Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

A Whole Recombinant Yeast-Based Therapeutic Vaccine Elicits HBV X,S and Core Specific T Cells in Mice and Activates Human T Cells Recognizing Epitopes Linked to Viral Clearance

Chronic hepatitis B infection (CHB) is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen) platform was used to make a vaccine candidate expressing hepatitis B virus (HBV) X, surface (S), and Core antigens (X-S-Core). Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS), or tumor challenge assays. Both CD4⁺ and CD8⁺ T cell responses were observed. Human T cells transduced with HBc18–27 and HBs183–91 specific T cell receptors (TCRs) produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs). Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs) isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core) resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Activation of cytotoxic T lymphocytes against CML28-bearing tumors by dendritic cells transduced with a recombinant adeno-associated virus encoding the CML28 gene

Induction of anti-tumor immune responses by dendritic cells (DCs) transduced with a recombinant adeno-associated virus type 2 (rAAV2) encoding tumor antigens is considered a promising approach for cancer vaccine development. CML28, a novel antigen with the properties of cancer/testis (CT) antigens, is an attractive target for antigen-specific immunotherapy. Here we investigated the feasibility of inducing CML28-specific cytotoxic T lymphocyte (CTL) responses using DCs transduced with the rAAV2 vectors containing the CML28 gene (rAAV/CML28). Using an adenovirus-free packaging system, rAAV/CML28 was generated. The transduction efficiency of rAAV/CML28 in DCs increased in a multiplicity of infection (MOI)-dependent manner. The rAAV/CML28 transduction did not impair DC maturation, but even enhanced the CD80 expression. The rAAV/CML28-transduced DCs induced CML28-specific CTLs which exhibited a MHC class I-mediated antigen-specific lytic activity against CML28-bearing tumor cell lines (HepG2 and MCF-7) as well as the primary leukemia blasts. These findings suggest that rAAV/CML28-transduced DCs vaccine may serve as a feasible approach for the treatment of CML28-associated cancers.     

Dendritic cell-tumor fusion vaccine prevents tumor growth in vivo

Dendritic cells (DCs) are potent antigen presenting cells that are uniquely effective in generating primary immune responses. DCs that are manipulated to present tumor antigens induce antitumor immunity in animal models and preclinical human studies. A myriad of strategies have been developed to load tumor antigen effectively onto DCs. DC-tumor fusion presents a spectrum of tumor-associated antigens to helper T- and cytotoxic T-cell populations in the context of DC-mediated costimulatory signals. In this study, fusion cells (FCs) were generated with MCA-102 fibrosarcoma cells and murine bone marrow-derived myeloid DCs. The FCs coexpressed the DC-derived MHC class II and costimulatory molecules. The FCs also retained the functional properties of DCs and stimulated syngeneic T cell proliferation and interferon-gamma (IFN-gamma) production. Significantly, the results show that syngeneic T cells are primed by FCs to induce MHC class I-dependent lysis of MCA-102 fibrosarcoma. These findings indicate that fusions of tumor cells and DCs activate T-cell responses against syngeneic tumors.     

融合表达小鼠IgG2b-Fc可增强人类免疫缺陷病毒1型Gag DNA疫苗的免疫原性

为阐明小鼠IgG2b-Fc对DNA疫苗免疫原性的增强作用,首先构建表达人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)CN54Gag基因的DNA疫苗及表达Gag与小鼠IgG2b-Fc融合基因的DNA疫苗,限制性酶切和DNA测序结果表明这两个疫苗均构建成功,蛋白免疫印迹结果也显示其正确表达。然后,利用上述DNA疫苗接种C57BL/6小鼠,比较两个DNA疫苗所诱导的特异性体液免疫反应和特异性细胞免疫反应。结果显示,融合表达小鼠IgG2b-Fc对特异性细胞免疫和体液免疫反应均有增强作用,但只有对特异性体液免疫反应的增强作用有统计学意义。     

Generation of potent anti-tumor immunity in mice by interleukin-12-secreting dendritic cells

To induce cytolytic immunity, dendritic cells (DCs) need to release bioactive interleukin-12 (IL-12) p70 heterodimeric molecules. To study the role of IL-12 for the generation of an anti-tumor immune response, we generated two classes of DCs. (1) DCs were initiated to secrete IL-12 by exposure to LPS/IFN- for 2 h resulting, as demonstrated in vitro, in continued IL-12 release for another 24 h (termed active DCs). (2) DCs were exposed to LPS/IFN- for 24 h and injected into mice at a time point when IL-12 production had ceased (termed exhausted DCs). These two classes of DCs were probed for their capacity to induce a cytolytic anti-tumor immune response in vivo in a syngeneic mouse tumor model. The mouse tumor cell line K-Balb was engineered to express neomycin phosphotransferase (NPT) as a model tumor antigen. DCs were charged with various NPT-derived antigens, including recombinant NPT protein, whole tumor cell lysate and NPT-derived synthetic peptides, and the induction of in vivo anti-tumor immunity was determined by measuring tumor growth. Only the injection of active DCs, i.e., cells that maintained the capacity to secrete IL-12, but not exhausted DCs that had lost the ability to produce IL-12, resulted in a measurable deceleration of growth of K-Balb-NPT tumors. This anti-tumor immune response was most pronounced when using recombinant protein as an antigen source, which was evident in a prophylactic as well as in a therapeutic setting. The absence of a response to parental K-Balb tumors confirmed the antigen specificity of the anti-tumor immune response. Together these data provide evidence for the unique capacity of actively IL-12 secreting DCs to trigger effective anti-tumor immunity using exogenous tumor antigens.     

Identification of a Human Cyclin D1-Derived Peptide that Induces Human Cytotoxic CD4 T Cells

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and ⁵¹Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.     

Induction of antigen-specific tumor immunity by genetic and cellular vaccines against MAGE: enhanced tumor protection by coexpression of granulocyte-macrophage colony-stimulating factor and B7-1.

BACKGROUND: A number of tumors express antigens that are recognized by specific cytotoxic T cells. The normal host immune responses, however, are not usually sufficient to cause tumor rejection. Using appropriate immunization strategies, tumor-specific antigens may serve as targets against which tumor-destructive immune responses can be generated. MAGE-1 and MAGE-3 are two clinically relevant antigens expressed in many human melanomas and other tumors, but not in normal tissues, except testis. Here, we have investigated whether DNA and cellular vaccines against MAGE-1 and MAGE-3 can induce antigen-specific anti-tumor immunity and cause rejection of MAGE-expressing tumors. MATERIALS AND METHODS: Mice were immunized against MAGE-1 and MAGE-3 by subcutaneous injection of genetically modified embryonic fibroblasts or intramuscular injection of purified DNA. Mice were injected with lethal doses of B16 melanoma cells expressing the corresponding MAGE antigens or the unrelated protein SIV tat, and tumor development and survival were monitored. RESULTS: Intramuscular expression of MAGE-1 and MAGE-3 by plasmid DNA injection and subcutaneous immunization with syngeneic mouse embryonic fibroblasts transduced with recombinant retroviruses to express these antigens induced specific immunity against tumors expressing MAGE-1 and MAGE-3. Both CD4+ and CD8+ T cells were required for anti-tumor immunity. Coexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) or B7-1 significantly increased anti-tumor immunity in an antigen-specific manner and resulted in a considerable proportion of mice surviving lethal tumor challenge. CONCLUSIONS: Our results suggest that genetic and cellular vaccines against MAGE and other tumor antigens may be useful for the therapy of tumors expressing specific markers, and that GM-CSF and B7-1 are potent stimulators for the induction of antigen-specific tumor immunity.     

Stimulatory effects of B7-related protein-1 on cellular and humoral immune responses in mice

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-gamma, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.     

Dendritic cell-based cancer immunotherapy targeting MUC-1

Vaccination therapy using dendritic cells (DC) as antigen presenting cells (APC) has shown significant promise in laboratory and animal studies as a potential treatment for malignant diseases. Pulsing of autologous DCs with tumor-associated antigens (TAA) is a method often used for antigen delivery and choice of suitable antigens plays an important role in designing an effective vaccine. We identified two HLA-A2 binding novel 9-mer peptides of the TAA MUC1, which is overexpressed on various hematological and epithelial malignancies. Cytotoxic T cells generated after pulsing DC with these peptides were able to induce lysis of tumor cells expressing MUC1 in an antigen-specific and HLA-restricted fashion. Within two clinical studies, we demonstrated that vaccination of patients with advanced cancer using DCs pulsed with MUC1 derived peptides is well tolerated without serious side effects and can induce immunological responses. Of 20 patients with metastatic renal cell carcinoma, 6 patients showed regression of metastases with 3 objective responses (1 CR, 2 PR). Furthermore, we found that in patients responding to treatment T cell responses for antigens not used for treatment occurred suggesting that antigen spreading in vivo might be a possible mechanism of mediating antitumor effects. These results demonstrate that immunotherapy in patients with advanced malignancies using autologous DCs pulsed with MUC1 derived peptides can induce immunological and clinical responses. However, further clinical studies are needed to identify the most potent treatment regimen that can consistently mediate an antitumor immune response in vivo. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004.     

Preassociation of IL-15 with IL-15R alpha-IgG1-Fc enhances its activity on proliferation of NK and CD8+/CD44high T cells and its antitumor action

In the induction of an immune response, IL-15Ralpha on APCs transpresents IL-15 to NK and CD8(+)/CD44(high) T cells that express the IL-2/15Rbeta and gammac subunits only. In this study, we show data mimicking this transpresentation by using IL-15 preassociated with a chimeric protein that is comprised of the extracellular domain of murine IL-15Ralpha and the Fc portion of human IgG1. When tested in vitro, IL-15Ralpha-IgG1-Fc strongly increased the IL-15-mediated proliferation of murine NK and CD8(+)/CD44(high) T cells. The effect of IL-15Ralpha-IgG1-Fc was dependent on the presence of both IgG1-Fc and IL-15Ralpha. When injected into mice, IL-15Ralpha-IgG1-Fc enhanced the capacity of IL-15 to expand the number of NK and CD8(+)/CD44(high) T cells. The effect on cell numbers in vivo also depended on Fc receptor binding because reduced expansion was observed in FcRgamma(-/-) mice. NK cells cultured in IL-15/IL-15Ralpha-IgG1-Fc complex gained cytotoxic activity toward a number of NK-sensitive targets. When mice bearing the NK-sensitive syngeneic tumor B16 were treated, the presence of IL-15Ralpha-IgG1-Fc increased the antitumor activity of IL-15. Thus, a preassociation with IL-15Ralpha-IgG1-Fc enhances the activities of IL-15 in vivo and in vitro that may be useful in the treatment of tumors.     

Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity

Systemic IL-2 is currently employed in the therapy of several tumor types, but at the price of often severe toxicities. Local vector mediated delivery of IL-2 at the tumor site may enhance local effector cell activity while reducing toxicity. To examine this, a model using CEA-transgenic mice bearing established CEA expressing tumors was employed. The vaccine regimen was a s.c. prime vaccination with recombinant vaccinia (rV) expressing transgenes for CEA and a triad of costimulatory molecules (TRICOM) followed by i.t. boosting with rF-CEA/TRICOM. The addition of intratumoral (i.t.) delivery of IL-2 via a recombinant fowlpox (rF) IL-2 vector greatly enhanced anti-tumor activity of a recombinant vaccine, resulting in complete tumor regression in 70–80% of mice. The anti-tumor activity was shown to be dependent on CD8⁺ cells and NK1.1⁺. Cellular immune assays revealed that the addition of rF-IL-2 to the vaccination therapy enhanced CEA-specific tetramer⁺ cell numbers, cytokine release and CTL lysis of CEA⁺ targets. Moreover, tumor-bearing mice vaccinated with the CEA/TRICOM displayed an antigen cascade, i.e., CD8⁺ T cell responses to two other antigens expressed on the tumor and not the vaccine: wild-type p53 and endogenous retroviral antigen gp70. Mice receiving rF-IL-2 during vaccination demonstrated higher avidity CEA-specific, as well as higher avidity gp70-specific, CD8⁺ T cells when compared with mice vaccinated without rF-IL-2. These studies demonstrate for the first time that the level and avidity of antigen specific CTL, as well as the therapeutic outcome can be improved with the use of i.t. rF-IL-2 with vaccine regimens.     

Induction of tumor-specific T-cell responses by vaccination with tumor lysate-loaded dendritic cells in colorectal cancer patients with carcinoembryonic-antigen positive tumors

Background Dendritic cells (DCs) are the most effective antigen-presenting cells. In the last decade, the use of DCs for immunotherapy of cancer patients has been vastly increased. High endocytic capacity together with a unique capability of initiating primary T-cell responses have made DCs the most potent candidates for this purpose. Although DC vaccination occasionally leads to tumor regression, clinical efficacy, and immunogenicity of DCs in clinical trials has not been yet clarified. The present study evaluated the safety and effectiveness of tumor-lysate loaded DC vaccines in advanced colorectal cancer (CRC) patients with carcinoembryonic antigen (CEA) positive tumors. Results Six patients HLA-A*0201-positive were vaccinated with autologous DCs loaded with tumor lysates (TL) together with tetanus toxoid antigen, hepatitis B, and influenza matrix peptides. Two additional patients were injected with DCs that were generated from their sibling or parent with one haplotype mismatch. All patients received the vaccines every 2 weeks, with a total of three intra-nodal injections per patient. The results indicated that DC vaccination was safe and well tolerated by the patients. Specific immune responses were detected and in some patients, transient stabilization or even reduction of CEA levels were observed. The injection of haplotype mismatched HLA-A*0201-positive DCs resulted in some enhancement of the anti-tumor response in vitro and led to stabilization/reduction of CEA levels in the serum, compared to the use of autologous DCs. Conclusion Altogether, these results suggest that TL-pulsed DCs may be an effective vaccine method in CRC patients. Elimination of regulatory mechanisms as well as adjustment of the vaccination protocol may improve the efficacy of DC vaccination. An erratum to this article can be found at     

Dendritic cells: Potential role in cancer therapy

Dendritic cells (DC) are extremely potent antigen presenting cells, uniquely capable of sensitizing naive T cells to protein antigens and eliciting antigen specific immune responses. Studies of human DC isolated from peripheral blood indicate that these cells can be used to stimulate and expand antigen specific CD4+ and CD8+ T cells, in vitro. On the basis of these findings we have initiated pilot clinical studies to investigate the ability of DC pulsed ex vivo with tumor associated proteins to stimulate host anti-tumor immunity when re-infused as a vaccine. In the first such study DC pulsed with tumor derived idiotype protein were infused into patients with low grade malignant B cell lymphoma who had failed conventional chemotherapy. The majority of treated patients developed T cell mediated anti-idiotype immune responses and some of the patients experienced tumor regression. These results suggest that DC based immunotherapy is a potentially useful approach to B cell lymphoma and raises the possibility that the approach may prove useful in the treatment of other tumors as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection

Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.     

How to connect an IgE-driven response with CTL activity?

One of the goals of cell-based immune therapy in cancer is the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses. To achieve this objective, the ability of dendritic cells (DC) to cross-present tumor antigens can be exploited. One of the most efficient pathways for the induction of CTLs by cross-presentation is mediated by immunoglobulins of the IgG class, which are used by DCs to sample antigen in the form of immune complexes via Fc-gamma receptors. Could DCs use an IgE-mediated cross-presentation mechanism in a comparable manner to induce CTLs? We here discuss the potential of two human IgE Fc receptors, FcεRI and FcεRII, to serve as antigen uptake receptors for IgE-mediated cross-presentation. We conclude that the existence of an IgE-mediated cross-presentation pathway would provide a direct link between IgE-driven immune responses and CTL activity.     

Activation of human peripheral IgM+ B cells is transiently inhibited by BCR-independent aggregation of Fc gammaRIIB

Immune complexes can trigger a SHIP-1-independent proapoptotic signal in mouse class-switched IgG(+) B cells and plasma cells by binding to Fc gammaRIIB, in the absence of concomitant coaggregation with BCR, hence regulating plasma cell survival and participating in the selection of B cells producing high affinity Abs during secondary Ab responses. By contrast, we demonstrate in the present study that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells does not induce apoptosis but transiently inhibits B cell proliferation and calcium influx triggered by BCR cross-linking. Using human peripheral B cells and IIA1.6 lymphoma B cells expressing wild-type human Fc gammaRIIB (IIA1.6-Fc gammaRIIB), we also show that the unique aggregation of human Fc gammaRIIB induces ITIM phosphorylation. This aggregation provokes the recruitment of phosphorylated SHIP-1 by Fc gammaRIIB and inhibits the constitutive phosphorylation of Akt in human IIA1.6-Fc gammaRIIB cells. This inhibitory signaling pathway is abrogated in IIA1.6 cells expressing ITIM-mutated Fc gammaRIIB (Fc gammaRIIB(Y292G)), suggesting that ITIM phosphorylation is necessary for Fc gammaRIIB-induced B cell blockade. Overall, we demonstrate that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells is sufficient to transiently down-regulate their activation without inducing apoptosis. Our results suggest that Fc gammaRIIB could negatively regulate IgM(+) B cells before class-switch occurrence and that its unique engagement by immune complexes represents a reversible checkpoint for peripheral IgM(+) B cells.     

T cell responses in melanoma patients after vaccination with tumor-mRNA transfected dendritic cells

We have developed an individualized melanoma vaccine based on autologous dendritic cells (DCs) transfected with autologous tumor-mRNA. The vaccine targets the unique spectrum of tumor antigens in each patient and may recruit multiple T cell clones. In a recent phase I/II trial, we demonstrated T cell responses against vaccine antigens in 9/19 patients evaluable by T cell assays. Here, we report a follow-up study that was conducted to characterize interesting T cell responses and to investigate the effects of long-term booster vaccination. Two patients were selected for continued vaccine therapy. The clinical follow-up suggested a favorable clinical development in both patients. The immunological data (T cell proliferation/IFNgamma ELISPOT/Bioplex cytokine assays) indicated sustained T cell responses and suggested an enhancing effect of booster vaccinations. Both CD4(+) and CD8(+) T cell responses were demonstrated. From post-vaccination samples, we generated 39 T cell clones that responded specifically to stimulation by mRNA-transfected DCs and 12 clones that responded to mock-transfected DCs. These data clearly indicate a two-component vaccine response, against transfected and non-transfected antigens. T cell receptor (TCR) clonotype mapping, performed on 11 tDC-specific clones, demonstrated that 10/11 clones had different TCRs. The results thus indicate a broad spectrum T cell response against antigens encoded by the transfected tumor-mRNA. We generally observed mixed Th1/Th2 cytokine profiles, even in T cell clones that were confirmed to be derived from a single cell. This finding suggests that cytokine patterns after cancer vaccination may be more complex than indicated by the classic Th1/Th2 dichotomy.     

Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine

Dendritic cells (DCs) play an important role in the induction of T cell responses. Fc gammaRs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual Fc gammaRs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that Fc gammaRI and Fc gammaRIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating Fc gammaRs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating Fc gammaRs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.