High-dimensional analyses reveal a distinct role of T-cell subsets in the immune microenvironment of gastric cancer

Abstract

Objectives: To facilitate disease prognosis and improve precise immunotherapy of gastric cancer (GC) patients, a comprehensive study integrating immune cellular and molecular analyses on tumor tissues and peripheral blood was performed.

Methods: The association of GC patients' outcomes and the immune context of their tumors was explored using multiplex immunohistochemistry (mIHC) and transcriptome profiling. Potential immune dysfunction mechanism/s in the tumors on the systemic level was further examined using mass cytometry (CyTOF) in complementary peripheral blood from selected patients. GC cohorts with mIHC and gene expression profiling data were also used as validation cohorts.

Results: Increased CD4⁺FOXP3⁺ T-cell density in the GC tumor correlated with prolonged survival. Interestingly, CD4⁺FOXP3⁺ T cells had a close interaction with CD8⁺ T cells rather than tumor cells. High densities of CD4⁺FOXP3⁺ T cells and CD8⁺ T cells (High-High) independently predicted prolonged patient survival. Furthermore, the interferon-gamma (IFN-γ) gene signature and PDL1 expression were up-regulated in this group. Importantly, a subgroup of genomically stable (GS) tumors and tumors with chromosomal instability (CIN) within this High-High group also had excellent survival. The High-High GS/CIN tumors were coupled with increased frequencies of Tbet⁺CD4⁺ T cells and central memory CD4⁺ T cells in the peripheral blood.

Conclusion: These novel findings identify the combination of CD8⁺ T cells and FOXP3⁺CD4⁺ T cells as a significant prognostic marker for GC patients, which also could potentially be targeted and applied in the combination therapy with immune checkpoint blockades in precision medicine.

Keywords: CD4+FOXP3+ T cells; CD8+ T cells; gastric cancer; interferon‐gamma gene signature.

Figure 1

Using the high‐dimensional immune context data as a prognostic indicator in gastric cancer. (a) Overview of the study design. Gastric tumors were profiled using matched multiplex immunohistochemistry (mIHC, n = 48) and gene expression microarrays (n = 36). The systemic immunity for the patients was studied using mass cytometry (CyTOF) from selected patients (n = 10). The data were then correlated with clinical parameters and patient outcomes to develop a prognostic indicator, which was further validated using public GC cohorts by mIHC (n = 84) and gene expression data (n = 876). (b) H&E sections were used to identify regions for subsequent mIHC analysis where tumor ‘edge’ represent the area within 1mm of the interface between tumor and normal sites, and tumor ‘core’ represent the tumor area proximal to the tumor edge. (c) Representative composite image showing the distribution of individual markers (CD3, CD4, CD8, FOXP3, CD56, AE1AE3 and DAPI) within a tumor region. (d) The same image was phenotyped, allowing the identification of different immune cell subtypes (DNT cell, CD4⁺ T cell, CD8⁺ T cell, CD4⁺FOXP3⁺ T cell, CD56⁺ cell, tumor cell and lineage⁻ cell).

Figure 2

Gastric tumor samples display heterogeneous immune cells distribution. (a) Schematic representation of the rationale to calculate the intercellular nearest distance between the reference cell (RC) and nearest cell (NC). Median Intercellular Nearest (MIN) distance was defined as the median value of the nearest distances. (b) The coloured lines represent the percentage of RC within a certain nearest distance (range from 10 to 150 μm) between RC and NC. Shown are pairs of MIN distance analyses from P2433 and P7422 where the RC is CD4⁺FOXP3⁺ T cells, CD8⁺ T cells or Tumor cells. Data are presented as the mean ± SD.

Figure 3

Higher densities of T‐cell subsets, especially CD4⁺FOXP3⁺ T cells, in the tumor core correlate with better patient survival. (a) Overall survival (OS; left) and relapse‐free survival (RFS; right) of all 48 patients based on densities of CD8⁺ T cells, CD4⁺ T cells, CD4⁺FOXP3⁺ T cells, DN T cells, CD56⁺ cells and Lineage⁻ cells. Individual immune infiltrate values were divided into higher (n = 24) or lower (n = 24) based on the median number of cells. (b) OS (left) and RFS (right) analysis of all 48 patients based on MIN distance to tumor cells from CD8⁺ T cells, CD4⁺ T cells, CD4⁺FOXP3⁺ T cells, DN T cells, CD56⁺ cells and Lineage⁻ cells. Distant (n = 24) and proximal (n = 24) distance values were defined based on the MIN distance. Hazard ratio (HR) is shown for lower compared to higher for density analysis, and distant compared to proximal for distance analysis. HR and 95% confidence interval are shown. Significance was determined using the log‐rank Mantel–Cox test. *P < 0.05, **P < 0.01. DNT cell: Double‐negative T cell.

Figure 4

Close interactions between CD4⁺FOXP3⁺ T cells and CD8⁺ T cells, but not tumor cells. (a) Correlation analysis (n = 48) between CD4⁺FOXP3⁺ T cells density and densities of tumor cells, CD8⁺ T cells, CD4⁺ T cells, DNT cells, CD56⁺ cells and Lineage⁻ cells. (b) Correlation analysis (n = 48) between the CD4⁺FOXP3⁺ T cells density and the MIN distance from CD4⁺FOXP3⁺ T cells (as the RC) to tumor cells, CD8⁺ T cells, CD4⁺ T cells, CD56⁺ cells, DNT cells and Lineage⁻ cells. Pearson correlation coefficient (r), 95% confidence interval and significance levels (P‐value) are shown. (c) Example image showing close interactions between CD4⁺FOXP3⁺ T cells (orange) and CD8⁺ T cells (green), not tumor cells (magenta). *P < 0.05, **P < 0.01, ***P < 0.001. DNT cell: Double‐negative T cell.

Figure 5

CD8 + CD4FOXP3 grouping as an independent biomarker of patients’ prognosis. (a) OS and RFS in the discovery cohort (n = 48) showing differential co‐existence of CD8⁺ T cells and CD4⁺FOXP3⁺ T cells, stratified into (CD8⁺)^Low(CD4⁺FOXP3⁺)^Low (the Low‐Low group), (CD8⁺)^High(CD4⁺FOXP3⁺)^Low (the High‐Low group), (CD8⁺)^Low(CD4⁺FOXP3⁺)^High (the Low‐High group) and (CD8⁺)^High(CD4⁺FOXP3⁺)^High (the High‐High group). Significance was determined using the log‐rank Mantel–Cox test. (b) OS of patients in an independent international validation cohort (n = 84) showing differential co‐existence of CD8⁺ T cells and CD4⁺FOXP3⁺ T cells, stratified into four and two groups, respectively. (c) Representative images of GC tumor showing differential co‐localisation of CD8⁺ (green) and CD4⁺FOXP3⁺ T cells (orange) were associated with short, median, and prolonged OS and RFS, respectively. (d) Subgroup analysis to estimate the OS and RFS of patients according to molecular subtypes (n = 39), histology subtypes (n = 30) and AJCC stage (n = 19), respectively. Significance was determined using the log‐rank Mantel–Cox test. (e) ROC curve using CD8 + CD4FOXP3 groups (Groups), T stages and AJCC stages to predict recurrence or survival. *P < 0.05, **P < 0.01, ***P < 0.001. DNT cell: Double‐negative T cell.

Figure 6

Association of interferon‐gamma (IFN‐γ) response and the High‐High GC tumors. (a) A volcano plot depicts the differentially expressed genes in the High‐High group (n = 13) when compared to the other three (CD8 + CD4FOXP3) groups (n = 23). Genes up‐regulated in the High‐High group and with P‐value < 0.01 are labelled in red. Genes associated with IFN‐γ response (CXCL9, CXCL10, IDO1, IFNG, HLA‐DRA and STAT1) are labelled. (b) IFN‐γ signature expression in four groups (n = 13, 6, 5, 12). (c) Nodes are representing enriched pathways with P‐value < 0.01 based on shared genes in pie chart format. (d) The kmplot interface 26 with overall survival data in two separate cohorts (n = 593 and n = 283) of GC patients was interrogated. Patients were classified into high (upper quartile) and low expressing groups based on the mean expression of the selected genes. HR is shown for high compared to the low mean expression of genes. *P < 0.05, **P < 0. 01, ***P < 0.001.

Figure 7

The High‐High GS/CIN tumors were associated with significantly increased Tbet⁺ CD4⁺ T cells and central memory CD4⁺ T cells in the peripheral blood. (a) PDL1 expression by cells in the peripheral blood was up‐regulated in the Low‐Low group using mass cytometry. Significance was determined using the two‐tailed Mann–Whitney U‐test. (b) viSNE illustration of 14 clusters identified by SPADE in the High‐High group (n = 5) and the Low‐Low group (n = 5). (c, d) Within the 14 clusters, Tbet⁺ CD4⁺ T cells (C6) and CCR7⁺CD45RA⁻ CD4⁺ T cells (C3) were significantly increased in the High‐High group (c), while two CD11c⁺ dendritic cells clusters were significantly increased in Low‐Low group (d). Significance was determined using the two‐tailed Mann–Whitney U‐test. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (e) Our working model depicts how immune tumor microenvironment and peripheral blood in the GC can be interpreted to enable prognostication of patients.