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We exploratively characterized T cell receptor (TCR) repertoires from occupational medicamentosa-like dermatitis due to trichloroethylene (OMDT) patients to better understand the underlying pathological mechanism. We used a combination of multiplex-PCR, Illumina sequencing and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR β-chain complementarity-determining region 3 (CDR3) gene in 10 OMDT cases and 10 trichloroethylene-exposed healthy tolerant controls. Compared with the tolerant controls, OMDT cases showed no significant difference in TCR repertoire diversity including repertoire breadth, highly expanded clone, and CDR3 length distribution. However, we observed several differences in TRBV/TRBJ usage and combination between the two groups, as well as some shared and unique T cell clones in the cases. The pilot study delineated some features of TCR repertoire in OMDT patients that warrant further investigation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There is increasing evidence that deep sequencing-based T cell repertoire can sever as a biomarker of immune response in cancer patients; however, the characteristics of T cell repertoire including diversity and similarity, as well as its prognostic significance in patients with cervical cancer (CC) remain unknown. In this study, we applied a high throughput T cell receptor (TCR) sequencing method to characterize the T cell repertoires of peripheral blood samples from 25 CC patients, 30 cervical intraepithelial neoplasia (CIN) patients and 20 healthy women for understanding the immune alterations during the cervix carcinogenesis. In addition, we also explored the signatures of TCR repertoires in the cervical tumor tissues and paired sentinel lymph nodes from 16 CC patients and their potential value in predicting the prognosis of patients. Our results revealed that the diversity of circulating TCR repertoire gradually decreased during the cervix carcinogenesis and progression, but the circulating TCR repertoires in CC patients were more similar to CIN patients than healthy women. Interestingly, several clonotypes uniquely detected in CC patients tended to share similar CDR3 motifs, which differed from those observed in CIN patients. In addition, the TCR repertoire diversity in sentinel lymphatic nodes from CC patients was higher than in tumor tissues. More importantly, less clonotypes in TCR repertoire of sentinel lymphatic node was associated with the poor prognosis of the patients. Overall, our findings suggested that TCR repertoire might be a potential indicator of immune monitoring and a biomarker for predicting the prognosis of CC patients. Although functional studies of T cell populations are clearly required, this study have expanded our understanding of T cell immunity during the development of CC and provided an experimental basis for further studies on its pathogenesis and immunotherapy.

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.

Background: Analytically sensitive techniques for measuring minimal residual disease (MRD) in multiple myeloma (MM) currently require invasive and costly bone marrow aspiration. These methods include immunohistochemistry (IHC), flow cytometry, quantitative PCR, and next-generation sequencing. An ideal MM MRD test would be a serum-based test sensitive enough to detect low concentrations of Ig secreted from multifocal lesions., Methods: Patient serum with abundant M-protein before treatment was separated on a 1-dimensional SDS-PAGE gel, and the Ig light-chain (LC) band was excised, trypsin digested, and analyzed on a Q Exactive mass spectrometer by LC-MS/MS. We used the peptide's abundance and sequence to identify tryptic peptides that mapped to complementary determining regions of Ig LCs. The clonotypic target tryptic peptides were used to monitor MRD in subsequent serum samples with prior affinity enrichment., Results: Sixty-two patients were tested, 20 with no detectable disease by IHC and 42 with no detectable disease by 6-color flow cytometry. A target peptide that could be monitored was identified in 57 patients (91%). Of these 57, detectable disease by LC-MS/MS was found in 52 (91%)., Conclusions: The ability to use LC-MS/MS to measure disease in patients who are negative by bone marrow-based methodologies indicates that a serum-based approach has more analytical sensitivity and may be useful for measuring deeper responses to MM treatment. The method requires no bone marrow aspiration., (© 2015 American Association for Clinical Chemistry.)

A number of antibodies that efficiently neutralize microbial targets contain long heavy chain complementarity determining region 3 (HCDR3) loops. For HIV, several of the most broad and potently neutralizing antibodies have exceptionally long HCDR3s. Two broad potently neutralizing HIV-specific antibodies, PG9 and PG16, exhibit secondary structure. Two other long HCDR3 antibodies, 2F5 and 4E10, protect against mucosal challenge with SHIV. Induction of such long HCDR3 antibodies may be critical to the design of an effective vaccine strategy for HIV and other pathogens, however it is unclear at present how to induce such antibodies. Here, we present genetic evidence that human peripheral blood antibodies containing long HCDR3s are not primarily generated by insertions introduced during the somatic hypermutation process. Instead, they are typically formed by processes occurring as part of the original recombination event. Thus, the response of B cells encoding antibodies with long HCDR3s results from selection of unusual clones from the naïve repertoire rather than through accumulation of insertions. These antibodies typically use a small subset of D and J gene segments that are particularly suited to encoding long HCDR3s, resulting in the incorporation of highly conserved genetic elements in the majority of antibody sequences encoding long HCDR3s.

Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples.

Rationale: Evaluation of the T-cell receptor (TCR) V beta-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V beta repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V beta primers instead of the conventionally labeled downstream C beta primer is described., Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C beta primer and an Omniscript RT kit. The 24 V beta primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 V beta primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C beta primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software., Results: V beta spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V beta primers. The resolution was superior to that obtained with the labeled C beta primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C beta labeling method, and the sample processing time was reduced by half., Conclusion: The method described for T-cell receptor V beta-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V beta repertoire analyses in clinical trials.

The skin and the intestinal mucosa form surfaces to external environments and share similarities in anatomic structure and immunologic defense. In healthy humans, intestinal gamma/delta T cells express a highly restricted gamma/delta T cell receptor repertoire whereas gamma/delta T cells of the skin were thought to express a polyclonal repertoire. Herein we report, using complementarity-determining region 3 size spectratyping and nucleotide sequencing of T cell receptor DV1 and DV2 rearrangements, that the human skin is also composed of clonally expanded gamma/delta T cells that are widely distributed. Identical complementarity-determining region 3 profiles and T cell receptor delta rearrangements were found in two separate skin samples that were obtained as far as 2-10 cm apart. Furthermore, analysis of peripheral blood mononuclear cells of these subjects clearly demonstrated that the skin harbors a unique population of gamma/delta T cells that is distinct from that in the peripheral blood. In addition comparable data were obtained irrespective of whether DNA or RNA was analyzed, indicating that the observed oligoclonality is not secondary to the expression of large amounts of mRNA from a few activated cells. Thus, gamma/delta T cells of the skin and the intestine both express an oligoclonal repertoire that enables them to respond to a variety of deleterious antigens without the need for diverse T cell receptors, possibly by recognition of stress-induced self-antigens or of conserved foreign antigens.

Intestinal T lymphocytes activated by antigen are suspected to play a key role in enterogenic spondyloarthropathies (SpA). Therefore, we aimed to identify and functionally characterize T-cell clones that are coexpanded in the intestinal mucosa and the synovium. Colon, peripheral blood, and synovium of a patient with enterogenic SpA were screened for clonal T-cell expansions by TCRB-CDR3 length analysis and sequencing. T-cell clones expanded in vivo were isolated from archived synovial cells by targeted T-cell cloning and characterized for phenotype, cytokine production, and antigen specificity. The synovial TCRBV18(+) T-cell repertoire of the patient was dominated by 2 CD8(+) T-cell clones using related CDR3. Both clones were expanded throughout the colon and were present in the peripheral blood. Upon in vitro stimulation with PDB/ionomycin, they showed predominantly interferon gamma and interleukin (IL)-4 but also tumor necrosis factor alpha and IL-10 production and did not specifically lyse autologous T-cell blasts, B-cell lines, or other autologous or allogeneic target or CD1d-transfected cells. These findings strongly suggest that T lymphocytes activated by antigen in the intestinal mucosa contribute to joint inflammation in enterogenic SpA by recognition of antigens specific for the inflamed synovium.

Objective: Autoreactive T cells may contribute to the pathogenesis of rheumatoid arthritis (RA). We studied the T-cell receptor (TCR) V-gene repertoire in the blood and synovium of early and chronic RA patients using polymerase chain reaction-enzyme-linked immunosorbent assay to evaluate possible differences between these patient groups., Results: Over-represented TCR V genes were observed in the synovium, but not in the blood of all RA patients (n = 38). The number of over-represented V genes was higher in the synovium of chronic RA patients (n = 31) than in that of early RA patients (n = 7). The V-gene profile was different among patients, and similar in the two knees for patients with bilateral synovitis (n = 5). The clonal composition of over-represented TCR BV genes in a patient with early RA and a patient with chronic RA was further studied by CDR3 region sequence analysis. A high level of clonal diversity was found in the joints and the blood of the early RA patient, suggesting a polyclonal T-cell expansion. In the chronic RA patient, predominant clonal expansions were observed in the blood and synovium, and some expanded clones were still present 2 yr later., Conclusions: The observation of similar T-cell populations in both joints in patients with bilateral synovitis and the persistence of clonally expanded T cells for more than 2 yr in the joints of a chronic RA patient may indicate a pathogenic role for these cells in the disease process.