Background:  HIV antigen-specific CD4⁺ T cells are preferentially targeted and deleted by cytopathic infection. In most cases, HIV-1 antigen-specific CD4⁺ T cells are barely detectable by proliferation assays or using major histocompatibility class (MHC) class II tetramers, and at low levels by interferon (IFN) -g production. We developed a novel assay of antigen-specific CD4⁺ T cells which does not rely on either proliferation or cytokine production, and reassessed the level of HIV-specific CD4⁺ T cells in untreated chronic infection.

Methods:  Samples of whole blood from 15 healthy adult controls and 13 consecutive untreated subjects with established HIV-1 infection (median:  158 CD4 cells/µL; 4.7 log RNA copies/mL) were incubated in vitro for 40 to 44 hours with:  culture medium alone; phytohemagglutinin (PHA); recall antigens including lysates of cytomegalovirus (CMV) and M. avium; ultraviolet (UV) -inactivated herpes simplex virus (HSV) -1; or a pool of overlapping 15-mer peptides covering HIV-1 Gag. The up-regulation of CD25 and the co-stimulatory molecule CD134 was measured on CD4⁺ T cells by flow cytometry. Results for subject groups are expressed as medians and interquartile ranges (IQR).

Results:  The background level of CD25⁺CD134⁺CD4⁺ T cells was very low, <0.03% of CD4⁺ T cells (mean + 3xSD). In healthy adult controls, recall responses to CMV, M. avium and HSV-1 were readily detected—some individual responses exceeded 10% of CD4 T cells—and correlated with lymphoproliferation in 7-day cultures with either ³H-thymidine or carboxy-fluorescein diacetate, succinimidyl ester (CFSE) readouts. In the cohort of HIV-infected subjects, responses to CMV, M. avium, and HSV-1 were also detected at high levels, as much as 18% of CD4⁺ T cells. Responses to HIV-1 Gag peptides were low in healthy adult controls (median 0.10%) but in the HIV-infected subjects responding cells were a median 0.80% of CD4⁺ T cells (*p <0.0001 compared with controls), with 6 of 13 subjects having much higher responses, ranging from 1.70 to 4.80% of CD4⁺ T cells.

Conclusions:  This novel assay has revealed relatively large populations of antigen-specific CD4⁺ T cells that respond to recall antigens, in both healthy adults and untreated chronically infected HIV-infected subjects. In particular, we have discovered that there are 5 to 10 times more HIV Gag-specific CD4⁺ T cells present in peripheral blood than previously estimated by proliferation, tetramers or IFN-g production.