Cellular Composition of Cerebrospinal Fluid in HIV-1 Infected and Uninfected Subjects Emily L. Ho 1 , Rollie Ronquillo 2 , Hermann Altmeppen 3 , Serena S. Spudich 4 , Richard W. Price 5 , Elizabeth Sinclair 6 * 1 Department of Neurology, Harborview Medical Center, University of Washington, Seattle, Washington, United States of America, 2 Department of Immunology and Microbiology, Rush University Medical Center, Chicago, Illinois, United States of America, 3 Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4 Department of Neurology, Yale University, New Haven, Connecticut, United States of America, 5 Department of Neurology, University of California San Francisco, San Francisco, California, United States of America, 6 Division of Experimental Medicine, University of California San Francisco, San Francisco, California, United States of America Abstract In order to characterize the cellular composition of cerebrospinal fluid (CSF) in a healthy state and in the setting of chronic pleocytosis associated with HIV-1 (HIV) infection, multi-parameter flow cytometry was used to identify and quantitate cellular phenotypes in CSF derived from HIV-uninfected healthy controls and HIV-infected subjects across a spectrum of disease and treatment. CD4+ T cells were the most frequent CSF population and the CD4:CD8 ratio was significantly increased in the CSF compared to blood (p = 0.0232), suggesting preferential trafficking of CD4+ over CD8+ T cells to this compartment. In contrast, in HIV-infection, CD8+ T cells were the major cellular component of the CSF and were markedly increased compared to HIV-uninfected subjects (p,0.001). As with peripheral blood, the CSF CD4:CD8 ratio was reversed in HIV-infected subjects compared to HIV-uninfected subjects. Monocytes, B cells and NK cells were rare in the CSF in both groups, although absolute counts of CSF NK cells and B cells were significantly increased in HIV-infected subjects (p,0.05). Our studies show that T cells are the major cellular component of the CSF in HIV-infected and uninfected subjects. The CSF pleocytosis characteristic of HIV infection involves all lymphocyte subsets we measured, except for CD4+ T cells, but is comprised primarily of CD8+ T cells. The reduced proportion of CD4+ T cells in the CSF may reflect both HIV-related peripheral loss and changes in trafficking patterns in response to HIV infection in the central nervous system. Citation: Ho EL, Ronquillo R, Altmeppen H, Spudich SS, Price RW, et al. (2013) Cellular Composition of Cerebrospinal Fluid in HIV-1 Infected and Uninfected Subjects. PLoS ONE 8(6): e66188. doi:10.1371/journal.pone.0066188 Editor: Clive M. Gray, University of Cape Town, South Africa Received February 7, 2012; Accepted May 7, 2013; Published June 18, 2013 Copyright: ß 2013 Ho 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. Funding: This study was funded by grants from the National Institutes of Health: R01 MH62701; R01 NS37660; R01 NS43103; K23 MH074466; P30AI027763 (UCSF- GIVI Center for AIDS Research); UL1RR024131 (NCRR UCSF-CTSI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: esinclair@sfgh.ucsf.edu Introduction HIV-1 (HIV) infection is frequently accompanied by cerebro- spinal fluid (CSF) pleocytosis that occurs early in infection and largely resolves with antiretroviral therapy (ART) [1,2,3,4,5,6]. The cellular composition of this pleocytosis has not been fully defined, particularly in patients with treated HIV infection and in comparison to HIV-uninfected individuals. Flow cytometry provides a powerful method for elucidating multiple cell charac- teristics in fluids, such as CSF [7,8,9,10,11]. Advances in polychromatic flow cytometry have increased the number of markers that can simultaneously be applied to a sample [12], allowing a more comprehensive analysis of CSF, with its relatively low number of cells, than was possible in earlier studies [8,10]. Flow cytometry has been used to evaluate CSF B and T cell subsets in various neurological conditions [13,14,15,16,17,18,19]. We used an eight-color flow cytometry panel combined with TruCount TM beads (for cell enumeration) to create ‘‘Flow Count,’’ a single platform assay for the determination of both the proportion and the absolute count of the major lymphocyte populations (CD4+ and CD8+ T cells, B cells, and NK cells) and that of monocytes and granulocytes. Flow Count was validated on whole blood samples of forty HIV-infected and ten HIV- uninfected subjects by comparison to standard clinical laboratory methods (described in Supporting Information, Text S1 and Figure S1). The relative frequencies and absolute counts of each cell population were determined in paired CSF and peripheral blood samples from both groups. To discover whether differen- tially effective levels of ART may alter the cellular composition of CSF in HIV infection, we compared the proportions and the absolute counts of CSF cell subsets between subjects on ART, with and without plasma viral suppression, and in subjects off therapy. This report defines both the proportion and absolute number of the CSF B cell, NK cell, and monocyte populations, in addition to the more abundant T cell populations in HIV-infected and uninfected individuals. Materials and Methods Ethics Statement This study involving human subjects was conducted according to the principles expressed in the Declaration of Helsinki. Protocols were approved by the UCSF Committee on Human PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e66188