Peripheral Blood Lymphoid Cells Research Articles

In vitro modelling of local gene therapy with IL-15/IL-15Rα and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4+ T cells

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with αPD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and αPD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-γ. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15Rα (IL-15/IL-15Rα) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4+ T cells or CD8+ T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8+ T cells. Gene-based delivery of IL-15/IL-15Rα to tumor cells, shows expansion of NK cells, activation of NK cells, CD4+ and CD8+ T cells, and killing of tumor spheroids. Both NK cells and CD8+ T cells are necessary for tumor cell killing and CD4+ T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4+ or CD8+ T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4+ T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15Rα to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with αPD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.

B-Cell Prolymphocytic Leukemia: Two Case Reports and Review of the Literature

B-cell prolymphocytic leukemia is a very rare mature B-cell leukemia occurring in elderly people diagnosed when prolymphocytes comprise more than 55% of the lymphoid cells in peripheral blood. Its evolution is most often aggressive. We report here the cases of two patients with clinical signs dominated by splenomegaly associated with significant hyperlymphocytosis. Their blood smears revealed extensive prolymphocyte invasion suggesting Prolymphocyte leukemia confirmed by immunophenotyping. Both purine analogues and monoclonal antibodies have shown promoting results, however, allogeneic hematopoietic stem cell transplantation remains the only therapeutic means allowing a lasting response. However, it is reserved for young patients with compatible donors. A good understanding of the pathogenesis and a better integration of the molecular data of this hemopathy open the way to the use of new targeted therapies. Prolymphocyte leukemia is a very rare, complex, and often aggressive, mature lymphoid hemopathy. The mean age of onset is 65 years with a predominance of males. We report here the case of a patient with splenomegaly and severe lymphocytosis in whom the smear showed an invasion of 90% of prolymphocytes suggesting Prolymphocyte leukemia confirmed by immunophenotyping.

Blockade of CD73 Increases the Cytotoxic Effects of Fludarabine in Chronic Lymphocytic Leukemia

Background: Ecto-5’-nucleotidase (CD73) is a membrane-bound enzyme that converts adenosine 5’-monophosphate into adenosine. It is proven that the presence of elevated levels of adenosine in the tumor microenvironment induces tumor growth while suppressing immune responses against tumor cells. According to several studies, it is reported that the expression of CD73 is increased in several types of malignancies. In chronic lymphocytic leukemia (CLL), a hematological malignancy characterized by the increased proliferation and accumulation of lymphoid cells in peripheral blood (PB) and lymphoid tissues, CD73 expression is associated with higher-risk disease. Moreover, it is reported that CD73 may contribute to fludarabine (a chemotherapeutic widely used for the treatment of CLL) resistance as well. Methods: We separated mononuclear cells obtained from PB and bone marrow (BM) from eleven CLL patients. Cells were treated individually or in combination with fludarabine and anti-CD73 siRNA (transfection by lipofectamine), and the effect of treatment on cell survival was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Results: We found that the highest level of apoptosis induction was observed in cells treated with fludarabine combined with CD73 inhibition, compared to the untreated (P<0.01). Our findings are in support of a study reporting the association between CD73 overactivation and fludarabine resistance. Conclusion: These findings suggest that CD73 inhibition can be a potential target for CLL treatment and can help overcome endurance towards fludarabine and can better the prognosis of CLL-affected individuals.

Diagnostic significance of TLR2 and TLR4 receptors on lymphoid cells as a marker of the progression of periodontal inflammation associated with key periodontal pathogenic species <i>F. alocis</i> and <i>P. gingivalis</i>

The aim of the work was to evaluate the diagnostic value of TLR2 and TLR4 expression on periodontal and peripheral blood lymphoid cells by immunofluorescence microscopy in patients with chronic periodontitis associated with key periodontal pathogenic species Filifactor alocis, Porphyromonas gingivalis.
 Materials and methods. The study included 150 patients 88 (59%) women and 62 (41%) men aged 18 to 73 years with chronic periodontitis in the acute phase (CP) and 32 people without signs of chronic periodontal inflammation. To confirm the diagnosis of periodontitis, the Multident-5 PCR kit was used (detection of P. gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans), as well as rt-PCR for F. alocis and P. gingivalis in the contents of the periodontal pocket (NPF GenLab, Russia). To evaluate cells carrying CD282 and CD284 markers, gingival fluid flushes from the periodontal pocket with Hanks' solution were used. The isolated cells were stained with antibodies to CD282 markers (corresponding to TLR2 receptor) or CD284 (corresponding to TLR4 receptor) labeled with FITC, and fixed with paraformaldehyde for subsequent immunofluorescence microscopy.
 Results. The expression of TLR2 and TLR4 on peripheral blood and gingival fluid leukocytes was studied in individuals with healthy periodontitis and patients with chronic periodontitis associated with F. alocis, P. gingivalis. According to the results of PCR, the detection rate of F. alocis and P. gingivalis was 64 and 62.7%, respectively, which confirmed their dominance in the microbial association. It was found that the expression of TLR2 and TLR4 on peripheral blood lymphoid cells varied in humans. The possible diagnostic significance of this phenomenon in assessing the progression of chronic periodontitis is discussed.
 Conclusion. In patients with chronic periodontitis associated with the dominance of periodontopathogenic species F. alocis, P. gingivalis, the multidirectional expression of TLR2 and TLR4 on peripheral blood cells was observed, which may have diagnostic significance in assessing the progression of periodontal diseases.

The MICA-NKG2D axis in clear cell renal cell carcinoma bolsters MICA as target in immuno-oncology

ABSTRACT NKG2D is a major natural killer (NK) cell-activating receptor that recognizes eight ligands (NKG2DLs), including MICA, and whose engagement triggers NK cell effector functions. As NKG2DLs are upregulated on tumor cells but tumors can subvert the NKG2D-NKG2DL axis, NKG2DLs constitute attractive targets for antibody (Ab)-based immuno-oncology therapies. However, such approaches require a deep characterization of NKG2DLs and NKG2D cell surface expression on primary tumor and immune cells. Here, using a bioinformatic analysis, we observed that MICA is overexpressed in renal cell carcinoma (RCC), and we also detected an association between the NKG2D-MICA axis and a diminished overall survival of RCC patients. Also, by flow cytometry (FC), we observed that MICA was the only NKG2DL over-expressed on clear cell renal cell carcinoma (ccRCC) tumor cells, including cancer stem cells (CSC) that also coexpressed NKG2D. Moreover, tumor-infiltrating leukocytes (TIL), but not peripheral blood lymphoid cells (PBL) from ccRCC patients, over-expressed MICA, ULBP3 and ULBP4. In addition, NKG2D was downregulated on peripheral blood NK cells (PBNK) from ccRCC patients but upregulated on tumor-infiltrating NK cells (TINK). These TINK exhibited impaired degranulation that negatively correlated with NKG2D expression, diminished IFN-γ production, upregulation of TIM-3, and an impaired glucose intake upon stimulation with cytokines, indicating that they are dysfunctional, display features of exhaustion and an altered metabolic fitness. We conclude that ccRCC patients exhibit a distorted MICA-NKG2D axis, and MICA emerges as the forefront NKG2DL for the development of targeted therapies in ccRCC.

Checkpoint Inhibitors Modulate Plasticity of Innate Lymphoid Cells in Peripheral Blood of Patients With Hepatocellular Carcinoma.

Innate lymphoid cells (ILC) are a heterogeneous and plastic population of cells of the innate immune system. Their role in cancer and specifically in hepatocellular carcinoma is unraveling. The presence of ILCs in peripheral blood of HCC patients has not been explored yet. Their role and function in response to checkpoint inhibitor therapy have also not been explored. Here, we characterized ILCs in PBMC of HCC patients at baseline and after treatment with immune checkpoint inhibitors (ICI) by flow cytometry and single-cell sequencing. Characterization of ILC subsets in PBMCs of HCC patients showed a significant increase in ILC1 and a decrease in ILC3 frequencies. Single-cell RNA-sequencing identified a subgroup of NK-like ILCs which expressed cytotoxicity markers as well as NKp80/KLRF1. This KLRF1high NK-like population showed low abundance in patients with HCC and was enhanced after combined anti-CTLA-4 and anti-PD-1immunotherapy. Trajectory analysis placed this population in between ILC1 and ILC3 cells. The transcriptomic signature of KLRF1high NK-like ILCs was associated with better progression-free survival in large HCC cohorts. This study shows a previously unknown effect of ICI on the composition and plasticity of ILCS in peripheral blood. Thus, ILCs from PBMC can be used to study changes in the innate immune system under immunotherapy.

MO220: Comparison of the Effects of Hydroxychloroquine and Glucocorticosteroids on Immunological Parametrs in Patients with IGA Nephropathy

Abstract BACKGROUND AND AIMS Current treatment strategies of IgA nephropathy (IgAN) primarily involve blood pressure control, renin–angiotensin–aldosterone system inhibitors (RAASIs), and glucocorticosteroids (GCs), which are recommended when the supporting therapies fail to reduce proteinuria (PU) levels below 1 g/day. Hydroxychloroquine (HCQ) is considered to have an immunomodulatory rather than an immunosuppressant effect. The immune pathways targeted by HCQ are not fully investigated during treatment of IgAN. The aim of this study was to compare effects of HCQ and GCs on immunological parameters in patients with IgAN. METHOD 32 IgAN patients aged 32.0 (26.7 ÷ 36.0) y.o., male/female ratio as 24/8 with PU > 1 g/day and estimated glomerular filtration rate (eGFR) 78 (61; 103) mL/min were divided into three treatment groups: RAASIs (n = 12); HCQ (n = 10); GCs Pozzi protocol (n = 10). The changes in clinical and immunological parameters were analyzed 6 months after treatment. Phenotypic and functional markers of peripheral blood lymphoid cells were identified by flow cytometry method (Cytoflex, «Beckman Coulter», USA). The concentrations of total immunoglobulins (Ig) class M, G, A and E were determined by ELISA method («VectorBest», Russia). Statistical analysis was done using Statistica 8.0. RESULTS After 6 months of treatment HCQ has shown effectiveness in decreasing of PU (P < .001), haematuria level (P <.01) as well as the same effect was noted in the other treatment groups after 6 months course. The renal function was stable in all investigated groups during follow up. There were no any adverse effects of the treatment. The decrease of potential cytotoxic γδTCR+Т-cells (P <.01) and CD3 + 56 + TNK-cells (P < .01) numbers in peripheral blood (Fig. 1) as well as the reduction of IgM (P < 0.05) and IgG (P < .05) serum levels (Table 1) were established in patients receiving HCQ. While GCs treatment resulted in CD3 + T-cells (P < .05) suppression (Fig. 1) mainly due to CD3 + CD4 + T-helpers down-regulation (P < .05) but stimulation of cytotoxic CD3 + CD8 + T-cells (P < .05) (data not shown) and increasing of IgE level (P < .05) (Table 1). It was not revealed any influence of RAASIs treatment either on the number of lymphoid cells populations (Fig. 1) or Ig levels. CONCLUSION This study demonstrates different ways of pathogenic treatment in IgAN. HCQ has exhibited an immunomodulatory action on the effector mechanisms of cellular cytotoxicity via minor lymphoid T-cells populations reduction as well as on productive phase of humoral immunity via IgM and IgG levels decreasing. While GCs have suppressed CD3 + T-cell and supported IgE-mediated autoinflammation.

Leucémie prolymphocytaire B

AbstractB-prolymphocytic leukemia (B-PLL) is a very rare mature B-cell leukemia occurring in elderly people. B-PLL can be diagnosed when prolymphocytes comprise more than 55% of the lymphoid cells in peripheral blood. The diagnosis can be challenging because B-PLL shares a number of biological features with other B-cell malignancies. The cytological diagnosis is difficult to establish, and may require a specialized review. It is imperative to perform immunophenotyping, because even if there is no typical profile, this analysis distinguishes B-PLL from chronic lymphocytic leukemia. Cytogenetic analysis is mandatory to guide the diagnosis and rule out a mantle cell lymphoma. There is no specific chromosomal abnormality. A complex karyotype and MYC gene abnormalities -translocation or more rarely gain and amplification, are observed in more than 70% of cases. Deletion of 17p including the TP53 gene is also common. The outcome is generally aggressive, with a poor response to chemotherapy. Three prognostic groups have recently been established according to the presence of an abnormality in the MYC gene and/or a 17p deletion. There is no consensus for treatment that relies on immunochemotherapy or targeted therapies such as inhibitors of the BCR signaling pathway or inhibitors of BCL2.

Innate lymphoid cells in peripheral blood of patients with American Cutaneous Leishmaniasis.

Innate lymphoid cells (ILCs) are classified by the expression of specific transcription factors: ILC1 depending on T-bet for IFN-γ production; ILC2 depending on GATA3 for IL-5 and IL-13; and ILC3 depending on ROR-γτ and AHR for IL-17 and IL-22. This study aimed to determine circulating ILCs in 23 patients with localized (LCL)=7, mucocutaneous (MCL)=10, intermediate (ICL)=3 and diffuse (DCL)=3 cutaneous leishmaniasis and 17 healthy controls from endemic area (EC)=9 and non-endemic area (HC)=8. Results evidenced a higher proportion of ILC1 in LCL than controls and MCL. ILC2 was higher in DCL compared with controls. ILC3s were abundant in MCL and DCL concerning controls. A prevalence ratio was calculated to approach cell plasticity: in LCL, the ratio showed a prevalence of ILC1/ILC3 (plasticity 1), in contrast to DCL, and controls, where ILC2/ILC3 (plasticity 3) is prevalent. Also, MCL and ICL showed higher ILC1/ILC2 (plasticity 2). These results suggest that ILC1 and ILC3 in LCL are associated with disease control and regulation of inflammation, while MCL and ICL are related to immunopathology and uncontrolled inflammation. In DCL, ILC2 is associated with the tolerogenic state of these patients.

Peripheral blood T lymphocytosis in thymoma: an insight into immunobiology

Peripheral blood T lymphocytosis (PBTL) is a rare, yet poorly understood manifestations of thymoma, which is postulated to be linked with autoimmune/paraneoplastic manifestations such as myasthenia gravis (MG), pure red cell aplasia (PRCA), etc.; more commonly encountered in this neoplasm. We aim to describe the flowcytometric immunophenotypic data of PBTL in a 43-year-old male; 6months after successful completion of chemoradiotherapy (CT/RT) for a large, invasive, and metastatic type B1 thymoma; and present a comprehensive review of all such cases reported over last 42years (N = 21) (1977-2019). A larger size of the tumors (≥ 10cm), presence of local invasion and/or distant metastasis, and type B (cortical or lymphocyte rich) histology were more likely to be associated with PBTL. Tumors associated with MG/PRCA (N = 9/21) tend to have lower PBTL compared to those without such manifestations; and PBTL subsided following thymectomy with or without CT/RT. Immunophenotypic analysis of PB revealed a CD8 + > CD4 + mature (naïve) polyclonal T cells resembling late cortical thymocytes. Thymic intratumoral microenvironment might influence occurrence PBTL that may have a pathophysiologic link to development of autoimmune manifestations. Immunophenotypic characteristics of peripheral blood lymphoid cells should be the clue for accurate characterization and to avoid a misdiagnosis of a lymphoproliferative neoplasm.

Increased group 2 innate lymphoid cells in peripheral blood of adults with mastocytosis

Systemic mastocytosis is a hematological disease in which aberrant mast cells accumulate because of gain-of-function mutations in the KIT receptor. Group 2 innate lymphoid cells (ILC2s) are effector cells of type 2 immune responses that also express KIT and colocalize with mast cells at barrier tissue sites. In mouse models, mast cell-ILC2 crosstalk can drive local inflammation. However, a possible role for ILC2s in the pathophysiology of mastocytosis remains unexplored. We sought to characterize circulating ILC2s in a clinically diverse cohort of patients with mastocytosis. We included 21 adults with systemic mastocytosis and 18 healthy controls. Peripheral blood ILC2 abundance and phenotype were analyzed by flow cytometry and correlated to clinical characteristics, including the presence of the D816V KIT mutation. ILC2 levels were significantly higher in D816V+ patients with mastocytosis compared with D816V- patients or healthy controls. We observed increased proportions of KIT+ ILC2s among patients with mastocytosis, regardless of D816V status. Patients with skin involvement and itch showed the highest levels of ILC2s, which was independent from atopy or serum tryptase levels. Allele-specific quantitative PCR showed that the vast majority of ILC2s did not carry the D816V mutation. Our findings suggest a role for ILC2s and pathogenic ILC2-mast cell crosstalk in mastocytosis. We hypothesize that a high cutaneous D816V+ mast cell burden alters the skin microenvironment to induce chronic local ILC2 activation and their dissemination into the circulation. Activated ILC2s could contribute to skin symptoms by producing inflammatory mediators and by further augmenting mast cell mediator release.

OMIP-066: Identification of Novel Subpopulations of Human Group 2 Innate Lymphoid Cells in Peripheral Blood.

This 14-color flow cytometry panel was designed to identify newly described subpopulations within human group 2 innate lymphoid cells (ILC2s) and other ILC subsets. This panel also allowed to identify recently reported subpopulations of peripheral blood CRTH2- c-Kit+ ILCs. We validated this panel mostly in human peripheral blood but also confirmed that the same panel and gating strategy works well in human tonsillar cells. The panel contains a few markers indicating the activation status of ILCs. In addition, phycoerythrin (PE) channel is available for the markers of interest in each study. In the validation studies described here, PE channel was used to test the expression of some markers. These features make this panel applicable for immunophenotyping of ILCs in various disease states. © 2020 International Society for Advancement of Cytometry.

Color clustering segmentation framework for image analysis of malignant lymphoid cells in peripheral blood.

Current computerized image systems are able to recognize normal blood cells in peripheral blood, but fail with abnormal cells like the classes of lymphocytes associated to lymphomas. The main challenge lies in the subtle differences in morphologic characteristics among these classes, which requires a refined segmentation. A new efficient segmentation framework has been developed, which uses the image color information through fuzzy clustering of different color components and the application of the watershed transformation with markers. The final result is the separation of three regions of interest: nucleus, entire cell, and peripheral zone around the cell. Segmentation of this zone is crucial to extract a new feature to identify cells with hair-like projections. The segmentation is validated, using a database of 4758 cell images with normal, reactive lymphocytes and five types of malignant lymphoid cells from blood smears of 105 patients, in two ways: (1) the efficiency in the accurate separation of the regions of interest, which is 92.24%, and (2) the accuracy of a classification system implemented over the segmented cells, which is 91.54%. In conclusion, the proposed segmentation framework is suitable to distinguish among abnormal blood cells with subtile color and spatial similarities. Graphical Abstract The segmentation framework uses the image color information through fuzzy clustering of different color components and the application of the watershed transformation with markers (Top). The final result is the separation of three regions of interest: nucleus, entire cell, and peripheral zone around the cell. The procedure is also validated by the implementation of a system to automatically classify different types of abnormal blood cells (Bottom).

New roles for B cell receptor associated kinases: when the B cell is not the target.

Targeting of B cell receptor associated kinases (BAKs), such as Bruton's tyrosine kinase (BTK) or phosphoinositol-3-kinase (PI3K) delta, by specific inhibitors has revolutionized the therapy of B lymphoid malignancies. BAKs are critical signaling transducers of BCR signaling and seem relevant in B cell lymphoma pathogenesis. The functional relevance of BTK for lymphoid malignancies is strongly supported by the observation that resistance to therapy in CLL patients treated with BTK inhibitors such as ibrutinib is often associated with mutations in genes coding for BTK or Phospholipase-C gamma (PLCɣ). In some contrast, next generation sequencing data show that BAKs are mutated at very low frequency in treatment-naïve B cell lymphomas. Therefore, it remains debatable whether BAKs are essential drivers for lymphoma development. In addition, results obtained by targeted deletion of BAKs such as Lyn and Btk in murine CLL models suggest that BAKs may be essential to shape the dialogue between malignant B cells and the tumor microenvironment (TME). Since BAKs are expressed in multiple cell types, BAK inhibitors may disrupt the lymphoma supportive microenvironment. This concept also explains the typical response to BAK inhibitor treatment, characterized by a long-lasting increase of peripheral blood lymphoid cells, due to a redistribution from the lymphoid homing compartments. In addition, BAK inhibitors have shown some efficacy in solid tumors, probably through mediator cells in the TME. This review summarizes and validates the evidence for BAK inhibitors being part of a class of agents that modulate the (hematopoietic) microenvironment of cancers.

Correlation between the changes of innate lymphoid cells in peripheral blood of systemic lupus erythematosus and its clinical significance

Objective: To explore the function and role of innate lymphoid cells in the pathogenesis of systemic lupus erythematosus (SLE) at different disease activity levels. Methods: From Nov 2017 to May 2018, 40 patients with SLE and 15 age-matched healthy non-immune-related diseases controls were enrolled from Anhui provincial hospital. According to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2K, the patients were divided into active group (n=20) and remission group (n=20). The frequency of ILCs, B cells, CD4+T and CD8+T cells from peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry. The subsets of ILCs in each group were compared with the subsets of B cells and T cell respectively. The levels of IL-4, IL-33 and IFN-γ in each group were tested by ELISA. Result: Compared with the control group, ILC1 percentage was significantly increased in SLE active group [(22.33%±2.52%) vs (14.56%±1.28%), P=0.018 1]; ILC2 percentage was decreased significantly in both remission group [(19.67%±1.83%) vs (42.48%±3.46%), P<0.000 1] and active group [(8.67%±0.83%) vs (19.67%±1.83%), P<0.000 1]; ILC3 percentage was decreased significantly in active group [(5.72%±1.08%) vs (14.35%±2.40%), P=0.001 3]. SLEDAI score was negatively correlated with the percentage of ILC2 (P=0.023 9) in all patients. The percentage of ILCs in the remission group (P=0.046 2) and activity group (P=0.003 7) were both increased significantly. Moreover, the percentage of ILC2 in active group was negatively correlated with CD4+T cells (P=0.030 8), and the serum IgG was negatively correlated with ILC2% in all patients (P=0.013 8). Compared with control group or remission group, the levels of IFN-γ (F=10.91, P=0.000 1) and IL-4 (F=6.046, P=0.004 7) in active group were remarkable higher. However, IL-33 was significantly reduced in active group (F=6.645, P=0.002 7). The percentage of ILC2 (r=0.154 3, P=0.028 8) and ILC3 (r=0.313 6, P=0.001 1) in all patients with SLE were positively correlated with the level of IL-4. Conclusion: The percentage of ILCs is related to disease activity, and ILCs play a "double-edged" role in the pathogenesis of SLE. Its function and mechanism are worth further exploration.

Abstract CT095: The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody

Abstract The goal of this current study was to clinically test the hypothesis that by targeting TRAIL-R2, myeloid-derived suppressor cells (MDSCs) can be selectively eliminated. MDSC) are one of the major contributors to immune suppression in cancer. We previously demonstrated in a preclinical study that MDSCs are sensitive to a TRAIL receptor 2 (TRAIL-R2) agonist. The TRAIL-R2 agonistic antibody (DS-8273a; provided by Daiichi Sankyo, Inc.) was tested in 16 patients with advanced solid cancers or lymphomas enrolled in a phase 1 trial. The antibody (24 mg/kg) was administered IV once every 3 weeks till disease progression, unacceptable toxicities, or withdrawal of consent. The safety and the presence of various populations of myeloid and lymphoid cells in peripheral blood and tumor tissues were evaluated using flow cytometry and immunohistochemistry. Overall, the treatment was well tolerated with only mild to moderate adverse events attributable to the study drug. Furthermore, treatment with DS-8273a resulted in reduction of the elevated numbers of MDSC in the peripheral blood of most patients to the levels observed in healthy volunteers. However, in several patients, MDSC rebounded back to the pre-treatment level by day 42. In contrast, DS-8273a did not affect the number of neutrophils, monocytes, and other populations of myeloid and lymphoid cells with decreases in MDSC levels inversely correlating with the length of progression-free survival. In tumors, DS-8273a treatment resulted in a decrease of MDSC in 50% of the patients who were able to provide pre- and on-treatment biopsies. In conclusion, targeting TRAIL-R2 using DS-8273a resulted in a temporary elimination of MDSCs without affecting mature myeloid or lymphoid cells, and these data support further use of this antibody in combination with current immmunotherapies of cancer. Citation Format: George A. Dominguez, Thomas Condamine, Sridevi Mony, Ayumi Hashimoto, Fang Wang, Qin Liu, Andres Forero, Johanna C. Bendell, Robert Witt, Neil Hockstein, Prasanna Kumar, Dmitry I. Gabrilovich. The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT095. doi:10.1158/1538-7445.AM2017-CT095

A “Killer Disease” Living up to its name

A 33-year old female presenting with hepatosplenomegaly and pancytopenia showed atypical lymphoid cells in peripheral blood and bone marrow. Immunophenotyping confirmed the diagnosis of Aggressive NK-cell leukemia. Despite treatment by chemotherapy and targeted therapy she deteriorated and died only 7 months after diagnosis.

Inhibition of Cdk2 promotes the generation of inducible CD8+ T regulatory cells by abrogating binding of the EZH2 methyltransferase on the Foxp3 promoter.

Abstract Cdk2 has a central role in cell cycle re-entry and Cdk2 inhibition is mandatory for induction of T cell tolerance. To determine the effect of Cdk2 inhibition on T cell alloresponses we used the B6D2F1 model of bone marrow transplantation (BMT) and two Cdk2 inhibitors (Cdk2i), CYC202 and CYC205. B6D2F1(Kd) recipients infused with BM and T cells from C57BL/6(Kb) donors were treated with each Cdk2i for 3 weeks and graft versus host disease (GvHD) was assessed over a 70-day period. Cdk2i treatment decreased GvHD mortality (p=0.0054). To examine if Cdk2i affected Treg differentiation we used T cells from Foxp3.GFP-KI mice during BMT. Cdk2i-treated recipients had no difference in CD4+GFP+ Treg compared to control groups in peripheral blood, splenocytes, lymph nodes and intestinal lymphoid cells (ILC) but had increased CD8+GFP+ Treg, predominantly in ILCs (p=0.05). In Treg polarizing culture Cdk2i had only minimal effect on CD4+GFP+ cells but induced significant increase of CD8+GFP+ cells. However, TGF-b-mediated signaling, as determined by Smad3 phosphorylation was unaffected. FOX family members are regulated by transcriptional and epigenetic mechanisms. The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2) induces histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. Cdk2 phosphorylates EZH2 Thr350, which is important for maintenance of H3K27me3 at EZH2 target loci. We determined that Cdk2i inhibited EZH2 Thr350 phosphorylation in CD8+ T cells, which coincided with increased Foxp3 expression. ChIP-PCR identified EZH2 binding on three sites of Foxp3 promoter, which was abrogated by Cdk2i. Thus, Cdk2 inhibition induces CD8+ Treg via EZH2-mediated epigenetic mechanisms.

Proximity-Based Single Cell Analysis of the Bone Marrow Niche Identifies Interleukin-18 As a Quiescence Regulator of Early Hematopoietic Progenitors

Background. Niche-derived signals are essential for maintenance and expansion of stem/progenitor cells in the bone marrow. Recently published studies used elegant genetic tools to dissect predominant cellular sources of known HSPC regulators, such as CXCL12 and kit-ligand (Ding et al, Nature 2012, Greenbaum et al, Nature 2013). However, strategies enabling identification of novel niche-derived factors are lacking. Here, we describe an approach which utilizes spatial proximity between osteolineage cells (OLC) and HSPC in the post-transplant bone marrow niche as a guide to niche factor discovery and reveals the role of Interleukin-18 (IL18) as a quiescence regulator of early hematopoietic progenitors.Results. We established a neonatal bone marrow (BM) transplantation model, in which adult BM-derived LKS CD34-Flk2- HSPCs fluorescently labeled with DiI were transplanted into irradiated newborn col2.3GFP recipients (in this mouse strain, the majority of OLCs are labeled with GFP). Forty-eight hours later, we obtained sections of femoral trabecular bone from transplanted mice and located rare OLCs harboring DiI-positive HSPCs in their immediate proximity. We harvested individual OLCs located within two cell diameters (proximal OLCs) and greater than five cell diameters (distal OLCs) from DiI-labeled HSPCs and performed comparative transcriptome analysis by single cell RNA-Seq. Remarkably, we found that proximal OLCs were distinct from their distal counterparts and showed a higher expression levels of known niche-associated molecules, as well as secreted factors not previously linked to regulatory niche function, including a pro-inflammatory cytokine IL18.Quantification of primitive subsets in the BM of IL18 knock-out (IL18KO) mice showed no abnormalities. However, cell cycle studies revealed that while long-term HSCs (which did not express the IL18 receptor) were unaffected by IL18 deletion, early progenitors - short-term HSC, multi-potent progenitor and common lymphoid progenitor – were more actively cycling. Accordingly, following sublethal exposure to 5-fluorouracil (5FU), IL18KO animals displayed a 2-fold increase in frequency of lin-kit+Sca1+ (LKS) cells, lin-kit+ myeloid progenitors and CLPs, as compared wild-type (WT) controls (Figure 1). On the other hand, exogenous administration of recombinant IL18 to 5FU-treated WT animals was associated with 25% mortality, while no deaths were observed in the vehicle-treated group. Taken together, these data illustrate that IL18 constrains the ability of the progenitor pool to respond to a genotoxic stress.Next, we tested if IL18 acts in a non cell-autonomous fashion. We transplanted progenitor-enriched bone marrow fraction (LKS cells) into IL18KO or WT hosts and observed an approximately 2-fold increase in both myeloid and lymphoid cells in peripheral blood of IL18KO animals. This finding was recapitulated in a reciprocal experiment, when LKS cells from IL18 receptor knock-out animals were transplanted into WT hosts, supporting the possibility that the effect of IL18 on progenitor proliferation is direct.Finally, we investigated if the loss of IL18-mediated quiescence can be therapeutically exploited in the transplant setting. To this end, we assessed survival of lethally irradiated WT and IL18KO mice following transplantation with a limiting BM dose, i.e. predicted to confer survival to 50% or less of WT animals. Notably, we observed reduced 30-day post-transplant mortality (33% versus 72%, p value = 0.05) in IL18KO animals, which likely resulted from accelerated progenitor expansion in the absence of IL18 in the host microenvironment (Figure 2).Conclusions.Our study demonstrates the capability of proximity-based single cell analysis of the post-transplant bone marrow microenvironment to identify a novel niche factor – IL 18. We show that IL18 specifically regulates quiescence of early hematopoietic progenitors, particularly under the conditions of stress hematopoiesis, such as exposure to 5FU or post-transplant hematopoietic expansion. The results presented here provide an insight into poorly understood molecular mechanisms of progenitor regulation by the niche. Moreover, improved post-transplant survival of animals in the absence of IL18 suggests that pharmacological inhibition of IL18 has the potential of improving clinical outcomes in transplant recipients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Conjugated bilirubin affects cytokine profiles in hepatitis A virus infection by modulating function of signal transducer and activator of transcription factors.

Hepatitis A virus (HAV) infection is the major cause of acute liver failure in paediatric patients. The clinical spectrum of infection is variable, and liver injury is determined by altered hepatic enzyme function and bilirubin concentration. We recently reported differences in cytokine profiles between distinct HAV-induced clinical courses, and bilirubin has been recognized as a potential immune-modulator. However, how bilirubin may affect cytokine profiles underlying the variability in the course of infection has not been determined. Herein, we used a transcription factor (TF) binding site identification approach to retrospectively analyse cytokine expression in HAV-infected children and to predict the entire set of TFs associated with the expression of specific cytokine profiles. The results suggested that modulation of the activity of signal transducers and activators of transcription proteins (STATs) may play a central role during HAV infection. This led us to compare the degree of STAT phosphorylation in peripheral blood lymphoid cells (PBLCs) from paediatric patients with distinct levels of conjugated bilirubin (CB). Low CB levels in sera were associated with increased STAT-1 and STAT-5 phosphorylation. A positive correlation was observed between the serum interleukin-6 (IL-6) content and CB values, whereas higher levels of CB correlated with reduced serum IL-8 values and with a reduction in the proportion of PBLCs positive for STAT-5 phosphorylation. When CB was used to stimulate patients' PBLCs in vitro, the levels of IL-6 and tumour necrosis factor-α were increased. The data showed that bilirubin plays a role in STAT function and affects cytokine profile expression during HAV infection.