We have developed a novel, highly efficient multiparameter flow cytometric assay that detects the rapid intracellular accumulation of cytokine(s) after short-term (6 hr.) in vitro Ag stimulation of CD4+ T cells. Responses in this assay are 1) restricted to the CD45RA(low) memory/effector subset, 2) dependent on class II MHC-mediated Ag presentation and appropriate costimulation, 3) correlate precisely with independent measures of sensitization history (e.g. seroreactivity), and 4) allow the simultaneous assessment of multiple cytokines on single effector T cells. Healthy HIV-individuals manifested an average of 0.71, 0.72, 0.38 and 0.06% CD4+ T cells responding to CMV with gamma-IFN, TNF-alpha, IL-2 and IL-4 production, respectively. Among these subjects, the most common phenotype of these CMV-specific effectors was gamma-IFN+, TNF-alpha+, IL-2+, IL-4-, but smaller populations exhibited nearly all possible combinations of these 4 cytokines on a single cell level. Surprisingly, 40% of 21 clinically diverse HIV+ subjects displayed strikingly higher frequencies of CMV-specific effectors (mean = 5.0%; range 2.3-7.6%) composed predominately of polarized gamma-IFN/TNF-alpha producing, IL-2/IL-4 non-producing cells. The remaining 60% of HIV+ subjects demonstrated CMV effector frequencies in the normal range. In contrast to their CMV responses, all HIV+ subjects showed diminished CD4+ effector frequencies (compared to HIV-subjects) for heterologous, non-ubiquitous viruses such as mumps. These data suggest the existence of homeostatic mechanisms capable of selectively preserving memory T cell populations reactive with ubiquitous, opportunistic pathogens such as CMV at the expense of memory populations reactive with more sporadically encountered infectious agents.
Keywords: AEGIS, Antigens, CD4, Cytomegalovirus, HIV, T-Lymphocytes, Immunologic Deficiency Syndromes, CD4-Positive T-Lymphocytes, Antineoplastic Combined Chemotherapy Protocols, Interleukin-2, Interleukin-4, Antigens, CD45, Cytokines, HIV Infections, HIV-1, CD8-Positive T-Lymphocytes, T-Lymphocyte Subsets, MEC protocol 1, In Vitro, AIDS
