TNFR2 is predominantly expressed by a subset of human and mouse CD4+CD25+FoxP3+ T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2+ Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30–40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2− Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4+CD25−TNFR2+ cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2+ Tregs was considerably more potent than that of reportedly highly suppressive CD103+ Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2+ Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.

Neutrophils are a vital component of immune protection, yet in cancer they may promote tumour progression, partly by generating reactive oxygen species (ROS) that disrupts lymphocyte functions. Metabolically, neutrophils are often discounted as purely glycolytic. Here we show that immature, c-Kit+ neutrophils subsets can engage in oxidative mitochondrial metabolism. With limited glucose supply, oxidative neutrophils use mitochondrial fatty acid oxidation to support NADPH oxidase-dependent ROS production. In 4T1 tumour-bearing mice, mitochondrial fitness is enhanced in splenic neutrophils and is driven by c-Kit signalling. Concordantly, tumour-elicited oxidative neutrophils are able to maintain ROS production and T cell suppression when glucose utilisation is restricted. Consistent with these findings, peripheral blood neutrophils from patients with cancer also display increased immaturity, mitochondrial content and oxidative phosphorylation. Together, our data suggest that the glucose-restricted tumour microenvironment induces metabolically adapted, oxidative neutrophils to maintain local immune suppression.

Summary Previously we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse regulatory T cells (Tregs). Here, we report that human peripheral blood (PB) FoxP3+ cells present in CD25high, CD25low and even CD25− subsets of CD4 cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4+CD25+ Tregs included all of FoxP3+ cells present in CD4+CD25high subset as well as a substantial proportion of FoxP3+ cells present in CD4+CD25low subset. CD4+CD25+TNFR2+ cells identified 5-fold greater number of PB CD4 lymphocytes as Tregs than identified by CD4+CD25high cells, and expressed comparable levels of FoxP3+ cells as reported CD4+CD25+CD127low/− Tregs. Furthermore, this population of cells exhibited the characteristic Treg phenotype, including expression of high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127. Upon TCR stimulation, human PB CD4+CD25+TNFR2+ cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T responder cells. In contrast, CD4+CD25+TNFR2− and CD4+CD25− TNFR2+ T cells did not show inhibitory activity. Since some non-Tregs express TNFR2, the combination of CD25 and TNFR2 must be used to identify larger population of human Tregs, which may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.

Our previous study showed that TNFR2 is preferentially expressed by CD4+FoxP3+ regulatory T cells (Tregs), and expression of this receptor identified maximally suppressive Tregs. TNFR2 is also expressed by a small fraction of CD4+FoxP3− conventional T cells (Tconvs) in normal mice, and its expression is upregulated by T cell activation. This raises questions about the role of TNFR2 signaling in the function of Tconv cells. In this study, by using FoxP3/gfp knock-in mice, we showed that TNFR2 signaling did not induce FoxP3− CD4 cells to become suppressive. Ki-67, a marker of proliferation, was concomitantly expressed with TNFR2 by CD4 cells, independent of forkhead box P3 expression, in normal mice and Lewis lung carcinoma-bearing mice. TNFR2 is associated with greater suppressive functions when expressed by Tregs and is associated with greater resistance to suppression when expressed by Tconv cells. In mice bearing 4T1 breast tumor or Lewis lung carcinoma, intratumoral Tconv cells expressing elevated levels of TNFR2 acquired the capacity to resist suppression by lymph node-derived Tregs. However, they remained susceptible to inhibition by more suppressive tumor-infiltrating Tregs,which expressed higher levels of TNFR2. Our data indicate that TNFR2 also costimulates Tconv cells. However, intratumoral Tregs expressing more TNFR2 are able to overcome the greater resistance to suppression of intratumoral Tconv cells, resulting in a dominant immunosuppressive tumor environment.