Natural Antigen-presenting Cells Research Articles

Shape matters: Biodegradable anisotropic nanoparticle artificial antigen presenting cells for cancer immunotherapy.

Artificial antigen presenting cells are biomimetic particles that recapitulate the signals presented by natural antigen presenting cells in order to stimulate T cells in an antigen-specific manner using an acellular platform. We have engineered an enhanced nanoscale biodegradable artificial antigen presenting cell by modulating particle shape to achieve a nanoparticle geometry that allows for increased radius of curvature and surface area for T cell contact. The non-spherical nanoparticle artificial antigen presenting cells developed here have reduced nonspecific uptake and improved circulation time compared both to spherical nanoparticles and to traditional microparticle technologies. Additionally, the anisotropic nanoparticle artificial antigen presenting cells efficiently engage with and activate T cells, ultimately leading to a marked anti-tumor effect in a mouse melanoma model that their spherical counterparts were unable to achieve. STATEMENT OF SIGNIFICANCE: Artificial antigen presenting cells (aAPC) can activate antigen-specific CD8+ T cells but have largely been limited to microparticle-based platforms and ex vivo T cell expansion. Although more amenable to in vivo use, nanoscale aAPC have traditionally been ineffective due to limited surface area available for T cell interaction. In this work, we engineered non-spherical biodegradable nanoscale aAPC to investigate the role of particle geometry and develop a translatable platform for T cell activation. The non-spherical aAPC developed here have increased surface area and a flatter surface for T cell engagement and, therefore, can more effectively stimulate antigen-specific T cells, resulting in anti-tumor efficacy in a mouse melanoma model.

Dendritic cell-mimicking scaffolds for ex vivo T cell expansion.

We propose an ex vivo T cell expansion system that mimics natural antigen-presenting cells (APCs) for adoptive cell therapy (ACT). Microfiber scaffolds coated with dendritic cell (DC) membrane replicate physicochemical properties of dendritic cells specific for T cell activation such as rapid recognition by T cells, long duration of T cell tethering, and DC-specific co-stimulatory cues. The DC membrane-coated scaffold is first surface-immobilized with T cell stimulatory ligands, anti-CD3 (αCD3) and anti-CD28 (αCD28) antibodies, followed by adsorption of releasable interleukin-2 (IL-2). The scaffolds present both surface and soluble cues to T cells ex vivo in the same way that these cues are presented by natural APCs in vivo. We demonstrate that the DC-mimicking scaffold promotes greater polyclonal expansion of primary human T cells as compared to αCD3/αCD28-functionalized Dynabead. More importantly, major histocompatibility complex molecules derived from the DC membrane of the scaffold allow antigen-specific T cell expansion with target cell-specific killing ability. In addition, most of the expanded T cells (∼97%) can be harvested from the scaffold by density gradient centrifugation. Overall, the DC-mimicking scaffold offers a scalable, modular, and customizable platform for rapid expansion of highly functional T cells for ACT.

Abstract B058: Rapid and controlled T-cell expansion using scaffolds that mimic antigen-presenting cells

Abstract Current approaches to expand T-cells ex vivo for therapeutic applications are limited by low expansion rates and often result in unpredictable expansion products of varying phenotype and function. Key attributes, such as a balanced ratio of CD4-to-CD8 T-cells, or a high central memory T-cell fraction are known to correlate with robust and durable therapeutic responses but remain difficult to reproducibly achieve. Strategies that allow one to precisely control the phenotype of expanded T-cell products may thus potentiate the effects of CAR-T-cell therapies, and understanding how T-cells respond to stimulation is a crucial step to this end. Recently, we described a 3D expansion system that mimics natural antigen presenting cells (APCs) and promotes greater polyclonal expansion of primary human T-cells than commercial expansion beads (Dynabeads). These biodegradable, APC-mimetic scaffolds (APC-ms) are composed of fluid lipid bilayers supported on mesoporous silica micro-rods, which enables precise control over the spatial organization of membrane-bound T-cell receptor (TCR) stimulatory and costimulatory cues (i.e., anti-CD3/anti-CD28). Here, we investigated the activation and memory signature of primary human T-cells over time in response to APC-ms with different densities of activating surface cues compared to Dynabead stimulation. Higher surface cue densities of anti-CD3/anti-CD28 promoted greater proportions of central memory T-cells, with a parallel increase in the CD4-to-CD8 T-cell ratio. T-cells stimulated with Dynabeads exhibited higher expression of CD25 and CD69 that peaked earlier than standard APC-ms formulations, suggesting that APC-ms promotes relatively more transient T-cell activation than Dynabeads, potentially mitigating overstimulation and exhaustion. Overall, these data demonstrate that APC-ms can be used to rapidly expand primary human T-cells and tune their phenotypic attributes (e.g., CD4-to-CD8 ratio and memory subpopulations). This technology has the potential to improve the production of T-cells for adoptive T-cell-based therapies. Citation Format: David K.Y. Zhang, Anna Vaynrub, David J. Mooney. Rapid and controlled T-cell expansion using scaffolds that mimic antigen-presenting cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B058.

Characterizing the Immunogenicity of DM-Sensitive and DM-Resistant Antigens

Introduction:The separation of graft-versus-leukemia (GvL) effect from graft-versus-host-disease (GvHD) is a major objective after allogeneic stem cell transplantation. We recently described two types of endogenous HLA class II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, presentation of DM-sensitive antigens rely on co-expression of HLA-DO - the natural inhibitor of HLA-DM. Since the expression of HLA-DO is not upregulated by inflammatory cytokines and restricted to B-cells, dendritic cells and thymic epithelial cells, DM-sensitive antigens cannot be presented on non-hematopoietic tissues. Therefore, usage of CD4 T-cells directed against DM-sensitive antigens might allow separation of GvL from GvHD. However, it remains elusive whether immunogenicity and anti-tumorigenic potential of DM-sensitive and DM-resistant antigens have comparable properties in vivo.Methods:Therefore, we sought to create an in vivo system using a DM-sensitive and a DM-resistant variant of the same model antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM or HLA-DO, respectively) to allocate the two model antigens ovalbumin (OVA) and murine Y-chromosome antigen DBY to their category. Furthermore, we introduced one to three amino acid substitutions within the MHC II restricted T-cell epitopes of the two antigens and tested DM-sensitivity or DM-resistance by T-cell activation using proliferation and IFN-g secretion as read-out in vitro. Finally, we vaccinated B6 mice with the generated epitope variants and measured expansion, phenotype and reactivity of OVA- or DBY-specific CD4 T-cells in vivo.Results:By testing T-cell recognition of OVA or DBY on murine B-cell lines overexpressing H2-M and H2-O, respectively, we could show that OVA leads to a more potent T-cell activation in the presence of H2-O demonstrating its DM-sensitive character. In contrast the wildtype epitope of DBY does not rely on H2-O expression for strong T-cell activation and was therefore assessed as DM-resistant antigen. By introducing one to three amino acid substitutions within the T-cell epitope we could generate one further DM-sensitive variant of OVA but also two DM-resistant counterparts. Likewise, we designed both DM-resistant and DM-sensitive epitope variants of murine DBY. To assess T-cell receptor avidity to our epitope variants presented on natural antigen presenting cells, titration of DM-sensitive and DM-resistant variants of the same antigen on untreated splenocytes from OVA or DBY T-cell receptor transgenic mice, respectively, were performed. We observed comparable activation of the same T-cell clone activated by either variant of the epitope as measured by proliferation and IFN-g secretion. Furthermore, upon vaccination of B6 mice with either variant of the epitope we could measure comparable expansion, phenotype, and reactivity of OVA- and DBY-specific T-cells both invivo and ex vivo.Conclusion:We successfully generated DM-sensitive and DM-resistant variants of the same epitope for the two model antigens OVA and murine DBY. With this tool we could demonstrate that DM-sensitive antigens are not inferior to their DM-resistant counterpart. Therefore, targeting DM-sensitive antigens after allogenic stem cell transplantation might be an interesting tool to improve the GvL effect with only limited GvHD. DisclosuresBernhardt:DFG TRR221/project A1 (German Research Foundation): Research Funding.

Engineering nanomaterials to address cell-mediated inflammation in atherosclerosis.

Atherosclerosis is an inflammatory disorder with a pathophysiology driven by both innate and adaptive immunity and a primary cause of cardiovascular disease (CVD) worldwide. Vascular inflammation and accumulation of foam cells and their products induce maturation of atheromas, or plaques, which can rupture by metalloprotease action, leading to ischemic stroke or myocardial infarction. Diverse immune cell populations participate in all stages of plaque maturation, many of which directly influence plaque stability and rupture via inflammatory mechanisms. Current clinical treatments for atherosclerosis focus on lowering serum levels of low-density lipoprotein (LDL) using therapeutics such as statins, administration of antithrombotic drugs, and surgical intervention. Strategies that address cell-mediated inflammation are lacking, and consequently have recently become an area of considerable research focus. Nanomaterials have emerged as highly advantageous tools for these studies, as they can be engineered to target specific inflammatory cell populations, deliver therapeutics of wide-ranging solubilities and enhance analytical methods that include imaging and proteomics. Furthermore, the highly phagocytic nature of antigen presenting cells (APCs), a diverse cell population central to the initiation of immune responses and inflammation, make them particularly amenable to targeting and modulation by nanoscale particulates. Nanomaterials have therefore become essential components of vaccine formulations and treatments for inflammation-driven pathologies like autoimmunity, and present novel opportunities for immunotherapeutic treatments of CVD. Here, we review recent progress in the design and use of nanomaterials for therapeutic assessment and treatment of atherosclerosis. We will focus on promising new approaches that utilize nanomaterials for cell-specific imaging, gene therapy and immunomodulation.

Erythrophagocytosis by plasmacytoid dendritic cells and monocytes is enhanced during inflammation.

Generation of antibodies against red blood cell (RBC) antigens can be a clinically significant problem. The underlying mechanisms that regulate the production of RBC antibodies are only partially understood; however, factors such as inflammation significantly increase the rates of RBC antibody generation. Humoral alloimmunization begins with consumption of transfused RBCs by antigen-presenting cells (APCs). Recently, it has become appreciated that there are multiple different types of APCs. The relative contribution of APC subsets to RBC antibodies has not been described in either the quiescent or the inflamed states. To evaluate the types of APCs that consume RBCs, and how inflammation affects this process, C56Bl/6 mice were treated with polyinosinic-polycytidylic acid (poly(I:C)) to induce an inflammatory response and/or were transfused with 3,3'-dihexadecyloxacarbocyanine perchlorate-labeled syngeneic RBCs. Erythrophagocytosis (both at baseline and during inflammation) was analyzed for different subsets of macrophages (MΦ), dendritic cells (DCs), B cells, and monocytes, by a combined approach using flow cytometry and fluorescent microscopy technology. In four independent experiments, erythrophagocytosis at baseline was predominately performed by red pulp MΦ; however, during inflammation both plasmacytoid DCs (pDCs) and monocytes increased RBC consumption. Furthermore, pDCs up regulated MHC-II and activation markers CD80 and CD86. In addition to changing patterns of erythrophagocytosis, inflammation also led to a significant decrease in CD11c+ conventional DC populations and an increase in granulocytes. The nature of APCs that consume transfused RBCs is changed by inflammation. Given that APCs initiate humoral immune responses, these findings provide potential mechanistic insight into how inflammation regulates RBC alloimmunization.

PATHWAYS OF RECOGNITION OF FOREIGN ANTIGENS IN THE ADAPTIVE IMMUNE RESPONSE TO ALLOGENEIC ORGAN TRANSPLANTATION

Immunological allograft rejection is based on the process of recognition of donor antigens by T-cell receptors. Antigens are generally represented by molecules of major hystocompatibility complex (MHC), expressed by the donor organ tissue cells. Immune response to allogeneic transplant in its activity is greatly higher than response to conventional infection wherein the basic mechanisms of recognition of infectious antigens and alloantigens seem to be similar. In this review we have analyzed three main pathways of recognition of alloantigens. Those pathways are differentiated by the nature of antigen presenting cells, kinetics and duration of its influence on the formation of alloimmunity: direct, indirect and semidirect. Establishing the relationship between mechanisms of allorecognition and manifestations of graft rejection is of great clinical importance.

B Cells and Programmed Death-Ligand 2 Signaling Are Required for Maximal Interferon-γ Recall Response by Splenic CD4⁺ Memory T Cells of Mice Vaccinated with Mycobacterium tuberculosis Ag85B.

CD4+ T cells producing interferon-γ are crucial for protection against Mycobacterium tuberculosis infection and are the cornerstone of tuberculosis vaccination and immunological diagnostic assays. Since emerging evidence indicates that B cells can modulate T cell responses to M. tuberculosis infection, we investigated the contribution of B cells in regulating interferon-γ recall response by memory Thelper1 cells specific for Ag85B, a leading candidate for tuberculosis sub-unit vaccines. We found that B cells were able to maximize the reactivation of CD4+ memory T cells and the interferon-γ response against ex vivo antigen recall in spleens of mice vaccinated with Ag85B. B cell-mediated increase of interferon-γ response was particular evident for high interferon-γ producer CD4+ memory T cells, likely because those T cells were required for triggering and amplification of B cell activation. A positive-feedback loop of mutual activation between B cells, not necessarily antigen-experienced but with integral phosphatidylinositol-3 kinase (PI3K) pathway and a peculiar interferon-γ-producing CD4highT cell subset was established. Programed death-ligand 2 (PD-L2), expressed both on B and the highly activated CD4high T cells, contributed to the increase of interferon-γ recall response through a PD1-independent pathway. In B cell-deficient mice, interferon-γ production and activation of Ag85B-specific CD4+ T cells were blunted against ex vivo antigen recall but these responses could be restored by adding B cells. On the other hand, B cells appeared to down-regulate interleukin-22 recall response. Our data point out that nature of antigen presenting cells determines quality and size of T cell cytokine recall responses. Thus, antigen presenting cells, including B cells, deserve to be considered for a better prediction of cytokine responses by peripheral memory T cells specific for M. tuberculosis antigens. We also invite to consider B cells, PD-L2 and PI3K as potential targets for therapeutic modulation of T cell cytokine responses for tuberculosis control.

Immunobiology of hepatitis B virus infection.

The adaptive immune response, particularly the virus-specific CD8(+) T-cell response, is largely responsible for viral clearance and disease pathogenesis during hepatitis B virus (HBV) infection. The HBV-specific CD8(+) T-cell response is vigorous, polyclonal and multispecific in acutely infected patients who successfully clear the virus and relatively weak and narrowly focused in chronically infected patients. The immunological basis for this dichotomy is unclear. A recent study using HBV transgenic mice and HBV-specific T-cell receptor transgenic mice suggests that intrahepatic antigen presentation by HBV positive hepatocytes suppresses HBV-specific CD8(+) T-cell responses through a co-inhibitory molecule, programmed cell death 1 (PD-1). In contrast, antigen presentation by activated professional antigen-presenting cells induces functional differentiation of HBV-specific CD8(+) T cells. These findings suggest that the outcome of T-cell priming is largely dependent on the nature of antigen-presenting cells. Another study suggests that the timing of HBV-specific CD4(+) T-cell priming regulates the magnitude of the HBV-specific CD8(+) T-cell response. Other factors that could regulate HBV-specific cellular immune responses are high viral loads, mutational epitope inactivation, T-cell receptor antagonism and infection of immunologically privileged tissues. However, these pathways become apparent only in the setting of an ineffective cellular immune response, which is therefore the fundamental underlying cause. Understanding the cellular and molecular mechanisms by which HBV evades host immune responses will eventually help develop new immunotherapeutic strategies designed to terminate chronic HBV infection.

Consequences of double negative regulatory T cell and antigen presenting cell interaction on immune response suppression

Recent studies have indicated that regulatory T cell (Treg)-mediated suppression may depend on interactions with antigen presenting cells (APCs). TCRαβ +CD3 +CD4 −CD8 −NK1.1 − double-negative (DN) Tregs have been shown to be able to suppress effector T cells in vitro in mice and humans, and control various diseases in an antigen (Ag)-specific manner in murine models. Studies on DN Tregs have been focused on their suppressive effect on T cells. However, the nature of APCs that can effectively activate DN Tregs as well as the effect of DN Tregs on APCs, have not previously been studied. In this report, we investigated the interactions of DN Tregs with APCs. We found that although stimulation with naïve allogeneic APCs could activate DN Tregs, it failed to induce proliferation of DN Tregs. Interestingly, stimulation with LPS-activated allogeneic APCs significantly augmented the proliferation of DN Tregs compared to naïve allogeneic APCs. Importantly, the expanded DN Tregs can maintain their suppressive function. Further, DN Tregs proliferated in the presence of LPS-activated B cells in an Ag-specific fashion. Although DN Tregs were not able to down regulate the expression of CD80 or CD86 on LPS-activated B cells, they could kill activated allogeneic as well as syngeneic B cells via a perforin-dependent pathway, indicating that eliminating activated B cells may contribute to DN Treg-mediated suppression. These data provide important insights into the interactions of DN Tregs with APCs and may facilitate production of functional Ag-specific DN Tregs in a clinical setting.

Dendritic cells and HNSCC: A potential treatment option? (Review)

Patient survival in head and neck squamous cell cancer (HNSCC) has not changed significantly in many years, despite progress in surgical, radiotherapy and chemotherapy techniques. Immunotherapy, a rapidly progressing alternative cancer treatment, aims to prompt or assist the body's immune system to combat the disease itself. A number of strategies exist including the use of dendritic cells, natural antigen presenting cells capable of stimulating an anti-tumor immune response. Encouraging work has been performed using these cells as vaccines against a number of tumors especially melanoma. Work with head and neck cancer is also encouraging, but less advanced. Dendritic cell presence in head and neck squamous cell cancers is associated with an improved prognosis, however due to immunosuppression, the exact mechanism of which remains poorly understood, these cells do not function efficiently. This prevents the stimulation of an effective anti-tumor immune response by the patient and allows tumor growth to continue. This review summarises the current level of understanding of dendritic cells and their relationship with HNSCC. It briefly summarises work with dendritic cells and other cancers where relevant to HNSCC; dendritic cells and head and neck cancer; the possible causes of dendritic cell impairment; the techniques used to restore their function and the methods used to prime the dendritic cells prior to their use as vaccines for the stimulation of an anti-tumor response.

Regulation of T helper cell differentiation in vivo by GP43 from Paracoccidioides brasiliensis provided by different antigen-presenting cells.

Paracoccidioidomycosis, endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis. The infection can evolve into different clinical forms that are associated with various degrees of suppressed cell-mediated immunity. Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated whether the nature of antigen-presenting cells (APCs) can influence the Th1/Th2 balance in vivo. It was observed that the injection of mature dendritic cells (DCs), macrophages and B cells primed the mice and induced a proliferation of T cells in vitro. It was seen that DCs from resistant mice stimulated predominantly interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), whereas macrophages activated IL-10, IL-4 and IFN-gamma-secreting T cells and B cells IL-4 and IL-10 only. Results presented here clearly demonstrate that DC drives the development of cells secreting Th1-derived cytokines, whereas B cells induce the differentiation of a Th2 phenotype in vivo.

Resident tissue macrophages within the normal rat iris lack immunosuppressive activity and are effective antigen-presenting cells

Despite extensive study of the numerous immunoregulatory mechanisms that contribute to the `immune-privileged'nature of the anterior chamber (AC) of the eye, little is known of the functional nature of antigen-presenting cells (APC) present in the tissues adjoining the AC. In the present study, we have compared the antigen-presenting capacity of dendritic cells (DC) and macrophages isolated from the normal rat iris. Whereas iris DC exhibited a potent ability to stimulate resting allogeneic T cells in MLR cultures (an in-vitro correlate of the ability to induce primary T cell responses), resident iris macrophages displayed negligible MLR-stimulatory capacity. Significantly, iris macrophages could efficiently elicit proliferation of primed antigen-specific T cells (an in-vitro correlate of the ability to act as local APC in secondary responses). This antigen-presenting activity was approximately half that of fully `mature' iris DC and considerably greater than that of freshly isolated iris DC. A key contributor to the effectiveness of resident iris macrophage antigen presentation was considered to be the absence of lymphocytostatic control of T cell proliferation exerted by these cells. The results indicate dichotomous but complementary roles for DC (immune surveillance) and macrophages (local antigen presentation in secondary responses) in this tissue.

Chronic allograft rejection. Do the Th2 cells preferentially induced by indirect alloantigen recognition play a dominant role?

Chronic rejection has been the major obstacle to the long-term allograft survival in the clinic. Although the etiology of this rejection reaction is multifactorial, alloantigen-specific immune activation plays the most critical role. We herein hypothesize that CD4+ Th2 cells that are preferentially induced by the indirect recognition of allogeneic histocompatibility antigens late in transplantation may play the most critical role in the initiation and/or maintenance of chronic allograft rejection. Immunosuppression used to prevent acute rejection and the nature of antigen-presenting cells and alloligands in the graft may all contribute to immune deviation to the Th2 response. This response may be further perpetuated by type 2 cytokines conceivably produced by activated macrophages, NK cells, and CD8+ T cells in the graft. Cytokines and growth factors induced by this type 2 response, in turn, allow for activation of B, endothelial, and smooth muscle cells that collectively contribute to the pathogenesis of chronic allograft rejection by producing alloantibodies and growth hormones required for interstitial fibrosis, extracellular matrix deposition, and vascular neointimal hyperplasia.

Advances in chronic inflammatory demyelinating polyneuropathy: disease variants and inflammatory response mediators and modifiers

Available data on the immunopathogenesis of chronic inflammatory demyelinating polyneuropathy remain still fragmentary and insufficient for a unified hypothesis. Macrophage-mediated demyelination appears to play a fundamental role and cytokines, especially tumour necrosis factor-alpha, participate in this process. The nature of antigen presenting cells, T-cell receptors, adhesion molecules between inflammatory cells and myelinated fibers and the apparent predominance of T helper cell 1-related cytokines need to be explored to design more specific immunotherapies. In chronic cases of chronic inflammatory demyelinating polyneuropathy, a concomitant axonal loss secondary to primary demyelination is common and should be taken into consideration in the design of future therapeutic strategies.

Expression and functional significance of an additional ligand for CTLA-4.

Effective T-cell activation requires antigen/major histocompatibility complex engagement by the T-cell receptor complex in concert with one or more costimulatory molecules. Recent studies have suggested that the B7 molecule, expressed on most antigen presenting cells, functions as a costimulatory molecule through its interaction with CD28 on T cells. Blocking the CD28/B7 interaction with CTLA4Ig inhibits T-cell activation in vitro and induces unresponsiveness. We demonstrate that another molecule(s), termed B7-2, is expressed constitutively on dendritic cells, is differentially regulated on B cells, and costimulates naive T cells responding to alloantigen. B7-2 is up-regulated by lipopolysaccharide in < 6 hr and is maximally expressed on the majority of B cells by 24 hr. In contrast, B7 is detected only on a subset of activated B cells late (48 hr) after stimulation. In addition, Con A directly induces B7-2 but not B7 expression on B cells. Finally, although both anti-B7 monoclonal antibodies and CTLA4Ig blocked T-cell proliferation to antigen-expressing B7 transfectants, only CTLA4Ig had any significant inhibitory effect on T-cell proliferation to antigens expressed on natural antigen presenting cells, such as dendritic cells. Thus, B7 is not the only costimulatory molecule capable of initiating T-cell responses since a second ligand, B7-2, can provide a necessary second signal for T-cell activation.