Human thymopoiesis produces polyspecific CD8+ α/ß T cells responding to multiple viral antigens.

T-cell receptors (TCRs) are formed by stochastic gene rearrangements, theoretically generating >1019 sequences. They are selected during thymopoiesis, which releases a repertoire of about 108 unique TCRs per individual. How evolution shaped a process that produces TCRs that can effectively handle a countless and evolving set of infectious agents is a central question of immunology. The paradigm is that a diverse enough repertoire of TCRs should always provide a proper, though rare, specificity for any given need. Expansion of such rare T cells would provide enough fighters for an effective immune response and enough antigen-experienced cells for memory. We show here that human thymopoiesis releases a large population of clustered CD8+ T cells harboring α/ß paired TCRs that (i) have high generation probabilities and (ii) a preferential usage of some V and J genes, (iii) which CDR3 are shared between individuals, and (iv) can each bind and be activated by multiple unrelated viral peptides, notably from EBV, CMV, and influenza. These polyspecific T cells may represent a first line of defense that is mobilized in response to infections before a more specific response subsequently ensures viral elimination. Our results support an evolutionary selection of polyspecific α/ß TCRs for broad antiviral responses and heterologous immunity.

T regulatory cells activation and distribution are modified in critically ill patients with acute respiratory distress syndrome: A prospective single-centre observational study.

BACKGROUNDS: Acute respiratory distress syndrome (ARDS) is a common and fatal inflammatory condition. Whether T regulatory cells (Tregs) are beneficial or detrimental remains controversial, and longitudinal studies are lacking. Phenotyping of Tregs activation markers has been poorly reported. We aimed to evaluate quantitative and functional alterations in blood and bronchoalveolar Treg phenotype of ARDS patients. METHODS: We performed a single-centre observational study in a French intensive care unit. The study enrolled 60 ARDS and 45 non-ARDS patients. Patients under 18years old or with immunosuppression (native or acquired) were excluded. Tregs phenotypes were assessed by flow cytometry, while cytokines were measured by multiplex-based assays in blood and bronchoalveolar samples collected over 3weeks after the onset of ARDS. RESULTS: Blood Tregs/CD4+ percentage (median %, 25-75% interquartile) was higher in ARDS patients than in non-ARDS patients: 12.1% [9.0-16.0] versus 9.9% [8.1-12.6], P=0.01. Alveolar Tregs/CD4+ percentage was lower in ARDS patients than in non-ARDS patients: 10.4% [6.3-16.6] versus 16.2% [12.4-21.1], P=0.03. In ARDS patients, Tregs activation was reduced in the blood and increased in the alveolus, compared to non-ARDS patients. ROC analysis revealed a threshold of 10.4% for the Tregs/CD4+ percentage in the blood collected within the first week of ARDS to discriminate between survivors and non-survivors (sensitivity: 75%; specificity 76%; area under the curve [95% confidence interval]: 0.72 [0.5-0.9]). CONCLUSIONS: Quantitative and functional alterations in Treg phenotype were observed in patients with ARDS. Whether rebalancing Tregs phenotype with therapeutic interventions would be beneficial deserves further investigations.

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials.

BACKGROUND: Quantification of regulatory T cells (Tregs) is crucial in immunomonitoring in clinical trials as this cell population has been shown to be involved in a wide range of diseases, including cancers, autoimmune diseases, infections, and allergies. Human Tregs are defined as CD4+ CD25+ CD127low FoxP3+ cells, and the standardization of their staining by flow cytometry is a challenge, especially in multicenter clinical trials, notably because of the intracellular location of FoxP3. METHOD: A flow cytometry staining procedure was settled and standardized to measure human Tregs in peripheral whole blood using precoated dried antibodies in ready-to-use tubes. It was compared with reference methods and implemented and validated to be suitable with different cytometer platforms. RESULTS: The standardized protocol developed with dried antibodies and reduced volumes of whole blood allows an optimal identification of Tregs. Compared with classical staining procedure, it reduces the number of steps required, in a very fast and simple technique. The accuracy of the method was confirmed by a multicenter comparison with different cytometer brands. CONCLUSIONS: Our results highlight the reliability of this high-standard protocol that could become a reference method for the monitoring of Tregs in clinical trials. © 2018 International Clinical Cytometry Society.