Suppress T-cell Responses Research Articles

Advances in Immune Microenvironment and Immunotherapy of Isocitrate Dehydrogenase Mutated Glioma.

The tumor immune microenvironment and immunotherapy have become current important tumor research concerns. The unique immune microenvironment plays a crucial role in the malignant progression of isocitrate dehydrogenase (IDH) mutant gliomas. IDH mutations in glioma can inhibit tumor-associated immune system evasion of NK cell immune surveillance. Meanwhile, mutant IDH can inhibit classical and alternative complement pathways and directly inhibit T-cell responses by metabolizing isocitrate to D-2-Hydroxyglutaric acid (2-HG). IDH has shown clinically relevant efficacy as a potential target for immunotherapy. This article intends to summarize the research progress in the immunosuppressive microenvironment and immunotherapy of IDH-mutant glioma in recent years in an attempt to provide new ideas for the study of occurrence, progression, and treatment of IDH-mutant glioma.

Immunometabolism of Myeloid-Derived Suppressor Cells: Implications for Mycobacterium tuberculosis Infection and Insights from Tumor Biology.

The field of immunometabolism seeks to decipher the complex interplay between the immune system and the associated metabolic pathways. The role of small molecules that can target specific metabolic pathways and subsequently alter the immune landscape provides a desirable platform for new therapeutic interventions. Immunotherapeutic targeting of suppressive cell populations, such as myeloid-derived suppressor cells (MDSC), by small molecules has shown promise in pathologies such as cancer and support testing of similar host-directed therapeutic approaches in MDSC-inducing conditions such as tuberculosis (TB). MDSC exhibit a remarkable ability to suppress T-cell responses in those with TB disease. In tumors, MDSC exhibit considerable plasticity and can undergo metabolic reprogramming from glycolysis to fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) to facilitate their immunosuppressive functions. In this review we look at the role of MDSC during M. tb infection and how their metabolic reprogramming aids in the exacerbation of active disease and highlight the possible MDSC-targeted metabolic pathways utilized during M. tb infection, suggesting ways to manipulate these cells in search of novel insights for anti-TB therapies.

Exosomes derived from γδ-T cells synergize with radiotherapy and preserve antitumor activities against nasopharyngeal carcinoma in immunosuppressive microenvironment

BackgroundRadiotherapy is the first-line treatment for patients nasopharyngeal carcinoma (NPC), but its therapeutic efficacy is poor in some patients due to radioresistance. Adoptive T cell-based immunotherapy has also shown promise...

Loss of Indoleamine-2,3-Dioxygenase-1 (IDO1) in Knockout Mice Does Not Affect the Development of Skin Lesions in the Imiquimod-Induced Mouse Model of Psoriasis.

Indoleamine-2,3-dioxygenase (IDO) degrades the essential amino acid tryptophan resulting in tryptophan depletion and the accumulation of catabolites such as kynurenine. The expression/activity of IDO in various cells, including macrophages and dendritic cells, results in an inhibition of T-cell responses in a number of situations, such as toward allogeneic fetuses and tissue grafts. Psoriasis is an immune-mediated skin disease involving T cells; kynureninase and its generation of catabolites downstream of IDO are reported to play an important role in this disease. We hypothesized that mice lacking the IDO1 gene would exhibit a hyperactive immune response and an exacerbation of skin lesions in the imiquimod-induced mouse model of psoriasis. Littermate wild-type and IDO1-knockout mice were treated with imiquimod for 5 days, and the severity of psoriasiform skin lesions assessed using the psoriasis area and severity index (PASI), ear edema measured using a digital caliper, and thickness of the epidermis determined by histology. Expression of pro-inflammatory mediators and tryptophan-metabolizing enzymes was monitored using quantitative RT-PCR. Imiquimod increased ear edema, PASI scores, and epidermal thickness in both WT and IDO1 knockout mice; however, there were no differences observed between the 2 genotypes. There were also no differences in imiquimod’s induction of skin inflammatory mediators, indicating no effect of IDO1 gene loss in this psoriasis model. Although these data suggest a lack of involvement of IDO1 in psoriatic skin inflammation, other possible mechanisms, such as compensatory changes in other pathways and the involvement of the IDO2 isoform, must also be considered.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

Nanotubes mediate mitochondrial transfer to tumor cells (by Michael Ströck via Wikimedia Commons)Tumors use various mechanisms to escape immune detection in the tumor microenvironment (TME). Saha et al. show that tumor cells form nanotubes that physically interact with immune cells in the breast cancer TME. Mitochondria are then transferred from immune cells to tumor cells, leading to tumor metabolic enhancement and loss of specific immune-cell subsets needed for antitumor responses. Targeting nanotube formation reversed these effects and when combined with immune checkpoint blockade, resulted in improved outcomes in a murine breast cancer model. The data highlight a new immune “hijacking” mechanism that tumors cells could use to promote cancer progression.Saha T, …, Sengupta S. Nat Nanotechnol 2021 Nov 18. DOI: 10.1038/s41565-021-01000-4.Lung cancers can become infiltrated by dysfunctional CD8+ T cells (by Eric Synder via NCI Visuals Online)Many non–small cell lung cancers (NSCLC) infiltrated with CD8+ T cells are resistant to immune checkpoint blockade. In an orthotopic NSCLC model, Horton et al. find lung tumors are infiltrated with CD8+ T cells but do not respond to combination anti–CTLA-4 and anti–PD-1. The CD8+ T cells have a dysfunctional phenotype molecularly distinct from T-cell exhaustion. The dysfunctional phenotype is induced during T-cell priming in lymph nodes and characterized by failure to acquire an effector program. Treatment with IL2 and IL12 restores effector T-cell differentiation. As CD8+ T cells from NSCLC patients have an analogous gene expression program of dysfunction, the data have translational potential.Horton BL, …, Spranger S. Sci Immunol 2021 Oct 29;6:eabi8800.γδ T cells infiltrate many tumor types and are highly cytotoxic (from Fig. 5C of Gherardin et al., Cancer Immunol Res 2021)Antibodies specific for butyrophilin 3A (BTN3A) can induce Vγ9Vδ2 T–cell cytolytic activity. De Gassart et al. have engineered a humanized BTN3A-specific antibody (ICT01) that induces Vγ9Vδ2 T–cell killing of several human tumor cell lines and primary tumor cells in vitro and xenografted tumors in mice following Vγ9Vδ2 T–cell transfer. Preliminary data from six patients with solid tumors administered ICT01 in the phase I/IIa EVICTION clinical trial (NCT04243499) show the antibody can be well tolerated, has caused no major safety events, and appears to induce broad immune-cell infiltration of tumors. Further studies should reveal the full potential of this strategy.De Gassart A, …, Frohna P. Sci Transl Med 2021 Oct 20;13:eabj0835.Histamine can suppress antitumor responses (by NEUROtiker via Wikimedia Commons)Identification of obstacles to effective antitumor immunity sometimes reveals surprising participants. Li et al. find that histamine, a mediator of allergic responses, plays an inhibitory role in antitumor responses. Histamine is frequently found in the tumor microenvironment and can activate macrophages via histamine receptor H1 (HRH1), promoting polarization to a suppressive M2-like phenotype and upregulation of the inhibitory checkpoint VISTA. Such macrophages suppress T-cell responses and promote tumor growth and therapy resistance. Knockout of HRH1 or use of antihistamines rescues experimental antitumor responses. The study highlights a role for histamine in modulating antitumor responses and suggests antihistamines as potential adjuvants for cancer immunotherapies.Li H, …, Yu D. Cancer Cell 2021 Nov 24. DOI: 10.1016/j.ccell.2021.11.002.PC-CAR T cells could unlock the immunotherapeutic potential of intracellular antigens (by Paris on Ponce & Le Maison Rouge via Flickr)Developing chimeric receptor (CAR) T cells specific for intracellular tumor-specific antigens is challenging because these antigens are only available to the immune system as peptides presented on the cell surface by MHC class I. Yarmarkovich et al. overcome this challenge by generating peptide-centric CARs (PC-CAR), which can recognize peptides derived from intracellular oncoproteins presented by multiple HLA types. PC-CAR T cells targeting a peptide from the neuroblastoma oncoprotein PHOX2B selectively kill neuroblastoma cells in vitro and clear tumors transplanted into mice. This study indicates PC-CARs have the potential to unlock the vast number of intracellular oncoproteins as potential targets for CAR T-cell immunotherapy.Yarmarkovich M, …, Maris JM. Nature 2021 Nov 3;599:477–84.TILs accumulate in ovarian cancer islets (from Fig. 1C of Heath et al., Cancer Immunol Res 2021)The tumor microenvironment (TME) factors that regulate exhaustion of CD8+ tumor-infiltrating lymphocytes (TIL) are not fully understood. Duraiswamy et al. found activated, tumor-specific CD8+ TILs concentrated in ovarian cancer islets, where they are closely associated with myeloid antigen-presenting cells (APC) but restrained by PD-1 signaling. Activated islet-resident APCs can provide CD28 costimulation, which is required for exhausted TILs to effectively respond to anti–PD-1 treatment in vitro. These findings suggest that a TME with insufficient CD28 signaling from myeloid APCs could explain poor responses to anti–PD-1 in tumors with substantial TIL populations and that activating the APCs with reagents such as anti-CD40 may rescue therapeutic responses.Duraiswamy J, …, Coukos G. Cancer Cell 2021 Dec 13;39:1623–42.e20.

Potent but transient immunosuppression of T-cells is a general feature of CD71+ erythroid cells

CD71+ erythroid cells (CECs) have been recently recognized in both neonates and cancer patients as potent immunoregulatory cells. Here, we show that in mice early-stage CECs expand in anemia, have high levels of arginase 2 (ARG2) and reactive oxygen species (ROS). In the spleens of anemic mice, CECs expansion-induced L-arginine depletion suppresses T-cell responses. In humans with anemia, CECs expand and express ARG1 and ARG2 that suppress T-cells IFN-γ production. Moreover, bone marrow CECs from healthy human donors suppress T-cells proliferation. CECs differentiated from peripheral blood mononuclear cells potently suppress T-cell activation, proliferation, and IFN-γ production in an ARG- and ROS-dependent manner. These effects are the most prominent for early-stage CECs (CD71highCD235adim cells). The suppressive properties disappear during erythroid differentiation as more differentiated CECs and mature erythrocytes lack significant immunoregulatory properties. Our studies provide a novel insight into the role of CECs in the immune response regulation.

Bone Marrow Neutrophils of Multiple Myeloma Patients Exhibit Myeloid-Derived Suppressor Cell Activity.

Activated normal density granulocytes (NDGs) can suppress T-cell responses in a similar way as myeloid-derived suppressor cells (MDSCs). In this study, we tested the hypothesis that NDGs from blood and bone marrow of multiple myeloma (MM) patients have the ability to suppress T-cells, as MDSC. MM is an incurable plasma cell malignancy of the bone marrow. Like most malignancies, myeloma cells alter its microenvironment to promote tumor growth, including inhibition of the immune system. We found that MM NDG from the bone marrow suppressed proliferation of T-cells, in contrast to healthy donors. The inhibitory effect could not be explained by changed levels of mature or immature NDG in the bone marrow. Moreover, NDG isolated from the blood of both myeloma patients and healthy individuals could inhibit T-cell proliferation and IFN-γ production. On the contrary to previous studies, blood NDGs did not have to be preactivated to mediate suppressive effects. Instead, they became activated during coculture, indicating that contact with activated T-cells is important for their ability to regulate T-cells. The inhibitory effect was dependent on the production of reactive oxygen species and could be reverted by the addition of its inhibitor, catalase. Our findings suggest that blood NDGs from MM patients are suppressive, but no more than NDGs from healthy donors. However, only bone marrow NDG from MM patients exhibited MDSC function. This MDSC-like suppression mediated by bone marrow NDG could be important for the growth of malignant plasma cells in MM patients.

Zap70 Regulates TCR-Mediated Zip6 Activation at the Immunological Synapse.

The essential microelement zinc plays immunoregulatory roles via its ability to influence signaling pathways. Zinc deficiency impairs overall immune function and resultantly increases susceptibility to infection. Thus, zinc is considered as an immune-boosting supplement for populations with hypozincemia at high-risk for infection. Besides its role as a structural cofactor of many proteins, zinc also acts as an intracellular messenger in immune cell signaling. T-cell activation instructs zinc influx from extracellular and subcellular sources through the Zip6 and Zip8 zinc transporters, respectively. Increased cytoplasmic zinc participates in the regulation of T-cell responses by modifying activation signaling. However, the mechanism underlying the activation-dependent movement of zinc ions by Zip transporters in T cells remains elusive. Here, we demonstrate that Zip6, one of the most abundantly expressed Zip transporters in T cells, is mainly localized to lipid rafts in human T cells and is recruited into the immunological synapse in response to TCR stimulation. This was demonstrated through confocal imaging of the interaction between CD4+ T cells and antigen-presenting cells. Further, immunoprecipitation assays show that TCR triggering induces tyrosine phosphorylation of Zip6, which has at least three putative tyrosine motifs in its long cytoplasmic region, and this phosphorylation is coupled with its physical interaction with Zap70. Silencing Zip6 reduces zinc influx from extracellular sources and suppresses T-cell responses, suggesting an interaction between Zip6-mediated zinc influx and TCR activation. These results provide new insights into the mechanism through which Zip6-mediated zinc influx occurs in a TCR activation-dependent manner in human CD4+ T cells.

Abstract 1604: Anticancer nanocage platforms for combined immunotherapy designed to harness immune checkpoints and deliver anticancer drugs

Abstract The interaction of programmed cell death 1 ligand 1 (PD-L1) with its receptor, programmed cell death 1 (PD-1), inhibits T-cell responses. Monoclonal antibodies that block this interaction have been shown effective as immunotherapy. However, only a subset of cancers exhibits a durable response to PD-1/PD-L1 blockade. Moreover, antibody-based immune checkpoint blockade is costly and is occasionally accompanied by systemic side effects. To overcome these limitations of antibody-based immune checkpoint blockade, an immune checkpoint-blocking ferritin nanocage displaying 24 PD-L1 binding peptides (PD-L1pep1) on its surface was designed and constructed. These ferritin nanocages displaying PD-L1pep1 (PpNF) specifically bind to PD-L1 expressed on cancer cells or to purified PD-L1 with a ~30 nM binding affinity. The addition of PpNF to cocultures of T cells and cancer cells inhibited PD-1/PD-L1 interactions and restored T-cell activities. In a mouse model of syngeneic colon cancer, PpNF specifically targeted tumors and showed antitumor activity. Moreover, PpNF nanocages encapsulating the chemotherapeutic drug doxorubicin had more potent antitumor activity than a monoclonal antibody against PD-L1. These results demonstrate that ferritin nanocages displaying surface PD-L1pep1 can be efficiently applied for immunotherapy, especially when encapsulating small chemotherapeutic drugs. These nanocages may have promise as an immunotherapeutic nanomedicine against various solid tumors. Citation Format: Soyoun Kim, Minseong Kim. Anticancer nanocage platforms for combined immunotherapy designed to harness immune checkpoints and deliver anticancer drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1604.

Contributions of PD-L1 reverse signaling to dendritic cell trafficking.

Programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) interactions are critical for dampening the immune response to both self and foreign antigens. The signaling of PD-L1 via its cytoplasmic domain, rather than through its interactions with PD-1 via the extracellular domain, has been termed PD-L1 reverse signaling. While this signaling is beneficial for cancer progression, little is understood about the consequences of PD-L1 reverse signaling in immune cells that express PD-L1 at steady state or in response to infection. Loss of PD-L1 during infection leads to unchecked T-cell proliferation and increased autoimmune T-cell responses. While the T-cell intrinsic role of PD-1 for inhibiting T-cell responses has been well explored, little to no effort has been directed at investigating the consequences of PD-L1 reverse signaling on the DCs interacting with PD-1+ T cells. We recently reported a defect in dendritic cell (DC) trafficking from the skin to the draining lymph node (LN) following immunization or infection in the absence of PD-L1. We demonstrated that a region within the cytoplasmic tail was responsible for the defect in DC trafficking. Here, we review the processes involved in DC trafficking and highlight what weknow about PD-L1 expression, PD-L1 post-translational modifications, PD-L1 intracellular interactions, and PD-L1 extracellular interactions.

Syngeneic Fecal Microbiota Transplant Effectively Attenuated Graft-Versus-Host Disease after Bone Marrow Transplantation in Mice

Introduction: The gut microbiota plays an important role in maintaining intestinal homeostasis and outcomes after allogeneic hematopoietic stem cell transplantation (allo-HCT). Post-transplant loss of microbiota diversity in patients has been associated with increased infections, graft-versus-host disease (GVHD), disease relapse, and treatment-related mortality. Case studies of fecal microbiota transplantation (FMT) from healthy donors into patients with steroid-resistant gastrointestinal GVHD suggest that FMT may restore the normal microbiota and improve GVHD. However, it remains unclear what constitutes the “optimal” target microbiota and how different changes in the microbiota may impact GVHD. A particularly interesting question is whether the goal of FMT should be to replicate the original recipient microbiota (recipient FMT) or to transfer the donor microbiota (donor FMT), given that the donor microbiota may be more compatible with the transplanted donor immune system.Methods: To test the hypothesis that donor FMT improves GVHD, we evaluated FMT in the context of our well-established allo-HCT acute GVHD model. T-cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into BALB/c mice without causing GVHD. T cells (1 x 106) from C57BL/6 donors were then be added back to induce GVHD. The BALB/c recipient microbiota was modified by FMT and/or antibiotics. FMT was performed by gavaging freshly collected stool from healthy mice of stem cell donor strain (B6, donor FMT) or recipient strain (BALB/c, recipient FMT). To avoid the potential mechanical injury caused by oral gavage that may affect results, we also evaluated another method of FMT in which allo-HCT recipients were housed with healthy mice, resulting in FMT via coprophagy. To test the effect of antibiotics, some recipient mice were supplied with imipenem and vancomycin in drinking water for three days before FMT. Proliferation of luciferase-labeled T cells was monitored using bioluminescent imaging.Results:As expected, BALB/c mice receiving TCD BM from C57BL/6 had no GVHD, while BALB/c mice receiving TCD BM with 1 x 106 T cells had significant GVHD-related mortality. Contrary to our original hypothesis, donor FMT significantly worsened mortality, while recipient FMT significantly improved mortality (Fig. 1a). The addition of antibiotics particularly exacerbated early morality in the donor FMT group (Fig. 1b) while antibiotics had minimal effect in the recipient FMT setting (Fig. 1c). Interestingly, even in the context of TCD BM alone, donor FMT worsened survival, and this effect was enhanced with the addition of antibiotics (Fig. 1d). Deaths were early and consistent with infection on necropsy.To better understand the mechanisms by which FMT affect survival, we observed T-cell proliferation using bioluminescent imaging of luciferase-labeled T cells. Mice that received recipient FMT had significantly lower luminescence in GVHD major target organs compared to the mice that received donor FMT, indicating less T-cell proliferation (Fig. 2), which is consistent with less T-cell activation and less GVHD.Similar results were seen in co-housing experiments, in which co-housing with healthy recipient animals improved body weight and GVHD scores (Fig. 3). Preliminary 16s ribosomal RNA sequencing results support our hypothesis that FMT changes the microbiome, and these changes in the microbiome are linked to clinical outcomes. We will also explore the pathological changes in the gut microenvironment and the potential mechanism.Conclusion: Recipient FMT in the context of allogeneic HCT helped restore the gut microbiota, suppress T-cell responses in GVHD-associated organs, and ameliorated GVHD. In contrast, donor FMT resulted in a high risk of bacterial infections as well as more severe GVHD. This study may provide crucial clinical guidance for using FMT as a potential treatment for GI GVHD. [Display omitted] DisclosuresSung:Novartis: Research Funding; Merck: Research Funding; Cellective: Research Funding.

Immunodepletion of MDSC By AMV564, a Novel Tetravalent Bispecific CD33/CD3 T Cell Engager Restores Immune Homeostasis in MDS in Vitro

Myelodysplastic syndromes (MDS) are age-dependent and genetically diverse hematopoietic neoplasms associated with inflammation, cytopenias, myeloid dysplasia and risk for acute myeloid leukemia (AML) progression. Mounting evidence indicates that emergence of somatic gene mutations arising in hematopoietic stem and progenitor cells (HSPC) with age plays an important role in MDS pathogenesis. Our investigations indicate that sustained activation of innate immune response directs inflammation in the bone marrow (BM) microenvironment that contributes to hematopoietic and immune impairment through local generation of inflammatory cytokines, expansion of regulatory T- cells, and up-regulation and activation of pattern recognition receptors. Critical cellular effectors are the inflammation-associated immature myeloid cells termed myeloid-derived suppressor cells (MDSCs). Human MDSCs, distinguished phenotypically by CD33high and HLA-DR−Lin−, expand and are activated in cancers. We previously reported that MDSCs are profoundly expanded in the local BM microenvironment of MDS where they contribute to MDS hematopoietic BM failure. MDSCs suppress T-cell responses and impair CD4+/CD8+ function, suggesting that this may be a key mechanism fostering clonal escape from anti-tumor immune response. Indeed, the MDSC population size limits the benefit of immune checkpoint antagonists in the treatment of solid tumors and effective strategies to deplete MDSCs are lacking. Therefore, we hypothesized the high CD33 surface density in MDSCs can be exploited for selective immune-depletion by the T-cell engager AMV564, a novel CD33/CD3 tetravalent bispecific antibody that recognizes both CD33 and CD3, currently in clinical development for AML (NCT03144245). Depletion of MDSCs may restore immune function, hematopoiesis and improve sensitivity to checkpoint inhibitors. Primary BM mononuclear cells (BMMNC) from 15 MDS patients were treated with AMV564 or isotype IgG control for 5-7 days in vitro . AMV564 treatment of MDS BMMNC eliminated CD33+ MDSCs in a dose-dependent manner and expanded CD4+ and CD8+ T-cells compared to controls (P ≤0.001). Proliferation of CD4+ and CD8+ T-cells, measured by Brdu incorporation, more than doubled with AMV564 treatment (P ≤0.001). IFNγ, a T-cell activation biomarker, dramatically increased in AMV564 treated cells indicating T-cells are fully activated by AMV564 at concentrations as low as 0.7 ng/ml (P ≤0.01). Moreover, depletion of MDSCs and immunological restoration was accompanied by significant improvement in colony-forming capacity after AMV564 treatment (P ≤0.01). Checkpoint inhibition alone with 10 µg/ml of anti-PD1 antibody did not result in reduction of MDSCs and T-cell activation in MDS BMMNCs, demonstrating that anti-PD1 alone is insufficient to appropriately activate T-cells or modulate MDSC cell number. Interestingly, combined treatment of MDS BMMNC with anti-PD1 and AMV564 yielded more than additive T-cell activation and increased colony forming capacity (P ≤0.01), indicating the potential to augment MDS-specific immune responses. Our findings demonstrate that AMV564 treatment selectively depletes MDSCs to reactivate T lymphocytes by improving both quantity and quality of immune responses leading to the improvement of hematopoiesis. These preclinical data provide a strong rationale for clinical investigation of this innovative approach in patients with MDS. DisclosuresGuenot:Amphivena Therapeutics, Inc.: Employment. Fox:Amphivena Therapeutics, Inc.: Consultancy.

Myeloid-Derived Suppressor Cells as a Potential Biomarker and Therapeutic Target in COVID-19.

Clinical presentations of COVID-19 are highly variable, yet the precise mechanisms that govern the pathophysiology of different disease courses remain poorly defined. Across the spectrum of disease severity, COVID-19 impairs both innate and adaptive host immune responses by activating innate immune cell recruitment, while resulting in low lymphocyte counts. Recently, several reports have shown that patients with severe COVID-19 exhibit a dysregulated myeloid cell compartment, with increased myeloid-derived suppressor cells (MDSCs) correlating with disease severity. MDSCs, in turn, promote virus survival by suppressing T-cell responses and driving a highly pro-inflammatory state through the secretion of various mediators of immune activation. Here, we summarize the evidence on MDSCs and myeloid cell dysregulation in COVID-19 infection and discuss the potential of MDSCs as biomarkers and therapeutic targets in COVID-19 pneumonia and associated disease.

Arginase 1+ IL‐10+ polymorphonuclear myeloid‐derived suppressor cells are elevated in patients with active pemphigus and correlate with an increased Th2/Th1 response

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which are characterized by their capability to suppress T-cell responses. While MDSCs have been traditionally associated with cancer diseases, their role as regulators of autoimmune diseases is emerging. Pemphigus is a chronic autoimmune blistering skin disease characterized by dysregulated T-cell responses and autoantibody production. The role of MDSCs in pemphigus disease has not been defined yet. The aim of this study was to characterize MDSCs in pemphigus patients and to dissect their relationship with CD4+ T-cell subsets and clinical disease assessments. For this purpose, we performed a cross-sectional analysis of 20 patients with pemphigus. Our results indicate that a population of CD66b+ CD11b+ polymorphonuclear-like MDSCs (PMN-MDSCs) is expanded in the peripheral blood mononuclear cell fraction of pemphigus patients compared to age-matched healthy donors. These PMN-MDSCs have the capability of suppressing allogeneic T-cell proliferation in vitro and show increased expression of characteristic effector molecules such as arginase I and interleukin-10. We further demonstrate that PMN-MDSCs are especially expanded in patients with active pemphigus, but not in patients in remission. Moreover, MDSC frequencies correlate with an increased Th2/Th1 cell ratio. In conclusion, the identification of a functional PMN-MDSC population suggests a possible role of these cells as regulators of Th cell responses in pemphigus.

Frequency and Functional Analysis of Myeloid-Derived Suppressor Cells (MDSCs) in the Peripheral Blood and Bone Marrow of Patients with Chronic Idiopathic Neutropenia (CIN)

Myeloid-derived suppressor cells (MDSCs) are myeloid cells with immunoregulatory properties characterized mainly by suppression of T-cell responses (Bizymi et al, HemaSphere 2019). They are divided in HLA-DRlow/-/CD11b+/CD33+/CD15+ polymorphonuclear (PMN-MDSCs) and HLA-DRlow/-/CD11b+/CD33+/CD14+ monocytic (M-MDSCs) subsets and they are implicated in inflammatory and malignant diseases. Chronic idiopathic neutropenia (CIN), is a (usually benign) neutrophil disorder characterized by persistent and unexplained neutropenia following a detailed clinical/laboratory investigation including anti-neutrophil antibody testing, bone marrow (BM) biopsy and karyotype (Dale & Bolyard, Curr Opin Hematol 2017). Previous studies have shown that neutropenia in CIN is associated with increased apoptosis of BM granulocytic progenitor cells due to an inflammatory BM microenvironment consisting of oligoclonal T-lymphocytes, proinflammatory monocytes and proapoptotic cytokines. The aim of the present study is to explore the possible involvement of the MDSCs in the pathophysiology of CIN by investigating their number in peripheral blood (PB) and BM in association with their functional characteristics. We have studied 100 CIN patients and 49 age- and sex-matched healthy controls. The patients fulfilled the previously described diagnostic criteria for CIN (Papadaki et al, Blood 2003) and had mean neutrophil counts 1095.67 ± 479.52 (median 1215, range 100-1700). MDSC subsets were quantitated by flow cytometry in the PB mononuclear cell (PBMC) fraction using the combination of CD33PC7/CD15PC5/HLA-DRECD/CD14PE/CD11bFITC monoclonal antibodies and the Kaluza analysis software. MDSC subsets were also studied in the BMMC fraction of 24 CIN patients and 8 healthy controls from the study population. The T-cell suppression function of patient MDSCs was evaluated in coculture experiments of immunomagnetically sorted, CFSE stained, normal CD3+ cells with immunomagnetically sorted M-MDSCs and PMN-MDSCs from 4 patients and 4 healthy donors using recombinant human IL-2 as activating factor. CFSE staining was detected in the CD3+ cells on day 0 and day 3 of coculture and analysis was performed with the Fcs Express 7 software. Statistical analysis was performed with the Statistica software. We found that the proportion of PB M-MDSCs was statistically significant lower in CIN patients (1.45% ± 1.82%) compared to controls (3.68% ± 3.12%, Mann-Whitney test, p < 0.0001) (Figure a) whereas the proportion of PB PMN-MDSCs, although lower in patients, did not differ significantly from the controls. The proportion of BM M-MDSCs did not differ significantly between CIN patients and controls whereas the proportion of BM PMN-MDSCs was statistically significant lower in patients (13.27% ± 11.27%) compared to controls (19.49% ± 4.46%; Mann-Whitney test, p = 0.0291) (Figure b). Paired analysis showed that the proportion of PMN-MDSCs were higher in the BMMC compared to PBMC fraction in both CIN patients (13.27% ± 11.27% vs 1.14% ± 1.64%, respectively; Wilcoxon test, p = 0.005) (Figure c) and healthy controls (19.49% ± 4.46% vs 9.92% ± 9.08%, respectively; Wilcoxon test, p = 0.0118). Interestingly, the proportion of increase of PMN-MDSCs (in BMMC vs PBMC fraction) was significantly higher in patients (86.71% ± 21.26%) compared to controls (55.95% ± 38.59%; Mann-Whitney test, p = 0.0357) (Figure d). The above data indicate low production of PMN-MDSCs in CIN patients compared to controls but a trend for accumulation of these cells in patients' BM. No statistically significant difference was documented in paired analysis of M-MDSCs between BMMC and PBMC fractions in either CIN patients or healthy controls. Patient PMN-MDSCs and M-MDSCs displayed normal capacity to suppress T-cell proliferation as was indicated by the T-cell generations in coculture experiments of normal CD3+ cells in the presence or absence of patient MDSCs (Figure e). In conclusion, CIN patients display low proportion of MDSCs in the PB and lower proportion of PMN-MDSC in the BM compared to normal individuals. Patient MDSCs display normal capacity to suppress T-cell activation. The low proportions of MDSCs may sustain the inflammatory process associated with CIN whereas the accumulation of PMN-MDSCs in the BM represents probably a compensatory mechanism to suppress the inflammatory processes within patients' BM microenvironment. Figure Disclosures Papadaki: Genesis pharma SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 598: Integrated computational and experimental design of a monobody targeting PDL1

Abstract Monobodies (Mb) are small, synthetic antibodies based on the human fibronectin type III domain that exhibit high thermodynamic stability without disulfide bonds. To explore suitability of these domains as cancer-targeting molecules, we apply computational and experimental methods to develop a Mb that targets programmed-death-ligand 1 (PDL1). As some tumors overexpress PDL1 to inhibit T-cell responses, a PDL1-binding Mb could be used to promote successful immunotherapy. More broadly, computational methods applied to developing a PDL1-binding Mb can also be applied to developing new Mbs that target cancer antigens. Starting from the G9 Mb (PDB: 1ttg), we grafted the CDR3 loop of a known PDL1-binder (PDB: 5jds) into the FG loop of the Mb. This design was displayed on the surface of yeast and shown to bind PDL1 better than the G9 Mb, though not as well as a high affinity variant of PD1, the cognate receptor of PDL1. Using molecular dynamics simulations, we evaluated structural features that are unlikely to contribute to PDL1-binding. Using this approach, we selected amino acid positions for site-saturated mutagenesis and constructed a yeast library displaying Mb isoforms. Through iterative rounds of selection, we identified a Mb isoform with further improved PDL1-binding. Citation Format: Reed E. Harrison, Chi-Wei Man, Yingxiao Wang. Integrated computational and experimental design of a monobody targeting PDL1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 598.

IL-6-induced CD39 expression on tumor-infiltrating NK cells predicts poor prognosis in esophageal squamous cell carcinoma.

Natural killer (NK) cells, a predominantinnatelymphocyte subset, mediates eradicating malignant cells. Purinergic signaling by ectonucleotidase CD39 can suppress T-cell response in caner. However, the role of CD39 in NK cells has not been fully elucidated. Here, we characterized CD39 expression on NK cells and its clinical relevance in esophageal squamous cell carcinoma (ESCC). Peripheral blood and tissue samples were collected from 36 ESCC patients. We observed that the proportion of NK cells significantly decreased but CD39 was obviously up-regulated on NK cells from cancerous tissues compared to paired peripheral blood in ESCC patients. Furthermore, tumor-infiltrating NK cells with high CD39 expression exhibited a phenotype of functional impairment. In vitro, conditioned media of ESCC cell lines could induce CD39 expression on peripheral NK cells from healthy donors. IL-6 was identified as the major cytokine produced by ESCC cell lines and also elevated in both tumor tissues and blood serum from ESCC patients. Recombinant IL-6 significantly induced surface CD39 expression in human NK cells, while IL-6-receptor antagonist tocilizumab prevented this effect. Finally, tumor-infiltrating CD39+ NK cells were correlated with poor prognosis in ESCC patients. Thus, tumor-derived IL-6 might impair NK cell functions through induction of CD39 expression. CD39+ NK cells may serve as a potential prognostic biomarker for ESCC patients.

Targeting of the A2A adenosine receptor counteracts immunosuppression in vivo in a mouse model of chronic lymphocytic leukemia.

Tumor immunosuppression is a major cause of treatment failure and disease relapse, both in solid tumors and leukemia. Local hypoxia is among the conditions that cause immunosuppression, acting at least in part through the upregulation of extracellular adenosine levels, which potently suppress T-cell responses and skew macrophages towards an M2 phenotype. Hence, there is intense investigation to identify drugs that target this axis. By using the TCL1 adoptive transfer chronic lymphocytic leukemia mouse model, we show that adenosine production and signaling are upregulated in the hypoxic lymphoid niches, where intense colonization of leukemic cells occurs. This leads to a progressive remodeling of the immune system towards tolerance, with expansion of T regulatory cells (Treg), loss of CD8+ T-cell cytotoxicity and differentiation of murine macrophages towards the patrolling (M2-like) subset. In vivo administration of SCH58261, an inhibitor of the A2A adenosine receptor, re-awakens T-cell responses, while limiting Treg expansion, and re-polarizes monocytes towards the inflammatory (M1-like) phenotype. These results show for the first time the in vivo contribution of adenosine signaling to immune tolerance in chronic lymphocytic leukemia, and the translational implication of drugs interrupting this pathway. Although the effects of SCH58261 on leukemic cells are limited, interfering with adenosine signaling may represent an appealing strategy for combination-based therapeutic approaches.

Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus-based immunosuppression with basiliximab induction.

Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non-rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy-proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL-2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first-week peripheral blood Treg frequency correlates with AR after LT under tacrolimus-based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.

Potential Role of Macrophage Migration Inhibitory Factor in the Pathogenesis of Marek's Disease.

IntroductionMarek’s disease virus (MDV) can cause malignant T-cell lymphomas and immunosuppression in chickens. Macrophage migration inhibitory factor (MIF) not only plays a critical role in inhibiting T-cell responses, but also contributes to multiple aspects of tumour progression. The aim of this study was to reveal the potential role of MIF in the pathogenesis of MDV infection.Material and MethodsMIF gene expression levels were measured by using real-time PCR. Expression was assayed at different times in chicken embryo fibroblast (CEF) cells and tissue samples of SPF chickens infected with different MDV strains and fold change was calculated by the 2–△△CT method.ResultsThe expression of MIF was significantly downregulated (p < 0.05 and FC > 2) in CEF cells infected with the very virulent MDV RB1B strain at 48 h post infection (hpi) and in the skin and spleen at 14 days post infection (dpi). The reduction of MIF expression was also found in CEF cells infected by reticuloendotheliosis virus (REV), avian leukosis virus subgroup J (ALV-J), and MDV vaccine strain CVI988 or in HD11 cells stimulated with TLR2, 3, 4, and 7 ligands. Interestingly, MIF expression decreased continuously from 7 to 28 dpi in the thymus after RB1B virus infection while it increased after CVI988 virus infection. Upregulated expression of MIF was found in CEF infected with RB1B at 96 hpi and in the spleen and skin at 21 and 28 dpi.ConclusionThe present study revealed the different expression pattern of MIF in response to MDV infection and indicated that MIF level may be associated with MDV pathogenesis.