Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes

Abstract

We report here the adoptive transfer, to patients with metastatic melanoma, of highly selected tumor-reactive T cells directed against overexpressed self-derived differentiation antigens after a nonmyeloablative conditioning regimen. This approach resulted in the persistent clonal repopulation of T cells in those cancer patients, with the transferred cells proliferating in vivo, displaying functional activity, and trafficking to tumor sites. This led to regression of the patients' metastatic melanoma as well as to the onset of autoimmune melanocyte destruction. This approach presents new possibilities for the treatment of patients with cancer as well as patients with human immunodeficiency virus-related acquired immunodeficiency syndrome and other infectious diseases.

Figures

Fig. 1

(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).

Fig. 2

(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.

Fig. 3

(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.