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Motivation:  Mass cytometry is a technique used to measure the intensity levels of proteins expressed by cells, at a single cell resolution. This technique is essential to characterize the phenotypes and functions of immune cell populations, but is currently limited to the measurement of 40 cell markers that restricts the characterization of complex diseases. However, algorithms and multi-tube cytometry techniques have been designed for combining phenotypic information obtained from different cytometric panels. The characterization of chronic HIV infection represents a good study case for multi-tube mass cytometry as this disease triggers a complex interactions network of more than 70 cell markers.   Method:  We collected whole blood from non-viremic HIV-infected patients on combined antiretroviral therapies and healthy donors. Leukocytes from each individual were stained using three different mass cytometry panels, which consisted of 35, 32, and 33 cell markers. For each patient and using the CytoBackBone algorithm, we combined phenotypic information from three different antibody panels into a single cytometric profile, reaching a phenotypic resolution of 72 markers. These high-resolution cytometric profiles were analyzed using SPADE and viSNE algorithms to decipher the immune response to HIV.   Results:  We detected an upregulation of several proteins in HIV-infected patients relative to healthy donors using our profiling of 72 cell markers. Among them, CD11a and CD11b were upregulated in PMNs, monocytes, mDCs, NK cells, and T cells. CD11b was also upregulated on pDCs. Other upregulated proteins included: CD38 on PMNs, monocytes, NK cells, basophils, B cells, and T cells; CD83 on monocytes, mDCs, B cells, and T cells; and TLR2, CD32, and CD64 on PMNs and monocytes. These results were validated using a mass cytometry panel of 25 cells markers.   Impacts:  We demonstrate here that multi-tube cytometry can be applied to mass cytometry for exploring, at an unprecedented level of details, cell populations impacted by complex diseases. We showed that the monocyte and PMN populations were strongly affected by the HIV infection, as CD11a, CD11b, CD32, CD38, CD64, CD83, CD86, and TLR2 were upregulated in these populations. Overall, these results demonstrate that HIV induced a specific environment that similarly affected multiple immune cells.

Characterization of Leukocytes From HIV-ART Patients Using Combined Cytometric Profiles of 72 Cell Markers