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CD8+T-cell activation has been demonstrated to distinguish patients with primary and infection-associated hemophagocytic lymphohistiocytosis (pHLH and iaHLH) from patients with early sepsis. We evaluated the activation profile of CD8+T cells in patients with various forms of secondary HLH (sHLH), including macrophage activation syndrome (MAS). Flow-cytometry analysis was performed on peripheral blood mononuclear cells isolated from children with inactive systemic juvenile idiopathic arthritis (sJIA, n=17), active sJIA (n=27), MAS in sJIA (n=14), iaHLH (n=7) and with other forms of sHLH (n=9). Compared to patients with active sJIA, in patients with MAS and sHLH of different origins, beside a significant increase in the frequency of CD38high/HLA-DR+CD8+T cells, we found a significant increase in the frequency of CD8+T cells expressing the CD4 antigen (CD4dimCD8+T cells). These cells not only expressed high levels of the activation markers CD38 and HLA-DR, suggesting that they were a subset of CD38high/HLA-DR+CD8+T cells, but also of the activation/exhaustion markers CD25, PD1, CD95, and IFNγ. The frequency of CD4dimCD8+T cells strongly correlated with most of the laboratory parameters of MAS severity and with levels of the MAS biomarkers CXCL9 and IL-18. These findings were confirmed in a prospective replication cohort, in which no expansion of particular TCR Vβ family in CD3+T cells of sHLH patients was found. Finally, frequency of CD4dimCD8+but not of CD38high/HLA-DR+CD8+T cells, significantly correlated with a clinical severity score. Altogether, our data, showing that CD4dimCD8+T cells are increased in patients with MAS/sHLH and associated with disease severity, strongly support their involvement in MAS/sHLH pathogenesis.

ABSTRACTThe cdk inhibitor p57kip2, encoded by the Cdkn1cgene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1csilencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1cexpression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1cinduction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1cpromoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1cpromoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1cis silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1cand, possibly, of its aberrant silencing in some pathological conditions.