Aging results in profound immune dysfunction, resulting in the decline of vaccine responsiveness previously attributed to irreversible defects in the immune system. In addition to increased interleukin-6 (IL-6), we found aged mice exhibit increased systemic IL-10 that requires forkhead box P3–negative (FoxP3−), but not FoxP3+, CD4+T cells. Most IL-10–producing cells manifested a T follicular helper (Tfh) phenotype and required the Tfh cytokines IL-6 and IL-21 for their accrual, so we refer to them as Tfh10 cells. IL-21 was also required to maintain normal serum levels of IL-6 and IL-10. Notably, antigen-specific Tfh10 cells arose after immunization of aged mice, and neutralization of IL-10 receptor signaling significantly restored Tfh-dependent antibody responses, whereas depletion of FoxP3+ regulatory and follicular regulatory cells did not. Thus, these data demonstrate that immune suppression with age is reversible and implicate Tfh10 cells as an intriguing link between “inflammaging” and impaired immune responses with age.

Background Siglec-8 is expressed on human eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-mediated cell death is markedly enhanced by the presence of the activation and survival factor IL-5 and becomes independent of caspase activity. Objective In this report we investigate the mechanism of Siglec-8-mediated cell death in activated eosinophils. Methods Human peripheral blood eosinophils were treated with agonistic anti-Siglec-8 antibody and IL-5, and cell death was determined by flow cytometry and morphology. Phosphorylation of MAPK was determined by phospho-luminex, flow cytometry, and Western blotting. ROS accumulation was determined by dihydrorhodamine (DHR) fluorescence. Results Co-stimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cells dying by necrosis accompanied by granule release as compared to stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in co-stimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 co-stimulation. Phosphorylation of ERK1/2 and MEK1 was significantly enhanced and sustained in co42 stimulated cells compared to cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors blocked anti-Siglec-8/IL-5-induced cell death. ROS accumulation was induced by Siglec-8 ligation in a MEK-independent manner. In contrast, ROS inhibitor prevented the anti-Siglec-8/IL-5-induced enhancement of ERK phosphorylation and cell death. Exogenous ROS mimicked stimulation by anti-Siglec-8 and was sufficient to induce enhanced cell death in IL-5-treated cells. Collectively, these data suggest that the enhancement of ERK phosphorylation is downstream of ROS generation. Conclusions In activated eosinophils, ligation of Siglec-8 leads to ROS-dependent enhancement of IL-5-induced ERK phosphorylation, which results in a novel mode of biochemically-regulated eosinophil cell death.

Regulatory T cells (Treg), a subset of CD4+ T cells, dramatically accumulate with age in humans and mice and contribute to age-related immune suppression. Recently, we showed that a majority of accumulating Treg in aged mice expressed low levels of CD25 and their accrual is associated with declining levels of IL-2 in aged mice. Here, we further investigated the origin of CD25lo Treg in aged mice. First, aged Treg had high expression of neuropilin-1 and Helios and had a broad Vβ repertoire. Next, we analyzed the gene expression profile of Treg, naïve, and memory T cells in aged mice. We found that the gene expression profile of aged CD25lo Treg were more related to young CD25lo Treg than to either naïve or memory T cells. Further, the gene expression profile of aged Treg was consistent with recently described “effector” Treg. Additional analysis revealed that nearly all Treg in aged mice were of an effector phenotype (CD44hiCD62Llo) and could be further characterized by high levels of ICOS and CD69. ICOS contributed to Treg maintenance in aged mice, as in vivo antibody blockade of ICOSL led to a loss of effector Treg, and this loss was rescued in Bim-deficient mice. Further, serum levels of IL-6 increased with age and contributed to elevated expression of ICOS on aged Treg. Finally, Treg accrual was significantly blunted in aged IL-6-deficient mice. Together, our data show a role for IL-6 in promoting effector Treg accrual with age likely through maintenance of ICOS expression.