Although Foxp3+ regulatory T cells (Tregs) require interleukin-2 (IL-2) for their development, it has been unclear whether continuing IL-2 signals are needed to maintain lineage stability, survival, and suppressor function in mature Tregs. We generated mice in which CD25, the main ligand-binding subunit of the IL-2 receptor, can be inducibly deleted from Tregs after thymic development. In contrast to Treg development, we find that IL-2 is dispensable for maintaining lineage stability in mature Tregs. Although continuous IL-2 signaling is needed for long-term Treg survival, CD25-deleted Tregs may persist for several weeks in vivo using IL-7. We also observe defects in glycolytic metabolism and suppressor function following CD25 deletion. Thus, unlike developing Tregs in which the primary role of IL-2 is to initiate Foxp3 expression, mature Tregs require continuous IL-2 signaling to maintain survival and suppressor function, but not to maintain lineage stability.

Regulatory T cells (Tregs) are required for the maintenance of immune tolerance and adoptive Treg infusion therapy has become a promising approach to suppress immune responses in diseases such as autoimmunity and transplant rejection. However, one critical challenge of Treg therapy is the requirement of in vitro expansion of functionally stable Tregs while preventing either the contamination of T effector and/or emergence of unstable pathogenic Tregs. Recent studies showing distinct metabolic requirements of T effectors and Tregs suggest that manipulation of cell metabolism may be an attractive strategy to achieve this goal. Here we show that human thymically derived Tregs (tTregs) and in vitro induced Tregs (iTregs) from naive T cells engage glycolysis equivalently upon activation. However, inhibiting glucose metabolism via 2-deoxy-D-glucose (2DG) has distinct effects on each of these subsets. While 2DG treatment at the onset of activation significantly reduced the proliferation and expression of suppressive molecules such as ICOS and CTLA-4 in tTregs, its effect on FOXP3 expression was small. In contrast, 2DG treatment during iTreg induction modestly decreased their proliferation but strongly reduced both ICOS and FOXP3 expression. Importantly, both Treg subsets became insensitive to 2DG after day 3 post activation with little effect on either proliferation or FOXP3 expression while T conventional Th0 cells showed reduced proliferation under the same conditions. Moreover, 2DG treatment at day 3 did not impair the suppressive capabilities of Treg subsets. Collectively, these findings suggest that there is a distinct temporal requirement of glycolysis in each of the activated human Treg subsets and T conventional cells. Furthermore, 2DG treatment at the onset as a strategy to impair contaminating T effector cell proliferation is unfavorable for optimal Treg generation as well.

Natural killer (NK) cells have a potential role in immune surveillance of hepatocellular carcinoma (HCC). Self-recognition of human leukocyte antigens (HLA) through killer immunoglobulin-like receptors (KIR) confers competence to NK cells—a process termed “licensing.” We investigated the effect of NK-cell licensing on the susceptibility of patients to HCC recurrence. A total of 170 Japanese patients with HCC who underwent primary curative hepatectomy between 1996 and 2010 were enrolled in this study. The median follow-up period was 5.4 years. We analyzed their KIR-HLA genotypes with sequence-specific polymorphism-based typing and estimated their susceptibility to HCC recurrence by performing propensity score–matching analyses. The presence of KIR2DL1-C2, KIR2DL2-C1, KIR3DL1-BW4, or KIR3DL2-A3/11, functional compound genotypes that intrinsically license NK cells, did not markedly affect HCC recurrence. However, the multiplicity of those compound KIR-HLA genotypes was significantly associated with the HCC recurrence rate, i.e., the cumulative risk of recurrence in patients with at least three compound genotypes was significantly lower than that in patients with one or two compound genotypes, suggesting that the effect of NK-cell licensing on HCC recurrence is quantitative. Patients at high risk of HCC recurrence after curative hepatectomy could be identified by KIR-HLA genotyping. Cancer Immunol Res; 2(12); 1142–7. ©2014 AACR.

Aim: Immunotherapies blocking the CD47‐SIRPα pathway by targeting CD47 enhance macrophage phagocytosis of neoplastic cells in mouse models. As SIRPα exhibits relatively restricted tissue expression, SIRPα antagonists may be better tolerated than agents targeting CD47, which is ubiquitously expressed in many tissues. Here, we investigated the therapeutic impact of monoclonal antibodies (mAbs) against CD47 and/or SIRPα on gastroenterological tumors in syngeneic immunocompetent mouse models.Methods: We used in vitro and in vivo phagocytosis assays in C57BL/6J (B6) mice to investigate anti‐CD47/SIRPα mAb effects on Hepa1‐6 and CMT93 originating from B6 mice. The influence of these mAbs on macrophage transmigration was also assessed. To investigate anti‐SIRPα mAb therapy‐induced inhibitory effects on sporadic colon cancer growth, we used a CDX2P9.5‐NLS Cre;APC⁺/FLOX (CPC‐APC) mouse model.Results: Systemic anti‐SIRPα mAb administration significantly increased Hepa1‐6 and CMT93 cell susceptibility to macrophage phagocytosis, both in vitro and in vivo. Conversely, similarly administered anti‐CD47 mAb did not promote macrophage phagocytosis of target cells, whereas cells incubated with anti‐CD47 mAb prior to inoculation were more susceptible to macrophage phagocytosis. In vitro cell migration assays revealed that binding with anti‐CD47 mAb inhibited macrophage transmigration. Anti‐SIRPα mAb treatment inhibited tumor progression in CPC‐APC mice and significantly improved overall survival. Anti‐CD47 mAb administration in vivo eliminated the phagocytosis‐promoting CD47 blockade effect, probably by inhibiting macrophage transmigration/chemotaxis. In contrast, anti‐SIRPα mAb exhibited enhanced macrophage phagocytic activity and marked anti‐tumor effects against gastroenterological malignancies.Conclusion: SIRPα mAb augmentation of macrophage phagocytic activity may represent an effective treatment strategy for human gastrointestinal tumors.

Recently, a new discipline termed “immunometabolism” has transformed the field of immunology. It encompasses the study of the intrinsic metabolic pathways of different immune subsets and their impact on cellular fate and function. For instance, broadly speaking, proinflammatory cells depend on glycolysis and glutamine oxidation, whereas cells involved in anti-inflammatory response, such as Foxp3+ regulatory T (Treg) cells, use predominantly fatty acid oxidation. However, although a useful paradigm, this actually is a reductionist view, and the engagement of these metabolic pathways is not mutually exclusive between these subsets. Over the past several years, new insights and new methods to better dissect, define, and harness the metabolic properties of immune cells for immunotherapeutic purposes have come to the forefront. In this review, we will discuss the metabolic heterogeneity of different T-cell subsets as well as basic principles of integrative technologies, such as metabolomics, which can be used to better understand the metabolic signatures of immune responses. Given the interest of exploiting this information in the context of transplantation, we will highlight the scope of immunometabolism in unraveling novel mechanisms of immune regulation that can be manipulated to promote Treg cell stability and function while inhibiting T effectors to establish long-term transplantation tolerance.