Background Blockade of co-inhibitory ‘checkpoint’ molecules, PD-1 and CTLA-4, has induced impressive clinical responses in advanced tumors; yet only in a subset of patients.^1–3 Limited success with checkpoint blockade therapy suggests other cell extrinsic or intrinsic mechanisms may be dampening an effective immune response. Cytotoxic CD8+ T cells (CTL) encountering chronic antigen and metabolic restriction can differentiate to a terminally exhausted state (Texh), marked by hyporesponsiveness and metabolic, epigenetic, and transcriptional dysfunction.^4–8 While enrichment of this population in tumor is a negative prognostic factor,^9–10 it remains unclear whether Texh are simply non-functional or instead possess tolerogenic or suppressive properties. Transcriptional profiling of tumor-infiltrating PD-1int (progenitor exhausted) CTL versus PD-1hiTIM-3+ (terminally exhausted; Texh), reveals that exhausted cells express a pattern of genes associated with immune suppression. We hypothesize that Texh potentiate the suppressive microenvironment of solid tumor by autoregulation and inhibition of local immune responses.

Methods T cell populations were isolated from murine melanoma–B16-F10 or a lab-generated melanoma clone of the spontaneous BREF/PTEN model–by expression of inhibitory receptors and assayed in tandem in microsuppression assays. Murine melanoma clones with inhibited oxidative metabolism were generated by CRISPR-Cas9 deletion and validated for ablated mitochondrial respiration by extracellular flux analysis. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery.

Results When sorted directly from tumor, PD-1hiTim3+ Texh, but not progenitor exhausted PD-1int CTL, induce marked suppression of T cell effector responses, comparable to Foxp3+ Treg from the same environment. Expression of the ectonucleotidase, CD39, is uniquely expressed in Texh and increases as T cells differentiate towards exhaustion. Genetic deletion of CD39 in Texh eliminates the regulatory phenotype of tumor-infiltrating Texh and enforced CD39 expression on effector T cells can inhibit T cell receptor signaling and downstream function. CD39 expression correlates with exposure to hypoxia and Texh sorted from tumors engineered to be less hypoxic displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, contributes to generation of immunosuppressive adenosine, and has been previously associated with terminal exhaustion.^11–13

Conclusions Our data support a model that as CTL progress to terminal exhaustion, hypoxic exposure enforces the upregulation of CD39, providing Texh a mechanism to suppress proinflammatory processes. These findings suggest Texh are not solely dysfunctional but rather are deleterious to anti-tumor immunity and may need to be drastically reprogrammed or deleted in order to alleviate immunosuppressive functions.

References

1. Wolchok JD. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med 2017; 377, 1345–1356.

2. Hellmann MD, et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012): results of an open-label, phase 1, multicohort study. Lancet Oncol 2017; 18, 31–41.

3. Robert C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 2015; 372, 2521–2532.

4. Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.

5. Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.

6. Blackburn SD, Shin H, Freeman GJ & Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc. Natl. Acad. Sci 2008;105:15016–15021.

7. Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165.

8. Najjar YG, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight. 2019; 4.

9. Loo K, et al. Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy. JCI Insight 2017; 2.

10. Daud AI, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. J. Clin. Invest 2016;126:3447–3452.

11. . Duhen T, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun 2018;9:2724.

12. Canale FP, et al. CD39 Expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.

13. Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11, e1005177.