High frequency of phenotypically naive circulating Melan-A–specific CD8 T Cells in HLA-A2 healthy individuals. (a) Purified CD8 T cells from individual HD 009 were stained with anti-CD3, anti-CD8 mAbs, and HLA-A2 multimers synthesized around peptides derived from self-proteins (Melan-A_26–35 A27L analogue, CEA_694–702, gp-100_209–217 T210M analogue, tyrosinase_368–376, MAGE-4_130–139, MAGE-10_254–262, or NY-ESO-1_157–165 C165A analogue) and influenza matrix protein (Flu-MA_58–66). The sequence of each peptide is shown on the top of the corresponding dot plot. Figures in upper right quadrants are the percentages of multimer⁺ cells in gated CD3⁺CD8⁺ cells. (b) Purified CD8 T cells from individual HD 009 were stained here with multimers synthesized around the Melan-A_26–35 natural peptide. (c) Purified Melan-A–specific CD8 T cells from individual HD 009, which had been expanded in vitro were stained with an anti-CD8 mAb, and multimers synthesized around the A27L Melan-A_26–35 A27L analogue (left), the Melan-A_26–35 natural peptide (middle), and the irrelevant influenza matrix_58–66 peptide (right). Dot plots are representative examples for six Melan-A–specific populations derived from PBMCs of adults and newborns, and from thymus. (d) Purified CD8 T cells from individual HD 009 were stained with A2/Melan-A multimers and a set of mAbs as indicated. Gated A2/Melan-A⁺ cells are painted in gray and negative controls are in white.

Melan-A–specific CD8 T cells in healthy individuals retain high TREC levels and long telomeres. (a) Purified CD8 T cells from HLA-A2 individuals were stained with anti-CD3 and anti-CD8 mAbs, together with PE-labeled A2/Melan-A and APC-labeled A2/Flu-MA multimers. Gated CD3⁺CD8⁺ cells were sorted into A2/Melan-A⁺ and A2/Flu-MA⁺ subsets. (b) Purified CD8 T cells were stained with anti-CD3, anti-CD8, anti-CCR7, and anti-CD45RA mAbs. Gated CD3⁺CD8⁺ cells were sorted into naive CCR7⁺CD45RA^high and memory/effector non-CCR7⁺CD45RA^high subsets. (c) Real time PCR quantification of TRECs was performed in whole CD8 T cells, in sorted naive and memory/effector subsets, and in sorted Melan-A and influenza-specific T cells (*, not detectable; ND, not done). Figures in the left of each bar are the percentage of the corresponding subsets in whole CD8 T cells. (d) Telomere fluorescence analysis was performed in sorted naive and memory/effector subsets, and Melan-A and influenza-specific CD8 T cells were obtained from individual HD 009. The telomere fluorescence was calculated by subtracting the mean background fluorescence from the mean fluorescence obtained with the telomere probe and is expressed in kilobase, as described in the Materials and Methods section.

High frequency of Melan-A–specific CD8 T cells in CB and thymus. (a and b) Purified CD8 T cells from 11 CB specimens were stained with anti-CD3, anti-CD8 mAbs, and A2/Melan-A (a), or A2/Flu-MA (b) multimers. The percentage of A2/peptide multimer⁺ cells is given in gated CD3⁺CD8⁺ lymphocytes. (c) Purified CB CD8 T cells were stained with A2/Melan-A multimers, anti-CD3, anti-CD8, and anti-CD45RA (left) or anti-CCR7 (right) mAbs. Histograms are shown for gated CD3⁺CD8⁺A2/Melan-A⁺ cells and are representative examples for seven individuals. Gated A2/Melan-A⁺ cells are painted in gray and negative controls are in white. (d) Real time PCR quantification of TRECs was performed in whole CB CD8 T cells and in Melan-A–specific T cells (0.08% of CD8 T cells) from CB15. TREC levels in CD8 T cells are representative for four individuals. (e and f) CD4-depleted thymocytes from nine thymus specimens were stained with anti-CD3, anti-CD4, anti-CD8 mAbs, and A2/Melan-A (e) or A2/Flu-MA (f) multimers. The percentage of A2/peptide multimer⁺ cells is given in CD3^highCD4^–CD8⁺ thymocytes.

CCR7⁺CD45RA^high CD8 T cells contain high levels of TRECs ex vivo. (a) Purified CD8 T cells were stained with anti-CD3, anti-CD8, anti-CCR7, and anti-CD45RA mAbs. Gated CD3⁺CD8⁺ cells were sorted into four subsets on the basis of CCR7 and CD45RA expression. (b) Real time PCR quantification of TRECs was performed in whole CD8 T cells (CD8⁺) and in CCR7^+/–CD45RA^high/low sorted subsets obtained from four individuals (HD 008, 009, 019, and 025). Figures in the left of each bar are the percentage of the corresponding subsets within whole CD8 T cells. TREC levels in whole CD8 T cells were also calculated by adding the TREC levels obtained for each CD8 T cell subset and multiplying by their relative frequency (reconstituted CD8⁺).

Memory/effector Melan-A–specific CD8 T cells display reduced TREC levels and shortened telomeres. (a) Purified CD8 T cells were obtained from PBMCs (top) and TILNs (bottom) of melanoma patient LAU 465. In the left panels, lymphocytes were stained with anti-CD3, anti-CD8, anti-CCR7, and anti-CD45RA mAbs, and dot plots are shown for gated CD3⁺CD8⁺ cells. In the right panels, lymphocytes were stained with anti-CCR7, anti-CD45RA mAbs, and A2/Melan-A multimers, and dot plots are shown for gated A2/Melan-A⁺ cells. (b) Real time PCR quantification of TRECs was performed in whole CD8 T cells, in sorted naive and memory/effector CD8 T cells from PBMCs, and in sorted Melan-A–specific T cells from both PBMCs and TILNs (*, not detectable). Figures in the left of each bar are the percentage of the corresponding subsets in CD8 T cells. (c) Telomere fluorescence analysis was performed in sorted naive and memory/effector CD8 T cells from PBMCs, and Melan-A–specific CD8 T cells from TILNs. Telomere fluorescence was calculated as in Fig. 3 d, and is expressed in kilobase (see Materials and Methods).

Functional potential of Melan-A–specific CD8 SP thymocytes. (a) Purified Melan-A–specific CD8 SP thymocytes, which had been expanded in vitro, were tested in a 4-h ⁵¹Cr-release assay for their cytolytic activity against A2⁺ TAP-deficient T2 cells in the presence of exogenously added Melan-A_26–35 (▴), Melan-A_26–35 A27L analogue (▪), or irrelevant influenza matrix_58–66 peptides (○). In lymphocyte to target cell ratio titrations (left), peptides were used at 1 μM. In peptide titrations (right), the lymphocyte to target cell ratio used was 10:1. Results are representative examples for six Melan-A–specific T cell populations derived from thymus and peripheral blood of newborns and adults. (b) Purified Melan-A–specific CD8 SP thymocytes, which had been expanded in vitro, were tested in a 4-h ⁵¹Cr-release assay for their cytolytic activity against Me 290 (•, A2⁺ / Melan-A⁺) and Na 8 (○, A2⁺ / Melan-A⁻) tumor cells. Results are representative examples for six Melan-A–specific T cell populations derived from thymus and peripheral blood of newborns and adults.