Heat Shock Protein Magic in Antigen Trafficking within Dendritic Cells:Implications in Antigen Cross-presentation in Immunity

Dendritic cells (DCs) take up soluble- or cell-associated antigens and digest them, delivering fragments to the MHC class I pathway to display antigenic peptides to CD8＋ T cells, a process known as cross-presentation. The pathway requires that, in order to be degraded by proteosomes, the extracellular antigens must have access to the cytosol across the endosomal membranes. Although the cross-presentation phenomena was first identified in the 1970s, the molecular mechanism responsible for the translocation is still not fully understood. In this context, we have recently found that cytosolic heat shock protein (HSP)90 translocates internalized antigen to the cytosol in DCs. Our results revealed the important role that cytosolic HSP90 plays in cross-presentation by pulling out endosomal antigen to the cytosol

Cancer immunotherapy with blocking of immune checkpoints

がんワクチンによる免疫治療では，如何にCD8T細胞を感作（プライミング）しその数を増やすか（免疫増強）という点に多大の努力が払われて来た．樹状細胞への抗原デリバリーと抗原プロセシング／提示，Toll様受容体などの刺激，即ち自然免疫系の活性化の併用などはそれに該当する．しかし十分に活性化されたＴ細胞をもってしても癌の拒絶は容易ではない．それには癌組織という特殊な環境が禍している．Ｔ細胞は癌塊内に入り込み莫大な数の癌細胞と遭遇する．癌組織内での繰り返す抗原認識の過程でＴ細胞は疲弊し，次第に本来あるべき機能を喪失していく．この疲弊（exhaustion）と呼ばれる現象は，Ｔ細胞に発現する複数の免疫抑制性分子―免疫チェックポイント分子―と腫瘍に発現するそのリガンドの結合によってもたらされる．代表的なチェックポイント分子の機能を抑制し，エフェクターＴ細胞が疲弊することなくその機能を長く維持できれば，これからのがん免疫治療に飛躍的な進展がみられるかもしれない

Nutrient Condition in the Microenvironment Determines Essential Metabolisms of CD8(+) T Cells for Enhanced IFN gamma Production by Metformin

Metformin (Met), a first-line drug for type 2 diabetes, lowers blood glucose levels by suppressing gluconeogenesis in the liver, presumably through the liver kinase B1-dependent activation of AMP-activated protein kinase (AMPK) after inhibiting respiratory chain complex I. Met is also implicated as a drug to be repurposed for cancers; its mechanism is believed identical to that of gluconeogenesis inhibition. However, AMPK activation requires high Met concentrations at more than 1 mM, which are unachievable in vivo. The immune-mediated antitumor response might be the case in a low dose Met. Thus, we proposed activating or expanding tumor-infiltrating CD8(+) T cells (CD8TILs) in a mouse model by orally administering Met in free drinking water. Here we showed that Met, at around 10 mu M and a physiologically relevant concentration, enhanced production of IFN gamma,TNF alpha and expression of CD25 of CD8(+) T cells upon TCR stimulation. Under a glucose-rich condition, glycolysis was exclusively involved in enhancing IFN gamma production. Under a low-glucose condition, fatty acid oxidation or autophagy-dependent glutaminolysis, or both, was also involved. Moreover, phosphoenolpyruvate carboxykinase 1 (PCK1), converting oxaloacetate to phosphoenolpyruvate, became essential. Importantly, the enhanced IFN gamma production was blocked by a mitochondrial ROS scavenger and not by an inhibitor of AMPK. In addition, IFN gamma production by CD8TILs relied on pyruvate translocation to the mitochondria and PCK1. Our results revealed a direct effect of Met on IFN gamma production of CD8(+) T cells that was dependent on differential metabolic pathways and determined by nutrient conditions in the microenvironment

Two Distinct Pathways Mediated by PA28 and hsp90 in Major Histocompatibility Complex Class I Antigen Processing

Major histocompatibility complex (MHC) class I ligands are mainly produced by the proteasome. Herein, we show that the processing of antigens is regulated by two distinct pathways, one requiring PA28 and the other hsp90. Both hsp90 and PA28 enhanced the antigen processing of ovalbumin (OVA). Geldanamycin, an inhibitor of hsp90, almost completely suppressed OVA antigen presentation in PA28α−/−/β−/− lipopolysaccharide blasts, but not in wild-type cells, indicating that hsp90 compensates for the loss of PA28 and is essential in the PA28-independent pathway. In contrast, treatment of cells with interferon (IFN)-γ, which induces PA28 expression, abrogated the requirement of hsp90, suggesting that IFN-γ enhances the PA28-dependent pathway, whereas it diminishes hsp90-dependent pathway. Importantly, IFN-γ did not induce MHC class I expressions in PA28-deficient cells, indicating a prominent role for PA28 in IFN-γ–stimulated peptide supply. Thus, these two pathways operate either redundantly or specifically, depending on antigen species and cell type

HSP70 and HSP90 Differentially Regulate Translocation of Extracellular Antigen to the Cytosol for Cross-Presentation

Antigens (Ag) from cancer or virus-infected cells must be internalized by dendritic cells (DCs) to be presented to CD8+ T cells, which eventually differentiate into Ag-specific cytotoxic T lymphocytes (CTLs) that destroy cancer cells and infected cells. This pathway is termed cross-presentation and is also implicated as an essential step in triggering autoimmune diseases such as Type I diabetes. Internalized Ag locates within endosomes, followed by translocation through a putative pore structure spanning endosomal membranes into the cytosol, where it is degraded by the proteasome to generate antigen peptides. During translocation, Ag is believed to be unfolded since the pore size is too narrow to accept native Ag structure. Here, we show that paraformaldehyde-fixed, structurally inflexible Ag is less efficient in cross-presentation because of diminished translocation into the cytosol, supporting the “unfolded Ag” theory. We also show that HSP70 inhibitors block both endogenous and cross-presentation. ImageStream analysis revealed that the inhibition in cross-presentation is not due to blocking of Ag translocation because a HSP70 inhibitor rather facilitates the translocation, which is in marked contrast to the effect of an HSP90 inhibitor that blocks Ag translocation. Our results indicate that Ag translocation to the cytosol in cross-presentation is differentially regulated by HSP70 and HSP90

HSP90α plays an important role in piRNA biogenesis and retrotransposon repression in mouse

HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α, one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA), which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog. Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28–32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24–32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α. Although DNA methylation and mRNA levels of L1 retrotransposon were largely unchanged in the Hsp90α mutant testes, the L1-encoded protein was increased, suggesting the presence of post-transcriptional regulation. This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals

Dysfunction of CD8+PD-1+T cells in type 2 diabetes caused by the impairment of metabolism-immune axis

The metabolic changes and dysfunction in CD8+T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8+splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8+splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8+PD-1+T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8+PD-1+T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity