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. 2021 May 7:12:660944.
doi: 10.3389/fimmu.2021.660944. eCollection 2021.

Hypoxia Supports Differentiation of Terminally Exhausted CD8 T Cells

Nadia Bannoud  1   2 , Tomás Dalotto-Moreno  3 , Lucía Kindgard  1 , Pablo A García  1 , Ada G Blidner  3 , Karina V Mariño  4 , Gabriel A Rabinovich  3   5 , Diego O Croci  1   6
Affiliations

Hypoxia Supports Differentiation of Terminally Exhausted CD8 T Cells

Nadia Bannoud et al. Front Immunol. .

Abstract

Hypoxia, angiogenesis, and immunosuppression have been proposed to be interrelated events that fuel tumor progression and impair the clinical effectiveness of anti-tumor therapies. Here we present new mechanistic data highlighting the role of hypoxia in fine-tuning CD8 T cell exhaustion in vitro, in an attempt to reconcile seemingly opposite evidence regarding the impact of hypoxia on functional features of exhausted CD8 T cells. Focusing on the recently characterized terminally-differentiated and progenitor exhausted CD8 T cells, we found that both hypoxia and its regulated mediator, vascular endothelial growth factor (VEGF)-A, promote the differentiation of PD-1+ TIM-3+ CXCR5+ terminally exhausted-like CD8 T cells at the expense of PD-1+ TIM-3- progenitor-like subsets without affecting tumor necrosis factor (TNF)-α and interferon (IFN)-γ production or granzyme B (GZMB) expression by these subpopulations. Interestingly, hypoxia accentuated the proangiogenic secretory profile in exhausted CD8 T cells. VEGF-A was the main factor differentially secreted by exhausted CD8 T cells under hypoxic conditions. In this sense, we found that VEGF-A contributes to generation of terminally exhausted CD8 T cells during in vitro differentiation. Altogether, our findings highlight the reciprocal regulation between hypoxia, angiogenesis, and immunosuppression, providing a rational basis to optimize synergistic combinations of antiangiogenic and immunotherapeutic strategies, with the overarching goal of improving the efficacy of these treatments.

Keywords: CD8 T cell exhaustion; Hypoxia; VEGF-A; anti cancer agents; immunosuppression.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Association between CD8 T cell exhaustion and hypoxia. (A) Schematic representation of progenitor and terminally exhausted T cell subsets. Although T cell exhaustion comprises a wide range of exhausted states, two major subsets of exhCD8 T cells have been studied in detail: the progenitor and the terminally exhausted T cell population. Whereas progenitor exhausted T cells exhibit proliferative potential, and stemness properties and can be rescued by immune checkpoint blockade (ICB) therapies, terminally exhausted T cells have higher cytotoxic potential but represent a terminal differentiation state and cannot be rescued by ICB. Proposed key molecules which discriminate these subpopulations are listed. (B) Modulation of CD8 T cell functions by hypoxia. Through HIF-1α and HIF2α- dependent mechanisms, hypoxic stimuli favor glycolytic anaerobic metabolism promoting T cell receptor (TCR) signaling. These include enhanced perforin and granzyme-B (GzmB) release as well as expression of immune checkpoint molecules (including both activators and inhibitors). Hypoxia inhibits expression of chemokine and cytokine receptors and adhesion molecules.
Figure 2
Figure 2
Hypoxia and VEGF-A promote differentiation of terminally exhCD8 T cells. (A) Schematic representation of workflow and gating strategy for in vitro differentiation of exhCD8 T cells. (B) Representative histograms showing intracellular staining of CD107a, Granzyme-B (GzmB), and TNF-α in progenitor (green) or terminally (pink) exhCD8 T cells. (C) Representative contour plots showing GzmB and CD107a expression in CD8+ PD-1+ T cells after 24 h exposure to hypoxia (1% O2) or normoxia (20% O2). (D) Differentiation of PD-1+TIM-3-CXCR5+ progenitor exhCD8 T cells and PD-1+TIM-3+ terminally exhCD8 T cells under normoxic (blue bars) or hypoxic (red bars) conditions. Left, percentage of each subpopulation. Right, representative density plots showing the subpopulations. Data are the mean ± SEM of five independent experiments. (E) Determination of cytokine expression by intracellular flow cytometry in different exhCD8 T cell subpopulations under normoxic or hypoxic conditions. Data are the mean ± SEM of 4 independent experiments. (F) Heat map representing normalized row Z-score of semiquantitative cytokine array analysis of angiogenic factors secreted by CD8+ T cells during the differentiation process. Data shows densitometric determinations and cluster analysis of pooled supernatants from five independent experiments. (G) Representation of the ratio of cytokines secreted by exhCD8 T cells under normoxic or hypoxic conditions. Heat maps represent the ratio of normalized densitometric data for each cytokine in normoxia versus hypoxia. (H) VEGF secretion by PD-1+TIM-3-CXCR5+ progenitor exhCD8 T cells and PD-1+TIM-3+ terminally exhCD8 T cells under normoxic (blue bars) or hypoxic (red bars) conditions. Data are the mean ± SEM of four independent experiments. (I) Differentiation of PD-1+TIM-3-CXCR5+ progenitor exhCD8 T cells and PD-1+TIM-3+ terminally exhCD8 T cells in the presence (orange bars) or in the absence (blue bars) of VEGF-A (50 ng/ml). Data are the mean ± SEM of five independent experiments. (J) Heat map representation of intracellular cytokines determined by flow cytometry in different exhCD8 T cells subpopulations in the presence or the absence of VEGF-A (50 ng/ml). Each row represents the mean ± SEM of the percentage of cells expressing each cytokine in four independent experiments. *p < 0.05, **p < 0.01.
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