Ectopic germinal centers in the thymus accurately predict prognosis of myasthenia gravis after thymectomy.

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.

Common gamma chain cytokines promote regulatory T cell development and survival at the CD4+ CD8+ stage in the human thymus.

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.

A normal T cell receptor beta CDR3 length distribution in patients with APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by mutations in the AIRE gene. Murine studies suggest that AIRE controls thymic expression of tissue-restricted antigens, its absence allowing nonselected autoreactive cells to escape. We tested this in humans using the TCRß CDR3 length repertoire as a surrogate of thymic selection, as it shortens during the process. Analysis of healthy thymuses showed an altogether 1.9 base pair shortening, starting at the CD4(+)CD8(+)CD3(low) stage and continuing until the CD4(+) subset, likely encompassing both the positive and negative selection. Comparison of five APECED patients with eight healthy controls showed a skewed repertoire with oligoclonal expansions in the patients' CD4(+) and CD8(+) populations. The average CDR3 length, however, was normal and unaffected by the skewing. This was also true of the hypothesized autoreactive CD8(+)CD45RA(+) population. We failed to detect a subset with an abnormally long CDR3 repertoire, as would be predicted by a failure in selection.

High-sequence diversity and structural conservation in the human T-cell receptor ß junctional region during thymic development.

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 ß 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.

Cervical lymph node diameter reflects disease progression in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease against the central nervous system (CNS), where B cells activate in the deep cervical lymph nodes (CLNs) before migrating to the CNS. CLN diameter in head magnetic resonance imaging (MRI) is an unexplored possible biomarker for disease activity. METHODS: We measured CLN axial diameter from head MRIs of patients with active stable relapsing-remitting MS (a-RRMS-stable, n = 26), highly active stable RRMS (ha-RRMS-stable, n = 23), RRMS patients directly after a relapse (RRMS-relapse, n = 64) and follow-up MRIs from the same patients (r-RRMS-follow-up, n = 26). MRIs of primary headache syndrome patients (n = 38) served as a control group. We evaluated the correlation between CLN diameter and clinical data. RESULTS: Increases in EDSS in approximately 2 year-follow up after imaging was connected to smaller CLN diameter at imaging (correlation coefficient -0.305, p = 0.009). In a regression model, age did not show a significant effect to CLN diameter in MS patients. Enlarged CLNs of over 10 mm diameter were more common in patients with shorter disease duration (p = 0.013). The largest CLN axial diameter in RRMS-relapse group was smaller than in the control group (p = 0.005), whereas MS subgroups of the study did not differ in CLN diameter. CONCLUSIONS: CLN diameter appears to reflect disease duration and disease progression in MS, in line with compartmentalization of immunological activity to the CNS in time. Decrease in CLN diameter was seen also during relapse. CLN axial diameter in MRI shows promise as a feasible biomarker for assessing MS disease activity.

Indoleamine 2,3-dioxygenase activity is associated with regulatory T cell response in acute Puumala hantavirus infection.

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.

Interleukin-7 promotes human regulatory T cell development at the CD4+CD8+ double-positive thymocyte stage.

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.

Regulatory T cell response correlates with the severity of human hantavirus infection.

OBJECTIVES: Hantaviruses are an important group of emerging zoonotic pathogens, with significant mortality rates. Immunopathology is thought to be important in hantaviral disease, but the balance between protective and harmful responses is unknown. We studied Puumala hantavirus (PUUV) infection, which causes hemorrhagic fever with renal syndrome (HFRS) with a generally mild but highly variable clinical course. METHODS: Clinical data and blood samples were collected from 24 patients with acute PUUV infection, and analyzed by flow cytometry and quantitative PCR. RESULTS: The patients had a significantly increased frequency of CD4(+) and CD8(+) cells expressing the cell cycle marker Ki-67, but the magnitude of the effector T cell response did not correlate with disease severity. The frequency of FOXP3(+) regulatory T (Treg) cells expressing Ki-67 was also increased, and likewise did not correlate with disease outcome. In contrast, the level of FOXP3 expression, a surrogate of the suppressive phenotype, had a strong positive correlation with disease severity. This correlation was also found in samples taken 6-12 months after the HFRS. CONCLUSIONS: The best predictor of a severe disease course in HFRS was the FOXP3(+) Treg cell response, suggesting that the role of Treg cells in acute human hantaviral infections may be deleterious.