Selective growth, in vitro and in vivo, of individual T cell clones from tumor-infiltrating lymphocytes obtained from patients with melanoma

Abstract

In recent clinical trials in patients with metastatic melanoma, adoptive transfer of tumor-reactive lymphocytes mediated the regression of metastatic tumor deposits. To better understand the role of individual T cell clones in mediating tumor regression, a 5' RACE technique was used to determine the distribution of TCR beta-chain V region sequences expressed in the transferred cells as well as in tumor samples and circulating lymphocytes from melanoma patients following adoptive cell transfer. We found that dominant T cell clones were present in the in vitro-expanded and transferred tumor-infiltrating lymphocyte samples and certain T cell clones including the dominant T cell clones persisted at relatively high levels in the peripheral blood of the patients that demonstrated clinical responses to adoptive immunotherapy. However, these dominant clones were either undetected or present at a very low level in the resected tumor samples used for tumor-infiltrating lymphocyte generation. These data demonstrated that there was selective growth and survival, both in vitro and in vivo, of individual T cell clones from a relatively small number of T cells in the original tumor samples. These results suggest that the persistent T cell clones played an active role in mediating tumor regression and that 5' RACE analysis may provide an important tool for the analysis of the role of individual T cell clones in mediating tumor regression. A similar analysis may also be useful for monitoring autoimmune responses.

FIGURE 1

Diversity of TRBV sequences expressed in the TIL and uncultured tumor samples from melanoma patient 1803. TRBV sequences amplified from TIL 1803 and the uncultured 1803 tumor (1803Tu) were aligned to germline TRBV sequences and the percentage of sequences corresponding to each other was determined. The absence of a bar indicates that none of the sequences corresponded to that particular TRBV gene. The datum next to each bar represents the number of distinct sequences with the same TRBV sequence that were identified by comparison of CDR3 junctional region sequences. A total of 96 clones were sequenced from TIL 1803 and 96 clones were sequenced from 1803Tu.

FIGURE 2

Alignment of the VDJ junctional region of TRBV20-1 sequences amplified from the 1803Tu sample and the TIL 1803 sample. Dots represent identical bases and dashes represent gaps introduced to align the D and J region sequences. The sequences in D region are noted in bold. The dominant TRBV20-1 sequence from the TIL 1803 sample was not detected in the uncultured 1803 tumor sample.

FIGURE 3

Diversity of TRBV sequences expressed in the TIL and uncultured tumor samples from melanoma patient 2023. TRBV sequences amplified from TIL 2023 and the uncultured 2023 tumor (2023Tu) were aligned to germline TRBV sequences and the percentage of sequences corresponding to each other was determined. The absence of a bar indicates that none of the sequences corresponded to that particular TRBV gene. The datum next to each bar represents the number of distinct sequences with the same TRBV sequence that were identified by comparison of CDR3 junctional region sequences. A total of 192 clones were sequenced from TIL 2023 and 96 clones were sequenced from 2023Tu.

FIGURE 4

Persistence of T cell clones in melanoma patients 1803 and 2023 after adoptive cell transfer. The TRBV sequences expressed in TIL as well as PBL samples obtained at 1, 2, 4, and 8 wk following adoptive TIL transfer were analyzed and compared. 2023Tu4w was the tumor sample obtained from patient 2023 at 4 wk following adoptive transfer and analyzed by 5′ RACE analysis of TRBV gene expression, and the TRBV sequences expressed in 2023Tu4w were also compared with those in TIL 2023 and PBL samples. Two T cell clones, 1803BV7-2 and 1803BV20-1, derived from the TRBV7-2 and TRBV20-1 germline genes persisted in patient 1803. Two T cell clones, 2023BV24-1 and 2023BV29-1, derived from the TRBV24-1 and TRBV29-1 germline genes persisted in patient 2023.

FIGURE 5

Tumor reactivity of TIL and T cell clones derived from TIL 1803 (A) and TIL 2023 (B). 1803BV7-2 and 1803BV20-1 T cells were sorted from TIL 1803 using corresponding Abs. Tumor reactivity of TIL 1803 cells was tested against uncultured 1803 tumor cells and HLA-A1-matched allogeneic 888mel as well as 888EBV-B cells. Tumor reactivity of 1803BV7-2, 1803BV20-1, and their depleted T cell samples were tested against 888mel only because of limited 1803Tu cells. 2023BV24-1 and 2023BV30 T cells were cloned from TIL 2023 by limiting dilution, and their tumor reactivity was tested against autologous 2034mel as well as HLA-A2-matched allogeneic 624mel, 1088me, 1907mel, and 1909mel.

FIGURE 6

Clinical response in melanoma patients after adoptive cell transfer. A, Computed axial tomography scans in patient 1803. Left panels, Disease status before adoptive cell transfer. Right panels, Disease status after treatment. Panels in the top row show the metastases in the lung. Panels in the second row show a metastasis in right axillary lymph node. Panels in the third row show a metastasis in the intra-abdominal site. Panels in the last row show a metastasis in the muscles of the thigh site. B, Computed axial tomography scans illustrating the s.c. metastases in patient 2023. Top panels, Disease status before treatment. Bottom panels, Disease status after the treatment. C, Computed axial tomography scans illustrating a metastasis in the left cervical lymph node in patient 2023. Left panels, Disease status before the treatment. Right panels, Disease status after the treatment. Arrows show sites of metastases.