Expansion and characterization of human melanoma tumor-infiltrating lymphocytes (TILs)

Abstract

Background: Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.

Principal findings: TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days.

Conclusions: TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT.

Figure 1. Growth of TILs in vitro.

Single cell suspensions or tissue fragments from melanoma specimens were cultured in medium containing 10% “in-house” human plasma and 6000 IU/ml IL-2. (A) For each tissue specimen (x-axis), the total number of TILs from all cultures that exhibited “rapid growth” (≥30×10⁶ TILs within 4 weeks) were enumerated. (B) The proportion of wells (plated on day 0) that exhibited rapid TIL growth (≥30×10⁶ TILs within 4 weeks), intermediate TIL growth (<30×10⁶ TILs within 4 weeks) and no TIL growth are shown for each tissue specimen. (C) Growth curves for seven TIL cultures from a representative tissue specimen (M35) are shown. Each line represents an independent TIL culture.

Figure 2. Immunohistochemistry for CD3.

A piece of each tissue specimen from which TIL culturing was attempted was stained for CD3. Left column: dense TIL infiltration in representative tissue specimens that yielded robust TIL growth in vitro (M28, M32, M41). Right column: paucity of TILs in tissue specimens that yielded no or poor TIL growth in vitro (M30, M39, M43). Note that the dark staining in M39 is from pigmented melanoma cells and melanophages and the dark staining in M43 is from melanophages. Original magnification: 20x.

Figure 3. Flow cytometric analysis of TILs.

(A) Profiles of representative TIL cultures (specimen M27, TIL cultures 2 and 8) stained for CD3, CD56, CD19, CD14, CD4 and CD8 are shown. Profiles from peripheral blood cells (PBCs) from a healthy donor are shown for comparison (“normal blood”). PBC profiles are gated on either lymphocytes (CD56xCD3, CD19, CD4xCD8) or total PBCs (CD14). TIL profiles are gated on live TILs based on forward scatter and side scatter for all plots. All CD4xCD8 profiles are also gated on CD3+ cells. (B) The proportion of CD4+ and CD8+ T cells (gated on CD3+ cells) are shown for all six independent TIL cultures derived from tissue specimen M27.

Figure 4. Cytotoxicity assay.

Six TIL cultures derived from specimen M25 were assayed for cytotoxic activity against the indicated ⁵¹chromium-loaded target cells. Melan-A/MART-1- and gp100-specific T cell lines were used as positive controls.

Figure 5. Tumor reactivity of TILs.

(A) Summary of data from all HLA-A*0201+ TIL cultures that exhibited reactivity against melanoma cells (i.e. autologous tumor cells or HLA-A*0201+ melanoma cell lines) and/or peptide-pulsed T2 cells (i.e. MART-1 or gp100 peptides). The proportion of TIL cultures showing reactivity against MART-1/gp100-pulsed T2 cells only, melanoma cells only, or both MART-1/gp100-pulsed and melanoma cells is shown. IFN-γ data are from 17 TIL cultures; CTL data are from 26 TIL cultures. (B) Results from functional assays of all TIL cultures (for which relevant targets were available) are summarized. The number and proportion of TIL cultures exhibiting tumor reactivity (based on IFN-γ production, CTL activity, or both) or not exhibiting tumor reactivity are shown.

Figure 6. Rapid expansion protocol (REP).

(A) The expansion rate of the six TIL cultures derived from specimen M32 is shown. REPs were initiated with 2×10⁵ TILs on day 0 (with 4×10⁷ irradiated allogeneic PBMCs, 30 ng/ml OKT3 and 3000 IU/ml IL-2) and maintained as described in the Materials and Methods. Each line represents an individual TIL culture. (B) The fold-expansion over 14 days is shown for 38 consecutive REPs. Each symbol shows the fold-expansion for one REP. The solid line shows the mean fold-expansion.