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Background:  HIV-specific CD4 T helper (T_H) cells are critical for effective immune control of HIV infection. Direct ex vivo phenotypic and functional characterization of low-frequency epitope-specific T_H cells is difficult. MHC class II tetramers have been mostly used so far for quantitation of T_H cells and phenotyping based on cell surface molecules.

Methods:  Epitope-specific cell lines were used to optimize the assays. PBMC from 5 DRB1*0401⁺ HIV-infected subjects were then assessed. One individual (AC04) was longitudinally followed before and after treatment interruption. We used DRB1*0401 class II tetramers based on the endogenous epitope GNQWVGYDNQESVKSK (control, cTet) and the HIV epitopes LNKIVRMYSPTSILD (Tet37) and DRFYKTLRAEQASQEV (Tet41). Tetramer staining was followed by intracellular staining for IL2 or Ki67 and IFN-g. Flow cytometry was performed on an LSRII (BD).

Results:  Optimal results were obtained by using 2 exclusion channels, apoptotic/dead cells and multilineage. Frequency of Tet⁺ CD4 cells ranged from < 1/50,000 to 1/780 (median 1/4260). Intracellular staining following tetramer staining resulted in frequencies 30 to 40% lower than tetramer surface staining alone. Gating on Tet⁺, cytokine-secreting CD4 cells allowed highly sensitive and specific detection of T_H cells (median cTet⁺IFN-g⁺ background:  1/76,000). Frequency of Tet⁺IFN-g⁺ cells in the Tet⁺ population ranged 0 to 64% (median 19%; n = 14). Results correlated with IFN-g ELISpot performed with CD8-depleted PBMC. Tet⁺IL2⁺ cells were not detectable in most cases (range:  0 to 10%). Rebound of viremia in subject AC04 after treatment interruption was accompanied by a marked but transient raise in Tet41⁺Ki67⁺ cells (up to 23% of Tet⁺ cells, vs 0.6% in total CD4 cell population) and by a more sustained increase in Tet⁺IFN-g⁺ cells. More than 90% of the Tet41⁺Ki67⁺ cells produced neither IFN-g nor IL2.

Conclusions:  The use of an optimized assay combining class II tetramer staining and intracellular staining allows assessment of HIV-specific T_H cells with sensitivity comparable to ELISpot assays while allowing characterization of both cytokine- and non-cytokine-secreting T_H cells. At the time points choosen for study, frequencies of Tet⁺ were low, with a higher fraction of IFN-g⁺ cells in the presence of viremia. Early after recurrence of viremia, a high proportion of T_H cells specific for some HIV epitopes can be activated and enter cell cycle. These cells may be particularly vulnerable to infection by HIV.

Keywords: CD4 T cells; HIV-specific immune responses; antiviral therapy