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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.

The ability of thymic histopathology to predict the long-term impact of thymectomy in non-thymomatous myasthenia gravis (NTMG) is mainly uncharted. We applied digital pathology to quantitatively characterize differences of thymic histology between early-onset (EOMG) and late-onset MG (LOMG) and to investigate the role of thymic changes for thymectomy outcomes in MG. We analyzed 83 thymic H&E slides from thymectomized NTMG patients, of which 69 had EOMG and 14 LOMG, using digital pathology open-access software QuPath. We compared the results to the retrospectively assessed clinical outcome at two years after thymectomy and at the last follow-up visit where complete stable remission and minimal use of medication were primary outcomes. The automated annotation pipeline was an effective and reliable way to analyze thymic H&E samples compared to manual annotation with mean intraclass correlation of 0.80. The ratio of thymic tissue to stroma and fat was increased in EOMG compared to LOMG (p = 8.7e-07), whereas no difference was observed in the ratio of medulla to cortex between these subtypes. AChRAb seropositivity correlated with the number of ectopic germinal centers (eGC; p = 0.00067) but not with other histological areas. Patients with an increased number of eGCs had better post-thymectomy outcomes at two years after thymectomy (p = 0.0035) and at the last follow-up (p = 0.0267). ROC analysis showed that eGC area predicts thymectomy outcome in EOMG with an AUC of 0.79. Digital pathology can thus help in providing a predictive tool to the clinician, the eGC number, to guide the post-thymectomy treatment decisions in EOMG patients.

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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.

The T-cell repertoire depends on intrathymic genetic rearrangement events in the T-cell receptor (TCR) locus, followed by positive and negative selection. The repertoire thus generated is highly diverse, but recent data indicate that the recombination of gene segments is less stochastic than previously suggested. Very little is known of the junctional complementarity determining region 3 (CDR3), which is to a large degree not germline encoded. We have analyzed the development of the human TCR β CDR3 repertoire, from the nonselected CD4 + CD8 + CD3 − cells up to the fully selected CD4 + CD8 − thymocytes. In addition to spectratyping, a fraction of the CDR3 repertoire was sequenced and a structural in silico analysis of the CDR3 loop characteristics performed. Our data show that the thymic TCR repertoire is extremely diverse, and the effect of the selection events can be detected as a measurable loss of polyclonality in the CDR3 loop. However, the main physicochemical features of the CDR3 loop were found already at the nonselected repertoire and showed no progressive changes during the selection. Thus, the main structural characteristics of the CDR3 loop were already determined by the recombination process and not significantly affected by the extensive thymocyte death associated with selection in the thymus. Keywords: T cells · TCR · Thymic selection IntroductionThe creation of a mature αβ T-cell repertoire is dependent on genetic rearrangement events, capable of creating an immenseCorrespondence: Dr. Anni Tuulasvaara e-mail: anni.tuulasvaara@helsinki.fi amount of diversity. Most of this diversity is concentrated on the junctional area, formed in the TCR β locus by the joining of Vβ, Dβ, and Jβ gene segments, and in the TCR α locus by Vα and Jα segments [1,2]. At the joining sites, the addition and deletion of nucleotides adds greatly to the potential diversity, estimated theoretically to be up to 10 16 different αβ combinations [3]. After successful rearrangement of both the TCR α and β loci, the emergent repertoire is subject to selection events, which shape a safe Eur. J. Immunol. 2013Immunol. . 43: 2185Immunol. -2193 and effective repertoire and finally direct the developing thymocytes to either CD4 + or CD8 + single positive (SP) lineage [4].However, most of what we know about the development of TCR repertoire is based on genetically manipulated murine models, while the human thymocyte maturation and shaping of the preimmune repertoire have been studied much less. The rearrangement of the TCR β locus begins already at the CD4 − CD8 − double negative stage, and continues until the cells become CD4 + CD8 + double positive (DP) [5,6]. Functional β chain expression, as tested by pairing with the surrogate preTα chain, is necessary for cell survival and further maturation. This process of β selection ends recombination of the β locus [7]. The cells begin to proliferate, until recombination activating enzymes RAG-1 and RAG-2 are re-expressed and recombination of the α locus begins [8]. After productive rearrange...

High indoleamine 2,3-dioxygenase (IDO) activity is associated with clinically severe acute infection caused by Puumala hantavirus. The immunoregulatory effects of IDO can be mediated either through metabolic control of effector T cells, caused by depletion of the essential amino acid tryptophan, or intercellular signaling and activation of regulatory T cell responses. Here, we have studied 24 patients with acute Puumala hantavirus infection to distinguish between these possibilities. Maximum IDO activity showed a significant positive correlation with FOXP3 expression levels in regulatory T cells, a quantitative surrogate marker for suppressive capability. In contrast, IDO activity did not correlate with the frequency of CD8 effector cells in cell cycle. The data suggest that in Puumala infection, the mechanism responsible for the suppressive effect of IDO is not metabolic control of effector cells but rather the signaling mediated by tryptophan breakdown products, such as kynurenine.

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