Role of NKG2D in Cytokine-Induced Killer (CIK) Cells Against Multiple Myeloma Cells

Abstract Abstract 5119 Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells. Disclosures: No relevant conflicts of interest to declare.

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Regulation of Human NK Cell Activation by Expression of HLA Class I Molecules in Pig Endothelial Cells

Proceedings of IMPRS ◽

10.18060/25715 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Benjamin Obando ◽

Arthur Cross-Najafi ◽

Kevin Lopez ◽

Deepthi Thadasina ◽

Wenjun Zhang ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Human Peripheral Blood ◽

Flow Cytometric Analysis ◽

Organ Shortage ◽

Major Barrier ◽

Xenograft Rejection ◽

Hla Class I Molecules

Background: Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically engineered pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute xenograft rejection (AXR) remains a major barrier to clinical XTx. AXR is mediated by multiple immune cells, of which natural killer (NK) cells play a crucial role. Previous studies have shown that human HLA-E suppresses NK cell activation through the inhibitory receptor NKG2A. We seek to improve pig-to-human compatibility by expressing HLA-E in a genetically modified pig endothelial cell (pEC) line. This cell line 5GKO/ HLA-G+ has mutations in five genes encoding for xenoantigens and expresses HLA-G, an inhibitory ligand of the NK cell receptor KIR2DL4. In this study, the 5GKO/HLA-G+/HLA-E+ pEC line was established to examine whether co-expression of inhibitory ligands promotes NK cell tolerance. Methods: The HLA-Eα/pCDNA3.1 plasmid containing the HLA-E α-chain (HLA-Eα) cDNA driven by a CMV promoter was linearized and introduced into 106 cells of the 5GKO/HLA-G+ pEC line by electroporation. After 48 hours, HLA-E expression was analyzed by flow cytometry. HLA-E+ pECs were isolated by flow cytometry sort and co-cultured with human peripheral blood mononuclear cells (PBMCs) stimulated by IL-2. NK cell degranulation was compared between the 5GKO/HLA-G+ and 5GKO/HLA-G+/HLA-E+ pEC lines by measuring CD107a expression in the CD3- CD56+ cell population. Results: HLA-E molecules were successfully expressed on the pECs surface, indicating the HLA-E a chain can pair with the existing b2-microglobulin (B2M). The transfection efficiency was 38.2%. Three weeks later, the 5GKO/HLA-G+/HLA-E+ pEC was successfully established, confirming via flow cytometric analysis. The analysis of NK cell degranulation (CD107a) is underway. Conclusion: We established a 5GKO/HLA-G+/HLA-E+ pEC line, which is a valuable tool to study human-to-pig xenoreactive immune response in vitro, with the goal of improving pig-to-human xenograft immunotolerance.

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CD138-Independent Strategy for Detecting Residual and Circulating Myeloma Plasma Cells

Blood ◽

10.1182/blood.v124.21.2077.2077 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2077-2077

Author(s):

Barbara Muz ◽

Feda Azab ◽

de la Puente Pilar ◽

Jacob Paasch ◽

Justin King ◽

...

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Residual Disease ◽

Positive Control ◽

The Other ◽

Myeloma Cells ◽

New Strategy

Abstract INTRODUCTION: Flow cytometry has been used extensively to detect MM cells in the bone marrow (BM), micro-residual disease and circulating myeloma cells. Accumulating literature defines MM cells as CD138+/CD38+ for the primary gating of plasma cells; however, several studies demonstrated the presence of clonogenic CD138-negative MM cells, and that hypoxia can decrease the expression of CD138 in MM cells. We propose a novel set of biomarkers to detect MM cells regardless of their CD138 expression or hypoxic status. METHODS and RESULTS: We have tested the effect of hypoxia on the expression of the MM markers, CD138 and CD38, and found that hypoxia decreased the expression of CD138, therefore it cannot be used as a universal marker for MM cells. Hypoxia did not alter the expression of CD38 in MM cells; however, CD38 is a general leukocyte marker and cannot be used alone to identify MM cells, since it is expressed on multiple other leukocytes including T-cells, B-cells, monocytes, NK cells, and dendritic cells. Therefore, we negatively selected these cells by flow cytometry using the specific markers for each of these populations including CD3, CD19, CD14, CD16, and CD123; respectively. Therefore, we detected MM cells as CD38+/CD3-/CD19-/CD14-/CD16-/CD123-. We used CD38 antibody conjugated with APC and FITC-conjugated antibodies for all the other markers, thus MM cells were defined as APC+/FITC- population. To compare traditional method (CD138-based) with our strategy to detect hypoxic and normoxic MM cells, MM cell lines were stained with a cocktail of CD38-APC, FITC-antibodies, and CD138-V450, and analyzed by flow cytometry. The use of CD138+ as a universal marker for MM cells detected 85-100% of the normoxic cells, and only 60-75% of the hypoxic MM cells. While APC+/FITC- strategy detected close to a 100% of the MM cells independent of the cells’ normoxic/hypoxic status or expression of CD138. The ability of the new strategy to detect hypoxic and normoxic MM cells in the peripheral blood was tested by staining hypoxic and normoxic MM cells with cell-tracker Calcein-Red-Orange (as a positive control), spiked 104 MM cells into 106 mononuclear cells from a healthy donor (1% MM in total), and the percentile of Calcein-Red-Orange+ (as a positive control), APC+/FITC-, and CD138+ populations, were analyzed by flow cytometry. Calcein-Red-Orange staining showed exact 1% of MM cells detected in total mononuclear cells for both hypoxic and normoxic MM cells; detection with CD138+ showed 0.95% for normoxic and 0.45% for hypoxic cells; and detection of MM cells using the APC+/FITC- strategy showed 1.05% for normoxic and 1.1% for hypoxic MM cells. Hence, the new strategy detects MM cells selectively and independently of their CD138 expression or hypoxic status. We have used the APC+/FITC- strategy to detect MM cells in BM CD138-negative fractions of 20 MM patients. The APC+/FITC- strategy was able to detect a range of 1.6-44% myeloma cells in the CD138-negative population of BM mononuclear cells isolated from MM patients. Moreover, we have assessed the clonality of this population using APC+/FITC- in the CD138-negative fractions of MM patients. We found that the clonality of the APC+/FITC- population was similar to the clonality of the original disease (CD138+ cells) in 70% of the cases. The other 30% of the cases showed very low involvement of myeloma population in the BM and showed Kappa/Lambda ratio within normal range. Analyzing the prevalence of circulating MM cells in the peripheral blood from 12 MM patients showed that all patients had a higher number of circulating MM cells as detected by APC+/FITC- strategy compared to CD138+, and the fold change ranged from 1.5 to 86 times. CONCLUSION: We found that CD138 cannot be used as a universal marker to detect MM cells. Moreover, we developed a novel strategy to detect MM cells independent of their CD138 expression or hypoxic status; and we used CD38+/CD3-/CD19-/CD14-/CD16-/CD123- population as an alternative set of biomarkers to detect MM cells. This strategy was able to detect a clonal MM cell population in the CD138-negative fraction of BM mononuclear cells isolated from MM patients, as well as in the peripheral blood. Currently, we are exploring the ability of this strategy to predict relapse in MM patients whose BM was defined a CD138-negative. More investigation to characterize this population and the role in tumor recurrence and drug resistance in MM is warranted. Disclosures No relevant conflicts of interest to declare.

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Study On Immune Mechanism of Human Marrow Mesenchymal Stem Cells Adjusting Dendritic Cells for Treatment of Aplastic Anemia

Blood ◽

10.1182/blood.v116.21.5137.5137 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5137-5137

Author(s):

Yang Xiao ◽

Leqin Zhang

Keyword(s):

Flow Cytometry ◽

T Lymphocytes ◽

Surface Antigen ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Human Peripheral Blood ◽

Interleukin 4 ◽

Peripheral Blood Flow ◽

Inhibiting Effect

Abstract Abstract 5137 Objective (1)To explore whether MSC has inhibiting effect on the proliferation ofAA patients' Tcell;(2)To discuss whether MSC affects T cell's proliferation via adjusting the growth of DCs. Materials and Methods (1) MSC were separated and cultured in vitro. Cell morphology was observed and the cell surface antigen was determined by flow cytometry.(2) Peripheral blood mononuclear cells were extracted from 20 patients suffered AA and then T lymphocytes were separated by nylon fiber column. Flow cytometry was applied to determine the surface antigen and subpopulation of T cell.(3)mononuclear cells were separated from normal human peripheral blood. DCs were prepared under the culture condition of recombination human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (IL-4). After acquiring the mature DCs induced by LPS, the phenotype analysis of DCs before and after culture was examined by flow cytometry, respectively. Results (1) After co-culture of MSC and T lymphocytes from AA peripheral blood, flow cytometry showed that the ratio of D8+ in T cells reduced significantly from 38.7% to 29.7 % (p < 0.05), whereas the CD4+ ratio increased from 24.9% to 34.9% significantly (p < 0.05). Meanwhile, ELISA analysis indicated that the concentration of IL-2 and IFN-γ were significantly decreased from 38.9 and 38.5 ng/L to 6.8 and 6.6 ng/L, respectively (p < 0.05). However, IL-4 and IL-10 increased from 2.8 and 2.9 to 5.3 and 8.3 ng/L, respectively (p < 0.05).(2) After the induction of immature DCs by LPS, flow cytometry showed that the expression of CD1a increased from 2.4% to 68.4% in the treatment without MSC, while that of CD14+ decreased from 83.6% to 3.5% (p < 0.05).(3) After the co-culture of mature DCs and MSC, the expression of CD14+ increased from 5.8% to 62.8% when the expression of CD1a, CD83 and CD80 decreased from 48.6%, 60.8% and 50.2% to 30.7%,40.9% and 20.3%, respectively. Conclusions Our study shows that (1)MSC inhibits the proliferation of T lymphocytes from AA p-atients by regulating CD8+ and CD4+; (2)futher study indicates that MSC can inhibit the growth of DCs and reverse the status of DCs from matureness to immatureness; (3)thus suggests the possible mechanism of MSC's inhibiting effect as follows: MSC decreases the liveness by controlling the growth of DCs and further inhibits its proliferation. Disclosures: No relevant conflicts of interest to declare.

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Resveratrol Enhances NKG2D-Mediated Cytotoxicity Against Leukemia Cells by Upregulating Both NKG2D Receptor on NK Cells As Well As NKG2D Ligands on Target Cells,

Blood ◽

10.1182/blood.v118.21.3236.3236 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3236-3236

Author(s):

Luis J. Espinoza ◽

Akiyoshi Takami ◽

Katsuya Nakata ◽

Ly Quoc Trung ◽

Kayoko Yamada ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Specific Inhibitor ◽

Receptor Expression ◽

Leukemia Cells ◽

Target Cells ◽

Lymphoid Leukemia ◽

Nkg2d Ligands ◽

Nkg2d Receptor

Abstract Abstract 3236 NKG2D is a powerful activating receptor expressed by natural killer (NK) cells that promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). Pharmacological induction of NKG2D-Ls in malignant cells has been an attractive therapeutic approach but has gained poor clinical utility because currently available NKG2D-Ls inducers have been hampered either by their limited efficacy or by their associated toxicity. Resveratrol (RVT), a compound derived from several natural sources, has proved in vivo and in vitro potent anti-tumor effects against various cancers. Extensive research in the last decade has shown that such effects are mediated by targeting various molecules involved in the regulation of proliferation and cell survival and those include, NFκB, STAT3, ATM/ATR and ERK1/2. To date, it is unknown whether RVT has any effect on NKG2D-Ls expression. We report here NKG2D-Ls induction by RVT in a broad range of leukemia cells. RVT upregulated the NKG2D-Ls MICA/B, ULBP1, ULBP2 and ULBP3 in the myeloid leukemia cells OUN-1, NB4, THP-1 and KG1 and upregulated MICA/B, ULBP-1 and ULBP3 ligands in the lymphoid leukemia cells Jurkat and Molt-4. The upregulation of NKG2D-Ls by RVT was associated with increased transcription of each NKG2D-L gene. Ligand upregulation induced by RVT was prevented by cell pretreatment with caffeine, and inhibitor of ATM/ATR, which is the main signal regulator of NKG2D-Ls expression. Leukemia cells treated with RVT were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the treatment of NK cells with anti-NKG2D monoclonal antibodies. Interestingly, the same concentration of RVT that effectively induced NKG2D-Ls in tumor cells, consistently upregulated NKG2D receptor expression in primary NK cells from healthy individuals and in the NK cell lines NKL and NK-92 and this effect was also associated with enhanced NKG2D-mediated NK cells cytotoxicity. RVT-induced NKG2D receptor enhancement in NK cells associated with the activation of the MAP kinase ERK1/2 and was prevented by the ERK1/2 specific inhibitor PD98059. Thus, RVT represents the first identified agent capable of activating both arms of the NKG2D axis. Since several clinical trials on RVT are ongoing, these previously unrecognized properties of this non toxic compound have an attractive immunotherapeutic potential. Disclosures: No relevant conflicts of interest to declare.

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Essential Roles of Monocytes in Stimulating Human Peripheral Blood Mononuclear Cells with Lactobacillus casei To Produce Cytokines and Augment Natural Killer Cell Activity

Clinical and Vaccine Immunology ◽

10.1128/cvi.00076-06 ◽

2006 ◽

Vol 13 (9) ◽

pp. 997-1003 ◽

Cited By ~ 62

Author(s):

Kan Shida ◽

Tomomi Suzuki ◽

Junko Kiyoshima-Shibata ◽

Shin-ichiro Shimada ◽

Masanobu Nanno

Keyword(s):

Lactobacillus Casei ◽

Mononuclear Cells ◽

Nk Cell ◽

Human Peripheral Blood ◽

Cell Activity ◽

Nk Cell Activity ◽

Peripheral Blood Mononuclear ◽

Tnf Α ◽

Blood Mononuclear Cells ◽

Ifn Γ

ABSTRACT We examined the effect of a probiotic strain, Lactobacillus casei strain Shirota, on cytokine production and natural killer (NK) cell activity in human peripheral blood mononuclear cells (PBMNC). The cellular mechanisms of immunoregulation by L. casei strain Shirota were also investigated. L. casei strain Shirota stimulated PBMNC to secrete interleukin-12 (IL-12), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10. However, depletion of monocytes from PBMNC eliminated the induction of these cytokines. L. casei strain Shirota was phagocytosed by monocytes and directly stimulated them to secrete IL-12, TNF-α, and IL-10. IFN-γ production was diminished by the addition of anti-IL-12 antibody to the PBMNC cultures. Purified T cells, but not NK cells, produced IFN-γ effectively when stimulated with L. casei strain Shirota in the presence of monocytes, indicating that monocytes triggered by L. casei strain Shirota help T cells to produce IFN-γ through secreting IL-12. In addition, NK cell activity and CD69 expression on NK cells increased after cultivation of PBMNC with L. casei strain Shirota. When monocytes were depleted from PBMNC, L. casei strain Shirota did not enhance NK cell activity. These results demonstrate that monocytes play critical roles in the induction of cytokines and following the augmentation of NK cell activity during the stimulation of human PBMNC with L. casei strain Shirota.

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Donor and Host Coexpressing KIR Ligands Promote NK Education Post Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood-2019-129609 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4519-4519

Author(s):

Xingxing Yu ◽

Xiang-Yu Zhao ◽

Zhengli Xu ◽

Xunhong Cao ◽

Mingrui Huo ◽

...

Keyword(s):

Nk Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Hla Typing ◽

Target Cells ◽

Hematopoietic Stem ◽

Hla Ligands ◽

Ifn Γ ◽

Hla Genotype

Background:Educated NK cells prevent autoreactive behavior but also permit cytotoxicity against target cells that have down-regulated HLA class I expression. When and how the process of education occurs has not been clearly discerned. Several groups reported that both the donor and host MHC could influence NK cell education in mouse models. In humans, Dulphy et al demonstrated that NK-cell education is shaped by donor HLA genotype. Moreover, our previous study found NK-cell education was shaped by host HLA genotype post allo-HSCT. However, due to the lack of single-KIR+ NK cells, functional analysis limited the full evaluation of the interaction between donor/host HLA and donor inhibitory KIR, so the contribution of donor HLA could not be excluded. Aims: In this research, we have investigated the relative contributions of donor and recipient HLA to NK cells education, the interplay between functional reconstitution and the involvement of donor/host HLA interaction in NK cell control of leukemia cells. Methods: Two cohorts of patients were enrolled in this study. We first prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore NK cell phenotypes and functional reconstitution. From June 2012 to April 2016, 276 AML/MDS patients that underwent haploidentical transplantation were enrolled in the second cohort to analyze the effect of donor-host KIR-HLA combinations on relapse post transplantation. Molecular HLA typing and KIR genotyping were performed according to the manufacturer's instructions (One Lambda, Canoga Park, CA, USA). Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry. The cytotoxicity and cytokine secretion of NK cells was determined using CD107a expression and IFN-γ production against the K562 cell line. Single-KIR+ NK cells were grouped into the following groups: (A) nsKIR: where both hosts and donors lacked HLA ligands for one donor KIR; (B) d-rsKIR, where donors and hosts, encoded HLA ligands for donor KIRs; (C) dsKIR, where donors, but not hosts, encoded HLA ligands for donor KIR; and (D) rsKIR, where hosts, but not donors, encoded HLA ligands for donor KIR. Results: 1. Donor KIR ligated by both donor and host HLA is associated with better single-KIR+ NK cell education among the same patients. KIR2DL2/L3 single+ NK cell exhibited higher reactivity compared to KIR2DL1 single+ NK cell in pairs of donors C1C1 or C1C2 and host C1C1. KIR2DL2/L3 single+ NK cell exhibited higher reactivity than KIR3DL1 single+ NK cell in pairs of donors Bw4C1Cx and host C1Cx. KIR2DL1 single+ NK cell exhibited comparable reactivity with KIR2DL2/L3 single+ NK cell in pairs of donor Bw4C1C2 and host Bw4C1C2. 2. Donor KIR ligated by both donor and host HLA contribute to better single-KIR+ NK cell education among the same single-KIR+ NK cells. KIR2DL2/L3 single+ NK cell in the group of d-rsKIR (C1Cx-C1Cx) exhibit higher reactivity compared with other groups (dsKIR (C1Cx-C2C2), rsKIR (C2C2- C1Cx)). KIR2DL1 single+ NK cells in group of d-rsKIR (C2Cx-C2Cx) exhibited higher reactivity compared with other groups (nsKIR (C1C1-C1C1), dsKIR (C2Cx-C1C1), rsKIR (C1C1-C2Cx)). KIR3DL1 single+ NK cells in groups of d-rsKIR (Bw4Bwx-Bw4Bwx) exhibited higher reactivity compared with other groups (nsKIR (Bw6Bw6-Bw6Bw6), dsKIR (Bw4Bwx-Bw6Bw6), rsKIR (Bw6Bw6-Bw4Bwx)). 3. Both donor and host HLA must coexist for maximum education of NK cells given donor 3 inhibitory KIRs. When both of donor and host presenting all HLA (Bw4C1C2), we showed a remarkable hierarchy of responses among NK populations. NK cells with two inhibitory KIRs for self-HLA exhibited higher NK responsiveness (CD107α and IFN-γ) compared with single KIR+ NK cells. NK cells with 3 inhibitory KIRs for self-HLA exhibited maximum responsiveness. 4. Both donor and host exhibiting all HLA (Bw4C1C2) for donor 3 inhibitory KIRs contributes to least relapse following haploidentical allo-HSCT. In the second cohort, the lowest relapse rate was found in d-rsKIR group (n=31, 0%) compared with rsKIR group (n=55 ,0% vs. 10.0±4.9%, P=0.115), dsKIR group (n=33, 0% vs 14.9%±7.0%, P=0.039), or nsKIR group (n=156, 0% vs. 18%±3.5%, P=0.022). Summary: This study demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients. Disclosures No relevant conflicts of interest to declare.

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Evaluation of Immunostimulatory Activities of Synthetic Mannose-Containing Structures Mimicking the β-(1→2)-Linked Cell Wall Mannans of Candida albicans

Clinical and Vaccine Immunology ◽

10.1128/cvi.00298-12 ◽

2012 ◽

Vol 19 (11) ◽

pp. 1889-1893 ◽

Cited By ~ 14

Author(s):

Kaarina Ranta ◽

Kaisa Nieminen ◽

Filip S. Ekholm ◽

Moniká Poláková ◽

Mattias U. Roslund ◽

...

Keyword(s):

Candida Albicans ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Peripheral Blood Mononuclear ◽

Content Type ◽

Mouse Macrophages ◽

Ifn Γ ◽

Low Levels ◽

Necrosis Factor

ABSTRACTImmunostimulatory properties of synthetic structures mimicking the β-(1→2)-linked mannans ofCandida albicanswere evaluatedin vitro. Contrary to earlier observations, tumor necrosis factor (TNF) production was not detected after stimulation with mannotetraose in mouse macrophages. Divalent disaccharide 1,4-bis(α-d-mannopyranosyloxy)butane induced TNF and some molecules induced low levels of gamma interferon (IFN-γ) in human peripheral blood mononuclear cells (PBMC).

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Therapeutic Targeting of CCR1 to Prevent Dissemination of Multiple Myeloma Plasma Cells

Blood ◽

10.1182/blood-2019-121974 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3099-3099

Author(s):

Mara N Zeissig ◽

Duncan R Hewett ◽

Krzysztof M Mrozik ◽

Vasilios Panagopoulos ◽

Monika Engelhardt ◽

...

Keyword(s):

Flow Cytometry ◽

Disease Progression ◽

Mononuclear Cells ◽

Plasma Cells ◽

Primary Tumour ◽

Nsg Mice ◽

Rpmi 8226 ◽

Retention Signal

Introduction:Multiple myeloma (MM) disease progression is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow (BM), re-enter the peripheral blood (PB) and disseminate to other BM sites. Previous studies show that expression of CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome this retention signal enabling MM PC egress and dissemination via the PB are poorly understood. Previous studies in haematopoietic progenitor cells have demonstrated that CCL3 overcomes the CXCL12 retention signal to drive mobilisation to the PB (Lord et al. Blood 1995). Here, we examined the role of the CCL3 chemokine receptor CCR1 in driving MM PC dissemination. Methods and results: Initially, we assessed the expression of CCR1 protein on CD138+CD38++CD45loCD19- PC from 28 MM, 8 MGUS and 2 SMM patients by flow cytometry. Results show CCR1 expression is significantly increased in newly diagnosed MM compared with premalignant MGUS and SMM patients (p=0.03; CCR1 MFI mean±SEM, MGUS: 53.0±33.6; SMM: 37.6±8.9 MM: 250.9±71.6). Furthermore, CCR1 expression on PB MM PC positively correlated with PB MM PC numbers (p=0.03; n=11 patients). To identify mechanistically how CCR1 may promote dissemination, the effect of CCL3 on the response to CXCL12 in human myeloma cell lines (HMCL) was assessed in vitro. The migration of RPMI-8226 and OPM2 cells was induced by CCL3 or CXCL12 chemoattractant in a transwell assay. Notably, pre-treatment of RPMI-8226 or OPM2 with CCL3 abrogated migration towards CXCL12 and blocked F-actin remodelling in response to CXCL12 in vitro. These findings suggest that CCL3 can desensitise cells to exogenous CXCL12, providing a potential mechanism facilitating loss of the CXCL12 retention signal. To confirm whether CCR1 is required for driving MM PC dissemination, homozygous CCR1 knockout (KO) cells were generated using a lentiviral CRISPR/Cas9 system in OPM2 cells. CCR1-KO OPM2 cells were confirmed to have no detectable CCR1 expression by flow cytometry and could no longer migrate towards CCL3 in vitro. Empty vector (EV) or CCR1-KO OPM2 MM PC were injected into the tibia of immune-compromised NOD-scidgamma (NSG) mice. After 4 weeks, primary tumour within the injected tibia and disseminated tumour in the PB and the contralateral tibia and femur was assessed by flow cytometry. We found that mice bearing CCR1-KO cells have a 45.5% decrease in primary tumour growth (p=0.008; % GFP+ of total mononuclear cells, EV: 77.2±17.2; CCR1-KO: 42.1±24.4), a 97.8% reduction in PB MM PC (p<0.0001; EV: 1.39±0.7; CCR1-KO: 0.03±0.046) anda 99.9% reduction in BM tumour dissemination (p<0.0001; EV: 49.5±17; CCR1-KO: 0.019±0.013), compared with controls. In a supportive study, CCR1 was expressed in the murine MM cell line 5TGM1 using lentiviral transduction. 5TGM1-CCR1 cells were confirmed to express CCR1 by qPCR and were able to migrate towards CCL3 in vitro. 5TGM1-CCR1 or EV cells were injected into the tibiae of C57BL/KaLwRij mice and allowed to initiate systemic MM disease for 3.5 weeks. Importantly, while 55% of control mice exhibited disseminated tumours, this increased to 92% with CCR1 expression (p<0.0001; n=12/group). These data suggest that CCR1 expression on MM PC may play an important role in MM PC dissemination. To determine whether therapeutic inhibition of CCR1 prevents dissemination, the effect of a small molecule CCR1 inhibitor, CCR1i, was assessed in vivo. OPM2 EV or RPMI-8226 cells were injected into the tibia of NSG mice and, after 3 days, mice were treated with CCR1i (15mg/kg) or vehicle twice daily by oral gavage for 25 days. OPM2-inoculated CCR1i-treated mice had 66.1% lower PB MM PC (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 23.9±7.2; CCR1i: 8.1±3.8) and a 22.1% reduction in BM dissemination (p=0.0002; vehicle: 78.1±4.8;CCR1i: 60.8±7.1) compared with controls. Similarly, CCR1i treatment reduced BM dissemination by 59.6% in RPMI-8226 bearing mice (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 0.86±0.15; CCR1i: 0.26±0.05). This suggests that CCR1 inhibition can slow tumour dissemination in vivo. Conclusion:This study identified CCR1 as a novel driver of MM PC dissemination in vivo, at least in part by overcoming the CXCL12 retention signal. Importantly, this study demonstrated for the first time that targeting CCR1 can be a viable therapeutic strategy to limit dissemination and potentially slow disease progression. Disclosures Croucher: Trovagene: Employment.

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T Cell Subsets and Associated Cytokines in Chronic Graft-Versus-Host-Disease Using G-CSF Primed Bone Marrow in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.4580.4580 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4580-4580

Author(s):

Monica M Rivera Franco ◽

Eucario Leon Rodriguez ◽

Diana Gomez Martin ◽

Javier Merayo Chalico ◽

Jorge Alcocer Varela

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Th17 Cells ◽

Mononuclear Cells ◽

Chronic Gvhd ◽

The Other ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Ifn Γ

Abstract Background Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. It is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Regulatory T (T regs) cells and T helper 17 (Th17) cells are two recently described lymphocyte subsets with opposing actions. Both can develop from naïve CD4+ T cell precursors under the influence of TGFβ1. Th17 lymphocytes, are key effector cells in rodent models of human diseases including GVHD. The other subset, T regs, is essential for dominant immunologic tolerance. At our institution, patients transplanted using G-CSF primed bone marrow (G-BM), have a lower incidence of acute and chronic GVHD when compared to those transplanted with peripheral blood and not primed bone marrow. Some microenvironment characteristics of this hematopoietic stem cells (HSC) source remain unknown, as well as the difference between Tregs, Th17 and cytokine levels in patients who develop GVHD and those who do not. Objective To analyze the characteristics of thirty-eight G-BM donor samples, identifying lymphocytes subsets and associated cytokines, and comparing patients who developed chronic GVHD (cGVHD) and those who did not. Materials and Methods A prospective analysis was performed in 38 G-BM samples from donors from 1999 to 2016. Mononuclear cells were defrosted, counted, and viability was evaluated. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay). From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN-γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Patients (recipients) information was analyzed using SPSS v.21. Results GVHD incidence was reported as following: Three (8%) patients developed acute GVHD (2 grade II, and 1 grade IV), 11 patients (29%) developed chronic GVHD (9% extensive, and 91% limited), and 24 patients did not present either. Mononuclear cells from G-BM from donors of patients who developed cGVHD showed a pro inflammatory response, characterized by an increased concentration of IL-17A (15.5 vs 0.71 pg/mL, p=0.013), TNF-α (80.27 vs 0.13 pg/mL, p=0.001), and IL-6 (4953.6 vs 11.75 pg/mL, p=0.025), after a mitogenic stimulation, compared to cells from donors of patients who did not developed GVHD. On the other hand, a decreased IL-10 production (2.62 vs 52.81 pg/mL, p=0.001) was documented in mononuclear cells from donors of patients who developed chronic GVHD, compared to donor cells of patients who did not. No significant difference in the production of IL-2, IL-4, and IFN-γ was observed. There was no difference in Th1 and Th2 between both groups, but mononuclear cells from donors of patients who developed chronic GVHD had a higher percentage of Th17 (1.02% vs 0.46%, p<0.001), and less Tregs (0.88% vs 1.95%, p<0.001), compared to those who did not developed GVHD. Conclusions Patients who develop cGVHD (29%) are characterized by a pro inflammatory response with an increased production of IL-17A, IL-6, and IFN-γ, and also a major percentage of Th17 cells. Also, a decreased suppressive response was documented with reduced IL-10 and Tregs levels. The low incidence of cGVHD show that G-CSF primed bone marrow is an excellent source for allogeneic HSC transplantations, and would be useful to compare these results with other HSC sources. Disclosures No relevant conflicts of interest to declare.

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Aberrant anti-viral response of natural killer cells in severe asthma

European Respiratory Journal ◽

10.1183/13993003.02422-2018 ◽

2020 ◽

Vol 55 (5) ◽

pp. 1802422

Author(s):

Justine Devulder ◽

Cécile Chenivesse ◽

Valérie Ledroit ◽

Stéphanie Fry ◽

Pierre-Emmanuel Lobert ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Severe Asthma ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Asthma Patients ◽

Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

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