T Cells Expressing Checkpoint Receptor TIGIT Are Enriched in Follicular Lymphoma Tumors and Characterized by Reversible Suppression of T-cell Receptor Signaling

Abstract

Purpose: T cells infiltrating follicular lymphoma (FL) tumors are considered dysfunctional, yet the optimal target for immune checkpoint blockade is unknown. Characterizing coinhibitory receptor expression patterns and signaling responses in FL T-cell subsets might reveal new therapeutic targets.Experimental Design: Surface expression of 9 coinhibitory receptors governing T-cell function was characterized in T-cell subsets from FL lymph node tumors and from healthy donor tonsils and peripheral blood samples, using high-dimensional flow cytometry. The results were integrated with T-cell receptor (TCR)-induced signaling and cytokine production. Expression of T-cell immunoglobulin and ITIM domain (TIGIT) ligands was detected by immunohistochemistry.Results: TIGIT was a frequently expressed coinhibitory receptor in FL, expressed by the majority of CD8 T effector memory cells, which commonly coexpressed exhaustion markers such as PD-1 and CD244. CD8 FL T cells demonstrated highly reduced TCR-induced phosphorylation (p) of ERK and reduced production of IFNγ, while TCR proximal signaling (p-CD3ζ, p-SLP76) was not affected. The TIGIT ligands CD112 and CD155 were expressed by follicular dendritic cells in the tumor microenvironment. Dysfunctional TCR signaling correlated with TIGIT expression in FL CD8 T cells and could be fully restored upon in vitro culture. The costimulatory receptor CD226 was downregulated in TIGIT⁺ compared with TIGIT^- CD8 FL T cells, further skewing the balance toward immunosuppression.Conclusions: TIGIT blockade is a relevant strategy for improved immunotherapy in FL. A deeper understanding of the interplay between coinhibitory receptors and key T-cell signaling events can further assist in engineering immunotherapeutic regimens to improve clinical outcomes of cancer patients. Clin Cancer Res; 24(4); 870-81. ©2017 AACR.

Figure 1. Skewing towards PD-1^int phenotype and reduced IFN-γ production in CD8 FL T cells

Single cell suspensions from FL LN and healthy donors (tonsils and PBMC) were analyzed by fluorescence flow cytometry. A, CD8 and CD4 T cells were divided into subsets based on expression of PD-1 and ICOS. B, Distribution of T-cell subsets gated in (A) in FL (n = 14) compared to tonsils (n = 11) and PBMC (n = 7). C, Cells were cultured with or without PMA and ionomycin, and intracellular IFN-γ was measured by flow cytometry. Each data point represents a single donor. FL (n = 9), tonsils (n = 13), PBMC (n = 7). Statistical differences calculated using Mann-Whitney non-parametric test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2. Intra-tumor FL T cells are distinguished by low levels of TCR-induced distal signaling

Single cell suspensions from FL LN (n = 9), and healthy donor tonsils (n = 11) and PBMC (n = 9) were cultured with or without α-CD3+α-CD28 antibodies for 2 minutes, followed by avidin crosslinking for 1, 4 or 10 minutes and then assayed for TCR-induced phosphorylation of CD3ζ, SLP76 and ERK using phospho-flow cytometry. A, Schematic overview of TCR signaling. B, Representative histograms of TCR-induced phosphorylation in CD3⁺ T cells from one FL patient sample compared to one healthy donor tonsil. Shown is median fold change (FC) induction relative to unstimulated cells, using arcsinh transformed data. C, TCR-induced p-ERK (4′), p-SLP76 (1′) and p-CD3ζ (1′) in CD8 and CD4 T-cell subsets shown as median FC induction relative to unstimulated cells. Each data point represents a single donor. Statistical differences calculated using Mann-Whitney non-parametric test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3. Expression patterns of co-inhibitory receptors in CD8 and CD4 T-cell subsets

11-parameter fluorescence flow cytometry was used to identify co-inhibitory receptor expression in conventional T-cell subsets from FL LN (n = 4) and healthy donor tonsils (n = 2), using single cell suspensions. Results are visualized by viSNE (gating shown in supplementary Fig. S4). Scale maximum is set to highest measured signal for each marker, or a minimum of 3000. The manually added line in the viSNE plots marks the distinction between CD8 and CD4 T cells.

Figure 4. TIGIT is frequently expressed in FL T_E, T_EM, T_FH and T_regs

Surface expression of co-inhibitory receptors was analyzed in single cell suspensions from FL LN, and healthy donor controls (tonsils and PBMC) by fluorescence flow cytometry. A, Plots show CD3⁺ T cells. B–C, Co-inhibitory receptor expression was measured in conventional CD8 and CD4 T-cell subsets. Each data point represents a single donor. FL (n = 14), tonsils (n = 11), PBMC (n = 7). Statistical differences calculated using Mann-Whitney non-parametric test; *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001. D, FL LN samples (n = 3) were assayed for the contribution of TIGIT⁺ T_regs. Tonsils and PBMC from healthy donors were included for comparison. Bar graph shows mean ± SEM.

Figure 5. TIGIT ligands are expressed in FL and TIGIT⁺ CD8 T cells are CD226^low

A, FL tissue sections were stained with antibodies against CD155, CD112 and CD21. The tissue sections are closely neighbored to each other, enabling the comparison of identical structures. Staining pattern of CD155 and CD112 in follicles (arrows) suggests expression by FDC, confirmed by staining of the same follicles with FDC marker CD21. Endothelium (arrow heads) also expressed CD155 and CD112. Image objective x10. B–C, TIGIT and CD226 expression was measured in CD8 and CD4 T cells from FL LN (n = 7) using flow cytometry. Healthy donor PBMC was included for comparison. Bargraphs show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann-Whitney test.

Figure 6. Dysfunctional TCR distal signaling in FL CD8 TIGIT⁺ T cells can be restored

Single cell suspensions from FL LN were assayed for TCR-induced signaling and analyzed by phospho-flow cytometry at day 0 and after 48h in vitro culture. The cryopreserved cell suspensions contained T cells and tumor cells, while FDC were not detectable in these cultures. Signaling was induced using α-CD3+α-CD28 antibodies for 2 minutes, followed by avidin crosslinking for 1 or 4 minutes, and is shown as median fold change (FC) induction relative to unstimulated cells, using arcsinh transformed data. A, TCR-induced p-ERK (4′) in TIGIT⁻ and TIGIT⁺ CD8 T cells from one representative FL sample at day 0. B, Levels of TCR-induced p-ERK (4′) and p-SLP76 (1′) in CD8 T cells from FL LN (n = 6) at day 0. *p < 0.05 by paired t-test. C, Schematic overview of in vitro cultures. TCR signaling was induced in single cell suspensions from FL LN at day 0 and after 2 days culture. D, TCR-induced signaling was measured in the same FL specimens (n = 4) at day 0 and after 48h in vitro culture in the presence of low IL-2. Bar graphs show mean ′ SEM. *p < 0.05 by paired t-test. E–F, TCR-induced p-ERK (4′) was measured in TIGIT⁻ and TIGIT⁺ CD8 T cells from the same FL specimens at day 0 and after 48 h in vitro culture (in medium only). E, Histograms show one representative FL sample. F, Recovery of TCR-induced p-ERK by in vitro culture shown in TIGIT⁻ and TIGIT⁺ CD8 T cells from FL LN (n = 4). **p < 0.01, ****p < 0.0001 by paired t-test.