Functional T cells targeting tumor-associated antigens are predictive for recurrence-free survival of patients with radically operated non-small cell lung cancer

Abstract

In this prospective study, we examined postoperative follow-up and preoperative IFN-γ T cell responses against 14 non-small cell lung cancer (NSCLC)-associated antigens in the blood of 51 patients with NSCLC, 7 patients with benign pulmonary tumors, and 10 tumor-free patients by enzyme-linked immunospot assay. The phenotype and function of T cells specific for tumor-associated antigens (TAAs) in the blood or tumor tissue of 9 NSCLC patients were characterized in detail using TNF-α, IL-2, and IFN-γ cytokine capture assays. We found that circulating TAA-specific T cells were significantly enriched in NSCLC compared with tumor-free patients. The most frequently recognized TAAs were Aurora kinase A, HER2/neu, NY-ESO-1, and p53. TNF-α was the most abundant cytokine secreted by TAA-specific T cells in the blood as well as by in situ-activated tumor-infiltrating lymphocytes, most of which were effector memory cells. The absence of TAA-reactive T cells identified patients at higher risk of tumor recurrence, irrespective of tumor stage (OR = 8.76, 95% CI: 1.57-34.79, p = 0.008). We conclude that pre-existing TAA-reactive circulating T cells are a strong independent prognostic factor for recurrence-free survival. These data may help discriminating high-risk from low-risk patients, improving prognostication, and redirecting adjuvant therapy. Our findings suggest the therapeutic relevance of Aurora kinase A, HER2/neu, NY-ESO-1, and p53 as targets for immunotherapy. This study is registered on Clinicaltrials.gov with trial identification number: NCT02515760.

Keywords: Immunotherapy; memory T cells; non-small cell lung cancer; survival; tumor-associated antigens.

Figures

Figure 1.

NSCLC patients have higher numbers of TAA-specific T cells than tumor-free patients. (A) Peripheral blood-derived dendritic cells (PBDCs) were pulsed with 14 polypeptides derived from TAAs or a peptide derived from human IgG as a peptide negative control. As positive controls, PBDCs + cytomegalovirus/adenovirus (CMV/AdV) and PBDCs + staphylococcal enterotoxin B (SEB) were used and compared with PBDCs alone and peripheral blood-derived T cells (PBTCs). The co-cultures were performed in triplicate. A patient was defined as responder against a TAA (indicated by the asterisks) when the triplicate IFN-γ spot counts for a specific peptide were significantly different compared with the IgG control peptide (p-value < 0.05 by t-test). Compared with the IgG control, this patient showed statistically significant responses against MUC-1, p53, Heparanase, EGFR, Survivin, RHAMM, CEA, NY-ESO-1, WT-1, and HER2/neu. (B) Response rate of PBTCs to NSCLC-associated antigens in 51 patients with NSCLC and 10 tumor-free patients categorized by the number of different TAAs recognized. (C) Fold increase in mean IFN-γ spot counts compared with mean IgG controls for all TAA-containing wells from NSCLC and tumor-free patients. p-Values were determined using a 2-tailed Mann–Whitney and Wilcoxon matched-pairs signed rank tests. (D) Response rate for each of the 14 TAA-specific peptides in NSCLC patients compared with tumor-free patients. Numerical data “X/Y” indicate the number of patients with responsive PBTCs for a given TAA (X) relative to the total number of patients tested for TAA responsiveness (Y). (E) Fold increase in mean TAA-specific IFN-γ spot counts relative to mean IgG IFN-γ spot counts. Data from all NSCLC (n = 51) and tumor-free (n = 10) responders and non-responders are presented. p-values were determined using a 2-tailed Mann–Whitney test.

Figure 2.

Gating strategy for T cell subsets. (A) Representative data from one NSCLC patient showing CD4+ (upper line) and CD8+ (lower line) T cell subset populations in blood and tumor. Tumor-infiltrating lymphocytes (TILs), immediately isolated from freshly resected NSCLC tumor tissue and peripheral blood-derived T cells (PBTCs) were used for flow cytometry to define T cell subsets (for cell preparation, see Patients and Methods section). The same gating strategy was used for TILs and PBTCs. First, using forward (FSC-A) vs. side scatter (SSC-A), the cells were gated for lymphocytes (plot marked with asterisk) and singlets (FSC-H vs. FSC-A). The viability of the gated cells was further analyzed using a live/dead stain, taking only the pacific orange-low population. CD4+ and CD8+ surface expression was used to determine the CD4+ and CD8+ T cell populations. By using CD62L and CD45RA, the different T cell subsets were identified from CD4+ and CD8+ populations as follows: CD62L+/CD45RA− cells (central memory T cell; TCM), CD62L+/CD45RA+ cells (naïve/stem-like memory T cell; TN/SCM), CD62L−/CD45RA+ cells (effector T cell; Teff), and CD62L−/CD45RA− cells (effector memory T cell; TEM). The numbers in the dot plots indicate the proportions (%) of gated populations among gated cells (B) Relative proportions of CD4+ and CD8+ cell subsets enumerated in freshly prepared PBTCs (PB, n = 4) and NSCLC tissue (TILs, n = 6) immediately processed after resection by flow cytometry. (C) The CD4/CD8 T cell ratio is shifted toward CD8+ predominance in TILs compared with PBTCs as determined by flow cytometry. (D) Effector to naïve/TSCM ratios for CD4+ and CD8+ PBTCs and TILs. (C–D) Each symbol represents data from an individual patient. Black lines indicate mean values. Error bars show mean ± SEM. p-values were determined by an unpaired 2-tailed t-test.

Figure 3.

Increased TNF-α secretion in activated T cells. (A) Frequencies of TNF-α-, IFN-γ-, and IL-2-producing CD4+ and CD8+ peripheral blood-derived T cells (PBTCs) activated with synthetic long peptides derived from the TAAs p53, heparanase, EGFR, and MUC1 or stimulated with an IgG-derived peptide. TNF-α secretion was detected by flow cytometry after applying cytokine capture assays with bispecific antibodies (for assay preparation see Material and Methods section) in a total of 5 patients with NSCLC and was increased in response to the TAA-derived peptides but not the IgG-derived peptide. (B) Frequencies of IFN-γ-producing PBTCs in 5 NSCLC patients. C Frequencies of IL-2-producing PBTCs in 4 NSCLC patients. Flow-cytometric data from one patient were excluded because of a high background signal for IL-2. (A–C) All measurements were performed in freshly prepared PBTCs stimulated with TAA or IgG control. Error bars show mean ± SEM. *p < 0.05 as determined by an unpaired 2-tailed t-test. (D) A representative flow cytometry plot of an NSCLC patient demonstrating high frequencies of TNF-α-secreting CD8+ and CD4+ PBTCs within the TEM compartment following stimulation with TAAs, but not with IgG control. The numbers in the dot plots indicate the proportions (%) of TNF-α-positive cells among the gated cells. IFN-γ, interferon gamma; IL-2, interleukin 2; PBTC, peripheral blood-derived T cell; TNFα, tumor necrosis factor α. Individual (E) and cumulative (F–G) data from TNF-α cytokine capture assays in PBTCs showing TAA-specific responsiveness of T cell subgroups in 4 NSCLC patients. (H) Contribution of CD4+ and CD8+ PBTC subsets to total TNF-α expression demonstrating that the TN/SCM compartment, due to the over-representation of CD4+ TCs and TN/SCM and the low numbers of TEM, represents a major source of TAA-reactive TNF-α secretion in NSCLC patients (n = 4). TNF-α, tumor necrosis factor α; TCM, T central memory cells; TEM, T effector memory cells; Teff, effector T cells; TN/SCM, naïve/ stem-like memory T cells.

Figure 4.

Increased cytokine expression in TILs. (A) Frequencies of TNF-α-producing unstimulated CD4+ and CD8+ T effector memory TILs were detected by ex vivo flow cytometry of NSCLC tissue processed immediately after tumor resection. Data from one representative patient are shown. The numbers in the dot plots indicate the proportion (%) of cytokine-expressing among gated cells. Cytokine secretion assays for TNF-α- (B), IFN-γ- (C), and IL-2- (D) are shown. For (B–D) each symbol represents data from an individual patient. Black lines indicate mean values. Error bars show mean ± SEM. (E) Contribution of CD4+ and CD8+in situ-activated TIL subsets to total cytokine expression showing that the TEM compartment is nearly the sole source of cytokine secretion in NSCLC patients (n = 6). FMO, fluorescence-minus-one; IFN-γ, interferon gamma; IL2, interleukin 2; TNF-α, tumor necrosis factor α; TCs, T cells; TCM, T central memory; TEM, T effector memory cells; Teff, effector T cells; TIL, tumor-infiltrating lymphocytes; *p < 0.05, as determined by Wilcoxon matched-pairs signed rank test.

Figure 5.

Preoperative T cell analysis correlates with recurrence-free survival (RFS). From the 51 patients assessed using ELISPOT analyses, 10 were excluded from survival analysis as outlined in Suppl. Fig. 1. (A) Survival according to the presence vs. absence of TAA-responsive peripheral blood-derived T cells (PBTCs). (B) Survival according to the number of TAAs recognized by PBTCs per patient. (C–D) The level of IFN-γ spot counts correlates significantly with RFS. From all 51 patients, a median value for IFN-γ spot counts was calculated. Survival based on IFN-γ spot counts above vs. below the median was correlated with PBTCs responsiveness using 2 different modes of spot count calculation: either by division of TAA-specific spot counts by the corresponding IgG control counts (C) or by subtracting the IgG control counts from the corresponding TAA-specific spot counts (D). (E–F) Survival according to the presence or absence of PBTCs responsive to NY-ESO-1 (E) or to one of the TAAs p53, NY-ESO-1, Her2/neu or Aurora kinase A (F). Survival curves are compared using the log-rank test.