Phenotype Of Antigen-presenting Cells Research Articles

High-throughput screening identification of novel immunomodulatory combinations for the generation of tolerogenic dendritic cells.

Tolerogenic Dendritic Cells (tolDCs) have an exceptional promise as a potential therapy for autoimmune disease and transplantation rejection. TolDCs are a unique phenotype of antigen presenting cells (APCs) that can influence naïve T cells into antigen specific T regulatory cells (Tregs), which can re-establish tolerance against auto/allo-antigens in the long term. Despite their promise, tolDCs have not found clinical success. Most strategies seek to generate tolDCs ex vivo by differentiating naïve dendritic cells (DCs) with immunosuppressive agents. Recently, we developed a tolDC generation strategy, which we call Push/Pull Immunomodulation (PPI). In PPI, DCs are treated with combinations of toll-like-receptor (TLR) agonists and immunomodulatory agents, which generate more robust, Treg-inducing tolDCs than previous strategies. Here, we seek to identify more potent and clinically viable PPI formulations using data from a high-throughput screening project. Over 40,000 combinations of pathogen-associated molecular patterns (PAMPs) and immunomodulatory small molecules were screened using a modified murine macrophage line, RAW dual cells, to observe the effect of these combinations on two major immune regulatory transcription factors, NF-κB and IRF. Combinations were further screened for inflammatory cytokine activity using a human monocyte cell line, THP-1, then on murine DCs. Leading candidates were co-cultured with T cells to assess antigen specific T cell responses. From this data, we identified 355 combinations that showed low or moderate IRF activity, low NF-κB activity, low inflammatory cytokine generation and good viability: all hallmarks of tolerogenic potential. We further screened these 355 combinations using bone marrow derived DCs (BMDCs) and identified 10 combinations that demonstrated high IL-10 (tolerogenic) and low TNF-α (inflammatory) secretion. After further optimizing these combinations, we identified two combinations that generate robust tolDCs from BMDCs ex vivo. We further show that these PPI-tolDCs can also generate antigen specific Tregs but do not increase overall Treg populations. These second-generation PPI formulations have significant potential to generate robust tolDCs and strong antigen specific Tregs.

NK cells reduce anergic T cell development in early-stage tumors by promoting myeloid cell maturation.

Studies of NK cells in tumors have primarily focused on their direct actions towards tumor cells. We evaluated the impact of NK cells on expression of homing receptor ligands on tumor vasculature, intratumoral T cell number and function, and T cell activation in tumor draining lymph node. Using an implantable mouse model of melanoma, T cell responses and homing receptor ligand expression on the vasculature were evaluated with and without NK cells present during the early stages of the tumor response by flow cytometry. NK cells in early-stage tumors are one source of IFNγ that augments homing receptor ligand expression. More significantly, NK cell depletion resulted in increased numbers of intratumoral T cells with an anergic phenotype. Anergic T cell development in tumor draining lymph node was associated with increased T-cell receptor signaling but decreased proliferation and effector cell activity, and an incomplete maturation phenotype of antigen presenting cells. These effects of NK depletion were similar to those of blocking CD40L stimulation. We conclude that an important function of NK cells is to drive proper APC maturation via CD40L during responses to early-stage tumors, reducing development of anergic T cells. The reduced development of anergic T cells resulting in improved tumor control and T cell responses when NK cells were present.

Impaired functionality of antigen presenting cells in HIV- exposed uninfected infants in the first six months of life.

HIV-exposed uninfected infants (HEU) have increased morbidity and mortality due to infections in the first 6 months of life that tapers down to 2 years of life. The underlying immunologic defects remain undefined. We investigated antigen-presenting cells (APC) by comparing the phenotype of unstimulated APC, responses to toll-like receptor (TLR) stimulation, and ability to activate natural killer (NK) cells in 24 HEU and 64 HIV-unexposed infants (HUU) at 1-2 days of life (birth) and 28 HEU and 45 HUU at 6 months of life. At birth, unstimulated APC showed higher levels of activation and cytokine production in HEU than HUU and stimulation with TLR agonists revealed lower expression of inflammatory cytokines and activation markers, but similar expression of IL10 regulatory cytokine, in APC from HEU compared to HUU. Differences were still present at 6 months of life. From birth to 6 months, APC underwent extensive phenotypic and functional changes in HUU and minimal changes in HEU. TLR stimulation also generated lower NK cell expression of CD69 and/or IFNγ in HEU compared with HUU at birth and 6 months. In vitro experiments showed that NK IFNγ expression depended on APC cytokine secretion in response to TLR stimulation. Ex vivo IL10 supplementation decreased APC-mediated NK cell activation measured by IFNγ expression. We conclude that APC maturation was stunted or delayed in the first 6 months of life in HEU compared with HUU. Deficient inflammatory APC responses and/or the imbalance between inflammatory and regulatory responses in HEU may play an important role in their increased susceptibility to severe infections.

Investigation of the Molecular Profile of Granular Cell Tumours and Schwannomas of the Oral Cavity.

Granular cell tumours (GCTs) are rare submucosal lesions, thought to develop from Schwann cells, characterised by large polygonal cells with abundant lysosomes. The objectives of this study are to investigate whether GCTs have an antigen-presenting cell (APC) phenotype or a neural crest phenotype using immunohistochemistry and to compare expression profiles with Schwannomas. Immunoreactivity to CD68, HLA-DR, CD163, CD40 and CD11c (APC phenotype) and markers of neural crest cell (NCC) origin S100, SOX10, NSE and GAP43 in 23 cases of GCTs and 10 cases of Schwannomas were evaluated. RT-qPCR was used to identify a possible NCC developmental phenotype in 6 cases of GCTs. GAP43 was identified as a new NCC marker for GCTs, and some evidence was found for an APC phenotype from CD68 and HLA-DR immunoreactivity. RT-qPCR failed to identify an NCC developmental phenotype of GCTs, likely due to technical issues.

Ultrasound combined with nanobubbles promotes systemic anticancer immunity and augments anti-PD1 efficacy

BackgroundThe poor immunogenicity of solid tumors limits the efficacy ofanti-programmed cell death protein 1 (anti-PD1)-based immune checkpoint blockade (ICB); thus, less than 30% of patients with cancer exhibit a response....

A Novel Role for Histone Deacetylase 10 (HDAC10) in the Regulation of PD-L1 Expression and Immune Tolerance Mediated By Antigen Presenting Cells (APCs)

Antigen-presenting cells (APCs) play a central role in determining T-cell activation versus T-cell anergy in the context of tumor growth. The inflammatory status of APCs at the time of tumor antigen presentation has been proposed as explanation for the induction of such divergent T-cell outcomes. This overall inflammatory status of APCs can be regulated epigenetically.HDAC10 is a member of the class IIb family of HDACs that represses gene transcription independent of its deacetylase activity when tethered to a promoter. It is normally expressed in liver, kidney, pancreas and spleen and its overexpression has been shown to accelerate cancer development. While we previously demonstrated a role for HDAC6, the other member of the class IIb family, in control of APC inflammatory vs tolerogenic status (Cheng F et al, J Immunol 2014), little is known about the role (if any) of HDAC10 in regulating APC's function.Here we show for the first time that knockdown of HDAC10 in APCs, using shRNA specific for murine HDAC10 or non-target control (NT), resulted in the induction of inflammatory APCs displaying an 20-30% increased expression of MHC class II molecules and abrogation of PD-L1 gene transcription (4 fold decreased) and expression as compared to controls. To further confirm these results in vivo, HDAC10 knock out (KO) mice were used. These HDAC10 KO mice, as compared to wild type (WT) mice, reveal no gross macroscopic abnormalities in any lymphoid organs. Macrophages isolated from HDAC10 KO mice demonstrated increased expression of MHC II molecules and decreased expression of PD-L1, analogous to the shRNA data. Furthermore, in vitro co-culture of these macrophages with antigen-specific CD4+ T cells lead to effective priming of naïve CD4+ T-cells and restoration of the responsiveness of tolerant T-cells, as determined by their 3 fold increased production of IL-2 and IFN-g in response to cognate antigen. This demonstrates that APCs lacking HDAC10 are more immunogenic than control cells. Finally, to assess the role of HDAC10 in tumor immunity, we challenged HDAC10KO mice as well as WT control with murine mantle cell lymphoma (MCL) cells (FC-muMCL1 cells) given s.c. and measured tumor growth. A significant delay in MCL growth was observed in HDAC10KO mice as compared to WT controls (p<0.05). Studies are ongoing to assess the phenotype and function of APCs and T-cells isolated from these MCL-bearing mice.In summary, our studies have unveiled previously unknown immunoregulatory properties of HDAC10. In particular, its regulatory effects upon the expression of the tolerogenic molecule PD-L1, opens new frontiers for the understanding of PD-L1 regulation and points to disruption of the HDAC10/PD-L1 axis as a novel epigenetic target for cancer immunotherapy. DisclosuresSmith:Gilead: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite Pharma: Research Funding; Morphosys: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees.

Co-delivery of Phagocytosis Checkpoint Silencer and Stimulator of Interferon Genes Agonist for Synergetic Cancer Immunotherapy

Efficient capture and presentation of tumor antigens by antigen-presenting cells (APCs), especially dendritic cells (DCs), are crucial for activating the anti-tumor immunity. However, APCs are immunosuppressed in the tumor microenvironment, which hinders the tumor elimination. To reprogram APCs for inducing strong anti-tumor immunity, we report here a co-delivery immunotherapeutic strategy targeting the phagocytosis checkpoint (signal regulatory protein α, SIRPα) and stimulator of interferon genes (STING) of APCs to jointly enhance their ability of capturing and presenting tumor antigens. In brief, a small interfering RNA targeting SIRPα (siSIRPα) and a STING agonist (cGAMP) were co-delivered into APCs by the encapsulation into poly(ethylene glycol)-b-poly(lactide-co-glycolide)-based polymeric nanoparticles (NPsiSIRPα/cGAMP). siSIRPα-mediated SIRPα silence promoted APCs to actively capture tumor antigens by engulfing tumor cells. The cGAMP-stimulated STING signaling pathway further enhanced the functions of APCs, thereby increased the activation and expansion of CD8+ T cells. Using ovalbumin (OVA)-expressing melanoma as a model, we demonstrated that NPsiSIRPα/cGAMP stimulated the activation of OVA-specific CD8+ T cells and induced holistic anti-tumor immune responses by reversing the immunosuppressive phenotype of APCs. Collectively, this co-delivery strategy synergistically enhanced the functions of APCs and can be extended to the treatment of tumors with poor immunogenicity.

Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90.

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms – a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition – apart from its radiosensitizing potential – obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.

Tregitopes regulate the tolerogenic immune response and decrease the foetal death rate in abortion-prone mouse matings

The imbalance in immune tolerance may cause the variety of reproductive failures. An intravenous immunoglobulin infusion (IVIg) therapy is used to improve the live birth rate in women suffering from recurrent pregnancy loss, recurrent spontaneous abortions and recurrent implantation failures. However, the results of IVIg studies are still inconclusive as IVIg infusion in women suffering from pregnancy loss is sometimes ineffective. One of the mechanisms of action of this treatment is inhibition of B cells differentiation and expansion of Tregs and secretion of interleukin 10. It was proposed that immunomodulatory effects of IVIg may be attributed to tregitopes - self-IgG-derived epitopes present in the structure of immunoglobulins. Similarly to IVIg, tregitopes cause the expansion of Tregs and secretion of antigen-specific effector cytokine response. Here, we studied whether the administration of mouse tregitope 167 and/or 289 can prevent abortions in mouse abortion-prone mouse matings. We revealed that tregitopes reduce the foetal death rate. This may be driven by observed higher pool of peripheral Tregs, increased production of IL-10 by Tregs and Bregs and/or maintaining the tolerogenic phenotype of antigen-presenting cells. We believe that our findings may indicate a potential alternative to IVIg for therapeutic intervention in case of pregnancy failures.

Design of biodegradable nanoparticles to modulate phenotypes of antigen-presenting cells for antigen-specific treatment of autoimmune disease

Current therapeutic options for autoimmune diseases, such as multiple sclerosis (MS), often require lifelong treatment with immunosuppressive drugs, yet strategies for antigen-specific immunomodulation are emerging. Biodegradable particles loaded with disease-specific antigen, either alone or with immunomodulators, have been reported to ameliorate disease. Herein, we hypothesized that the carrier could impact polarization of the immune cells that associate with particles and the subsequent disease progression. Single injection of three polymeric carriers, 50:50 poly (DL-lactide-co-glycolide) (PLG) with two molecular weights (Low, High) and poly (DL-lactide) (PLA), loaded with the disease-specific antigen, proteolipid protein (PLP139-151), were investigated for the ability to attenuate clinical scores in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. At a low particle dose, mice treated with PLA-based particles had significantly lower clinical scores at the chronic stage of the disease over 200 days post immunization, while neither PLG-based particles nor OVA control particles reduced the clinical scores. Compared to PLG-based particles, PLA-based particles were largely associated with Kupffer cells and liver sinusoidal endothelial cells, which had a reduced co-stimulatory molecule expression that correlated with a reduction of CD4+ T-cell populations in the central nervous system. Delivery of PLA-based particles encapsulated with higher levels of PLP139-151 at a reduced dose were able to completely ameliorate EAE over 200 days along with inhibition of Th1 and Th17 polarization. Collectively, our study demonstrates that the carrier properties and antigen loading determine phenotypes of immune cells in the peripheral organs, influencing the amelioration of both acute and chronic stages of autoimmunity.

Streptococcal Pyrogenic Exotoxin A-Stimulated Monocytes Mediate Regulatory T-Cell Accumulation through PD-L1 and Kynurenine

Bacterial superantigens (SAgs) are exotoxins that promote a fulminant activation of the immune system. The subsequent intense release of inflammatory cytokines often results in hypotension, shock, and organ failure with high mortality rates. In the current paradigm, the direct and simultaneous binding of SAgs with T-cell receptor (TCR)-bearing Vβ regions and conserved structures on major histocompatibility complex class II (MHC class II) on antigen-presenting cells (APCs) induces the activation of both cell types. However, by crosslinking MHC class II molecules, APCs can be activated by SAgs independently of T lymphocytes. Recently, we showed that streptococcal pyrogenic exotoxin A (SPEA) of Streptococcus pyogenes stimulates an immunogenic APC phenotype with upregulated costimulatory molecules and inflammatory cytokines. Additionally, we revealed that SPEA triggers immunosuppressive programs in monocytes that facilitate the accumulation of regulatory T cells (Tregs) in in vitro monocyte/CD4+ T-cell cocultures. Immunosuppressive factors include anti-inflammatory interleukin 10 (IL-10), co-inhibitory surface molecule programmed cell death 1 ligand 1 (PD-L1), and the inhibitory indoleamine 2,3-dioxygenase (IDO)/kynurenine effector system. In the present study, we investigated the underlying mechanism of SPEA-stimulated monocyte-mediated accumulation of Tregs. Blood-derived monocytes from healthy donors were stimulated with SPEA for 48 h (SPEA-monocytes). For the evaluation of SPEA-monocyte-mediated modulation of CD4+ T lymphocytes, SPEA was removed from the culture through extensive washing of cells before adding allogeneic CD3/CD28-activated T cells. Results: In coculture with allogeneic CD4+ T cells, SPEA-monocytes mediate apoptosis of CD4+Foxp3− lymphocytes and accumulation of CD4+Foxp3+ Tregs. PD-L1 and kynurenine are critically involved in the mediated cell death because blocking both factors diminished apoptosis and decreased the proportion of the CD25+/Foxp3+ Treg subpopulation significantly. Upregulation of PD-L1 and kynurenine as well as SPEA-monocyte-mediated effects on T cells depend on inflammatory IL-1β. Our study shows that monocytes activated by SPEA mediate apoptosis of CD4+Foxp3− T effector cells through PD-L1 and kynurenine. CD4+Foxp3+ T cells are resistant to apoptosis and accumulate in SPEA-monocyte/CD4+ T-cell coculture.

Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance.

Background: Cancer-induced immunosuppression is antigen-specific rather than systemic and the mechanisms for the antigen specificity are incompletely understood. Here we explore the option that tumor-associated antigens (TAAs) may be transferred to antigen-presenting cells (APCs), together with immunosuppressive molecules, through cancer-derived small extracellular vesicles (sEVs), such as exosomes. Stimulation of a suppressive phenotype in the very same APCs that take up TAAs may yield antigen-specific tolerance. Methods: sEVs isolated from patient-derived or well-established melanoma cell lines were used to demonstrate the transfer of major histocompatibility complex (MHC) molecules to the surface of APCs. The immunosuppressive influence of sEVs was assessed by flow cytometry analysis of activation markers, cytokine expression, and mixed lymphocyte reactions. Results: MHC class I molecules were transferred from melanoma cells to the cell surface of APCs by sEVs. Concomitantly, CD86 and CD40 co-stimulatory molecules were down-regulated and IL-6 production was strongly induced. TGF-β transported by sEVs contributed to the promotion of a suppressive phenotype of APCs. Conclusion: The presented results indicate the existence of a hitherto undescribed mechanism that offers an explanation for antigen-specific tolerance induction mediated by cancer-derived sEVs.

Abstract LB-278: Vaccination with CD47-/- whole tumor cell vaccines creates a homogenous tumor microenvironment with distinct suppressor cell phenotypes

Abstract Cancer recurrence and metastasis accounts for approximately 90% of cancer-related deaths, despite which effective prophylactic measures are unavailable. Vaccination is a powerful tool for generating tumor-specific responses by exposing masked tumors to the immune system. Using CD47, a cell-surface protein that interacts with SIRP-α on macrophages as the target, we developed whole-cell cancer vaccines for the subcutaneous mouse melanoma. We immunized C57BL/6 mice using non-replicating CD47-/- B16F10 cells and challenged the mice with B16F10 tumors a week later. We showed that the vaccines effectively suppressed tumor growth: 33% of vaccinated mice remained tumor-free 90 days post tumor challenge, and the rest showed 2-fold reduced tumor growth rates and 5-fold reduced tumor sizes. Immunophenotyping showed a dramatic increase in activated effector cell subsets and M1-type macrophages aided by a significant reduction in the tumor-associated macrophage and myeloid-derived suppressor cell compartments. Additionally, we found that CD47 was down regulated in the vaccinated mice significantly. It has been emphasized in previous research that common characteristics of tumors that have been irreversible thus far like metastatic potential, downregulation of MHC genes, and overexpression of evasion markers, can be attributed to the specific suppressive phenotypes of antigen presenting cells in the tumor microenvironment (TME). In the tumors that did escape after vaccination, the TME was analyzed for immune phenotypes at three different stages of tumor growth - small (200-300mm3), medium (500-600mm3), and large (800-900mm3) tumor volumes. In comparison with the vehicle (PBS) vaccinated tumors, we found that in the CD47-/- B16F10 vaccinated tumors, the immune cells infiltrating the tumors maintain significantly homogenous levels throughout the different tumor growth stages. This was true especially for the suppressive phenotypes like the myeloid derived suppressor cells (MDSCs), the tumor-associated macrophages (TAMs), and the PD-1+ exhausted T cell subsets. Additionally, we performed deeper mechanistic analyses of the various TAM subsets infiltrating all the tumors and found that the CD47-/- B16F10 vaccinated tumors had significantly less TAMs, except for one specific subset - Ly6Chi MHC-IIlow TAMs. These TAMs are known to infiltrate in hypoxic conditions and we found corresponding increase in neo-vasculature around these tumors. These TAMs were also found to express high levels of Ly6G, the neutrophil lineage marker. Overall, our preliminary characterization showed that vaccination with syngeneic CD47-/- tumor cells orchestrates the levels and enhances distinct subtypes of tumor infiltrating suppressive lymphocytes, opening up avenues for further combinatorial therapy regimes and aiding the process of turning “cold” tumors “hot”. Citation Format: Subhadra Jayaraman Rukmini, Huanjing Bi, Puloma Sen, Benjamin Everhart, Busra Buyuk, Sha Jin, Kaiming Ye. Vaccination with CD47-/- whole tumor cell vaccines creates a homogenous tumor microenvironment with distinct suppressor cell phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-278.

Bidirectional regulatory potentials of short-chain fatty acids and their G-protein-coupled receptors in autoimmune neuroinflammation

Microbial metabolites, produced in the intestine, have significant effects on inflammatory diseases throughout the body. Short-chain fatty acids (SCFAs) have protective effects on experimental autoimmune encephalitis (EAE) responses but the detailed roles of SCFAs and their receptors in regulating autoimmune CNS inflammation have been unclear. SCFAs metabolically regulate T cells and change the phenotype of antigen presenting cells to efficiently induce IL-10+ regulatory T cells. In line with the overall protective effect, blood levels of major SCFAs, such as acetate, propionate and butyrate, are significantly decreased in long-term active progressive multiple sclerosis (MS) patients. Importantly, SCFAs can induce CD4+ effector T cells, which are highly inflammatory when transferred into mice, suggesting that the direct effect of SCFAs on T cells can even be pro-inflammatory in the CNS. In contrast to the moderate protective effect of SCFAs, mice deficient in GPR41 or GPR43 are more resistant to EAE pathogenesis. Thus, despite the overall protective function of SCFAs, SCFAs and their receptors have the potential to regulate autoimmune CNS inflammation both positively and negatively.

Regional and functional heterogeneity of antigen presenting cells in the mouse brain and meninges

The central nervous system (CNS) is considered to be immune privileged, owing in part to the absence of major histocompatibility (MHC) class II+ cells in the healthy brain parenchyma. However, systemic inflammation can activate microglia to express MHC class II, suggesting that systemic inflammation may be sufficient to mature microglia into functional antigen presenting cells (APCs). We examined the effects of systemic lipopolysaccharide (LPS)-induced inflammation on the phenotype and function of putative APCs within the mouse brain parenchyma, as well as its supporting tissues-the choroid plexus and meninges. Microglia isolated from different regions of the brain demonstrated significant heterogeneity in their ability to present antigen to naïve OT-II CD4+ T cells following exposure to systemic LPS. Olfactory bulb microglia (but not cortical microglia) intimately interacted with T cells in vivo and stimulated T cell proliferation in vitro, albeit in the absence of co-stimulation. In contrast, myeloid cells within the choroid plexus and meninges were immunogenic and upregulated the co-stimulatory molecule CD80 following systemic inflammation. Dural APCs, which clustered around LYVE-1+ lymphatics, were more efficient at stimulating naïve T cell proliferation than choroid plexus APCs, suggesting that the dura may be an under-appreciated site for immune interactions. This study has highlighted the functional diversity of myeloid cells within the sub-compartments of the CNS and its supporting tissues. Furthermore, these findings demonstrate that systemic inflammation can mature selected microglia populations and choroid plexus/meningeal myeloid cells into functional APCs, which may contribute to the pathogenesis of neuroinflammation and neurodegenerative diseases.

Bone marrow endothelial cells sustain a tumor-specific CD8+ T cell subset with suppressive function in myeloma patients

ABSTRACT Endothelial cells (EC) line the bone marrow microvasculature and are in close contact with CD8+ T cells that come and go across the permeable capillaries. Because of these intimate interactions, we investigated the capacity of EC to act as antigen-presenting cells (APC) and modulate CD8+ T cell activation and proliferation in bone marrow of patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance. We found that EC from MM patients show a phenotype of semi-professional APC given that they express low levels of the co-stimulatory molecules CD40, CD80 and CD86, and of the inducible co-stimulator ligand (ICOSL). In addition, they do not undergo the strong switch from immunoproteasome to standard proteasome subunit expression which is typical of mature professional APC such as dendritic cells. EC can trap and present antigen to CD8+ T cells, stimulating a central memory CD8+ T cell population that expresses Foxp3 and produces high amounts of IL-10 and TGF-β. Another CD8+ T cell population is stimulated by professional APC, produces IFN-γ, and exerts antitumor activity. Thus, two distinct CD8+ T cell populations coexist in the bone marrow of MM patients: the first population is sustained by EC, expresses Foxp3, produces IL-10 and TGF-β, and exerts pro-tumor activity by negatively regulating the second population. This study adds new insight into the role that EC play in MM biology and describes an additional immune regulatory mechanism that inhibits the development of antitumor immunity and may impair the success of cancer immunotherapy.

Antigen-Presenting Cell Characteristics of Human γδ T Lymphocytes in Chronic Myeloid Leukemia

ABSTRACTHuman γδ T lymphocytes play a role in the immune system defense against cancer. Their broad anti-cancer activity against different types of cancers makes them outstanding candidates for cancer immunotherapy. An issue of recent interest is whether their antigen presentation features are similar to mature dendritic cells. The antigen-presenting cell (APC)-like phenotype and function of γδ T lymphocytes have been confirmed in many clinical trials. In this study, to support the strong role played by Vγ9Vδ2 T cells against cancer, we provide evidence that Vγ9Vδ2 T cells activated with chronic myeloid leukemia (CML) cell lysate antigens can efficiently express an APC phenotype and function. Vγ9Vδ2 T cells derived from normal peripheral blood mononuclear cells were activated with tumor cell lysate, and the tumor-activated Vγ9Vδ2 T cells could recognize and kill CML through their cytotoxic activity. In conclusion, the Vγ9Vδ2 T cells activated by cancer cell lysate showed APC characteristics, and this may greatly increase interest in investigating their therapeutic potential in hematologic malignancies.Abbreviations: CML: chronic myeloid leukemia; APC: antigen-presenting cell; TCR: T cell receptor; MHC: major histocompatibility complex; N-BPs: nitrogen-containing bisphosphonates; IPP: isopentenyl pyrophosphate; PBMC: peripheral blood mononuclear cells; NKG2D: natural killer receptor group 2, member D; TRAIL: tumor necrosis factor-related apoptosis-inducing ligand.

Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.

Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.

Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells.

MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs), that are facilitated to engulf antigen and transport it to draining lymph node (dLN) where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs). Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE) as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of vaccine adjuvants based on emulsions.

Pro-inflammatory chitosan/poly(γ-glutamic acid) nanoparticles modulate human antigen-presenting cells phenotype and revert their pro-invasive capacity.

The immune system is responsible to detect and destroy abnormal cells preventing the development of cancer. However, the immunosuppressive tumor microenvironment can compromise the immune response favoring tumor progression. Thus, immune system modulation towards an immunostimulatory profile can improve anticancer therapies. This research focus on the development of chitosan/poly(γ-glutamic acid) nanoparticles (NPs) to modulate human antigen-presenting cells (APCs) phenotype and to counteract their pro-invasive capacity. Interestingly, Ch/γ-PGA NPs had a prominent effect in inducing macrophages and dendritic cells immunostimulatory phenotype, thus favoring T cell proliferation and inhibiting colorectal cancer cell invasion. We propose that their combination with other immunomodulatory drugs or conventional anticancer therapies can improve patients' outcome.