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Larsen M.,French Institute of Health and Medical Research | Larsen M.,University Pierre and Marie Curie | Sauce D.,French Institute of Health and Medical Research | Deback C.,University Pierre and Marie Curie | And 19 more authors.
PLoS Pathogens | Year: 2011

Systemic Lupus Erythematosus (SLE) pathology has long been associated with an increased Epstein-Barr Virus (EBV) seropositivity, viremia and cross-reactive serum antibodies specific for both virus and self. It has therefore been postulated that EBV triggers SLE immunopathology, although the mechanism remains elusive. Here, we investigate whether frequent peaks of EBV viral load in SLE patients are a consequence of dysfunctional anti-EBV CD8 + T cell responses. Both inactive and active SLE patients (n = 76 and 42, respectively), have significantly elevated EBV viral loads (P = 0.003 and 0.002, respectively) compared to age- and sex-matched healthy controls (n = 29). Interestingly, less EBV-specific CD8 + T cells are able to secrete multiple cytokines (IFN-γ, TNF-α, IL-2 and MIP-1β) in inactive and active SLE patients compared to controls (P = 0.0003 and 0.0084, respectively). Moreover, EBV-specific CD8 + T cells are also less cytotoxic in SLE patients than in controls (CD107a expression: P = 0.0009, Granzyme B release: P = 0.0001). Importantly, cytomegalovirus (CMV)-specific responses were not found significantly altered in SLE patients. Furthermore, we demonstrate that EBV-specific CD8 + T cell impairment is a consequence of their Programmed Death 1 (PD-1) receptor up-regulation, as blocking this pathway reverses the dysfunctional phenotype. Finally, prospective monitoring of lupus patients revealed that disease flares precede EBV reactivation. In conclusion, EBV-specific CD8 + T cell responses in SLE patients are functionally impaired, but EBV reactivation appears to be an aggravating consequence rather than a cause of SLE immunopathology. We therefore propose that autoimmune B cell activation during flares drives frequent EBV reactivation, which contributes in a vicious circle to the perpetuation of immune activation in SLE patients. © 2011 Larsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Larsen M.,French Institute of Health and Medical Research | Larsen M.,University Pierre and Marie Curie | Arnaud L.,French Institute of Health and Medical Research | Hie M.,French Institute of Health and Medical Research | And 15 more authors.
European Journal of Immunology | Year: 2011

The ontogenic relationship between pro-inflammatory populations of interleukin-17 (IL-17A)- and/or IL-22-producing T cells and other T-cell subsets is currently unclear in humans. To appreciate T helper cell-lineage commitment, we combined cytokine production profiles of in vitro expanded T-cell clones with T-cell receptor (TCR) clonotypic signatures. Moreover, ex vivo cytokine production profiles at the single-cell level were analyzed using an original approach based on the hierarchical cluster analysis of multiparametric flow cytometry data. These combined approaches enabled the delineation of distinct functional T-cell subsets, including Th1, Th2, Tr1, Th17 cells and a highly polyfunctional IL-22-producing T-cell population. Cluster analysis highlighted that the IL-22-producing T-cell population should be considered independently from the Th17 and Th1 subsets, although it was more closely related to the former. In parallel, we observed extensive TCRαβ sharing across all five subsets defined. The strategy described here allows the objective definition of cellular subsets and an unbiased insight into their similarities. Together, our results underscore the ontogenic plasticity of CD4 + T-cell progenitors, which can adopt a differentiation profile irrespective of antigen specificity. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Larsen M.,French Institute of Health and Medical Research | Larsen M.,University Pierre and Marie Curie | Sauce D.,French Institute of Health and Medical Research | Sauce D.,University Pierre and Marie Curie | And 9 more authors.
PLoS ONE | Year: 2012

Functional evaluation of naturally occurring or vaccination-induced T cell responses in mice, men and monkeys has in recent years advanced from single-parameter (e.g. IFN-γ-secretion) to much more complex multidimensional measurements. Co-secretion of multiple functional molecules (such as cytokines and chemokines) at the single-cell level is now measurable due primarily to major advances in multiparametric flow cytometry. The very extensive and complex datasets generated by this technology raise the demand for proper analytical tools that enable the analysis of combinatorial functional properties of T cells, hence polyfunctionality. Presently, multidimensional functional measures are analysed either by evaluating all combinations of parameters individually or by summing frequencies of combinations that include the same number of simultaneous functions. Often these evaluations are visualized as pie charts. Whereas pie charts effectively represent and compare average polyfunctionality profiles of particular T cell subsets or patient groups, they do not document the degree or variation of polyfunctionality within a group nor does it allow more sophisticated statistical analysis. Here we propose a novel polyfunctionality index that numerically evaluates the degree and variation of polyfuntionality, and enable comparative and correlative parametric and non-parametric statistical tests. Moreover, it allows the usage of more advanced statistical approaches, such as cluster analysis. We believe that the polyfunctionality index will render polyfunctionality an appropriate end-point measure in future studies of T cell responsiveness. © 2012 Larsen et al.

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