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Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and haveled the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation.We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.

The relationship between the T cell receptor (TCR) repertoires used by self-reactive transcription factor Foxp3-positive (Foxp3(+)) CD4(+) regulatory T cells (T(reg) cells) and nonregulatory T cells with autoimmune potential is unclear. Here we found that the TCR repertoire of thymic T(reg) cells in TCRbeta-transgenic mice was diverse and was more similar to that of peripheral T(reg) cells than that of nonregulatory T cells, suggesting that thymic T(reg) cells make a substantial contribution to the peripheral T(reg) cell population. Activated T cells in Foxp3-deficient mice, which lack T(reg) cells, 'preferentially' used TCRs found in the TCR repertoire of T(reg) cells in Foxp3-sufficient TCRbeta-transgenic mice, suggesting that these self-reactive TCRs contribute to the pathology of Foxp3-deficient mice. Our analyses suggest that T(reg) cells and potentially pathogenic autoimmune T cells use overlapping pools of self-reactive TCRs.

Naturally arising CD25+ CD4+ regulatory T cells (TR) play an important role in the prevention of autoimmunity. TCR specificity is thought to play a critical role in TR development and function, but the repertoire and specificity of TR TCRs remain largely unknown. We find by sequencing of TRAV14 (Valpha2) TCRalpha chains associated with a transgenic TCRbeta chain that the TRand CD25- CD4+ TCR repertoires are similarly diverse, yet only partially overlapping. Retroviral expression of TCRalpha genes in TCR transgenic RAG-deficient T cells revealed that a high frequency of TCRs derived from CD25+ but not CD25- CD4+ T cells confers the ability to rapidly expand upon transfer into a lymphopenic host. Thus, these data show that a large proportion of naturally arising TR have substantially more efficient interactions with MHC class II bound peptides from the peripheral self than CD25- T cells.

SummaryThe homeostasis of multicellular organisms requires terminally differentiated cells to preserve their lineage specificity. However, it is unclear whether mechanisms exist to actively protect cell identity in response to environmental cues that confer functional plasticity. Regulatory T (Treg) cells, specified by the transcription factor Foxp3, are indispensable for immune system homeostasis. Here, we report that conserved noncoding sequence 2 (CNS2), a CpG-rich Foxp3 intronic cis-element specifically demethylated in mature Tregs, helps maintain immune homeostasis and limit autoimmune disease development by protecting Treg identity in response to signals that shape mature Treg functions and drive their initial differentiation. In activated Tregs, CNS2 helps protect Foxp3 expression from destabilizing cytokine conditions by sensing TCR/NFAT activation, which facilitates the interaction between CNS2 and Foxp3 promoter. Thus, epigenetically marked cis-elements can protect cell identity by sensing key environmental cues central to both cell identity formation and functional plasticity without interfering with initial cell differentiation.

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3Cpro, that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3Cpro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3Cpro and downstream 3Dpol sequence, but not 3Dpol polymerase activity. Cleavage occurs at two non-canonical 3Cpro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3Dpol sequence modulates the substrate specificity of the upstream 3Cpro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3Cpro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate., Author Summary While viruses that target the liver often cause lengthy infections with considerable morbidity, there is limited understanding of how they evade host responses. We have studied hepatitis A virus (HAV), an important cause of acute hepatitis in humans. Although HAV infection typically results in hepatic inflammation, there is no disease in the liver during the first weeks of infection despite robust virus replication. This suggests that HAV either fails to stimulate or efficiently evades recognition by host innate immune sensors. Our prior work showed HAV disrupts RIG-I/MDA5 signaling by targeting MAVS, an essential adaptor protein, for degradation by 3ABC, a precursor of the only HAV protease, 3Cpro. Here, we show here that a distinct viral processing intermediate, the 3CD protease-polymerase, disrupts TLR3 signaling by degrading its adaptor protein, TRIF. HAV has evolved a novel strategy to target two different host adaptor proteins with a single protease, using its 3Dpol RNA polymerase to modify the substrate specificity of its 3Cpro protease when fused to it in the 3CD precursor, thus allowing it to target non-canonical 3Cpro recognition sequences in TRIF. This remarkable example of viral adaptation allows the virus to target two different host adaptor proteins with a single viral protease.

Solubilization of bone mineral by osteoclasts depends on the formation of an acidic extracellular compartment through the action of a V-proton pump that has not yet been characterized at the molecular level. We previously cloned a gene (Atp6i, for V-proton pump, H+ transporting (vacuolar proton pump) member I) encoding a putative osteoclast-specific proton pump subunit, termed OC-116kD (ref. 4). Here we show that targeted disruption of Atp6i in mice results in severe osteopetrosis. Atp6i-/- osteoclast-like cells (OCLs) lose the function of extracellular acidification, but retain intracellular lysosomal proton pump activity. The pH in Atp6i-/- liver lysosomes and proton transport in microsomes of Atp6i-/- kidney are identical to that in wild-type mice. Atp6i-/- mice exhibit a normal acid-base balance in blood and urine. Our results demonstrate that Atp6i is unique and necessary for osteoclast-mediated extracellular acidification.

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer., Author Summary Persons infected with hepatitis C virus (HCV) are at increased risk for liver cancer. This is remarkable because HCV is an RNA virus with replication confined to the cytoplasm and no potential for integration of its genome into host cell DNA. While it is likely that chronic inflammation contributes to liver cancer, prior studies with HCV transgenic mice indicate that the viral proteins are intrinsically carcinogenic. In this study, we have examined the interaction of one of these, the RNA-dependent RNA polymerase nonstructural protein 5B, with an important cellular tumor suppressor protein, the retinoblastoma protein (pRb). pRb is a master regulator of the cell cycle, and altered expression of some of the many genes it regulates may lead to cancer. We show that the abundance of pRb is strongly downregulated in cells infected with HCV, and that nonstructural protein 5B targets pRb for destruction via the cell's normal protein degradation machinery. The E6-associated protein appears to play a role in this process, which is interesting as it also mediates the degradation of another tumor suppressor, p53, by papillomaviruses. The loss of pRb function in HCV-infected cells likely promotes hepatocellular proliferation as well chromosomal instability, factors important for the development of liver cancer.