1. Differences in the frequency and function of HHV8-specific CD8 T cells between asymptomatic HHV8 infection and Kaposi sarcoma
by Marion Lambert, Monique Gannagé, Alexandre Karras, Michal Abel, Christophe Legendre, Delphine Kerob, Felix Agbalika, Pierre-Marie Girard, Celeste Lebbe, and Sophie Caillat-Zucman
Blood
Volume 108(12):
December 1, 2006
©2006 by American Society of Hematology

2. The frequencies of HHV8-specific responses depend on the context and course of infection.
The frequencies of HHV8-specific responses depend on the context and course of infection. (A) The number of patients with HHV8-specific CD8 T-cell responses were compared in 12 transplant recipients (□), 24 patients with AIDS-related KS (▪) and 12 patients with classical KS (▦) by chi-square test. The histograms show the percentages of patients with positive responses to the indicated peptide. (B) For each peptide, a representative example of the staining of the tetramer+ population is shown in the left panels, and grouped data for the frequency of the corresponding tetramer+ CD8 T cells are compared in the right panels. Boxes in the left panels delineate the tetramer+ CD8 T-cell population. The cut-off value for positive staining was defined by the mean + 2 SD frequency of tetramer+ cells in HLA-A2–/HHV8+ patients. Horizontal bars represent the median percentages of tetramer+ cells. P values are shown for the comparison between the 3 patient groups (nonparametric Kruskall-Wallis test). (C) Comparison of anti-HHV8 responses in patients without KS (n = 8; □), patients with KS with persistent lesions (n = 18; ▪) and patients with KS in remission (n = 22; ▦). The results are the percentages of patients with positive responses to the indicated peptide. P values are shown for the comparison between the 3 groups.
Marion Lambert et al. Blood 2006;108:
©2006 by American Society of Hematology

3. Phenotype of HHV8-specific cells according to the outcome of infection.
Phenotype of HHV8-specific cells according to the outcome of infection. (A) Tetramer+ cells were compared for the presence of CD45RA, CCR7, and CD27 markers in 14 patients from whom enough material was available (□ indicates 6 patients without KS; ▪, 7 patients with ongoing KS; and ▦, 5 patients in KS remission). The histograms show the percentages of tetramer+ cells (grouped data) with the markers indicated (mean ± SEM). (B) Flow cytometric analysis of CD45RA and CCR7 or CD27 coexpression gated on CD8+ tetramer+ cells. Each display represents results from a representative patient in each group. (C) Intracellular perforin content was determined in tetramer+ cells. Dot plots are gated on live CD8 T cells and show tetramer and perforin staining. The percentages indicated in the quadrants are representative of grouped data for patients without KS (median, 63%; range, 12%-99%), patients in KS remission (median, 48%; range, 25%-88%, nonsignificant), and patients with ongoing KS (median, 33%; range, 13%-66%, P = .04 for comparison with patients without KS).
Marion Lambert et al. Blood 2006;108:
©2006 by American Society of Hematology

4. In situ tetramer staining of HHV8-specific CD8 T cells in KS biopsies.
In situ tetramer staining of HHV8-specific CD8 T cells in KS biopsies. CD8 cells appear in red; HHV8-specific tetramer+ cells appear in green; and LANA-specific cells appear in blue. Large 3-channel merged images are shown. Compared with large tetramer– CD8 T-cell infiltrates in the vicinity of LANA+ tumoral cells (left panel), double-stained HHV8-specific CD8 T cells appear as isolated yellow cells (arrow) scarcely dispersed within the LANA– tissue (middle panel). Detection of LANA+ cells by immunohistochemistry is shown as control (right panel). Magnification, × 63.
Marion Lambert et al. Blood 2006;108:
©2006 by American Society of Hematology

5. Time course of HHV8-specific CD8 T cells in a transplant recipient with recurring KS. The figure illustrates the dynamics of HHV8 tetramer+ cells tested at approximately 3-month intervals over 2 years (beginning 4 years after grafting, indicated as M0), and...
Time course of HHV8-specific CD8 T cells in a transplant recipient with recurring KS. The figure illustrates the dynamics of HHV8 tetramer+ cells tested at approximately 3-month intervals over 2 years (beginning 4 years after grafting, indicated as M0), and shows major clinical events and viral load variations during the same period. × indicates gB; ▴, ORF65; ○, k12; ⋄, ORF6; and ▪, ORF61. • indicates the time course of HHV8 DNA load (copies/mL). Clinical relapses are indicated by arrows. The quadrant shows the phenotype profile and perforin expression of ORF6 tetramer+ cells at the indicated time.
Marion Lambert et al. Blood 2006;108:
©2006 by American Society of Hematology

6. HHV8-specific CD8 T cells in a seronegative transplant recipient exposed to the virus through the graft.
HHV8-specific CD8 T cells in a seronegative transplant recipient exposed to the virus through the graft. The figure shows the staining of the tetramer+ population for the indicated peptides (A), their perforin expression (B), and their phenotype profile (C), performed 12 months after transplantation with a kidney graft from an HHV8-infected donor. (A) Boxes delineate the tetramer+ CD8 T-cell population.
Marion Lambert et al. Blood 2006;108:
©2006 by American Society of Hematology