Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr+) but not from tyrosinase-negative (HSP70-PC/tyr−) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-α), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.

Heat shock proteins (HSP) when released into the extracellular milieu can act simultaneously as a source of antigen due to their ability to chaperone peptides and as a maturation signal for dendritic cells, thereby inducing DCs to cross-present antigens to CD8+ T-cells. HSP can also act independently from associated peptides, stimulating the innate immune system. Previous results regarding the activation of NK cells by HSP70 cell surface expression on tumour cells and soluble HSP70 will be further covered elsewhere within this issue. For cross-presentation, HSP70-peptide complexes (HSP70-PC) were used from two human melanoma cell lines that differ in the expression of the tumour-associated antigen tyrosinase. Purified HSP70-PC consists of both the constitutively expressed HSC70 and the inducible HSP70. HSP70-peptide complexes purified from tyrosinase positive (HSP70-PC/tyr+) human melanoma cells, incubated with immature DCs, results in the activation of HLA-*A0201-restricted tyrosinase peptide-specific T-cells. Receptor-mediated uptake of HSP70-PC by DCs and intracellular transport are required for efficient MHC class I restricted cross-presentation of chaperoned peptides. Demonstration of HSP70-PC mediated cross-presentation of such non-mutated naturally expressed tumour antigens is of special clinical interest with regard to hyperthermia. Tumour regression and improved local control have been shown within clinical phase II/III trials integrating regional hyperthermia combined with radiation and/or chemotherapy in multimodal treatment strategies. According to the proposed concept, local necrosis induced by hyperthermic treatment induces the release of HSPs, followed by uptake, processing and presentation of associated peptides by DCs. By acting as chaperone and a signal for DC maturation, HSP70-PC might efficiently prime circulating T-cells. Therefore, upregulating HSP70 and causing local necrosis in tumour tissue by hyperthermia offers great potential as a new approach to directly activate the immune system.

BACKGROUNDHeat shock proteins (HSPs) play important roles in tumor immunity. The authors prospectively investigated the correlation between the tumor‐specific Hsp70 membrane expression as an independent clinicopathological marker and overall survival in tumor entities that differ in their route of metastasis.METHODSHsp70 membrane expression was examined by flow cytometry in 58 colon, 19 gastric, 54 lower rectal carcinoma, and 19 squamous cell carcinoma specimens and the corresponding normal tissues at time of first diagnosis. Kaplan‐Meier survival curves were analyzed to determine the relation of Hsp70 expression to the patients' prognosis.RESULTSAn Hsp70 membrane‐positive phenotype was found in 40% (colon), 37% (gastric), 43% (lower rectal), and 42% (squamous cell) of the analyzed tumor specimens. None of the corresponding normal tissues was found to be Hsp70 membrane‐positive. In patients with colon (P = .032) and gastric (P = .045) carcinomas, an Hsp70 membrane expression correlated significantly with an improved overall survival; a negative association was seen in lower rectal (P = .085) and squamous cell carcinoma (P = .048).CONCLUSIONSThe authors hypothesized that differing relations between surface expression of Hsp70 on tumor cells and clinical outcomes may reflect differences in the route of metastases. Colon and gastric carcinomas metastasize into the liver where hepatic natural killer cells may have the capacity to recognize and kill Hsp70 membrane‐positive tumor cells and thus account for a better overall survival. Cancer 2007; 110:926–35. © 2007 American Cancer Society.

We show first clinical data of G plus Cis with RHT being clinically active in G-pretreated APC with low toxicity. A prospective controlled phase II second-line clinical trial (EudraCT: 2005-003855-11) and a randomised phase III adjuvant clinical trial offering this treatment (HEAT; EudraCT: 2008-004802-14) are currently open for recruitment.

IntroductionIfosfamide, which is a commonly used antineoplastic agent in the treatment of malignancies such as lymphomas, breast cancer, and sarcomas [1][2][3] has been shown to reduce glutathione (GSH) levels in various cell types. 4,5 Previously, we demonstrated that ifosfamideinduced GSH depletion in T cells and natural killer (NK) cells was accompanied by a corresponding decrease in T-cell function and NK-cell cytotoxicity. 6,7 In vivo ifosfamide is metabolically activated by hepatic mixedfunction oxidases into 4-hydroxyifosfamide (4-OH-IF). In our studies we used the prodrug 4-hydroperoxyifosfamide (4-OOH-IF), which in aqueous solution spontaneously gives rise to pharmacologically equivalent amounts of 4-OH-IF, the activated form of ifosfamide. 8 GSH is a tripeptide consisting of glutamate, cysteine, and glycine, and it takes part in many metabolic and cell-cycledependent functions such as detoxification of free radicals and exogenous toxins, protein synthesis, transport, and maintenance of intracellular redox balance. 9 In addition to housekeeping properties, GSH influences lymphocyte proliferation, 10-12 polymorphonuclear cell function, 13 and chemokine and cytokine production by different cell types. [14][15][16] Antigen-presenting cells (APCs) derived from murine spleen have been shown to direct T-cell responses toward a T H 1-or T H 2-type response, depending on the level of intracellular glutathione. T H 2 responses occur when glutathione levels are low. 17 Suppression of tumor cell growth is favored by a T H 1 response in which cytokines such as interferon-␥ (IFN-␥) and interleukin-12 (IL-12) play important roles. 18,19 Dendritic cells (DCs) are the most potent APCs in the body, and their unique ability to stimulate a primary T-cell response places them at the center of an immune response toward tumors. 20,21 Immature DCs specialize in capturing and processing antigens, whereas mature DCs present antigenic peptides to T cells. 22 We have therefore made a comparison of the effect of 4-OH-IF on GSH levels in immature and mature DCs and monocytes with GSH levels in T cells and NK cells. In addition, we studied the effect of 4-OH-IF-induced GSH depletion on DC functional properties. We found that when comparing cells from the same donors, DCs had higher constitutive levels of GSH than T cells and NK cells and were less sensitive to GSH depletion by 4-OH-IF. Treatment of DCs with 4-OH-IF or L-buthioninesulfoximine (BSO) (which inhibits GSH biosynthesis by blocking ␥-glutamyl-cysteine synthetase) reduced the ability of immature DCs to stimulate an allogeneic response but had no effect on the ability of mature DCs to present exogenously added tyrosinase peptide to tyrosinase-specific cytotoxic T lymphocytes (CTLs). IFN-␥ production was also reduced in allogeneic cultures containing 4-OH-IF-treated DCs. 4-OH-IF also impaired the ability of DCs to produce IL-12p70 after lipopolysaccharide (LPS) and IFN-␥ stimulation. The allostimulatory capacity of DCs and the cytokine production by peripheral blood leukocyte...