Expression Of Co-stimulatory Markers Research Articles

Differential Immune Response Patterns Induced by Anionic and Cationic Lipid Adjuvants in Intranasal Anti-Influenza Immunization.

Currently, vaccine development against different respiratory diseases is at its peak. It is of utmost importance to find suitajble adjuvants that can increase the potency of the vaccine candidates. This study aimed to determine the systemic and splenic immune mechanisms in mice models induced by anionic and cationic lipid adjuvants in the presence of the vaccine-candidate influenza antigen hemagglutinin (HA). In the presence of the HA antigen, the cationic adjuvant (N3) increased conventional dendritic cell 1 (cDC1) abundance with enhanced MHCI and CD80-CD86 costimulatory marker expression, and significantly higher CD8T and Th17 populations with enhanced interferon-gamma (IFNγ) expression in CD8T and CD4T populations. Conversely, the anionic adjuvant (L3) increased the cDC2 population percentage with significantly higher MHCII and DEC205 expression, along with an increase in the CD4T and regulatory T cell populations. The L3-treated group also exhibited higher percentages of activated B and plasma cell populations with significantly higher antigen-specific IgG and IgA titer and virus neutralization potential. While the anionic adjuvant induced significantly higher humoral responses than the cationic adjuvant, the latter influenced a significantly higher Th1/Th17 response. For customized vaccine development, it is beneficial to have alternative adjuvants that can generate differential immune responses with the same vaccine candidate antigen. This study will aid the selection of adjuvants based on their charges to improve specific immune response arms in the future development of vaccine formulation.

A Cancer Nanovaccine for Co-Delivery of Peptide Neoantigens and Optimized Combinations of STING and TLR4 Agonists.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8+ T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8+ cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8+ T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines.

Dominance and improved survivability of human γδT17 cell subset aggravates the immunopathogenesis of pemphigus vulgaris.

Human γδ T cells are highly enriched in epithelial cell-dominated compartments like skin. Nonetheless, their function in the pathogenesis of pemphigus vulgaris (PV), an autoimmune skin disorder, is lacking. Therefore, we investigated the functional expression of human γδT cell subsets along with their homing chemokine receptor-ligand and inflammatory cytokines in the immunopathogenesis of PV. Estimation of the frequency of γδT cell subsets by flow cytometry revealed four major subsets of γδ T cells (γδT1, γδT2, γδT17, γδTreg) in both control and PV circulation. The elevated frequency of γδT17 cells producing IL17 and expressing CCR6 receptor suggests their inflammatory and migratory potential in PV. In vitro culture of γδ T cells from patients showed increased mRNA expression of inflammatory cytokines IL17, RORγt, IL23, IL1, and co-stimulatory markers, CD27 and CD70. These findings correlated the role of IL1 and IL23 cytokines that alleviate the Th17 population in PV. Cytotoxic activities of γδ T cells were higher and inflammatory γδT17 cells were localized in the skin of PV whereas γδTreg cells associated TGFβ and FOXP3 were lowered. Hyperinflammatory phenotype of the γδT17 cell subset and its migratory potential might be aiding in the pathogenesis of PV, whereas γδTreg cells fail to suppress these inflammatory responses. Hence, γδT17 cell-associated markers can be targeted for identifying novel therapeutics in PV.

Tailored PGE2 Immunomodulation of moDCs by Nano-Encapsulated EP2/EP4 Antagonists.

Prostaglandin E2 (PGE2) is an important maturation mediator for dendritic cells (DCs). However, increased PGE2 levels in the tumor exert immunosuppressive effects on DCs by signaling through two E-Prostanoid (EP) receptors: EP2 and EP4. Blocking EP-receptor signaling of PGE2 with antagonists is currently being investigated for clinical applications to enhance anti-tumor immunity. In this study, we investigated a new delivery approach by encapsulating EP2/EP4 antagonists in polymeric nanoparticles. The nanoparticles were characterized for size, antagonist loading, and release. The efficacy of the encapsulated antagonists to block PGE2 signaling was analyzed using monocyte-derived DCs (moDCs). The obtained nanoparticles were sized between 210 and 260 nm. The encapsulation efficacy of the EP2/EP4 antagonists was 20% and 17%, respectively, and was further increased with the co-encapsulation of both antagonists. The treatment of moDCs with co-encapsulation EP2/EP4 antagonists prevented PGE2-induced co-stimulatory marker expression. Even though both antagonists showed a burst release within 15 min at 37 °C, the nanoparticles executed the immunomodulatory effects on moDCs. In summary, we demonstrate the functionality of EP2/EP4 antagonist-loaded nanoparticles to overcome PGE2 modulation of moDCs.

Environmentally weathered polystyrene particles induce phenotypical and functional maturation of human monocyte-derived dendritic cells

Micro- and nanoplastics (MNP) are ubiquitously present in the environment due to their high persistence and bioaccumulative properties. Humans get exposed to MNP via various routes and consequently, they will encounter dendritic cells (DC) which are antigen-presenting cells involved in regulating immune responses. The consequences of DC exposure to MNP are an important, yet understudied, cause of concern. Therefore, this study aimed to assess the uptake and effect of MNP in vitro by exposing human monocyte-derived dendritic cells (MoDC) to virgin and environmentally weathered polystyrene (PS) particles of different sizes (0.2, 1, and 10 µm), at different concentrations ranging from 1 to 100 µg/ml. The effects of these particles were examined by measuring co-stimulatory surface marker (i.e. CD83 and CD86) expression. In addition, T-cell proliferation was measured via a mixed-leukocyte reaction (MLR) assay. The results showed that MoDC were capable of absorbing PS particles, and this was facilitated by pre-incubation in heat-inactivated (HI) plasma. Furthermore, depending on their size, weathered PS particles in particular caused increased expression of CD83 and CD86 on MoDC. Lastly, weathered 0.2 µm PS particles were able to functionally activate MoDC, leading to an increase in T-cell activation. These in vitro data suggest that, depending on their size, weathered PS particles might act as an immunostimulating adjuvant, possibly leading to T-cell sensitization.

Treatment of in vitro generated Langerhans cells with JAK-STAT inhibitor reduces their inflammatory potential.

Alopecia areata (AA) is a condition in which hair is lost in small regions or over the entire body. It has a prevalence of 1 in 1000 and has a great impact on psychological wellbeing. AA is generally considered an autoimmune disease in which a collapse of the immune privilege systemof the hair follicle has shown to play an important role, potentially driven by interferon gamma (IFN-γ). The most prominent cells located in or around the hair follicle in AA are Langerhans cells, CD4+ or CD8+ T cells, macrophages and mast cells. Langerhans cells, specialized dendritic cells, are resident in the epidermis and are known to associate with hair follicles. Therefore, we aimed to develop in vitro generated Langerhans cells contributing as an in vitro model of disease. In vitro models provide insight into the behaviour of cells and are a valuable tool before being in need of an animal model or patient samples. For this, Langerhans-like cells were generated from CD14+ monocytes in the presence of GM-CSF and TGF-β. After 10days of cell culture, Langerhans-like cells express CD207 and CD1a but lack CD209 expression as well as Birbeck granules. Next, Langerhans-like cells were exposed to inflammatory conditions and the effect of different AA treatments was investigated. All treatments-diphencyprone contact immunotherapy, UV-B light therapy and JAK-STAT inhibition-affect the expression of costimulatory and skin-homing markers on Langerhans-like cells. Importantly, also the T cell stimulatory capacity of Langerhans-like cells was significantly reduced following treatment under inflammatory conditions. Noteworthy, JAK-STAT inhibition outperformed conventional AA treatments. In conclusion, our findings demonstrate that in vitro generated Langerhans-like cells can be used as a model of disease. Moreover, JAK-STAT inhibition may become a valuable new approach for the treatment of AA.

NSAIDs affect dendritic cell cytokine production.

BackgroundImmunotherapy is now considered as the new pillar in treatment of cancer patients. Dendritic cells (DCs) play an essential role in stimulating anti-tumor immune responses, as they are capable of cross-presenting exogenous tumor antigens in MHCI complexes to activate naïve CD8+ T cells. Analgesics, like non-steroid anti-inflammatory drugs (NSAIDs), are frequently given to cancer patients to help relieve pain, however little is known about their impact on DC function.MethodsHere, we investigated the effect of the NSAIDs diclofenac, ibuprofen and celecoxib on the three key processes of DCs required for proper CD8+ cytotoxic T cell induction: antigen cross-presentation, co-stimulatory marker expression, and cytokine production.ResultsOur results show that TLR-induced pro- and anti-inflammatory cytokine excretion by human monocyte derived and murine bone-marrow derived DCs is diminished after NSAID exposure.ConclusionsThese results indicate that various NSAIDs can affect DC function and warrant further investigation into the impact of NSAIDs on DC priming of T cells and cancer immunotherapy efficacy.

Copper homeostasis is critical for T cell activation

Abstract Elevated copper levels are associated with autoimmune disease, infection, and tumors in both humans and mice. Modulating copper availability is a promising potential intervention to treat these diseases. However, the mechanisms underlying the beneficial impact of copper modulation in disease are poorly understood. Moreover, the role of copper homeostasis in immune function, including in T cell responses, is not clear. Copper is a required cofactor for proteins involved in essential T cell processes, including extracellular matrix regulation, signaling pathways, mitochondrial metabolism, autophagy, and maintenance of reactive oxygen species. To investigate the impact of copper levels on T cell function, we examined T cell activation during altered copper availability. We found that copper chelation limited T cell proliferation and reduced expression of activation markers following stimulation. We also found that both activation and copper availability altered copper transporter expression in T cells. Additionally, copper modulation substantially impacted the transcriptional profiles of activated T cells, including expression of transcription factors, co-stimulatory markers, cytokines, and metabolic genes. Overall, our data demonstrate that altered copper levels impair T cell activation. Further, our results reveal new pathways impacted by copper homeostasis in T cells, which will be key to both understanding the role of copper in disease and leveraging this role for therapeutic development. Studies to evaluate the role of copper homeostasis in the immune response in vivo are ongoing. Supported by funding from ALSAC.

Adenosine deaminase-1 (ADA-1) induces maturation and skewing of cytokine and chemokine production in dendritic cells to improve adaptive immune responses.

Abstract Dendritic cells (DCs) are critical mediators of antigen-specific immunity through their ability to present antigen and drive T cell differentiation into different effector subsets such as follicular helper T cells (TFH). TFH cells provide both physical and cytokine-mediated stimuli to B cells resulting in somatic hypermutation and class-switching of B cell receptors. Molecules that target TFH cells, such as adenosine deaminase-1 (ADA-1), would improve humoral immunity. We have previously demonstrated that co-delivery of plasmid-encoded adenosine deaminase with an HIV-1 envelope and SARS-CoV-2 DNA vaccines in vivo, enhanced both humoral and cellular responses. However, the mechanism by which ADA-1 is acting as an adjuvant remains to be elucidated. To this end, we treated monocyte-derived DCs from healthy human donors in vitro with recombinant ADA-1 protein and evaluated the expression of maturation markers, cytokines and chemokines. ADA-1-treated DCs had a significantly increased expression of CD40 and CD86 as well as HLA-DR compared to their unstimulated, immature counterparts. The level of co-stimulatory marker and HLA-DR expression on ADA-1-treated DCs was similar to that on LPS/IFN-γ-treated DCs, indicating ADA-1-mediated DC maturation. ADA-1-treated DCs also exhibited a significant increase in IL-6, IL-1β and CXCL13 expression. IL-6 is a key pro-TFH cytokine and IL-1β and CXCL13 may play a role in TFH cell differentiation, function, and proliferation. Ongoing studies are aimed to drive ADA-1 overexpression in DCs and evaluate effects on antigen presentation. Overall, elucidating the mechanism of ADA-1’s adjuvanticity will allow for its progression as a clinical adjuvant. This work was supported by funding to Dr. Elias Haddad from NIH 5RO1AI106482-01A and 1U19 AI128910-01.

Aggregates Associated with Instability of Antibodies during Aerosolization Induce Adverse Immunological Effects.

Background: Immunogenicity refers to the inherent ability of a molecule to stimulate an immune response. Aggregates are one of the major risk factors for the undesired immunogenicity of therapeutic antibodies (Ab) and may ultimately result in immune-mediated adverse effects. For Ab delivered by inhalation, it is necessary to consider the interaction between aggregates resulting from the instability of the Ab during aerosolization and the lung mucosa. The aim of this study was to determine the impact of aggregates produced during aerosolization of therapeutic Ab on the immune system. Methods: Human and murine immunoglobulin G (IgG) were aerosolized using a clinically-relevant nebulizer and their immunogenic potency was assessed, both in vitro using a standard human monocyte-derived dendritic cell (MoDC) reporter assay and in vivo in immune cells in the airway compartment, lung parenchyma and spleen of healthy C57BL/6 mice after pulmonary administration. Results: IgG aggregates, produced during nebulization, induced a dose-dependent activation of MoDC characterized by the enhanced production of cytokines and expression of co-stimulatory markers. Interestingly, in vivo administration of high amounts of nebulization-mediated IgG aggregates resulted in a profound and sustained local and systemic depletion of immune cells, which was attributable to cell death. This cytotoxic effect was observed when nebulized IgG was administered locally in the airways as compared to a systemic administration but was mitigated by improving IgG stability during nebulization, through the addition of polysorbates to the formulation. Conclusion: Although inhalation delivery represents an attractive alternative route for delivering Ab to treat respiratory infections, our findings indicate that it is critical to prevent IgG aggregation during the nebulization process to avoid pro-inflammatory and cytotoxic effects. The optimization of Ab formulation can mitigate adverse effects induced by nebulization.

Tumor-Infiltrating T Cells Can Be ExpandedSuccessfully from Primary Uveal Melanoma after Separation from Their TumorEnvironment.

PurposeTo evaluate whether expanded tumor-infiltrating lymphocytes (TILs) can be obtained from primary uveal melanoma (UM) for potential use as adjuvant treatment in patients at risk of developing metastatic disease.DesignExperimental research study.ParticipantsFreshly obtained primary UM from 30 patients.MethodsThree different methods were used to expand TILs: (1) direct culture from small fragments of fresh tumor tissue, (2) single-cell tissue preparation by enzymatic digestion and subsequent enrichment of mononuclear cells, and (3) selection of CD3+ T cells using magnetic beads. Surface expression of costimulatory and inhibitory T-cell markers and T-cell reactivity against autologous tumor cells was assessed. Clinical, histopathologic, genetic, and immunologic characteristics of the tumors were compared with the capacity to expand TILs and with their reactivity against autologous tumor cells.Main Outcome MeasuresThe feasibility of expanding TILs from primary UM, testing their reactivity to autologous UM cells, and evaluating the impact of an immunomodulatory environment.ResultsDirect culture of tumor parts led to successful TIL culture in 4 of 22 tumors (18%), enrichment of mononuclear cells gave rise to TILs in 5 of 12 tumors (42%), while preselection of CD3+ T cells with magnetic beads resulted in TIL expansion in 17 of 25 tumors (68%). In 8 of 17 tumors (47%), the TIL cultures comprised UM-reactive T cells. The presence of UM-reactive T cells among TILs was not related to clinical, histologic, genetic, or immunological tumor characteristics. Interestingly, RNA-Seq analysis showed that approximately half of the UM tumors displayed an increased expression of immunomodulatory molecules related to T-cell suppression, such as galectin 3, programmed death-ligand 1, cytotoxic T-lymphocyte-associated protein 4, indoleamine 2,3-dioxygenase 1, and lymphocyte activating 3, potentially explaining why T cells require optimal removal of tumor components for expansion.ConclusionsThe need to separate TILs from their tumor microenvironment for their successful expansion and the presence of UM-reactive T cells among TILs suggests that these UM-reactive T cells are strongly suppressed in vivo and that UM is immunogenic. These findings indicate that adoptive TIL therapy could be an option as an adjuvant treatment in primary UM patients at high risk of developing metastatic disease.

Chronic Lymphocytic Leukemia Actively Induces T-Cell Dysfunction By Contact-Dependent Signaling Via CD24 and CD52

IntroductionSuccess rates of autologous T cell-based therapies, such as CAR-T cell therapy, in chronic lymphocytic leukemia (CLL) have been suboptimal and correlate with failure of activation and proliferation of T cells in vitro and in vivo. Previous data showing that impaired CD8 T-cell activation, proliferation and metabolic reprogramming could be restored by purifying CLL T cells via cell-sorting (van Bruggen et al., Blood, 2019) indicating that an as yet unknown, CLL-derived factor is responsible for acquired T-cell dysfunction. In this study we aim to elucidate the mechanistic basis of CLL-mediated T-cell dysfunction.ResultsDynamic analysis of αCD3/CD28 stimulated autologous T cells in presence of CLL cells over a period of 9 days revealed that T-cell activation (CD25, CD71, CD95 and PD-1) in CLL is in fact not impaired but occurs in a delayed fashion. CLL T cells reached peak activation after 5-6 days in contrast to 2-3 days for age-matched healthy donors. (Fig. 1A). This delayed T cell receptor-induced T cell activation was largely normalized with tumor cell depletion by flow-sorting prior to activation. Accordingly, in absence versus presence of autologous CLL cells, CAR-T cells derived from CLL patients showed enhanced proliferation, cytokine production and cytotoxicity, indicating potential clinical relevance. These findings show that T cells in CLL are not (terminally) exhausted but that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We attempted to identify the mechanism of action in which CLL cells induce T cell dysfunction and whether these suppressive effects are mediated through a soluble factor secreted by CLL cells or by contact-dependent mechanisms.Previous studies have shown that CD40 activation of CLL cells results in increased expression of key surface-expressed adhesion and costimulatory molecules, but also in alterations of immune-modulatory cytokines secretion. This model was therefore used to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation in a contact-dependent manner (based on trans-well experiments; Fig. 1B ).Several clinically approved kinase inhibitors were tested to identify signaling cascades involved in CD40-mediated alleviation of T-cell dysfunction. Only pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce expression of co-stimulatory markers on CD40-activated CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. RNA sequencing of CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL, which we confirmed at the protein level. We also found increased expression of Siglec-10 on CLL T cells, suggesting a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, inhibition of Siglec-10 ligation by blocking CD24, and CD52 antibodies subsequently improved T-cell activation despite presence of CLL cells (Fig. 1C).ConclusionThese results demonstrate that T cells derived from CLL patients are not terminally dysfunctional and can be revived. Our observations indicate that CLL cells actively suppress (CAR) T-cell function in a contact-dependent fashion through CD24- and CD52-mediated Siglec-10 ligation. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell function in CLL. [Display omitted] DisclosuresKater: Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.

β2-Integrin Adhesion Regulates Dendritic Cell Epigenetic and Transcriptional Landscapes to Restrict Dendritic Cell Maturation and Tumor Rejection.

Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow-derived DCs (BM-DC) expressing dysfunctional β2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8+ T-cell response. BM-DCs expressing dysfunctional β2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo-cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, β2-integrin-mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting β2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.

Convection enhanced delivery of light responsive antigen capturing oxygen generators for chemo-phototherapy triggered adaptive immunity

In recent years, combination therapy has emerged as the cornerstone of clinical practice in treating glioblastoma multiforme. However, their ability to trigger and leverage the body's adaptive immunity has rarely been studied. Tumour heterogeneity, the presence of the blood-brain barrier, and an immunosuppressive tumor microenvironment play a crucial role in the 90% local tumor recurrence post-treatment. Herein, we report an improved combination therapy approach capable of stimulating an immune response that utilizes Light responsive antigen-capturing oxygen generators (LAGs). The engineered LAGs loaded with a non-genotoxic molecule, Nutlin-3a, and a photosensitizer, Protoporphyrin IX, can release the payload on-demand when exposed to light of a specific wavelength. The in-situ oxygen generation capability of LAGs enables tumor oxygenation enhancement, thereby alleviating the tumor hypoxia and enhancing the efficacy of chemo-photodynamic therapy. Furthermore, by modulating the surface properties of LAGs, we demonstrated that the tumor-derived protein antigens released can be captured and retained in-situ, which improves antigen uptake and presentation by the antigen-presenting cells. Dual drug-loaded LAGs (DD-LAGs) upregulated the expression of cell surface CD83 maturation and CD86 costimulatory markers on monocyte-derived-dendritic cells, suggesting intrinsic immune adjuvancy. In the presence of 3D printed hypoxic U87 spheroids (h-U87), DD-LAGs induced cancer cell death, upregulated IL-1β, and downregulated IL-10 resulting in CD3+, helper CD4+, and cytotoxic CD8+ proliferation. Finally, we have investigated convection-enhanced delivery as a potential route of administration for DD-LAGs. Our work presents a novel strategy to induce tumor cell death both during and post-treatment, thereby reducing the possibility of recurrence.

Thymopoiesis, Alterations in Dendritic Cells and Tregs, and Reduced T Cell Activation in Successful Extracorporeal Photopheresis Treatment of GVHD

Acute graft-versus-host disease (aGVHD) is a significant complication of allogeneic hematopoietic stem cell transplant (HSCT) and negatively affects T cell reconstitution. Extracorporeal photopheresis (ECP) reduces aGVHD, but the mechanisms remain incompletely understood. Our objective was to examine the impact of ECP on thymopoiesis in pediatric aGVHD and the mechanisms at a cellular and transcriptional level. Sixteen pediatric HSCT patients were recruited: 6 with ECP-treated aGVHD, 5 without aGVHD, and 5 with aGVHD treated with corticosteroids only. Thymopoiesis was evaluated by measuring naive T cells, TRECs, IL-7, and T cell receptor repertoire diversity. Regulatory T cell (Treg) enumeration and function and dendritic cell (DC) enumeration and phenotype were analyzed using flow cytometry. T cell transcriptome analysis was performed on ECP patients after treatment and responders pre- and post-treatment. Four ECP responders demonstrated thymic-dependent T cell recovery, and superior median naïve T cell numbers at 8 and 12 months post-HSCT compared to the aGVHD corticosteroid group. Increased Tregs and Treg suppressive function, reduced cDC/pDC and DC co-stimulatory marker expression in ECP responders suggest upregulated peripheral tolerance; these findings were not observed in partial responders. Responder post-ECP CD3+ T cell transcriptional profile demonstrated 3333 downregulated and 364 upregulated genes, with significant downregulation of ERRα and GαS pathways, and reduced expression of pro-inflammatory and adhesion proteins.Thymic function improves with successful ECP treatment. ECP reduces T cell activation and impacts peripheral tolerance via DCs and Tregs. Differences in thymic recovery, DC, and Treg cellular patterns and the T cell transcriptome were observed between ECP responders and partial responders and require further validation and investigation in additional patients.

Co-delivery of Peptide Neoantigens and Stimulator of Interferon Genes Agonists Enhances Response to Cancer Vaccines.

Cancer vaccines targeting patient-specific neoantigens have emerged as a promising strategy for improving responses to immune checkpoint blockade. However, neoantigenic peptides are poorly immunogenic and inept at stimulating CD8+ T cell responses, motivating a need for new vaccine technologies that enhance their immunogenicity. The stimulator of interferon genes (STING) pathway is an endogenous mechanism by which the innate immune system generates an immunological context for priming and mobilizing neoantigen-specific T cells. Owing to this critical role in tumor immune surveillance, a synthetic cancer nanovaccine platform (nanoSTING-vax) was developed that mimics immunogenic cancer cells in its capacity to efficiently promote co-delivery of peptide antigens and the STING agonist, cGAMP. The co-loading of cGAMP and peptides into pH-responsive, endosomolytic polymersomes promoted the coordinated delivery of both cGAMP and peptide antigens to the cytosol, thereby eliciting inflammatory cytokine production, co-stimulatory marker expression, and antigen cross-presentation. Consequently, nanoSTING-vax significantly enhanced CD8+ T cell responses to a range of peptide antigens. Therapeutic immunization with nanoSTING-vax, in combination with immune checkpoint blockade, inhibited tumor growth in multiple murine tumor models, even leading to complete tumor rejection and generation of durable antitumor immune memory. Collectively, this work establishes nanoSTING-vax as a versatile platform for enhancing immune responses to neoantigen-targeted cancer vaccines.

Feasibility of dendritic cell-based vaccine against glioblastoma by using cytoplasmic transduction peptide (CTP)-fused protein antigens combined with anti-PD1

ABSTRACT Recent clinical trials utilizing antigen-pulsed dendritic cells (DCs) have demonstrated increased survival of vaccinated cancer patients. Besides, the cytoplasmic transduction peptide (CTP) not only has an excellent transcellular efficiency but also shows a strong tendency to remain in the cytoplasm after transduction, without migrating into the nucleus. In this study, we investigated the effectiveness of immunotherapy against malignant gliomas using DCs pulsed with CTP-fused protein antigens combined with programmed cell death protein 1 blockade (anti-PD1). The expression of tumor associated antigen (WT1 and BIRC5) and PDL1 on glioblastoma (GBM) target cells was confirmed by western blot. The effect of CTP-fused protein antigens on mature DCs (VaxDCs) was determined. The immunophenotypes of VaxDCs pulsed with CTP-fused protein antigens was confirmed by flow cytometry and the cytokine production levels of T helper polarization were measured by enzyme-linked immunosorbent (ELISA) assay. The IFN-γ-enzyme linked immunospot and lactate dehydrogenase release assays were performed to estimate the cytotoxic activity of antigen-specific cytotoxic T lymphocytes (CTLs), stimulated by VaxDCs pulsed with CTP-fused protein antigens and anti-PD1, against malignant glioma cells expressing target antigens. VaxDCs pulsed with CTP-fused protein antigens showed enhanced expression of major histocompatibility complex (MHC) and co-stimulatory markers of DCs and resulted in Th1 cytokine polarization. The increase in the number of IFN-γ+ effector T cells paralleled with the enhanced percent specific lysis of GBM targets cells by antigen-specific CTLs. Our study suggested that using CTP-fused protein antigens for DC vaccine preparation along with PD1 blockade could be an effective immunotherapy strategy for GBM.

In Vitro Immunological Effects of CXCR3 Inhibitor AMG487 on Dendritic Cells.

AMG 487 is the targeted blocker of chemokine receptor CXCR3 and improves inflammatory symptoms by blocking the inflammatory cycle. Here we investigated whether AMG 487 affects dendritic cell (DC) biology and function. The expression of co-stimulatory markers on DCs was reduced, indicating the semi-mature state of DC when AMG 487 was added throughout the in vitro differentiation period. Additionally, when added solely during the final lipopolysaccharide-induced activation step, AMG 487 inhibited DC activation, as demonstrated by a decreased expression of activation markers. AMG487 also promoted the expression of PD-L2 and impaired the ability to induce antigen-specific T cell responses. Our results demonstrated that AMG 487 significantly affects DC maturity in vitro and function leading to impaired T cell activation, inducing DCs to have characteristics similar to tolerogenic DCs. AMG 487 may directly play an immunomodulatory role during DC development and functional shaping.

Role of CD28 Signaling on T cell Phenotype following Lymphodepletion and Homeostatic Reconstitution

Calcineurin inhibitors, the current standard of care in immunosuppression for transplant patients, leads to nephrotoxicity and loss of graft function over time. Preclinical studies have shown selective CD28 blockade (CD28 dAb, Lulizumab) to be superior to calcineurin inhibitors in reducing toxicity and prolonging allograft survival. Emory University will soon commence a Phase 2 clinical trial testing the efficacy of a Lulizumab‐based immunosuppression regimen in kidney transplantation. Patients in the trial will be T cell depleted (TCD), rendering them lymphopenic at the time of transplant and will therefore undergo homeostatic reconstitution in the weeks following transplantation. Lulizumab prevents T cell activation by selectively targeting CD28 signaling, but it is presently unclear what effect blocking this pathway will have on T cell phenotypes following homeostatic reconstitution (HR) in these patients. Previous studies have shown that HR leads to increased differentiation of naïve T cells into memory cells and to premature senescence. We hypothesized that blocking CD28 signaling in T cells during this process could prevent memory differentiation and senescence. We investigated this question using a murine model of TCD and skin transplantation. Four groups of mice were established: no treatment control, TCD alone, CD28 dAb alone, and both TCD and CD28 dAb (n = 4). Mice were sacrificed at 6 weeks post‐depletion and transplantation, and blood and tissues were collected for flow cytometric analysis. Using both traditional flow cytometry analysis and an unsupervised machine‐learning approach, we found that TCD CD4+ T cells have significantly elevated expression of the memory markers CD27, CD103, and CD69 when compared to no treatment control CD4+ T cells, indicating HR‐induced differentiation into memory T cells. In addition, TCD CD4+ T cells also expressed significantly higher levels of PD‐1, TIGIT, and CD226 (DNAM‐1) when compared to no treatment control CD4+ T cells, indicating a higher degree of senescence. Finally, we found that treating TCD mice with CD28 dAb restored expression of both memory markers and costimulatory markers to baseline levels. As such, we believe CD28 dAb presents a viable method to control HR‐induced differentiation and senescence of CD4+ T cells. These results were consistent across the blood, lung, and spleen. Our future directions include using murine models to determine whether the selective CD28 blockade‐mediated rescue of the HR‐induced phenotypic changes significantly affects protective immunity to infection.Support or Funding InformationResearch reported here was supported by the National Institutes of Health under award number R25 DK 101390 ‐ Title: Summer Undergraduate Program in Renal Research (SUPERR).

B Cell Development and T-Dependent Antibody Response Are Regulated by p38γ and p38δ.

p38MAP kinase (MAPK) signal transduction pathways are important regulators of inflammation and the immune response; their involvement in immune cell development and function is still largely unknown. Here we analysed the role of the p38 MAPK isoforms p38γ and p38δ in B cell differentiation in bone marrow (BM) and spleen, using mice lacking p38γ and p38δ, or conditional knockout mice that lack both p38γ and p38δ specifically in the B cell compartment. We found that the B cell differentiation programme in the BM was not affected in p38γ/δ-deficient mice. Moreover, these mice had reduced numbers of peripheral B cells as well as altered marginal zone B cell differentiation in the spleen. Expression of co-stimulatory proteins and activation markers in p38γ/δ-deficient B cells are diminished in response to B cell receptor (BCR) and CD40 stimulation; p38γ and p38δ were necessary for B cell proliferation induced by BCR and CD40 but not by TLR4 signaling. Furthermore, p38γ/δ-null mice produced significantly lower antibody responses to T-dependent antigens. Our results identify unreported functions for p38γ and p38δ in B cells and in the T-dependent humoral response; and show that the combined activity of these kinases is needed for peripheral B cell differentiation and function.