Cross-presentation Research Articles

Biodegradable polymer, PLGA, is an efficient vehicle for class II MHC-restricted antigen presentation and the induction of DC maturation (106.15)

Abstract The antigen presentation and maturation activities of antigen-encapsulated biodegradable nanoparticles were compared with that of antigen-antibody immune complexes. Dendritic cells (DCs) were allowed to uptake ovalbumin (OVA) via pinocytosis, phagocytosis or endocytosis, and then the levels of MHC-restricted OVA peptide presentation were measured using OVA specific CD4 and CD8 T cells. Poly(D,L-lactic acid-co-glycolic acid, PLGA)-encapsulated OVA (pOVA) and OVA-IgG complexes (OVA-IC) were equally efficient in inducing MHC class I and II-restricted OVA peptide presentation. Comparison of the amount of OVA peptide presentation showed that class II-restricted presentation pathways were robust process compared to class I-restricted presentation pathways. Maturation inducing activities of pOVA and OVA-IC were also examined. pOVA was as efficient as or better compared to OVA-IC in inducing the expression of cell surface molecules such as class II, CD80, CD86, and the secretion of cytokines IL-6, IL-1β and TNF-α. These results show that the biodegradable polymer, PLGA, is an efficient vehicle not only for class II-restricted antigen presentation but also for induction of DC maturation. The present study also shows that in exogenous antigen presentation by DCs, cross presentation is a much weaker phenomenon compared to MHC class II-restricted presentation.

Abstract 1578: MTBhsp70 fusion protein targeting mesothelin augments tumor specific T cell responses and prolongs survival in a murine model of ovarian cancer

Abstract Background and Purpose: There is a clear logic to adjuvant immunotherapy to enhance conventional treatment of ovarian cancer. Mesothelin (MSLN) is a surface glycoprotein expressed at high levels by ovarian cancers and is already a clinical target for immunotherapy. We have created a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting MSLN to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is known as a potent immune activator. This fusion protein binds MSLN on tumor cells and activates antigen presenting cells (APCs) through the interaction of MTBhsp70 with CD40 on APCs, thus promoting tumor antigen presentation and cross presentation and adjuvanting antitumor specific CD4 and CD8 T cell responses. We tested the hypothesis that the MSLN targeted MTBhsp70 fusion protein would improve survival and increase antitumor immune responses. Experimental Procedures: The efficacy of the MSLN targeted fusion protein was evaluated in a syngeneic mouse model of papillary ovarian cancer in immune competent FVB/NJ mice. Mice received 4 intraperitoneal (ip) treatments with experimental or control proteins from 7 days post ip injection of BR5FVB ovarian cancer cells. Survival time was compared from the day of tumor inoculation to that of the onset of disease (clinically evident ascites). In immunological studies, mice were sacrificed 26 days after tumor cell inoculation. Splenocytes were stained for CD3, CD4, CD8, CD25 and Foxp3, and examined by flow cytometry. Splenocytes were stimulated with MSLN or Her2/neu peptide, or mitomycin C-treated BR5FVB cells. IFNα-generated CD3+CD8+ T cells and CD3+CD8+ T Cell-surface exposure of CD107a/b were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo antibody depletion experiments using standard methodologies. Results: MSLN targeted MTBhsp70 fusion protein significantly delayed the onset of disease compared to saline (p=0.0025) or an unfused mixture of the component proteins, MTBhsp70 and scFv(MSLN) (p=0.0149). The survival advantage of the fusion protein treatment was abrogated by CD8 T cell depletion in tumor bearing mice. The fusion protein also reduced the proportion of CD4+CD25+Foxp3 Treg cells (p=0.0159) and increased the proportion of CD3+CD8+ T cells (p=0.0079) in the spleen compared to controls. In preliminary studies, significantly greater CD8 T cell responses were demonstrated in the spleen from the majority of fusion protein-treated mice compared to controls. The MSLN targeted fusion protein was also shown to evoke dendritic cell activation as well as antigen presentation and cross presentation in vitro. Conclusion: MSLN targeted MTBhsp70 fusion protein treatment of ovarian cancer significantly enhances survival and slows tumor growth while augmenting anti-tumor antigen responses in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1578. doi:1538-7445.AM2012-1578

Abstract 1571: Developing antigen-specific Th1 epitope based vaccines by excluding immunosuppressive peptide sequences

Abstract We have determined that IGFBP-2 is immunogenic in ovarian cancer. Our aim is to develop a multi-epitope vaccine that will elicit Th1 immunity to IGFBP-2. Antigen specific Th1 can modulate the tumor microenvironment to enhance cross priming, supporting the proliferation of cytotoxic T cells which are capable of eradicating ovarian cancer cells. Although some clinical benefit has been demonstrated with this strategy, recent ex vivo analyses of human PBMC have described the presence of antigen-specific CD4+ T regulatory cell (Tregs) in cancer patients that were not detected in healthy individuals. Thus, we questioned if we could optimize our vaccine to include only Th1-stimulating epitopes. Using a combined scoring system from five algorithms for predicting class II binding to determine Th epitopes, we identified 14 IGFBP-2 peptides. Th1 immunogenicity (IFN-gamma) and potential immunosuppression (IL-10) was evaluated by ELISPOT for 40 different donors. Twenty-two percent of the donors only responded with IL-10 secretion to any peptide, 22% only responded with IFN-gamma and 53% of the patients had a mixed IFN-gamma and IL-10 response. To determine which peptides would induce a predominantly Th1 response in the greatest number of people, we used a ratio of IFN-gamma to IL-10 and analyzed both the magnitude and frequency of ELISPOT responses for each of peptides using the following algorithm: (corrected mean SPW) x (percent of responding donors). The peptides were then ranked from highest IL-10 response to highest IFN-gamma response. Interestingly, 6 of the 14 peptides demonstrated a preference to secrete IFN-gamma over IL-10 and are only located in the N-terminus (amino acids 1-163) of IGFBP-2; the remaining potentially immunosuppressive peptides are located in the C-terminus (amino acids 164-328). Vaccination with p1-163 in MMTV-neu mice demonstrated a robust Th1 response (p=0.03) and concomitant inhibition of tumor growth by 70% compared to adjuvant only control animals, p164-328-vaccinated or full length protein-vaccinated mice (p<0.001 for all). Vaccination with p164-328 or full length protein did not inhibit tumor growth. These data suggest that more effective vaccines can be designed when both Th1 epitopes and immunosuppressive epitopes are screened simultaneously and epitopes that are most likely to induce robust Th1 responses in the majority of individuals can be identified and included as vaccine components. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1571. doi:1538-7445.AM2012-1571

Glycan based DC targeting strategies that enhance cross-presentation for improved vaccination

Glycan based DC targeting strategies that enhance cross-presentation for improved vaccination

Ubiquitin-receptor CXCR4 promotes cross presentation through facilitated uptake of poly-ubiquitylated antigens

Ubiquitin-receptor CXCR4 promotes cross presentation through facilitated uptake of poly-ubiquitylated antigens

Role of UNC93B1 in the MHC class I cross presentation pathway

Role of UNC93B1 in the MHC class I cross presentation pathway

TAP1-deficiency does not alter atherosclerosis development in Apoe-/- mice.

Antigen presenting cells (APC) have the ability to present both extra-cellular and intra-cellular antigens via MHC class I molecules to CD8+ T cells. The cross presentation of extra-cellular antigens is reduced in mice with deficient Antigen Peptide Transporter 1 (TAP1)-dependent MHC class I antigen presentation, and these mice are characterized by a diminished CD8+ T cell population. We have recently reported an increased activation of CD8+ T cells in hypercholesterolemic Apoe−/− mice. Therefore, this study included TAP1-deficient Apoe−/− mice (Apoe−/−Tap1−/−) to test the atherogenicity of CD8+ T cells and TAP1-dependent cross presentation in a hypercholesterolemic environment. As expected the CD8+ T cell numbers were low in Apoe−/−Tap1−/− mice in comparison to Apoe−/− mice, constituting ∼1% of the lymphocyte population. In spite of this there were no differences in the extent of atherosclerosis as assessed by en face Oil Red O staining of the aorta and cross-sections of the aortic root between Apoe−/−Tap1−/− and Apoe−/− mice. Moreover, no differences were detected in lesion infiltration of macrophages or CD3+ T cells in Apoe−/−Tap1−/− compared to Apoe−/− mice. The CD3+CD4+ T cell fraction was increased in Apoe−/−Tap1−/− mice, suggesting a compensation for the decreased CD8+ T cell population. Interestingly, the fraction of CD8+ effector memory T cells was increased but this appeared to have little impact on the atherosclerosis development.In conclusion, Apoe−/−Tap1−/− mice develop atherosclerosis equal to Apoe−/− mice, indicating a minor role for CD8+ T cells and TAP1-dependent antigen presentation in the disease process.

Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5′ ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism – single crossing CPA – and provide details on alternative mechanisms.

Sec22b in cross-presentation

Sec22b in cross-presentation

Phenotype and functions of conventional dendritic cells are not compromised in aged mice

Aging has profound effects on the immune system, including thymic involution, reduced diversity of the T cell receptor repertoire, reduced effector T cell and B cell function and chronic increase of proinflammatory cytokine production by innate immune cells. The precise effects of aging on conventional dendritic cells (cDC), the main antigen presenting cells of the immune system, however, are not well understood. We found that in aged mice the number of cDC in the spleen and lymph nodes remained stable, whereas the number of cDC in the lungs increased with age. Whereas cDC in mice showed similar cycling kinetics in all organs tested, cDC reconstitution by aged bone marrow precursors was relatively higher than that of their young counterparts. With the exception of CD86, young and aged cDC did not differ in their expression of co-stimulatory molecules at steady state. Most toll-like receptor (TLR) ligands induced comparable upregulation of co-stimulatory molecules CD40, CD86 and B7H1 on young and aged cDC, whereas TLR2 and TLR5 stimulation resulted in reduced upregulation of CD80 and CD86 on aged cDC in vitro. In vivo, influenza infection-induced upregulation of CD86, but not other co-stimulatory molecules, was lower in aged DC. Young and aged DC were equally capable of direct and cross presentation of antigens in vitro. Transcriptome analysis did not reveal any significant difference between young and aged cDC. These data show that unlike T and B cells, the maintenance of cDC throughout the life of a healthy animal is relatively robust during the aging process.

Low Levels of 25-Hydroxyvitamin D Prior to Allogeneic Transplantation Correlate with the Development of Chronic Graft-Versus-Host Disease

Abstract 4063 Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. GVHD is mediated by the activation and expansion of donor derived alloreactive lymphocytes stimulated in part by residual host dendritic cells (DCs) and donor DCs with cross presentation of alloantigens. Vitamin D is a hormone essential for calcium homeostasis and exhibits potent immunomodulatory characteristics. We have demonstrated that vitamin D exposure inhibits DC maturation and DC mediated stimulation of allogeneic T cells. As such, we hypothesized that vitamin D deficiency may result in the enhanced capacity of DCs to induce GVHD. In this study, we performed a retrospective case-control study to evaluate whether monohydroxyvitamin D levels measured in serum samples obtained from patients prior to allogeneic transplantation were predictive of the risk for GVHD. Fifty-three patients aged 18–60 who underwent a myeloablative matched related or matched unrelated donor transplant between December 2000 and December 2009 at Dana Farber Cancer Institute (DFCI) were included in the analysis. 25-OH vitamin D concentrations were measured by an ELISA assay on serum samples that had been cryopreserved between one and three months prior to transplantation. All patients were alive and evaluable at day 100. The median 25-OH vitamin D level was 21.9 ng/mL (range 7.8–45.7). Patients were divided into two cohorts based on a vitamin D level: < 25 ng/mL (32 patients) or ≥25 ng/mL (21 patients). There was no difference between the groups with respect to patient or donor age, donor type (matched related versus unrelated donor), cell source (peripheral blood versus bone marrow), patient-donor sex matching, myeloid versus lymphoid malignancy, risk status, patient/donor CMV status, conditioning regimen, or GVHD prophylaxis regimen. The cumulative incidence of grades II-IV acute GVHD at 100 days was 53.1% in the group with vitamin D level <25 versus 33.3% in the group with vitamin D≥25 (p=0.13). Remarkably, the cumulative incidence (CI) of chronic GVHD (cGVHD) at 2 years in patients with 25-OH vitamin D <25 was 63.8%, compared to 23.8% in patients with vitamin D levels ≥ 25 (p=0.009). Similarly the 2 year CI of extensive cGVHD was significantly greater in patients with vitamin D levels <25 compared to those with ≥25 (54.5% versus 14.3%, p=0.005). This result is consistent in multivariable competing risks model for which patient and transplant related factors mentioned above were adjusted (HR 5.26, p=0.02). [Figure 1][1] represents the CI of cGVHD with death and relapse as a competing risk. Three-year progression-free survival and overall survival were similar between the two groups (51% vs. 47%, p=0.61; and 53% vs. 50%, p=0.57 respectively). In summary, vitamin D deficiency prior to allogeneic transplantation is associated with a significantly increased risk of chronic GVHD. A prospective clinical trial evaluating the use of vitamin D for the prevention of chronic GVHD is planned. ![Figure 1:][2] Figure 1: Cumulative Incidence of cGVHD with Death/Relapse as a Competing Risk Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:yes

Antigen delivery for cross priming via the emulsion vaccine adjuvants

The function of emulsion adjuvants in vaccine antigen delivery remains unclear. To investigate the roles of emulsion adjuvants in cross presentation of exogenous antigens, a series of emulsions were prepared for both in vitro and in vivo studies. Bone marrow-derived dendritic cells (BMDCs) were treated with the adjuvants and analyzed by flow cytometry for the expression of costimulatory molecules. The activation of antigen-specific T cells in vitro was determined with B3Z cells. Antibody secretion in the draining lymph nodes of emulsion adjuvant-treated animals was measured by enzyme-linked immuno-spot (ELISPOT) assays, and antigen-specific proliferation of cells was conducted to examine the roles of emulsion adjuvants in antigen delivery. Data on phagocytosis of adjuvant-treated cells correlated well with the degree of cell death induced by the emulsion adjuvants. Significant inflammatory infiltration and cell death were observed in vivo at the adjuvant injection sites, as demonstrated by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Ovalbumin (OVA)-based ELISPOT assays showed that L121-adjuvant, containing Pluronic L121, induced the most significant cell death also stimulated the strongest antibody-producing response in the draining lymph nodes, consistent with the data on the proliferation of antigen-specific T cells and activation of B3Z cells in vitro. Results presented in this study have demonstrated the roles of emulsion adjuvants in induction of cell death and delivery of exogenous antigens for cross-priming, leading to stimulation of antigen-specific immune responses.

In vitro Ag Cross-presentation and in vivo Ag Cross-presentation by Dendritic Cells in the Mouse.

Antigen cross presentation is important for effective immune responses to tumors and viral infections. Dendritic cells are professional antigen presenting cells and are unique in their ability to cross-present exogenous antigens on MHC class I molecules and activate antigen specific cytotoxic T cells. This protocol describes antigen cross presentation by dendritic cells (DCs) (bone marrow derived DCs and splenic DCs) in an in vitro and in an in vivo assay system using soluble ovalbumin protein.

The Tricky Balancing Act of using Heat Shock Proteins for Cross-Presentation

The Tricky Balancing Act of using Heat Shock Proteins for Cross-Presentation

The Role of Heat Shock Proteins in Antigen Cross Presentation

Heat shock proteins (HSPs) are molecular chaperones that bind tumor antigens and mediate their uptake into antigen presenting cells. HSP–antigen complexes are then directed toward either the MHC class I pathway through antigen cross presentation or the conventional class II pathway, leading to activation of T cell subsets. Uptake of HSP-chaperoned polypeptides can involve both receptor-mediated and receptor-independent routes, and mechanisms of antigen sorting between the Class I and II pathways after uptake are currently under investigation. The processes involved in internalization of HSP–antigen complexes differ somewhat from the mechanisms previously determined for (unchaperoned) particulate and free soluble antigens. A number of studies show that HSP-facilitated antigen cross presentation requires uptake of the complexes by scavenger receptors (SR) followed by processing in the proteasome, and loading onto MHC class I molecules. In this review we have examined the roles of HSPs and SR in antigen uptake, sorting, processing, cell signaling, and activation of innate and adaptive immunity.

LAH4 enhances CD8+ T cell immunity of protein/peptide-based vaccines

It is now clear that CD8+ T cells are crucial for therapeutic immunity against chronic viral infections and/or tumors. We reason that a strategy capable of improving CD8+ T cell activation would improve the efficacy of protein-based vaccines, which predominantly generate CD4+ T cell-mediated responses. Herein, we explore the ability of a novel cell-penetrating peptide (CPP), LAH4, to facilitate intracellular delivery of protein-based vaccines adjuvanted with Toll-like receptor 9 agonist CpG oligonucleotide (CpG) to generate enhanced CD8+ T cell immune responses and antitumor effects. LAH4 was found to mediate the intracellular delivery of both protein and nucleotide cargo and facilitate protein internalization using mechanisms involving endosomal acidification and processing through the proteasome pathway, leading to enhanced cross presentation of protein antigen by dendritic cells to CD8+ T cells. LAH4 also improved the internalization of CpG, resulting in NFkB activation, thus potentiating the adjuvant effect of CpG. We found that protein-based vaccine comprised of LAH4 mixed with model antigen and CpG generated significantly improved antigen-specific CD8+ T cell immune responses and/or antitumor effects. Furthermore, we found that LAH4 was able to enhance the ability of a tyrosinase-related protein 2 (TRP-2) peptide-based vaccine to generate TRP2-specific CD8+ T cells and antitumor effects against TRP2-expressing tumors. Thus, our results suggest that CPP technology using LAH4 is able to enhance both protein-based and peptide-based vaccine potency to generate antigen-specific CD8+ T cells and antitumor effects. Our findings serve as an important foundation for future clinical applications of CPP technology to improve protein/peptide-based vaccine potency.

Low Levels of 25-Hydroxyvitamin D Prior to Allogeneic Transplantation Correlate with the Development of Chronic Graft-Versus-Host Disease,

Abstract 4063Graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. GVHD is mediated by the activation and expansion of donor derived alloreactive lymphocytes stimulated in part by residual host dendritic cells (DCs) and donor DCs with cross presentation of alloantigens. Vitamin D is a hormone essential for calcium homeostasis and exhibits potent immunomodulatory characteristics. We have demonstrated that vitamin D exposure inhibits DC maturation and DC mediated stimulation of allogeneic T cells. As such, we hypothesized that vitamin D deficiency may result in the enhanced capacity of DCs to induce GVHD. In this study, we performed a retrospective case-control study to evaluate whether monohydroxyvitamin D levels measured in serum samples obtained from patients prior to allogeneic transplantation were predictive of the risk for GVHD. Fifty-three patients aged 18–60 who underwent a myeloablative matched related or matched unrelated donor transplant between December 2000 and December 2009 at Dana Farber Cancer Institute (DFCI) were included in the analysis. 25-OH vitamin D concentrations were measured by an ELISA assay on serum samples that had been cryopreserved between one and three months prior to transplantation. All patients were alive and evaluable at day 100. The median 25-OH vitamin D level was 21.9 ng/mL (range 7.8–45.7). Patients were divided into two cohorts based on a vitamin D level: < 25 ng/mL (32 patients) or ≥25 ng/mL (21 patients). There was no difference between the groups with respect to patient or donor age, donor type (matched related versus unrelated donor), cell source (peripheral blood versus bone marrow), patient-donor sex matching, myeloid versus lymphoid malignancy, risk status, patient/donor CMV status, conditioning regimen, or GVHD prophylaxis regimen. The cumulative incidence of grades II-IV acute GVHD at 100 days was 53.1% in the group with vitamin D level <25 versus 33.3% in the group with vitamin D≥25 (p=0.13). Remarkably, the cumulative incidence (CI) of chronic GVHD (cGVHD) at 2 years in patients with 25-OH vitamin D <25 was 63.8%, compared to 23.8% in patients with vitamin D levels ≥ 25 (p=0.009). Similarly the 2 year CI of extensive cGVHD was significantly greater in patients with vitamin D levels <25 compared to those with ≥25 (54.5% versus 14.3%, p=0.005). This result is consistent in multivariable competing risks model for which patient and transplant related factors mentioned above were adjusted (HR 5.26, p=0.02). Figure 1 represents the CI of cGVHD with death and relapse as a competing risk. Three-year progression-free survival and overall survival were similar between the two groups (51% vs. 47%, p=0.61; and 53% vs. 50%, p=0.57 respectively). In summary, vitamin D deficiency prior to allogeneic transplantation is associated with a significantly increased risk of chronic GVHD. A prospective clinical trial evaluating the use of vitamin D for the prevention of chronic GVHD is planned. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Induction and maintenance of protective CD8+ T cells against malaria liver stages: implications for vaccine development

CD8+ T cells against malaria liver stages represent a major protective immune mechanism against infection. Following induction in the peripheral lymph nodes by dendritic cells (DCs), these CD8+ T cells migrate to the liver and eliminate parasite infected hepatocytes. The processing and presentation of sporozoite antigen requires TAP mediated transport of major histocompatibility complex class I epitopes to the endoplasmic reticulum. Importantly, in DCs this process is also dependent on endosome-mediated cross presentation while this mechanism is not required for epitope presentation on hepatocytes. Protective CD8+ T cell responses are strongly dependent on the presence of CD4+ T cells and the capacity of sporozoite antigen to persist for a prolonged period of time. While human trials with subunit vaccines capable of inducing antibodies and CD4+ T cell responses have yielded encouraging results, an effective anti-malaria vaccine will likely require vaccine constructs designed to induce protective CD8+ T cells against malaria liver stages.

292. Direct Presentation, Not Cross Presentation of AAV Capsids, Render Hepatocytes Target for CTLs in a Humanized Mouse Model

292. Direct Presentation, Not Cross Presentation of AAV Capsids, Render Hepatocytes Target for CTLs in a Humanized Mouse Model