A novel 8-color flow cytometry panel to study activation, maturation and senescence of CD4 and CD8 T lymphocytes in HIV-infected individuals at different stages of disease.

Multicolor flow cytometry allows to study the markers differentially expressed during maturation, activation, function and senescence on immune cells. Despite the availability of reagents and technology, scarce agreement has been gained regarding phenotypic markers of HIV disease progression other than CD4 T-cell count. In this work, we present a novel high-throughput global analysis of CD4 and CD8 T-lymphocyte profiles by standardized 8-color combinations of antibodies aimed at analyzing HIV disease course progression. For this purpose, two tubes with lyophilized reagent cocktails (CD4- and CD8-specific tubes) were designed to compare the immunological characteristics of HIV-infected persons (37 "high CD4" HAART-treated and 32 "low CD4" naïve or failed-treatment patients) with healthy donors (HD). In particular, T-cell activation (CD25, CD38, CD69), differentiation (CD45RA, CCR7), apoptosis (CD95) and immune suppression profiles (CD25(high)CD127-) in HIV+ patients were compared with HD. Statistical analysis was performed by identifying the parameters associated with disease progression, namely markers that were found to be significantly different between groups with high CD4 counts (including HD) and low CD4 counts (restricted to HIV patients) but not between the HD and the "high CD4" group. This set of markers, including those identifying different maturation and senescence subtypes of CD4 and CD8 T cells, was found to be associated with therapy failure, and it is in fact evaluated in an ongoing study aimed to verify its prognostic value. This robust assay was found feasible on a semi-routine scale for HIV-infected persons, and allows for broader clinical studies aimed at defining markers associated with treatment outcome, possibly having a high impact on the clinical management of HIV disease.