Assessment of Immune Function by Lymphoproliferation Underestimates Lymphocyte Functional Capacity in HIV Patients Treated with Highly Active Antiretroviral Therapy

Abstract

The objective of this study was to evaluate T cell responses in HIV-infected patients after highly active antiretroviral therapy (HAART), using four assays of immune function, and to determine which best reflects the presence of CD4⁺ T cells able to respond to CMV antigen. Peripheral blood mononuclear cells from 41 HIVinfected patients and 31 healthy HIV-seronegative controls were cultured with mitogen (PMA/Ca²⁺ ionophore) or antigen (CMV). Production of interferon γ (IFN-γ) determined by ELISpot assay was compared with lymphoproliferation, IFN-γ production assessed by ELISA, and CD69 expression and intracellular IFN-γ assessed by flow cytometry. Cells from patients whose CD4⁺ T cells counts increased 4-fold or to >200 cells/μl after HAART responded as well as control cells when assessed by IFN-γ production and CD69 expression after mitogenic stimulation, but lymphoproliferation responses were depressed by about 52%. Patients who did not meet these criteria for immune reconstitution had lymphoproliferative responses up to 30-fold lower than control subjects, while intracellular IFN-γ and CD69 expression and ELISpot counts were less than 3-fold lower. Responses to CMV antigen could not be detected by flow cytometry, but were readily detected by ELISpot in CMV-seropositive patients whose CD4⁺ T cell counts had increased after HAART. This included patients with low responses assessed by lymphoproliferation. Moreover, ELISpot responses measured with fresh and frozen cells were comparable, while lymphoproliferation assays required fresh cells. In conclusion, the ELISpot assay is a sensitive and efficient technique for detecting CMV-specific IFN-γ responses in samples that display poor responses when assessed by lymphoproliferation assays.