Suppression Of Natural Killer Cell Cytotoxicity Research Articles

Intratumoral CD38+CD19+B cells associate with poor clinical outcomes and immunosuppression in patients with pancreatic ductal adenocarcinoma

The widespread involvement of tumor-infiltrating B cells highlights their potential role in tumor behavior. However, B cell heterogeneity in PDAC remains unexplored. Studying TIL-Bs in PDAC aims to identify new treatment strategies. We performed single-cell RNA sequencing to study the heterogeneity of B cells in PDAC. The prognostic and immunologic value of the identified CD38+ B cells was explored in FUSCC (n=147) and TCGA (n=176) cohorts. Flow cytometry was conducted to characterize the relationship between CD38+ B cells and other immune cells, as well as their phenotypic features. Invitro and in vivo experiments were performed to assess the putative effect of CD38+ B cells on antitumor immunity. The presence of CD38+ B cells in PDAC was associated with unfavorable clinicopathological features and poorer overall survival (p<0.001). Increased infiltration of CD38+ B cells was accompanied by reduced natural killer (NK) cells (p=0.021) and increased regulatory T cells (p=0.016). Molecular profiling revealed high expression of IL-10, IL-35, TGF-β, GZMB, TIM-1, CD5 and CD21, confirming their putative regulatory B cell-like features. Co-culture experiments demonstrated suppression of NK cell cytotoxicity by CD38+ B cell-derived IL-10 (p<0.001). Finally, invivo experiments suggested adoptive transfer of CD38+ B cells reduced antitumor immunity and administration of a CD38 inhibitor hampered tumor growth (p<0.001). We discovered regulatory B cell-like CD38+ B cell infiltration as an independent prognostic factor in PDAC. The use of CD38 inhibitor may provide new possibilities for PDAC immunotherapy. This study was supported by the National Natural Science Foundation of China (U21A20374), Shanghai Municipal Science and Technology Major Project (21JC1401500), Scientific Innovation Project of Shanghai Education Committee (2019-01-07-00-07-E00057), Special Project for Clinical Research in the Health Industry of the Shanghai Health Commission (No. 20204Y0265) and Natural Science Foundation of Shanghai (23ZR1479300).

Long-chain polyunsaturated omega-3 fatty acids reduce multiple myeloma exosome-mediated suppression of NK cell cytotoxicity.

Despite the advances in the treatment of multiple myeloma (MM), complete remission is usually challenging. The interactions between tumor and host cells, in which exosomes (EXs) play critical roles, have been shown to be among the major deteriorative tumor-promoting factors herein. Therefore, any endeavor to beneficially target these EX-mediated interactions could be of high importance. a) To investigate the effects of myeloma EXs on natural killer (NK) cell functions. b) To check whether treatment of myeloma cells with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), two polyunsaturated omega-3 fatty acids with known anti-cancer effects, can modify myeloma EXs in terms of their effects on natural killer functions. L363 cells were treated with either EPA or DHA or left untreated and the released EXs (designated as E-EX, D-EX and C-EX, respectively) were used to treat NK cells for functional studies. Myeloma EXs (C-EXs) significantly reduced NK cytotoxicity against K562 cells (P ≤ 0.05), while the cytotoxicity suppression was significantly lower (P ≤ 0.05) in the (E-EX)- and (D-EX)-treated NK cells compared to the (C-EX)-treated cells. The expression of the activating NK receptor NKG2D and NK degranulation, after treatment with the EXs, were both altered following the same pattern. However, C-EXs could increase IFN-γ production in NK cells (P < 0.01), which was not significantly affected by EPA/DHA treatment. This indicates a dual effect of myeloma EXs on NK cells functions. Our observations showed that myeloma EXs have both suppressive and stimulatory effects on different NK functions. Treatment of myeloma cells with EPA/DHA can reduce the suppressive effects of myeloma EXs while maintaining their stimulatory effects. These findings, together with the previous findings on the anti-cancer effects of EPA/DHA, provide stronger evidence for the repositioning of the currently existing EPA/DHA supplements to be used in the treatment of MM as an adjuvant treatment. EXs released from L363 (myeloma) cells in their steady state increase IFN-γ production of NK cells, while reduce their cytotoxicity against the K562 cell line (right blue trace). EXs from L363 cells pre-treated with either EPA or DHA are weaker stimulators of IFN-γ production. These EXs also increase NK cytotoxicity and NKG2D expression (left brown trace) compared to the EXs obtained from untreated L363 cells. Based on these findings, myeloma EXs have both suppressive and stimulatory effects on different NK functions depending on the properties of their cells of origin, which can be exploited in the treatment of myeloma.

The PI3K/Akt/GSK-3β/ROS/eIF2B pathway promotes breast cancer growth and metastasis via suppression of NK cell cytotoxicity and tumor cell susceptibility

ObjectiveTo examine the effect of pSer9-GSK-3β on breast cancer and to determine whether the underlying metabolic and immunological mechanism is associated with ROS/eIF2B and natural killer (NK) cells.MethodsWe employed TWS119 to inactivate GSK-3β by phosphorylating Ser9 and explored its effect on breast cancer and NK cells. The expression of GSK-3β, natural killer group 2 member D (NKG2D) ligands, eIF2B was quantified by PCR and Western blot. We measured intracellular reactive oxygen species (ROS) and mitochondrial ROS using DCFH-DA and MitoSOXTM probe, respectively, and conducted quantitative analysis of cellular respiration on 4T1 cells with mitochondrial respiratory chain complex I/III kits. ResultsOur investigation revealed that TWS119 downregulated NKG2D ligands (H60a and Rae1), suppressed the cytotoxicity of NK cells, and promoted the migration of 4T1 murine breast cancer cells. Nevertheless, LY290042, which attenuates p-GSK-3β formation by inhibiting the PI3K/Akt pathway, reversed these effects. We also found that higher expression of pSer9-GSK-3β induced higher levels of ROS, and observed that abnormality of mitochondrial respiratory chain complex I/III function induced the dysfunction of GSK-3β-induced electron transport chain, naturally disturbing the ROS level. In addition, the expression of NOX3 and NOX4 was significantly up-regulated, which affected the generation of ROS and associated with the metastasis of breast cancer. Furthermore, we found that the expression of pSer535-eIF2B promoted the expression of NKG2D ligands (Mult-1 and Rae1) following by expression of pSer9-GSK-3β and generation of ROS.ConclusionsThe PI3K/Akt/GSK-3β/ROS/eIF2B pathway could regulate NK cell activity and sensitivity of tumor cells to NK cells, which resulted in breast cancer growth and lung metastasis. Thus, GSK-3β is a promising target of anti-tumor therapy.

Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity.

BackgroundMesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells.ResultsUC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity.ConclusionAlthough the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0063-9) contains supplementary material, which is available to authorized users.

Role of gamma-secretase in human umbilical-cord derived mesenchymal stem cell mediated suppression of NK cell cytotoxicity

Mesenchymal stem cells (MSCs) are increasingly considered to be used as biological immunosuppressants in hematopoietic stem cell transplantation (HSCT). In the early reconstitution phase following HSCT, natural killer (NK) cells represent the major lymphocyte population in peripheral blood and display graft-vs-leukemia (GvL) effects. The functional interactions between NK cells and MSCs have the potential to influence the leukemia relapse rate after HSCT. Until date, MSC-NK cell interaction studies are largely focussed on bone marrow derived (BM)-MSCs. Umbilical cord derived (UC)-MSCs might be an alternative source of therapeutic MSCs. Thus, we studied the interaction of UC-MSCs with unstimulated allogeneic NK cells. UC-MSCs could potently suppress NK cell cytotoxicity in overnight cultures via soluble factors. The main soluble immunosuppressant was identified as prostaglandin (PG)-E2. Maximal PGE2 release involved IL-1β priming of MSCs after close contact between the NK cells and UC-MSCs. Interestingly, blocking gamma-secretase activation alleviated the immunosuppression by controlling PGE2 production. IL-1 receptor activation and subsequent downstream signalling events were found to require gamma-secretase activity. Although the role of PGE2 in NK cell-MSC has been reported, the requirement of cell-cell contact for PGE2 induced immunosuppression remained unexplained. Our findings shed light on this puzzling observation and identify new players in the NK cell-MSC crosstalk.

Interleukin-15 enhances rituximab-dependent cytotoxicity against chronic lymphocytic leukemia cells and overcomes transforming growth factor beta-mediated immunosuppression

Chemoimmunotherapy with anti-CD20 monoclonal antibody rituximab is increasingly used for the treatment of patients with chronic lymphocytic leukemia (CLL). Antibody-dependent cytotoxicity (ADCC) is one of the most important mechanisms of action of rituximab against B-cell malignancies. We studied ways to increase the cytotoxic effect of rituximab on CLL cells by enhancing ADCC. Peripheral blood mononuclear cell (PBMC) or purified natural killer (NK) cells from healthy donors were activated with interleukin-15 (IL-15) and cultured with rituximab-coated CLL cells, and ADCC was evaluated using a (51)chromium release assay. The IL-15 significantly enhanced invitro ADCC against CLL cells, and this effect was mainly mediated by NK cells. The IL-15 treated effector cells with the low affinity FcγRIIIA receptor (158FF) had an ADCC comparable to those with the high affinity FcγRIIIA form (158VF). Inaddition, IL-15 enhanced rituximab-mediated ADCC of CLL cells in the presence of transforming growth factor-beta. The IL-15 increases rituximab-mediated ADCC against CLL, and supports the use of such agents with the goal of improving clinical response to chemoimmunotherapy in patients with CLL.

ORIGINAL ARTICLE: A High Dose of Intravenous Immunoglobulin Increases CD94 Expression on Natural Killer Cells in Women with Recurrent Spontaneous Abortion

Problem A high dose of intravenous immunoglobulin (HIVIg) therapy is effective in various diseases such as autoimmune diseases, and also is expected to have efficacy in recurrent spontaneous abortion (RSA). The aim of this study was to understand immunological mechanisms of this therapy.Method of study By flowcytometric analyses, we examined phenotypic changes of a variety of immunological cells including natural killer (NK) cells, cytotoxic T cells, regulatory T cells and macrophages in peripheral blood of RSA women with HIVIg therapy (n = 8).Results Expression percentages of inhibitory CD94 on NK cells significantly (P = 0.01) increased after the therapy (58.8 ± 21.4% versus 71.0 ± 17.6%).Conclusion Mechanisms of possible efficacy of HIVIg therapy for RSA may include enhancement of CD94 expression and subsequent suppression of NK cell cytotoxicity.

Reversal of human immunodeficiency virus type 1 protein-induced inhibition of natural killer cell activity by alpha interferon and interleukin-2.

A recombinant fusion peptide, Env-Gag, derived from the human immunodeficiency virus type 1 (HIV-1) genome corresponding to a defined portion of the envelope (Env) and internal core (Gag) proteins was examined for immunoregulatory effects on the cytotoxic activity of natural killer (NK) cell-enriched, large granular lymphocytes (LGL) from healthy donors. Percoll-separated, NK cell-enriched LGL precultured for 24 h with Env-Gag at 10- and 50-ng/ml concentrations, which significantly stimulated lymphocyte proliferation, caused significant suppression of NK cell activity. Denatured Env-Gag did not cause any effect on the NK cell activity of LGL. Two other control peptides, one derived from the Escherichia coli vector used to clone the HIV Env-Gag fusion peptide and the other derived from a non-HIV-1 viral antigen (rubeola virus), did not produce any observable effect on the NK cell activity of LGL, demonstrating the specificity of the effect produced by Env-Gag. Subsequent treatment of LGL with alpha interferon (IFN-alpha) or interleukin 2 (IL-2) alone partially reversed the Env-Gag-induced suppression of NK cell activity. However, LGL treated with both IFN-alpha and IL-2 completely reversed the suppression of NK cell cytotoxicity by Env-Gag. The combined effect of IFN-alpha and IL-2 in enhancing NK cell activity may provide a novel therapeutic approach to the restoration of depressed NK cell activity observed in HIV-infected patients.

Severity, Time, and β-Adrenergic Receptor Involvement in Surgery-Induced Immune Alterations

Although investigations of surgical stress in animals have reported immune alterations, surprisingly little is known about the variables or mechanisms contributing to the effect. Thus, we completed a series of experiments investigating the immune-altering effects of surgery severity, time of maximal immune alterations, and recovery, as well as the involvement of β-adrenergic receptors in surgery-induced immune alterations in Lewis rats. Immune alterations included natural killer (NK) cell cytotoxicity as well as B- and T-cell proliferation. Results showed increased immune suppression with larger incisions (6 cm > 3 cm > anesthesia > saline). In addition, maximal immune alterations induced by surgery occurred after 24 h; anesthesia effects predominated at the earlier time points. Recovery of immune status varied depending on the immunological measure of interest. Although NK cell cytotoxicity returned to control values within 2 days, B-cell proliferation remained suppressed for at least 8 days, and T-cell proliferation did not begin to recover until 4–8 days following the surgical procedure. To assess the mechanisms involved in surgery-induced immune alterations, follow-up assessments evaluated the effect of nadolol, a β-adrenergic receptor antagonist, on surgery-induced immune alterations. Results show that nadolol blocks the surgery-induced reduction in B- and T-cell proliferation but has no effect on the suppression of NK cell cytotoxicity. These results indicate the need to consider surgical severity and postoperative time of immune assessment when investigating the immune-altering effects of surgery. Importantly, activation of β-adrenergic receptors appears to play a modulatory role in surgery-induced immune alterations.

The effect of biochanin A on immune function in rats and tumor cell growth and viability

Animals exposed to a single dose of biochanin A (BA) in vivo (8 mg kg‐1 by gavage or 4 mg kg‐1 intraperitoneal) had natural killer cell (NKC) activity similar to the control when observed 20 h after treatment. No effect on NKC response, antibody production or delayed‐type hypersensitivity reactions was seen in rats treated with the same BA doses administered three times a week for 3 weeks. Therefore, in vivo treatment of rats with BA in this study had little or no effect on the immune responses tested. NKC cytotoxicity was significantly enhanced in rat splenocytes exposed in vitro to 1 X 10‐5‐1 X 10‐12 M‐BA or genistein (GEN) in 2.5% dimethylsulfoxide (DMSO). However, the NKC response in the 2.5% DMSO vehicle‐treated controls was significantly suppressed. When the DMSO concentration was reduced to 0.5%, no effects were seen in BA, GEN or vehicle cultures. This suggests that BA and GEN treatment protected against suppression of NKC cytotoxicity by DMSO. Biochanin A treatment of tumor cells, YAC‐1 or EL4, in vitro did not affect viability, however, cell growth was significantly inhibited in both cell lines.

Morphine inhibits spontaneous and cytokine-enhanced natural killer cell cytotoxicity in volunteers.

Opioids are used by patients who have conditions ranging from the acute pain of surgery and chronic cancer pain to substance abuse. Despite their widespread use and considerable experimental data about them, little is known about how opioids may alter in vivo immunity in humans. This study was designed to evaluate the in vivo effect of morphine on human peripheral blood immune functions. Healthy volunteers underwent continuous exposure to morphine for 36 h including a 24-h intravenous infusion in the hospital. Peripheral blood was drawn for immune function studies at five measurement times before, during, and after morphine exposure. Peripheral blood mononuclear cells were tested for acute and gamma-interferon-stimulated natural killer cell cytotoxicity (NKCC), antibody-dependent cell cytotoxicity, antibody Fc receptor expression, and human immunodeficiency virus infectivity. Significant suppression of NKCC was observed at 2 and 24 h after the onset of intravenous morphine exposure. Suppression of NKCC persisted for 24 h after termination of morphine infusion in a "high"-dose study group. gamma-Interferon-stimulated NKCC and antibody-dependent cell cytotoxicity were also decreased after 24 h of intravenous morphine exposure. No effect on Fc receptor expression was observed. Mean virus antigen production after lymphocyte infection with human immunodeficiency virus was not increased (p24 100 ng/ml after morphine vs. 43 ng/ml before morphine; P = 0.17). These results suggest that morphine administration, at doses within the range of analgesic use, can cause measurable suppression of some components of the human cellular immune system.

Stress-induced suppression of natural killer cell cytotoxicity in the rat: A naltrexone-insensitive paradigm.

The suppression of natural killer (NK) cell cytotoxicity by footshock stress can be attenuated by opioid antagonists, implicating endogenous opioids in its mediation. A stress paradigm that induces NK suppression that is not blocked by the opioid antagonist naltrexone is reported. This stress paradigm is also shown to cause analgesia and elevated plasma corticosterone levels that are not attenuated by naltrexone. In the first experiment, a significant suppression of NK cell cytotoxicity after forced swimming was demonstrated in Fischer 344 rats treated with either saline or naltrexone, compared with nonstressed controls. Significantly higher corticosterone levels were evident in both stressed groups. In the second experiment, the same stress paradigm was shown to cause significant analgesia in the tail-flick test, whereas no differences were seen between groups pretreated with saline and naltrexone. It is concluded that opioids need not always be involved in the suppression of NK cell cytotoxicity by stress.