Claudia Rieser

CELLULAR AND HUMORAL IMMUNE RESPONSES IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA AFTER VACCINATION WITH ANTIGEN PULSED DENDRITIC CELLS

PURPOSE Dendritic cells are the most potent stimulators of immune responses including antitumor responses. We performed a pilot study of cultured antigen loaded dendritic cells in patients with metastatic renal cell carcinoma. MATERIALS AND METHODS Dendritic cells were obtained by culturing plastic adherent mononuclear cells from peripheral blood for 5 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. Day 5 dendritic cells were loaded with cell lysate from cultured autologous tumor cells and with the immunogenic protein keyhole-limpet hemocyanin (KLH) which serves as a helper antigen and as a tracer molecule. During the antigen pulse dendritic cells were activated with a combination of tumor necrosis factor-alpha and prostaglandin E2. Dendritic cells were administered by 3 intravenous infusions at monthly intervals. Cellular and humoral immune responses to KLH and cell lysate were measured in vitro before and after the vaccinations. RESULTS Preparation of 12 dendritic cell vaccines from patients with advanced renal cell carcinoma was successful. Treatment with fully activated CD83+ dendritic cells was well tolerated with moderate fever as the only side effect. Potent immunological responses to KLH and, most importantly, against cell lysate could be measured in vitro after the vaccinations. CONCLUSIONS Our data demonstrate that a dendritic cell based vaccine can induce antigen specific immunity in patients with metastatic renal cell carcinoma. Dendritic cell based immunotherapy represents a feasible, well tolerated and promising new approach for the treatment of advanced renal cell carcinoma.

CD83+ blood dendritic cells as a vaccine for immunotherapy of metastatic renal-cell cancer

1 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392: 245–52. 2 Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptideor tumor lysate-pulsed dendritic cells. Nat Med 1998; 4: 328–32. 3 Murphy G, Tjoa B, Ragde H, Kenny G, Boynton A. Phase I clinical trial: T-cell therapy for prostate cancer using autologous dendritic cells pulsed with HLA-A0201-specific peptides from prostate-specific membrane antigen. Prostate 1996; 29: 371–80. 4 Rieser C, Bock G, Klocker H, Bartsch G, Thurnher M. Prostaglandin E2 and tumor necrosis factor alpha cooperate to activate human dendritic cells: synergistic activation of interleukin 12 production. J Exp Med 1997; 186: 1603–08. 5 Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma. N Engl J Med 1996; 335: 865–75.

Mature Dendritic Cells Induce T-Helper Type-1-Dominant Immune Responses in Patients with Metastatic Renal Cell Carcinoma

We performed a pilot study on a dendritic cell (DC)-based vaccine in 4 patients with advanced renal cell carcinoma. The vaccine consisted of cultured blood DCs loaded with autologous tumor cell lysate plus keyhole limpet hemocyanin (KLH) and matured with a combination of tumor necrosis factor α and prostaglandin E2. We describe the immune response against KLH induced by DC-based immunization in a patient undergoing an objective partial response and compare it with the responses observed in patients with either stable or progressive disease. The patient with the clinical response developed strong delayed-type hypersensitivity (DTH) against KLH after a single vaccination with antigen-loaded DCs, whereas the other patients failed to develop DTH reactivity even after repeated vaccinations. Antigenic stimulation of mononuclear cells (MNCs) induced proliferation and IFN-γ but not IL-4 production as well as expression of the chemokine receptor CXCR3 consistent with a T-helper (Th) type-1 bias. Exogenous IL-12 enhanced and exogenous IL-4 diminished IFN-γ production. In the 2 patients with stable disease two or more vaccinations were required to induce maximal MNC responses. In the patient with progressive disease MNC responses were hardly detectable. Anti-KLH antibodies appeared with different kinetics but could be detected in the serum of all patients. Isotype analysis revealed the presence of IgM, IgG1, IgG2 and IgG3 as well as IgA and complete absence of IgE. The patient with progressive disease also developed IgG4 antibodies indicative of a deviation towards Th2. Cultured blood DCs can be a potent vaccine for the antigen-specific immunization of patients with advanced kidney cancer. KLH serves as a tracer molecule which allows determination of the magnitude, kinetics and Th bias of the cellular and humoral immune response induced by DC-based immunization. The data also suggest that Th type-1-dominant immune responses involving DTH reaction are required for the induction of tumor regression.

Interleukin 1beta mediates the modulatory effects of monocytes on LNCaP human prostate cancer cells.

Proliferative and secretory responses in androgen-sensitive prostate cancer LNCaP cells are regulated by steroid and peptide hormones and by differentiation-promoting substances. In the present study, we evaluated whether peripheral blood monocytes that exhibit anti-tumour activity in haematopoietic and solid tumours influence growth and secretion in the LNCaP cell line. For this purpose, LNCaP cells were incubated with monocyte-conditioned medium (MCM), and proliferation as well as expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) were assessed. Conditioned medium from monocytes reduced proliferation in a dose-dependent manner. Incubation with 40% MCM caused a 50% reduction in cell proliferation. AR protein decreased by 70% and PSA levels in supernatants from LNCaP cells were reduced by approximately 80% following treatment with MCM. We focused on the contribution of two major products of activated monocytes, prostaglandin E2 and interleukin 1beta (IL-1beta), to the MCM modulatory action. LNCaP cells treated with prostaglandin E2 showed neither a reduction in proliferation nor a down-regulation of AR and PSA levels. The effects of MCM on cellular proliferation, AR protein and PSA secretion were abolished by pretreatment of MCM with a neutralizing anti-IL-1beta antibody. In addition, recombinant IL-1beta was able to replace MCM for the inhibition of proliferation and down-regulation of AR and PSA proteins. LNCaP cells were shown to express the IL-1beta receptor type 1, which transduces IL-1beta signal. Our findings reveal that monocyte-derived IL-1beta inhibits the proliferation of androgen-responsive prostate tumour cells and reduces AR and PSA levels.

Human monocyte‐derived dendritic cells produce macrophage colony‐stimulating factor: enhancement of c‐fms expression by interleukin‐10

Human monocyte‐derived dendritic cells (DC) generated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IL‐4 express c‐fms (CD115), the receptor for macrophage‐CSF (M‐CSF). Expression of c‐fms on monocyte‐derived DC has been interpreted as the susceptibility of these cells to M‐CSF‐induced macrophage development. We show here that homogenous cultures of CD14− DC constitutively produced large amounts of M‐CSF. However, presence of M‐CSF neither induced macrophage development nor did it prevent terminal maturation into CD83+ DC. M‐CSF production by DC was driven by GM‐CSF and inhibited by the specific phosphatidylinositol 3‐kinase inhibitor wortmannin. M‐CSF synthesis was rapidly induced during the first 24 h of DC culture and then declined during the 5‐day culture period. Replating of the cells, which was associated by a transient adherence, always induced a strong up‐regulation of M‐CSF synthesis. Addition of recombinant IL‐10 to DC cultures enhanced c‐fms expression and induced macrophage development as measured by the strong up‐regulation of CD14 expression as well as by enhanced expression of the Fcγ receptors I, II, and III (CD64, CD32, CD16). Our data demonstrate that immature monocyte‐derived DC produce M‐CSF which does not induce macrophage development, despite the surface expression of c‐fms on DC. IL‐10 appears to induce macrophage development by up‐regulating c‐fms and, thereby, enhancing the sensitivity of the cells to endogenously produced M‐CSF.

ACTIVATION OF HUMAN DENDRITIC CELLS BY BACILLUS CALMETTE-GUERIN

PURPOSE Dendritic cells are the most potent antigen presenting cells capable of initiating antitumor immune responses. We previously showed that bacillus Calmette-Guerin (BCG) stimulates cultured human dendritic cells. We extended these studies and tested the ability of cultured human dendritic cells to express interleukin IL-8 in response to BCG. We also investigated the T cell stimulatory potential of BCG treated dendritic cells in mixed leukocyte reactions. MATERIALS AND METHODS Dendritic cells were obtained by culturing plastic adherent mononuclear cells from peripheral blood for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and IL-4. Spontaneous and BCG stimulated IL-8 protein release into culture supernatants was measured by a quantitative immunoassay. IL-8 gene transcription was assessed by reverse transcription-polymerase chain reaction. Untreated and BCG exposed dendritic cells were compared as stimulators of allogeneic T cell proliferation, measured as [3H]thymidine incorporation. RESULTS BCG stimulated IL-8 messenger ribonucleic acid expression and IL-8 protein release. IL-8 secretion occurred in a dose and time dependent fashion. BCG induced IL-8 release was further enhanced in the presence of indomethacin. BCG treated dendritic cells were much more potent T cell stimulators than untreated dendritic cells. CONCLUSIONS These data demonstrate that BCG enhances the production of IL-8, a potent chemokine of T cells and granulocytes, as well as the T cell stimulatory potential of human dendritic cells.

Oral immunization with poly-(D,L-lactide-Co-glycolide) and poly-(L-lactic acid) microspheres containing pneumotropic bacterial antigens

Encouraged by recent findings showing the usefulness of nonreplicating antigen delivery systems in the induction ofmucosal immune responses, we investigated microspheres as a means to deliver LW 50020, an immunomodulator consisting of lysates of seven common respiratory pathogens. BALB/c mice were orally immunized with LW 50020 encapsulated into poly-(D,L-lactide-co-glycolide) (PLG) and poly-(L-lactic acid) (PLA) microspheres prepared by either a solvent-evaporation or a solvent-extraction double-emulsion technique. Particle uptake into intestinal Peyers patches, induction of antibodies in sera and secretion of immunoglobulins by isolated Peyers patches, lungs and spleen lymphocytes were investigated. Our results revealed size and surface characteristic-dependent uptake of microspheres into Peyers patches. Microsphere translocation into Peyers patches was efficient for 0.8-microm microspheres, but poor for 2.0-microm and surface-modified microspheres. We showed an enhanced immune response in the lungs and sera following oral immunization with 0.8-microm PLG solvent-evaporation microspheres. The immunomodulation was statistically significant as compared to free LW 50020. In contrast, oral immunization with other preparations caused reduced or absent modulation of the immune response compared to 0.8-microm microspheres and free antigen. These findings indicate that microspheres displaying small particle sizes, rapid antigen release and high antigen content provide optimal tools to deliver orally applied antigens.

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyers patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PPs and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.

Nordihydroguaiaretic acid blocks secretory and endocytic pathways in human dendritic cells

Nordihydroguaiaretic acid (NDGA), an antioxidant and inhibitor of the lipoxygenase arm of the arachidonic acid metabolism, was recently demonstrated to inhibit transport of secretory proteins to the Golgi complex. We have investigated the effects of NDGA on the secretory and endocytic activity of cultured human blood dendritic cells (DC). Treatment with NDGA strongly diminished cytokine secretion by DC. Moreover, NDGA reduced in a dose‐ and time‐dependent fashion fluid phase as well as receptor‐mediated endocytosis in DC. Zileuton and MK‐886, specific inhibitors of 5‐lipoxygenase and 5‐lipoxygenase‐activating protein, respectively, had no effect. Likewise, N‐acetyl‐l‐cysteine, a thiol antioxidant precursor of glutathione, did not affect DC function. Finally, serum remarkably protected the cells from the inhibitory effects of NDGA. Our data demonstrate that NDGA not only disrupts vesicular transport along the secretory route but is also a potent inhibitor of the endocytic pathways in human DC and that NDGA has inhibitory properties different from those described. J. Leukoc. Biol. 64: 747–752; 1998.

The role of CD11c+ cells as possible candidates for immature dendritic cells in the murine Peyer's patches.

The gastroinstestinal mucosa is continously exposed to food-derived antigens, resident microorganisms and invading pathogens. Correct antigen sampling and handling is essential for regulation and maintenance of mucosal immune responses against these factors However, antigen transport through the epithelium, uptake, processing and presentation by specialized cells in vivo are important steps in the initiation of a defined immune response (or tolerance) in the gut.