Assessment of immune function by lymphoproliferation underestimates lymphocyte functional capacity in HIV patients treated with highly active antiretroviral therapy

The objective of this study was to evaluate T cell responses in HIV-infecte d patients after highly active antiretroviral therapy (HAART), using four a ssays of immune function, and to determine which best reflects the presence of CD4(+) T cells able to respond to CMV antigen. Peripheral blood mononuc lear cells from 41 HIV-infected patients and 31 healthy HIV-seronegative co ntrols were cultured with mitogen (PMA/Ca2+ ionophore) or antigen (CMV). Pr oduction of interferon gamma (IFN-gamma) determined by ELISpot assay was co mpared with lymphoproliferation, IFN-gamma production assessed by ELISA, an d CD69 expression and intracellular IFN-gamma assessed by flow cytometry. C ells from patients whose CD4(+) T cells counts increased 4-fold or to >200 cells/mul after HAART responded as well as control cells when assessed by I FN-gamma production and CD69 expression after mitogenic stimulation, but ly mphoproliferation responses were depressed by about 52%. Patients who did n ot meet these criteria for immune reconstitution had lymphoproliferative re sponses up to 30-fold lower than control subjects, while intracellular IFN- gamma and CD69 expression and ELISpot counts were less than 3-fold lower. R esponses to CMV antigen could not be detected by flow cytometry, but were r eadily detected by ELISpot in CMV-seropositive patients whose CD4(+) T cell counts had increased after HAART. This included patients with low response s assessed by lymphoproliferation. Moreover, ELISpot responses measured wit h fresh and frozen cells were comparable, while lymphoproliferation assays required fresh cells. In conclusion, the ELISpot assay is a sensitive and e fficient technique for detecting CMV-specific IFN-gamma responses in sample s that display poor responses when assessed by lymphoproliferation assays.