Recruitment Of Antigen-presenting Cells Research Articles

Co-administration of GM-CSF expressing RNA is a powerful tool to enhance potency of SAM-based vaccines

Self-amplifying mRNAs (SAM)-based vaccines have been shown to induce a robust immune response in various animal species against both viral and bacterial pathogens. Due to their synthetic nature and to the versatility of the manufacturing process, SAM technology may represent an attractive solution for rapidly producing novel vaccines, which is particularly critical in case of pandemic infections or diseases mediated by newly emerging pathogens. Recent published data support the hypothesis that Antigen Presenting Cells (APCs) are responsible for CD8+ T-cell priming after SAM vaccination, suggesting cross-priming as the key mechanism for antigen presentation by SAM vaccines. In our study we investigated the possibility to enhance the immune response induced in mice by a single immunization with SAM by increasing the recruitment of APCs at the site of injection. To enhance SAM immunogenicity, we selected murine granulocyte-macrophage colony-stimulating factor (GM-CSF) as a model chemoattractant for APCs, and developed a SAM-GM-CSF vector. We evaluated whether the use of SAM-GM-CSF in combination with a SAM construct encoding the Influenza A virus nucleoprotein (NP) would lead to an increase of APC recruitment and NP-specific immune response. We indeed observed that the administration of SAM-GM-CSF enhances the recruitment of APCs at the injection site. Consistently with our hypothesis, co-administration of SAM-GM-CSF with SAM-NP significantly improved the magnitude of NP-specific CD8+ T-cell response both in terms of frequency of cytotoxic antigen-specific CD8+ T-cells and their functional activity in vivo. Furthermore, co-immunization with SAM-GM-CSF and SAM-NP provided an increase in protection against a lethal challenge with influenza virus. In conclusion, we demonstrated that increased recruitment of APCs at the site of injection is associated with an enhanced effectiveness of SAM vaccination and might be a powerful tool to potentiate the efficacy of RNA vaccination.

Seminal Plasma Exposures Strengthen Vaccine Responses in the Female Reproductive Tract Mucosae.

HIV-1 sexual transmission occurs mainly via mucosal semen exposures. In the female reproductive tract (FRT), seminal plasma (SP) induces physiological modifications, including inflammation. An effective HIV-1 vaccine should elicit mucosal immunity, however, modifications of vaccine responses by the local environment remain to be characterized. Using a modified vaccinia virus Ankara (MVA) as a vaccine model, we characterized the impact of HIV-1+ SP intravaginal exposure on the local immune responses of non-human primates. Multiple HIV-1+ SP exposures did not impact the anti-MVA antibody responses. However, SP exposures revealed an anti-MVA responses mediated by CD4+ T cells, which was not observed in the control group. Furthermore, the frequency and the quality of specific anti-MVA CD8+ T cell responses increased in the FRT exposed to SP. Multi-parameter approaches clearly identified the cervix as the most impacted compartment in the FRT. SP exposures induced a local cell recruitment of antigen presenting cells, especially CD11c+ cells, and CD8+ T cell recruitment in the FRT draining lymph nodes. CD11c+ cell recruitment was associated with upregulation of inflammation-related gene expression after SP exposures in the cervix. We thus highlight the fact that physiological conditions, such as SP exposures, should be taken into consideration to test and to improve vaccine efficacy against HIV-1 and other sexually transmitted infections.

Recruitment of Antigen Presenting Cells to Skin Draining Lymph Node From HPV16E7-Expressing Skin Requires E7-Rb Interaction.

“High-risk” human papillomaviruses (HPV) infect keratinocytes of squamous epithelia. The HPV16E7 protein induces epithelial hyperplasia by binding Rb family proteins and disrupting cell cycle termination. Murine skin expressing HPV16E7 as a transgene from a keratin 14 promoter (K14.E7) demonstrates epithelial hyperplasia, dysfunctional antigen presenting cells, ineffective antigen presentation by keratinocytes, and production of immunoregulatory cytokines. Furthermore, grafted K14.E7 skin is not rejected from immunocompetent non-transgenic recipient animals. To establish the contributions of E7, of E7-Rb interaction and of epithelial hyperplasia to altered local skin immunity, K14.E7 skin was compared with skin from K14.E7 mice heterozygous for a mutant Rb unable to bind E7 (K14.E7xRbΔL/ΔL mice), that have normoplastic epithelium. Previously, we demonstrated that E7-speicfic T cells do not accumulate in K14.E7xRbΔL/ΔL skin grafts. Here, we further show that K14.E7xRbΔL/ΔL skin, like K14.E7 skin, is not rejected by immunocompetent non-transgenic animals. There were fewer CD11b+ antigen presenting cells in skin draining lymph nodes from animals recipient of K14.E7xRbΔL/ΔL grafts, when compared with animals receiving K14.E7 grafts or K5mOVA grafts. Maturation of migratory DCs derived from K14.E7xRbΔL/ΔL grafts found in the draining lymph nodes is significantly lower than that of K14.E7 grafts. Surprisingly, K14.E7xRbΔL/ΔL keratinocytes, unlike K14.E7 keratinocytes, are susceptible to E7 directed CTL-mediated lysis in vitro. We conclude that E7-Rb interaction and its associated epithelial hyperplasia partially contribute to the suppressive local immune responses in area affected by HPV16E7 expression.

Non-thermal plasma induces immunogenic cell death in vivo in murine CT26 colorectal tumors

ABSTRACTImmunogenic cell death is characterized by the emission of danger signals that facilitate activation of an adaptive immune response against dead-cell antigens. In the case of cancer therapy, tumor cells undergoing immunogenic death promote cancer-specific immunity. Identification, characterization, and optimization of stimuli that induce immunogenic cancer cell death has tremendous potential to improve the outcomes of cancer therapy. In this study, we show that non-thermal, atmospheric pressure plasma can be operated to induce immunogenic cell death in an animal model of colorectal cancer. In vitro, plasma treatment of CT26 colorectal cancer cells induced the release of classic danger signals. Treated cells were used to create a whole-cell vaccine which elicited protective immunity in the CT26 tumor mouse model. Moreover, plasma treatment of subcutaneous tumors elicited emission of danger signals and recruitment of antigen presenting cells into tumors. An increase in T cell responses targeting the colorectal cancer-specific antigen guanylyl cyclase C (GUCY2C) were also observed. This study provides the first evidence that non-thermal plasma is a bone fide inducer of immunogenic cell death and highlights its potential for clinical translation for cancer immunotherapy.

Enhanced antigen presenting and T cell functions during late-phase allergic responses in the lung.

Allergic inflammation is a common feature of asthma and may contribute to both development and perpetuation of disease. The interaction of antigen-presenting cells (APC) with sensitized helper T lymphocytes (TC) producing Th2 cytokines may determine the inflammatory response. Recruitment of APC and TC to the lung during allergic responses has been demonstrated, but functional studies in humans have been limited. This study examined the function of APC and TC accumulating at sites of inflammation after segmental allergen challenge (SAC). Fifteen allergic patients underwent SAC, and cells from bronchoalveolar lavage (BAL) were collected after 24 hours. APC and TC from the blood and BAL were purified based on expression of the monocyte marker, CD14; the plasmacytoid dendritic cell (pDC) marker, BDCA4, identifying neuropilin-1 (NRP1); and the helper T cell marker, CD4. Functional activity was assessed using allergen-induced T cell proliferation. Flow cytometry identified cells expressing CD14 and NRP1. SAC resulted in a 12-fold increase in mononuclear cells having the morphologic appearance of blood monocytes. Most of these cells co-expressed CD14 and NRP1. After saline challenge, BAL mononuclear cells demonstrated little APC function. Following SAC, BAL mononuclear cells showed function equal to pDC from blood and greater than blood monocytes. Purified NRP1+ cells from BAL had even greater function than pDC cells from blood (P = .008). Using consistent sources of APC, enhanced proliferation of TC from lung compared to blood was also demonstrated (P = .002). The marked increase in APC function for allergen-specific TC proliferation during allergic inflammation is largely due to the recruitment of monocytes and dendritic cells. There is also an enhanced response in the lung TC population, consistent with recruitment of allergen-specific T cells. Interactions between recruited APC and TC may occur as an early event promoting allergic airway inflammation.

CCL3 augments tumor rejection and enhances CD8+ T cell infiltration through NK and CD103+ dendritic cell recruitment via IFNγ

ABSTRACTInflammatory chemokines are critical contributors in attracting relevant immune cells to the tumor microenvironment and driving cellular interactions and molecular signaling cascades that dictate the ultimate outcome of host anti-tumor immune response. Therefore, rational application of chemokines in a spatial-temporal dependent manner may constitute an attractive adjuvant in immunotherapeutic approaches against cancer. Existing data suggest that the macrophage inflammatory protein (MIP)-1 family and related proteins, consisting of CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES), can be major determinant of immune cellular infiltration in certain tumors through their direct recruitment of antigen presenting cells, including dendritic cells (DCs) to the tumor site. In this study, we examined how CCL3 in a murine colon tumor microenvironment, CT26, enhances antitumor immunity. We identified natural killer (NK) cells as a major lymphocyte subtype that is preferentially recruited to the CCL3-rich tumor site. NK cells contribute to the overall IFNγ content, CD103+ DC accumulation, and augment the production of chemokines CXCL9 and CXCL10 for enhanced T cell recruitment. We further demonstrate that both soluble CCL3 and CCL3-secreting irradiated tumor vaccine can effectively halt the progression of established tumors in a spatial-dependent manner. Our finding implies an important contribution of NK in the CCL3 – CD103+ DC – CXCL9/10 signaling axis in determining tumor immune landscape within the tumor microenvironment.

Increased migration of antigen presenting cells to newly-formed lymphatic vessels in transplanted kidneys by glycol-split heparin.

BackgroundChronic renal transplant dysfunction is characterized by loss of renal function and tissue remodeling, including chronic inflammation and lymph vessel formation. Proteoglycans are known for their chemokine presenting capacity. We hypothesize that interruption of the lymphatic chemokine–proteoglycan interaction interferes with the lymphatic outflow of leukocytes from the renal graft and might decrease the anti-graft allo-immune response.MethodsIn a rat renal chronic transplant dysfunction model (female Dark-Agouti to male Wistar Furth), chemokines were profiled by qRT-PCR in microdissected tubulo-interstitial tissue. Disruption of lymphatic chemokine–proteoglycan interaction was studied by (non-anticoagulant) heparin-derived polysaccharides in vitro and in renal allografts. The renal allograft function was assessed by rise in plasma creatinine and urea.ResultsWithin newly-formed lymph vessels of transplanted kidneys, numerous CD45+ leukocytes were found, mainly MHCII+, ED-1-, IDO-, HIS14-, CD103- antigen presenting cells, most likely representing a subset of dendritic cells. Treatment of transplanted rats with regular heparin and two different (non-)anticoagulant heparin derivatives revealed worsening of kidney function only in the glycol-split heparin treated group despite a two-fold reduction of tubulo-interstitial leukocytes (p<0.02). Quantitative digital image analysis however revealed increased numbers of intra-lymphatic antigen-presenting cells only in the glycol-split heparin group (p<0.01). The number of intra-lymphatic leukocytes significantly correlates with plasma creatinine and urea, and inversely with creatinine clearance.ConclusionsTreatment of transplanted rats with glycol-split heparin significantly increases the number of intra-lymphatic antigen presenting cells, by increased renal diffusion of lymphatic chemokines, thereby increasing the activation and recruitment of antigen presenting cells towards the lymph vessel. This effect is unwanted in the transplantation setting, but might be advantageous in e.g., dendritic cell vaccination.

The TLR2 Binding Neisserial Porin PorB Enhances Antigen Presenting Cell Trafficking and Cross-presentation

TOLL-like receptor (TLR) ligands activate both innate and adaptive immune cells, while modulating the cellular immune response. The outer membrane protein (OMP) from Neisseria meninigitidis, PorB, is a naturally occurring TLR2 ligand and functions as an adjuvant. Here, we demonstrate that PorB increases the level of OVA in the endo-/lysosomal cellular compartment of BMDCs, increases antigen presenting cell (APC) trafficking to draining lymph nodes, and enhances antigen cross-presentation. PorB is capable of mounting an antigen specific T cell response by efficiently stimulating antigen cross-presentation in vivo and in vitro assessed by BMDC OT-I cocultivation assays. The enhanced antigen cross-presentation and the increased APC recruitment to secondary lymphoid tissues expand the scope of known adjuvant effects of PorB on the immune system. Our findings lead to a better understanding of how TLR-ligand based adjuvants can alter and modulate immune responses.

Abstract B50: Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors

Abstract Development of effective and durable antitumor immunity requires the activation, expansion and maintenance of function of tumor antigen-specific effector T cells. The mechanism of action of recently FDA approved monoclonal antibody (mAb) therapies which target immune checkpoints such as PD-1 and CTLA-4 and uncouple inhibitory pathways from the activation of antigen-specific T cells underlines the critical requirement for antigen-specific priming to elicit potent and long-lasting anti-tumor immunity. However, immune checkpoint blockade results in significant clinical benefit largely in malignancies with high mutational burden, where the tumor itself can initiate T cell priming, but is less effective when used as a single agent among cancers with lower mutational burden, associated typically with a low level of lymphocyte infiltration into the tumor microenvironment (TME). We are evaluating clinical immunotherapy regimens combining immune checkpoint blockade with recombinant live-attenuated double-deleted strains of the intracellular bacterium Listeria monocytogenes (LADD), based on a hypothesis that effective immunotherapy will result from agents that in combination re-polarize the TME to facilitate immune effector cell function, prime functional tumor-specific T cells in the appropriate context, and block inhibitory signaling pathways. Here we show that tumor antigen-expressing LADD therapy enhanced CD8+ T cell effector function, resulting in significant tumor eradication in several preclinical mouse models. LADD treatment in addition induced favorable changes in the TME, as shown by enhanced CD8+ T effector function, recruitment of antigen presenting cells and reduction of regulatory T cells. Treatment regimens combining LADD-based immunotherapy with PD-1 immune checkpoint blockade significantly enhanced antitumor efficacy in CT26, 4T1 and MC38 tumor models. Together these data support the rationale for integrating LADD-based immunotherapy into clinical regimens with immune checkpoint blockade on the basis that TME modification and priming of tumor Ag-specific T cells significantly enhances the activity of mAb therapies blocking T cell inhibitory pathways. Citation Format: Weiwen Deng, Weiqun Liu, Thomas Hudson, Chris S. Rae, Ed Lemmens, Anthony Desbien, Bill Hanson, Pete Lauer, Thomas W. Dubensky, Jr., Meredith Leong. Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B50.

Misbalanced CXCL12 and CCL5 Chemotactic Signals in Vitiligo Onset and Progression

Generalized nonsegmental vitiligo is often associated with the activation of melanocyte-specific autoimmunity. Because chemokines play an important role in the maintenance of immune responses, we examined chemotactic signatures in cultured vitiligo melanocytes and skin samples of early (≤2 months) and advanced (≥6 months) vitiligo. Analysis showed that melanocytes in early lesions have altered expression of several chemotaxis-associated molecules, including elevated secretion of CXCL12 and CCL5. Higher levels of these chemokines coincided with prominent infiltration of the skin with antigen presenting cells (APCs) and T cells. Most of the intralesional APCs expressed the CD86 maturation marker and co-localized with T cells, particularly in early vitiligo lesions. These observations were confirmed by invivo animal studies showing preferential recruitment of APCs and T cells to CXCL12- and CCL5-expressing transplanted melanocytes, immunotargeting of the chemokine-positive cells, continuous loss of the pigment-producing cells from the epidermis, and development of vitiligo-like lesions. Taken together, our studies show that melanocyte-derived CXCL12 and CCL5 support APC and T-cell recruitment, antigen acquisition, and T-cell activation in early vitiligo and reinforce the role of melanocyte-derived CXCL12 and CCL5 in activation of melanocyte-specific immunity and suggest inhibition of these chemotactic axes as a strategy for vitiligo stabilization.

Phase II trial of albumin-bound paclitaxel and granulocyte macrophage colony-stimulating factor as an immune modulator in recurrent platinum resistant ovarian cancer

BackgroundGranulocyte macrophage colony-stimulating factor (GM-CSF) stimulates immunity via recruitment of antigen presenting cells and tumor specific T-cell stimulation. Albumin-bound paclitaxel (nab-paclitaxel) followed by GM-CSF may enhance antitumor responses and prolong remissions in ovarian cancer. Immune phenotypes present before treatment may identify responders to chemo-immunotherapy. MethodsRecurrent platinum-resistant ovarian, peritoneal, or fallopian tube cancer patients received nab-paclitaxel, 100mg/m2 days 1, 8, 15 followed by GM-CSF 250μg days 16–26 every 28days for 6 planned cycles. The primary endpoint was remission duration compared to immediate prior remission. Peripheral blood was evaluated by flow cytometry and interferon-γ ELISPOT. ResultsTwenty-one patients were enrolled. Six patients (29%) achieved a biochemical complete response and 9 (43%) a partial response for an overall response rate of 72%. Median time to progression was 4months and 10% of patients achieved longer remissions than the immediate prior regimen. Median overall survival (OS) was 16.8months. Fewer myeloid derived suppressor cells (MDSC) at enrollment significantly associated with complete response (p=0.05). T-cell responses to IGF1R-p1332–1346 (r=0.827, p=0.0003) and IGF1R-p1242–1256 (r=0.850, p=0.0001) during treatment correlated with time to progression. ConclusionsNab-paclitaxel combined with GM-CSF demonstrated biochemical responses in a majority of patients, although responses were not sustained. This combination did not demonstrate an advantage in OS over prior studies of nab-paclitaxel monotherapy. Agents that modulate MDSC should be studied as potential adjuvants to therapy. Strategies to expand T cells recognizing tumor-associated antigens biologically significant in ovarian cancer should also continue to be investigated.

IL-10 differentially controls the infiltration of inflammatory macrophages and antigen-presenting cells during inflammation.

The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen‐presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte‐derived macrophages we noted differential responses to the immune‐modulatory cytokine IL‐10. In contrast to the suppressive actions of IL‐10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL‐10 may thus have limited impact on development of potentially tissue‐damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune‐mediated membrane dysfunction, treatment failure, and increased morbidity.

An ex vivo model for studying hepatic schistosomiasis and the effect of released protein from dying eggs.

BackgroundWe report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis.Methods and FindingsUsing thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5.ConclusionsThis ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs.

Current concepts on the pathogenesis of type 1 diabetes--considerations for attempts to prevent and reverse the disease.

It may be considered unusual to consider a period of three decades “historical.” Yet, the evolution for our understanding of the natural history and pathogenesis of type 1 diabetes has been greatly advanced by a vast number of studies aimed at validating a model (1), proposed by the late Dr. George Eisenbarth in 1986 (2). As a result of this work, the majority of current conventional wisdom portrays type 1 diabetes as a T cell–mediated autoimmune disease involving the specific destruction of insulin-producing pancreatic β-cells. In this model, persons destined to develop type 1 diabetes are assumed to begin life with a full cadre of β-cells. However, a “triggering” insult, likely environmental, initiates a process involving the recruitment of antigen-presenting cells. Antigen-presenting cells sequester self-antigens released by injured β-cells, followed by their transport to pancreatic lymph nodes where they are subsequently presented to autoreactive T cells. These T cells, rogue constituents brought to life due to genetically driven failures of thymic deletion (i.e., central tolerance) combined with defects in mechanisms designed to induce peripheral immune tolerance, come into play (3). This toxic duo, imparting lack-of-tolerance formation, again in the context of genetic susceptibility, allows for migration of self-reactive T cells to islets, mediating β-cell killing and promoting further inflammation (4). When 85–90% of pancreatic β-cells meet their demise, symptoms of the disease occur. In the final stage of the model, the autoimmune process ends with the complete elimination of β-cells. While this concept still forms the prevailing intellectual dogma for the majority of individuals associated with diabetes care and research today, a series of recent observations has challenged multiple aspects of this long-standing model (5). Many of these evolving concepts will be presented in this Perspective, with a discussion of how our understanding of models of type 1 diabetes pathogenesis …

Role of C-C motif ligand 2 and C-C motif receptor 2 in murine pulmonary graft-versus-host disease after lipopolysaccharide inhalations.

Environmental exposures are a potential trigger of chronic pulmonary graft-versus-host disease (pGVHD) after successful recovery from hematopoietic cell transplant (HCT). We hypothesized that inhalations of LPS, a prototypic environmental stimulus, trigger pGVHD via increased pulmonary recruitment of donor-derived antigen-presenting cells (APCs) through the C-C motif ligand 2 (CCL2)-C-C motif receptor 2 (CCR2) chemokine axis. B10.BR(H2(k)) and C57BL/6(H2(b)) mice underwent allogeneic (Allo) or syngeneic (Syn) HCT with wild-type (WT) C57BL/6, CCL2(-/-), or CCR2(-/-) donors. After 4 weeks, recipient mice received daily inhaled LPS for 5 days and were killed at multiple time points. Allo mice exposed to repeated inhaled LPS developed prominent lymphocytic bronchiolitis, similar to human pGVHD. The increase in pulmonary T cells in Allo mice after LPS exposures was accompanied by increased CCL2, CCR2, and Type-1 T-helper cytokines as well as by monocytes and monocyte-derived dendritic cells (moDCs) compared with Syn and nontransplanted controls. Using CCL2(-/-) donors leads to a significant decrease in lung DCs but to only mildly reduced CD4 T cells. Using CCR2(-/-) donors significantly reduces lung DCs and moDCs but does not change T cells. CCL2 or CCR2 deficiency does not alter pGVHD pathology but increases airway hyperreactivity and IL-5 or IL-13 cytokines. Our results show that hematopoietic donor-derived CCL2 and CCR2 regulate recruitment of APCs to the Allo lung after LPS exposure. Although they do not alter pathologic pGVHD, their absence is associated with increased airway hyperreactivity and IL-5 and IL-13 cytokines. These results suggest that the APC changes that result from CCL2-CCR2 blockade may have unexpected effects on T cell differentiation and physiologic outcomes in HCT.

CD40 ligand and tdTomato-armed vaccinia virus for induction of antitumor immune response and tumor imaging

Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides co-stimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed an oncolytic vaccinia virus expressing hCD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA expressing the tdTomato fluorochrome for detection of virus, potentially important for biosafety evaluation. We show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model, CD40L expression did not add potency to vaccinia oncolysis. Tumors treated with vvdd-mCD40L-tdtomato showed enhanced efficacy in a syngenic mouse model and induced recruitment of antigen-presenting cells and lymphocytes at the tumor site. In summary, oncolytic vaccinia virus coding for CD40L mediates multiple antitumor effects including oncolysis, apoptosis and induction of Th1 type T-cell responses.

Chemokines as Cancer Vaccine Adjuvants.

We are witnessing a new era of immune-mediated cancer therapies and vaccine development. As the field of cancer vaccines advances into clinical trials, overcoming low immunogenicity is a limiting step in achieving full success of this therapeutic approach. Recent discoveries in the many biological roles of chemokines in tumor immunology allow their exploitation in enhancing recruitment of antigen presenting cells (APCs) and effector cells to appropriate anatomical sites. This knowledge, combined with advances in gene therapy and virology, allows researchers to employ chemokines as potential vaccine adjuvants. This review will focus on recent murine and human studies that use chemokines as therapeutic anti-cancer vaccine adjuvants.

Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

Deletion of Parasite Immune Modulatory Sequences Combined with Immune Activating Signals Enhances Vaccine Mediated Protection against Filarial Nematodes

BackgroundFilarial nematodes are tissue-dwelling parasites that can be killed by Th2-driven immune effectors, but that have evolved to withstand immune attack and establish chronic infections by suppressing host immunity. As a consequence, the efficacy of a vaccine against filariasis may depend on its capacity to counter parasite-driven immunomodulation.Methodology and Principal FindingsWe immunised mice with DNA plasmids expressing functionally-inactivated forms of two immunomodulatory molecules expressed by the filarial parasite Litomosoides sigmodontis: the abundant larval transcript-1 (LsALT) and cysteine protease inhibitor-2 (LsCPI). The mutant proteins enhanced antibody and cytokine responses to live parasite challenge, and led to more leukocyte recruitment to the site of infection than their native forms. The immune response was further enhanced when the antigens were targeted to dendritic cells using a single chain Fv-αDEC205 antibody and co-administered with plasmids that enhance T helper 2 immunity (IL-4) and antigen-presenting cell recruitment (Flt3L, MIP-1α). Mice immunised simultaneously against the mutated forms of LsALT and LsCPI eliminated adult parasites faster and consistently reduced peripheral microfilaraemia. A multifactorial analysis of the immune response revealed that protection was strongly correlated with the production of parasite-specific IgG1 and with the numbers of leukocytes present at the site of infection.ConclusionsWe have developed a successful strategy for DNA vaccination against a nematode infection that specifically targets parasite-driven immunosuppression while simultaneously enhancing Th2 immune responses and parasite antigen presentation by dendritic cells.

Endogenous IL-21 regulates pathogenic mucosal CD4 T-cell responses during enhanced RSV disease in mice.

A role for interleukin-21 (IL-21) has recently been found in several diseases, but contribution to mucosal defences has not been described. In BALB/c mice infected with respiratory syncytial virus (RSV), IL-21 depletion had little effect in primary infection. However, depletion of mice during priming with recombinant vaccinia expressing RSV G protein (which primes RSV-specific T helper type 2 cells and causes lung eosinophilia during RSV infection) further exacerbated pathology during RSV challenge, with reduced viral clearance and impaired virus-specific serum antibody responses. This enhancement was accompanied by lymphocyte, neutrophil, and antigen-presenting cell recruitment to the lungs, with increased bronchoalveolar lavage interferon-γ and IL-17 levels. Adoptive transfer of splenic CD4 T cells from depleted mice into naive recipients replicated these effects, indicating that IL-21 mediates its effects via CD4 T cells. Endogenous IL-21, therefore, has potent and specific effects on mucosal antiviral responses, assisting viral clearance, regulating pulmonary T- and B-cell responses, and inhibiting IL-17 production.