Modified Hypoxia Inducible Factor expression in CD8+ T cells increases anti-tumor efficacy

Abstract

<jats:title>Abstract</jats:title><jats:p>Adoptive transfer of anti-tumor cytotoxic T cells is a novel form of cancer immunotherapy, and a key challenge is to ensure the survival and function of the transferred T cells. Immune cell survival requires adaptation to different micro-environments, and particularly to the hypoxic milieu of solid tumors. The HIF transcription factors are an essential aspect of this adaptation, and we undertook experiments to define structural determinants of HIF that would potentiate anti-tumor efficacy in cytotoxic T cells. We created retroviral vectors to deliver ectopic expression of HIF-1ɑ and HIF-2ɑ in mouse CD8+ T cells, together or individually, and with or without sensitivity to their oxygen-dependent inhibitors Von Hippel-Lindau (VHL) and Factor Inhibiting HIF (FIH). We found that HIF-2ɑ, but not HIF-1ɑ, drives broad transcriptional changes in CD8+ T cells, resulting in increased cytotoxic differentiation and cytolytic function against tumor targets. We further found that a specific mutation replacing the hydroxyl group acceptor site for FIH in the HIF-2ɑ isoform gives rise to the most effective anti-tumor T cells after adoptive transfer in vivo. Lastly, we show that co-delivering an FIH-insensitive form of HIF-2ɑ with an anti-CD19 chimeric antigen receptor greatly enhances cytolytic function of human CD8+ T cells against lymphoma cells. These experiments provide a means to increase the anti-tumor efficacy of therapeutic CD8+ T cells via ectopic expression of the HIF transcription factor.</jats:p><jats:sec><jats:title>Graphical abstract</jats:title><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="159137v2_ufig1" position="float" orientation="portrait" /></jats:fig></jats:sec>

Oxygen levels at the time of activation determine T cell persistence and immunotherapeutic efficacy

“…T cell activation in low oxygen increased the production of IL-2 when compared to cells activated in ambient oxygen (Figure 4K). While FG-4592 treatment did not impact IL-2 production in T cells, we have previously found that Il2 gene expression is increased by ectopic HIF-1α in mouse T cells (Veliça et al, 2021). In fact, we observed that the increase of IL-2 production following 24 hours of T cell activation in hypoxia was accompanied by an increase in STAT5 phosphorylation (Y694) and in the expression of IL-2 receptor subunit CD25 (Figure 4L).…”

“…Factor Interleukin 3 Regulated (NFIL3), previously reported to be increased following ectopic expression of HIF-α or culture in 1% O 2 for 24 hours (Ross et al, 2021;Veliça et al, 2021). As NFIL3 has been linked to perforin production by NK cells, this transcription factor could underpin the increased T cell production of effector molecules following low oxygen conditioning or increased HIF signalling (Rollings et al, 2018).…”

“…In both preclinical studies and in clinical manufacturing, CD8 + T cells are typically cultured at atmospheric sea level O 2 conditions (approximately 20.5%); however, reports have shown that physiologically relevant oxygen tensions (corresponding to 1%-5% O 2 ) can profoundly shift T cell differentiation and function (Atkuri et al, 2007;Caldwell et al, 2001;Gropper et al, 2017;Larbi et al, 2010;Loeffler et al, 1990;Naldini et al, 1997;Palazon et al, 2017;Ross et al, 2021;Veliça et al, 2021). Lower oxygen levels alter CD8 + T cell effector differentiation in a process that requires hypoxia-inducible factors (HIFs), the key transcriptional regulators of cellular responses to low oxygen (Finlay et al, 2012;Palazon et al, 2017).…”

Vitamin B6 Metabolism Determines T Cell Anti-Tumor Responses

“…PDXP regulates vitamin B6-dependent enzymes by removing a phosphate group from pyridoxal-5-phosphate (PLP), the active form of vitamin B6, and thus inactivates it ( Figure 5F ). To confirm which of the two major HIF isoforms, HIF1 or HIF2, is responsible for driving expression of Pdxp , we assessed the expression of the PLP regulator genes Pdxp , Pdxk and Pnpo in mouse CD8+ T cells that ectopically expressed varying levels of HIF1 or HIF2 (GSE166758) ( 7 ). Ectopic expression of HIF1, but not HIF2, significantly increased the expression of Pdxp in a dose-dependent manner, while Pdxk and Pnpo were not significantly affected by either HIF isoform ( Figure 5G and Supplementary Figure 4A ).…”

“…In T cells, the hypoxia-inducible factor (HIF) transcription factors are master regulator of metabolic genes that direct cell fate and function (3)(4)(5)(6)(7). Metabolite availability, along with activation signals downstream of the T cell receptor (TCR) and co-stimulatory receptors, dynamically modulate HIF activity at the transcriptional, translational and post-translational level (8).…”