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. 1998 Nov 2;188(9):1641–1650. doi: 10.1084/jem.188.9.1641

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Direct identification of HLA-A2/tumor peptide antigen tetramer binding lymphocytes in TILNs. (A) Four TILNs obtained from three HLA-A2 melanoma patients were stained, after overnight culture, with either A2/Melan-A_26–35 A27L or A2/tyrosinase_368–376 tetramers together with anti-CD3^PerCP mAb and anti-CD8^FITC mAb. Dot plots are shown for gated CD8⁺ LN cells. (B) Ex vivo A2/Melan-A tetramer⁺ TILNs have an activated phenotype. Cell suspensions prepared from normal (NLN) or metastatic (TILN) LNs from patient LAU 267 were directly analyzed by three-color flow cytometry using anti-CD8^PerCP mAb, A2/Melan-A tetramers, and either anti-CD45RA^FITC mAb (top) or anti-CD45RO^FITC mAb (bottom). Dot plots are shown for gated CD8⁺ LN cells. (C) Enumeration and phenotype of tetramer⁺ cells in ex vivo circulating lymphocytes. Highly homogeneous CD8⁺ lymphocyte populations (>98%) were obtained from PBMC of patient LAU 267, or from a healthy donor (HD), by two rounds of positive selection with magnetic cell sorting. The lymphocyte preparation was then stained with A2/Melan-A or A2/influenza matrix tetramers and anti-CD45RA^Cychrome and analyzed immediately by flow cytometry. The histograms on top of dot plots show the intensity of the fluorescence signal on the FL-3 channel of gated Melan-A/tetramer⁺ or influenza tetramer⁺ lymphocytes. (D) Summary of phenotyping data obtained from NLNs, TILNs, and PBMC from patients LAU 181 and LAU 267. The LN cell suspensions were stained either immediately after cell suspension preparation (LAU 267) or after overnight culture of previously cryopreserved uncultured LN cells (LAU 181) with either anti-CD45RA^FITC or anti-CD45RO^FITC in combination with anti-CD8^PerP and A2/Melan-A tetramers. The results for the CD45RA and CD45RO triple staining of NLNs and TILN-2 from LAU 267 are illustrated in B. Additionally, PBMC available from the same time of the LN dissection from patient LAU 267 were stained as illustrated in C. The percentages of either CD45RO⁺ or CD45RA⁻ in gated A2/Melan-A tetramer⁺ LN cells were calculated with CellQuest™ software.

Direct identification of HLA-A2/tumor peptide antigen tetramer binding lymphocytes in TILNs. (A) Four TILNs obtained from three HLA-A2 melanoma patients were stained, after overnight culture, with either A2/Melan-A_26–35 A27L or A2/tyrosinase_368–376 tetramers together with anti-CD3^PerCP mAb and anti-CD8^FITC mAb. Dot plots are shown for gated CD8⁺ LN cells. (B) Ex vivo A2/Melan-A tetramer⁺ TILNs have an activated phenotype. Cell suspensions prepared from normal (NLN) or metastatic (TILN) LNs from patient LAU 267 were directly analyzed by three-color flow cytometry using anti-CD8^PerCP mAb, A2/Melan-A tetramers, and either anti-CD45RA^FITC mAb (top) or anti-CD45RO^FITC mAb (bottom). Dot plots are shown for gated CD8⁺ LN cells. (C) Enumeration and phenotype of tetramer⁺ cells in ex vivo circulating lymphocytes. Highly homogeneous CD8⁺ lymphocyte populations (>98%) were obtained from PBMC of patient LAU 267, or from a healthy donor (HD), by two rounds of positive selection with magnetic cell sorting. The lymphocyte preparation was then stained with A2/Melan-A or A2/influenza matrix tetramers and anti-CD45RA^Cychrome and analyzed immediately by flow cytometry. The histograms on top of dot plots show the intensity of the fluorescence signal on the FL-3 channel of gated Melan-A/tetramer⁺ or influenza tetramer⁺ lymphocytes. (D) Summary of phenotyping data obtained from NLNs, TILNs, and PBMC from patients LAU 181 and LAU 267. The LN cell suspensions were stained either immediately after cell suspension preparation (LAU 267) or after overnight culture of previously cryopreserved uncultured LN cells (LAU 181) with either anti-CD45RA^FITC or anti-CD45RO^FITC in combination with anti-CD8^PerP and A2/Melan-A tetramers. The results for the CD45RA and CD45RO triple staining of NLNs and TILN-2 from LAU 267 are illustrated in B. Additionally, PBMC available from the same time of the LN dissection from patient LAU 267 were stained as illustrated in C. The percentages of either CD45RO⁺ or CD45RA⁻ in gated A2/Melan-A tetramer⁺ LN cells were calculated with CellQuest™ software.