T-cell Localization, Activation, and Clonal Expansion in Human Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to most therapies, including immune checkpoint blockade. To elucidate mechanisms of immunotherapy resistance, we assessed immune parameters in resected human PDA. We demonstrate significant interpatient variability in T-cell number, localization, and phenotype. CD8⁺ T cells, Foxp3⁺ regulatory T cells, and PD-1⁺ and PD-L1⁺ cells were preferentially enriched in tertiary lymphoid structures that were found in most tumors compared with stroma and tumor cell nests. Tumors containing more CD8⁺ T cells also had increased granulocytes, CD163⁺ (M2 immunosuppressive phenotype) macrophages, and FOXP3⁺ regulatory T cells. PD-L1 was rare on tumor cells, but was expressed by CD163⁺ macrophages and an additional stromal cell subset commonly found clustered together adjacent to tumor epithelium. The majority of tumoral CD8⁺ T cells did not express molecules suggestive of recent T-cell receptor (TCR) signaling. However, 41BB⁺PD-1⁺ T cells were still significantly enriched in tumors compared with circulation. Tumoral CD8⁺PD-1⁺ T cells commonly expressed additional inhibitory receptors, yet were mostly T-BET^hi and EOMES^lo, consistent with a less terminally exhausted state. Analysis of gene expression and rearranged TCR genes by deep sequencing suggested most patients have a limited tumor-reactive T-cell response. Multiplex immunohistochemistry revealed variable T-cell infiltration based on abundance and location, which may result in different mechanisms of immunotherapy resistance. Overall, the data support the need for therapies that either induce endogenous, or provide engineered, tumor-specific T-cell responses, and concurrently relieve suppressive mechanisms operative at the tumor site. Cancer Immunol Res; 5(11); 978-91. ©2017 AACR.

Figure 1

Effector and regulatory T-cell abundance and localization in PDA. A, Immune cell frequency in normal individual blood (Nl blood), PDA patient blood (PDA blood) and resectable tumor specimens (PDA) was assessed by flow cytometric analysis of mononuclear cells. B, CD8⁺, CD4⁺Foxp3^- and CD4⁺Foxp3⁺ Treg cell number in Nadj and resected tumors normalized to gram of tissue. C, Immunohistochemistry (IHC) for CD8 and Foxp3 in representative Nadj pancreas, 2 tumors that differ in the CD8 T-cell infiltrate (PDA-1 and PD1-2) and TLS in PDA. Ac, acinar; TEC, tumor epithelial cell nests; St, stroma; TLS, tertiary lymphoid structures; and black line, demarcating TEC vs. stroma vs. TLS. Scale bar represents 25 μm. D, CD8⁺ T-cell number per mm² in Nadj, tumor epithelial cell nests (TEC), stroma (St) and TLS. Each circle represents the mean number of cells per mm² in the specified location for each resected specimen. E, Foxp3⁺ T-cell number per mm² in Nadj, tumor epithelial cell nests (TEC), stroma (St) and TLS. Each circle is the mean number of cells per mm² in the specified location for each resected specimen. F, Immunofluorescent staining of TLS in PDA for CD31 and PNAd for HEVs. Arrowhead, CD31⁺ vessel in tumor stroma; arrow, CD31⁺PNAd⁺ HEVs in TLS; and dashed white line demarcates TLS vs. stroma. Scale bar represents 25 μm. G, CD8:Treg ratio was determined in TLS and in tumor stroma that were either TLS⁺ or TLS^- by IHC. H, Mean fluorescence intensity (MFI) of Foxp3 in Treg isolated from Nadj pancreas and tumors was determined by flow cytometry. I, Correlation between CD8 T-cell and Treg numbers in PDA was determined by flow cytometry. Green circles represent tumors with TLS.

Figure 2

Activation marker and inhibitory receptor expression on tumor-infiltrating T cells. A, Naïve (CD45RO^-CCR7⁺), effector memory (CD45RO⁺CCR7^-) and central memory (CD45RO⁺CCR7⁺) CD8⁺ T-cell frequency in normal blood (Nl), circulation of PDA patients (PDA blood) and in tumors (PDA). Percentages were determined by flow cytometric analysis of CD8⁺ T cells. Each symbol represents an independent sample. B, Representative histogram of CD45RO on CD8⁺ T cells in blood, spleen and TLS^- or TLS⁺ tumors. C, Percentage of CD8⁺ T-cell that express the indicated markers in normal blood (Nl blood), PDA patient blood (PDA blood), PDA patient spleens (PDA spleen) and tumors (PDA) was determined by flow cytometry. Each symbol represents an independent sample. D, Flow plots of CD8⁺ T cells from patient in which the majority CD8⁺ T cells expressed PD1 in tumors. E, CD8⁺PD1⁺ T cells in PDA co-express 41BB, Lag3 and to a lesser extent Tim3. F, Histogram overlay of 41BB and PD1 by CD8⁺ T cells isolated from TLS^- or TLS⁺ tumor. G, Percentage of CD8⁺ T cells that express 41BB or PD1 in TLS^- or TLS⁺ tumors. H, Flow plots demonstrating Eomes and T-bet expression by CD8⁺PD1⁺ T cells from 5 different tumors. I, Percentage of CD8⁺PD1⁺ T cells in PDA that are T-bet^hiEomes^lo or T-bet^loEomes^hi subset. Each dot represents an independent sample.

Figure 3

Deep-sequencing of TCRβ CDR3 and expression of T-cell activation and inhibitory genes in PDA. A, Clonality of T cells in blood (B) vs. PDA. B, Number of productive TCRβ CDR3 templates in blood (B) vs PDA. C, Number of productive unique TCRβ CDR3 templates in blood (B) vs. PDA. D, Frequency of the top 15 T-cell clones (○) and clonality scores ( ) from 9 different tumors. Presence of TLS is shown. Dashed line represents a clonality score of 0.10; blue arrows, tumors with a clonality of >0.10. E-F, Gene expression in Nadj pancreas, tumors with clonality >0.10 (n=5) or <0.10 (n=4) was determined by Nanostring.

Figure 4

Antigen-presentation molecule and inhibitory ligand expression in human pancreatic tumors. A, Representative IHC of MHC class I (HLA-A), MHC class II (HLA-DR), Galectin-3 and Galectin-9 in Nadj pancreas, PDA and TLS. is, islet; Ac, acinar; d, normal duct. Scale bar represents 25 μm. B, Quantification of IHC staining in acinar cells (Ac), islets (Is) and ducts (D) from Nadj pancreas, PNET tumor epithelial cells (TEC, Pn), PDA TEC and PDA TLS. C, Evaluation of the correlation between CD8 T-cell staining and ligand expression in PDA.

Figure 5

Spectrum of inflammatory monocyte and granulocyte accumulation in PDA and correlation with CD8⁺ T cells. A, CD14⁺HLA-DR^low monocyte frequency in circulation (Bl) of normal (Nl) individuals and in circulation (Bl), Nadj pancreas and tumors (Tu) from PDA patients. B, Number of CD14⁺HLA-DR^low in Nadj pancreas and PDA normalized to gram of tissue. C, Representative FACs plots of CD33⁺CD15⁺ granulocytes (gated on CD45⁺ cells) from a patient. D, CD11b⁺CD33⁺CD15⁺ granulocyte frequency in circulation (Bl) of normal (Nl) individuals and in circulation (Bl), Nadj pancreas and tumors (Tu) from patient samples. E, CD11b⁺CD33⁺CD15⁺ granulocyte number in Nadj pancreas and PDA normalized to gram of tissue. F, CD8⁺ T-cell number and myeloid subsets were determined by flow cytometric analysis. G, Multicolor immunofluorescence of CD8⁺ T cells, CD15⁺ granulocytes and cytokeratin (CK⁺) tumor cells in two PDAs that differ in the T-cell stromal infiltrate. Representative PNET sample is shown. Scale bar represents 25 μm. H, Expression of granulocyte genes in tumors that had a clonality score of >0.10 or <0.10. I, Analysis of TCGA dataset show a correlation between CD8 T-cell transcripts and genes involved in granulocyte trafficking in PDA.

Figure 6

In situ staining of immune cells and PD-1/PD-L1 stratify patient samples based on the abundance and localization of the T-cell infiltrate. A, Representative staining of cells in PDA. Pan-cytokeratin (CK). Scale bars, 100 μm. Arrows, CD163⁺PD-L1⁺ cells. *, CK⁺PD-L1⁺ cells. B, Percentage of nucleated cells (total), tumor cells (CK) and CD163⁺ macrophages that express PD-L1. C, Representative TLS in PDA demonstrating CD8⁺PD-1⁺ cells (arrow), CD8^-PD-1⁺ cells (arrowheads). Scale bar, 100 μm. D, Frequency of CD8⁺ cells that are PD-1⁺ in the indicated region. Each symbol is the average number of cells in each independent tumor sample. Each circle for TLS represents an independent TLS from 6 patient tumors. E, Number of the indicated cell subset in each compartment normalized to area was determined by averaging the means of independent tumor samples (n=10). F, Number of CD8⁺ T cells in stroma (blue) vs. tumor cell nests (red) in independent tumor samples. P-values are shown for the comparison of T cells in tumor vs. stroma. G, Tumor cell to T-cell ratio in individual tumors corresponding to tumors in 6F. Each circle is an independent region. H, Representative staining of patient tumors stratified from Fig. 6F. Scale bars, 100 μm. I, Correlation between CD8⁺ T-cell infiltration and neutrophils, macrophages and PD-L1 in PDA. Each dot is the number of cells normalized to area in an independent region.