Serial study of lymph node cell subsets using fine needle aspiration in pigtail macaques

Abstract

Lymphoid tissues are of intense interest for studies of the pathogenesis of human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques but are relatively difficult to sample non-invasively. Fine needle aspiration (FNA) cytology, conventionally a diagnostic procedure for lymphadenopathy, can be used for longitudinal study of tissue cell subsets during HIV/SIV infection. In this study, we serially sampled lymph node (LN) FNA from pigtail macaques and studied cell subsets in the aspect of absolute count, frequency, and functionality by flow cytometry. The median recovered lymphocyte count from FNA samples was 2.01×105 (3.0×103 to 2.25×106, n=38) and median CD4+ T cell subset recovered was 5.94×104 (277 to 6.17×105, n=38). Although we observed a relatively large variation in the frequencies of cell subsets of FNA samples taken from different time points, the cell subset composition of FNA samples, in particular T cell and CD4+ T cell frequencies, was broadly comparable to whole excised LNs (n=6) and distinct from peripheral blood. A subset of CD4+ T cells that is located almost exclusively in secondary lymphoid tissues, T follicular helper (TFH) cells, was readily identifiable in LN FNAs and the TFH cell frequencies were strongly correlated with B cell frequencies. In vitro functionality of FNA lymphocytes was demonstrated using polyclonal SEB stimulation, resulting in a median 6% of responding CD4+ T cells, comparable to circulating CD4+ T lymphocytes. We conclude that serial sampling of macaque LNs using FNA is a potentially useful method to study the immunopathogenesis of SIV infection and may be extended to HIV infection.