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.immune exhaustion | CD8T cells | antitumor immunity | tumor microenvironment | multifunctionality

Heat shock protein (HSP) preparations derived from cancer cells and virus-infected cells have been shown previously to elicit cancer-specific or virus-specific immunity. The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs. The studies reported here demonstrate that immunogenic HSP–peptide complexes can also be reconstituted in vitro. The studies show that (a) complexes of hsp70 or gp96 HSP molecules with a variety of synthetic peptides can be generated in vitro; (b) the binding of HSPs with peptides is specific in that a number of other proteins tested do not bind synthetic peptides under the conditions in which gp96 molecules do; (c) HSP–peptide complexes reconstituted in vitro are immunologically active, as tested by their ability to elicit antitumor immunity and specific CD8+ cytolytic T lymphocyte response; and (d) synthetic peptides reconstituted in vitro with gp96 are capable of being taken up and re-presented by macrophage in the same manner as gp96– peptides complexes generated in vivo. These observations demonstrate that HSPs are CD8+ T cell response–eliciting adjuvants.

SummaryVaccination of mice with heat shock protein 70 (hsp70) preparations derived from the Meth A sarcoma, but not from normal tissues, renders the mice immune to a substantial challenge with Meth A sarcoma. The immunogenicity is dose dependent and tumor specific. Treatment of an antigenically active hsp70 preparation with ATP followed by removal of low-molecular weight material leaves hsp70 intact, as judged by SDS-PAGE but results in loss of antigenicity, as judged by tumor rejection assays. Separation of this low-molecular weight material on a C18 reverse-phase column shows a diverse array of peptides with molecular mass between 1,000 and 5,000 daltons. Our data indicate that antigenicity of hsp70 preparations derives, not from hsp70 per se, but from associated peptides. These observations may suggest a novel method of using the peptide-binding property of hsp70 for specific vaccination against cancer and infectious diseases.

Recently emerging evidence indicates that the heat shock proteins (HSPs) gp96, hsp90, and hsp70 associate with antigenic peptides derived from cellular proteins. This evidence forms the basis of the following two hypotheses: 1) that HSPs constitute a relay line in which the peptides, after generation in the cytosol by the action of proteases, are transferred from one HSP to another, until they are finally accepted by MHC class I molecules in the endoplasmic reticulum, and 2) that the binding of peptides by HSPs constitutes a key step in the priming of cytotoxic T lymphocytes (CTLs) in vivo. The following chain of events is suggested: HSPs are released from virus-infected cells or tumor cells in vivo during lysis of cells during infection or by the action of antibodies or nonspecific effectors. The HSPs, which are now complexed with antigenic peptides derived from the cognate cells, are taken up by macrophage or other specialized antigen-presenting cells, possibly by a receptor-mediated mechanism. The HSP-borne peptide is then routed to the endogenous presentation pathway in the antigen-presenting cell and is displayed in the context of that cell's MHC class I, where it is finally recognized by the precursor CTLs. Thus it is suggested that, as with antigen presentation by MHC class II molecules, presentation by MHC class I molecules is also carried out primarily by the host antigen-presenting cells. This mechanism explains the phenomenon of cross-priming and has implications for the development of immunological strategies against cancer and infectious diseases.

ABSTRACTwhole irradiated tumor cells shows a different profile ofcellular requirements. In contrast to immunization with gp96, depletion of CD4+, but not CD8+, T cells during priming with whole tumor cells abrogates tumor immunity. Further, ablation of macrophage function during priming or effector phases has no effect on tumor immunity elicited by whole cells. Our results suggest the existence of a macrophagedependent and a macrophage-independent pathway of tumor immunity Our observations also show that in spite of exogenous a nistration, vaccination with gp96 preparations elicits a CD8+ T-cell response in vivo, and it is therefore a useful method ofvaccination against cancer and infectious diases.