The Pleiotropic Immunosuppressive Role of Osteoclasts in Multiple Myeloma

Abstract Skeletal related events impact both quality of life and overall survival of multiple myeloma (MM) patients. The underlying cause of osteolytic bone disease is an imbalance between bone-resorbing osteoclasts and bone-forming osteoblasts. Bone cells are regulated by a complex milieu of bone marrow microenvironmental components including the immune system via the effect of cytokines, signaling molecules and regulatory proteins. For example, osteoclastogenesis is tightly regulated by T cells through signaling crosstalk between RANKL and IFN-γ (Takayanagi H et al., Nature 2000). However, the effect of osteoclasts (OCs) on the immune system is less well defined. Here we investigated the effect of osteoclasts on immune cells including T cells, Th17 cells, NK cells and myeloid-derived suppressor cells (MDSCs) in MM. To gain insight into the functional impact of OCs on the immune cells, each type of immune cell was isolated from peripheral blood mononuclear cells (PBMNCs) and examined further in co-cultures with OCs. Our preliminary data showed that the frequency of NK cells and MDSCs in PBMNCs derived from myeloma patients increased when co-cultured with autologous OCs. Activation of the inhibitory immune checkpoints suppresses T cell-mediated antitumor immunity. Up-regulation of several co-inhibitory molecules has also been shown in MM (Kwon M et al., J Immunol 2017). We, therefore, assessed the expression of various immune checkpoint receptors, such as PD-1, LAG-3 and B7-H3, on T cells in co-culture with or without OCs by multi-color flow cytometry. OC co-culture significantly increased PD-1 expression in both CD4+and CD8+ T cell populations. The presence of OCs also enhanced PD-L1 protein expression on MM cells. Th17 cells, a newsubset of helper T cells, have recently been identified as immunosuppressive cells. Th17 cells are increased in myeloma; morevover they secrete IL-17 which promotes myeloma cell growth (Prabhala RH et al., Blood 2010). Interestingly, we found that naïve CD4 T cells have a higher propensity to differentiate into Th17 lineage in the presence of Th17 differentiation cytokines when co-cultured with OCs. Furthermore, we also observed an increased expansion of Th17 cells in co-culture with OCs. NK cell cytotoxic function is severely impaired in myeloma (Fauriat C et al., Leukemia. 2006). In our experiments, NK cells co-cultured in the presence of OCs demonstrated significant decrease in NK cell cytotoxicity, despite increase in NK cell numbers. Our study demonstrated that OCs suppress the cytotoxic function of T cells and NK cells and induce the expansion of immunosuppressive Th17 cells and MDSCs through direct contact. These findings indicate that OCs play an important role in tumor progression in part by enhancing immune suppression. Targeting OCs represent an attractive therapeutic option not only for controlling osteolytic bone disease but also for restoring the impaired immune surveillance in MM. Ongoing studies will focus on understanding direct-contact-mediated interactions between OCs and immune cells. Furthermore, the effect of OC inhibition using anti-resorptive drugs on the immune system will be interrrogated. Disclosures Raje: Medscape: Honoraria; Research to Practice: Honoraria; AstraZeneca: Research Funding; Takeda: Consultancy; Merck: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Amgen Inc.: Consultancy.

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Osteoclast-Induced Immunosuppression Occurs through Dysregulation of Immune Checkpoint Axes in Multiple Myeloma

Blood ◽

10.1182/blood-2019-130462 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4382-4382

Author(s):

Kenta Mukaihara ◽

Mattia D'Agostino ◽

Cristina Panaroni ◽

Keertik Fulzele ◽

Tomoaki Mori ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Immune Cells ◽

Immune Checkpoint ◽

Th17 Cells ◽

Immune Surveillance ◽

Color Flow ◽

Osteolytic Bone Disease ◽

Ifn Γ

Osteolytic bone disease has a significant impact on both quality of life and overall survival of patients with multiple myeloma (MM). Given that bone and immune cells share the same microenvironment and interact with each other via cytokines, signaling molecules and regulatory proteins in the bone marrow (Tsukasaki M et al., Nat Rev Immunol 2019), we sought to elucidate the effect of osteoclasts (OCs) on the immune microenvironment. Specifically, T cells significantly suppress differentiation of OCs through signaling crosstalk between RANKL and IFN-γ (Takayanagi H et al., Nature 2000). However, little is known regarding the effect of OCs on the immune system. Here we investigated the effect of OCs on immune cells, especially on T cells. Dysregulation of several checkpoint molecules has been shown in MM (Kwon M et al., J Immunol 2017). We, therefore, assessed the expression of various immune checkpoint receptors, such as PD-1, TIGIT, OX40 and CD137 on T cells in co-culture with or without autologous OCs by multi-color flow cytometry. OC co-culture significantly increased co-inhibitory checkpoint (PD-1 and TIGIT) through direct contact, while decreasing co-stimulatory checkpoint (OX40 and CD137) in CD3+ T cells. Expression of the checkpoint ligand, PD-L1 was significantly increased on MM cells in the presence of both T cells and OCs compared to the presence of T cells alone. Conversely, OCs in the absence of T cells did not induce significant increase of PD-L1 on MM cells. According to previous literature, PD-L1 expression on MM cells is induced in part by IFN-γ (Liu J et al., Blood 2007). Interestingly, our data demonstrated that OCs activated IFN-γ producing T cells in co-culture conditions. Furthermore, we observed that T cell and OC-mediated upregulation of PD-L1 on MM cells was partially reversed by using IFN-γ neutralizing antibody. This implies that OCs indirectly induce PD-L1 upregulation on MM cells by enhancing IFN-γ secretion from T cells. In addition, we found that naïve CD4+ T cells have a higher propensity to differentiate into Th17 lineage in the presence of Th17 differentiation cytokines when co-cultured with OCs. Moreover, we observed an increased expansion of Th17 cells in co-culture with OCs. Those Th17 cells also showed a similar pattern of dysregulation of immune checkpoint axes. Our study demonstrates that OCs positively regulate co-inhibitory checkpoint molecules and negatively regulate co-stimulatory molecules on T cells. These findings indicate that OCs play an important role in inhibiting T cell-mediated antitumor immunity. Targeting OCs may help restore impaired immune surveillance in MM in addition to their critical role in preventing lytic bone lesions in MM. These data may support the role of antiresorptives in immune surveillance with indirect anti-tumor effects. Disclosures No relevant conflicts of interest to declare.

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Prognostic significance of natural killer cell-associated markers in gastric cancer: quantitative analysis using multiplex immunohistochemistry

Journal of Translational Medicine ◽

10.1186/s12967-021-03203-8 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hee Young Na ◽

Yujun Park ◽

Soo Kyung Nam ◽

Jiwon Koh ◽

Yoonjin Kwak ◽

...

Keyword(s):

Gastric Cancer ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Natural Killer ◽

Immune Cells ◽

Nk Cell ◽

Critical Role ◽

Killer Cell ◽

Prognostic Significance

Abstract Background Natural killer (NK) cells mediate the anti-tumoral immune response as an important component of innate immunity. The aim of this study was to investigate the prognostic significance and functional implication of NK cell-associated surface receptors in gastric cancer (GC) by using multiplex immunohistochemistry (mIHC). Methods We performed an mIHC on tissue microarray slides, including 55 GC tissue samples. A total of 11 antibodies including CD57, NKG2A, CD16, HLA-E, CD3, CD20, CD45, CD68, CK, SMA, and ki-67 were used. CD45 + CD3-CD57 + cells were considered as CD57 + NK cells. Results Among CD45 + immune cells, the proportion of CD57 + NK cell was the lowest (3.8%), whereas that of CD57 + and CD57- T cells (65.5%) was the highest, followed by macrophages (25.4%), and B cells (5.3%). CD57 + NK cells constituted 20% of CD45 + CD57 + immune cells while the remaining 80% were CD57 + T cells. The expression of HLA-E in tumor cells correlated with that in tumoral T cells, B cells, and macrophages, but not CD57 + NK cells. The higher density of tumoral CD57 + NK cells and tumoral CD57 + NKG2A + NK cells was associated with inferior survival. Conclusions Although the number of CD57 + NK cells was lower than that of other immune cells, CD57 + NK cells and CD57 + NKG2A + NK cells were significantly associated with poor outcomes, suggesting that NK cell subsets play a critical role in GC progression. NK cells and their inhibitory receptor, NKG2A, may be potential targets in GC.

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Long Lasting Alteration of Natural Killer Cells Post-Chemotherapy in Elderly Patients with Acute Myeloid Leukemia (AML).

Blood ◽

10.1182/blood.v114.22.1653.1653 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1653-1653

Author(s):

Daniel Olive ◽

Nicolas Anfossi ◽

Pascale Andre ◽

Jerome Rey ◽

Florence Orlanducci ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

Elderly Patients ◽

Nk Cells ◽

Natural Killer ◽

Cd8 T Cells ◽

Nk Cell ◽

Time Points ◽

Early Stages ◽

Cell Counts

Abstract Abstract 1653 Poster Board I-679 Background The immune system is involved in AML control and Natural Killer (NK) cells are among the most promising effectors. The therapeutic potential of NK cells has been revealed by the Killer Immunoglobulin Receptor (KIR) mismatch allogeneic transplant model where the anti-leukemic effect of the graft, is due to unleashed NK cells towards AML blasts, as suggested by enhanced in vitro lytic activity of KIR-HLA mismatched donor NK cells against recipient blasts (Miller et al. 2005; Ruggeri et al. 2002). Receptors involved in the function of NK cells against AML blasts have been identified (Pende et al., 2005). Some of these receptors are altered in AML patients at diagnosis and might be involved in the immune escape of AML blasts (Costello et al., 2002). However, the status of NK cells during early stages of patient's chemotherapy (CT) treatment is unknown. The present study monitored status of NK cells during early stages following patient's remission after CT that may be critical for their long lasting clinical response, and results might provide new targets for immunotherapy. Methods We enrolled 20 elderly patients (60 to 80 years old) with non promyelocytic AML in first CR following induction and pre-consolidation CT with normal renal and hepatic functions. Patient peripheral NK, gd T and CD8 T cells were analyzed before consolidation CT and every other week after treatment for 8 weeks. 6-colors flow cytometry was performed to investigate the expression of MHC receptors (CD158a, b, e, i, CD85j and NKG2A), activating receptors (NKp30, NKp46, NKG2D, CD16, DNAM-1, 2B4) as well as their differentiation status (perforin and granzyme expression). Their function, as determined by cytotoxicity (51Cr release and CD107 expression) and cytokine production (intracellular staining of IFN-g), was analyzed using purified NK cells stimulated by K562, or in redirected assays using NKp30, NKp46 and CD16 mAbs. Results NK cell counts were depressed away from the induction and pre-consolidation CT as compared to NK cell counts of age-matched controls (ctl) (95±107 NK/μL vs 229±91 NK/μL respectively); they were further depressed during the first 2 weeks post-consolidation CT (55±57 NK/μL), but were back to pre-consolidation CT level at 4 weeks (105±102 NK/μL). In contrast, CD8 T cells and gd T cells counts were normal even at early times post-CT. Expression of 2B4 was depressed at all time points. In contrast, NKp30 expression was lower at diagnosis and close to ctl level post-consolidation CT (p=0.0003) and NKp46 expression increased after CT (p<0.0001). Sizes of NK cell subsets expressing CD158a or CD158b in patients post-induction and consolidation CT were smaller than those of ctl (% CD158a+: p=0.003; % CD158b+: p=0.014). In contrast the NKG2A or CD85j positive NK cell subsets were either unchanged or slightly increased respectively at all time points (p=0.0015 for CD85j+). Moreover, sizes of perforin or granzyme positive NK cell subsets were increased in treated AML patients (% Granzyme+: p= 0.0125 and % Perforin+: p=0.0268). In addition, we observed an important heterogeneity in the expression of the surface receptors among patients that is currently analyzed with respect to the duration of the CR. Finally, NK cell cytoxicity was comparable at all time points to the one of age-matched ctl. In contrast, IFN-g secretion was decreased, at all time points, against K562 or in redirected assays using CD16 mAb and almost abolished using redirected assay with NKp30 mAb. Conclusions This study demonstrates that in elderly AML patients in CR after CT (1) several alterations are detected at all time points, (2) NK cell number is lower and (3) IFN-g secretion is impaired. However NK cytotoxic function is comparable to age-matched controls. The likely basis of the complex pattern of modifications might rely on an interplay between the direct and indirect effects of chemotherapy, activation of immune system, NK cell differentiation and its interaction with AML blasts. Altogether this study indicates that new immunotherapeutic approaches might be used to increase NK cell numbers and functions (cytotoxicity and IFN-g secretion) at early times post-CT in elderly patients with AML. Disclosures Romagne: Innate Pharma: Employment.

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Activation of HIF Signaling in Mononuclear Phagocyte System Causes Hemophagocytic Lymphohistiocytosis in C57/BL6 Mice

Blood ◽

10.1182/blood.v128.22.3675.3675 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3675-3675

Author(s):

Rui Huang ◽

Yoshihiro Hayashi ◽

Xiaomei Yan ◽

Michael Jordan ◽

Gang Huang

Keyword(s):

T Cells ◽

Nk Cells ◽

Immune Cells ◽

Nk Cell ◽

Macrophage Polarization ◽

Mononuclear Phagocyte ◽

Mononuclear Phagocyte System ◽

Cell Numbers ◽

Ifn Γ

Abstract Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by an overwhelming activation of diverse immune cells. Chemotherapy based regimen as a first-line therapy for HLH has a substantial risk of mortality. Thus, exploring a less toxic therapy is urgently needed. Accumulating evidence suggests that hypoxia-inducible factors (HIFs) play an important role in the regulation of the immune system. Hypoxia, as well as inflammation, infectious microorganisms, and cancer, triggers HIF expression and stabilization in immune cells. HIFs activation enhances phagocyte capacity, drives T cell differentiation and increases cytotoxic activity. HIFs also regulate the cellular metabolism of immune cells to dictate their fate, development, and function. However, little is known about HIFs' function in HLH pathogenesis. To determine whether activation of HIF-1α/HIF-1b in hematopoietic cells is sufficient to induce HLH phenotypes, we generated transgenic mice with doxycycline-inducible HIF-1α/HIF-1b expression. Using a Vav1-Cre/Rosa26-LSL-rtTA driver, a doxycycline-inducible expression of both a stable and constitutively active human HIF-1α triple-point-mutation (TPM) and wild-type HIF-1β was achieved. All the inducible HIF-1α/HIF-1b mice developed HLH phenotypes in C57/BL6background and died within three weeks. They quickly developed severe anemia, thrombocytopenia, multi-organ failure, splenomegaly, and hemophagocytosis. Total and type-1 polarized macrophages were significantly increased in the bone marrow (BM) and spleen (SP) of HLH mice compared to the controls. To determine the phagocytic activity of the type-1 polarized macrophages, we generated type-1 and type-2 polarized macrophages from BM mononuclear cells in vitro with M-CSF/IFNγ or M-CSF/IL-4 respectively. We found that type 1 rather than type 2 macrophages engulfed erythroblasts in an in vitro co-culture assay. IFN-γ signaling is critical for Type-1 macrophage polarization. We generated IFN-γ receptor-/- /Vav1-Cre/LSL/TPM mice and found that knockout of IFN-γ receptor completely blocked the macrophage activation and HLH development. Interestingly, the serum IFN-γ level was only slightly upregulated in Vav1-Cre/LSL/TPM mice, suggesting that IFN-γ locally, but not systemically, exerts its function in our HLH model. However IFN-γ expression in NK cells and CD8+ T cells did not increase. Thus, the source of the IFN-γ for macrophage polarization is still unclear. In primary HLH, defective cytotoxic function in NK and T cells is important for HLH development. We found that the cell surface CD107a (degranulation) and NK46p (activating receptor) expression didn't change in these HLH mice. Interestingly, the absolute numbers of total NK cells and DX5+ mature cytotoxic NK cells were significantly reduced in the PB, SP, and BM from HLH mice. However, the frequencies of CD8+ T cells, CD4+ T cells, Th17 cells, and total T cells did not change in HLH mice. In order to dissect the contribution of individual immune cell subpopulations to the HLH pathogenesis, diverse lineage specific Cre transgenic alleles were used. Induction of TPM allele in myeloid cells (LysM-Cre), mature T cells (dLck-Cre), NK cells (NCR-Cre), or multi-lineages (LysM-Cre/dLck-Cre) did not cause HLH. Interestingly, induction of TPM allele in the mononuclear phagocyte system (monocyte, macrophage, and dendritic cells) with Cx3cr1-Cre could give rise to HLH phenotypes, as well as macrophage activation and reduced NK cell numbers, which are similar to the Vav1-Cre model. These results indicate that activation of HIF-1α/HIF-1b in the mononuclear phagocyte system is sufficient to polarize macrophages and induce HLH phenotype. The reduced NK cell numbers may be secondary to macrophage/dendritic cell activation in this HLH model. In conclusion, we found that; 1) induction of stable and constitutively active form of HIF-1α/HIF-1β expression in hematopoietic cells, especially in the mononuclear phagocyte system, polarizes macrophages and causes HLH, 2) IFN-γ signaling is required for HLH development and macrophages/dendritic cells are critical immune cell populations in this model, 3) Reduced NK cell numbers may be a secondary phenomenon in this model. This new HLH model recapitulates the features of secondary HLH in human, provides a unique model for dissecting the detail mechanisms, and helps in testing new therapies for sHLH. Disclosures No relevant conflicts of interest to declare.

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Pre-Clinical and Clinical Immunomodulatory Effects of Pomalidomide or CC-92480 in Combination with Bortezomib in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153994 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1613-1613

Author(s):

Chad C Bjorklund ◽

Michael Amatangelo ◽

Hsiling Chiu ◽

Jian Kang ◽

Tiziana Civardi ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Granzyme B ◽

Target Cells ◽

Publicly Traded ◽

Dose Dependent ◽

Privately Held

Abstract Background: Pomalidomide (POM) is an established agent in relapsed/refractory (R/R) multiple myeloma (MM) with direct cytotoxicity against MM cells and immunostimulatory activities in multiple cell types including T cells and NK cells. CC-92480 is a novel Aiolos/Ikaros degrading cereblon E3 ligase modulator (CELMoD ®) agent is currently being investigated in combination with the proteasome inhibitor (PI) bortezomib (BTZ) and corticosteroid dexamethasone (DEX), or with DEX only in R/R MM (CC-92480-MM-002 and CC-92480-MM-001). Previous results indicate that triplet combination of POM/BTZ/DEX may enhance some T, B and NK cell subpopulations, overcoming immunosuppression when compared to BTZ/DEX-only treated patients (Rao et al, 2019). Mechanisms of action (MOA) of CC-92480- and POM-mediated substrate depletion occurs via ubiquitination and proteasome degradation, where BTZ has been speculated as potentially antagonistic as a PI. Here, we report pre-clinical and clinical observations of an immune MOA of CC-92480 or POM in combination with BTZ. Results: To mimic the clinical pharmacokinetics, BTZ was utilized as a high-dose pulse method alone and in combination with POM or CC-92480, followed by flow cytometric measurements of Aiolos and Ikaros protein abundance in healthy donor (HD) T cells. The addition of BTZ modestly delayed CRBN-dependent substrate depletion compared to single agent POM or CC-92480; however, this effect was only apparent at early time points (1-6 hr) where the effect was negligible by 24 hr. To understand the functional implications of BTZ combination, we conducted CD3-stimulated PBMC-mediated cytotoxicity assay against H929 MM target cells in a co-culture model. The efficiency of POM or CC-92480 induced PBMC-mediated killing in a dose dependent manner (~65% increase compared to DMSO) were similar at a 100-fold lower dose range of CC-92480 compared to POM, with the effect not being altered by co-treatment with BTZ. These data were replicated with a POM or CC-92480 treated supernatant stimulation of purified NK cells co-culture, which induced an 80% reduction in target cell viability with the BTZ combination having no negative effects on CELMoD-mediated activity. Cytokine analysis on PBMC supernatants treated with either POM or CC-92480 in the absence or presence of BTZ-pulse showed a dose-dependent increase in IL-2 (>2.4-fold) and Granzyme B (>3.1-fold), which were not impacted by BTZ co-treatment. As a secondary readout on activation status, we measured multiple signaling molecules and activation markers on the cell surface of T and NK cell subsets in CD3 stimulated HD PBMCs treated with dose-dependent POM or CC-92480 with or without co-treatment of BTZ. Compared to DMSO controls, elevated expression levels of CD25 (IL2RA), CD278 (ICOS), Granzyme B, CD134 (OX40R) and HLA-DR were observed with both POM and CC-92480 on CD4, CD8 and NK cells demonstrating a CELMoD-mediated increase in immune activation. These effects were not impacted by the co-treatment of BTZ. Examination of peripheral blood samples from MM patients enrolled in the CC-92480-MM-001/002 (NCT03374085/NCT03989414) clinical trials revealed that CC-92480 promoted potent immunomodulation when administered in combination with DEX and with BTZ/DEX. These data included increased numbers of activated and central memory T cells, as well as increased Ki67+ proliferating T and NK cell populations compared to samples collected during the screening period before any drugs had been administered, consistent with earlier observation of POM in combination with BTZ/DEX treated patients. Conclusions: Taken together, these data demonstrate that POM and CC-92480 are potent immunomodulatory agents with enhanced induction of PBMC and NK mediated cell killing of MM tumor cells and activation of T and NK cells, at 100-fold lower concentrations of CC-92480 compared to POM. Additionally, we showed that combination with BTZ in preclinical assays and in the clinical setting did not antagonistically affect the immunostimulatory ability of POM or CC-92480. Disclosures Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Amatangelo: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Chiu: Bristol Myers Squibb: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kang: BMS: Current equity holder in publicly-traded company. Civardi: Bristol Myers Squibb: Current Employment. Katz: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Maciag: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hagner: BMS: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Bahlis: Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Richardson: Oncopeptides: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Protocol Intelligence: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; AbbVie: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

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Immunomodulation of human natural killer cell cytotoxic function by organochlorine pesticides

Human & Experimental Toxicology ◽

10.1191/0960327104ht477oa ◽

2004 ◽

Vol 23 (10) ◽

pp. 463-471 ◽

Cited By ~ 34

Author(s):

Adrian Reed ◽

Leticia Dzon ◽

Bommanna G Loganathan ◽

Margaret M Whalen

Keyword(s):

T Cells ◽

Nk Cells ◽

Tumor Cells ◽

Organochlorine Pesticides ◽

Mononuclear Cells ◽

Viral Infections ◽

Nk Cell ◽

Cell Preparation ◽

Lytic Function ◽

Cytotoxic Function

Organochlorine pesticides are used worldwide. To our knowledge there have been no studies dealing with the effects of these agents under in vitro conditions on human natural killer (NK) cell cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of organochlorine pesticides and some of their breakdown products on the ability of human NK cells to lyse tumor cells. A total of 11 compounds were tested. The compounds were tested in both purified NK cells as well as a cell preparation that contained other mononuclear cells (predominantly T cells) and NK lymphocytes (referred to as T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 hour to 6 days. Exposure of highly purified NK cells to 5 μ M α-chlordane, γ-chlordane, 4,4'-DDT, heptachlor, oxychlordane, or pentachlorophenol (PCP) inhibited their ability to destroy K562 tumor-cells by 88±5, 92±8, 61±13%, 64±10%, 69±11%, 76±12%, respectively, after a 24h exposure. The loss of cytotoxic function seen with α-and γ-chlordane remained essentially constant out to 6 days, while that seen with 4,4'-DDT, oxychordane and PCP increased with longer exposures (6 d). PCP was the most effective of the compounds tested at decreasing NK function. Of the compounds that caused decreased lytic function when tested in purified NK cells, only PCP and oxychordane decreased the lytic function of the T/NK cell preparation after any exposure. The results provide evidence of relative toxic potential for the 11 compounds and their immunomodulatory effects on other mononuclear cells (such as T-cells, B-cells, and monocytes) as well as NK lymphocyte function.

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Effect of Elotuzumab on Circulating Lymphocytes, Chemokines, and Cytokines In Multiple Myeloma Patients

Blood ◽

10.1182/blood.v116.21.4070.4070 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4070-4070 ◽

Cited By ~ 1

Author(s):

Lauri Neyer ◽

Han Ding ◽

Doreen Chen ◽

James P Sheridan ◽

Audie Rice ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Cell Surface ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Phase 1 ◽

Lymphocyte Trafficking ◽

Transient Decrease ◽

Combination Study

Abstract Abstract 4070 Background: Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein which is highly and uniformly expressed on malignant plasma cells in multiple myeloma (MM). CS1 is also expressed at a lower level on the cell surface of natural killer (NK) cells, natural killer T-cells (NKT), and on a subset of CD8 positive T-cells, but not resting B cells, monocytes, or CD4 positive T-cells. Preclinical studies have previously demonstrated that elotuzumab kills myeloma cells via NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, in whole blood assays elotuzumab treatment resulted in the elevation of chemokines and cytokines in culture supernatants. Elotuzumab is being studied in three phase 1 clinical trials in relapsed and/or refractory MM: 1) a monotherapy dose-escalation study, 2) a combination study with bortezomib, and 3) a combination study with lenalidomide and low-dose dexamethasone. Given that NK cells express CS1, there was a concern that elotuzumab treatment could potentially lead to NK cell depletion. Therefore, the pharmacodynamic goals of these clinical studies were to examine the effects of elotuzumab treatment on lymphocyte counts (in particular NK cells), chemokine levels, and cytokine levels. Methods: Absolute lymphocyte counts were determined in peripheral blood samples using the TruCOUNT™ flow cytometry assay. Serum levels of chemokines and cytokines were measured using a multiplex, bead-based assay (Luminex®). Results: In all 3 studies, we observed no depletion of total lymphocytes or lymphocyte subsets, including CS1 positive NK cells, with elotuzumab dosing either alone or in combination with bortezomib or lenalidomide/dexamethasone. A transient decrease in the absolute number of circulating total lymphocytes (approximately 75%–90% reduction from baseline) upon first elotuzumab dose was observed, followed by a recovery of these lymphocyte counts to baseline or near baseline levels as dosing cycles continued. The transient decrease in lymphocytes included both CS1 positive and CS1 negative cell subsets. This transient decrease in lymphocyte counts was associated with increased levels of circulating chemokines and cytokines following dosing. Post-dose serum samples from study subjects had a median 16-fold (range 1.3–270-fold) elevated levels of interferon inducible protein 10 (IP-10), a chemokine well known to induce lymphocyte trafficking. Other serum analytes were elevated following the initial elotuzumab dosing, including MCP-1, IL-6, and TNF-α, also known for their role in chemotaxis and inflammatory processes. The elevated levels of chemokines and cytokines were generally not seen at subsequent doses. Conclusions: A transient decrease in both CS1 positive and CS1 negative lymphocyte counts was observed following the first dose of elotuzumab in all phase 1 studies in patients with relapsed and/or refractory MM, which resolved during subsequent dosing. This transient decrease appears to be due to lymphocyte trafficking resulting from release of chemokines and cytokines. No evidence of elotuzumab-mediated depletion of CS1 positive lymphocytes was observed. Elotuzumab treatment thus does not appear to be associated with NK cell depletion. Disclosures: Neyer: Facet Biotech: Employment. Ding:Facet Biotech: Employment. Chen:Facet Biotech: Employment. Sheridan:Facet Biotech: Employment. Rice:Facet Biotech: Employment. Balasa:Facet Biotech: Employment. Keller:Facet Biotech: Employment. Fang:Facet Biotech: Employment. Albano:Facet Biotech: Employment. Tran:Facet Biotech: Employment. Zhao:Facet Biotech: Employment. Afar:Facet Biotech: Employment.

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CD80-IL2 Expressing Myeloma Cells for Immune Gene Therapy of Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.4718.4718 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4718-4718

Author(s):

Giulia Giunti ◽

David Malone ◽

Lucas Chan ◽

Darling David ◽

Shahram Y Kordasti ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Nk Cells ◽

Nk Cell ◽

Cytolytic Activity ◽

Immune Gene ◽

Moderate Increase ◽

Myeloma Cells ◽

Cell Functions

Abstract Abstract 4718 Improved experimental therapies are needed for Multiple Myeloma (MM). Despite major progress in treatment and initial induction of remission, myeloma remains an incurable disease. Although immunotherapy and, in particular, the employment of NK cells offers an approach of interest for the treatment of Multiple Myeloma (MM), recent studies have shown that myeloma cells utilise a number different mechanisms to impair NK and T cell functions. Important amongst these mechanisms is the reduced expression of CD80 in the sub-populations of PBMC isolated from myeloma patients. We have previously demonstrated CD80/IL-2 mediated stimulation of NK and T cells isolated from AML patients (as measured by proliferation, cytokine release and target cell specific cytolytic activity). In the present study we have examined the ability of genetically modified MM cells engineered to express CD80 and IL-2 to stimulate NK cell functions. These studies confirm the ability of MM cells to suppress NK cell functions in healthy PBMC and show that in contrast to the unmodified MM cells, the CD80/IL-2 expressing MM cells are able to stimulate a moderate increase in NK and T cell numbers and a significant increase in the fraction of NK cells with activatory receptors (NKp44, NKp30, NKp46) and activation markers (CD69) on the cell surface of both NK and T cells. More importantly for potential therapeutic applications the stimulated NK cells show increased cytolytic activity against the unmodified MM cells. This data suggest that CD80/IL-2 MM cells may be able to overcome the immune suppressive functions of unmodified MM cells and to stimulate NK, and T cell mediated responses against the unmodified MM cells. Therefore CD80/IL-2 expressing MM cells may provide a suitable cellular vaccine for NK cell stimulation and possibly the induction of broader ranging immunological responses against multiple myeloma cells. Disclosures: No relevant conflicts of interest to declare.

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Age-related sexual dimorphism on the longitudinal progression of blood immune cells in BALB/cByJ mice

The Journals of Gerontology Series A ◽

10.1093/gerona/glab330 ◽

2021 ◽

Author(s):

Cláudia Serre-Miranda ◽

Susana Roque ◽

Palmira Barreira-Silva ◽

Claudia Nobrega ◽

Neide Vieira ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

Sexual Dimorphism ◽

B Cells ◽

Nk Cells ◽

Mouse Models ◽

Immune Cells ◽

Age Related ◽

The Impact ◽

Over Time

Abstract The study of immune system aging is of relevance, considering its myriad of interactions and role in protecting and maintaining body homeostasis. While mouse models have been extensively used to study immune system aging, little is known on how the main immune populations progress over time and what is the impact of sex. To contribute to filling this gap, male and female BALB/cByJ mice were longitudinally evaluated, from 3 to 18 months old, for the main blood populations, assessed by flow cytometry. Using linear mixed-effect models, we observed that the percentages of neutrophils, monocytes, eosinophils and total natural killer (NK) cells increase with aging; while those of B cells, T cells (including CD4 + and CD8 + subsets) and Ly6C + NK cells, decrease. Males present higher percentages of neutrophils and classical monocytes Ly6C high over time, while females present higher percentages of total T cells, both CD4 + and CD8 +, eosinophils and NK cells. Males and females display similar percentages of B cells, even though with opposite accelerated progressions over time. This study revealed that mouse models recapitulate what is observed in humans during aging: an overall proportional decrease in the adaptive and an increase in the innate immune cells. Additionally, it uncovers an age-related sexual dimorphism in the proportion of immune cells in circulation, further strengthening the need to explore the impact of sex when addressing immune system aging using mouse models.

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Natural Killer Cells: The Linchpin for Successful Cancer Immunotherapy

Frontiers in Immunology ◽

10.3389/fimmu.2021.679117 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kari A. Shaver ◽

Tayler J. Croom-Perez ◽

Alicja J. Copik

Keyword(s):

T Cells ◽

Immune System ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Natural Killer ◽

Nk Cell ◽

Checkpoint Inhibitors ◽

Cytotoxic T Cells ◽

Current Evidence ◽

Cell Therapies

Cancer immunotherapy is a highly successful and rapidly evolving treatment modality that works by augmenting the body’s own immune system. While various immune stimulation strategies such as PD-1/PD-L1 or CTLA-4 checkpoint blockade result in robust responses, even in patients with advanced cancers, the overall response rate is low. While immune checkpoint inhibitors are known to enhance cytotoxic T cells’ antitumor response, current evidence suggests that immune responses independent of cytotoxic T cells, such as Natural Killer (NK) cells, play crucial role in the efficacy of immunotherapeutic interventions. NK cells hold a distinct role in potentiating the innate immune response and activating the adaptive immune system. This review highlights the importance of the early actions of the NK cell response and the pivotal role NK cells hold in priming the immune system and setting the stage for successful response to cancer immunotherapy. Yet, in many patients the NK cell compartment is compromised thus lowering the chances of successful outcomes of many immunotherapies. An overview of mechanisms that can drive NK cell dysfunction and hinder immunotherapy success is provided. Rather than relying on the likely dysfunctional endogenous NK cells to work with immunotherapies, adoptive allogeneic NK cell therapies provide a viable solution to increase response to immunotherapies. This review highlights the advances made in development of NK cell therapeutics for clinical application with evidence supporting their combinatorial application with other immune-oncology approaches to improve outcomes of immunotherapies.

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