Shusaku Mizukami

Immune-mediated antitumor effect by type 2 diabetes drug, metformin

Significance The multifunctional ability of CTLs is downregulated by interaction between immune-checkpoint molecules expressed on CTLs and their ligands expressed on cancer cells, referred to as immune exhaustion. The antibody-mediated, immune-checkpoint blockade turned out to a promising method for immunotherapy against advanced melanoma. Metformin, a drug prescribed for patients with type 2 diabetes, has been recognized to have anti-cancer effect. We found that CD8+ tumor infiltrating lymphocytes (TILs) is a target of metformin. CD8+ TILs inevitably undergo immune exhaustion, characterized by diminished production of multiple cytokines such as IL-2, TNFα, and IFNγ, followed by elimination with apoptosis. Metformin is able to counter the state. Along with conventional therapy, treatment of cancer patients with metformin may have a great advantage for cancer therapy. Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell–deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8+ tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8+ TILs capable of producing multiple cytokines were mainly PD-1−Tim-3+, an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8+ TIL multifunctionality. The adoptive transfer of antigen-specific CD8+ T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8+ T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment.

Heat shock protein 90 (HSP90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells

In antigen (Ag) cross-presentation, dendritic cells (DCs) take up extracellular Ag and translocate them from the endosome to the cytosol for proteasomal degradation. The processed peptides can enter the conventional MHC I pathway. The molecules responsible for the translocation of Ag across the endosomal membrane into the cytosol are unknown. Here we demonstrate that heat shock protein 90 (HSP90) is critical for this step. Cross-presentation and -priming were decreased in both HSP90α-null DCs and mice. CD8α+ DC apoptosis mediated by translocation of exogenous cytochrome c to the cytosol was also eliminated in HSP90α-null mice. Ag translocation into the cytosol was diminished in HSP90α-null DCs and in DCs treated with an HSP90 inhibitor. Internalized Ag was associated with HSP90 and translocated to the cytosol, a process abrogated by the HSP90 inhibitor. Ag within purified phagosomes was released in an HSP90-dependent manner. These results demonstrate the important role of HSP90 in cross-presentation by pulling endosomal Ag out into the cytosol.

Essential Role of Endogenous Heat Shock Protein 90 of Dendritic Cells in Antigen Cross-Presentation

Extracellular HSP90 associated with Ag peptides have been demonstrated to efficiently cross-prime T cells, following internalization by dendritic cells (DCs). In addition, the nature of cell-associated Ags required for cross-priming is implicated as peptides and proteins chaperoned by heat shock protein (HSP). However, the role of endogenous HSP in DCs during cross-presentation remains elusive. In this paper, we show that endogenous HSP90 is essential for cross-presentation of both soluble and cell-associated Ags in DCs. Cross-presentation of soluble OVA and OVA-loaded transporter associated with Ag processing-1–deficient cells by bone marrow-derived DCs and DC-like cell line DC2.4 was profoundly blocked by HSP90 inhibitors, whereas presentation of endogenously expressed OVA was only partially suppressed. Assays using small interfering RNA and heat shock factor-1–deficient DCs (with defective expression of HSP90α) revealed the pivotal role of HSP90α in cross-presentation. The results suggest that in addition to HSP90 in Ag donor cells, endogenous HSP90 in DCs plays an essential role during Ag cross-presentation and, moreover, points to a link between heat shock factor-1–dependent induction of HSP90α within DC and cytotoxic T cell immunity.

Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors

Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP‐associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant‐free manner and is mediated by CD8+ T cells, indicating that HSP can act as a natural adjuvant and cross‐prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8+ T cell epitope fused to the carboxyl‐terminus of hsc70 and demonstrated efficient generation of antigen‐specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70‐CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4+ T cell epitope to the amino terminus of the hsc70‐CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)–derived 8 mer peptide, OVA257‐264, and 16mer peptide, OVA265‐280, were used as CD8+ and CD4+ T cell epitopes, respectively. Vaccination with hsc70‐OVA257‐264 generated peptide specific CTL more effectively than a peptide plus incomplete Freunds adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA265‐280 to the amino‐terminus of hsc70‐OVA257‐264 (OVA265‐280‐hsc70‐OVA257‐264) enhanced the generation of the OVA257‐264‐specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70‐OVA257‐264. Our results suggest that fusion of both CD4+ and CD8+ T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8+ T cell epitope alone. (Cancer Sci 2008; 99: 1008–1015)

Hsp90-mediated Assembly of the 26 S Proteasome Is Involved in Major Histocompatibility Complex Class I Antigen Processing

Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90α to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90α- and PA28α-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90α and PA28α, whereas two different epitopes were identified in the hsp90α- and PA28α-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.

Allele-Selective Effect of PA28 in MHC Class I Antigen Processing

PA28 is an IFN-γ-inducible proteasome activator and its genetic ablation causes complete loss of processing of certain Ags, but not all of them. The reason why this occurs and how PA28 influences the formation of peptide repertoires for MHC class I molecules remains unknown. In this study, we show the allele-specific role of PA28 in Ag processing. Retrovirus-transduced overexpression of PA28α decreased expression of Kd (Dd) while it increased Kb and Ld on the cell surface. By contrast, overexpression of PA28αΔC5, a mutant carrying a deletion of its five C-terminal residues and capable of attenuating the activity of endogenous PA28, produced the opposite effect on expression of those MHC class I molecules. Moreover, knockdown of both PA28α and β by small-interfering RNA profoundly augmented expression of Kd and Dd, but not of Ld, on the cell surface. Finally, we found that PA28-associated proteasome preferentially digested within epitopic sequences of Kd, although correct C-terminal flankings were removed, which in turn hampered production of Kd ligands. Our results indicate that whereas PA28 negatively influences processing of Kd (Dd) ligands, thereby, down-regulating Ag presentation by those MHC class I molecules, it also efficiently produces Kb (Ld) epitopes, leading to up-regulation of the MHC molecules.

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.

Differential MyD88/IRAK4 requirements for cross-priming and tumor rejection induced by heat shock protein 70-model antigen fusion protein.

Priming of CD8+ T cells requires two signals, one produced by T‐cell receptor recognition of antigen, and a second that is often provided by the innate immune response. In this context, antigens non‐covalently or covalently associated with heat shock proteins (HSP) are internalized and processed in antigen‐presenting cells (APC) to be presented by MHC I molecules to CD8+ T cells, thus, signal 1 has been well characterized in this pathway of cross‐presentation. Signal 2 is not fully understood, although there are reports that Toll‐like receptors (TLRs) interact with HSP and activate APC. The ability of HSP to activate APC through TLRs is, however, controversial because of the possibility of endotoxin contamination. Using a variety of TLR KO mice, we present evidence that TLRs (TLR2, 3, 4, 7, and 9) and their adaptor molecules MyD88 and IRAK4 are dispensable in cross‐priming by a mycobacterial HSP70–antigen (ovalbumin as a model antigen) fusion protein; in contrast, MyD88/IRAK4, but not TLRs, are required for tumor rejection induced by the same reagent. Our results indicate that HSP‐mediated cross‐priming uses a second signal produced by mechanisms other than TLR cascades. We hypothesize that efficient cross‐priming by HSP70 alone is insufficient for tumor rejection and that MyD88/IRAK4‐dependent inflammatory stimulation, which might contribute to maintenance of the initially primed effector cells, is required to eradicate tumor burden. (Cancer Sci 2012; 103: 851–859)

Heat shock proteins in antigen trafficking--implications on antigen presentation to T cells.

Heat shock proteins (HSP) are molecular chaperones implicated in facilitation of protein folding and translocation between distinct compartments, and hence in preventing protein from aggregation. In terms of proteolysis, HSP act as a double-edged sword, stimulating proteasome-dependent proteolysis while preventing the degradation of the same proteins, even though in both cases association of unfolded proteins with HSP is the initial step. The proteasomal degradation products are utilised as ligands of major histocompatibility complex (MHC) class I molecules to be recognised by CD8+ T cells, leading to activation of cytotoxic T cell immunity indispensable in fighting virus infections and cancers. In this context, HSP-mediated antigen traffic towards proteasomal degradation is coupled with acquired T cell immunity. In addition, exogenous antigens internalised by dendritic cells (DC) are also forwarded to the proteasome, possibly through the ER-associated degradation (ERAD) system, based on the fusion of the ER-membrane to the endosome containing the antigens. Thus, antigens within endosomes might be translocated to the cytosol, possibly through the Sec61 complex recruited from ER and degraded by the proteasome, rendering their peptides presentable by MHC class I molecules, a process known as cross-presentation. Since binding protein (Bip) facilitates degradation of most ER luminal soluble proteins in yeast, it is possible that endosomal HSP in DC, mimicking the action of Bip, facilitate the degradation of internalised soluble antigens. This may explain why the HSP-peptide/protein complex is extremely efficient in terms of cross-presentation ability. In this review, we discuss how HSP are linked to the ubiquitin-dependent proteasome system to generate peptides presentable by MHC molecules.

Immunogenicity and anti-fecundity effect of nanoparticle coated glutathione S-transferase (SjGST) DNA vaccine against murine Schistosoma japonicum infection.

There is still urgent need for a vaccine against schistosomiasis, especially in Schistosoma japonicum endemic areas where even a vaccine that will interrupt zoonotic transmission will be potentially effective as an intervention tool. We had developed a novel nanoparticle gene delivery system, which has proven efficacious in gene transfection to target immune cells with complementary adjuvant effect and high protective efficacy in several diseases. Here, we applied this nanoparticle system in combination with S. japonicum glutathione S-transferase (SjGST) DNA vaccine to show the immunogenicity and anti-fecundity effect of the nanoparticle coated vaccine formulation against murine schistosomiasis. The nanoparticle-coated DNA vaccine formulation induced desired immune responses. In comparison with the nanoparticle coated empty vector, it produced significantly increased antigen-specific humoral response, T-helper 1 polarized cytokine environment, higher proportion of IFN-γ producing CD4(+) T-cells and the concomitant decrease in IL-4 producing CD4(+) T-cells. Although there was no effect on worm burden, we recorded a marked reduction in tissue egg burden. There was up to 71.3% decrease in tissue egg burden and 55% reduction in the fecundity of female adult worms. Our data showed that SjGST DNA vaccine, delivered using the nanoparticle gene delivery system, produced anti-fecundity effect on female adult schistosomes as previously described by using conventional subunit vaccine with adjuvant, proving this DNA vaccine formulation as a promising candidate for anti-pathology and transmission blocking application.