<b.Background:  We and others have demonstrated a better survival of GBV-C⁺ HIV-infected patients. However, the mechanism is unknown.

Methods: We studied 82 HIV-infected patients in 3 different sets of experiments with 3 independent cohorts for each experiment. 34 individuals tested GBV-C RNA⁺ by PCR while 48 patients were GBV-C RNA⁺. PBMC were analyzed directly ex vivo for the frequency of apoptotic cells, CD4⁺CD25⁺ T cells and intracellular perforin expression by flow cytometry. Cells were then cultured in different conditions including stimulation with IL-2 and superantigens. The number of living cells was determined after 1, 2, 3, and 5 days. Proliferation was studied by 3H-thymidin assays and on single cell level by staining with CFSE. IL-2 receptor expression on CD4⁺ T cells was tested for each culture condition on day 5.

Results: In vitro cell survival of PBMC significantly differed between GBV-C^- and GBV-C⁺ individuals. This was confirmed in all 3 independent experiments (cell counting on day 5 of culture p =0.02, p =0.005, and p = 0.04 for the 3 cohorts). The difference became evident already on day 2 of culture (p =0.6, p = 0.03, p = 0.02 and p =0.05 for days 1,2,3, and 5, respectively) and could not be explained by discrepancies in cell proliferation as determined by thymidin assays and CFSE staining. The difference in in vitro cell survival remained highly significant even after the addition of IL-2 or PHA to the culture (p <0.005). Ex vivo, CD4⁺ T helper cells of GBV-C⁺ HIV-infected patients tented to test less often positive for Annexin (32% vs. 41%, p = 0.15). In addition, T regulatory CD4⁺CD25⁺ T cells tended to be less frequent in GBV-C⁺ individuals than in GBV-C^- patients (10.6% vs 17.2%, respectively; p = 0.11). Intracellular perforin staining showed no difference in perforin⁺ CD4⁺ T cells, perforin⁺ CD8⁺ T cells or perforin⁺ CD4^-/CD8^- lymphoid cells between the 2 groups (p = 0.62, p = 0.80, and p = 0,73, respectively). IL-2 receptor expression after in vitro stimulation was similar in both groups. T-cell responses to the recall antigen tetanus toxoid were stronger in GBV-C⁺ patients.

Conclusions:  In vitro cell survival differs between GBV-C⁺ and GBV-C^- HIV-infected patients. The mechanisms responsible for our observation are not due to differences in cell proliferation but can be explained rather by a different apoptosis rate. The role of T regulatory T cells requires further investigation.

Keywords: GB Virus C; apoptosis; cell survival