349 Repertoire of HIV-1 p24 Epitopes Targeted by CD4+ T-cells in HIV-1 Infected Subjects Either Receiving or Not Receiving Antiretroviral Therapy E. Boritz*, B. Palmer, C. Wilson Univ of Colorado Hlth Sci Ctr, Denver
Background: CD4+ T-cell recognition of multiple HIV-1-derived epitopes during HIV-1 infection may contribute to effective HIV-1-specific immunity. In a pilot study, we found a diverse repertoire of HIV-1 p24-specific CD4+ T-cell clones in a subject who began ART during advanced HIV-1 disease. To pursue this further, we analyzed the repertoire of HIV-1 p24 peptides recognized by CD4+ T-cells from additional subjects who started Antiretroviral Therapy (ART) during advanced disease, and we are developing methods to measure peptide-specific CD4+ T-cell responses from untreated subjects.
Methods: HIV-1-infected study subjects were untreated or "suppressed" (> 6 months of ART, plasma HIV-1 RNA < 400 copies/ml). HIV-1 p24-specific CD4+ T-cells in CD8-depleted peripheral blood mononuclear cells (PBMC) were quantified by IFN-gamma ELISpot. CD4+ T-cell responses to 25 individual overlapping p24 peptides were measured in IFN-gamma ELISpots using cells previously expanded by p24 antigen stimulation. CD4+ T-cell proliferation was quantified by flow cytometry of CFSE-labeled cells cultured for 6-7 days with p24 antigen. In some assays, cells were cultured with p24 plus CD28 antibody, CD40 ligand trimer (CD40LT), IL2, IL12, or didanosine.
Results: In 9 study subjects (6 suppressed, 3 untreated), we detected a median of 19 p24-specific spot forming cells per million fresh, CD8-depleted PBMC (range 0-46). To facilitate detection of CD4+ T-cell responses to individual p24 peptides, we successfully expanded the p24-specific population from suppressed subjects by culture with p24 antigen. In IFN-gamma ELISpots measuring p24 peptide-specific responses by expanded cells, 7 suppressed subjects responded to a median of 5 peptides/subject (range 3-7). Because in vitro proliferation of HIV-1-specific CD4+ T-cells from untreated subjects is often weak, we attempted to expand these cells under modified conditions. When CD8-depleted PBMC were used for culture, expansion of IFN-gamma-secreting, p24-specific CD4+ T-cells was detected in 7/10 untreated subjects. CD40LT and IL12 also occasionally increased the magnitude of these responses.
Conclusions: These results confirm that CD4+ T-cells from HIV-1-infected subjects first treated with ART during advanced HIV-1 disease may target multiple HIV-1-derived epitopes. These methods will also enable analysis of the epitopes targeted by CD4+ T-cells from untreated subjects, allowing repertoire changes that result from viral suppression to be tracked directly.
