Expanding Multiparametric Flow Cytometry Capacity for the DC D-CFAR

Douglas F. Nixon, MD, PhD

August 29, 2013

The immune response to infections such as HIV, vaccination to infectious agents, or other immunological settings of atopy, autoimmunity or primary immunodeficiency, can be characterized by examining changes in the expression of proteins on leukocytes or other cells (either antigen-specific B- or T-cells, innate immune cells such as macrophages, NK or NKT cells, or globally). These proteins identify a large range of cell types. Our knowledge of which subsets of these cells are clinically relevant is limited. Sometimes, the
immunologically relevant cell subset represents a small minority of the total bulk cell population. Gross measurements taken from heterogeneous samples (for example, as generally performed with microarrays) can mask immunologically or clinically significant signals. This limitation can be overcome with polychromatic (>7 color) or multiparametric flow cytometry (MFC), where protein expression can be assessed among a large number of cell subsets, at the single cell level.

MFC has been particularly valuable in the study of HIV infection. Simple measurements of CD4+ T cell counts have been a crucial measurement of disease outcome, but have provided little help in identifying those individuals who would benefit from early initiation of highly active anti-retroviral therapy (HAART). Recent studies in the simian immunodeficiency virus (SIV) infection of non-human primates showed that the level of central memory T-cells could be a relevant predictor of the need for early therapy, and the presence of longlived T-cells during early infection correlated with long-term progression, as does the absence of proliferating cells. Measurements of polyfunctional T-cells (simultaneously producing at least three of the following: IFNγ,IL-2, CD107a, MIP-1β and TNFα) are relevant in individuals whose disease progresses. Importantly, enumeration of central memory, long-lived, proliferating or polyfunctional cells requires MFC technology, since many markers are needed to discriminate each of these cell types from other populations of leukocytes. Highly multiplexed approaches such as MFC are critical exploratory tools to identify potential correlates of pathogenesis.

Currently, there are limited flow cytometric facilities at GW or DC D-CFAR sites for HIV investigators. GW has a BD FACSAria with three lasers at excitation wavelengths of 488 nm, 633 nm and 407 nm, but this only provides capability of detecting up to 9 fluorochromes. In addition, the flow cytometry usage exceeds capacity.