Bone marrow expands the repertoire of functional T cells targeting tumor-associated antigens in patients with resectable non-small-cell lung cancer

Abstract

The efficacy of cancer immunotherapy may be improved by increasing the number of circulating tumor-reactive T cells. The bone marrow is a priming site and reservoir for such T cells. The characteristics of bone marrow-derived tumor-reactive T cells are poorly understood in patients with non-small-cell lung cancer (NSCLC). To compare the responsiveness of tumor antigen-reactive T cells from the bone marrow with matched peripheral blood samples in patients with resectable NSCLC, we used flow cytometry, cytokine capture assays and enzyme-linked immunospot assays to examine the responsiveness of T cells to 14 tumor antigens in matched bone marrow and peripheral blood samples from patients with resectable NSCLC or benign tumors and tumor-free patients. T cells with reactivity to tumor antigens were detected in the bone marrow of 20 of 39 (51%) NSCLC patients. The panel of tumor antigens recognized by bone marrow-derived T cells was distinct from that recognized by peripheral blood-derived T cells in NSCLC patients. Unlike for peripheral blood T cells, the presence of tumor-reactive T cells in the bone marrow did not correlate with recurrence-free survival after curative intent resection of NSCLC. T cells with reactivity to tumor antigens are common in the bone marrow of patients with NSCLC. Tumor-reactive T cells of the bone marrow have the potential to significantly broaden the total repertoire of tumor-reactive T cells in the body. To clarify the role of tumor-reactive T cells of the bone marrow in T cell-based immunotherapy approaches, clinical studies are needed (ClinicalTrials.gov: NCT02515760).

Keywords: Lung cancer; T cells; bone marrow; immunotherapy.

© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.

Figures

Figure 1.

(A) Response rates of PBTCs (PB samples available from n = 51 patients) and BMTCs (BM samples available from n = 39 patients) to TAs in the patients with NSCLC or benign tumors or tumor-free patients categorized by the number of different TAs recognized, as determined using the ELISPOT assay. (B) Higher frequencies of TA-specific TCs were observed in the PB than in the BM in NSCLC patients. The fold increase was calculated by comparing the mean IFN-γ spot count with the count of the IgG controls for all TA-containing wells used to assess samples from the patients with NSCLC or benign tumors or tumor-free patients with ELISPOT. P-values were determined using two-tailed Mann-Whitney and Wilcoxon matched-pairs signed rank tests. (C) – (D) Data from the 39 patients with PB and corresponding BM samples illustrating the response rate in responders alone, as determined using the ELISPOT assay (C), and the total number of TAs recognized per patient in the different compartments (D). (A) – (D) For a better visualization of TC responsiveness in BM versus PB, we show PB data that were published in our previous study.8P-values were determined using two-tailed Wilcoxon matched-pairs signed rank tests. Ad/CMV, adenovirus/cytomegalovirus; BM, bone marrow; ELISPOT, enzyme-linked immunospot; PB, peripheral blood; PBTCs, peripheral blood-derived T cells; TA, tumor-associated antigen.

Figure 2.

(A) – (C) Response rates for each of the 14 TA-specific peptides in the BM from the patients with NSCLC compared with those for patients with benign tumors or tumor-free patients. (D) – (F) Response rates for each of the 14 TA-specific peptides in the PB and BM from the patients with NSCLC or benign tumors or the tumor-free patients. Numerical data “X/Y” indicate the number of patients with PBTCs responding to a particular TA (X) relative to the total number of patients tested for TA responsiveness (Y). (D) – (F) For a better visualization of TC responsiveness in BM and PB, we show PB data that were published in our previous study.8.

Figure 3.

(A) Responses of PBTCs (gray points) and the corresponding BMTCs (blue points) from 39 patients with NSCLC to any of the 14 tested NSCLC-associated antigens. The gray line between two points indicates the TA-specific spot counts of the PBTCs and BMTCs from the same patient. The mean TA-specific IFN-γ spot counts minus the mean IgG (negative control) IFN-γ spot counts are shown. P-values were determined using two-tailed Wilcoxon matched-pairs signed rank tests. For a better visualization of TC responsiveness in BM and PB, we show PB data that were published in our previous study.8 (B) Scatterplot showing the cumulative BMTC data in relation to PBTC responsiveness to any of the 14 tested TAs in 39 patients with NSCLC. BMTC responsiveness increased as PBTC responsiveness increased (two-tailed p < .0001, Pearson’s correlation coefficient r = 0.325, R2 = 0.106). (C) We defined early recurrence as tumor recurrence within 11 months after surgery.4 Scatterplot showing the cumulative BMTC data in relation to PBTC responsiveness to any of the 14 tested TAs in the patients with NSCLC recurrence following curative intent surgery within 11 months postsurgery (defined as early recurrence) compared with >11 months postsurgery (defined as late recurrence or tumor-free). The linear regression line of the patients with early recurrence (green line) was not significantly different from the linear regression line of the patients with late recurrence or tumor-free patients (blue line). (B) – (C) The frequencies of TA-specific TCs are shown as the fold increases in the mean TA-specific IFN-γ spot counts (calculated relative to the mean IgG control spot counts). The values in the lower left quadrants were not considered TA-specific responses and were excluded (n = 238 in (B) and n = 278 in (C)) from the regression analysis. Only the TC responses of 2 or more IFN-γ spot counts greater than the mean IgG control spot counts (n = 199 in (B) and n = 160 in (C)) were deemed positive and included in the regression analysis. The black line represents the linear regression line. BMTCs, bone marrow-derived T cells; NSCLC, non-small-cell lung cancer; PBTC, peripheral blood-derived T cell; TA, tumor-associated antigen.

Figure 4.

(A) Cumulative BMTC data in relation to PBTC responsiveness in the patients with TA-specific responses in the PB but not in the BM (PB+BM-; blue line; Pearson’s correlation coefficient r = 0.333, R2 = 0.111, p = .033, n = 41) compared with patients with TA-specific responses in the BM but not the PB (PB-BM+; yellow line; Pearson’s correlation coefficient r = 0.266, R2 = 0.071, p = .122, n = 35) or patients with TA-specific responses in the PB and BM (PB+BM+; red line; Pearson’s correlation coefficient r = 0.433, R2 = 0.187, p = .140, n = 13). The frequencies of TA-specific TCs are shown as the fold increases in the mean TA-specific IFN-γ spot counts (calculated relative to the mean IgG control spot counts). TA-specific responsiveness was tested against 14 TAs in patients with NSCLC. RFS was not different between the patients with NSCLC with or without preoperative TA-responsive BMTCs. (B). Survival curves are plotted for all four scenarios of present/absent TA-specific BMTCs and PBTCs. (C) – (D). Patients were classified as TA responders when the triplicate IFN-γ spot counts for a specific peptide were significantly different from the counts for the IgG control peptide (p-value<0.05 by a t-test). Kaplan-Meier survival curves were compared using the log-rank test (B) – (D). TA-specific responsiveness to 14 TAs was assessed in patients with NSCLC. BMTC, bone marrow-derived T cell; NSCLC, non-small-cell lung cancer; PBTC, peripheral blood-derived T cell; RFS, recurrence-free survival; TA, tumor-associated antigen.

Figure 5.

(A) and (C) Scatterplots showing the cumulative BMTC data in relation to PBTC responsiveness to any of the 14 tested TAs in patients with NSCLC (A) younger than 64 years and patients older than 63 years and (C) in patients with pathologically confirmed lymph node metastasis in the final pathology assessment (yellow linear regression line) compared with patients with no lymph node metastasis (black linear regression line). (A) Linear regression analyses showed a statistically significant difference between the slopes (p < .001). In the patients younger than 64 years, BMTC responsiveness increased as PBTC responsiveness increased (two-tailed p < .0001, Pearson’s correlation coefficient r = 0.456, R2 = 0.208). (C) Linear regression analyses showed a statistically significant difference between the slopes (p = .003). In the patients with no lymph node metastasis, BMTC responsiveness increased as PBTC responsiveness increased (two-tailed p < .0001, Pearson’s correlation coefficient r = 0.468, R2 = 0.219). (A) and (C) The frequencies of TA-specific TCs are shown as the fold increase in the mean TA-specific IFN-γ spot counts (calculated relative to the mean IgG control spot counts). The values in the lower left quadrants were excluded, n = 240 in (A) and n = 239 in (C). (B) and (D) Kaplan-Meier survival curves suggested (B) improved RFS in the younger patients (blue) compared with patients older than 63 years of age (gray) and (D) improved RFS in the patients with no lymph node metastasis (blue) compared with patients with pN1 or pN2 lymph node metastasis (gray) (two-tailed p = .046, log-rank test). BMTC, bone marrow-derived T cell; NSCLC, non-small-cell lung cancer; PBTC, peripheral blood-derived T cell; TA, tumor-associated antigen.