Differential effects of ifosfamide on dendritic cell-mediated stimulation of T cell interleukin-2 production, natural killer cell cytotoxicity and interferon-gamma production.

Ifosfamide is a DNA-alkylating agent used frequently in chemotherapy of human malignancies. Ifosfamide and its major decomposition products deplete intracellular glutathione (GSH). Glutathione is the major intracellular thiol reductant that protects cells against oxidative injury. Ifosfamide depletion of intracellular GSH in human dendritic cells (DC), T cells and natural killer (NK) cells impairs their functional activity which can be restored by reconstituting GSH. Here we assessed the effect of ifosfamide on DC-mediated stimulation of NK cell proliferation via T cells and on direct DC stimulation of NK cell cytotoxicity and interferon (IFN)-gamma production. Indirect DC stimulation of NK cell proliferation via T cells and T cell-derived interleukin (IL)-2 were reduced by ifosfamide treatment of DC and reconstitution of GSH in DC restored both responses. When DC and NK cells were treated with ifosfamide, DC could overcome the negative effect of ifosfamide on NK cytotoxic function whereas NK cell IFN-gamma production was less efficiently restored. The ability of IL-2 activated NK cells to kill autologous immature DC or to induce DC maturation was reduced moderately by treatment of both cell types with ifosfamide. Overall, our results suggest that DC may stimulate anti-tumour effector cells in patients even if they had received treatment with chemotherapeutic agents such as ifosfamide.

Expression of the chemokine receptor CXCR1 in human glomerular diseases.

Leukocyte infiltration, a hallmark of renal diseases, is orchestrated in part by the actions of chemokines. The chemokine CXCL8/interleukin (IL)-8 is expressed during renal diseases and allograft rejection, whereas the corresponding receptor CXCR1 has not been described previously. Expression of CXCR1 was characterized in peripheral blood using multicolor fluorescence-activated cell sorter analysis (FACS). CXCR1 was localized in 81 formalin-fixed, paraffin-embedded renal specimens by immunohistochemistry using a monoclonal antibody against human CXCR1. Included were biopsies with crescentic glomerulonephritis (CGN, n = 22), immunoglobulin (Ig) A nephropathy (n = 15), membranoproliferative glomerulonephritis (MPGN, n = 17), lupus nephritis (n = 12), membranous nephropathy (n = 11), and non-involved parts of tumor nephrectomies (n = 4). Consecutive tissue sections of human tonsils, allograft explants, and renal biopsies were stained for CD15- and CD68-positive cells. Expression of CXCR1 and CXCL8/IL-8 mRNA was quantified by real-time reverse transcriptase-polymerse chain reaction of microdissected renal biopsies (n = 35) of the same disease entities. By FACS CXCR1 expression was found on polymorphonuclear CXCR1 expression by polymorphonuclear leukocytes (PMNs), natural killer cells, and a subpopulation of monocytes. By immunohistochemistry, CXCR1 expression was found on infiltrating inflammatory cells (predominantly PMNs), as well as on intrinsic renal cells (arterial smooth muscle cells, endothelial cells of peritubular capillaries). The distribution pattern of CXCR1 differed between disease entities. The highest numbers of glomerular CXCR1-positive cells were present in biopsies with MPGN, followed by lupus nephritis, and CGN. CXCR1 might be involved in the recruitment of PMNs to the glomerular tuft, which could be targeted by CXCR1-blocking agents.

GPI-anchored TIMP-1 treatment renders renal cell carcinoma sensitive to FAS-meditated killing.

The resistance of tumours to immune-mediated lysis has been linked to the biology of matrix metalloproteinases (MMPs), and specifically to the cell surface expression of MMPs by the tumour cell. The endogenous tissue inhibitors of metalloproteinases (TIMPs) exhibit diverse physiological/biological functions including the moderation of tumour growth, metastasis and apoptosis. These biologic activities are mediated in part by the stoichiometry of TIMP/MMP/cell surface protein interactions. A glycosylphosphatidylinositol (GPI) anchor was fused to TIMP-1 to focus defined concentrations of this inhibitory protein on the surface of three renal cell carcinoma (RCC) cell lines (RCC-26, RCC-53 and A498) independently of cell surface protein-protein interactions. Exogenously added TIMP-1-GPI efficiently inserted into the RCC cell membrane and dramatically altered the association of MMPs with the cell surface. TIMP-1-GPI treatment inhibited RCC proliferation and rendered the normally FAS-resistant RCC cells sensitive to FAS-induced apoptosis but did not alter perforin-mediated lysis by cytotoxic effector cells. The increased sensitivity to FAS-mediated apoptosis correlated with an alteration in the balance of pro- and antiapoptotic BCL-2-family proteins. By interfering with the proliferative capacity and inducing sensitivity to immune effector mechanisms GPI-anchored TIMP-1 may represent a more effective version of the TIMP-1 protein for therapeutic strategies.

Heat shock protein 70: role in antigen presentation and immune stimulation.

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.

HLA-DR53 molecules restrict glutamic acid decarboxylase peptide presentation to T cells of a Type I diabetes patient: specification of the trimolecular HLA-peptide/T-cell receptor complex.

AIMS/HYPOTHESIS: Our aim was to define the molecular specificity of glutamic acid decarboxylase-specific T-cells isolated from a patient (patient 40) with recent onset Type I (insulin-depent) diabetes mellitus. METHODS: The peptide epitope was defined using synthetic peptides to identify the minimal sequence required for T-cell activation and to determine the amino acids that contribute either to MHC binding or T-cell receptor signaling. The MHC class II-restricted peptide presentation was determined using a panel of allogeneic antigen-presenting cells and murine fibroblast-cell lines transfected to express individual human class II alleles and by blocking studies with monoclonal antibodies. The T-cell receptor was also molecularly characterized. RESULTS: Despite that patient 40 carries high-risk alleles of the DRB1 and DQB1 loci, his T-cells recognize a glutamic acid decarboxylase-derived peptide in association with class II, DR53, molecules. Although anchor residues for DR53 molecules have not yet been determined, it was possible to model epitope binding based on sequence comparisons with other class II molecules associated with susceptibility or protection for Type I diabetes. CONCLUSION/INTERPRETATION: The complete molecular specification of the MHC-peptide ligand and the T-cell receptor complex of glutamic acid decarboxylase-specific T-cells will enable analysis of strategies designed to alter T-cell function. For example, the role of altered peptide ligands or T-cell receptor-specific peptides can be studied using a model whose components reflect the natural affinities of MHC-peptide and T-cell receptor-ligand interactions selected in response to this important autoantigen.

Molecular cloning and immunogenicity of renal cell carcinoma-associated antigen G250.

The molecular cloning of the cDNA and gene encoding the renal cell carcinoma (RCC)-associated protein G250 is described. This protein is one of the best markers for clear cell RCC: all clear-cell RCC express this protein, whereas no expression can be detected in normal kidney and most other normal tissue. Antibody studies have indicated that this molecule might serve as a therapeutic target. In view of the induction/up-regulation of G250 antigen in RCC, its restricted tissue expression and its possible role in therapy, we set out to molecularly define the G250 antigen, which we identified as a transmembrane protein identical to the tumor-associated antigen MN/CAIX. We determined, by FISH analysis, that the G250/MN/CAIX gene is located on chromosome 9p12-13. In view of the relative immunogenicity of RCC, we investigated whether the G250 antigen can be recognized by TIL derived from RCC patients. The initial characterization of 18 different TIL cultures suggests that anti-G250 reactivity is rare.