS but not in NCA patients., Conclusions: This study revealed the distinct TCR repertoires in patients with ACS and demonstrated the presence of disease associated T-cell clonotypes. These findings suggested a role of T cells in ACS and provided a new way to explore the mechanisms of ACS.

The TCR is consisted of four chains: α (TCRα), β (TCRβ), γ (TCRγ), and δ (TCRδ) that are present in all jawed vertebrates. Birds are very important in terms of evolutionary aspects of the adaptive immune system, in which it bridges the evolutionary gap between mammals and other vertebrates. To gain better understanding into the genomic organization and complexity of birds' TCR loci, we applied cross-reference error-correction sequencing approach by using Illumina and single-molecule real-time sequencing technology to resequence genomic regions of chicken TCR loci based on 10 mapped bacterial artificial chromosome clones. We did de novo classification of V and J genes for all four chains of the TCR loci according to our sequencing results using the Immunogenetics nomenclature. In sum, we identified 85, 8, and 37 TCR V gene segments in the chicken TCRα/TCRδ , TCRβ , and TCRγ loci, respectively. The phylogenetic analysis showed the Vα 7 and Vα family 4 gene sequences shared greater sequence similarity with mammalian species, whereas the other Vα segment sequences are evolutionary closer with sequences from bony fishes. The organization of chicken TCRβ locus is more similar to fish TCRβ locus over mammalian species, as chicken TCRβ locus has a single translocon of its V-D-J-C and exhibits significantly fewer Vβ gene segments. In this study, we present a highly precise genomic map for chicken TCR loci and phylogenetic relationships of TCR variable gene segments against other animal species and verified the relative stability of the receptor structure during evolutional process., (Copyright © 2020 The Authors.)

Background: Overall survival (OS) is a key endpoint measure in the management of patients with cancer. Immunotherapy has become a dominant strategy in cancer therapy. To investigate the relationship between OS and the immune system, we assessed the role of immune genes in OS in 8648 patients across 22 cancer types., Methods: Gene expression data and clinical information were collected from The Cancer Genome Atlas (TCGA) and cBioPortal. Survival analysis was performed with a Cox proportional hazards regression model., Results: (1) The number of prognostic genes, prognostic immune genes (PIGs) and the hazard ratio (HR) of PIGs in different cancer types all varied greatly; (2) KEGG pathway enrichment analyses indicated that the prognostic genes of 6 cancer types were significantly enriched in multiple (≥5) immune system-related pathways. Of the PIGs in these 6 cancer types, we screened 48 common PIGs in at least 5 cancer types. Eleven out of the 48 PIGs were found to participate in the T cell receptor (TCR) signaling pathway according to the STRING database. Among these genes, ZAP70, CD3E, CD3G, CD3D, and CD247 were part of the TCR 'signal-triggering module'; (3) High expression of the PIGs involved in the TCR signaling pathway was associated with improved OS in 5 cancer types (breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), and sarcoma (SARC)), but was associated with decreased OS in brain lower-grade glioma (LGG)., Conclusions: The TCR signaling pathway played a distinct role in the OS of these 6 cancer types.

Antigen-specific memory T cells persist for years after exposure to a pathogen and provide effective recall responses. Many memory T cell subsets have been identified and differ in abundance throughout tissues. This study focused on CD4 and CD8 memory T cells from paired human lung and lung draining lymph node (LDLN) samples and identified substantial differences in the transcriptional landscape of these subsets, including higher expression of an array of innate immune receptors in lung T cells which were further validated by flow cytometry. Using T cell receptor analysis, we determined the clonal overlap between memory T cell subsets within the lung and within the LDLN, and this was greater than the clonal overlap observed between memory T cell subsets compared across tissues. Our results suggest that lung and LDLN memory T cells originate from different precursor pools, recognize distinct antigens and likely have separate roles in immune responses., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

The genes coding for the antigenic T cell receptor (TR) subunits are assembled in thymocytes from discrete V, D, and J genes by a site-specific recombination process. A tight control of this activity is required to prevent potentially detrimental recombination events. V, D, and J genes are flanked by semi-conserved nucleotide motives called recombination signal sequences (RSSs). V(D)J recombination is initiated by the precise introduction of a DNA double-strand break exactly at the border of the genes and their RSSs by the RAG recombinase. RSSs are therefore physically separated from the coding region of the genes before assembly of a rearranged TR gene. During a high throughput profiling of TRB genes in mice, we identified rearranged TRB genes in which part or all of a flanking RSS was retained in V-D or D-J coding joints. In some instances, this retention of germline DNA resulted from the use of an upstream alternative RSS. However, we also identified TRB sequences where retention of germline DNA occurred in the absence of alternative RSS, suggesting that RAG activity was mis-targeted during recombination. Similar events were also identified in human rearranged TRB and TRG genes. The use of alternative RSSs during V(D)J recombination illustrates the complexity of RAG-RSSs interactions during V(D)J recombination. While the frequency of errors resulting from mis-targeted RAG activity is very low, we believe that these RAG errors may be at the origin of oncogenic translocations and are a threat for genetic stability in developing lymphocytes., (Copyright © 2019 Mika, Kabacik, Badie, Polanska and Candéias.)

Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features 1-3 . When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells 4 . As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease 5 . Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens 6 . We therefore isolated a high-affinity Wilms' Tumor Antigen 1-specific TCR (TCR C4 ) from HLA-A2 + normal donor repertoires, inserted TCR C4 into Epstein-Bar virus-specific donor CD8 + T cells (T TCR-C4 ) to minimize graft-versus-host disease risk and enhance transferred T cell survival 7,8 , and infused these cells prophylactically post-HCT into 12 patients ( NCT01640301 ). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). T TCR-C4 maintained TCR C4 expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse.

Mammals inhabit a wide variety of ecological niches, which in turn can be affected by various ecological factors, especially in relation to immunity. The canonical TRC repertoire ( TRAC, TRBC, TRGC , and TRDC ) codes C regions of T cell receptor chains that form the primary antigen receptors involved in the activation of cellular immunity. At present, little is known about the correlation between the evolution of mammalian TRC genes and ecological factors. In this study, four types canonical of TRC genes were identified from 37 mammalian species. Phylogenetic comparative methods (phyANOVA and PGLS) and selective pressure analyses among different groups of ecological factors (habitat, diet, and sociality) were carried out. The results showed that habitat was the major ecological factor shaping mammalian TRC repertoires. Specifically, trade-off between TRGC numbers and positive selection of TRAC and the balanced evolutionary rates between TRAC and TRDC genes were speculated as two main mechanisms in adaption to habitat and sociality. Overall, our study suggested divergent mechanisms for the evolution of TRCs, prompting mammalian immunity adaptions within diverse niches.

Human cytomegalovirus (HCMV) infection is a leading cause of morbidity and mortality in immunocompromised patients, but no specific therapeutic strategy is effective clinically, despite recent achievements. HCMV-specific T-cell therapy was thought to be helpful for the management of HCMV infection. To conduct a deep exploration, we investigated the possibility of engineering peripheral blood mononuclear cells (PBMCs) from immunocompetent and immunocompromised subjects with specific T-cell receptor (TCR) genes. CD8-positive T cells that specifically bind to NLV pentamers could be generated by transferring TCR genes to PBMCs from immunocompetent and immunocompromised subjects. The generation of functional T cells varied among transduction of different PBMCs. The numbers of IFN-γ-secreting T cells increased significantly in immunocompetent and immunodeficient PBMCs, but were unchanged in immune-reconstituted PBMCs. TCR gene transfer is a potential therapeutic strategy for controlling HCMV infection in immunocompromised patients. The transfer of TCR genes into immunocompetent and immunodeficient PBMCs would be more meaningful in response to HCMV infection than would the transfer into immune-reconstituted PBMCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: High-affinity tumor antigen-specific T-cell receptor (TCR) gene is required to engineer potent T cells for therapeutic treatment of cancer patients. However, discovery of suitable therapeutic TCR genes is hampered by the fact that naturally occurring tumor antigen-specific TCRs are generally of low-affinity, and artificial modification of TCRs can mediate cross-reactivity to other antigens expressed in normal tissues. Here, we discovered a naturally occurring T-cell clone which expressed high-affinity HLA-A*02:01 (A*02)-restricted TCR against NY-ESO-1 from a patient who had NY-ESO-1-expressing ovarian tumor., Methods: A*02-restricted NY-ESO-1-specific T-cell clones were established from peripheral blood of patients who had NY-ESO-1-expressing ovarian tumors. TCR α and β chain genes were retrovirally transduced into polyclonally activated T cells. Phenotype and function of the parental and TCR-transduced T cells were analyzed by flow cytometry, ELISA and cytotoxicity assay. In vivo therapeutic efficacy was investigated in a xenograft model using NOD/SCID/IL-2Rγ-deficient (NSG) mice., Results: A rare population of NY-ESO-1-specific T cells, which we named 19305DP, expressed cell surface CD4, CD8α, and CD8β but not CD56 and recognized A*02 + NY-ESO-1 + cancer cell lines in a CD4- and CD8-independent manner. 19305DP showed a gene expression profile that is consistent with a mixed profile of CD4 + and CD8 + single-positive T cells. Both CD4 + and CD8 + T cells that were retrovirally transduced with 19305DP-derived TCR gene (19305DP-TCR) showed strong reactivity against A*02 + NY-ESO-1 + cancer cells, whereas TCR genes from the conventional A*02-restricted NY-ESO-1-specific CD8 + single-positive T-cell clones functioned only in CD8 + T cells. Both 19305DP-TCR gene-engineered CD4 + and CD8 + T cells eliminated A*02 + NY-ESO-1 + tumor xenografts in NSG mice. Finally, based on reactivity against a series of alanine-substituted peptides and a panel of normal human tissue-derived primary cells, 19305DP-TCR was predicted to have no cross-reactivity against any human non-NY-ESO-1 proteins., Conclusion: Together, our results indicate that the naturally occurring 19305DP-TCR derived from CD4 + CD8 + double-positive αβ T cells, is a promising therapeutic TCR gene for effective and safe adoptive T-cell therapy in A*02 + patients with NY-ESO-1-expressing tumor.

Squamata are reptiles that diverged from mammals 300 million years ago. During this period, the immunoglobulin (IG) and T lymphocyte receptor (TCR) genes evolved parallel to mammals. However, unlike mammals whose IG/TCR locus has retained a constant structure throughout evolution, Squamata have witnessed duplications, losses, and/or gains in the domains of their immunoglobulin genes. The recent genome sequencing of Shinisaurus crocodilurus, a representative species of the oldest reptiles, provides an opportunity to contrast the structure of IG and TCR genes from previously studied Squamata. This study revealed ten immunoglobulin genes: five genes for immunoglobulin M (IgM), two for immunoglobulin D (IgD), one for immunoglobulin D2 (IgD2), and two for immunoglobulin Y (IgY). As in other Squamata, there are genes for the λ light chain (IGLV) but not for the κ chain (IGKV). Here, the data shows that in some IgM genes, the cysteine needed to bind the λ chains does not exist, but we present evidence for possible non-covalent binding to the light chain. With respect to TCR, one gene is detected for the α constant chain (TRAC) and two genes for the β constant chain (TRBC); one of which is located in the locus of the variable regions of the heavy chain. As in the rest of the Squamata, genes for the γ/δ T cell receptor were not found. The V gene repertoire is found to be consistent with all other Squamata with few V genes for beta chain of TCR., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aims: Refractory coeliac disease type II (RCDII), a rare complication of coeliac disease (CD) associated with high morbidity, requires identification of a clonal population of phenotypically aberrant intraepithelial lymphocytes (IELs) for diagnosis. However, data regarding the frequency and significance of clonal T cell receptor (TCR) gene rearrangements (TCR-GRs) in small bowel (SB) biopsies of patients without RCDII are limited., Methods: We analysed results of TCR-GR analyses performed on SB biopsies at our institution over a 3-year period, which were obtained from eight active CD, 172 CD on gluten-free diet (GFD), 33 RCDI, and three RCDII patients and 14 patients without CD. TCR-GR patterns were divided into clonal, polyclonal and prominent clonal peaks (PCPs) and these patterns were correlated with clinical and pathological features., Results: Clonal TCR-GR products were detected in biopsies from 67% of patients with RCDII, 17% of patients with RCDI and 6% of patients with GFD. PCPs were observed in all disease phases (range 12%-33%). There was no significant difference in the TCR-GR patterns between the non-RCDII disease categories (p=0.39). A higher frequency of surface CD3(-) IELs was noted in cases with clonal TCR-GR, but the PCP pattern did not show associations with any clinical or pathological feature. Persistence of clonal or PCP pattern on repeat biopsy was seen for up to 2 years without evidence of RCDII., Conclusions: Clonal TCR-GRs are not infrequent in cases lacking features of RCDII, while PCPs are frequent in all disease phases. TCR-GR results should be assessed in conjunction with immunophenotypic, histological and clinical findings for appropriate diagnosis and classification of RCD., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Objective: To investigate the clinicopathological features of EBV-positive T/NK cell lymphoproliferative diseases (EBV(+) T/NK-LPD). Methods: The clinical characteristics of 156 cases of EBV(+) T/NK-LPD were collected from August 2002 to March 2015 at Beijing Friendship Hospital, Capital Medical University. Immunohistochemical staining, EBER in situ hybridization and clonal analysis of TCR gene were performed. All patients were followed up. Results: There were 106 male and 50 female patients; patients' age ranged from 1 to 75 years (median 20 years). The course of the diseases before diagnosis ranged from 2 to 540 months (median 20 months). Fever was noted in 122 patients (78.2%), 108 patients had lymphadenopathy (69.2%), and 75 patients had hepatosplenomegaly (48.1%). Thirty-three cases were grade 1, 68 cases were grade 2, and 55 cases were grade 3. TCR gene arrangement analysis was performed in 45 cases, and 33 cases (73.3%) showed clonal rearrangement. The follow-up period ranged from 1-134 months, and 44 patients (28.2%) died. There was a trend of increased death rate associated with increasing grade ( P >0.05). Conclusions: There are many types of EBV(+) T/NK-LPD, and they can be classified as systemic, relatively localized and localized. The prognosis should be based on a comprehensive analysis of pathology and clinical data. There is no significant correlation between morphological grade and mortality. An important goal of therapy is to prevent serious complications.

Wilms' tumor 1 (WT1) is constantly expressed in leukemic cells of acute leukemia and myelodysplastic syndrome (MDS). A T-cell receptor (TCR) that specifically reacts with WT1 peptide in the context of HLA-A*24:02 has been identified. We conducted a first-in-human trial of TCR-gene transduced T-cell (TCR-T-cell) transfer in patients with refractory acute myeloblastic leukemia (AML) and high-risk MDS to investigate the safety and cell kinetics of the T cells. The WT1-specific TCR-gene was transduced to T cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes. The T cells were transferred twice with a 4-week interval in a dose-escalating design. After the second transfer, sequential WT1 peptide vaccines were given. Eight patients, divided into 2 dose cohorts, received cell transfer. No adverse events of normal tissue were seen. The TCR-T cells were detected in peripheral blood for 8 weeks at levels proportional to the dose administered, and in 5 patients, they persisted throughout the study period. The persisting cells maintained ex vivo peptide-specific immune reactivity. Two patients showed transient decreases in blast counts in bone marrow, which was associated with recovery of hematopoiesis. Four of 5 patients who had persistent T cells at the end of the study survived more than 12 months. These results suggest WT1-specific TCR-T cells manipulated by ex vivo culture of polyclonal peripheral lymphocytes survived in vivo and retained the capacity to mount an immune reaction to WT1. This trial was registered at www.umin.ac.jp as #UMIN000011519., (© 2017 by The American Society of Hematology.)

The adoptive genetic transfer of T cell receptors (TCRs) has been shown to be overall feasible and offer clinical potential as a treatment for different types of cancer. However, this promising clinical approach is limited by the serious potential consequence that exogenous TCR mispairing with endogenous TCR chains may lead to the risk of self-reactivity. In the present study, domain‑exchange and three‑dimensional modeling strategies were used to create a set of chimeric TCR variants, which were used to exchange the partial or complete constant region of αβTCR with corresponding γδTCR domains. The expression, assembly and function of the chimeric TCR variants were examined in Jurkat T cells and peripheral mononuclear blood cells (PBMCs). Genetically‑encoded chimeras were fused with a pair of fluorescent proteins (ECFP/EYFP) to monitor expression and the pairing between chimeric TCRα chains and TCRβ chains. The fluorescence energy transfer based on confocal laser scanning microscopy showed that the introduction of γδTCR constant sequences into the αβTCR did not result in a global reduction of mispairing with endogenous TCR. However, the TCR harboring the immunoglobulin‑like domain of the γδTCR constant region (i.e., TCR∆IgC), showed a higher expression and preferential pairing, compared with wild‑type (wt)TCR. The function analysis showed that TCR∆IgC exhibited the same levels of interferon-γ production and cytotoxic activity, compared with wtTCR. Furthermore, these modified TCR-transduced T cells retained the classic human leukocyte antigen restriction of the original TCR. The other two chimeric TCRs, had either exchange of the cp+tm+ic domain or exchange of the whole C domain (Fig. 1). Ultimately, exchange of these domains demonstrated defective function in the transduced T cells. Taken together, these findings may provide further understanding of the γδTCR constant domain with implications for the improvement of TCR gene transfer therapy.

Understanding tumor-resident T cells is important for cancer prognosis and treatment options. Conventional, solid tumor specimen exome files can be searched directly for recombined T cell receptor (TcR)-α segments; RNASeq files can include TcR-β VDJ recombinations. To learn whether there are medically relevant uses of exome-based detection of TcR V(D)J recombinations in the tumor microenvironment, we searched cancer genome atlas and Moffitt Cancer Center, tumor specimen exome files for TcR-β, TcR-γ, and TcR-δ recombinations, for bladder and stomach cancer. We found that bladder cancer exomes with productive TcR-β recombinations had a significant association with No Subsequent Tumors and a positive response to drug treatments, with p < 0.004, p < 0.05, and p < 0.004, depending on the sample sets examined. We also discovered the opportunity to detect productive TcR-γ and TcR-δ recombinations in the tumor microenvironment, via the tumor specimen exome files.

The T-cell receptor (TCR) is the key player within the so called immunological synapse and the analysis of its repertoire offers a picture of both versatility and wideness of the whole immune T-cell compartment. Among the different approaches applied to its study the so-called spectratyping identifies the pattern of the third complementarity determining region (CDR3) length distribution in each one of the beta variable (TRBV) subfamilies encoded by the corresponding genes. This technique consists in a CDR3 fragment analysis through capillary electrophoresis, performed after cell separation, RNA extraction and reverse transcriptase PCR. This review will run through the most relevant studies which have tried to dissect the TCR repertoire usage in patients with different immune-mediated and infective diseases as well as solid or haematologic malignancies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy., Competing Interests: MTB, MHG and DB: Co-inventor on a patent application concerning the described technology (USPTO patent application 20150197771). The other author declares that no competing interests exist.

Real-time quantitative polymerase chain reaction (RQ-PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow-up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ-PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases. The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value., (© 2016 John Wiley & Sons Ltd.)

Somatic mosaicism, the presence of genetically distinct cells within an organism, has been increasingly associated with human morbidity, ranging from being a cause of rare syndromes to a risk factor for common disorders such as malignancy and cardiovascular disease. Previous studies interrogating the normal prevalence of somatic mosaicism have focused on adults. We here present an estimate of the baseline frequency of somatic mosaic copy number variation (CNV) at the time around birth, by sampling eight different organs from a total of five fetuses and newborns. Overall we find a significantly lower frequency of organ specific (i.e. mosaic) CNVs as compared to adults (p = 0.003; Mann-Whitney U-test). The rate of somatic CNV in adults has been estimated to around 2.2 CNV per organ assayed. In contrast, after stringent filtering, we found no organ-private CNVs in fetuses or newborns with exception of the thymus. This organ exhibited a specific genome profile in the form of deletions resulting from polyclonal T-cell receptor rearrangements. This implies that somatic non-immune related CNVs, if present at birth, are typically confined to very small cell populations within organs.

Background: Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma (CTCL), followed by CD30+ lymphoproliferative disorders, including lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large cell lymphoma (pcALCL). The objective was to report on a series of patients with different types of CTCL at different times in their clinical course, with a focus on clonality studies., Methods: Four patients with multiple diagnoses of CTCLs were identified. The clinical information, treatment interventions, and histopathology were reviewed. T-cell receptor (TCR) gene rearrangement studies were performed on all available specimens., Results: The four patients carried diagnoses of: (1) pcALCL and MF; (2) pcALCL, LyP, and pcALCL; (3) LyP, MF, and pcALCL; (4) LyP, pcALCL, and MF; each with characteristic presentation and histopathologic findings. The results of the TCR polymerase chain reaction showed that all tumors expressed and retained a TCR clone(s) as follows: (1) biallelic clone; (2) single clone; (3) biallelic clone with additional clone; and (4) single clone, respectively., Conclusion: We report a series of four cases of individual patients with coexisting diagnoses of some combination of MF, LyP, and pcALCL, whose lesions presented in nontraditional sequence and demonstrated a retained clone by gene rearrangement analysis., (© 2015 The International Society of Dermatology.)

T-cell lymphoma pathogenesis and classification have, until recently, remained enigmatic. Recently performed whole-exome sequencing and gene-expression profiling studies have significant implications for their classification and treatment. Recurrent genetic modifications in antigen ("signal 1"), costimulatory ("signal 2"), or cytokine receptors ("signal 3"), and the tyrosine kinases and other signaling proteins they activate, have emerged as important therapeutic targets in these lymphomas. Many of these genetic modifications do not function in a cell-autonomous manner, but require the provision of ligand(s) by constituents of the tumor microenvironment, further supporting the long-appreciated view that these lymphomas are dependent upon and driven by their microenvironment. Therefore, the seemingly disparate fields of genomics and immunology are converging. A unifying "3 signal model" for T-cell lymphoma pathogenesis that integrates these findings will be presented, and its therapeutic implications briefly reviewed., (© 2015 Wiley Periodicals, Inc.)

To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/β genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.

Purpose: The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells., Experimental Design: T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells., Results: We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines., Conclusions: These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers., (©2015 American Association for Cancer Research.)

Background & Aims: Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice., Methods: We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells., Results: Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice., Conclusions: We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoHtg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoHtg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4+ CD8+ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2-/-RhoHtg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.

Among Epstein-Barr virus (EBV)-positive cytotoxic T/NK-cell lymphoma, there are only a few reports on the clinicopathologic features of patients with primary nodal presentation (nodal EBV cytotoxic T-cell lymphoma [CTL]). Here, we compared the clinicopathologic profiles of 39 patients with nodal EBV CTL with those of 27 cases of "extranasal" NK/T-cell lymphoma of nasal type (ENKTL), especially addressing their T-cell receptor (TCR) phenotype. Histologically, 22 of 39 nodal EBV CTL cases (56%) were unique in having centroblastoid appearance, which was contrasted with the lower incidence of this feature in ENKTL (15%, P=0.001). In contrast, pleomorphic appearance was more frequently seen in ENKTL than in nodal EBV CTL (67% vs. 23%, P=0.001). Thirty-three of 39 nodal EBV CTL cases (85%) were of T-cell lineage on the basis of TCR expression and/or TCRγ gene rearrangement; in detail, 18 cases (46%) were TCRβ positive (αβ T), 5 (13%) were TCRγ and/or δ positive (γδ T), and 10 (26%) were TCR-silent type with clonal TCRγ gene rearrangement but no expression of TCRβ, γ, or δ. These results were clearly contrasted by a lower incidence of T-cell lineage in ENKTL (7 cases, 26%, P<0.001). Notably, the survival time of the 5 nodal lymphoma patients with γδ T-cell phenotype was within 3 months, which was inferior to those of αβ T and TCR-silent types (P=0.003), and 3 of those with available clinical information were all found to be associated with autoimmune diseases. These data suggest that nodal EBV CTL is distinct from ENKTL.

Background/aim: Wilms' tumor gene 1 (WT1) product is a pan-tumor-associated antigen. We previously identified WT1 protein-derived promiscuous helper peptide, WT1332. Therefore, isolation and characterization of the WT1332-specific T-cell receptors (TCRs) are useful to develop broadly applicable TCR gene-based adoptive immunotherapy., Materials and Methods: A novel HLA-DRB1*04:05-restricted WT1332-specific TCR gene was cloned and transduced into human CD4+ T-cells by using a lentiviral vector., Results: The WT1332-specific TCR-transduced CD4+ T-cells showed strong proliferation and Th1-cytokine production in an HLA-DRB1*04:05-restricted, WT1332-specific manner. Furthermore, the WT1332-specific TCR-transduced CD4+ T-cells could lyse HLA-DRB1*04:05-positive, WT1-expressing leukemia cells in vitro., Conclusion: The novel TCR gene cloned here should be a promising tool to develop adoptive immunotherapy of WT1332-specific TCR-transduced CD4+ T-cells for the treatment of WT1-expressing cancer, such as leukemia., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

IMGT(®), the international ImMunoGeneTics information system(®)(http://www.imgt.org) is the global reference in immunogenetics and immunoinformatics. By its creation in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS), IMGT(®) marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT(®) is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH) and proteins of the IgSF and MhSF superfamilies. IMGT(®) is built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences and 3D structures. The concepts include the IMGT(®) standardized keywords (identification), IMGT(®) standardized labels (description), IMGT(®) standardized nomenclature (classification), IMGT unique numbering and IMGT Colliers de Perles (numerotation). IMGT(®) comprises 7 databases, 17 online tools and 15,000 pages of web resources, and provides a high-quality and integrated system for analysis of the genomic and expressed IG and TR repertoire of the adaptive immune responses, including NGS high-throughput data. Tools and databases are used in basic, veterinary and medical research, in clinical applications (mutation analysis in leukemia and lymphoma) and in antibody engineering and humanization. The IMGT/mAb-DB interface was developed for therapeutic antibodies and fusion proteins for immunological applications (FPIA). IMGT(®) is freely available at http://www.imgt.org., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Biomarkers of T-cell receptor excision circles (TRECs) and immunoglobulin κ-deleting recombination excision circles (KRECs) reflect naïve T and B cell emigrants. This study assessed the biomarkers in patients with primary immunodeficiency diseases (PIDs) to determine the lymphocyte output disturbance and the correlation to lymphocytes., Methods: A standard plasmid was constructed to calculate TRECs and KRECs in 250 ng genomic DNA from whole blood of PIDs patients. These were correlated to naïve and memory lymphocytes for further classification and adequate treatment., Results: In 69 studied patients, the low TRECs mainly included those with severe combined T and B immunodeficiency (SCID, 7/8), combined immunodeficiency (CID, 4/4), and common variable immunodeficiency (CVID, 6/7). The diminished KRECs was in SCID (4/8), CID (4/4), CVID (7/7), Bruton's tyrosine kinase mutation (Btk, 3/4), anti-B cell deletion (by anti-CD20 antibody in 1), and Behçet syndrome under steroid treatment (1). The TRECs and KRECs positively correlated to absolute naïve T (CD4 + CD45RA+) and naïve B (CD19 + CD27-), and to memory B (CD19 + CD27+) numbers, respectively., Conclusion: This study validates that low TRECs and KRECs values reflect low naïve T and B lymphocytes in 'combined immunodeficiencies' and in some CVID patients with the potential to develop the CID phenotype.

Objective: To investigate the cytotoxicity of normal CD8(+) T lymphocytes retrovirally transduced with WT1 peptide-specific T-cell receptor (TCR) genes against human lung cancer cells., Methods: HLA-A*2402-restricted and WT1 peptide-specific TCR-α/β genes were cloned from a cytotoxic T lymphocyte clone and inserted into a retroviral TCR expression vector. The cytotoxicity of normal peripheral CD8⁺ T cells transduced with the WT1-TCR genes against human lung cancer cells was evaluated using a standard ⁵¹Cr release assay., Results: The WT1-TCR gene-modified T cells recognized the peptide-pulsed target cells but not the non-pulsed cells. TCR-redirected CD8⁺ T cells lysed WT1-overexpressing human lung cancer cells in an HLA-A*2402-restricted manner, but did not kill normal cells positively expressing HLA-A*2402., Conclusion: These data demonstrate the feasibility of adoptive immunotherapy with TCR-redirected T cell for the treatment of lung cancer.

The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BAC probes and by sequencing of PCR products, and the frequencies of recombination were assessed in horses and 4 other equids. The presence of several trans-rearrangement products between the TRA and TRG genes was verified by PCR in all investigated equids. Frequencies of trans-rearrangements in horses are higher than in humans, and colocalization of the TCR genes on the same chromosome increases the incidence of trans-rearrangements between them. The orientation of the TCR genes does not impact interlocus recombination itself but does affect the viability of cells carrying its products and consequently the number of trans-rearrangements observed in lymphocytes.

Antigen-specific T cell therapy, or T cell receptor (TCR) gene therapy, is a promising immunotherapy for infectious diseases and cancers. However, a suitable rapid and direct screening system for antigen-specific TCRs is not available. Here, we report an efficient cloning and functional evaluation system to determine the antigen specificity of TCR cDNAs derived from single antigen-specific human T cells within 10 d. Using this system, we cloned and analyzed 380 Epstein-Barr virus-specific TCRs from ten healthy donors with latent Epstein-Barr virus infection and assessed the activity of cytotoxic T lymphocytes (CTLs) carrying these TCRs against antigenic peptide-bearing target cells. We also used this system to clone tumor antigen-specific TCRs from peptide-vaccinated patients with cancer. We obtained 210 tumor-associated antigen-specific TCRs and demonstrated the cytotoxic activity of CTLs carrying these TCRs against peptide-bearing cells. This system may provide a fast and powerful approach for TCR gene therapy for infectious diseases and cancers.

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.