An atlas of gene regulatory networks for memory CD4 + T cells in youth and old age.

Aging profoundly affects immune-system function, promoting susceptibility to pathogens, cancers and chronic inflammation. We previously identified a population of IL-10-producing, T follicular helper-like cells (" Tfh10 "), linked to suppressed vaccine responses in aged mice. Here, we integrate single-cell ( sc )RNA-seq, scATAC-seq and genome-scale modeling to characterize Tfh10 - and the full CD4 + memory T cell ( CD4 + TM ) compartment - in young and old mice. We identified 13 CD4 + TM populations, which we validated through cross-comparison to prior scRNA-seq studies. We built gene regulatory networks ( GRNs ) that predict transcription-factor control of gene expression in each T-cell population and how these circuits change with age. Through integration with pan-cell aging atlases, we identified intercellular-signaling networks driving age-dependent changes in CD4 + TM. Our atlas of finely resolved CD4 + TM subsets, GRNs and cell-cell communication networks is a comprehensive resource of predicted regulatory mechanisms operative in memory T cells, presenting new opportunities to improve immune responses in the elderly.

Elevated CD153 Expression on Aged T Follicular Helper Cells is Vital for B cell Responses.

Our recent data showed that an aberrant IL-10-producing T follicular helper population (Tfh10) accumulates dramatically with age and is associated with age-related declines in vaccine responsiveness. Through single cell gene expression and chromatin accessibility analysis of IL-10+ and IL-10- memory CD4+ T cells from young and aged mice, we identified increased expression of CD153 on aged Tfh and Tfh10 cells. Mechanistically, we linked inflammaging (increased IL-6 levels) to elevated CD153 expression of Tfh cells through c-Maf. Surprisingly, blockade of CD153 in aged mice significantly reduced their vaccine-driven antibody response, which was associated with decreased expression of ICOS on antigen-specific Tfh cells. Combined, these data show that an IL-6/c-Maf/CD153 circuit is critical for maintaining ICOS expression. Thus, although overall Tfh-mediated B cell responses are reduced in the context of vaccines and aging, our data suggest that elevated expression of CD153 on Tfh cells potentiates the remaining Tfh function in aged mice.

Aging mitigates the severity of obesity-associated metabolic sequelae in a gender independent manner.

BACKGROUND: Understanding gender-associated bias in aging and obesity-driven metabolic derangements has been hindered by the inability to model severe obesity in female mice. METHODS: Here, using chow- or high fat diet (HFD)-feeding regimens at standard (TS) and thermoneutral (TN) housing temperatures, the latter to model obesity in female mice, we examined the impact of gender and aging on obesity-associated metabolic derangements and immune responsiveness. Analysis included quantification of: (i) weight gain and adiposity; (ii) the development and severity of glucose dysmetabolism and non-alcoholic fatty liver disease (NAFLD); and (iii) induction of inflammatory pathways related to metabolic dysfunction. RESULTS: We show that under chow diet feeding regimen, aging was accompanied by increased body weight and white adipose tissue (WAT) expansion in a gender independent manner. HFD feeding regimen in aged, compared to young, male mice at TS, resulted in attenuated glucose dysmetabolism and hepatic steatosis. However, under TS housing conditions only aged, but not young, HFD fed female mice developed obesity. At TN however, both young and aged HFD fed female mice developed severe obesity. Independent of gender or housing conditions, aging attenuated the severity of metabolic derangements in HFD-fed obese mice. Tempered severity of metabolic derangements in aged mice was associated with increased splenic frequency of regulatory T (Treg) cells, Type I regulatory (Tr1)-like cells and circulating IL-10 levels and decreased vigor of HFD-driven induction of inflammatory pathways in adipose and liver tissues. CONCLUSION: Our findings suggest that aging-associated altered immunological profile and inflammatory vigor may play a dominant role in the attenuation of obesogenic diet-driven metabolic dysfunction.

IL-10-producing Tfh cells accumulate with age and link inflammation with age-related immune suppression.

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.

Interleukin 2 Promotes Hepatic Regulatory T Cell Responses and Protects From Biliary Fibrosis in Murine Sclerosing Cholangitis.

In the multidrug resistance protein 2 (Mdr2)-/- mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2-/- mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14-30 of life. Antibody-mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro-fibrogenic cytokine. Depletion of intrahepatic Tregs with anti-CD25 antibody between days 7-30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti-interleukin 2 immune complexes (IL-2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL-2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro-inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2-/- mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg-associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL-2c, suggesting that Treg-directed low-dose Il-2 treatment may be considered as therapy for SC.

Tissue-specific control of latent CMV reactivation by regulatory T cells.

Cytomegalovirus (CMV) causes a persistent, lifelong infection. CMV persists in a latent state and undergoes intermittent subclinical viral reactivation that is quelled by ongoing T cell responses. While T cells are critical to maintain control of infection, the immunological factors that promote CMV persistence remain unclear. Here, we investigated the role of regulatory T cells (Treg) in a mouse model of latent CMV infection using Foxp3-diphtheria toxin receptor (Foxp3-DTR) mice. Eight months after infection, MCMV had established latency in the spleen, salivary gland, lung, and pancreas, which was accompanied by an increased frequency of Treg. Administration of diphtheria toxin (DT) after establishment of latency efficiently depleted Treg and drove a significant increase in the numbers of functional MCMV-specific CD4+ and CD8+ T cells. Strikingly, Treg depletion decreased the number of animals with reactivatable latent MCMV in the spleen. Unexpectedly, in the same animals, ablation of Treg drove a significant increase in viral reactivation in the salivary gland that was accompanied with augmented local IL-10 production by Foxp3-CD4+T cells. Further, neutralization of IL-10 after Treg depletion significantly decreased viral load in the salivary gland. Combined, these data show that Treg have divergent control of MCMV infection depending upon the tissue. In the spleen, Treg antagonize CD8+ effector function and promote viral persistence while in the salivary gland Treg prevent IL-10 production and limit viral reactivation and replication. These data provide new insights into the organ-specific roles of Treg in controlling the reactivation of latent MCMV infection.

IL-6 and ICOS Antagonize Bim and Promote Regulatory T Cell Accrual with Age.

Regulatory T cells (Tregs), 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 Tregs in aged mice expressed low levels of CD25, and their accrual is associated with declining levels of IL-2 in aged mice. In this study, we further investigated the origin of CD25(lo) Tregs in aged mice. First, aged Tregs had high expression of neuropilin-1 and Helios, and had a broad Vß repertoire. Next, we analyzed the gene expression profile of Tregs, naive T cells, and memory T cells in aged mice. We found that the gene expression profile of aged CD25(lo) Tregs were more related to young CD25(lo) Tregs than to either naive or memory T cells. Further, the gene expression profile of aged Tregs was consistent with recently described "effector" Tregs (eTregs). Additional analysis revealed that nearly all Tregs in aged mice were of an effector phenotype (CD44(hi)CD62L(lo)) and could be further characterized by high levels of ICOS and CD69. ICOS contributed to Treg maintenance in aged mice, because in vivo Ab blockade of ICOSL led to a loss of eTregs, 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 Tregs. Finally, Treg accrual was significantly blunted in aged IL-6-deficient mice. Together, our data show a role for IL-6 in promoting eTreg accrual with age likely through maintenance of ICOS expression.

Mechanism of Siglec-8-mediated cell death in IL-5-activated eosinophils: role for reactive oxygen species-enhanced MEK/ERK activation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (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 using flow cytometry and morphology. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by using phosphoLuminex, flow cytometry, and Western blotting. Reactive oxygen species (ROS) accumulation was determined by using dihydrorhodamine fluorescence. RESULTS: Costimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cell death by means of necrosis accompanied by granule release compared with that seen after stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in costimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 costimulation. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and MAPK-ERK kinase (MEK) 1 was significantly enhanced and sustained in costimulated cells compared with that seen in 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, an 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.