Enhanced Ag Presentation Research Articles

Mycobacterium tuberculosis Rv3628 is an effective adjuvant via activation of dendritic cells for cancer immunotherapy

Tumor antigens (Ags) are weakly immunogenic and elicit inadequate immune responses, thus induction of antigen-specific immune activation via the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we examined the effect of Rv3628 from Mycobacterium tuberculosis (Mtb) on activation of DCs and anti-tumor immunity in vivo. Intravenous injection of mice with Rv3628 promoted DC activation of spleen and lymph nodes. More importantly, Rv3628 also induced activation of DCs and enhanced Ag presentation in tumor-bearing mice. In mice bearing ovalbumin (OVA)-expressing tumors, combination treatment with Rv3628 and OVA peptide promoted OVA-specific T cell activation and accumulation of interferon (IFN)-γ and tumor necrosis factor (TNF)-α-producing OT-I and OT-II cells in tumor-draining lymph nodes. Moreover, three different tumor Ags in three different tumor models showed enhanced anti-tumor activity with Rv3628 as adjuvant, including inhibition of growth of OVA-expressing B16 melanoma, CT26 carcinoma, and B16 melanoma tumors, and a synergistic effect with anti-programmed cell death protein 1 (PD-1) antibody treatment. Additionally, potential application against human tumors was indicated by similar activation of human peripheral blood DCs by Rv3628. Taken together, these data demonstrate that Rv3628 could be an effective adjuvant in tumor immunotherapy via enhanced capacity of DC activation and Ag presentation.

Enhancing Antigen Presentation and Inducing Antigen-Specific Immune Tolerance with Amphiphilic Peptides.

Ag-specific immunotherapy to restore immune tolerance to self-antigens, without global immune suppression, is a long-standing goal in the treatment of autoimmune disorders such as type 1 diabetes (T1D). However, vaccination with autoantigens such as insulin or glutamic acid decarboxylase have largely failed in human T1D trials. Induction and maintenance of peripheral tolerance by vaccination requires efficient autoantigen presentation by APCs. In this study, we show that a lipophilic modification at the N-terminal end of CD4+ epitopes (lipo-peptides) dramatically improves peptide Ag presentation. We designed amphiphilic lipo-peptides to efficiently target APCs in the lymph nodes by binding and trafficking with endogenous albumin. Additionally, we show that lipophilic modification anchors the peptide into the membranes of APCs, enabling a bivalent cell-surface Ag presentation. The s.c. injected lipo-peptide accumulates in the APCs in the lymph node, enhances the potency and duration of peptide Ag presentation by APCs, and induces Ag-specific immune tolerance that controls both T cell- and B cell-mediated immunity. Immunization with an amphiphilic insulin B chain 9-23 peptide, an immunodominant CD4+ T cell epitope in NOD mice, significantly suppresses the activation of T cells, increases inhibitory cytokine production, induces regulatory T cells, and delays the onset and lowers the incidence of T1D. Importantly, treatment with a lipophilic β-cell peptide mixture delays progression to end-stage diabetes in acutely diabetic NOD mice, whereas the same doses of standard soluble peptides were not effective. Amphiphilic modification effectively enhances Ag presentation for peptide-based immune regulation of autoimmune diseases.

Hypomethylating Agent Leads to Enhanced Immunogenicity of a Dendritic Cell/Acute Myeloid Leukemia Fusion Vaccine and Prolonged Survival in an Immunocompetent Mouse Model

Immunotherapy for acute myeloid leukemia (AML) holds great promise as a modality to overcome tolerance and elicit memory responses needed for durable remissions. Our group pioneered a novel personalized vaccine that targets a broad array of leukemic antigens (Ags) by fusing whole patient-derived AML cells to autologous dendritic cells (DCs). In a clinical trial of AML patients in chemotherapy-induced remission, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Enhancing response to vaccination relies on increasing Ag presentation and overcoming factors in the microenvironment that contribute to tolerance.While the mechanism of action of hypomethylating agents (HMAs) is multifactorial, HMAs are increasingly recognized as exhibiting immunomodulatory functions thought to contribute to therapeutic efficacy. We have previously shown that the HMA SGI-110 augments AML Ag processing and increases AML susceptibility to T cell-mediated killing. In the present study, we aimed to elucidate the effect of HMAs on enhancing Ag presentation and modulating the AML microenvironment as well as to investigate the impact of HMA on the immunogenicity of a DC/AML vaccine.We first investigated the ability of SGI-110 to reverse critical aspects of the AML immunosuppressive milieu and enhance vaccine efficacy. C57BL/6J mice were retro-orbitally inoculated with 1x105 syngeneic TIB-49 AML cells and treated with low-dose SGI-110 (1mg/kg) to assess immunologic effects without preventing engraftment. AML engraftment in the spleen and bone marrow (BM) was similarly observed in treated and control animals. However, SGI-110-treated animals showed decreased PD-1 expression on CD4 T cell splenocytes (mean 3.1% vs. 6.3%; n=4; p=0.01). SGI-110 therapy was also associated with decreased myeloid-derived suppressor cell (MDSC) burden in the BM (n=5; p=0.01) and spleen (n=4; p=0.03) compared to control.Consistent with these findings, treated mice demonstrated increased AML-specific immunity via increased T cells isolated from the BM or spleen that expressed intracellular IFNϒ following ex-vivo exposure to TIB-49 lysate (mean 3% vs. 0.5%; n=5; p=0.02).We then examined the potential synergy of SGI-110 + vaccine in this model. Mice treated with SGI-110 + vaccine showed a significant increase in BM and spleen CD4 (n=5; p=0.05) and CD8 (n=5; p=0.02) T cell IFNϒ in response to tumor lysate as compared to vaccine or SGI-110 alone.To further elucidate the mechanism behind the immune activating effects of HMAs, we investigated the effect of HMA on the endogenous retroviral (ERV) pathway. Activation of this pathway results in increased IFN ß expression leading to increased MHC HLA Class I expression and subsequently, immune stimulation in a solid tumor model (Chiappinelli et al. Cell . 2015). MOLM-14 cells were exposed to low-dose HMA daily X 3 days and analyzed for ERV-3 expression using western blotting (WB). The results showed significant upregulation of ERV-3 expression after treatment. Due to activation of the ERV pathway, increased IFN ß expression was observed with WB analysis. Indeed, analysis of an HLA-A2 positive human AML cell line, THP1, showed significant HLA-A2 upregulation after HMA exposure. Most importantly, HLA-A2 upregulation led to a significant rise in tumor Ag presentation, detected by measuring the binding capacity of a unique T-cell receptor-like antibody using flow cytometric analysis in THP-1 cells.Given these effects, we sought to examine whether treatment with vaccine + SGI-110 would enhance survival in this model. Mice were treated with: vehicle, SGI-110 X 5 days, vaccine, or both agents. Monotherapy with either agent led to a survival advantage compared to untreated mice (p<0.05). Notably, the combination arm further prolonged survival as compared to untreated mice (p<0.05).In conclusion, SGI-110 modulates critical factors contributing to the immunosuppressive milieu in AML by decreasing T cell PD-1 expression and promoting MDSC expansion, resulting in enhanced anti-AML immunity. Moreover, SGI-110 augments the immune response to an autologous DC/AML vaccine in a murine model resulting in a survival benefit. In addition, HMA enhances Ag presentation, in part due to activation of the ERV pathway. The combination of a novel personalized DC/AML fusion vaccine and HMA holds great potential and a clinical trial is being initiated. DisclosuresAvigan:Genus Oncology: Research Funding.

JNK2 modulates the CD1d-dependent and -independent activation of iNKTcells.

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti-tumor, and anti-microbial immune responses, and are activated by glycolipids presented by the MHC class I-like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)-dependent iNKTcell activation is not well-known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d-mediated Ag presentation in APCs, but also contributes to CD1d-independent iNKTcell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKTcells in a CD1d-independent, but IL-12-dependent manner, we found the virus-induced loss of iNKTcells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild-type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKTcells were found to express less surface IL-12 receptors. As with a VV infection, an IL-12 injection also resulted in a smaller decrease in JNK2 KO iNKTcells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d-mediated Ag presentation and contributes to IL-12-induced iNKTcell activation and loss during viral infections.

Abstract 19: Hypomethylating agent, SGI-110, alters the immunosuppressive milieu in acute myeloid leukemia (AML) and enhances the immunogenicity of a dendritic cell/AML fusion vaccine

Abstract Acute myeloid leukemia (AML) is associated with an immunosuppressive tumor microenvironment (TME) that prevents immune recognition and fosters disease growth. In an attempt to reverse this immune suppression, we developed a personalized vaccine in which patient-derived AML cells are fused with autologous dendritic cells (DCs) such that a broad array of leukemic antigens (Ags) is presented in the context of DC-mediated costimulation. We have examined strategies to enhance vaccine efficacy. In this regard, we previously demonstrated that the hypomethylating agent (HMA), SGI-110, augments leukemic Ag processing and increases AML cell susceptibility to T cell-mediated killing. In the present study, we investigated if SGI-110 enhances leukemic Ag presentation by measuring the binding capacity of a unique T-cell receptor-like (TCRL) antibody (Ab) to AML cells in vitro. The TCRL Ab is a TCR mimic that recognizes the PR1-HLA-A2-MHC complex on the surface of leukemic cells where PR-1 is a known leukemic Ag. Following exposure of SGI-110 to the AML cell line, THP-1 which endogenously expresses HLA-A2, we noted enhanced tumor Ag presentation and HLA-A2 upregulation via FACS analysis. Aside from facilitating Ag presentation, we investigated if SGI-110 affects myeloid-derived suppressor cell (MDSC) burden in the TME where MDSCs have immunosuppressive capabilities. In an in vitro assay where healthy donor-derived peripheral blood mononuclear cells were cocultured with 2 AML cell lines, SGI-110 treatment led to a significant decrease in MDSC expansion compared to control. Based on these in vitro data, we investigated the ability of SGI-110 to reverse critical aspects of the immunosuppressive milieu in AML and enhance vaccine efficacy in vivo. C57BL/6J mice were retro-orbitally inoculated with 1x105 syngeneic TIB-49 AML cells and treated with low-dose SGI-110 (1 mg/kg) to assess immunologic effects while not preventing engraftment. AML engraftment in the spleen and bone marrow (BM) was similarly observed in treated and control animals. However, SGI-110-treated animals showed decreased PD-1 expression on CD4 T cell splenocytes (mean 3.1% vs. 6.3%; n=4; p=0.01). Similarly, SGI-110 therapy was associated with a decrease in MDSC burden in the BM (n=5; p=0.01) and spleen (n=4; p=0.03) compared to control mice. Consistent with these findings, SGI-110-treated mice demonstrated increased AML-specific immunity as manifested by increased T cells isolated from the BM or spleen that expressed intracellular IFNγ following ex vivo exposure to TIB-49 lysate (mean 3% vs. 0.5%; n=5; p=0.02). We subsequently examined the potential synergy of SGI-110 plus the vaccine in this model. Mice treated with SGI-110 + vaccine showed a significant increase in BM and spleen CD4 (n=5; p=0.05) and CD8 (n=5; p=0.02) T cell IFNγ in response to tumor lysate as compared to vaccine or SGI-110 alone. Given these effects, we sought to examine whether treatment with the vaccine and SGI-110 would enhance survival in this model. Mice were treated with vehicle, SGI-110 X 5 days, vaccine, or both agents. Monotherapy with either agent led to a statistically significant survival advantage as compared to untreated mice; yet the combination arm further prolonged survival as compared to either monotherapy arm. In conclusion, SGI-110 enhances Ag presentation and modulates the AML-associated immunosuppressive milieu by decreasing both T-cell PD-1 expression and MDSC burden with a notable increase in AML-specific immunity. Moreover, SGI-110 increases the immunogenicity of the DC/AML vaccine through an enhanced AML-specific response with a survival benefit in a murine model. This combination strategy has promising translational potential and a phase 1 clinical trial with this synergistic approach is planned. Citation Format: Myrna Rita Nahas, Dina Stroopinksy, Anna Sergeeva, Athalia Pyzer, Abigail Washington, Leandra Cole, Salvia Jain, Malgorzata McMasters, James D. Levine, Rebecca Karp Leaf, Shira Orr, Matthew Weinstock, Jeffrey J. Molldrem, David Avigan, Jacalyn Rosenblatt. Hypomethylating agent, SGI-110, alters the immunosuppressive milieu in acute myeloid leukemia (AML) and enhances the immunogenicity of a dendritic cell/AML fusion vaccine [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 19.

Modulation of Protective Efficacy of Fracisella tularensis Immunogens by Differential Cultivation in Mueler Hinton Broth Versus Brain Heart Infusion Medium

Abstract Francisella tularensis (Ft) can survive in a variety of habitats with a remarkable ability to adapt to changing environmental conditions. For example, Ft expresses a distinct set of antigens (Ags) in the macrophage, which are not expressed in Ft grown in vitro with Mueler Hinton Broth (MHB) medium. In contrast, Ft grown in Brain Heart Infusion (BHI) medium exhibits an antigenic profile more closely resembling that of in vivo growth conditions. In addition, studies also suggest an alteration in immunological properties of Ft when grown in BHI versus MHB medium. Thus, we investigated the protective efficacy of BHI and MHB-grown Ft, when inactivated and used as an immunogen to protect against Ft LVS challenge. Inactivated Ft (iFt) generated in MHB (iFt-MHB) was found to have superior protective efficacy as compared to that of the iFt generated in BHI (iFt-BHI). The enhanced protection generated by iFt- MHB-immunized mice was also associated with lower bacterial burden and sepsis-like conditions in the lungs and spleens of iFt-MHB versus iFt-BHI-immunized mice. Further evaluation of early immunological events revealed, in the case of iFt-MHB immunogen, superior Ag binding to Ag-presenting dendritic cells (DCs), increased MHC class II expression by DCs, and enhanced Ag presentation by DCs, as apposed to the use of iFt-BHI. Also, iFt-MHB induced increased levels of Ft-specific IgG as well as IFNg and IL17 production versus that of iFt-BHI. These studies thus demonstrate the critical importance of considering bacterial growth medium in Ft vaccine development.

ERAP1-ERAP2 dimerization increases peptide-trimming efficiency.

The endoplasmic reticulum aminopeptidases (ERAP)1 and ERAP2 play a critical role in the production of final epitopes presented by MHC class I molecules. Formation of heterodimers by ERAP1 and ERAP2 has been proposed to facilitate trimming of epitope precursor peptides, but the effects of dimerization on ERAP function remain unknown. In this study, we produced stabilized ERAP1-ERAP2 heterodimers and found that they produced several mature epitopes more efficiently than a mix of the two enzymes unable to dimerize. Physical interaction with ERAP2 changes basic enzymatic parameters of ERAP1 and improves its substrate-binding affinity. Thus, by bringing the two enzymes in proximity and by producing allosteric effects on ERAP1, dimerization of ERAP1/2 creates complexes with superior peptide-trimming efficacy. Such complexes are likely to enhance Ag presentation by cells displaying coordinated expression of the two enzymes.

Antigen-conjugated human IgE induces antigen-specific T cell tolerance in a humanized mouse model.

Dendritic cells (DCs) play an important role in immune homeostasis through their ability to present Ags at steady state and mediate T cell tolerance. This characteristic renders DCs an attractive therapeutic target for the induction of tolerance against auto-antigens or allergens. Accordingly, Ag-conjugated DC-specific Abs have been proposed to be an excellent vehicle to deliver Ags to DCs for presentation and tolerance induction. However, this approach requires laborious reagent generation procedures and entails unpredictable side effects resulting from Ab-induced crosslinking of DC surface molecules. In this study, we examined whether IgE, a high-affinity, non-cross-linking natural ligand of FcεRI, could be used to target Ags to DCs and to induce Ag-specific T cell tolerance. We found that Ag-conjugated human IgE Fc domain (Fcε) effectively delivered Ags to DCs and enhanced Ag presentation by 1000- to 2500-fold in human FcεRIα-transgenic mice. Importantly, this presentation resulted in a systemic deletion of Ag-specific T cells and prevented these mice from developing delayed-type hypersensitivity, which is critically dependent on Ag-specific T cell immunity. Thus, targeting FcεRI on DCs via Ag-Fcε fusion protein may serve an alternative method to induce Ag-specific T cell tolerance in humans.

Regulation of the Actin Cytoskeleton by Rho Kinase Controls Antigen Presentation by CD1d

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.

Type I IFN drives a distinctive dendritic cell maturation phenotype that allows continued MHC-II synthesis and antigen processing (106.21)

Abstract Microbial molecules or cytokines can stimulate dendritic cell (DC) maturation, which involves DC migration to lymph nodes and enhanced presentation of Ag to launch T cell responses. Toll-like receptor (TLR) agonists are the most studied inducers of DC maturation and promote a burst of Ag processing with enhanced expression of peptide-MHC-II complexes and co-stimulatory molecules. Subsequently, MHC-II localization is shifted from vacuolar compartments to the surface and synthesis is inhibited, thus decreasing Ag processing and limiting presentation to a cohort of Ags kinetically associated with the maturation signal. Type I interferon (IFN-I) also promotes DC maturation, but the phenotypic changes induced by IFN-I are poorly defined. Our studies show that treatment of DC with IFN-I also increases MHC-II Ag presentation, but, in contrast to TLR-induced maturation, allows for continued Ag processing. IFN-I treated DC expressed high levels of MHC-II at the surface as well as in vacuolar compartments, consistent with sustained Ag processing function. Expression of mRNA for MHC-II and CIITA was inhibited in DC treated with TLR agonists, but maintained in DC matured with IFN-I. Similar to TLR agonists, however, IFN-I induced changes in MARCH1 expression and MHC-II ubiquitination. Our findings suggest that IFN-I drives a distinctive DC maturation program that enhances Ag presentation to T cells without a shutdown of Ag processing, allowing continued sampling of Ags for presentation.

Type I IFN Drives a Distinctive Dendritic Cell Maturation Phenotype That Allows Continued Class II MHC Synthesis and Antigen Processing

Microbial molecules or cytokines can stimulate dendritic cell (DC) maturation, which involves DC migration to lymph nodes and enhanced presentation of Ag to launch T cell responses. Microbial TLR agonists are the most studied inducers of DC maturation, but type I IFN (IFN-I) also promotes DC maturation. In response to TLR stimulation, DC maturation involves a burst of Ag processing with enhanced expression of peptide-class II MHC complexes and costimulator molecules. Subsequently, class II MHC (MHC-II) synthesis and expression in intracellular vacuolar compartments is inhibited, decreasing Ag processing function. This limits presentation to a cohort of Ags kinetically associated with the maturation stimulus and excludes presentation of Ags subsequently experienced by the DC. In contrast, our studies show that IFN-I enhances DC expression of MHC-II and costimulatory molecules without a concomitant inhibition of subsequent MHC-II synthesis and Ag processing. Expression of mRNA for MHC-II and the transcription factor CIITA is inhibited in DCs treated with TLR agonists but maintained in cells treated with IFN-I. After stimulation with IFN-I, MHC-II expression is increased on the plasma membrane but is also maintained in intracellular vacuolar compartments, consistent with sustained Ag processing function. These findings suggest that IFN-I drives a distinctive DC maturation program that enhances Ag presentation to T cells without a shutdown of Ag processing, allowing continued sampling of Ags for presentation.

Role of T cells in ovariectomy induced bone loss—revisited

Role of T cells in ovariectomy induced bone loss—revisited

TRAIL/DR5 Plays a Critical Role in NK Cell-Mediated Negative Regulation of Dendritic Cell Cross-Priming of T Cells

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.

Reactive Carbonyls Are a Major Th2-Inducing Damage-Associated Molecular Pattern Generated by Oxidative Stress

Oxidative stress is widespread and entwined with pathological processes, yet its linkage to adaptive immunity remains elusive. Reactive carbonyl (RC) adduction, a common feature of oxidative stress, has been shown to target proteins to the adaptive immune system. Because aldehydes are important mediators of carbonylation, we explored the immunomodulatory properties of model Ags modified by common bioactive aldehyde by-products of oxidative stress: 4-hydroxy-2-nonenal, malondialdehyde, and glycolaldehyde. Ag modification with all three aldehydes resulted in Ag-specific IgG1-dominated responses in adjuvant-free murine immunizations in an RC-dependent manner. The central role of RCs was confirmed, as their reduction into nonreactive groups abrogated all adaptive responses, despite the presence of other well-known aldehyde-driven adducts such as N(ε)-carboxymethyllysine and glycolaldehyde-pyridine. Moreover, Ag-specific Ab responses robustly correlated with the extent of RC adduction, regardless of the means of their generation. T cell responses mirrored the Th2-biased Ab isotypes by Ag-specific splenocyte production of IL-4, IL-5, and IL-13, but not IFN-γ. The RC-induced Th2 response was in sharp contrast to that induced by Th1/Th2 balanced or Th1-biasing adjuvants and was maintained in a range of mouse strains. In vitro studies revealed that RC adduction enhanced Ag presentation with Th2 polarization in the absence of conventional dendritic cell activation. Taken together, these data implicate commonly occurring RC as an important oxidation-derived Th2 immunomodulatory damage-associated molecular pattern with potentially important roles in health and disease.

IFN-α Enhances Peptide Vaccine-Induced CD8+ T Cell Numbers, Effector Function, and Antitumor Activity

Type I IFNs, including IFN-alpha, enhance Ag presentation and promote the expansion, survival, and effector function of CD8(+) CTL during viral infection. Because these are ideal characteristics for a vaccine adjuvant, we examined the efficacy and mechanism of exogenous IFN-alpha as an adjuvant for antimelanoma peptide vaccination. We studied the expansion of pmel-1 transgenic CD8(+) T cells specific for the gp100 melanocyte differentiation Ag after vaccination of mice with gp100(25-33) peptide in IFA. IFN-alpha synergized with peptide vaccination in a dose-dependent manner by boosting relative and absolute numbers of gp100-specific T cells that suppressed B16 melanoma growth. IFN-alpha dramatically increased the accumulation of gp100-specific, IFN-gamma-secreting, CD8(+) T cells in the tumor through reduced apoptosis and enhanced proliferation of Ag-specific CD8(+) T cells. IFN-alpha treatment also greatly increased the long-term maintenance of pmel-1 CD8(+) T cells with an effector memory phenotype, a process that required expression of IFN-alpha receptor on the T cells and IL-15 in the host. These results demonstrate the efficacy of IFN-alpha as an adjuvant for peptide vaccination, give insight into its mechanism of action, and provide a rationale for clinical trials in which vaccination is combined with standard-of-care IFN-alpha therapy for melanoma.

Heat Shock Proteins HSP27 and HSP70 Are Present in the Skin and Are Important Mediators of Allergic Contact Hypersensitivity

Proteomic analysis of murine skin has shown that a variety of heat shock proteins (HSPs) are constitutively expressed in the skin. Using murine allergic contact hypersensitivity as a model, we investigated the role of two heat shock proteins, HSP27 and HSP70, in the induction of cutaneous cell-mediated immune responses. Immunohistochemical examination of skin specimens showed that HSP27 was present in the epidermis and HSP70 was present in both the epidermis and dermis. Inhibition of HSP27 and HSP70 produced a reduction in the 1-fluoro-2,4-dinitrobenzene contact hypersensitivity response and resulted in the induction of Ag-specific unresponsiveness. Treatment of dendritic cell cultures with recombinant HSP27 caused in the up-regulation of IL-1beta, TNF-alpha, IL-6, IL-12p70, and IL-12p40 but not IL-23p19, which was inhibited when Abs to HSP27 were added. The 1-fluoro-2,4-dinitrobenzene-conjugated dendritic cells that had been treated with HSP27 had an increased capacity to initiate contact hypersensitivity responses compared with control dendritic cells. This augmented capacity required TLR4 signaling because neither cytokine production by dendritic cells nor the increased induction of contact hypersensitivity responses occurred in TLR4-deficient C3H/HeJ mice. Our findings indicate that a cascade of events occurs following initial interaction of hapten with the skin that includes increased activity of HSPs, their interaction with TLR4, and, in turn, increased production of cytokines that are known to enhance Ag presentation by T cells. The results suggest that HSPs form a link between adaptive and innate immunity during the early stages of contact hypersensitivity.

Enhancement of DNA Vaccine Potency through Coadministration of CIITA DNA with DNA Vaccines via Gene Gun

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.

C-Reactive Protein Enhances Immunity to Streptococcus pneumoniae by Targeting Uptake to FcγR on Dendritic Cells

C-reactive protein (CRP) is an acute phase reactant with roles in innate host defense, clearance of damaged cells, and regulation of the inflammatory response. These activities of CRP depend on ligand recognition, complement activation, and binding to FcgammaR. CRP binds to phosphocholine in the Streptococcus pneumoniae cell wall and provides innate defense against pneumococcal infection. These studies examine the effect of this early innate defense molecule on the development of Abs and protective immunity to S. pneumoniae. Dendritic cells (DC) initiate and direct the adaptive immune response by integrating innate stimuli with cytokine synthesis and Ag presentation. We hypothesized that CRP would direct uptake of S. pneumoniae to FcgammaR on DC and enhance Ag presentation. CRP opsonization of the R36a strain of S. pneumoniae increased the uptake of bacteria by DC. DC pulsed with untreated or CRP-opsonized R36a were transferred into recipient mice, and Ab responses were measured. In mice challenged with free R36a, CRP opsonization resulted in higher secondary and memory IgG responses to both phosphocholine and pneumococcal surface protein A. Furthermore, mice immunized with DC that had been pulsed with CRP-opsonized R36a showed increased resistance to intranasal infection with virulent S. pneumoniae. The effects of CRP on Ag uptake, Ab responses, and protection from infection all required FcR gamma-chain expression on DC. The results indicate that innate recognition by CRP enhances effective uptake and presentation of bacterial Ags through FcgammaR on DC and stimulates protective adaptive immunity.

CD4 T Cell-Induced, Bid-Dependent Apoptosis of Cutaneous Dendritic Cells Regulates T Cell Expansion and Immune Responses

The fate of dendritic cells (DCs) after Ag presentation may be DC subset-specific and controlled by many factors. The role of activation-induced apoptosis in regulating DC function is not clear. We investigated the fate of cutaneous DCs (cDCs), specifically Langerhans cells (LCs), and observed that they undergo apoptosis after successful Ag presentation to CD4 T cells. Caspase-specific inhibitors revealed that LC lines use a type II apoptosis pathway in response to CD4 T cells. In support of this, BH3-interacting domain (Bid) protein was present at high levels and specifically cleaved in the presence of Ag-specific T cells. Significant resistance to apoptosis by OT-2 CD4 cells was also observed for Bid knockout (KO) LCs in vitro. To test whether Bid was required to regulate LC function in vivo, we measured contact sensitization and topical immunization responses in Bid KO mice and observed markedly enhanced ear swelling and proliferation responses compared with wild-type mice. Furthermore, when Ag-pulsed Bid KO migratory cDCs were inoculated into wild-type recipients, an increase in both the rate and percentage of expanded OT-2 T cells expressing IFN-gamma was observed. Thus, enhanced Ag presentation function was intrinsic to Bid KO cDCs. Therefore, Bid is an important regulator of LC viability and Ag presentation function.

Salmonella escape from antigen presentation can be overcome by targeting bacteria to Fc gamma receptors on dendritic cells.

Dendritic cells (DCs) are professional APCs with the unique ability to activate naive T cells, which is required for initiation of the adaptive immune response against pathogens. Therefore, interfering with DC function would be advantageous for pathogen survival and dissemination. In this study we provide evidence suggesting that Salmonella enterica serovar typhimurium, the causative agent of typhoid disease in the mouse, interferes with DC function. Our results indicate that by avoiding lysosomal degradation, S. typhimurium impairs the ability of DCs to present bacterial Ags on MHC class I and II molecules to T cells. This process could correspond to a novel mechanism developed by this pathogen to evade adaptive immunity. In contrast, when S. typhimurium is targeted to FcgammaRs on DCs by coating bacteria with Salmonella-specific IgG, bacterial Ags are efficiently processed and presented on MHC class I and class II molecules. This enhanced Ag presentation leads to a robust activation of bacteria-specific T cells. Laser confocal microscopy experiments show that virulent S. typhimurium is rerouted to the lysosomal degradation pathway of DCs when internalized through FcgammaR. These observations are supported by electron microscopy studies demonstrating that internalized S. typhimurium shows degradation signs only when coated with IgG and captured by FcgammaRs on DCs. Therefore, our data support a potential role for bacteria-specific IgG on the augmentation of Ag processing and presentation by DCs to T cells during the immune response against intracellular bacteria.