Phagocytic Antigen-presenting Cells Research Articles

Abstract 6438: Next-generation pan-cancer immunotherapy with patient-derived APC

Abstract Harnessing phagocytic antigen presenting cells (APC) and tumor neoantigen-specific T cells represent the ultimate power of immunotherapy against cancer. The promises of phagocytic APCs lie in their abilities of not only initiating phagocytosis towards cancer cells (to “eat off” tumor) but also, through immunogenic antigen presentation, activating neoantigen-specific Tcells that systemically and durably eliminate cancer cells, as well as the establishment of a long-lasting cellular and hormonal immunity that prevents tumor recurrence. However, the efforts of developing APCs -based therapy have been hindered by the current lack of technology that robustly produce APCs from cancer patients, as none of the traditional methods for differentiating DC or macrophage APCs are effective for cancer patients. Through a proprietary process, we have created a unique reagent combo that enables robust differentiation of cancer patients’ peripheral monocytes (cMo) to be potent APC, termed κAPC. κAPC display a unique gene expression profile, separating from classical DC or macrophage, and profound proinflammatory phenotype with enhanced phagocytic capability and elevated immunogenic antigen presentation machinery. In both in vitro and in vivo settings, κAPC demonstrate direct uptake of tumor antigens and presentation to T cells, the latter leading to activation and large expansion of multi-targeting, polyclonal tumor-specific CD4 and CD8 T populations - termed NeoT- from circulating and tumor-infiltrating lymphocytes. Theexpanded CD8 NeoT are highly tumoricidal and once adoptively transferred in vivo, potently eliminated late-stage metastatic solid tumors, whereas CD4 NeoT contribute to immune memory and repolarize a proinflammatory TME favorable for tumor elimination. Key words: κAPC, NeoT, NeoT adoptive cell therapy, κAPC-based cancer vaccine, combination therapies Citation Format: Zhen Bian, Lei Shi, Yuan Liu, harry Stylli. Next-generation pan-cancer immunotherapy with patient-derived APC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6438.

Abstract CT110: Intratumoral injection of SYNB1891, a synthetic biotic designed to activate the innate immune system, demonstrates target engagement in humans including intratumoral STING activation

Abstract Introduction: SYNB1891 is a live, modified strain of the probiotic E. coli Nissle engineered to produce cyclic dinucleotides under hypoxia leading to stimulator of interferon genes (STING)-activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. Methods: This first-in-human study (NCT04167137) is enrolling patients with refractory advanced solid tumors or lymphoma to receive an intratumoral (IT) injection of SYNB1891 on Days 1, 8 and 15 of the first 21-day cycle and then on Day 1 of each subsequent cycle. Dose escalation is planned across 7 cohorts (1x106 - 1x109 live cells) with Arm 1 consisting of escalating doses of SYNB1891 as monotherapy, and Arm 2 in combination with atezolizumab. The primary objective is to determine the single-agent maximum tolerated dose as monotherapy and the recommended Phase 2 dose in combination with atezolizumab. Other objectives include SYNB1891 kinetics in blood and the injected tumor, STING-target engagement as assessed by IT gene expression and serum cytokines, and tumor responses. Results: This interim analysis includes the first 11 patients across 4 cohorts dosed at 1x106, 3x106, 1x107, or 3x107 live cells, with a total of 59 doses administered. The mean (range) age was 56 (25-70); 9 patients were female. There were no dose-limiting toxicities, SYNB1891-related infections or discontinuations due to adverse events. There was one SYNB1891-related serious adverse event in a patient who experienced a grade 2 cytokine release syndrome that resolved within one day, and one patient experienced a grade 2 injection site reaction of erythema which resolved. SYNB1891 was not detected in the blood at 6 or 24 hours after the first dose or intratumorally 7 days following the first dose. Treatment with SYNB1891 demonstrated activation of the STING pathway and target engagement as assessed by upregulation of interferon-stimulated genes (ISG15, IFIT1, IFIt2), chemokines/cytokines (CXCL9, CXCL10, TNFRS18, TNFSF10) and T-cell response genes (GZMA, CD4, PD-L2) in core biopsies obtained pre-dose and 7 days following the third weekly dose. In addition, there was a dose-response increase in serum cytokines at dose levels of 1x107 and 3x107 live cells (IL-6, TNFα, IFNγ, IL-1Ra) at 6 hours post-dose. Durable, stable disease was observed in two patients refractory to prior PD-1/L1 antibodies with vulvar melanoma (1x106 live cells) and small cell lung cancer (1x107 live cells). Conclusions: Repeat IT injection of SYNB1891 as monotherapy is safe and well-tolerated up to at least 3x107 cells and shows evidence of STING pathway target engagement. These data support the continued dose escalation of SYNB1891 as monotherapy, and initiation of Arm 2 in combination with atezolizumab. Citation Format: Filip Janku, Jason J. Luke, Aoife Brennan, Richard Riese, Mary Varterasian, Michael B. Armstrong, Karen L. Kuhn, Anna Sokolovska, James F. Strauss. Intratumoral injection of SYNB1891, a synthetic biotic designed to activate the innate immune system, demonstrates target engagement in humans including intratumoral STING activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT110.

A THP-1 Cell Line-Based Exploration of Immune Responses Toward Heat-Treated BLG.

Allergen recognition and processing by antigen presenting cells is essential for the sensitization step of food allergy. Macrophages and dendritic cells are both phagocytic antigen presenting cells and play important roles in innate immune responses and signaling between the innate and adaptive immune system. To obtain a model system with a homogeneous genetic background, we derived macrophages and dendritic cells from THP-1 monocytes. The difference between macrophages and dendritic cells was clearly shown by differences in their transcription response (microarray) and protein expression levels. Their resemblance to primary cells was analyzed by comparison to properties as described in literature. The uptake of β-lactoglobulin after wet-heating (60°C in solution) by THP-1 derived macrophages was earlier reported to be significantly increased. To analyse the subsequent immune response, we incubated THP-1 derived macrophages and dendritic cells with native and differently processed β-lactoglobulin and determined the transcription and cytokine expression levels of the cells. A stronger transcriptional response was found in macrophages than in dendritic cells, while severely structurally modified β-lactoglobulin induced a more limited transcriptional response, especially when compared to native and limitedly modified β-lactoglobulin. These results show that processing is relevant for the transcriptional response toward β-lactoglobulin of innate immune cells.

Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity

Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.

Abstract 2150: engEx: A novel exosome engineering platform enabling targeted transfer of pharmacological molecules

Abstract Exosomes are natural and abundant nanoscale vesicles for intercellular communication, capable of transferring biological instructions between neighboring and distant cell types. Translational research efforts have focused on exploiting this communication mechanism to deliver exogenous pharmacologic payloads to treat a variety of diseases including cancer. Functionalization of the exosome surface with proteins and peptides is an important strategy to maximize the potential of exosomes as therapeutics. Comparative proteomic analysis (LC/MS) of stringently purified exosomes led to the identification of several highly enriched and unique proteins, including a transmembrane glycoprotein (Protein X, PrX), belonging to the immunoglobulin superfamily. Stable expression of PrX in a producer cell line resulted in a 200-fold increase of PrX on the secreted exosomes. Protein X was extensively characterized and the minimum structural requirements for exosome enrichment were determined. With our engExTM platform, we developed precision engineered exosome therapeutics using PrX as a scaffold to enable high-density exosome surface display of an array of structurally and biologically diverse proteins, including enzymes, antibodies, type I cytokines, and TNF superfamily members. These proteins were genetically fused to PrX and overexpressed in a producer cell. Significantly higher transgene expression on secreted exosomes was achieved compared to conventional scaffolds, including the tetraspanins CD9/CD63/CD81 and LAMP2B. Oligomerization of PrX coupled with avidity effects inherent in exosome surface display resulted in a clear activity advantage compared to free protein. Protein X-mediated display of CD40L on exosomes resulted in a 20-fold potency increase in B cell activation over recombinant CD40L. Furthermore, expression of CD40L redirected exosome uptake from phagocytic antigen presenting cells (APCs) to B cells, demonstrating exosome surface modifications can alter cellular tropism. We also evaluated the functionality of IL-12 tethered to the exosome surface and demonstrated superior tumor retention compared to free cytokine, resulting in robust anti-tumor activity in anti-PD-1 refractory B16F10 tumor models. These results demonstrate the potential of the engExTM platform to generate novel exosome therapeutics. Citation Format: Kevin Dooley, Ke Xu, Sonya Haupt, Nuruddeen Lewis, Rane Harrison, Shelly Martin, Christine McCoy, Chang Ling Sia, Su Chul Jang, Katherine Kirwin, Russell McConnell, Bryan Choi, Adam T. Boutin, Damian Houde, Jorge Sanchez-Salazar, Agata Villiger-Oberbek, Kyriakos D. Economides, John D. Kulman, Sriram Sathyanarayanan. engEx: A novel exosome engineering platform enabling targeted transfer of pharmacological molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2150.

A malaria vaccine adjuvant based on recombinant antigen binding to liposomes

Pfs25 is a malaria transmission-blocking vaccine antigen candidate, but its apparently limited immunogenicity in humans has hindered clinical development. Here, we show that recombinant, his-tagged Pfs25 can be mixed at the time of immunization with pre-formed liposomes containing cobalt-porphyrin-phospholipid (CoPoP), resulting in spontaneous nanoliposome antigen particleization (SNAP). Antigens are stably presented in uniformly-oriented display via his-tag insertion in the CoPoP bilayer, without covalent modification or disruption of antigen conformation. SNAP immunization of mice and rabbits is well tolerated with minimal local reactogenicity and results in orders-of-magnitude higher functional antibody generation compared to other “mix-and-inject” adjuvants. Serum-stable antigen-binding during transit to draining lymph nodes leads to enhanced antigen uptake by phagocytic antigen presenting cells, with subsequent generation of long-lived, antigen-specific plasma cells. Seamless multiplexing with four additional his-tagged Plasmodium falciparum polypeptides induces strong and balanced antibody production, illustrating the simplicity of developing multi-stage particulate vaccines with SNAP immunization.

Submicron-sized hydrogels incorporating cyclic dinucleotides for selective delivery and elevated cytokine release in macrophages

Despite the emerging evidences supporting the potential of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) as a vaccine adjuvant, few properly designed micro-/nanocarriers for the delivery of cyclic dinucleotides have been developed. In this study, we formulated cGAMP within linear polyethyleneimine (LPEI)/hyaluronic acid (HA) hydrogels via inverse water-in-oil (W/O) emulsion/crosslinking. Spherical and cationic LPEI/HA hydrogels (LH gels) with a size of 455.3±3.1nm and a surface charge of 48.7±3.7mV were selectively and efficiently delivered into phagocytic macrophage cells, which are one type of antigen-presenting cells (APCs), but not into non-phagocytic fibroblast cells. LH gels incorporating cGAMP (LH/cGAMP gels) elicited excellent induction of the cytokines interferon-β (IFN-β) and interleukin-6 (IL-6). In particular, the amount of IFN-β released by LH hydrogels was significantly increased by 2.5-fold compared to that released by conventional cationic liposomes, such as Lipofectamine. In addition, fabricated LH gels showed superior biocompatibility in phagocytic cell lines and primary bone marrow-derived macrophages (BMDMs). After intramuscular injection with ovalbumin into C57BL/6 mice, LH/cGAMP gels exhibited significantly elevated levels of anti-ovalbumin total IgG in serum and IFN-β mRNA in spleens. Thus, the newly designed cGAMP-incorporating hydrogels can serve as safe and potent adjuvants for vaccination and immunotherapy. Statement of significanceSince cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) was first found as a second messenger of immune signaling in human systems in February 2013 (Science, 15, 826), several scientific studies have been reported related to the potential of cGAMP as a vaccine adjuvant or additive for immunotherapy. However, only naked cGAMP without carriers were studied via intramuscular or intranasal administration so far. In our study, we first investigated the feasibility of polymeric hydrogels incorporating cGAMP in terms of selective uptake into phagocytic antigen presenting cells (APCs), induction of cytokines, production of target antibodies, and biocompatibility for vaccination and immunotherapy in vitro and in vivo. Therefore, we believe this manuscript would be of great interest to the biomaterial communities especially who are studying immunotherapy.

Identification of Drosophila Yin and PEPT2 as Evolutionarily Conserved Phagosome-associated Muramyl Dipeptide Transporters

NOD2 (nucleotide-binding oligomerization domain containing 2) is an important cytosolic pattern recognition receptor that activates NF-kappaB and other immune effector pathways such as autophagy and antigen presentation. Despite its intracellular localization, NOD2 participates in sensing of extracellular microbes such as Staphylococcus aureus. NOD2 ligands similar to the minimal synthetic ligand muramyl dipeptide (MDP) are generated by internalization and processing of bacteria in hydrolytic phagolysosomes. However, how these derived ligands exit this organelle and access the cytosol to activate NOD2 is poorly understood. Here, we address how phagosome-derived NOD2 ligands access the cytosol in human phagocytes. Drawing on data from Drosophila phagosomes, we identify an evolutionarily conserved role of SLC15A transporters, Drosophila Yin and PEPT2, as MDP transporters in fly and human phagocytes, respectively. We show that PEPT2 is highly expressed by human myeloid cells. Ectopic expression of both Yin and PEPT2 increases the sensitivity of NOD2-dependent NF-kappaB activation. Additionally, we show that PEPT2 associates with phagosome membranes. Together, these data identify Drosophila Yin and PEPT2 as evolutionarily conserved phagosome-associated transporters that are likely to be of particular importance in delivery of bacteria-derived ligands generated in phagosomes to cytosolic sensors recruited to the vicinity of these organelles.

Antitumor Activity of G3139 Lipid Nanoparticles (LNPs)

G3139, an antisense oligodeoxyribonucleotide (ODN) against Bcl-2, contains two CpG dinucleotides and has shown immunostimulatory activities in preclinical studies. It has been suggested that immunoactivation, rather than antisense activity, is primarily responsible for the therapeutic efficacy of G3139. Nanoparticle formulations naturally target phagocytic antigen presenting cells and therefore might enhance the immunological effects of G3139. In this study, a novel formulation of lipid nanoparticles (LNPs) encapsulating G3139 was synthesized and evaluated in mice bearing L1210 subcutaneous tumors. Intravenous injection of G3139-LNPs into mice led to increased serum levels of IL-6 and IFN-gamma, promoted proliferation of natural killer (NK) cells and dendritic cells (DCs), and triggered a strong antitumor immune response in mice. The observed effects were much greater than those induced by free G3139. Correspondingly, the G3139-LNPs more effectively inhibited tumor growth and induced complete tumor regression in some mice. In contrast, free G3139 was ineffective in tumor growth inhibition and did not prolong survival of the tumor-bearing mice. These results suggest that G3139-LNPs are a potential immunomodulatory agent and may have applications in cancer therapy.

Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts

The induction of alloantigen-specific unresponsiveness remains an elusive goal in organ transplantation. Here we identify plasmacytoid dendritic cells (pDCs) as phagocytic antigen-presenting cells essential for tolerance to vascularized cardiac allografts. Tolerizing pDCs acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting pDCs induced the generation of CCR4+ CD4+ CD25+ Foxp3+ regulatory T cells (Treg cells). Depletion of pDCs or prevention of pDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized pDCs induced Treg cell development and prolonged graft survival. Thus, alloantigen-presenting pDCs home to the lymph nodes in tolerogenic conditions, where they mediate alloantigen-specific Treg cell development and allograft tolerance.

Regulation of cytokine production during phagocytosis of apoptotic cells

Loss of self-tolerance and expansion of auto-reactive lymphocytes are the basis for autoimmunity. Apoptosis and the rapid clearance of apoptotic cells by phagocytes usually occur as coordinated processes that ensure regulated cellularity and stress response with non-pathological outcomes. Defects in clearance of apoptotic cells would contribute to the generation of self-reactive lymphocytes, which drive autoimmune disorders such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The IL-12 family of cytokines (IL-12, IL-23, and IL-27) and IL-10 are produced by phagocytic macrophages and play critical roles in the regulation of antigen-presenting cells (APCs) and effector lymphocytes during an immune response to pathogens. Inappropriate expression of these cytokines and their dysregulated activities have been strongly implicated in the pathogenesis of several autoimmune diseases. The production of pro- and anti-inflammatory cytokines by phagocytic APCs is delicately regulated during the ingestion of apoptotic cells as part of an intrinsic mechanism to prevent inflammatory autoimmune reactions. How apoptotic cell-derived signals regulate cytokine production is poorly understood. A recent study by our group demonstrated that phagocytosis of apoptotic cells by activated macrophages results in strong inhibition of IL-12 p35 gene expression by activating a novel transcription repressor, which we named GC-binding protein (GC-BP), through tyrosine dephosphorylation. We are also beginning to understand the molecular mechanisms underlying apoptotic cell-triggered production of IL-10 by phagocytes. These studies will help to elucidate some novel immune regulatory mechanisms and explore the regulation of immune responses to autoantigens with potentials to discover new therapeutic targets for the treatment of autoimmune disorders.

Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines.

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-beta amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.

Immunity to Salmonella from a dendritic point of view.

Dendritic cells (DC) are the key link between innate and adaptive immunity. Features of DC, including their presence at sites of antigen entry, their ability to migrate from peripheral sites to secondary lymphoid organs, and their superior capacity to stimulate naïve T cells places them in this pivotal role in the immune system. DC also produce cytokines, particularly IL-12, upon antigen encounter and can thus influence the ensuing adaptive immune response. As DC are phagocytic antigen-presenting cells located at sites exposed to bacterial invaders, studies have been performed to gain insight into the role of DC in combating bacterial infections. Indeed, studies with Salmonella have shown that DC can internalize and process this bacterium for peptide presentation on MHC-II as well as MHC-I. DC can also act as bystander antigen--presenting cells by presenting Salmonella antigens after internalizing neighbouring cells that have undergone Salmonella-induced apoptotic death. DC also produce IL-12 and TNF-alpha upon Salmonella encounter. Moreover, studies in a murine infection model have shown that splenic DC increase surface expression of co-stimulatory molecules during infection, and DC contain intracellular bacteria. In addition, quantitative changes occur in splenic DC numbers in the early stages of oral Salmonella infection, and this is accompanied by redistribution of the defined DC subsets in the spleen of infected mice. DC from Salmonella-infected mice also produce cytokines and can stimulate bacteria-specific T cells upon ex vivo co-culture. In addition, DC may play a role in the traversal of bacteria from the intestinal lumen. Studying the function of DC during Salmonella infection provides insight into the capacity of this sophisticated antigen-presenting cell to initiate and modulate the immune response to bacteria.

Immunohistology of antigen-presenting cells in vivo: a novel method for serial observation of fluorescently labeled cells.

Dendritic cells and macrophages are phagocytic antigen-presenting cells that bridge the innate and acquired immune systems. The coexistence of subtypes of dendritic cells and macrophages with overlapping properties complicates resolution of their precise roles in an immune response within a given tissue. This report documents a method to identify and observe these cells over time in a living animal and thereby to visualize them during a dynamic immune response. To label potential antigen-presenting cells, fluorescently tagged ovalbumin was injected into the anterior chambers of mouse eyes. Fluorescently tagged antibodies to cell surface proteins were injected to label specific cell types. Intravital fluorescence microscopy with digital image recording was used to visualize the labeled cells in the irises at various times after the injection. The pattern and density (116-148 cells/mm(2)) of cells labeled in vivo by fluorescent ovalbumin or F4/80 antibodies were similar to that identified by conventional wholemount immunostaining for macrophages and dendritic cells. Fluorescent antibodies specific for CD11b, CD11c, CD80, CD86, or major histocompatibility complex (MHC) class II protein each labeled selective populations of cells in vivo. In contrast to conventional histology, in vivo immunohistology permitted serial observations. The phenotype of cells labeled by fluorescent ovalbumin was not the same at 6 (95% CD11c(+)) and 24 hours (24% CD11c(+)) after injection. This method of in vivo immunohistology provides a tool for studying cell kinetics and dynamic interactions that cannot be assessed by conventional immunohistology. Furthermore, it avoids potential artifacts from tissue fixation and may work with antibodies that label cells poorly in vitro.

Evaluation of cationic solid lipid microparticles as synthetic carriers for the targeted delivery of macromolecules to phagocytic antigen-presenting cells

Biodegradable microparticles represent a promising carrier system for the efficient delivery of therapeutic macromolecules to phagocytic professional antigen-presenting cells (APC). Solid lipid microparticles (SLM) consisting of a tripalmitin matrix were prepared using a novel micromixer-based solvent extraction process. A positive surface charge was introduced by the incorporation of cationic lipids into the formulation. All obtained SLM were efficiently phagocytosed by primary macrophages in vitro. Complete intracellular degradation was observed already within 24 h, making SLM a suitable carrier for the immediate delivery of therapeutics to APC. Cationic SLM adsorbed plasmid DNA and bovine serum albumin (BSA) used as a model protein, and triggered the cellular internalization of the macromolecules by phagocytic macrophages. Surprisingly, the cationic SLM also triggered the internalization of these molecules by non-phagocytic 293 cells. This was probably due to the detachment of nanocomplexes formed of cationic lipid and DNA or BSA, respectively, from the surface of DNA- or BSA-loaded SLM and their subsequent uptake into the cells. Transfection efficiency of the DNA-loaded SLM was most pronounced in non-phagocytic cells and was not detected in the macrophage cell line or in primary macrophages. Our further studies revealed that cytotoxic effects of cationic SLM were more pronounced in the phagocytic cells, which could be explained by the very rapid uptake and degradation of the cationic SLM in these cells. In conclusion, SLM may provide a new, efficient means for the immediate intracellular delivery of therapeutic macromolecules into APC. Caution is warranted for cationic carriers, which may accentuate cytotoxic effects in the phagocytic cells.

Receptor-mediated targeting of spray-dried lipid particles coformulated with immunoglobulin and loaded with a prototype vaccine.

Spray-dried lipid-based microparticles (SDLM) serve as a platform for delivery of a wide variety of compounds including peptides, proteins, and vaccines to the respiratory mucosa. In the present study, we assessed the impact of IgG-mediated targeting to phagocytic cells of inactivated influenza virus formulated in SDLM, on subsequent immune responses. SDLM were produced containing inactivated influenza virus strain A/WSN/32/H1N1 (WSN), with or without IgG. Using phagocytic antigen presenting cells (APC) and a T cell hybridoma (TcH) line specific for a dominant influenza virus epitope, we compared the in vitro responses elicited by ligand-formulated (SDLM-IgG-WSN) and non-ligand particles (SDLM-WSN). The effect of including the IgG ligand in the formulation was further characterized by measuring the immune responses of rodents vaccinated with SDLM. SDLM-IgG-WSN were internalized in an Fc receptor (FcR)-dependent manner by phagocytic APC that were then able to effectively present a dominant, class II-restricted epitope to specific T cells. While SDLM-WSN elicited a lower response than administration of plain inactivated virus in saline, the level of the T cell response was restored both in vitro and in vivo by incorporating the APC FcR ligand, IgG, in the SDLM. Incorporation of FcR ligand (IgG) in SDLM restored the limited ability of formulated virus to elicit T-cell immunity, by receptor-mediated targeting to phagocytes.

Delivery of lipids and liposomal proteins to the cytoplasm and Golgi of antigen-presenting cells

Liposomes have the well-known ability to channel protein and peptide antigens into the MHC class II pathway of phagocytic antigen-presenting cells (APCs) and thereby enhance the induction of antibodies and antigen-specific T cell proliferative responses. Liposomes also serve as an efficient delivery system for entry of exogenous protein and peptide antigens into the MHC class I pathway and thus are very efficient inducers of cytotoxic T cell responses. Soluble antigens that are rendered particulate by encapsulation in liposomes are localized both in vacuoles and in the cytoplasm of bone marrow-derived macrophages. Utilizing fluorophore-labeled proteins encapsulated in liposomes we have addressed the question of how liposomal antigens enter the MHC class I pathway. After phagocytosis of the liposomes, the fluorescent liposomal protein and liposomal lipids enter the cytoplasm where they are processed by the proteasome complex. The processed liposomal protein is then transported via the TAP complex into the endoplasmic reticulum and the Golgi complex. Both the liposomal lipids and the liposomal proteins appear to follow the same intracellular route and they are processed as a protein–lipid unit. In the absence of a protein antigen (empty liposomes), there is no organelle-specific localization of the liposomal lipids. In contrast, when a protein is encapsulated in these liposomes, the distribution of the liposomal lipids is dramatically affected and the liposomal lipids localize to the trans-Golgi area. Localization of the protein in the trans-Golgi area requires liposomal lipids. Similarly, for the localization of liposomal lipids in the trans-Golgi area, there is an obligatory requirement for protein. Therefore, the intracellular trafficking patterns of liposomal lipids and liposomal protein are reciprocally regulated. Presence of both liposomal lipids and liposomal protein in the trans-Golgi therefore facilitates the entry of liposomal antigens into the MHC class I pathway. It is also possible that liposomal lipids are presented to T cells via the recently described CD1 pathway for lipid antigens. Because liposome-formulated vaccines have the potential to stimulate antibody as well as cellular immune responses to protein and lipid components, this approach could prove to be extremely useful in designing vaccine strategies.

Potentiality of gelatin microsphere as immunological adjuvant

This paper describes a new attempt to enhance the production of antibody by delivery of an antigen to phagocytic antigen-presenting cells (e.g. macrophages) using gelatin microspheres. A model protein antigen, human gamma globulin (HGG), was incorporated into microspheres composed of gelatin which have an opsonic ability for macrophage phagocytosis. Subcutaneous injection of the microspheres induced the production of HGG-specific IgG antibody in the mouse serum to a great extent compared with that of HGG in soluble form or in Freund's incomplete adjuvant (FIA) form. There was an optimal concentration of cross-linking agent (glutaraldehyde) for the highest production of antibody. When gelatin microspheres were cross-linked at lower concentrations of glutaraldehyde, they were more extensively swollen in an aqueous solution, leading to an increase in the size of hydrated microsphers because of their lower cross-linking densities. The increased size of microspheres caused a decrease in their macrophage phagocytosis, whereas the release rate of HGG from the microspheres increased as the concentration of cross-linking agent became low. The balance of the two factors, the microsphere susceptibility to macrophage phagocytosis and the rate of HGG release, seemed to affect the efficacy of gelatin microspheres to enhance the antibody production. In addition, incorporation of HGG into gelatin microspheres enhanced the delayed-type hypersensitivity reaction. Moreover, the microspheres developed a strong secondary response in comparison with FIA. The gelatin microspheres induced a minimal inflammatory response around the injection site in contrast to FIA. These findings demonstrate that the gelatin microsphere is promising as an adjuvant to enhance both humoral and cellular immune responses to antigen.

Analysis of T lymphocyte reactivity to complex antigen mixtures by the use of proteins coupled to latex beads

This report describes a suitable model for analysing heterogeneous T cell responses to complex foreign antigens using coated polystyrene beads. The advantage of this technique is that it allows the simple removal of detergents from bound antigen so that biochemically separated antigens or crude antigen mixtures can be used. Furthermore, due to the enhanced uptake of latex-bound antigens by phagocytic antigen-presenting cells (APC), very small amounts of antigen will suffice for activation of T cells in vitro. The potential use of this technique to analyse relevant T cell responses to antigens which are difficult to obtain purified in bulk quantities, is discussed.