Direct ex vivo analysis of CD8⁺ T cells from PBMCs by flow cytometry. (A) Representative examples of BTLA expression by Melan-A^MART-1– and virus-specific (EBV, CMV, and influenza virus [FLU]) CD8⁺ T cells from healthy donors (HD) and melanoma patients. BTLA⁺ T cells were distinguished from BTLA^– T cells using a threshold established according to the autologous naive T cell subset, which is always BTLA positive. Numbers indicate percentage of positive or negative cells. (B) Comparison of BTLA expression on total CD8⁺ and virus- and Melan-A–specific T cells, from healthy individuals and unvaccinated melanoma patients. **P < 10^–2 for each of the 2 populations of Melan-A–specific T cells compared with all 5 other populations. (C–E) BTLA expression in subsets of CD8⁺ T cells of healthy individuals (n = 58), upon definition of early and late differentiation stages by CD45RA and CCR7 expression (i.e. naive [N, CD45RA⁺CCR7⁺], central memory [CM, CD45RA^–CCR7⁺], effector memory [EM, CD45RA^–CCR7^–], and effector memory RA⁺ [EMRA, CD45RA⁺CCR7^–] cells), gated as shown in C; a representative example demonstrates the narrow positive peak observed in phenotypic naive cells. (D) Statistical assessment of BTLA⁺ CD8⁺ T cell subsets. (E) GMFI data, showing results compatible with those presented in D. Overall GMFI was determined by including the whole range from BTLA-negative to -positive cells, as shown in C. GMFI data were normalized to values determined in autologous naive CD8⁺ T cells.

(A) Representative examples of BTLA, LIGHT, and HVEM expression by the melanoma cell lines Me 280, Me 261, and Me 290. Cells were stained with isotype control (open histograms) or with anti-HVEM, anti-BTLA, or anti-LIGHT mAb (filled histograms) (B) Relative expression of HVEM, BTLA, and LIGHT by melanoma cell lines, expressed as ratio fluorescence intensity (RFI), i.e., MFI with specific mAb/MFI with isotype control. Of 40 melanoma cell lines analyzed, 19 were highly positive (white dots), 7 weakly positive (gray dots), and 14 negative for HVEM expression (black dots). None of the melanoma cell lines expressed BTLA or LIGHT (black dots). (C) HVEM expression (in reddish brown) detected in paraffin-embedded tumor sections from 16 tumors of 14 melanoma patients. Examples show tumor tissues that were HVEM-negative, weakly positive (<10% HVEM⁺ tumor cells) or strongly positive (>50% positive tumor cells). Original magnification, ×200. (D) Melan-A^MART-1/HLA-A*0201–specific CD8⁺ T cell clones (cl.) 618-45, 618-4, and 618-420 were stimulated by melanoma cell lines Me 275 and Me 290 (both Melan-A⁺, HLA-A*0201⁺, HVEM⁺). The graph shows fold increase in IFN-γ production by Melan-A^MART-1–specific clones in the presence of blocking antibody BTLA-8.2, relative to isotype control antibody. IFN-γ production was determined by ELISA in supernatants of 24-hour cultures. BTLA expression by T cell clones was assessed by flow cytometry with BTLA-specific antibody (filled histograms) and isotype control (open histograms). GMFI is indicated in parentheses.

(A) Representative dot plots of A2/Melan-A^MART-1 tetramer and BTLA staining (gated on CD8⁺ T cells) of PBMCs from 2 patients, before vaccination and after 16 vaccinations, respectively. Numbers indicate percentages of positive or negative cells. (B and C) Correlations between numbers of vaccinations without or with CpG and percentages of BTLA⁺ T cells among (B) Melan-A–specific CD8⁺ T cells and whole CD8⁺ T cells and (C) subsets of Melan-A–specific CD8⁺ T cells.

(A) Representative example of BTLA downregulation by BTLA⁺ EBV-specific CD8⁺ T cells from PBMCs of healthy donor BCB136 upon EBV peptide stimulation with target cells expressing HVEM or not. Histograms are gated on CD8⁺ EBV⁺ T cells. Percentages of BTLA⁺ cells (and GMFI in parentheses) are indicated. (B) Percentages of BTLA expression by antigen-specific CD8⁺ T cells after stimulation with SKMEL37 cells expressing HVEM or not. Each dot represents a single experiment with Melan-A^MART-1– or EBV-specific T cells. (C and D) Kinetic analysis of surface BTLA expression (C) and expansion (D) of EBV-specific CD8⁺ T cells upon stimulation with irradiated target cells expressing HVEM or not, in the presence or absence of EBV peptide. Results for 2 of 5 representative healthy donors tested are shown.

(A) Analysis of BTLA expression by Melan-A^MART-1–specific T cells depending on differentiation. Representative example of PBMCs from an unvaccinated melanoma patient. (B) Percentages of BTLA⁺ CD8⁺ T cells from healthy individuals (gray, n = 58) and unvaccinated melanoma patients (white, n = 18); and from Melan-A^MART-1 tetramer⁺ CD8⁺ T cells from unvaccinated patients (black, n = 14). Data from healthy donors (same as in Figure 1D) are shown again for comparison. Circulating central memory Melan-A^MART-1–specific cells were rare, precluding analysis for BTLA expression. (C and D) BTLA expression by CD45RA^+/– and CCR7^+/– T cells from normal lymph nodes (NLN, n = 18) and tumor-infiltrated lymph nodes (TILN, n = 6) from melanoma patients. (C) Representative example and (D) comparison of BTLA expression in CD8⁺ T cells and Melan-A^MART-1–specific T cells. In NLNs, Melan-A^MART-1–specific T cells are all naive. Light gray, whole CD8⁺ T cells in NLNs (n = 18); white, Melan-A^MART-1 tetramer⁺ CD8⁺ T cells in NLNs (n = 18); black, whole CD8⁺ T cells in TILNs (n = 6); dark gray, Melan-A^MART-1 tetramer⁺ CD8⁺ T cells in TILNs (n = 6).

(A) Representative examples of A2/Melan-A^MART-1 tetramer and BTLA staining in unvaccinated and vaccinated patients. Numbers indicate percentages of positive or negative cells. w/o, without. (B) Comparison of BTLA expression in Melan-A^MART-1–specific CD8⁺ T cells from 14 unvaccinated patients, 24 patients vaccinated with peptide but without CpG, and 16 patients vaccinated with peptide plus CpG. (C) Expression of BTLA by Melan-A^MART-1–specific CD8⁺ T cells (black) and whole CD8⁺ T cells (white) according to CD8 differentiation stages after vaccinations without CpG (upper panel; n = 24) or with CpG (lower panel; n = 16).

(A) Direct ex vivo analysis of percentage of A2/Melan-A^MART-1 tetramer⁺ CD8⁺ T cells among PBMCs, in relation to their BTLA expression, after vaccination without and with CpG. (B) In vitro expansion of Melan-A^MART-1 tetramer⁺ T cells from healthy donors, after 10 days of stimulation with peptide-loaded DCs, in the absence versus presence of blocking mAb BTLA-8.2. (C and D) IFN-γ (C) and TNF-α (D) production by BTLA⁺ antigen-specific CD8⁺ T cells after 4 hours of peptide stimulation of PBMCs assessed ex vivo, i.e., without prior in vitro cultivation. Representative histograms of PBMCs from patient LAU 618 (after 2 vaccinations, with CpG) and from healthy donor BCB122 stimulated by SKMEL37 cells expressing HVEM or not, loaded with Melan-A^MART-1 or EBV peptides, respectively. Histograms are gated on tetramer⁺ T cells. Percentages of cytokine-positive cells are indicated. (E) Significant correlation between percentages of BTLA⁺ tetramer⁺ T cells and percentages of IFN-γ production inhibition (i.e., reduction when stimulated with HVEM⁺ SKMEL37 cells as compared with HVEM^– SKMEL37 cells). Each dot represents a single experiment with Melan-A^MART-1– or EBV-specific T cells.