A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCRα and TCRβ gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1 to 3×10(6) unique TCRβ and 0.5×10(6) TCRα sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3×10(6) unique TCRβ sequences and 3.7×10(6) TCRα sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results provide an estimate of a thymic repertoire consisting of 40 to 70×10(6) unique TCRβ sequences and 60 to 100×10(6) TCRα sequences. The thymic repertoire is thus extremely diverse. Moreover, extrapolation of the data and comparison with earlier estimates of peripheral diversity also suggest that the thymic repertoire is transient, with different clones produced at different times.
Although mature human FOXP3(+) regulatory T cells are CD127 (IL-7Rα) negative, CD4(+)CD8(+) FOXP3(+) thymocytes express relatively high levels of CD127 and are responsive to IL-7. However, the role of IL-7 in human regulatory T cell development is poorly known. We show that at the CD4(+)CD8(+) stage, FOXP3(+) thymocytes are highly susceptible to apoptosis, and IL-7 selectively rescues them from death, leading to an increased frequency of FOXP3(+) cells. IL-7 also promotes the development of regulatory T cell phenotype by inducing up-regulation of FOXP3(+) and CTLA-4 expression. In contrast, IL-7 does not enhance proliferation of FOXP3(+)thymocytes or induce demethylation of FOXP3(+) regulatory T cell-specific demethylated region. After the CD4(+)CD8(+) stage, the FOXP3(+) thymocytes down-regulate CD127 expression but despite very low levels of CD127, remain responsive to IL-7. These results suggest that IL-7 affects human regulatory T cell development in the thymus by at least 2 distinct mechanisms: suppression of apoptosis and up-regulation of FOXP3(+) expression.
Thymic commitment of human FOXP3 regulatory T cells begins at the double positive (DP) CD4 CD8 stage. In the current study we show that interleukin-2 promotes the development of FOXP3 thymocytes and enhances their survival at the DP phase. IL-2 increases the frequency of FOXP3 cells and promotes the Treg phenotype after TCR-mediated positive selection at the most mature DP stage. However, it has no effect on FOXP3 cells at the earlier maturation steps before positive selection. DP FOXP3 are highly susceptible to cell death but IL-2 promotes their survival. The anti-apoptotic protein BCL-2 (B Cell Lymphoma 2) is also upregulated by IL-2 at the most mature DP stage. In addition to IL-2, we identify IL-15 to have a significant role in the upregulating FOXP3 and survival of Tregs at the DP phase. IL-7 also increases the expression of BCL-2 in the DP FOXP3 thymocytes. Our results indicate that common gamma chain cytokines IL-2, IL-7 and IL-15 promote the development of regulatory T cells at the most mature DP stage after TCR-mediated positive selection through suppressing cell death. This article is protected by copyright. All rights reserved.
We have analyzed T cell receptor repertoires in a unique set of thymus samples from a pair of monozygotic twins. While genetics affect the V(D)J rearrangement and generation of junctional sequences, the thymic selections seem largely stochastic and import no detectable inheritable effect at clonal level.
The IKAROS family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but IKAROS family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency yet. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2, encoding the translational activator and repressor HELIOS which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8+ T cell lineage. Protein-protein interaction analysis revealed that the variant abolished HELIOS dimerizations as well as binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, as well as chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis we show that reduced HELIOS expression was associated with chronic T cell activation and increased production of pro-inflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed profoundly reduced circulating MAIT cell numbers. In summary, we show that this novel loss-of-function variant in HELIOS leads to an immunodeficiency with signs of immune overactivation.
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