A reversion of an IL2RG mutation in combined immunodeficiency providing competitive advantage to the majority of CD8+ T cells

Mutations in the common gamma chain (γc, CD132, encoded by the IL2RG gene) can lead to B+T-NK-X-linked severe combined immunodeficiency, as a consequence of unresponsiveness to γc-cytokines such as interleukins-2, -7 and -15. Hypomorphic mutations in CD132 may cause combined immunodeficiencies with a variety of clinical presentations. We analyzed peripheral blood mononuclear cells of a 6-year-old boy with normal lymphocyte counts, who suffered from recurrent pneumonia and disseminated mollusca contagiosa. Since proliferative responses of T cells and NK cells to γc -cytokines were severely impaired, we performed IL2RG gene analysis, showing a heterozygous mutation in the presence of a single X-chromosome. Interestingly, an IL2RG reversion to normal predominated in both naïve and antigen-primed CD8+ T cells and increased over time. Only the revertant CD8+T cells showed normal expression of CD132 and the various CD8+T cell populations had a different T-cell receptor repertoire. Finally, a fraction of γδ+T cells and differentiated CD4+CD27-effector-memory T cells carried the reversion, whereas NK or B cells were repeatedly negative. In conclusion, in a patient with a novel IL2RG mutation, gene-reverted CD8+T cells accumulated over time. Our data indicate that selective outgrowth of particular T-cell subsets may occur following reversion at the level of committed T progenitor cells

Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction

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

Regulatory T Cells Expanded from Hiv-1-Infected Individuals Maintain Phenotype, Tcr Repertoire and Suppressive Capacity

While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4+ Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.Elizabeth Glaser Pediatric AIDS Foundation (Pediatric HIV Vaccine Program Award MV-00-9-900-1429-0-00)Massachusetts General Hospital. Executive Committee on Research (MGH/ECOR Physician Scientist Development Award)National Institutes of Health (U.S.) (NIH NIAID (KO8 AI074405))National Institutes of Health (U.S.) (NIH NIAID AI074405-03S1)Massachusetts General Hospital (William F. Milton Fund)Harvard University. Center for AIDS Research (CFAR Scholar Award)Massachusetts General Hospital. Center for the Study Inflammatory Bowel Disease (P30DK043351)Harvard University. Center for AIDS Research (NIH funded program (5P30AI060354-09

Nationwide Association of Surgical Performance of Minimally Invasive Esophagectomy With Patient Outcomes

IMPORTANCE: Suboptimal surgical performance is hypothesized to be associated with less favorable patient outcomes in minimally invasive esophagectomy (MIE). Establishing this association may lead to programs that promote better surgical performance of MIE and improve patient outcomes.OBJECTIVE: To investigate associations between surgical performance and postoperative outcomes after MIE.DESIGN, SETTING, AND PARTICIPANTS: In this nationwide cohort study of 15 Dutch hospitals that perform more than 20 MIEs per year, 7 masked expert MIE surgeons assessed surgical performance using videos and a previously developed and validated competency assessment tool (CAT). Each hospital submitted 2 representative videos of MIEs performed between November 4, 2021, and September 13, 2022. Patients registered in the Dutch Upper Gastrointestinal Cancer Audit between January 1, 2020, and December 31, 2021, were included to examine patient outcomes.EXPOSURE: Hospitals were divided into quartiles based on their MIE-CAT performance score. Outcomes were compared between highest (top 25%) and lowest (bottom 25%) performing quartiles. Transthoracic MIE with gastric tube reconstruction.MAIN OUTCOME AND MEASURE: The primary outcome was severe postoperative complications (Clavien-Dindo ≥3) within 30 days after surgery. Multilevel logistic regression, with clustering of patients within hospitals, was used to analyze associations between performance and outcomes.RESULTS:In total, 30 videos and 970 patients (mean [SD] age, 66.6 [9.1] years; 719 men [74.1%]) were included. The mean (SD) MIE-CAT score was 113.6 (5.5) in the highest performance quartile vs 94.1 (5.9) in the lowest. Severe postoperative complications occurred in 18.7% (41 of 219) of patients in the highest performance quartile vs 39.2% (40 of 102) in the lowest (risk ratio [RR], 0.50; 95% CI, 0.24-0.99). The highest vs the lowest performance quartile showed lower rates of conversions (1.8% vs 8.9%; RR, 0.21; 95% CI, 0.21-0.21), intraoperative complications (2.7% vs 7.8%; RR, 0.21; 95% CI, 0.04-0.94), and overall postoperative complications (46.1% vs 65.7%; RR, 0.54; 95% CI, 0.24-0.96). The R0 resection rate (96.8% vs 94.2%; RR, 1.03; 95% CI, 0.97-1.05) and lymph node yield (mean [SD], 38.9 [14.7] vs 26.2 [9.0]; RR, 3.20; 95% CI, 0.27-3.21) increased with oncologic-specific performance (eg, hiatus dissection, lymph node dissection). In addition, a high anastomotic phase score was associated with a lower anastomotic leakage rate (4.6% vs 17.7%; RR, 0.14; 95% CI, 0.06-0.31).CONCLUSIONS AND RELEVANCE: These findings suggest that better surgical performance is associated with fewer perioperative complications for patients with esophageal cancer on a national level. If surgical performance of MIE can be improved with MIE-CAT implementation, substantially better patient outcomes may be achievable.</p

Advances in rheumatology: new targeted therapeutics

Treatment of inflammatory arthritides - including rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis - has seen much progress in recent years, partially due to increased understanding of the pathogenesis of these diseases at the cellular and molecular levels. These conditions share some common mechanisms. Biologic therapies have provided a clear advance in the treatment of rheumatological conditions. Currently available TNF-targeting biologic agents that are licensed for at east one of the above-named diseases are etanercept, infliximab, adalimumab, golimumab, and certolizumab. Biologic agents with a different mechanism of action have also been approved in rheumatoid arthritis (rituximab, abatacept, and tocilizumab). Although these biologic agents are highly effective, there is a need for improved management strategies. There is also a need for education of family physicians and other healthcare professionals in the identification of early symptoms of inflammatory arthritides and the importance of early referral to rheumatologists for diagnosis and treatment. Also, researchers are developing molecules - for example, the Janus kinase inhibitor CP-690550 (tofacitinib) and the spleen tyrosine kinase inhibitor R788 (fostamatinib) - to target other aspects of the inflammatory cascade. Initial trial results with new agents are promising, and, in time, head-to-head trials will establish the best treatment options for patients. The key challenge is identifying how best to integrate these new, advanced therapies into daily practice

Alterations of the Synovial T Cell Repertoire in Anti-Citrullinated Protein Antibody-Positive Rheumatoid Arthritis

Objective. The association of HLA-DRB1 alleles with anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) suggests the potential involvement of T lymphocytes in ACPA-seropositive disease. The purpose of this study was to investigate this hypothesis by systematic histologic and molecular analyses of synovial T cells in ACPA+ versus ACPA- RA patients. Methods. Synovial biopsy samples were obtained from 158 RA patients. Inflammation was determined histologically and immunohistochemically. RNA was extracted from peripheral blood mononuclear cells and synovial tissues obtained from 11 ACPA+ RA patients, 7 ACPA- RA patients, and 10 spondylarthritis (SpA) patients (arthritis controls). T lymphocyte clonality was studied by combined quantitative and qualitative T cell receptor CDR3 length distribution (LD) analysis and direct sequencing analysis. Results. ACPA+ and ACPA- RA patients were similar at both the clinical and histologic levels. At the molecular level, however, patients with ACPA+ synovitis displayed a marked elevation of qualitative CDR3 LD alterations as compared with those with ACPA- synovitis and with the SpA controls. These differences in CDR3 LD were not observed in the peripheral blood, indicating a selective recruitment and/or local expansion of T cells in the synovial compartment. The CDR3 LD alterations reflected true monoclonal or oligoclonal expansions, as confirmed by direct sequencing of the T cell receptor. The CDR3 LD alterations in RA synovium did not correlate with B cell clonal expansions but were inversely associated with synovial lymphoid neogenesis. Conclusion. The T cell repertoire is specifically restricted in RA patients with ACPA+ synovitis. Whereas the origin and role of these clonal alterations remain to be determined, our data suggest the preferential involvement of T lymphocytes in ACPA-seropositive R

Immunoglobulin G4+ clones identified by next-generation sequencing dominate the B cell receptor repertoire in immunoglobulin G4 associated cholangitis

Immunoglobulin G4 (IgG4)-associated cholangitis (IAC) is a manifestation of the recently discovered idiopathic IgG4-related disease. The majority of patients have elevated serum IgG4 levels and/or IgG4-positive B-cell and plasma cell infiltrates in the affected tissue. We hypothesized that clonally expanded, class-switched IgG4-positive B cells and plasma cells could be causal to these poorly understood phenomena. In a prospective cohort of six consecutive IAC patients, six healthy controls, and six disease controls, we used a novel next-generation sequencing approach to screen the B-cell receptor (BCR) repertoires, in blood as well as in affected tissue, for IgG4+ clones. A full repertoire analysis of the BCR heavy chain was performed using GS-FLX/454 and customized bioinformatics algorithms (>10,000 sequences/sample; clones with a frequency ≥0.5% were considered dominant). We found that the most dominant clones within the IgG+ BCRheavy repertoire of the peripheral blood at baseline were IgG4+ only in IAC patients. In all IAC patients, but none of the controls, IgG4+ BCR clones were among the 10 most dominant BCR clones of any immunoglobulin isotype (IgA, IgD, IgM, and IgG) in blood. The BCR repertoires of the duodenal papilla comprised the same dominant IgG4+ clones as the paired peripheral blood samples. In all IAC patients, after 4 and 8 weeks of corticosteroid therapy the contribution of these IgG4+ clones to the IgG+ repertoire as well as to total BCR repertoire was marginalized, mirroring sharp declines in serum IgG4 titers and regression of clinical symptoms. Conclusion: The novel finding of highly abundant IgG4+ BCR clones in blood and tissue of patients with active IAC, which disappear upon corticosteroid treatment, suggests that specific B cell responses are pivotal to the pathogenesis of IAC

The Costimulatory Molecule CD27 Maintains Clonally Diverse CD8+ T Cell Responses of Low Antigen Affinity to Protect against Viral Variants

SummaryCD70 and CD27 are costimulatory molecules that provide essential signals for the expansion and differentiation of CD8+ T cells. Here, we show that CD27-driven costimulation lowered the threshold of T cell receptor activation on CD8+ T cells and enabled responses against low-affinity antigens. Using influenza infection to study in vivo consequences, we found that CD27-driven costimulation promoted a CD8+ T cell response of overall low affinity. These qualitative effects of CD27 on T cell responses were maintained into the memory phase. On a clonal level, CD27-driven costimulation established a higher degree of variety in memory CD8+ T cells. The benefit became apparent when mice were reinfected, given that CD27 improved CD8+ T cell responses against reinfection with viral variants, but not with identical virus. We propose that CD27-driven costimulation is a strategy to generate memory clones that have potential reactivity to a wide array of mutable pathogens

Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction

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

Dominant B cell receptor clones in peripheral blood predict onset of arthritis in individuals at risk for rheumatoid arthritis

The onset of seropositive rheumatoid arthritis (RA) is preceded by the presence of specific autoantibodies in the absence of synovial inflammation. Only a subset of these at-risk individuals will develop clinical disease. This impedes efforts to implement early interventions that may prevent onset of clinically manifest disease. Here we analyse whether clonal changes in the B cell receptor (BCR) repertoire can reliably predict onset of signs and symptoms. In a prospective cohort study in 21 individuals at risk for RA based on the presence of autoantibodies, the BCR repertoire of paired peripheral blood and synovial tissue samples was analysed using next-generation BCR sequencing. BCR clones that were expanded beyond 0.5% of the total repertoire were labelled dominant. The relative risk (RR) for onset of arthritis was assessed using the presence of ≥5 dominant BCR clones as cut-off. Findings in peripheral blood were validated in an independent prospective cohort of 50 at-risk individuals. Based on the test cohort, individuals in the validation cohort were considered positive if peripheral blood at study entry showed ≥5 dominant BCR clones. Both in the test and validation cohort, the presence of ≥5 dominant BCR clones in peripheral blood was significantly associated with arthritis development after follow-up (validation cohort RR 6.3, 95% CI 2.7 to 15, p <1×10(-4)). Even when adjusted for a recently described clinical prediction rule the association remained intact (RR 5.0, 95% CI 1.2 to 20, p=0.024). When individuals developed arthritis, dominant BCR clones disappeared from peripheral blood and appeared in synovial tissue, suggesting a direct role of these clones in disease pathogenesis. Dominant BCR clones in peripheral blood predict onset of clinical signs and symptoms of RA in at-risk individuals with high accuracy. Our data suggest that during onset of RA these clones shift from peripheral blood to the target tissu