Rapid Screen for Antiviral T-Cell Immunity with Nanowire Electrochemical Biosensors.

The ability to rapidly assess and monitor patient immune responses is critical for clinical diagnostics, vaccine design, and fundamental investigations into the presence or generation of protective immunity against infectious diseases. Recently, findings on the limits of antibody-based protection provided by B-cells have highlighted the importance of engaging pathogen-specific T-cells for long-lasting and broad protection against viruses and their emergent variants such as in SARS-CoV-2. However, low-cost and point-of-care tools for detecting engagement of T-cell immunity in patients are conspicuously lacking in ongoing efforts to assess and control population-wide disease risk. Currently available tools for human T-cell analysis are time and resource-intensive. Using multichannel silicon-nanowire field-effect transistors compatible with complementary metal-oxide-semiconductor, a device designed for rapid and label-free detection of human T-cell immune responses is developed. The generalizability of this approach is demonstrated by measuring T-cell responses against melanoma antigen MART1, common and seasonal viruses CMV, EBV, flu, as well as emergent pandemic coronavirus, SARS-CoV-2. Further, this device provides a modular and translational platform for optimizing vaccine formulations and combinations, offering quick and quantitative readouts for acquisition and persistence of T-cell immunity against variant-driven pathogens such as flu and pandemic SARS-CoV-2.

Detection of Immunogenic Cell Death in Tumor Vaccination Mouse Model.

Immunogenic cell death (ICD) is a form of regulated cell death that is capable of eliciting an immune response. In cancer, tumor cells undergoing ICD are known to emit damage associated molecular patterns (DAMPs) that are capable of recruiting and activating antigen presenting cells (APCs), which ultimately lead to the activation of an antitumor immune response. Surface translocation of intracellular chaperones such as calreticulin, release of TLR agonists such as high mobility box 1, and the secretion of type I IFN are some of the hallmark features seen in tumors succumbing to ICD. While detection of these molecules is suggestive of ICD induction, which alone does not certify that the treatment is an ICD inducer, an in vivo vaccination assay using injured tumor cells remains to be the gold standard method to functionally verify ICD. This chapter will discuss the necessary steps required to conduct an in vivo vaccination assay, focusing on the preparation of vaccine using treated tumor cells, and how these cells are then utilized in the animal model.

Transimmunization restores immune surveillance and prevents recurrence in a syngeneic mouse model of ovarian cancer.

Ovarian cancer accounts for most deaths from gynecologic malignancies. Although more than 80% of patients respond to first-line standard of care, most of these responders present with recurrence and eventually succumb to carcinomatosis and chemotherapy-resistant disease. To improve patient survival, new modalities must, therefore, target or prevent recurrent disease. Here we describe for the first time a novel syngeneic mouse model of recurrent high-grade serous ovarian cancer (HGSOC), which allows immunotherapeutic interventions in a time course relevant to human carcinomatosis and disease course. Using this model, we demonstrate the efficacy of Transimmunization (TI), a dendritic cell (DC) vaccination strategy that uses autologous and physiologically derived DC loaded with autologous whole tumor antigens. TI has been proven successful in the treatment of human cutaneous T cell lymphoma and we report for the first time its in vivo efficacy against an intra-peritoneal solid tumor. Given as a single therapy, TI is able to elicit an effective anti-tumor immune response and inhibit immune-suppressive crosstalks with sufficient power to curtail tumor progression and establishment of carcinomatosis and recurrent disease. Specifically, TI is able to inhibit the expansion of tumor-associated macrophages as well as myeloid-derived suppressive cells consequently restoring T cell immune-surveillance. These results demonstrate the possible value of TI in the management of ovarian cancer and other intra-peritoneal tumors.

Extracorporeal Photochemotherapy: Mechanistic Insights Driving Recent Advances and Future Directions.

Dendritic cells (DCs) are professional antigen-presenting cells, necessary for the initiation and maintenance of antigen-specific immunity and tolerance. Decades of research have been driven by hopes to harness the immunological capabilities of DCs and achieve physiological partnership with the immune system for therapeutic ends. Potential applications for DC-based immunotherapy include treatments for cancer, autoimmune disorders, and infectious diseases. However, DCs have poor availability in peripheral and lymphoid tissues and have poor survivability in culture, leading to the development of multiple strategies to generate and manipulate large numbers of DCs ex vivo. Among these is Extracorporeal Photopheresis (ECP), a widely used cancer immunotherapy. Recent advancements have uncovered that stimulation of monocyte-to-DC maturation via physiologic inflammatory signaling lies at the mechanistic core of ECP. Here, we describe the landscape of DC-based immunotherapy, the historical context of ECP, the current mechanistic understanding of ex vivo monocyte-to-DC maturation in ECP, and the implications of this understanding on making scientifically driven improvements to modern ECP protocols and devices.

Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes.

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.

Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy.

Generation of large numbers of dendritic cells (DC) for research or immunotherapeutic purposes typically involves in vitro conversion of murine bone marrow precursors or human blood monocytes to DC via cultivation with supraphysiologic concentrations of cytokines such as GM-CSF and IL-4 for up to 7 days. Alternatively, our group has recently established a new approach, based on the underlying mechanism of action of a widely used cancer immunotherapy termed Extracorporeal Photochemotherapy (ECP). Our method of rapid and cytokine-free production of therapeutically relevant DC populations, leveraging the innate physiologic programs likely responsible for DC differentiation from blood monocytes in vivo, potentially offers a novel, inexpensive, and easily accessible source of DC for clinical and research uses. This approach involves ex vivo physiologic reprogramming of blood monocytes to immunologically tunable dendritic antigen-presenting cells, which we term "phDC," for physiological DC. To facilitate access and utilization of these new DC populations by the research community, in this chapter, we describe the use of a scaled-down version of the clinical ECP leukocyte-treatment device termed the Transimmunization (TI) chamber or plate, suitable for processing both mouse and human samples. We highlight the methodological sequences necessary to isolate mouse or human peripheral blood mononuclear cell (PBMC) from whole blood, and to expose those PBMC to the TI chamber for facilitating monocyte activation and conversion to physiological DC (phDC) through interaction with blood proteins and activated platelets under controlled flow conditions. We then provide sample protocols for potential applications of the generated DC, including their use as vaccinating antigen-presenting cells (APC) in murine in vivo antitumor models, and in human ex vivo T-cell stimulation and antigen cross-presentation assays which mimic clinical vaccination. We additionally highlight the technical aspects of loading mouse or human phDC with tumor-associated antigens (TAA) in the form of peptides or apoptotic tumor cells. We provide a simple and clinically relevant means to reprogram blood monocytes into functional APC, potentially replacing the comparatively expensive and clinically disappointing cytokine-derived DC which have previously dominated the dendritic cell landscape.

Ex vivo dendritic cell generation-A critical comparison of current approaches.

Dendritic cells (DCs) are professional antigen-presenting cells, required for the initiation of naïve and memory T cell responses and regulation of adaptive immunity. The discovery of DCs in 1973, which culminated in the Nobel Prize in Physiology or Medicine in 2011 for Ralph Steinman and colleagues, initially focused on the identification of adherent mononuclear cell fractions with uniquely stellate dendritic morphology, followed by key discoveries of their critical immunologic role in initiating and maintaining antigen-specific immunity and tolerance. The medical promise of marshaling these key capabilities of DCs for therapeutic modulation of antigen-specific immune responses has guided decades of research in hopes to achieve genuine physiologic partnership with the immune system. The potential uses of DCs in immunotherapeutic applications include cancer, infectious diseases, and autoimmune disorders; thus, methods for rapid and reliable large-scale production of DCs have been of great academic and clinical interest. However, difficulties in obtaining DCs from lymphoid and peripheral tissues, low numbers and poor survival in culture, have led to advancements in ex vivo production of DCs, both for probing molecular details of DC function as well as for experimenting with their clinical utility. Here, we review the development of a diverse array of DC production methodologies, ranging from cytokine-based strategies to genetic engineering tools devised for enhancing DC-specific immunologic functions. Further, we explore the current state of DC therapies in clinic, as well as emerging insights into physiologic production of DCs inspired by existing therapies.

Extracorporeal photochemotherapy induces bona fide immunogenic cell death.

Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells. However, only a fraction of WBCs is exposed to ECP, and 8-MOP is inactive in the absence of UVA light, implying that other mechanisms underlie the anticancer effects of ECP. Recently, ECP has been shown to enable the physiological differentiation of monocytes into dendritic cells (DCs) that efficiently cross-present tumor-associated antigens (TAAs) to CD8+ T lymphocytes to initiate cognate immunity. However, the source of TAAs and immunostimulatory signals for such DCs remains to be elucidated. Here, we demonstrate that 8-MOP plus UVA light reduces melanoma cell viability along with the emission of ICD-associated danger signals including calreticulin (CALR) exposure on the cell surface and secretion of ATP, high mobility group box 1 (HMGB1) and type I interferon (IFN). Consistently, melanoma cells succumbing to 8-MOP plus UVA irradiation are efficiently engulfed by monocytes, ultimately leading to cross-priming of CD8+ T cells against cancer. Moreover, malignant cells killed by 8-MOP plus UVA irradiation in vitro vaccinate syngeneic immunocompetent mice against living cancer cells of the same type, and such a protection is lost when cancer cells are depleted of calreticulin or HMGB1, as well as in the presence of an ATP-degrading enzyme or antibodies blocking type I IFN receptors. ECP induces bona fide ICD, hence simultaneously providing monocytes with abundant amounts of TAAs and immunostimulatory signals that are sufficient to initiate cognate anticancer immunity.

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP's clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity.

Extracorporeal photochemotherapy (ECP) is a cancer immunotherapy for cutaneous T-cell lymphoma (CTCL) operative in more than 350 centers worldwide. Although its efficacy and favorable safety profile have driven its widespread use, elucidation of its underlying mechanism has been difficult. In this study, we identify the principal contributors to the anticancer immunotherapeutic effects of ECP, with the goal of enhancing potency and broadening applicability to additional malignancies. First, we scaled down the clinical ECP leukocyte-processing device to mouse size. Second, we used that miniaturized device to produce a cellular vaccine that regularly initiated therapeutic antimelanoma immunity. Third, we individually subtracted key factors from either the immunizing inoculum or the treated animal to ascertain their contribution to the in vivo antimelanoma response. Platelet-signaled monocyte-to-dendritic cell (DC) differentiation followed by sorting/processing/presentation of tumor antigens derived from internalized apoptotic tumor cells were absolute requirements. As in clinical ECP, immunogenic cell death of tumor cells was finely titrated by DNA cross-linkage mediated by photoactivated 8-methoxypsoralen (8-MOPA). ECP-induced tumor-loaded DC were effective immunotherapeutic agents only if they were spared exposure to 8-MOPA, indicating that healthy DC are required for ECP. Infusion of responder T cells into naïve tumor-challenged mice established the protective role of stimulated T-cell antitumor immunity. Collectively, these results reveal that selective antitumor effects of ECP are initiated by tumor antigen-loaded, ECP-induced DC, which promote potent collaboration between CD4 and CD8 tumor-specific T cells. These mechanistic insights suggest potential therapeutic applicability of ECP to solid tumors in addition to CTCL.Significance: These findings identify principal cellular contributors to the anticancer immunotherapeutic impact of ECP and suggest this treatment may be applicable to a broad spectrum of immunogenic malignancies. Cancer Res; 78(14); 4045-58. ©2018 AACR.

Quantifying in vivo murine antigen-specific T cell responses without requirement for prior knowledge of antigen identity.

Extracorporeal Photochemotherapy (ECP) is a widely applied anti-cancer immunotherapy for patients with cutaneous T cell lymphoma (CTCL). By using apoptotic malignant cells as a source of patient-specific tumor antigen, it enables clinically relevant and curative anti-CTCL immunity, with potential efficacy in other tumors. Currentmethods to track patient-specific responses are tedious, and new methods are needed to assess putative global immunity. We developed a clinically practical method to assess antigen-specific T cell activation that does not rely on knowledge of the particular antigen, thereby eliminating the requirement for patient-specific reagents. In the OT-I transgenic murine system, we quantified calcium flux to reveal early T cell engagement by antigen presenting cells constitutively displaying a model antigenic peptide, ovalbumin (OVA)-derived SIINFEKL. We detected calcium flux in OVA-specific T cells, triggered by specific T cell receptor engagement by SIINFEKL peptide-loaded DC. This approach led to sensitive detection of antigen-specific calcium flux (ACF) down to a peptide-loading concentration of ∼10-3uM and at a frequency of ∼0.1% OT-I cells among wild-type (WT), non-responding cells. Antigen-specific T cells were detected in spleen, lymph nodes, and peripheral blood after adoptive transfer into control recipient mice. Methods like this for assessing therapeutic response are lacking in patients currently on immune-based therapies, such as ECP, where assessment of clinical response is made by delayed measurement of the size of the malignant clone. These findings suggest an early, practical way to measure therapeutically-induced anti-tumor responses in ECP-treated patients that have been immunized against their malignant cells.

Configuration-dependent Presentation of Multivalent IL-15:IL-15Rα Enhances the Antigen-specific T Cell Response and Anti-tumor Immunity.

Here we report a "configuration-dependent" mechanism of action for IL-15:IL-15Rα (heterodimeric IL-15 or hetIL-15) where the manner by which IL-15:IL-15Rα molecules are presented to target cells significantly affects its function as a vaccine adjuvant. Although the cellular mechanism of IL-15 trans-presentation via IL-15Rα and its importance for IL-15 function have been described, the full effect of the IL-15:IL-15Rα configuration on responding cells is not yet known. We found that trans-presenting IL-15:IL-15Rα in a multivalent fashion on the surface of antigen-encapsulating nanoparticles enhanced the ability of nanoparticle-treated dendritic cells (DCs) to stimulate antigen-specific CD8(+) T cell responses. Localization of multivalent IL-15:IL-15Rα and encapsulated antigen to the same DC led to maximal T cell responses. Strikingly, DCs incubated with IL-15:IL-15Rα-coated nanoparticles displayed higher levels of functional IL-15 on the cell surface, implicating a mechanism for nanoparticle-mediated transfer of IL-15 to the DC surface. Using artificial antigen-presenting cells to highlight the effect of IL-15 configuration on DCs, we showed that artificial antigen-presenting cells presenting IL-15:IL-15Rα increased the sensitivity and magnitude of the T cell response, whereas IL-2 enhanced the T cell response only when delivered in a paracrine fashion. Therefore, the mode of cytokine presentation (configuration) is important for optimal immune responses. We tested the effect of configuration dependence in an aggressive model of murine melanoma and demonstrated significantly delayed tumor progression induced by IL-15:IL-15Rα-coated nanoparticles in comparison with monovalent IL-15:IL-15Rα. The novel mechanism of IL-15 transfer to the surface of antigen-processing DCs may explain the enhanced potency of IL-15:IL-15Rα-coated nanoparticles for antigen delivery.

Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen.

Targeting antigen to dendritic cells (DCs) is a powerful and novel strategy for vaccination. Priming or loading DCs with antigen controls whether subsequent immunity will develop and hence whether effective vaccination can be achieved. The goal of our present work was to increase the potency of DC-based antitumor vaccines by overcoming inherent limitations associated with antigen stability and cross-presentation. Nanoparticles prepared from the biodegradable polymer poly(lactic-co-glycolic acid) have been extensively used in clinical settings for drug delivery and are currently the subject of intensive investigation as antigen delivery vehicles for vaccine applications. Here we describe a nanoparticulate delivery system with the ability to simultaneously carry a high density of protein-based antigen while displaying a DC targeting ligand on its surface. Utilizing a targeting motif specific for the DC-associated surface ligand DEC-205, we show that targeted nanoparticles encapsulating a MART-127-35 peptide are both internalized and cross-presented with significantly higher efficiency than isotype control-coated nanoparticles in human cells. In addition, the DEC-205-labeled nanoparticles rapidly escape from the DC endosomal compartment and do not colocalize with markers of early (EEA-1) or late endosome/lysosome (LAMP-1). This indicates that encapsulated antigens delivered by nanoparticles may have direct access to the class I cytoplasmic major histocompatibility complex loading machinery, overcoming the need for "classical" cross-presentation and facilitating heightened DC stimulation of anti-tumor CD8(+) T-cells. These results indicate that this delivery system provides a flexible and versatile methodology to deliver melanoma-associated antigen to DCs, with both high efficiency and heightened potency.

Optimization of stability, encapsulation, release, and cross-priming of tumor antigen-containing PLGA nanoparticles.

PURPOSE: In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP. METHODS: Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate. RESULTS: Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80 KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate. CONCLUSIONS: Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80 KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses.

Enhanced stimulation of anti-ovarian cancer CD8(+) T cells by dendritic cells loaded with nanoparticle encapsulated tumor antigen.

PROBLEM: Dendritic cell (DC)-based cancer therapies are favored approaches to stimulate anti-tumor T-cell responses. Unfortunately, tolerance to tumor antigens is difficult to overcome. Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) are effective reagents in the delivery of drugs and tumor-associated antigens (TAA). In this study, we assessed the capacity of a PLGA NP-based delivery system to augment CD8 T-cell responses to ovarian cancer TAA. METHOD OF STUDY: Human DC were generated from blood monocytes by conventional in vitro differentiation and loaded with either soluble tumor lysate or NP/lysate conjugates (NPL). These antigen-loaded DC were then used to stimulate autologous CD8(+) T cells. Cytokine production and activation markers were evaluated in the CD8(+) T cells. RESULTS: DC loading with NPL increased cytokine production by stimulated CD8 T cells and induced T-cell expression of cell surface co-stimulatory molecules, typical of anti-tumor immune responses. In contrast, delivery of naked tumor lysate antigens preferentially induced a T-cell profile characteristic of tolerization/exhaustion. CONCLUSION: These findings indicate that delivery of TAA in NP enables DC to efficiently activate anti-tumor CD8(+) T cells. PLGA NP encapsulation of tumor-derived lysate protein antigens is an encouraging new preparative methodology for DC-based vaccination meriting clinical testing.

Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy.

Encapsulation of tumor-associated antigens in polymer nanoparticles (NP) is a promising approach to enhance efficiency of antigen delivery for anti-tumor vaccines. Head and neck squamous carcinoma (HNSCC) cell lines were initially used to generate tumor-associated antigens (TAA)-containing poly (lactic-co-glycolic acid) (PLGA) NP; encapsulation efficiency and release kinetics were profiled. Findings were adopted to entrap fresh tumor lysate from five patients with advanced HNSCC. To test the hypothesis that NP enhance antigen presentation, dendritic cell (DC) produced from patient blood monocyte precursors were loaded with either the un-encapsulated or NP-encapsulated versions of tumor lysates. These were used to stimulate freshly-isolated autologous CD8+ T cells. In four of five patients, anti-tumor CD8+ T cells showed significantly increased immunostimulatory IFN-γ (p=0.071) or decreased immmunoinhibitory IL-10 production (p=0.0004) associated with NP-mediated antigen delivery. The observations represent an enabling step in the production of clinically-translatable, inexpensive, highly-efficient, and personalized polymer-based immunotherapy for solid organ malignancies. FROM THE CLINICAL EDITOR: Enhancing the antigen presentation may be a viable approach to increase the efficiency of tumor cell directed cytotoxicity via immune mechanisms. This study presents an example for this using head and neck cancer cell lines and nanotechnology-based encapsulated antigen presentation to dendritic cells. The observed CD8+ T-cell response was significantly enhanced. This method may pave the way to a highly efficient cancer cell elimination method with minimal to no toxicity.

Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles.

CD8(+) T-cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8(+) T cells against these cell-associated antigens, exogenous antigens must be cross-presented by professional antigen-presenting cells (APCs). While cross-presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein-based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow-derived dendritic cells (BMDCs), the MHC class I presentation of PLGA-encapsulated ovalbumin (OVA) stimulated T cell interleukin-2 secretion at 1000-fold lower concentration than soluble antigen and 10-fold lower than antigen-coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead-associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle-mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and hIL-4.

The in vitro generation of dendritic cells (DCs) from either blood or bone marrow has been accomplished for humans and a number of other species. This ability has facilitated the opportunity to test the efficacy of DC vaccines in various tumor models. The cottontail rabbit papillomavirus (CRPV) model is the most clinically relevant animal model for human papillomavirus (HPV)-associated carcinogenesis. The CRPV model has been used to test various preventative and therapeutic vaccination strategies, and the availability of rabbit DCs would further expand its utility. However, to date, rabbit DCs have not been phenotypically and/or functionally characterized. Here we show that DCs can be generated in vitro from rabbit bone marrow mononuclear cells (BMMCs) cultured in the presence of the human cytokines GM-CSF and IL-4 and matured with lipopolysaccharide (LPS). These cells show upregulation of MHC class II and CD86, as well as downregulation of CD14, do not have non-specific esterase activity, are able to perform receptor-mediated endocytosis, and are potent stimulators of allogeneic T cell proliferation in mixed lymphocyte reactions. The ability to generate rabbit DCs makes it possible to test the efficacy of DC vaccination in the prevention and treatment of CRPV-induced lesions, which may provide useful preclinical data regarding the use of DC vaccines for HPV-associated lesions, including cervical cancer.

Transimmunization, a novel approach for tumor immunotherapy.

This review describes our experience with the development of a novel form of immunotherapy that may represent the first practical and effective means of performing tumor-loaded dendritic cell (DC) immunotherapy. We have modified the highly successful extracorporeal photopheresis (ECP) treatment that has been used in the therapy of cutaneous T cell lymphoma (CTCL). autoimmune disease, transplantation rejection episodes and graft-versus-host disease to enhance its efficacy by the addition of an overnight incubation period. This adaption of ECP is termed "transimmunization (TI)" since the new therapy permits transfer of tumor antigens that have been previously poorly recognized to potent antigen presenting cells where the tumor epitopes can be displayed in the full context of major histocompatibility, co-stimulatory and adhesion molecules. The TI modification of ECP is a practical and safe means of rapidly inducing DC differentiation from peripheral blood monocytes in the presence of apoptotic tumor cells. Uptake of the apoptotic CTCL cells by the immature DC, in the presence of inflammatory cytokines, further drives their maturation into potent antigen presenting cells. Reinfusion of these tumor-loaded DC, that have access to the full spectrum of tumor antigens, has the potential to invoke an anti-tumor immune response in the recipient. Standard ECP has been a very useful form of immunotherapy and a modification of this approach that can enhance its ellicacy and utility should broaden its application to a larger variety of disorders including potentially the treatment of solid tumors and the modulation of the immune response in graft-versus-leukemia and graft-versus-host transplantation regimens. An understanding of the mechanism of ECP and TI will provide the physician with the ability to more finely tune the desired immune response and thereby, provide an enhanced immunotherapy for malignancy and other disorders of immunocompetence.

The growth of cutaneous T-cell lymphoma is stimulated by immature dendritic cells.

In the initial stage of cutaneous T-cell lymphoma (CTCL), proliferating CTCL cells are concentrated in the epidermis in close association with an immature dendritic cell (DC), the Langerhans cell. Because long-term in vitro culture of CTCL cells has proven difficult, the in vivo association with the major antigen-presenting cell (APC) of the epidermis has been postulated to play a role in directly stimulating the clonal T-cell proliferation. We report that CTCL cells can be reproducibly grown in culture for 3 months when cocultured with immature DCs. CTCL cells retain the phenotype and genotype of the initial malignant clone, whereas the APCs are a mixture of immature and mature DCs. CTCL cell and DC survival was dependent on direct membrane contact. Growth was inhibited by antibodies that bound to the T-cell receptor (TCR) or interfered with the interaction of CD40 with its ligand on the CTCL cell. Addition of antibody to CD3 or the clonotypic TCR caused rapid CTCL cell apoptosis followed by engulfment by avidly phagocytic immature DCs and subsequent DC maturation. The opportunity to study CTCL cells and immature DCs for prolonged periods will facilitate studies of tumor cell biology and will allow investigation of the intriguing hypothesis that CTCL cell growth is driven through TCR recognition of class II-presented self-peptides. In addition, the culture of CTCL cells will permit evaluation of therapies in vitro before clinical intervention, thereby improving safety and efficacy.