PD-1-induced proliferating T cells exhibit a distinct transcriptional signature

Abstract

Ligation of the inhibitory receptor PD-1 on T cells results in the inhibition of numerous cellular functions. Despite the overtly inhibitory outcome of PD-1 signalling, there are additionally a collection of functions that are activated. We have observed that CD4⁺ T cells stimulated through the T-cell receptor and PD-1 primarily do not proliferate; however, there is a population of cells that proliferates more than T-cell receptor stimulation alone. These highly proliferating cells could potentially be associated with PD-1-blockade unresponsiveness in patients. In this study, we have performed RNA sequencing and found that following PD-1 ligation proliferating and non-proliferating T cells have distinct transcriptional signatures. Remarkably, the proliferating cells showed an enrichment of genes associated with an activated state despite PD-1 signalling. Additionally, circulating follicular helper T cells were significantly more prevalent in the non-proliferating population, demonstrated by enrichment of the associated genes CXCR5, CCR7, TCF7, BCL6 and PRDM1 and validated at the protein level. Translationally, we also show that there are more follicular helper T cells in patients that respond favourably to PD-1 blockade. Overall, the presence of transcriptionally and functionally distinct T cell populations responsive to PD-1 ligation may provide insights into the clinical differences observed following therapeutic PD-1 blockade.

Keywords: PD-1; RNA sequencing; T cells; proliferation.

FIGURE 1

Proliferating T cells following anti‐CD3, anti‐CD28 and PDL1/L2 stimulation have a different transcriptional signature compared to anti‐CD3 and anti‐CD28. (ai). Flow cytometry of CFSE stained CD4⁺ human T cells stimulated with soluble anti‐CD28 and plate bound anti‐CD3 with or without PDL1 and PDL2. (b) The percentage of cells with 0–1 doublings is shown. (c) The median fluorescent intensity (MFI) of cells that underwent two or more doublings is shown. Data shown in A are representative of 15 independent donors and experiments. (d) Principle component analysis (PCA) of transcriptional profiles of proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28, and PDL1 or PDL2. (e) Heatmap of significant DEG based on the same threshold criteria; proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28 and PDL1. (f) Heatmap of significant DEG based on the same threshold criteria; proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28 and PDL2. (g) Volcano plot displaying differentially expressed genes (DEG) between proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28 and PDL1. Thresholds are set at adjusted P‐value 0·02 and log2 fold change ±1, and significant DEG are in blue. (h) Volcano plot displaying DEG between proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28 and PDL2. Thresholds are set at adjusted P‐value 0·02 and log2 fold change ±1, and significant DEG are in blue. (i) Comparisons of PDL1 and PDL2 significant DEG based on the same threshold criteria. DEG are either decreased (‘lower’) or increased (‘higher’) in proliferating cells. (j‐k) Volcano plot displaying DEG between non‐proliferating (j) or proliferating (k) cells stimulated with anti‐CD3, anti‐CD28 and either PDL1 or PDL2. Thresholds are set at adjusted P‐value 0·02 and log2 fold change ±1, and significant DEG are in red with the significant gene names indicated. Bars represent mean ±SEM; * P ≤ 0·05

FIGURE 2

T cells that proliferate following PDL1/L2 stimulation have an activated transcriptional signature. (a‐b) Heatmap displaying significant DEG between proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28, and PDL1 or PDL2 that are within the indicated gene sets. Thresholds are set at adjusted P‐value 0·02 and log2 fold change ±1

FIGURE 3

Differences in cytokine inhibition by PD‐1 are associated with functional heterogeneity. (a) Secreted levels of IL‐2 and IL‐15 from primary human CD4⁺ T cells stimulated with anti‐CD3 and anti‐CD28 with or without PDL2 quantified by Luminex. (b) Heatmaps displaying DEG between proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28, and PDL1 or PDL2. Thresholds are set at log2 fold change ±1, and significant DEG with adjusted P‐value 0·02 are indicated by *

FIGURE 4

T cells that proliferate following PDL1/L2 stimulation have decreased expression of Tfh cell associated genes. (a‐b) Heatmaps displaying DEG between proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28, and PDL1 or PDL2. Thresholds are set at log2 fold change ±1, and significant DEG with adjusted P‐value 0·02 are indicated by *. (c–d) Volcano plot (c) displaying DEG between proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28 and PDL1. Thresholds are set at adjusted P‐value 0·02 and log2 fold change ±1, and significant DEG are in blue. DEG from the Tfh subset are indicated in red with those that are significant shown in the heatmap (d). (e–f). Volcano plot (e) displaying DEG between proliferating and non‐proliferating cells stimulated with anti‐CD3, anti‐CD28 and PDL2. Thresholds are set at adjusted P‐value 0·02 and log2 fold change ±1, and significant DEG are in blue. DEG from the Tfh subset are indicated in red with those that are significant shown in the heatmap (f).

FIGURE 5

Tfh cell‐associated proteins are enriched within the proliferating population. (a) Flow cytometry data with events first gated on CD3⁺ events followed by CD4⁺ events followed by CFSE expression level. Proliferating cells were gated as the top 5% of proliferating cells based on CFSE fluorescent intensity. Shown is the per cent of CD4⁺ cells positive for each of the indicated proteins that were proliferating or non‐proliferating. Note TCF1 protein is encoded for by TCF7, and Blimp1 protein is encoded for by PRDM1. (b) Flow cytometry data with events again gated on CD3⁺ events followed by CD4⁺ events then CCR7⁺CXCR5⁺TCF1⁺Blimp1⁻ to identify the Tfh cells. CFSE fluorescence is shown to compare proliferation of the total CD4⁺ population to the Tfh population. (c) The percent of cells from the identified Tfh population that were proliferating (top 5%) or non‐proliferating. *P ≤ 0·05, **P ≤ 0·01

FIGURE 6

Patients that respond favourably to PD‐1 blockade have more Tfh cells. (a) tSNE (t‐distributed stochastic neighbour embedding) plot of CD45⁺ cells of melanoma tumours from patients treated with pembrolizumab retrieved from data set GSE120575. Different colours represent 9 clusters (cell types) defined by Bio Turing Browser. Position of Tfh cells from the tSNE plot is shown in purple. (b) tSNE plots of Tfh cells corresponding to responders (red; left) and non‐responders (blue; right), respectively. Bar representing the percentage of Tfh cells among responders vs. non‐responders is shown. (c) Bar graph representing Tfh cell counts as per cent of total cell counts, with pre‐ and post‐treatment values indicated as grey and green, respectively. R = responder, NR = non‐responder. % values are shown beneath the bar graph in the heatmap