Adaptive resistance to anti-PD1 therapy by Tim-3 upregulation is mediated by the PI3K-Akt pathway in head and neck cancer

Abstract

Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are immune checkpoint receptors that are expressed on tumor-infiltrating lymphocytes (TIL) in tumor-bearing mice and humans. As anti-PD-1 single agent response rates are only <20% in head and neck squamous cell carcinoma (HNSCC) patients, it is important to understand how multiple inhibitory checkpoint receptors maintain suppressed cellular immunity. One such receptor, Tim-3, activates downstream proliferative pathways through Akt/S6, and is highly expressed in dysfunctional TIL. We observed that PD-1 and Tim-3 co-expression was associated with a more exhausted phenotype, with the highest PD-1 levels on TIL co-expressing Tim-3. Dampened Akt/S6 phosphorylation in these PD-1⁺Tim-3⁺ TIL, when the PD-1 pathway was ligated, suggested that signaling cross-talk could lead to escape through Tim-3 expression. Indeed, PD-1 blockade of human HNSCC TIL led to further Tim-3 upregulation, supporting a circuit of compensatory signaling and potentially permitting escape from anti-PD-1 blockade in the tumor microenvironment. Also, in a murine HNC tumor model that is partially responsive to anti-PD-1 therapy, Tim-3 was upregulated in TIL from persistently growing tumors. Significant antitumor activity was observed after sequential addition of anti-Tim-3 mAb to overcome adaptive resistance to anti-PD-1 mAb. This increased Tim-3-mediated escape of exhausted TIL from PD-1 inhibition that was mediated by phospho-inositol-3 kinase (PI3K)/Akt complex downstream of TCR signaling but not cytokine-mediated pathways. Taken together, we conclude that during PD-1 blockade, TIL upregulate Tim-3 in a PI3K/Akt-dependent manner, providing further support for dual targeting of these molecules for more effective cancer immunotherapy.

Keywords: Head and neck cancer; PD-1; Tim-3; immunotherapy; monoclonal antibody.

Figure 1.

PD-1⁺Tim-3⁺ TIL subsets are dampened in IFNγ and TNF-α secretion upon TCR stimulation. Freshly isolated TIL from HNSCC patients were sorted based on PD-1, Tim-3 expression. Sorted cells were stimulated in vitro with anti-CD3/CD28 beads and protein transport inhibitor (BD GolgiPlug™) for 6 h. Cells were then stained intracellularly for cytokines. (A) Intracellular staining results of TNFα and IFNγ in sorted CD8⁺ T cells are shown. (n = 3) Significance were calculated by RM-one-way ANOVA, *p < 0.05.

Figure 2.

Tim-3 is upregulated after PD-1 blockade in vitro in human TIL. Freshly isolated tumor-infiltrating lymphocytes from HNSCC patient were treated with anti-PD-1 mAb—Nivolumab, or anti-CTLA-4 mAb—Ipilumumab, and IgG4/IgG1 as isotype control. All the treatments were given at the concentration of 10 µg/mL in vitro for 24 h. Tim-3, CTLA-4, TIGIT expressions were assessed by flow cytometry. (A) Representative flow plots showing Tim-3 expression in CD8⁺ and CD4⁺CD25^lo/− cells. (B) Summary data showing MFI fold change of Tim-3, CTLA-4 and TIGIT. MFI of isotype control group is normalized to 1. (n = 6) Significance was calculated with multiple t test, *p < 0.05. (C) Summary data showing Tim-3 MFI fold change in isotype, anti-PD-1(nivolumab) or anti-CTLA-4 (ipilumumab) treatment. (n = 5) Significance was calculated with RM-one-way ANOVA, *p < 0.05. All data represent average ± SEM.

Figure 3.

PD-1 blockade induced upregulation of IFNγ and TNF-α expression are mostly seen in Tim-3- cells. Freshly isolated tumor-infiltrating lymphocytes from HNSCC patient were treated with anti-PD-1 mAb—Nivolumab and IgG4 as an isotype control. All treatments were given at the concentration of 10 µg/mL in vitro for 48 h. Anti-CD3/CD28 beads and protein transport inhibitor (BD GolgiPlug™) were added according to manufacture's instruction at least 6 h of cell culture for cytokine detection. Cytokine expressions were assessed by flow cytometry. (A) Summary data of baseline PD-1 expression in Tim-3+ and Tim-3- subsets.(n = 5) (B) Flow plot of IFNγ, TNFα expression in Tim-3⁺ and Tim-3⁻ subsets. (C) Summary data of IFNγ, TNFα expression in Tim-3⁻ and Tim-3⁺ subsets. (n = 5) All data represent average ± SEM. Significance were calculated by two-way ANOVA, *p < 0.05, **p < 0.001.

Figure 4.

Tim-3 is upregulated in TIL after in vivo anti-PD-1 treatment, and is further upregulated upon in vitro PD-1 blockade in murine HNC model. C57BL/6 mouse were injected 1 × 10⁶ MEER cells in the neck, anti-mouse PD-1 mAb was given as treatment; all the mouse were scarified around day 36 after injection of tumor cells. Freshly isolated tumor-infiltrating lymphocytes from murine HNC model were analyzed for Tim-3 expression by flow cytometry. (A) Representative flow plots of Tim-3 expression pattern in no treatment group and anti-PD-1 treated group were shown in CD8⁺ and CD4⁺CD25^lo/− T cells. (B, C) Summary data of Tim-3 MFI and %Tim-3⁺ cells in tumor-infiltrating CD8⁺ and CD4⁺CD25^lo/− T cells. (n = 6) Significance were calculated by unpaired t test, *p < 0.05, **p < 0.001. (D) Freshly isolated splenocytes were incubated with anti-PD-1 mAb or IgG4 for 48 h and Tim-3 expression was assessed by flow cytometry. Summary data of Tim-3 MFI in CD8⁺ and CD4⁺ T cells from splenocytes.(n = 6) Significance were calculated by two-way ANOVA, *p < 0.05, **p < 0.001.

Figure 5.

Sequential anti-Tim-3 treatment suppresses tumor growth and show better antitumor immune response. C57BL/6 mouse were injected 1 × 10⁶ MEER cells in the neck, after tumor progression (day 10) anti-mouse PD-1 mAb was given at five doses every 2 d, sequential anti-Tim-3 mAb treatment were given to one group of mice starting from day 22 for four doses every 2 d. Tumor volume were measured every 2 d, Mice TIL were harvested at day 31, and HPV E7-tetramer and PD-1 expression were assessed by flow cytometry. (A) Tumor growth curve up to day 31 was shown, (n = 5) significance was calculated by two-way ANOVA, *p < 0.05. (B) Representative flow plots and summary data showing %E7-tetramer⁺ cells in CD8⁺ T cells. (n = 5) Significance was calculated by ordinary one-way ANOVA, *p < 0.05.

Figure 6.

Tim-3 upregulation after PD-1 blockade is PI3K/Akt pathway dependent. TIL from HNSCC patient were treated with anti-CD3/CD28 beads in combination with DMSO, broad PI3K inhibitor (LY294002), PI3K p110α/mTOR inhibitor (CAY10626), Akt inhibitor, PI3K p110δ inhibitor (GS-1101) and PI3K p110γ inhibitor (AS041164) with or without anti-PD-1 mAbs (Nivolumab) for 48 h. Cells are collected and Tim-3, p-S6 expressions were assessed by flow cytometry. (A) Summary data showing Tim-3 and p-S6 MFI, (n = 4), significance were calculated with ordinary one-way ANOVA, *p < 0.05, **p < 0.001. (B) Summary data showing Tim-3 MFI fold change in the presence of selective PI3K inhibitors with anti-PD-1 or IgG4. (n = 4) Significance was calculated with unpaired t test, *p < 0.05.