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Background: Pityriasis lichenoides (PL) is a papulosquamous disease that affects both adults and children. Previous studies have shown a subset of this entity to have clonal T-cell populations via PCR-based assays. In this study, we sought to implement next-generation sequencing (NGS) as a more sensitive and specific test to examine for T-cell clonality within the pediatric population., Methods: We identified 18 biopsy specimens from 12 pediatric patients with clinical and histopathologic findings compatible with PL. Patient demographics, clinical features, management, and histopathologic findings were reviewed. All specimens were analyzed for clonality with NGS of T-cell receptor beta (TRB) and gamma (TRG) genes., Results: Of the 12 patients, 9 (75%) had complete resolution of lesions at the time of data collection (mean follow-up 31 months). The remaining three patients significantly improved with methotrexate (with or without acitretin). Interestingly, 7 of 12 patients (58%) and 9 of 17 biopsy specimens (53%) showed evidence of T-cell clonality. Two patients showed matching TRB clones from different anatomic sites., Conclusions: T-cell clonality is a common finding in PL, probably representing a "reactive clonality" rather than a true lymphoproliferative disorder. Clonality alone cannot be used as a means to distinguish PL from lymphomatoid papulosis or cutaneous lymphoma., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Gluten-specific CD4+ T cells are drivers of celiac disease (CeD). Previous studies of gluten-specific T-cell receptor (TCR) repertoires have found public TCRs shared across multiple individuals, biased usage of particular V-genes and conserved CDR3 motifs. The CDR3 motifs within the gluten-specific TCR repertoire, however, have not been systematically investigated. In the current study, we analyzed the largest TCR database of gluten-specific CD4+ T cells studied so far consisting of TCRs of 3122 clonotypes from 63 CeD patients. We established a TCR database from CD4+ T cells isolated with a mix of HLA-DQ2.5:gluten tetramers representing four immunodominant gluten epitopes. In an unbiased fashion we searched by hierarchical clustering for common CDR3 motifs among 2764 clonotypes. We identified multiple CDR3α, CDR3β, and paired CDR3α:CDR3β motif candidates. Among these, a previously known conserved CDR3β R-motif used by TRAV26-1/TRBV7-2 TCRs specific for the DQ2.5-glia-α2 epitope was the most prominent motif. Furthermore, we identified the epitope specificity of altogether 16 new CDR3α:CDR3β motifs by comparing with TCR sequences of 231 T-cell clones with known specificity and TCR sequences of cells sorted with single HLA-DQ2.5:gluten tetramers. We identified 325 public TCRα and TCRβ sequences of which 145, 102 and 78 belonged to TCRα, TCRβ and paired TCRαβ sequences, respectively. While the number of public sequences was depended on the number of clonotypes in each patient, we found that the proportion of public clonotypes from the gluten-specific TCR repertoire of given CeD patients appeared to be stable (median 37%). Taken together, we here demonstrate that the TCR repertoire of CD4+ T cells specific to immunodominant gluten epitopes in CeD is diverse, yet there is clearly biased V-gene usage, presence of public TCRs and existence of conserved motifs of which R-motif is the most prominent., Competing Interests: SD-K, LR, RN, AC, KL, S-WQ, GS and LS are holders of a patent application entitled "Method of diagnosing celiac disease" (US20210010077A1) on the use of the gluten-specific T cell receptor sequences described in the current work for diagnosis of celiac disease., (Copyright © 2021 Dahal-Koirala, Risnes, Neumann, Christophersen, Lundin, Sandve, Qiao and Sollid.)

Restoration of T cell repertoire diversity after allogeneic bone marrow transplantation (allo-BMT) is crucial for immune recovery. T cell diversity is produced by rearrangements of germline gene segments (V (D) and J) of the T cell receptor (TCR) α and β chains, and selection induced by binding of TCRs to MHC-peptide complexes. Multiple measures were proposed for this diversity. We here focus on the V-gene usage and the CDR3 sequences of the beta chain. We compared multiple T cell repertoires to follow T cell repertoire changes post-allo-BMT in HLA-matched related donor and recipient pairs. Our analyses of the differences between donor and recipient complementarity determining region 3 (CDR3) beta composition and V-gene profile show that the CDR3 sequence composition does not change during restoration, implying its dependence on the HLA typing. In contrast, V-gene usage followed a time-dependent pattern, initially following the donor profile and then shifting back to the recipients' profile. The final long-term repertoire was more similar to that of the recipient's original one than the donor's; some recipients converged within months, while others took multiple years. Based on the results of our analyses, we propose that donor-recipient V-gene distribution differences may serve as clinical biomarkers for monitoring immune recovery.

Within each individual, the adaptive immune system generates a repertoire of cells expressing receptors capable of recognizing diverse potential pathogens. The theoretical diversity of the T-cell receptor (TCR) repertoire exceeds the actual size of the T-cell population in an individual by several orders of magnitude - making the observation of identical TCRs in different individuals extremely improbable if all receptors were equally likely. Despite this disparity between the theoretical and the realized diversity of the repertoire, these 'public' receptor sequences have been identified in autoimmune, cancer and pathogen interaction contexts. Biased generation processes explain the presence of public TCRs in the naive repertoire, but do not adequately explain the different abundances of these public TCRs. We investigate and characterize the distribution of genomic TCR-β sequences of naive CD8+ T cells from three genetically identical mice, comparing non-productive (non-functional sequences) and productive sequences. We find public TCR-β sequences at higher abundances compared with unshared sequences in the productive, but not in the non-productive, repertoire. We show that neutral processes such as recombination biases, codon degeneracy and generation probability do not fully account for these differences, and conclude that thymic or peripheral selection plays an important role in increasing the abundances of public TCR-β sequences., (© 2021 John Wiley & Sons Ltd.)

Whether T cells promote bone loss following immobilization after spinal cord injury (SCI) remains undetermined. Therefore, wild-type (WT) and T cell-deficient (Tcrb -/- ) male mice underwent sham or contusion SCI to cause hindlimb paralysis. Femurs were isolated and distal and midshaft regions were evaluated by microcomputed tomography scanning. Bone marrow (BM) levels of bone turnover markers, as well as receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG), were measured by ELISA. At 2 weeks post-SCI, immobilization resulted in marked reduction in trabecular fractional bone volume (55%), thickness (40%), connectivity, and cortical thickness only in the Tcrb -/- animals (interaction with P < 0.05). BM analysis revealed lower bone formation (procollagen type 1 intact N-terminal propeptide), higher bone resorption (tartrate-resistant acid phosphatase-5b), and a higher RANKL/OPG ratio in the Tcrb -/- SCI animals. At 5 weeks post-SCI, while both WT and Tcrb -/- paralyzed animals showed deterioration of all indices of bone structure, they were more severe in Tcrb -/- animals. In summary, unlike other skeletal disorders, loss of αβ T cells compromises, rather than preserves, skeletal integrity under conditions of immobilization., (Published 2020. This article is a U.S. government work and is in the public domain in the USA.)

T cells must display diversity regarding both the cell state and T-cell receptor (TCR) repertoire to provide effective immunity against pathogens; however, the generation and evolution of cellular T-cell heterogeneity in the adaptive immune system remains unclear. In the present study, a combination of multiplex PCR and immune repertoire sequencing (IR-seq) was used for a standardized analysis of the TCR β-chain repertoire of CD4 + naive, CD4 + memory, CD8 + naive and CD8 + memory T cells. We showed that the T-cell subsets could be distinguished from each another with regard to the TCR β-chain (TCR-β) diversity, CDR3 length distribution and TRBV usage, which could be observed both in the preselection and in the post-selection repertoire. Moreover, the Dβ-Jβ and Vβ-Dβ combination patterns at the initial recombination step, template-independent insertion of nucleotides and inter-subset overlap were consistent between the pre- and post-selection repertoires, with a remarkably positive correlation. Taken together, these results support differentiation of the CD4 + and CD8 + T-cell subsets prior to thymic selection, and these differences survived both positive and negative selection. In conclusion, these findings provide deeper insight into the generation and evolution of TCR repertoire generation., (© 2020 John Wiley & Sons Ltd.)

EBV is one of the most common viruses found in humans and is prototypic of a persistent viral infection characterized by periods of latency. Across many HLA class I molecules, the latent-specific CD8 + T cell response is focused on epitopes derived from the EBNA-3 protein family. In the case of HLA-B*07:02 restriction, a highly frequent class I allele, the T cell response is dominated by an epitope spanning residues 379-387 of EBNA-3 (RPPIFIRRL [EBV RPP ]). However, little is known about either the TCR repertoire specific for this epitope or the molecular basis for this observed immunodominance. The EBV RPP CD8 + T cell response was common among both EBV-seropositive HLA-B*07:02 + healthy and immunocompromised individuals. Similar TCRs were identified in EBV RPP -specific CD8 + T cell repertoires across multiple HLA-B7 + individuals, indicating a shared Ag-driven bias in TCR usage. In particular, TRBV4-1 and TRAV38 usage was observed in five out of six individuals studied. In this study, we report the crystal structure of a TRBV4-1 + TCR-HLA-B*07:02/EBV RPP complex, which provides a molecular basis for the observed TRBV4-1 bias. These findings enhance our understanding of the CD8 + T cell response toward a common EBV determinant in HLA-B*07:02 + individuals., (Copyright © 2020 by The American Association of Immunologists, Inc.)

During human pregnancy, proinflammatory responses in the placenta can cause severe fetal complications, including growth restriction, preterm birth, and stillbirth. Villitis of unknown etiology (VUE), an inflammatory condition characterized by the infiltration of maternal CD8 + T cells into the placenta, is hypothesized to be secondary to either a tissue rejection response to the haploidentical fetus or from an undiagnosed infection. In this study, we characterized the global TCR β-chain profile in human T cells isolated from placentae diagnosed with VUE compared with control and infectious villitis-placentae by immunoSEQ. Immunosequencing demonstrated that VUE is driven predominantly by maternal T cell infiltration, which is significantly different from controls and infectious cases; however, these T cell clones show very little overlap between subjects. Mapping TCR clones to common viral epitopes (CMV, EBV, and influenza A) demonstrated that Ag specificity in VUE was equal to controls and significantly lower than CMV-specific clones in infectious villitis. Our data indicate VUE represents an allograft response, not an undetected infection. These observations support the development of screening methods to predict those at risk for VUE and the use of specific immunomodulatory therapies during gestation to improve outcomes in affected fetuses., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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 thymus is the main organ of the lymphatic system, in which T cells undergo a rigorous selection to ensure that their receptors (TCRs) will be functional and will not react against the self. Genes encoding for TCR chains are fragmented and must be rearranged by a process of somatic recombination generating TCR rearrangement excision circles (TRECs). We recently documented coxsackievirus B4 (CV-B4) infection of Swiss albino mouse thymus in the course of in utero transmission. In the current study, we intended to evaluate thymic output in this experimental model. For this purpose, pregnant Swiss albino mice were inoculated with CV-B4 at day 10 or 17 of gestation, and thymus and spleen were sampled from offspring at different time points and then subjected to quantification of TREC molecules and Ptk7 gene expression. Results showed a pronounced effect of in utero CV-B4 infection on the thymus with an increase in the cellularity and, consequently, the weight of the organ. sj and DβTREC analysis, by real-time PCR, revealed a significant decrease following CV-B4 infection compared to controls, a decrease which gets worse as time goes by, both in the thymus and in the periphery. Those observations reflect a disturbance in the export of T cells to the periphery and their accumulation within the thymus. The evaluation of Ptk7 transcripts in the thymus, for its part, showed a decrease in expression, especially following an infection at day 10 of gestation, which supports the hypothesis of T cell accumulation in a mature stage in the thymus. The various effects observed correlate either negatively or positively with the viral load in the thymus and spleen. Disruption in thymic export may indeed interfere with T cell maturation. We speculate that this may lead to a premature release of T cells and the possibility of circulating autoreactive or proliferation-impaired T cell clones., (Copyright © 2020 Halouani, Jmii, Bodart, Michaux, Renard, Martens, Aouni, Hober, Geenen and Jaïdane.)

Based on the latest assembly of the red jungle fowl (Gallus gallus) genome sequence, we characterized the detailed genomic organization of the T cell receptor beta (TCRβ) locus of chicken. The chicken TCRβ locus spans approximately 210 kb, and is organized in a typical translocon organization as previously reported. Within this locus, a total of 16 germline Vβ gene segments were classified into three subgroups, containing 11, four, and one members, respectively. Phylogenetic analysis revealed that the chicken Vβ3.1 segment was homologous with the duck Vβ1 subgroup, and further clustered with Vβ  segments from reptiles but not amphibians. We also identified nine protease serine 1 (PRSS1) and three protease serine 2 (PRSS2) genes, which were interspersed within the chicken TCRβ locus. Dot-plot analysis of the chicken TCRβ locus against itself revealed that the 5' part of the locus had arisen through a series of tandem duplication events. The homology units were composed of one Vβ1 segment followed by a PRSS1 gene, or one Vβ2 segment followed by a PRSS2 gene. This duplication pattern, in which the Vβ segments and trypsinogen genes form a duplication unit, was unique to TCRβ loci of chicken and duck, but not observed in TCRβ loci of other tetrapods studied thus far. By analyzing the cloned TCRβ cDNA sequences, we found that the usage pattern of Vβ segments was consistent with the results of previous studies. These studies showed that members of the Vβ1 subgroup are preferentially utilized in V-D-J recombination. Furthermore, we found that the Vβ3.1 segment participated into V-D-J recombination, but at a very low frequency. The length distribution of the chicken complementarity-determining region 3β (CDR3β) showed a tendency similar to that observed for the duck., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mammalian TCRβ loci contain 30 Vβ gene segments upstream and in the same transcriptional orientation as two DJCβ clusters, and a downstream Vβ (TRBV31) in the opposite orientation. The textbook view is upstream Vβs rearrange only by deletion and TRBV31 rearranges only by inversion to create VβDJCβ genes. In this study, we show in mice that upstream Vβs recombine through inversion to the DJCβ2 cluster on alleles carrying a preassembled Trbv31 -DJCβ1 gene. When this gene is in-frame, Trbv5 evades TCRβ-signaled feedback inhibition and recombines by inversion to the DJCβ2 cluster, creating αβ T cells that express assembled Trbv5 -DJCβ2 genes. On alleles with an out-of-frame Trbv31 -DJCβ1 gene, most upstream Vβs recombine at low levels and promote αβ T cell development, albeit with preferential expansion of Trbv1 -DJβ2 rearrangements. Finally, we show wild-type Tcrb alleles produce mature αβ T cells that express upstream Vβ peptides in surface TCRs and carry Trbv31 -DJβ2 rearrangements. Our study indicates two successive inversional Vβ-to-DJβ rearrangements on the same allele can contribute to the TCRβ repertoire., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Progressive multifocal leukoencephalopathy (PML) is a rare, potentially devastating myelin-degrading disease caused by the JC virus. PML occurs preferentially in patients with compromised immune system, but has been also observed in multiple sclerosis (MS) patients treated with disease-modifying drugs. We characterized T and B cells in 5 MS patients that developed PML, 4 during natalizumab therapy and one after alemtuzumab treatment, and in treated patients who did not develop the disease. Results revealed that: i) thymic and bone marrow output was impaired in 4 out 5 patients at the time of PML development; ii) T-cell repertoire was restricted; iii) clonally expanded T cells were present in all patients. However, common usage or pairings of T-cell receptor beta variable or joining genes, specific clonotypes or obvious "public" T-cell response were not detected at the moment of PML onset. Similarly, common restrictions were not found in the immunoglobulin heavy chain repertoire. The data indicate that no JCV-related specific T- and B-cell expansions were mounted at the time of PML. The current results enhance our understanding of JC virus infection and PML, and should be taken into account when choosing targeted therapies.

In multiple tumor types, prediction of response to immune therapies relates to the presence, distribution and activation state of tumor infiltrating lymphocytes (TILs). Although such therapies are, to date, unsuccessful in gliomas, little is known on the immune contexture of TILs in these tumors. We assessed whether low and high-grade glioma (LGG and HGG, grade II and IV respectively) differ with respect to number, location and tumor reactivity of TILs; as well as expression of molecules involved in the trafficking and activation of T cells. Intra-tumoral CD8 T cells were quantified by flow cytometry (LGG: n = 12; HGG: n = 8) and immunofluorescence (LGG: n = 28; HGG: n = 28). Neoantigen load and expression of Cancer Germline Antigens (CGAs) were assessed using whole exome sequencing and RNA-seq. TIL-derived DNA was sequenced and the variable domain of the TCRβ chain was classified according to IMGT nomenclature. QPCR was used to determine expression of T cell-related genes. CD8 T cell numbers were significantly lower in LGG and, in contrast to HGG, mainly remained in close vicinity to blood vessels. This was accompanied by lower expression of chemo-attractants CXCL9, CXCL10 and adhesion molecule ICAM1. We did not observe a difference in the number of expressed neoantigens or CGAs, nor in diversity of TCR-Vβ gene usage. In summary, LGG have lower numbers of intra-tumoral CD8 T cells compared to HGG, potentially linked to decreased T cell trafficking. We have found no evidence for distinct tumor reactivity of T cells in either tumor type. The near absence of TILs in LGG suggest that, at present, checkpoint inhibitors are unlikely to have clinical efficacy in this tumor type.

Preeclampsia (PE) is one of the major causes of maternal and perinatal mortality worldwide. This study aimed to determine the immunological characteristics of PE patients and normal pregnancy at the T cell receptor beta-chain (TRB) level by using high-throughput sequencing. High-throughput sequencing was performed to analyze the expression of TRB-CDR3 in circulating T cells. T cells were isolated from 36 healthy pregnant women, 24 patients with severe PE, and 18 patients with moderate PE. Rearranged mRNA sequences were assigned to their germline V, D, and J counterparts, and translated into proper amino acids by the IMGT database. In general, PE samples had more TRB-CDR3 reads and types than those of normal pregnant woman in the circulation, but the mean number of TRB-CDR3 reads and unique TRB-CDR3 reads in severe group was lower than that in the moderate group. In PE patients, the V7&#95;9 and V20&#95;1 gene loci were more prevalent than in healthy pregnant women. In addition, 4 kinds of TRB-CDR3 peptides were found to be highly relevant to the pathogenesis of PE. Of them, peptides matched to herpes simplex virus antigen-specific T cells were much lower in PE samples., (Copyright © 2019. Published by Elsevier Inc.)

With the prominent breakthrough in the field of tumor immunology, diverse cancer immunotherapies have attracted great attention in the last decade. The immune checkpoint inhibitors, adoptive cell therapies, and therapeutic cancer vaccines have already achieved impressive clinical success. However, the fact that only a small subset of patients with specific tumor types can benefit from these treatments limits the application of cancer immunotherapy. To seek out the molecular mechanisms behind this challenge and to select cancer precision medicine for different individuals, researchers apply the immune repertoire sequencing (IRS) to evaluate genetic responses of each patient to current immunotherapies. This review summarizes the technical advances and recent applications of IRS in cancer immunotherapy, indicates the limitations of this technique, and predicts future perspectives both in basic studies and clinical trials., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Precise clonal and functional assessments of the T cell receptor (TCR) repertoire diversity require paired TCRα and TCRβ gene sequence information at monoclonal level. However, available single-cell strategies are typically limited in throughput and often do not provide full-length DNA templates for direct gene cloning. Here, we describe a high-throughput strategy for the unbiased amplification and automated sequence analysis of paired TCRα and TCRβ genes from primary mouse T cells. The platform links cell phenotype and TCR gene sequence information at single-cell level. Furthermore, it enables direct functional analyses through the efficient cloning of both genes and the generation of stable TCR expressing cell lines. This highly efficient workflow is a powerful tool to determine the diversity and quality of the murine T-cell repertoire in various settings, for example in vaccine development, infectious diseases, autoimmunity, or cancer., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nasal polyps (NP) are associated with inflamed mucosa of unknown etiology. The role of T cells in nasal polyposis is unclear. Invariant natural killer T cells (iNKT) can promote Th2 responses and have been implicated in some types of asthma. As there are shared inflammatory pathways involved in asthma and NPs, we evaluated the frequency of iNKT in 17 patients with NPs, but without asthma. A median of 6% polyp cells were T lymphocytes, of which iNKT were 0 to 2.38% (mean 0.674%). In the matched group (n = 10), iNKT in NPs was significantly higher than PBMCs (1.057% vs 0.155%, P < 0.05). Relative expression of Vα24 to TCR-beta genes in polyps (n = 14) was higher than blood in matched samples (n = 4). The presence of greater proportions of iNKT in NPs than in blood suggests that iNKT may play a role in the pathogenesis of nasal polyposis., (Copyright © 2019. Published by Elsevier Inc.)

Background: Tuberculosis (TB) is a major global health problem and has replaced HIV as the leading cause of death from a single infectious agent., Methods: Here, we applied high throughput sequencing to study the immune repertoire of nine pulmonary tuberculosis patients and nine healthy control samples., Results: Tuberculosis patients and healthy controls displayed significantly different high express clones and distinguishable sharing of CDR3 sequences. The TRBV and TRBJ gene usage showed higher expression clones in patients than in controls and we also found specific high express TRBV and TRBJ gene clones in different groups. In addition, six highly expressed TRBV/TRBJ combinations were detected in the CD4 group, 21 in the CD8 group and 32 in the tissue group., Conclusion: In conclusion, we studied the patients with tuberculosis as well as healthy control individuals in order to understand the characteristics of immune repertoire. Sharing of CDR3 sequences and differential expression of genes was found among the patients with tuberculosis which could be used for the development of potential vaccine and targets treatment., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Background: The adaptive immune system maintains a diversity of T cells capable of recognizing a broad array of antigens. Each T cell's specificity for antigens is determined by its T cell receptors (TCRs), which together across all T cells form a repertoire of millions of unique receptors in each individual. Although many studies have examined how TCR repertoires change in response to disease or drugs, few have explored the temporal dynamics of the TCR repertoire in healthy individuals., Results: Here we report immunosequencing of TCR β chains (TCRβ) from the blood of three healthy individuals at eight time points over one year. TCRβ repertoires of all peripheral-blood T cells and sorted memory T cells clustered clearly by individual, systematically demonstrating that TCRβ repertoires are specific to individuals across time. This individuality was absent from TCRβs from naive T cells, suggesting that the differences resulted from an individual's antigen exposure history, not genetic background. Many characteristics of the TCRβ repertoire (e.g., diversity, clonality) were stable across time, although we found evidence of T cell expansion dynamics even within healthy individuals. We further identified a subset of "persistent" TCRβs present across all time points. These receptors were rich in clonal and highly public receptors and may play a key role in immune system maintenance., Conclusions: Our results highlight the importance of longitudinal sampling of the immune system, providing a much-needed baseline for TCRβ dynamics in healthy individuals. Such a baseline will improve interpretation of changes in the TCRβ repertoire during disease or treatment.

Allergic contact dermatitis caused by contact sensitizers is a T-cell-mediated inflammatory skin disease. The most prevalent contact allergens is nickel. Whereas, memory T cells from nickel-allergic patients are well-characterized, little is known concerning nickel-specific naïve T-cell repertoire. The purpose of this study was to identify and quantify naïve CD4+ and CD8+ T cells recognizing nickel in the general population. Using a T-cell priming in vitro assay based on autologous co-cultures between naïve T cells and dendritic cells loaded with nickel, we were able to detect a naïve CD4+ and CD8+ T-cell repertoire for nickel in 10/11 and 7/8 of the tested donors. We calculated a mean frequency of 0.49 nickel-specific naïve CD4+ T cells and 0.37 nickel-specific naïve CD8+ T cells per million of circulating naïve T cells. The activation of these specific T cells requires MHC molecules and alongside IFN-γ production, some nickel-specific T-cells were able to produce granzyme-B. Interestingly, nickel-specific naïve CD4+ and CD8+ T cells showed a low rate of cross-reactivity with cobalt, another metallic hapten, frequently mixed with nickel in many alloys. Moreover, naïve CD4+ T cells showed a polyclonal TCRβ composition and the presence of highly expanded clones with an enrichment and/or preferentially expansion of some TRBV genes that was donor and T-cell specific. Our results contribute to a better understanding of the mechanism of immunization to nickel and propose the T-cell priming assay as a useful tool to identify antigen-specific naïve T cells.

Cervical cancer is more strongly associated with a specific virus, Human Papilloma Virus (HPV), in otherwise healthy individuals, than is any other cancer. Thus, there is an expectation that an adaptive immune signature of cervical cancer would be highly apparent. Here we used a genomics approach to investigate the relationship between T-cell receptor (TCR) V and J usage and survival for patients diagnosed with cervical cancer, relying exclusively on tissue and blood exome files. Specific TCR V or J segments, identified in recombination reads recovered from the exome files, were combined with the patient HLA alleles to identify V or J, HLA allele combination groups associated with distinct survival rates. For examples, the T-cell receptor-β (TRB) V6-5, HLA-A*02:01 combination was associated with a positive outcome, and the TRBV6-1, HLA-A*01:01 combination was associated with a negative outcome. Overall, these results point to V or J usage, HLA allele combinations as survival biomarkers, likely conveniently accessible with a noninvasive procedure, and the results may point the way towards immunological reagents useful in therapy designs., (Copyright © 2019. Published by Elsevier Inc.)

The antigenic specificity of T cells occurs via generation and rearrangement of different gene segments producing a functional T cell receptor (TCR). High-throughput sequencing (HTS) allows in-depth assessment of TCR repertoire patterns. There are limited data concerning whether TCR repertoires are altered in inflammatory bowel disease. We hypothesized that pediatric ulcerative colitis (UC) patients possess unique TCR repertoires, resulting from clonotypical expansions in the gut. Paired blood and rectal samples were collected from nine newly diagnosed treatment-naive pediatric UC patients and four healthy controls. DNA was isolated to determine the TCR-β repertoire by HTS. Significant clonal expansion was demonstrated in UC patients, with inverse correlation between clinical disease severity and repertoire diversity in the gut. Using different repertoire variables in rectal biopsies, a clear segregation was observed between patients with severe UC, those with mild-moderate disease and healthy controls. Moreover, the overlap between autologous blood-rectal samples in UC patients was significantly higher compared with overlap among controls. Finally, we identified several clonotypes that were shared in either all or the majority of UC patients in the colon. Clonal expansion of TCR-β-expressing T cells among UC patients correlates with disease severity and highlights their involvement in mediating intestinal inflammation., (© 2018 British Society for Immunology.)

Objectives: T-cell receptor (TCR) gene rearrangement studies are widely used for assessing T-cell clonality. The frequency and significance of clonal peaks restricted to TCR β (TCRB) tube C are uncertain. We retrospectively reviewed 80 TCR studies performed on bone marrow/peripheral blood., Methods: TCRB and TCR γ (TCRG) analyses were performed using BIOMED-2 primers. A peak was considered clonal or atypical if it was reproducible and 5× or more or 3× to 5× polyclonal background, respectively., Results: TCRB analysis demonstrated 12 (15%) of 80 cases with one to four isolated peaks in tube C (>3×) with polyclonal pattern in tubes A and B. TCRG analysis was monoclonal in two cases (both definite T-cell neoplasms), polyclonal in four, and oligoclonal in six. Of the 10 cases without clone in TCRG, six had autoimmune disorder and none had T-cell neoplasm., Conclusions: Peaks restricted to TCRB tube C in the TCR analysis may be misleading, as it is often not indicative of an overt T-cell neoplasm., (© American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Systemic lupus is characterized by the expansion of a self-reactive repertoire of B cells and CD4 cells that together promote IgG Ab production against common nuclear Ags. Although several studies have suggested roles for CD8 + T cells in lupus, the full contribution of these lymphocytes to disease remains undefined. In particular, few studies have examined TCR clonotypes of the CD8 pool in lupus. We previously described activated but nonpathogenic CD8 + T cells in a mouse model of systemic autoimmune disease triggered by increased copy number of the tlr7 gene (TLR7tg mice), in which some of these T cells accumulate in the brain. In this article, we report, through the analysis of TCRβ sequences, that CD8 cells from TLR7tg animals are strongly selected for a small number of clones, some of them reaching 30% of the repertoire, compared with less than 0.4% for the top clone in any wild type mice. High frequency clones are variable in sequence among individual TLR7tg mice and are distinct from top clones in the control animals, whereas CDR3 sequences of spleen and brain-resident T cells from the same TLR7tg animals have perfect concordance. These results suggest that top CD8 clones are selected in stochastic fashion in each animal but limit further diversification, and that brain-infiltrating CD8 cells in TLR7tg mice are not selected by a common tissue Ag. This kind of extreme clonal dominance and narrowing of the CD8 + repertoire might impair anti-viral responses and should be considered as an additional detrimental feature of chronic autoimmune disease.

With age, the immune system becomes less effective, causing increased susceptibility to infection. Chronic CMV infection further impairs immune function and is associated with increased mortality in the elderly. CMV exposure elicits massive CD8 + T cell clonal expansions and diminishes the cytotoxic T cell response to subsequent infections, leading to the hypothesis that to maintain homeostasis, T cell clones are expelled from the repertoire, reducing T cell repertoire diversity and diminishing the ability to combat new infections. However, in humans, the impact of CMV infection on the structure and diversity of the underlying T cell repertoire remains uncharacterized. Using TCR β-chain immunosequencing, we observed that the proportion of the peripheral blood T cell repertoire composed of the most numerous 0.1% of clones is larger in the CMV seropositive and gradually increases with age. We found that the T cell repertoire in the elderly grows to accommodate CMV-driven clonal expansions while preserving its underlying diversity and clonal structure. Our observations suggest that the maintenance of large CMV-reactive T cell clones throughout life does not compromise the underlying repertoire. Alternatively, we propose that the diminished immunity in elderly individuals with CMV is due to alterations in cellular function rather than a reduction in CD8 + T cell repertoire diversity., (Copyright © 2019 The Authors.)

Allelic exclusion is a vital mechanism for the generation of monospecificity to foreign Ags in B and T lymphocytes. In this study, we developed a high-throughput barcoded method to simultaneously analyze the VDJ recombination status of both mouse TCR-β alleles in hundreds of single cells using next-generation sequencing., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The α/β T cell receptor (TR) is a complex heterodimer that recognizes antigenic peptides and binds to major histocompatibility complex (MH) molecules. Both α and β chains are encoded by different genes localized on two distinct chromosomal loci: TRA and TRB. The present study employed the recent release of the swine genome assembly to define the genomic organization of the TRB locus. According to the sequencing data, the pig TRB locus spans approximately 400 kb of genomic DNA and consists of 38 TRBV genes belonging to 24 subgroups located upstream of three in tandem TRBD-J-C clusters, which are followed by a TRBV gene in an inverted transcriptional orientation. Comparative analysis confirms that the general organization of the TRB locus is similar among mammalian species, but the number of germline TRBV genes varies greatly even between species belonging to the same order, determining the diversity and specificity of the immune response. However, sequence analysis of the TRB locus also suggests the presence of blocks of conserved homology in the genomic region across mammals. Furthermore, by analysing a public cDNA collection, we identified the usage pattern of the TRBV, TRBD, and TRBJ genes in the adult pig TRB repertoire, and we noted that the expressed TRBV repertoire seems to be broader and more diverse than the germline repertoire, in line with the presence of a high level of TRBV gene polymorphisms. Because the nucleotide differences seems to be principally concentrated in the CDR2 region, it is reasonable to presume that most T cell β-chain diversity can be related to polymorphisms in pig MH molecules. Domestic pigs represent a valuable animal model as they are even more anatomically, genetically and physiologically similar to humans than are mice. Therefore, present knowledge on the genomic organization of the pig TRB locus allows the collection of increased information on the basic aspects of the porcine immune system and contributes to filling the gaps left by rodent models.

Numerous studies of relatively few patients have linked T cell receptor (TCR) genes to psoriasis but have yielded dramatically conflicting results. To resolve these discrepancies, we have chosen to mine RNA-Seq datasets for patterns of TCR gene segment usage in psoriasis. A meta-analysis of 3 existing and 1 unpublished datasets revealed a statistically significant link between the relative expression of TRAJ23 and psoriasis and the psoriasis-associated cytokine IL-17A. TRGV5, a TCR-γ segment, was also associated with psoriasis but correlated instead with IL-36A, other IL-36 family members, and IL-17C (not IL-17A). In contrast, TRAJ39 was strongly associated with healthy skin. T cell diversity measurements and analysis of CDR3 sequences were also conducted, revealing no psoriasis-associated public CDR3 sequences. Finally, in comparison with the expression of TCR-αβ genes, the expression of TCR-γδ genes was relatively low but mildly elevated in psoriatic skin. These results have implications for the development of targeted therapies for psoriasis and other autoimmune diseases. Also, the techniques employed in this study have applications in other fields, such as cancer immunology and infectious disease.

Ankylosing spondylitis (AS) is a chronic and progressive autoimmune disease affecting the invasion of the spine, sacroiliac joints and peripheral joints. T cells play a vital role in the underlying pathogenesis of AS, which mediated autoimmune and inflammatory responses via specific recognition of autoantigen peptides presented by susceptibility HLA. Antigen-specific T cells triggered by HLA/antigen complexes will undergo a massive expansion that forming an uneven T cell repertoire. To enhance our understanding of T-cell-mediated autoimmune in AS, we applied TCR β chains high-throughput sequencing to AS patients for in-depth TCR repertoire analysis. A significantly lower TCR repertoire diversity was observed in peripheral blood of AS patients relative to controls. And severe patients in our AS cohort have a more restricted TCR repertoire than mild patients, suggesting that the TCR repertoire diversity might be associated with the clinical severity of disease. No V, J and VJ pairs with significant biased usage were identified, which indicated that the usage frequency deviation of certain V/J/V-J genes in AS patients is little. This is a pilot study with potentially interesting observation on reduced diversity of T cells repertoire in peripheral blood of AS patients and further studies are needed., (Copyright © 2018. Published by Elsevier Inc.)

Objectives: This study was performed to evaluate for a potentially important role of T cells in the pathophysiology and treatment sensitivity of large cell neuroendocrine lung carcinoma (LCNEC), an orphan disease with poor prognosis and scarce data to guide novel therapeutic strategies., Materials and Methods: We performed T-cell receptor (TCR) β-chain spectratyping on blood samples of patients treated within the CRAD001KDE37 trial (n = 35) using age-matched current or former (n = 11) and never smokers (n = 10) as controls. The data were analyzed in conjunction with the complete blood counts of the probands as well as the data about response to treatment and overall survival in the clinical trial., Results and Conclusion: Untreated stage IV LCNEC patients had significant T-cell repertoire alterations (p < 0.001) compared to age-matched smokers. These changes correlated positively with blood lymphocyte counts (r = 0.49, p < 0.01), suggesting antigen-induced T-cell proliferation as the causative mechanism. At the same time, LCNEC patients showed mild lymphopenia (1.54 vs. 2.51/nl in median, p < 0.01), which reveals a second, antigen-independent mechanism of systemic immune dysregulation. More pronounced T-cell repertoire alterations and higher blood lymphocyte counts at diagnosis were associated with a better treatment response by RECIST and with a longer overall survival (441 vs. 157 days in median, p = 0.019). A higher degree of T-cell repertoire normalization after 3 months of therapy also distinguished a patient group with more favourable prognosis (median overall survival 617 vs. 316 days, p = 0.036) independent of radiological response. Thus, LCNEC induces clinically relevant changes of the T-cell repertoire, which are measurable in the blood and could be exploited for prognostic, predictive and therapeutic purposes. Their pathogenesis appears to involve antigen-induced oligoclonal T-cell expansions superimposed on TCR-independent lymphopenia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The diversity of the T cell receptor (TCR) complementarity determining region 3 (CDR3) repertoire is the result of random combinations, insertions and deletions during recombination of the germline V, D and J gene fragments. During evolution, some human TCR beta chain variable (TRBV) pseudogenes have been retained. Many previous studies have focused on functional TRBV genes, while little attention has been given to TRBV pseudogenes. To describe the compositional characteristics of TRBV pseudogene rearrangements, we compared and analysed TRBV pseudogenes, TRBV open reading frames (ORFs) and functional TRBV genes via high-throughput sequencing of DNA obtained from the peripheral blood of 4 healthy volunteers and 4 patients. Our results revealed several differences in J and D gene usage. The V deletion distribution profile of the pseudogenes was significantly different from that of the ORFs and functional genes. In addition, arginine, lysine and cysteine were more frequently used in putative CDR3 pseudogene rearrangements, while functional rearrangements used more leucine. This study presents a comprehensive description of the compositional characteristics of peripheral TRBV pseudogene rearrangements, which will provide a reference for further research on TRBV pseudogenes.

Objective: Refractory coeliac disease (RCD) is a potentially hazardous complication of coeliac disease (CD). In contrast to RCD type I, RCD type II is a precursor entity of enteropathy-associated T-cell lymphoma (EATL), which is associated with clonally expanding T-cells that are also found in the sequentially developing EATL. Using high-throughput sequencing (HTS), we aimed to establish the small-intestinal T-cell repertoire (TCR) in CD and RCD to unravel the role of distinct T-cell clonotypes in RCD pathogenesis., Design: DNA extracted from duodenal mucosa specimens of controls (n=9), active coeliacs (n=10), coeliacs on a gluten-free diet (n=9), RCD type I (n=8), RCD type II (n=8) and unclassified Marsh I cases (n=3) collected from 2002 to 2013 was examined by TCRβ-complementarity-determining regions 3 (CDR3) multiplex PCR followed by HTS of the amplicons., Results: On average, 10 6 sequence reads per sample were generated consisting of up to 900 individual TCRβ rearrangements. In RCD type II, the most frequent clonotypes (ie, sequence reads with identical CDR3) represent in average 42.6% of all TCRβ rearrangements, which was significantly higher than in controls (6.8%; p<0.01) or RCD type I (6.7%; p<0.01). Repeat endoscopies in individual patients revealed stability of clonotypes for up to several years without clinical symptoms of EATL. Dominant clonotypes identified in individual patients with RCD type II were unique and not related between patients. CD-associated, gliadin-dependent CDR3 motifs were only detectable at low frequencies., Conclusions: TCRβ-HTS analysis unravels the TCR in CD and allows detailed analysis of individual TCRβ rearrangements. Dominant TCRβ sequences identified in patients with RCD type II are unique and not homologous to known gliadin-specific TCR sequences, supporting the assumption that these clonal T-cells expand independent of gluten stimulation., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Changes in coding and coverage generate an uncertain reimbursement environment for molecular pathology laboratories. We analyzed our experience with two representative molecular oncology tests: a T-cell receptor (TCR) β rearrangement test and a large (467-gene) cancer next-generation sequencing panel, the Columbia Combined Cancer Panel (CCCP). Before 2013, the TCR β test was coded using stacked current procedural terminology codes and subsequently transitioned to a tier 1 code. CCCP was coded using a combination of tier 1 and 2 codes until 2015, when a new Genomic Sequencing Procedure code was adopted. A decrease in reimbursement of 61% was observed for the TCR β test on moving from stacking to tier 1 codes. No initial increase in total rejection rate was observed, but a subsequent increase in rejection rates in 2015 and 2016 was noted. The CCCP test showed a similar decrease (48%) in reimbursement after adoption of the new Genomic Sequencing Procedure code and was accompanied by a sharp increase in rejection rates both on implementation of the new code and over time. Changes in coding can result in substantial decreases in reimbursement. This may be a barrier to patient access because of the high cost of molecular diagnostics. Revisions to the molecular code set will continue. These findings help laboratories and manufacturers prepare for the financial impact and advocate appropriately., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Superantigens (SAgs) produced by Staphylococcus aureus at high concentrations induce proliferation of T cells bearing specific TCR Vβ sequences and massive cytokinemia that cause toxic shock syndrome. However, the biological relevance of SAgs produced at very low concentrations during asymptomatic colonization or chronic infections is not understood. In this study, we demonstrate that suboptimal stimulation of human PBMCs with a low concentration (1 ng/ml) of staphylococcal enterotoxin C1, at which half-maximal T cell proliferation was observed, induced CD8 + CD25 + T cells expressing markers related to regulatory T cells (Tregs), such as IFN-γ, IL-10, TGF-β, FOXP3, CD28, CTLA4, TNFR2, CD45RO, and HLA-DR. Importantly, these CD8 + CD25 + T cells suppressed responder cell proliferation mediated in contact-dependent and soluble factor-dependent manners, involving galectin-1 and granzymes, respectively. In contrast, optimal stimulation of human PBMCs with a high concentration (1 μg/ml) of staphylococcal enterotoxin C1, at which maximal T cell proliferation was observed, also induced similar expression of markers related to Tregs, including FOXP3 in CD8 + CD25 + cells, but these T cells were not functionally immunosuppressive. We further demonstrated that SAg-induced TCR Vβ-restricted and MHC class II-restricted expansion of immunosuppressive CD8 + CD25 + T cells is independent of CD4 + T cells. Our results suggest that the concentration of SAg strongly affects the functional characteristics of activated T cells, and low concentrations of SAg produced during asymptomatic colonization or chronic S. aureus infection induce immunosuppressive CD8 + Tregs, potentially promoting colonization, propagation, and invasion of S. aureus in the host., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The nonsense-mediated mRNA decay (NMD) pathway degrades aberrant transcripts containing premature translation termination codons (PTCs) and also regulates the levels of many normal mRNAs containing NMD-inducing features. The activity of this pathway varies considerably in different cell types and can change in response to developmental and environmental cues. Modulating NMD activity represents a potential therapeutic avenue for certain genetic disorders and cancers. Simple reporter systems capable of faithfully assessing NMD activity in mammalian cells greatly facilitate both basic and translational research on NMD. Here we describe a simple and effective method for assaying NMD specifically and quickly in live mammalian cells using a multicolored bioluminescence-based reporter system. This reporter can be transiently or stably introduced into cultured cells as well as animals, and NMD activity can be accurately assessed by bioluminescence imaging, western blot, or RT-qPCR.

Objective: We investigated the proportion of Vβ T cell receptor (TCR) gene expression in peripheral CD3+ lymphocytes in familial and non-familial systemic lupus erythematosus (SLE) patients., Method: The Vβ TCR repertoire was studied in 14 families in which several members had SLE. The Vβ TCR usage in SLE patients (n = 27) was compared with that in healthy members of these multiplex families (n = 47), in 37 sporadic SLE patients who had no relatives with SLE, and in 15 healthy unrelated controls. Vβ TCR repertoire expression was studied by multiparameter flow cytometry with the use of an array of 24 different Vβ TCR gene family-specific monoclonal antibodies., Results: We found the same Vβ TCR expression profile in the comparisons between sporadic SLE and familial SLE cases and healthy relatives, which included increased expression of Vβ 5.2, Vβ 11 and Vβ 16, and lower expression of Vβ 3, Vβ 4, Vβ 7.1 and Vβ 17. Interestingly, solely Vβ 17 was differentially expressed among sporadic and familial SLE. Also, increased expression of Vβ 9 was the hallmark among familial SLE (casesand h ealthy relatives) in comparison to controls., Conclusion: These results highlight the notion that the final profile of the Vβ TCR repertoire seen in familial and non-familial SLE seems to arise from the interaction of genetic, environmental, and immunoregulatory factors. Furthermore, it may contribute to the immunologic abnormalities affecting relatives of SLE patients., (Copyright: © 2018 SecretarÍa de Salud)

Celiac disease (CD) patients mount an abnormal immune response to gluten. T-cell receptor (TCR) repertoires directed to some immunodominant gluten peptides have previously been described, but the global immune response to in vivo gluten exposure in CD has not been systematically investigated yet. Here, we characterized signatures associated with gluten directed immune activity and identified gluten-induced T-cell clonotypes from total blood and gut TCR repertoires in an unbiased manner using immunosequencing. CD patient total TCR repertoires showed increased overlap and substantially altered TRBV-gene usage in both blood and gut samples, and increased diversity in the gut during gluten exposure. Using differential abundance analysis, we identified gluten-induced clonotypes in each patient that were composed of a large private and an important public component. Hierarchical clustering of public clonotypes associated with dietary gluten exposure identified subsets of highly similar clonotypes, the most proliferative of which showing significant enrichment for the motif ASS[LF]R[SW][TD][DT][TE][QA][YF] in PBMC repertoires. These results show that CD-associated clonotypes can be identified and that common gluten associated immune response features can be characterized in vivo from total repertoires, with potential use in disease stratification and monitoring.

Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3β. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCRβ chain libraries pre-enriched for target antigen specificity. Several TCRβ chains paired with a transgenic TCRα chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.

Objectives: The imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation., Methods: We compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions., Results: We found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells., Conclusions: Using an innovative antigen-agnostic approach, we uncovered a population of bona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: The mouse strain BALB/c deficient in IL-1 receptor antagonist protein (Il-1ra) develops spontaneous arthritis disease (SAD) while the strain DBA/1 IL1rn (-/-) with the same deficiency does not. Previously, we mapped a QTL on chromosome 1 for SAD and then developed a congenic mouse strain BALB.D1-1(-/-) that contains the QTL genomic fragment associated with resistance from DBA/1(-/-) on a BALB/c(-/-) background. The congenic strain was relatively resistant to spontaneous arthritis and had delayed onset and reduced severity of disease. We obtained whole genome expression profiles from the spleen of the congenic strain BALB.D1-1(-/-) and four other strains, the wild type BALB/c, DBA/1 and the deficient DBA/1 IL1rn (-/-) and the BALB/c IL1rn (-/-). We then compared the similarities and differences between the congenic strain and the four parental strains. Here we report the selected potential causal genes based on differential expression levels as well as function of genes., Results: There is a considerable number of genes that are differentially expressed between the congenic strain and the three parental strains, BALB/c, DBA/1, and DBA/1(-/-). However there only a few differentially expressed genes were identified by comparing the congenic strain and the BALB/c(-/-)strain. These differentially expressed genes are mainly from T-cell receptor beta chain (Tcrb) and interferon-activatable protein (Ifi) genes. These genes are also differentially expressed between congenic strain and BALB/c strains. However, their expression levels in the congenic strain are similar to that in DBA/1 and DBA/1(-/-). The expression level of Tcrb-j gene is positively associated with two genes of Ifi gene 200 cluster., Conclusions: Decreased expression levels of Ifi genes is associated to the increased resistance to spontaneous arthritis disease and with down regulation of expressions of Tcrb genes in the mouse congenic strain. Ifi genes may play an important role in the susceptibility to SAD in mice.

Tumor infiltrating lymphocytes (TILs) have been associated with favorable prognosis in multiple tumor types. The Cancer Genome Atlas (TCGA) represents the largest collection of cancer molecular data, but lacks detailed information about the immune environment. Here, we show that exome reads mapping to the complementarity-determining-region 3 (CDR3) of mature T-cell receptor beta (TCRB) can be used as an immune DNA (iDNA) signature. Specifically, we propose a method to identify CDR3 reads in a breast tumor exome and validate it using deep TCRB sequencing. In 1,078 TCGA breast cancer exomes, the fraction of CDR3 reads was associated with TILs fraction, tumor purity, adaptive immunity gene expression signatures and improved survival in Her2+ patients. Only 2/839 TCRB clonotypes were shared between patients and none associated with a specific HLA allele or somatic driver mutations. The iDNA biomarker enriches the comprehensive dataset collected through TCGA, revealing associations with other molecular features and clinical outcomes.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML., (© 2016 by The American Society of Hematology.)

NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR β-chains, including Vβ8, Vβ7, and Vβ2 in mice and Vβ11 in humans, joined to a range of TCR Dβ and Jβ genes. In this study, we demonstrate that TCR β-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vβ7(+) NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vβ8(+) NKT cells from mice. We show that the influence of the TCR β-chain is due to a combination of Vβ-, Jβ-, and CDR3β-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3β-encoded residues determined Ag preference. These findings indicate that NKT TCR β-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both α and β chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and β chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.

Motivation: Recombined T- and B-cell receptor repertoires are increasingly being studied using next generation sequencing (NGS) in order to interrogate the repertoire composition as well as changes in the distribution of receptor clones under different physiological and disease states. This type of analysis requires efficient and unambiguous clonotype assignment to a large number of NGS read sequences, including the identification of the incorporated V and J gene segments and the CDR3 sequence. Current tools have deficits with respect to performance, accuracy and documentation of their underlying algorithms and usage., Results: We present IMSEQ, a method to derive clonotype repertoires from NGS data with sophisticated routines for handling errors stemming from PCR and sequencing artefacts. The application can handle different kinds of input data originating from single- or paired-end sequencing in different configurations and is generic regarding the species and gene of interest. We have carefully evaluated our method with simulated and real world data and show that IMSEQ is superior to other tools with respect to its clonotyping as well as standalone error correction and runtime performance., Availability and Implementation: IMSEQ was implemented in C++ using the SeqAn library for efficient sequence analysis. It is freely available under the GPLv2 open source license and can be downloaded at www.imtools.org., Supplementary Information: Supplementary data are available at Bioinformatics online., Contact: lkuchenb@inf.fu-berlin.de or peter.robinson@charite.de., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Here we show that TCR ligation-anchored-magnetically captured PCR (TCR-LA-MC PCR) identifies TCR α- and β-chain diversity without sequence-associated or quantitative restrictions in healthy and diseased conditions. TCR-LA-MC PCR identifies convergent recombination events, classifies different stages of cutaneous T-cell lymphoma in vivo and demonstrates TCR reactivation after in vitro cytomegalovirus stimulation. TCR-LA-MC PCR allows ultra-deep data access to both physiological TCR diversity and mechanisms influencing clonality in all clinical settings with restricted or distorted TCR repertoires.

Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vβ gene segments (Trbvs) and their DβJβ targets. Formation of a functional VβDβJβ join signals for robust proliferation of double-negative thymocytes and their differentiation into double-positive (DP) cells, where Trbv recombination is squelched (allelic exclusion). DP differentiation also is accompanied by decontraction of Tcrb, which has been thought to separate the entire Trbv cluster from DβJβ segments (spatial segregation-based model for allelic exclusion). However, DP cells also repress transcription of unrearranged Trbvs, which may contribute to allelic exclusion. We performed a more detailed study of developmental changes in Tcrb topology and found that only the most distal portion of the Trbv cluster separates from DβJβ segments in DP thymocytes, leaving most Trbvs spatially available for rearrangement. Preferential dissociation of distal Trbvs is independent of robust proliferation or changes in transcription, chromatin, or architectural factors, which are coordinately regulated across the entire Trbv cluster. Segregation of distal Trbvs also occurs on alleles harboring a functional VβDβJβ join, suggesting that this process is independent of rearrangement status and is DP intrinsic. Our finding that most Trbvs remain associated with DβJβ targets in DP cells revises allelic exclusion models from their current conformation-dominant to a transcription-dominant formulation., (Copyright © 2015 by The American Association of Immunologists, Inc.)