Immunotherapy-induced cytotoxic T follicular helper cells reduce numbers of retrovirus-infected reservoir cells in B cell follicles.

Antiretroviral therapy (ART) transformed HIV from a life-threatening disease to a chronic condition. However, eliminating the virus remains an elusive therapy goal. For several decades, Friend virus (FV) infection serves as a murine model to study retrovirus immunity. Similar to HIV, FV persists at low levels in lymph nodes B cell follicles avoiding elimination by immune cells. Such immune-privileged reservoirs exclude cytotoxic T cells from entry. However, CXCR5+ T cells are permitted to traffic through germinal centers. This marker is predominantly expressed by CD4+ follicular helper T cells (Tfh). Therefore, we explored immunotherapy to induce cytotoxic Tfh, which are rarely found under physiological conditions. The TNF receptor family member CD137 was first identified as a promising target for cancer immunotherapy. We demonstrated that FV-infected mice treatment with αCD137 antibody resulted in an induction of the cytotoxic program in Tfh. The therapy significantly increased numbers of cytotoxic Tfh within B cell follicles and contributed to viral load reduction. Moreover, αCD137 antibody combined with ART delayed virus rebound upon treatment termination without disturbing the lymph node architecture or antibody responses. Thus, αCD137 antibody therapy might be a novel strategy to target the retroviral reservoir and an interesting approach for HIV cure research.

HPV-associated head and neck cancer is characterized by distinct profiles of CD8+ T cells and myeloid-derived suppressor cells.

Patients with HPV--localized head and neck cancer (HNC) show inferior outcomes after surgery and radiochemotherapy compared to HPV-associated cancers. The underlying mechanisms remain elusive, but differences in immune status and immune activity may be implicated. In this study, we analyzed immune profiles of CD8+ T cells and myeloid-derived suppressor cells (MDSC) in HPV+ versus HPV- disease.The overall frequency of CD8+ T cells was reduced in HNC versus healthy donors but substantially increased after curative therapy (surgery and/or radiochemotherapy). In HPV+ patients, this increase was associated with significant induction of peripheral blood CD8+/CD45RA-/CD62L- effector memory cells. The frequency of HPV-antigen-specific CD8+ cells was low even in patients with virally associated tumors and dropped to background levels after curative therapy. Pre-therapeutic counts of circulating monocytic MDSC, but not PMN-MDSC, were increased in patients with HPV- disease. This increase was accompanied by reduced fractions of terminally differentiated CD8+ effector cells. HPV- tumors showed reduced infiltrates of CD8+ and CD45RO+ immune cells compared with HPV+ tumors. Importantly, frequencies of tumor tissue-infiltrating PMN-MDSC were increased, while percentages of Granzyme B+ and Ki-67+ CD8 T cells were reduced in patients with HPV- disease.We report differences in frequencies and relative ratios of MDSC and effector T cells in HPV- HNC compared with more immunogenic HPV-associated disease. Our data provide new insight into the immunological profiles of these two tumor entities and may be utilized for more tailored immunotherapeutic approaches in the future.

TNF-α and IL-1ß sensitize human MSC for IFN-γ signaling and enhance neutrophil recruitment.

During inflammatory processes, tissue environmental cues are influencing the immunoregulatory properties of tissue-resident mesenchymal stem/stromal cells (MSC). In this study, we elucidated one of the molecular and cellular responses of human MSC exposed to combinations of inflammatory cytokines. We showed that during multi-cytokine priming by TNF-α, IL-1ß, and IFN-γ, IL-1ß further augmented the well-established immunoregulatory activity induced by TNF-α/IFN-γ. On the molecular level, TNF-α and IL-1ß enhanced the expression of IFN-γ receptor (IFN-γR) via NF 'kappa-light-chain-enhancer' of activated B-cells (NF-κΒ) signaling. In turn, enhanced responsiveness to IFN-γ stimulation activated STAT5 and p38-MAPK signaling. This molecular feedback resulted in an increased IL-8 release and augmented recruitment of polymorphonuclear granulocytes (PMN). Our study suggests the possibility that responses of MSC to multi-cytokine priming regimens may be exploited therapeutically to fine-tune inflammatory activity in tissues. This study elucidates molecular mechanisms underlying the immunological priming of mesenchymal stromal cells (MSC) and their interaction with neutrophils.

Cutting edge: An inactive chromatin configuration at the IL-10 locus in human neutrophils.

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. We analyzed posttranslational modifications of histones associated with genes that are active, repressed, or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac, and H3K4me1 marks. Differently from autologous IL-10-producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils from healthy subjects or melanoma patients. By contrast, increased H3K4me3, H4Ac, H3K4me1, and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10.

Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors.

Precise molecular characterization of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is hampered by their mixed composition of mature and immature cells and lack of specific markers. Here, we focus on mature CD66b+CD10+CD16+CD11b+ PMN-MDSCs (mPMN-MDSCs) from either cancer patients or healthy donors receiving G-CSF for stem cell mobilization (GDs). By RNA sequencing (RNA-seq) experiments, we report the identification of a distinct gene signature shared by the different mPMN-MDSC populations under investigation, also validated in mPMN-MDSCs from GDs and tumor-associated neutrophils (TANs) by single-cell RNA-seq (scRNA-seq) experiments. Analysis of such a gene signature uncovers a specific transcriptional program associated with mPMN-MDSC differentiation and allows us to identify that, in patients with either solid or hematologic tumors and in GDs, CD52, CD84, and prostaglandin E receptor 2 (PTGER2) represent potential mPMN-MDSC-associated markers. Altogether, our findings indicate that mature PMN-MDSCs distinctively undergo specific reprogramming during differentiation and lay the groundwork for selective immunomonitoring, and eventually targeting, of mature PMN-MDSCs.

Immunological priming of mesenchymal stromal/stem cells and their extracellular vesicles augments their therapeutic benefits in experimental graft-versus-host disease via engagement of PD-1 ligands.

Mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) exert profound anti-inflammatory and regenerative effects in inflammation and tissue damage, which makes them an attractive tool for cellular therapies. In this study we have assessed the inducible immunoregulatory properties of MSCs and their EVs upon stimulation with different combinations of cytokines. First, we found that MSCs primed with IFN-γ, TNF-α and IL-1ß, upregulate the expression of PD-1 ligands, as crucial mediators of their immunomodulatory activity. Further, primed MSCs and MSC-EVs, compared to unstimulated MSCs and MSC-EVs, had increased immunosuppressive effects on activated T cells and mediated an enhanced induction of regulatory T cells, in a PD-1 dependent manner. Importantly, EVs derived from primed MSCs reduced the clinical score and prolonged the survival of mice in a model of graft-versus-host disease. These effects could be reversed in vitro and in vivo by adding neutralizing antibodies directed against PD-L1 and PD-L2 to both, MSCs and their EVs. In conclusion, our data reveal a priming strategy that potentiates the immunoregulatory function of MSCs and their EVs. This concept also provides new opportunities to improve the clinical applicability and efficiency of cellular or EV-based therapeutic MSC products.

Subconscious olfactory influences of stimulant and relaxant odors on immune function.

Brain and immune system are linked by bidirectional pathways so that changes of the central nervous system may influence various immune functions. The olfactory system may be involved in this interaction. In most odor studies subjects are aware of an odor exposure, using frequently high odor concentrations or long-term exposures without controls. In this pilot study, the potential immune effects of short-term odor exposure were examined in 32 blinded subjects (16 male, 16 female). Subjects were exposed without their knowledge either to a stimulant essential oil (grapefruit, fennel, pepper), a no-odor control or a relaxant essential oil (lavender, patchouli, rose) during a set of psychological questionnaires for 30 min at three separate visits. Activity of neutrophil granulocytes (CXCL8 release, CD16) and peripheral blood concentrations of mainly neutrophil-related immunological markers were measured. We tested the triple of stimulant odor, control and relaxant odor for every subject in a model which assumed opposite effects of the stimulant and the relaxant odor. This hypothesis was falsified by our experimental data, as no significant effect was observed for the parameters tested. The human immune functions tested in our study are not modulated by short-term odor exposure in blinded subjects. Further studies should directly dissect possible differences between long-term and short-term exposures of non-blinded subjects versus blinded subjects.

Macrophage-Orbital Fibroblast Interaction and Hypoxia Promote Inflammation and Adipogenesis in Graves' Orbitopathy.

The inflammatory eye disease Graves' orbitopathy (GO) is the main complication of autoimmune Graves' disease. In previous studies we have shown that hypoxia plays an important role for progression of GO. Hypoxia can maintain inflammation by attracting inflammatory cells such as macrophages (MQ). Herein, we investigated the interaction of MQ and orbital fibroblasts (OF) in context of inflammation and hypoxia. We detected elevated levels of the hypoxia marker HIF-1α, the MQ marker CD68, and inflammatory cytokines TNFα, CCL2, CCL5, and CCL20 in GO biopsies. Hypoxia stimulated GO tissues to release TNFα, CCL2, and CCL20 as measured by multiplex enzyme-linked immunosorbent assay (ELISA). Further, TNFα and hypoxia stimulated the expression of HIF-1α, CCL2, CCL5, and CCL20 in OF derived from GO tissues. Immunofluorescence confirmed that TNFα-positive MQ were present in the GO tissues. Thus, interaction of M1-MQ with OF under hypoxia also induced HIF-1α, CCL2, and CCL20 in OF. Inflammatory inhibitors etanercept or dexamethasone prevented the induction of HIF-1α and release of CCL2 and CCL20. Moreover, co-culture of M1-MQ/OF under hypoxia enhanced adipogenic differentiation and adiponectin secretion. Dexamethasone and HIF-1α inhibitor PX-478 reduced this effect. Our findings indicate that GO fat tissues are characterized by an inflammatory and hypoxic milieu where TNFα-positive MQ are present. Hypoxia and interaction of M1-MQ with OF led to enhanced secretion of chemokines, elevated hypoxic signaling, and adipogenesis. In consequence, M1-MQ/OF interaction results in constant inflammation and tissue remodeling. A combination of anti-inflammatory treatment and HIF-1α reduction could be an effective treatment option.

Tumor-derived macrophage migration inhibitory factor modulates the biology of head and neck cancer cells via neutrophil activation.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been reported to enhance the aggressiveness and metastatic potential of tumor cells. However, the mechanisms through which MIF influences tumor development and progression are not understood. The objectives of our study were to assess the effects of tumor-derived MIF on neutrophils in head and neck cancer (HNC) and to identify possible feedback effects on tumor cells. To this end, we used an in vitro system to model the interaction between human HNC cells and neutrophils. In addition, we analyzed expression of MIF in tissues from HNC patients in relation to neutrophilic infiltration and clinical parameters. Our results show that human HNC is infiltrated by neutrophils proportional to the levels of tumoral MIF. Strong MIF expression by the tumor is associated with higher lymph node metastasis and reduced survival in HNC patients. In vitro, MIF modulated functions of human neutrophils by inducing chemokine CXC motif receptor 2(CXCR2)-dependent chemotaxis, enhancing neutrophil survival and promoting release of chemokine C-C Motif Ligand 4 (CCL4) and matrix metalloprotease 9(MMP9). Further, neutrophils activated with tumor-derived MIF enhanced migratory properties of HNC cells. In conclusion, our data indicate that the effects of tumor-derived MIF on neutrophils represent an additional mechanism by which MIF might contribute to tumor progression.

Polymorphonuclear granulocytes in human head and neck cancer: enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease.

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN-related alterations in HNSCC, the role of tumor-infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN-related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil-to-lymphocyte ratio and serum concentrations of CXCL8 (interleukin-8), CCL4 (MIP-1ß) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC-conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor-derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host-mediated changes in the tumor microenvironment.

Immunophenotyping of Circulating Myeloid-Derived Suppressor Cells (MDSC) in the Peripheral Blood of Cancer Patients.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of pathologically expanded myeloid cells with immunosuppressive activity. According to their phenotype, MDSC can be divided into three major subpopulations: early stage MDSC (e-MDSC), lacking myeloid lineage markers, monocytic MDSC (M-MDSC), and granulocytic MDSC (PMN-MDSC). Additionally, PMN-MDSC can be subdivided based on their activation and differentiation status, although it is not clear how this status contributes to immunosuppression and disease pathology. Here, we describe an immunophenotyping and gating strategy for the identification and isolation of MDSC subsets based on fluorescence-activated cell sorting. This method allows direct comparison of MDSC subsets in clinical settings.

Isolation of Human Circulating Myeloid-Derived Suppressor Cells and Analysis of Their Immunosuppressive Activity.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of myeloid cells with potent immunosuppressive activity and characterized by a pathological state of activation. The T cell suppression assay is the most common method to evaluate the suppressive capacity of MDSC. Identifying the suppressive potential of different MDSC subsets within individual donors is key for understanding the biology of MDSC and their clinical relevance. Here we describe assays to ascertain and quantify the suppression of autologous T cells by human MDSC. These include the dye dilution proliferation assay for flow cytometry and the detection of IFNγ production by T cells using flow cytometry and sandwich ELISA.

Proteomic and bioinformatic profiling of neutrophils in CLL reveals functional defects that predispose to bacterial infections.

Patients with chronic lymphocytic leukemia (CLL) typically suffer from frequent and severe bacterial infections. Although it is well known that neutrophils are critical innate immune cells facilitating the early defense, the underlying phenotypical and functional changes in neutrophils during CLL remain largely elusive. Using a murine adoptive transfer model of CLL, we demonstrate aggravated bacterial burden in CLL-bearing mice upon a urinary tract infection with uropathogenic Escherichia coli. Bioinformatic analyses of the neutrophil proteome revealed increased expression of proteins associated with interferon signaling and decreased protein expression associated with granule composition and neutrophil migration. Functional experiments validated these findings by showing reduced levels of myeloperoxidase and acidification of neutrophil granules after ex vivo phagocytosis of bacteria. Pathway enrichment analysis indicated decreased expression of molecules critical for neutrophil recruitment, and migration of neutrophils into the infected urinary bladder was significantly reduced. These altered migratory properties of neutrophils were also associated with reduced expression of CD62L and CXCR4 and correlated with an increased incidence of infections in patients with CLL. In conclusion, this study describes a molecular signature of neutrophils through proteomic, bioinformatic, and functional analyses that are linked to a reduced migratory ability, potentially leading to increased bacterial infections in patients with CLL.

Inflammation-driven activation of JAK/STAT signaling reversibly accelerates acute myeloid leukemia in vitro.

Acute myeloid leukemia (AML) is characterized by a high relapse rate and dismal long-term overall survival which is related to persistence of leukemia-initiating cells in their niche. Different animal models of myeloid malignancies reveal how neoplastic cells alter the structural and functional characteristics of the hematopoietic stem cell niche to reinforce malignancy. Understanding and disruption of the microenvironmental interactions with AML cells are a vital need. Malignant niches frequently go along with inflammatory responses, but their impact on cancerogenesis often remains unexplored. Here, we uncovered an aberrant production of inflammatory cytokines in untreated AML bone marrow that was proved to promote the proliferation of leukemia cells. This inflammatory response induced an activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway in AML blasts as well as bone marrow stromal cells that also fostered leukemia proliferation. Inhibition of JAK/STAT signaling using the selective JAK1/2 inhibitor ruxolitinib resulted in significant antileukemic activity in AML in vitro which is mediated through both cell-autonomous and microenvironment-mediated mechanisms. However, in a xenograft transplantation model, monotherapy with ruxolitinib did not achieve substantial antileukemic activity, possibly suggesting a complementary function of JAK1/2 inhibition in AML.

Protocol to assess the suppression of T-cell proliferation by human MDSC.

Inhibition of T-cell proliferation is the most common approach to assess human myeloid-derived suppressor cell (MDSC) functions. However, diverse methodologies hinder the comparison of results obtained in different laboratories. In this chapter, we present a T-cell proliferation assay procedure based on allogeneic MDSC and T-cells that is potentially suitable to multi-center studies. The T-cells are isolated from non-cancerous donors and frozen for later use in different research groups. We observed that pure thawed T-cells showed poor proliferative capacities. To retain proliferation, T-cell-autologous mature dendritic cells are supplemented after thawing. MDSC are isolated from clinical samples and represent the sole variant between assays. Flow cytometry is used to assess T-cell proliferation by the dilution of a tracking dye.

Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation.

BACKGROUND: Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. METHODS: We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. RESULTS: We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. CONCLUSIONS: This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation.

Chemoirradiated neutrophils and T cells differentially affect immune functions of APCs.

Extracorporeal photopheresis (ECP) is known as an immunomodulatory therapy with few side effects, which is mainly used in the treatment of cutaneous T cell lymphoma, graft-versus-host disease, and allograft rejection. During ECP, leukocytes are separated from whole blood by leukapheresis, subsequently chemoirradiated with 8-methoxypsoralen and UVA light, and re-infused into the patient. Although clinically effective, its mode of action has not been fully elucidated. In the present study, we analyzed the interaction of chemoirradiated neutrophils and CD3+ lymphocytes with APC in an in vitro model. We report that chemoirradiated CD3+ T cells induced increased expression of activation markers on dendritic cells (DC), macrophages, and monocytes. Coculture of chemoirradiated CD3+ T cells with these APC also led to significantly increased secretion of TNF-α. Although less pronounced, additional activation of APC took place when APC were stimulated with LPS or IFN-γ. In contrast, chemoirradiated neutrophils did not show activating effects on APC. The presence of chemoirradiated neutrophils during LPS and IFN-γ stimulation of DC rather diminished DC and macrophage activation. In line with these findings DC cocultured with chemoirradiated CD3+ T cells, but not neutrophils, showed significantly increased activation of CD3+ responder lymphocytes in a mixed lymphocyte reaction. With this study, we demonstrate that chemoirradiated leukocytes have differential indirect immunomodulatory effects. Whereas chemoirradiated CD3+ T cells activate APC, chemoirradiated neutrophils suppress activation of APC in the presence of other activating factors, suggesting that the composition of the ECP-treated buffy coat might be of importance for its immunomodulatory effects.

Multidimensional imaging provides evidence for down-regulation of T cell effector function by MDSC in human cancer tissue.

A high intratumoral frequency of neutrophils is associated with poor clinical outcome in most cancer entities. It is hypothesized that immunosuppressive MDSC (myeloid-derived suppressor cell) activity of neutrophils against tumor-reactive T cells contributes to this effect. However, direct evidence for such activity in situ is lacking. Here, we used whole-mount labeling and clearing, three-dimensional (3D) light sheet microscopy and digital image reconstruction supplemented by 2D multiparameter immunofluorescence, for in situ analyses of potential MDSC-T cell interactions in primary human head and neck cancer tissue. We could identify intratumoral hotspots of high polymorphonuclear (PMN)-MDSC and T cell colocalization. In these areas, the expression of effector molecules Granzyme B and Ki67 in T cells was strongly reduced, in particular for T cells that were in close proximity or physically engaged with PMN-MDSC, which expressed LOX-1 and arginase I. Patients with cancer with evidence for strong down-regulation of T cell function by PMN-MDSC had significantly impaired survival. In summary, our approach identifies areas of clinically relevant functional interaction between MDSC and T cells in human cancer tissue and may help to inform patient selection in future combination immunotherapies.

High Salt Inhibits Tumor Growth by Enhancing Anti-tumor Immunity.

Excess salt intake could affect the immune system by shifting the immune cell balance toward a pro-inflammatory state. Since this shift of the immune balance is thought to be beneficial in anti-cancer immunity, we tested the impact of high salt diets on tumor growth in mice. Here we show that high salt significantly inhibited tumor growth in two independent murine tumor transplantation models. Although high salt fed tumor-bearing mice showed alterations in T cell populations, the effect seemed to be largely independent of adaptive immune cells. In contrast, depletion of myeloid-derived suppressor cells (MDSCs) significantly reverted the inhibitory effect on tumor growth. In line with this, high salt conditions almost completely blocked murine MDSC function in vitro. Importantly, similar effects were observed in human MDSCs isolated from cancer patients. Thus, high salt conditions seem to inhibit tumor growth by enabling more pronounced anti-tumor immunity through the functional modulation of MDSCs. Our findings might have critical relevance for cancer immunotherapy.