Background:  Our goal was to evaluate the role of human herpesvirus-8 (HHV-8) T-cell responses in the pathophysiology of HHV-8-related multicentric Castleman disease (MCD).

Methods:  We matched for CD4 cell count, 11 HIV/HHV-8 co-infected asymptomatic patients and 11 patients with HIV-associated (n = 8) or classic (n = 3) MCD, all seropositive for HHV-8. T-cell responses to HHV-8 were first analyzed with an interferon-gamma (IFN-g) ELISpot assay using 56 peptides:  predicted LANA-1 epitopes binding HLA-A2, 15-mer peptides overlapping by 10 amino acids covering K12 protein and exons 1 to 3 in K15, and 4 previously described epitopes in lytic glycoproteins GpH, Gp35/37, and GpB. Threshold was 50 SFC/10⁶ peripheral blood mononuclear cells (PBMC) above background. HHV-8-specific T cells were then analyzed after peptide stimulation in 5-color flow cytometry using surface staining for CD45RA, CCR7, CD27, and CD28 molecules, and intracellular staining for IFN-g. Spearman non-parametric test was used for statistical analysis.

Results:  T-cell responses against HHV-8 could be detected in 6 of 11 (54.5%) MCD patients (2 classic and 4 HIV-related MCD) and in 6 of 11 (54.5%) asymptomatic patients in IFN-g ELISpot assay. The median sum of HHV-8-specific T cells detected was equivalent in both groups:  505 (range 50 to 1420) and 460 (range 125 to 1455) SFC/10⁶ PBMC, respectively. HHV-8-specific T-cell numbers were not correlated to CD4 cell counts (p = 0.433 and r = 0.171). Correlation with HHV-8 viral loads will be presented. Repertoire of T-cell responses was similar in both groups of patients and responses were mainly against K12 protein (4 MCD and 5 asymptomatic patients), and to a lesser extent against K15 (3 asymptomatic patients and 2 MCD), LANA (1 asymptomatic patient and 1 MCD), and lytic glycoproteins (4 asymptomatic patients and 2 MCD). These findings permitted the identification of a new 10-mer CD8 epitope predicted to bind B7 molecule in K15 (amino acids 166 to 175). PBMC from 1 MCD patient with positive responses to lytic glycoproteins in ELISpot were stimulated with those HHV-8 peptides and INF-g secreting cells (0.62% of lymphocytes in flow cytometry) showed mainly (90%) a terminal effector CD8⁺CCR7^­CD27^­CD45RA^­ T-cell phenotype. More data concerning phenotypic patterns of HHV-8-specific T cells in other patients responding in ELISpot will be presented.

Conclusions:  Patients with HHV-8-related MCD and asymptomatic HHV-8 carriers exhibit equivalent magnitude of HHV-8-specific T-cell responses. These results suggest that MCD is not linked with a quantitative impairment of HHV-8-specific T-cell responses.