Increased Proportions of CD39⁺ Infiltrating T-Cells Contribute to Adenosine Mediated T-Cell Hypo-Responsiveness within Human Follicular Lymphoma.

Abstract 758

The immunosuppressive nature of the tumor microenvironment may limit the efficacy of immunotherapy for patients with follicular lymphoma (FL). We previously showed that FL infiltrating T-cells (FL-T) are hypo-responsive to potent stimulation with plate bound anti-CD3/anti-CD28 antibodies as determined by proliferation and cytokine production. We showed that one mechanism for hypo-responsiveness was suppression by infiltrating CD4⁺CD25^HIGITR⁺ regulatory T-cells (Hilchey et al. J. Immunol. (2007) 178:4051-61). Here we identify an additional novel mediator of FL-T hypo-responsiveness: pericellular adenosine, generated through the hydrolysis of extracellular ATP, and mediating its effects through interaction with FL-T associated A_2A/B adenosine receptors (AR). We show that FL-T hypo-responsiveness is attenuated in a subset of FL patient samples by blocking the A_2A/B, but not the A₁ AR. Specifically, treatment of lymph node mononuclear cells (LNMC) derived from FL biopsy specimens with the specific A_2A/B AR antagonist SCH58261 results in an increase in IFN-g and/or IL-2 secretion upon soluble anti-CD3/anti-CD28 antibody stimulation (in 3 of 6 patient samples tested), whereas treatment with an A₁ AR antagonist, CPX, had no effect on cytokine secretion. In contrast to that seen with FL LNMC, SCH58261 treatment of stimulated LNMC derived from normal lymph nodes (NLN; n=5) had no effect on IFN-g and/or IL-2 secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ectoATPase CD39, we next determined the effect of inhibiting CD39 activity on stimulated FL-T cytokine production. Inhibiting CD39 function with the selective CD39 antagonist ARL 67156, partially overcomes FL-T hypo-responsiveness in a subset of patient samples (2 of the 5 patient samples tested). Given the pivotal role that CD39 plays in the ATP-CD39-adenosine-A_2AR pathway, we next determined whether the frequency of CD39 bearing T-cell populations differed in FL nodes as compared to that seen in NLN, reactive lymph nodes (RLN), or normal donor peripheral blood (PBMC), using 12 color flow cytometry. We show that CD4⁺CD39⁺ and CD8⁺CD39⁺ T-cell populations are overrepresented in FL as compared to that seen in NLN. In addition, 30% of the FL CD4⁺CD39⁺ T-cells have a regulatory T-cell (Treg) phenotype, co-expressing CD25^HI and FOXP3 suggesting that the increased numbers of CD4⁺CD39⁺ T-cells observed within FL, as compared to NLN or RLN, are accounted for in part by the increased numbers of Tregs infiltrating the FL. Finally we show that the FL and NLN T-cell associated CD39 is functional, as it hydrolyzes ATP in vitro, in a dose and time dependent fashion. Taken together, the data suggest that the ATP-CD39-adenosine-A_2AR pathway is one mechanism for T-cell hypo-responsiveness in FL. Indeed, to our knowledge, this is the first demonstration that this pathway is immunologically significant in any human tumor. The data also suggest that pharmacological inhibition of CD39 ecto-ATP-diphosphohydrolase activity, and/or blockade of the adenosine-A_2AR interaction may be rationale strategies to enhance the efficacy of immunotherapeutic treatment approaches for patients with FL.