Enrichment of CD8+ cells from melanoma tumor-infiltrating lymphocyte cultures reveals tumor reactivity for use in adoptive cell therapy

Abstract

Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for metastatic melanoma has shown objective response rates as high as 72%. The successful application of this therapy requires the selection of unique tumor-reactive lymphocyte cultures for each patient. This is a technically and logistically difficult undertaking, and patients who do not have tumor-reactive TIL are not considered eligible for treatment. To simplify the methods of TIL generation and extend TIL-based immunotherapy to additional patients, methods were developed to use unselected, minimally cultured ("young") TIL. Young TIL cultures contain a variable number of CD8(+), CD4(+), and CD3(-)CD56(+) natural killer cells. In this study we retrospectively investigated a role for these subsets in the clinical outcome of patients treated with TIL derived from selected microcultures. This analysis demonstrated a suggestive but nonsignificant association between the number of CD8(+) cells administered and tumor regression. We therefore investigated the feasibility of selecting CD8(+) cells from young TIL cultures for ACT therapy. The available methods for clinical scale CD8(+) enrichment proved inadequate for TIL, so an optimized CD8(+) enrichment method was developed and is reported here. We observed that CD8 (+)enrichment of some TIL cultures revealed in vitro tumor recognition that was not evident in bulk culture, and an improved in vitro recognition of tumor in other TIL cultures. In addition, the enriched CD8(+) young TIL expanded more reliably and predictably in rapid expansions than the bulk TIL. Thus, optimized CD8(+) selection combines the benefits of antigen-selected TIL and young TIL for generating lymphocyte cultures for ACT, and should be evaluated in cell transfer therapy protocols.

FIGURE 1.

Nonresponding patients generally received TIL with a higher percentage of CD4⁺ cells than responding patients. Ninety-two samples of tumor-reactive TIL that were administered to patients in Surgery Branch clinical protocols⁶ were evaluated for phenotype of lymphocyte composition by FACS. Percent of CD4⁺ cells is graphed for nonresponding patients and responding patients. Mean for each population is shown by the black bar. P = 0.090 (Student t test with allowance for unequal variance). FACS includes fluorescence-activated cell sorting; TIL, tumor-infiltrating lymphocytes.

FIGURE 2.

Process optimization was required for acceptable levels of magnetic enrichment of CD8⁺ cells. A, FACS profiles using nonoptimized CD8⁺ enrichment process. Upper left: starting TIL culture from patient 1. Upper middle: CD8⁺ fraction from nonoptimized protocol with 7.35% CD4⁺ cells. Upper right: CD8 depleted fraction from nonoptimized enrichment process. B, FACS profiles using optimized CD8⁺ enrichment process. Lower left: starting TIL from a patient enrolled in a current Surgery Branch clinical protocol using optimized CD8⁺ enrichment protocol. Lower middle: CD8⁺ enriched fraction from optimized protocol with 0.94% CD4⁺ cells. Lower right: CD8 depleted fraction from optimized process. FACS includes fluorescence-activated cell sorting; TIL, tumor-infiltrating lymphocytes.

FIGURE 3.

CD8⁺ enrichment using magnetic beads does not impair capacity for rapid expansion. To evaluate the ability of cells to undergo a REP at various times after magnetic enrichment, CD8⁺-enriched TIL cells were plated in complete media with IL-2 after purification. Immediately after purification (day 0) or at the indicated times, an aliquot of 1 × 10⁵ purified TIL was removed and added to a complete REP mix. At the indicated times after the initiation of the REP, cells were counted. These results are representative of 3 independent experiments. IL indicates interleukin; REP, rapid expansion protocol; TIL, tumor-infiltrating lymphocytes.

FIGURE 4.

IFN-γ release increases disproportionately to CD8⁺ T cell number after CD8⁺ enrichment. The label below the x-axis describes a patient and a tumor target from Table 4. Each pair of bars represents 2 ratios. The left (dark) bar of each pair (CD8⁺) represents the ratio of the number of CD8⁺ cells in a coculture assay after CD8⁺ enrichment compared with the number before enrichment. The right bar (light) of each pair (IFN-γ) represents the ratio of the IFN-γ release after enrichment compared with the IFN-γ release before enrichment. The numerical value of each of these rations is listed below each respective patient. For 4 samples of 5 shown, the increase in IFN-γ release cannot be explained simply by an increase in the number of CD8⁺ cells in the coculture assay after CD8⁺ enrichment. IFN-γ indicates interferon-γ.