(A) Patient 9 (left) and patient 10 (right) exhibited a profound lymphocytosis after treatment. ALCs (solid squares) and absolute neutrophil counts (ANCs) (open diamonds) are plotted overtime. The upperlimit of normal for ALCs is 4800 cells/mm³.(B) Individual Vβs that were overrepresented in the infused cells (TILs) of patients 9 and 10 dominated the TCR repertoire of PBLs sampled after treatment (11). Values in the “CD8” column indicate the absolute numberof CD8⁺ cells/mm³ of peripheral blood on the indicated day. PRE, pretreatment PBL; nd, not done. (C) T cell clones derived from the infused TILs by limiting dilution specifically recognized the MART-1: 27-35 peptide and HLA-A2⁺ melanoma cell lines by cytokine secretion assay (11). M1C3 is a Vβ12⁺ clone from patient 9 and S1A5 is a Vβ7⁺ clone from patient 10. (D) The DNA and predicted protein sequences of complementarity-determining region 3 (CDR3) of the TCR β chains of a Vβ12⁺ T cell clone from the infused TILs of patient 9 (left) ora Vβ7⁺ T cell clone derived from the TILs of patient 10 (right) are shown. Although both clones recognize the MART-1 antigen, no CDR3 sequence similarity between the clones is evident. (E) MART-1-reactive clones persisted as the predominant lymphocyte population in the peripheral blood for more than 4 months. PBL samples from patient 9 (left) or patient 10 (right) at the indicated days after cell transfer were analyzed by FACS (11) using antibodies specific for the indicated TCR Vβ families (Vβ7, solid diamonds; Vβ12, solid squares; Vβ14, solid triangles; average of other Vβs, open circles) or HLA-A2/MART-1: 26-35(27L) tetrameric complexes (A2/MART tetramer, solid circles). Lymphocytes from patient 10 reacted only with the native MART-1: 27-35 peptide, not the altered MART-1: 26-35(27L) peptide (14).

(A) Antibodies specific forCD8 and TCR Vβ reveal tissue infiltration by a limited repertoire of T cells (11). Twenty days aftercell transferin patient 9, a resected tumornodule contains diffusely infiltrating lymphocytes consisting of CD8⁺ Vβ12⁺ T cells, but no Vβ7⁺ T cells. Similarly, a residual tumornodule biopsied 14 days aftercell transferfrom patient 10 exhibited few viable tumor cells but contained areas densely infiltrated by CD8⁺ Vβ7⁺ T cells, with few Vβ12⁺ cells. (B) A tumor lesion excised from patient 9 before nonmyeloablating chemotherapy (“pretreatment”) exhibited scant CD8⁺ cells (left), strong stromal cell staining but weak staining of tumor cells with antibody to MHC class I (center), and sporadic cell staining with an antibody to MHC class II (probably tumor macrophages) but minimal staining of tumor cells (right). In contrast, a sample resected 57 days after cell transfer (“post treatment”) exhibited a dense, diffuse CD8⁺ infiltrate and ubiquitous expression of both MHC class I and class II molecules in tumor cells.

(A) The infused TILs (triangles) and PBLs collected 7 days after cell transfer (squares), but not pretreatment PBLs (circles), from patient 9 (left) and patient 10 (right) specifically lysed the HLA-A2⁺ melanoma cell line 526 (lowergraphs) but not the HLA-A2^- melanoma cell line 938 (uppergraphs) (11). (B) Blood smears obtained from patient 9 (left) and patient 10 (right) during the lymphocytic episodes showed the highly activated phenotype of the lymphocytes, which show irregular and hyperchromatic nuclei, a high nuclear-to-cytoplasmic ratio, toxic granulation, and the presence of Dohle bodies. (C) PBLs collected 9 days (patient 9, left) or8 days (patient, 10 right) after cell transfer were cultured overnight in the absence (striped boxes) or presence (solid boxes) of 600 IU/ml of IL-2, washed extensively, and tested forcytokine release when stimulated with the indicated target cells.