Natural Killer Cytotoxic Activity Research Articles

Abstract 2329: Disulfiram metabolite modulates NK and T cell cytotoxicity against metastatic colorectal cancer through tumor derived NKG2D ligands

Abstract Introduction: Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide, contributing to one million deaths yearly. While survival is expected in early-stage disease, the 5-year survival rate is only 15% in metastatic CRC (mCRC). Frequently patients develop resistance to their chemotherapy regimens resulting in CRC metastatic lesions to appear in the lungs, liver, and brain. In recent years, the development of immunotherapy has become a promising avenue for the treatment of cold-tumor environments such as CRC. Indeed, dense infiltration of lymphocytes in CRC correlates with longer patient survival. Thus, patients suffering from mCRC would benefit from the combination of classical chemotherapy/radiotherapy with small immunomodulatory molecules capable of overcoming cancer cell resistance to therapy through chemo- and radio-sensitization. Recently, Antabuse’s metabolite dithiocarb-copper complex (CuET) was found to have anti-cancer activity. Here, we report for the first time on the immunomodulatory properties of CuET in the context of T/NK cell antitumor response in mCRC. Methods: To assess CuET anticancer activity in vitro, we performed survival curve, colony-formation, and migration-invasion assays in the murine colorectal cancer cell lines MC-38 and CT-26, and in the human KRAS mutant HCT116 cell line. We assessed in vivo efficacy of CuET in ectopic and metastatic MC-38 and CT-26 models in respective C57BL/6 and BALB/c mice. We characterized tumor growth, mice survival, and tumor immunohistochemistry. We evaluated CuET’s immunomodulatory properties through immunophenotyping of mouse and human T/NK cell receptors as well as corresponding tumor cell ligands after CuET treatment. We assessed CuET’s effect on the functional anticancer cytotoxicity of human PBMC-derived T/NK cells. Results: In vitro, CuET significantly inhibits viability, clonogenicity, and migration of tumor cells MC-38, CT-26, and HCT116 with IC50 values in the nanomolar range. In vivo, systemic treatment with CuET significantly reduces tumor growth in mice and prolongs survival (p=0.0041) when compared to controls. CuET significantly inhibits MC-38 liver metastasis growth in C57BL/6 mice. CuET recruits macrophages and T lymphocytes inside tumor cores and induces apoptosis. CuET induces the expression of NKG2D activating receptors in NK and T lymphocytes, as well as the expression of the corresponding ligands MICA/B and ULBP1/2 on tumor cells, enhancing the T/NK cell cytotoxic response anticancer. Conclusion: CuET enhances the functional antitumor cytotoxic activity of NK and T lymphocytes in mice and in human PBMC. In summary, our findings demonstrate that Antabuse-derived copper-diethyldithiocarbamate, CuET, is a potent immunomodulator which could benefit patients with mCRC. Citation Format: Daciana Catalina Dumut, Ivo Frydrych, Miroslav Popper, Dusan Garic, Radu Alexandru Paun, Amanda Centorame, Olivia Canavan, Juhi Shah, Martin Mistrik, Petr Dzubak, Jiri Bartek, Marian Hajduch, Juan Bautista DeSanctis, Danuta Radzioch. Disulfiram metabolite modulates NK and T cell cytotoxicity against metastatic colorectal cancer through tumor derived NKG2D ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2329.

Removing the B (cell) STING to improve cancer immunotherapy.

Removing the B (cell) STING to improve cancer immunotherapy.

A cytotoxic effect of human lactoferrin fusion with Fc domain of IgG.

Lactoferrin (LTF) is a natural iron-binding protein with a potential for clinical utility in many human immune disorders, including cancer. A fusion of LTF with the Fc domain of IgG2 (FcLTF) was designed with inherent properties of an extended the half-life in circulation. Furthermore, the effects of LTF and FcLTF were assessed for influence on the activity of natural killer (NK) cells isolated from human peripheral blood, on the NK-92 cell line, and on human monocytes. The NK cytotoxic activity induced by LTF and FcLTF was determined against the human leukemia K562 cell line, and also for monocytes, by measuring TNFα and granzyme B production, and in an assay for Jurkat cell viability. Selected gene expression in NK-92 cells and monocytes, induced by LTF and FcLTF, was performed by Real Time PCR. No significant difference was observed in NK-92 cytotoxicity stimulated by LTF and FcLTF. The effects on NK cells isolated from the human peripheral blood were varied, possibly due to the immunoregulatory nature of LTF sensing the immune status of donors. Furthermore, only the FcLTF group strongly stimulated production of TNFα and granzyme B in isolated monocytes. In addition, only supernatants from the monocyte cultures treated with FcLTF decreased the viability of Jurkat cells. The ability of FcLTF to induce TNFα in monocytes was strongly inhibited by anti-CD32 and moderately inhibited by anti-CD14 antibody. Lastly, it was demonstrated that FcLTF, strongly induced expression of PI3K, with subsequent activation of AKT/mTOR signaling pathway. Overall, it was demonstrated that this novel fusion molecule may be a perferred choice for clinical utility than the wild type LTF.

EP16.03-042 BET Inhibitors Stimulate NK Cytotoxic Activity in NSCLC through Attenuation of YAP/TAZ and SMAD3 Transcriptional Programs

Over the past years, the introduction of immune therapy in the treatment of lung cancer represented one of the most groundbreaking advances obtained in the cure of such deadly disease. In the field of immune-mediated therapies, Natural Killer (NK) cells represent an innovative cellular anti-tumor therapy and therapeutic target. Their cytotoxic activity is relevant to maintain immune surveillance in NSCLC patients, where the overall survival is positively related to NK cell infiltration. NK cytotoxicity is regulated through a wide range of receptors that allow them to rapidly respond to the absence of self molecules on target cells.

THE EFFECT OF B. subtillis ІМV B-7724 LECTIN ON FUNCTIONAL ACTIVITY OF THE MAIN EFFECTORS OF ANTITUMOR IMMUNITY OF INTACT MICE

Aim. The purpose of the study was to evaluate the effect of B. subtilis IMV B-7724 lectin on the functional activity of macrophages (Mph) and natural killer cells (NK) of intact Balb/c mice. Materials and Methods. Balb/c mice were subjected to 10 consecutive administrations of the lectin in a dose of 1 mg/kg of body weight. The functional activity of peritoneal Mph and NK were studied. Statistical analysis of the results was performed according to the widely accepted methods of variational statistics. Results. Administration of bacterial lectin increased Mph and NK cytotoxic activity; maximal increase was registered after the complete course of injections. A significant increase in the NO production indicates the polarization of peritoneal Mph into pro-inflammatory type M1. The transcription factors of IRF (at the early stage) and STAT (at the latter stages) signalling pathways were involved in the process of Mph polarization. Conclusion. The ability of B. subtillis IMV B-7724 lectin to increase in vivo cytotoxic activity of innate immunity effectors and to maintain the long-term Mph M1 polarization urges further studies on the lectin effectiveness.

Enhanced half-life and antitumor activity of interleukin-15 through genetic fusion of a serum albumin-specific protein binder

Human interleukin-15 (hIL-15) has attracted a considerable attention as a promising cancer immunotherapeutic due to its function to directly stimulate the proliferation and cytotoxic activity of NK and T cells. Nevertheless, a relatively short half-life of hIL-15 requires repeated administration and higher doses, causing serious side effects. Here, we demonstrate an enhanced blood half-life and biological activity of hIL-15 through genetic fusion of a human serum albumin-specific protein binder (rHSA). The fusion construct (rHSA-IL15) was observed to maintain respective binding activities for both hIL-15 receptor α and human serum albumin. The rHSA-IL15 led to a significant increase in the secretion of Granzyme B and INF-γ by immune cells compare to free hIL-15, expanding the population of activated T cell subset such as CD4 + T and CD8+ T cells. The terminal half-life of the rHSA-IL15 was prolonged by around a 40-fold in transgenic mice expressing human serum albumin, compared to free hIL-15. The rHSA-IL15 resulted in distinct anti-tumor activities in xenograft SCC (squamous cell carcinoma) mouse and allograft melanoma mouse models through activation of NK and CD8+ T cells. The rHSA-IL15 is expected to be used in cancer immunotherapy, assisting in the development of other cytokines as immunotherapeutic agents with greater efficacy.

Abstract 2906: AGEN1571 is a novel high-affinity ILT2 antagonist antibody that promotes adaptive and innate immune responses

Abstract Background: Ig-Like Transcript 2 (ILT2, LILRB1) is an inhibitory receptor prominently expressed on resting macrophages, monocytes, and dendritic cells (DCs), with variable expression on CD4+ T cells, CD8+ T cells, B cells, NK cells, and NKT cells in the tumor microenvironment. Interaction of ILT2 with its ligands, HLA-G and classical MHC class I molecules, is reported to impair cytotoxic activity of NK and effector T cells, attenuate B cell function, inhibit antigen-presentation by dendritic cells, and promote immunosuppressive activity of myeloid cells. Further, high ILT2 expression has been associated with poor prognosis in several human malignancies. As a result, ILT2 has been proposed as a therapeutic target to promote antitumor immunity and to overcome resistance to checkpoint blockade. Here, we report that AGEN1571, a fully human anti-ILT2 monoclonal IgG4κ antibody, reversed ILT2-mediated immunosuppression and promoted activation of NK, NKT, T cells, and myeloid cells in a series of preclinical studies. Methods: AGEN1571 binding to ILT2 was analyzed by SPR and flow cytometry. HDX-MS was used to identify the AGEN1571 binding epitope on ILT2. Mechanisms of AGEN1571-mediated modulation of immune cell function were evaluated using reporter cell lines and primary cell-based in vitro and ex vivo assays. Results: AGEN1571 bound to recombinant ILT2 with an approximate monovalent binding affinity of 0.38 nM and to cell-expressed ILT2 with an EC50 of 0.07 - 0.55 nM, depending on cell type and donor. AGEN1571 did not cross-react with related LILRA and LILRB family members except with weak reactivity to ILT4 (~370 nM). Consistent with its binding epitope, AGEN1571 potently blocked ILT2 binding to HLA-A, -B, -C, and -G and restored FcγR signaling inhibited by ILT2-HLA-G engagement in dose-dependent manner. AGEN1571 also augmented NFκB and ISRE activation induced by TLR, FcγR, and STING agonists in myeloid cells, and promoted polarization of primary human macrophages towards a pro-inflammatory phenotype. ILT2 blockade with AGEN1571 alone and in combination with a PD-1 antagonist enhanced secretion of pro-inflammatory cytokines and activation of NK, NKT and CD8+ T cells in a PBMC tumor cell co-culture assay. AGEN1571 was designed to have minimal binding to FcγR and complement which has been confirmed by lack of FcγR signaling and complement cascade induction in vitro. An in silico T cell epitope screen predicted AGEN1571 to be non-immunogenic, while AGEN1571 showed no evidence of inducing systemic cytokine release in a cytokine release assay using whole blood and PBMC cultures. Conclusion: AGEN1571 is a novel high-affinity ILT2 antagonist antibody that effectively antagonizes ILT2 to enhance T cell, NK cell, NKT cell, and myeloid activity. These data support clinical development of AGEN1571 as a therapeutic agent for patients with solid tumors. Citation Format: Olga Udartseva, Malgorzata Pupecka-Swider, Pilar Garcia-Broncano, Naxin Guo, Spencer Campbell, Elizabeth Klakus, Rebecca Ward, Priyadarshini Iyer, Christine Brittsan, Beth Wensley, Benjamin Morin, Andrew Slee, Dhan Chand, Claire Galand, Olga Ignatovich, Emmanuel Briend, David Savitsky. AGEN1571 is a novel high-affinity ILT2 antagonist antibody that promotes adaptive and innate immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2906.

Cytotoxic Activity and Lymphocyte Subtypes in Mice Selected for Maximal and Minimal Inflammatory Response after Transplantation of B16F10 and S91 Melanoma Cells.

AIRmax and AIRmin mice strains were selected according to the intensity of their acute inflammatory responsiveness. Previous studies have shown that AIR mice differ in their resistance to chemically induced skin tumors and in the development of melanoma metastases, in addition to differences in neutrophil and NK cells activity. In the present work, we aimed to evaluate whether the difference of susceptibility to murine melanoma is associated with NK cytotoxic activity against Yac.1 cells and lymphocyte subsets. Mice were subcutaneously inoculated with B16F10 or S91 melanoma cells. After 7, 14, or 30 days, the animals were euthanized to analyze the number of lymphocyte subsets, cytotoxic activity, and number of cytokine-producing spleen cells. AIRmax mice presented a higher number of CD4+/CD25+ cells than that of AIRmin mice following inoculation of B16F10 cells, whereas inoculation of S91 cells reduced CD4+/CD25+ and increased TCD8+ cell subsets in the AIRmax mice. AIRmax mice had a higher number of interleukin (IL)-10- and IL-12-producing cells and a lower number of interferon-γ–producing cells than those of AIRmin mice at 30 days. The cytotoxic activity of nonadherent spleen cells was similar in both the AIR strains. These results suggest that melanoma cells can induce different responses in AIR mice, possibly owing to alterations in regulatory mechanisms, such as the action of CD4+/CD25+ regulatory T cells and IL-10, in AIRmax mice.

Chemical screening identifies novel small molecule activators of natural killer cell cytotoxicity against cancer cells.

Natural killer (NK) cells are cytotoxic lymphocytes that play a major role in the innate immune system. NK cells exhibit potent cytotoxic activity against cancer cells and virally infected cells without antigen priming. These unique cytotoxic properties make NK cells a promising therapeutic against cancer. Limitations of NK cell therapy include deficiencies in high clinical efficacy often due to a need for a high NK cell to target cell ratio to achieve effective killing. In order to address the suboptimal efficacy of current adoptive NK cell therapy, a high throughput screen (HTS) was designed and performed to identify drug-like compounds that increase NK cytotoxic activity against tumor cells without affecting the normal cells. This screen was performed in a 384-well plate format utilizing an expanded primary NK cell product and ovarian cancer cells as a target cell (TC) line. Of the 8000 diverse small molecules screened, 16 hits were identified (0.2% hit rate) based on both a robust Z (RZ) score < -3 and a greater than 10% increase in NK cell killing. A validation screen had a confirmation rate of 70%. Select compounds were further validated and characterized by additional cytotoxicity assays including activity against multiple blood cancer and solid tumor cell lines, with no effect on primary human T cells. This work demonstrates that high-throughput screening can be reliably used to identify compounds that increase NK tumoricidal activity in vitro that can be further investigated and translated for potential clinical application. Précis: Our work led to the identification of promising compound that potently increases NK cell-mediated killing of a variety of different cancer cells, but no impact on the killing of normal cells. This compound demonstrates the utility of this assay.

Restoring NK cells functionality via cytokine activation enhances cetuximab-mediated NK-cell ADCC: A promising therapeutic tool for HCC patients

Natural Killer (NK) cells are considered the first line of defense against viral infections and tumors. Several factors affect NK cytotoxic activity rendering it dysfunctional and thereby impeding the ability to scavenge abnormal cells as a part of immune escaping mechanisms induced by different types of cancers. NK cells play a crucial role augmenting the activity of various types of anticancer mAb since dysfunctional NK cells are the main reason for the low response to these therapies. To this light, we examined the phenotypic characters of the circulating NK cells isolated from HCC patients compared to healthy controls. Then, dysfunctional NK cells, from HCC patients, were reactivated with cytokines cocktail and their cytotoxic activity with the anti-EGFR mAb “cetuximab” was investigated. This showed a downregulation of patients NK cells activating receptors (NKP30, NKP46, NKG2D and CD16) as well as CD56 and up-regulation of NKG2A inhibitory receptor. We also reported an increase in aberrant CD56− NK cells subset in peripheral blood of HCC patients compared to healthy controls. Thus, confirming the dysfunctionality of peripheral NK cells isolated from HCC patients. Cytokines re-activation of those NK cells lead to upregulation of NK activating receptors and downregulation of inhibitory receptor. Moreover, the percentage of aberrant CD56− NK cells subset was reduced. Here, we proved that advanced HCC patients have an increased percentage of more immature and noncytotoxic NK cell subsets in their peripheral blood, which might account for the low cytotoxicity noticed in those patients. A significant improvement in the cytotoxicity against HCC was noticed upon using reactivated NK cells combined with cetuximab. Therefore, this study highlights the potential recruitment of NK immune cells along with cetuximab to enhance cytotoxicity against HCC.

Natural Killer Cell Cytotoxicity Is a Predictor of Outcome for Patients with High Risk Myelodysplastic Syndrome and Oligoblastic Acute Myeloid Leukemia Treated with Azacytidine

Introduction: Hypomethylating agents (HMA) have shown significant therapeutic efficacy in the treatment of patients with MDS and AML. Although it has been suggested, that HMA act through re-activation of tumor-suppressor genes, the exact mechanism of their action has not been completely elucidated yet. The aim of our study was to identify predictors of outcome for patients treated with azacytidine (AZA).Patients and Methods: Eighteen patients with high risk MDS (RAEB-II) and oligoblastic AML (20-30% bone marrow blasts) were included in this study. Patients were not eligible for intensive induction chemotherapy due to age, or presence of comorbidities. All patients were selected for treatment with AZA.NK-cytotoxicity against K562, Rajii, and the percentage of various immune regulatory subpopulations in the peripheral blood (PB) was measured after 3-6 cycles of AZA. A previous short exposure to AZA was required with the aim: 1) to examine the effect of AZA on these populations, and 2) to identify biomarkers with the potential to predict outcome after treatment with AZA.NK-cytotoxicity against K562 and Rajii was tested by using a flow cytometry based assay as previously described (1).Quantitative estimation of immune regulatory subpopulations in PB of patients was performed by using flow cytometry. The number of myeloid derived suppressor cells (MDSCs) (CD33+/CD11b+/CD14-/HLADRlow/-) was expressed as percentage among CD45+ PB monocytes. The number of Tregs (CD3+/CD4+/CD25+/Foxp3+) was expressed as percentage among CD3+/CD4+ PB lymphocytes. The number of double negative TCRαβ (CD3+/TCRαβ+/CD4-/CD8-) was expressed as percentage among PB CD3+ lymphocytes.Comparison between groups was performed by using Mann-Whitney test for continuous variables. Receiver operating curve analysis (ROC) was performed with the aim to determine the ability of various parameters for the prediction of 2-year survival. Kaplan-Meier and log rank test were used for estimating the probability of overall survival (OS).ResultsNK cytotoxicity: The median NK-cytotoxicity against K562 of patients and healthy donors was 21.8% (95% CI, 7.5 - 32.6) and 41.8% (95% CI, 11.4 - 56.1). Patients with MDS/AML after a short exposure to azacytidine had a significantly reduced NK-cytotoxicity against K562 as compared with NK cytotoxic activity of healthy donors (p=0.01). NK cytotoxic activity against Rajii of both patients and healthy donors was very low to absent (data not shown).Immune regulatory cells: The median MDSCs of patients and healthy donors was 0.41% (95% CI, 0.13 - 1.26) and 0.60% (95% CI, 0.30 - 1.94). There was no difference between MDSCs in the PB of patients and healthy donors. There was no difference in the number of Tregs and double negative TCRαβ cells between patients and donors (data not shown).NK cytotoxicity is a predictor of overall survival in patients treated with AZA: The ability of various parameters including the PB percentage of MDSCs, Tregs, and double negative TCRαβ, as well as of NK cytotoxic activity to predict survival at 2 years after treatment with AZA was examined by using ROC analysis. NK cytotoxic activity was the only predictor of OS for patients treated with AZA (AUC=0.875, 95% CI, 0.635 - 0.979, p<0.0001). NK-cytotoxicity above 20.85% was estimated as the best cut off with sensitivity and specificity 80% and 87% in predicting 2-year survival (Figure 1A). In multivariate analysis, NK cytotoxicity above 20.85% (high NK-activity) was the only parameter statistically associated with significantly improved OS [HR=0.10, 95% CI, 0.01 - 0.90, p=0.041] (Figure 1B).Conclusions: Our study showed that NK cytotoxicity is a predictor of the outcome for patients treated with AZA. Patients with high NK-activity after a short course of AZA had significantly improved OS as compared with patients with low NK-activity. The results of our study suggest that the therapeutic activity of AZA is at least partly mediated by an immunomodulatory effect.

Chimeric Virus-Like Particles of Prawn Nodavirus Displaying Hepatitis B Virus Immunodominant Region: Biophysical Properties and Cytokine Response.

Hepatitis B is a major global health challenge. In the absence of an effective treatment for the disease, hepatitis B vaccines provide protection against the viral infection. However, some individuals do not have positive immune responses after being vaccinated with the hepatitis B vaccines available in the market. Thus, it is important to develop a more protective vaccine. Previously, we showed that hepatitis B virus (HBV) ‘a’ determinant (aD) displayed on the prawn nodavirus capsid (Nc) and expressed in Spodoptera frugiperda (Sf9) cells (namely, Nc-aD-Sf9) self-assembled into virus-like particles (VLPs). Immunisation of BALB/c mice with the Nc-aD-Sf9 VLPs showed significant induction of humoral, cellular and memory B-cell immunity. In the present study, the biophysical properties of the Nc-aD-Sf9 VLPs were studied using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antigenicity of the Nc-aD-Sf9 VLPs, and multiplex ELISA was employed to quantify the cytokine response induced by the VLPs administered intramuscularly into BALB/c mice (n = 8). CD spectroscopy of Nc-aD-Sf9 VLPs showed that the secondary structure of the VLPs predominantly consisted of beta (β)-sheets (44.8%), and they were thermally stable up to ~52 °C. ELISA revealed that the aD epitope of the VLPs was significantly antigenic to anti-HBV surface antigen (HBsAg) antibodies. In addition, multiplex ELISA of serum samples from the vaccinated mice showed a significant induction (p < 0.001) of IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-12p70. This cytokine profile is indicative of natural killer cell, macrophage, dendritic cell and cytotoxic T-lymphocyte activities, which suggests a prophylactic innate and adaptive cellular immune response mediated by Nc-aD-Sf9 VLPs. Interestingly, Nc-aD-Sf9 induced a more robust release of the aforementioned cytokines than that of Nc-aD VLPs produced in Escherichia coli and a commercially used hepatitis B vaccine. Overall, Nc-aD-Sf9 VLPs are thermally stable and significantly antigenic, demonstrating their potential as an HBV vaccine candidate.

The effect of human leukocyte antigen A1 and B35-Cw4 on sustained BK polyomavirus DNAemia after renal transplantation.

Human leukocyte antigen (HLA) class I presentation pathway plays a central role in natural killer (NK) cell and cytotoxic T-cell activities against BK polyomavirus (BKPyV) DNAemia. We determined the risk of sustained BKPyV DNAemia in 175 consecutive renal transplant recipients considering the simultaneous effect of donor/recipient HLA class I antigens and pre- or post-transplant variables. Median (IQR) age was 53 (44-64) years, and 37% of patients were female. 40 patients (22.9%) developed sustained BKPyV DNAemia [median (IQR) viral load: 9740 (4350-17125) copies/ml]. In the Cox proportional hazard analysis, HLA-A1 (HR: 3.06, 95% CI: 1.51-6.17) and HLA-B35-Cw4 (HR: 4.63, 95% CI: 2.12-10.14) significantly increased the risk of sustained BKPyV DNAemia, while 2 HLA-C mismatches provided a marginally protective effect (HR: 0.32, 95% CI: 0.10-0.98). HLA-Cw4 is a ligand for NK cell inhibitory receptor, and HLA-B35 is in strong linkage disequilibrium with the HLA-Cw4 allele. The association between HLA-B35-Cw4 expression and sustained BKPyV DNAemia supports the important role of cytotoxic T cells and NK cells that would normally control BKPyV activation through engagement with immunoglobulin-like killerreceptors(KIRs). Further studies are required to investigate the effect of HLA-C alleles along with NK cell activity against BKPyV DNAemia.

Ma-Nuo-Xi decoction has an immunostimulating effect in cyclophosphamide-immunosuppressed mice

Ma-Nuo-Xi decoction (MNXD), as well as its hundreds of derivative preparations, has been used in Tibetan medicine since the 14th century. MNXD is in accordance with the theory of treatment determination based on syndrome differentiation. This study aimed to compare the effect of the auxiliary MNXD prescription (MNXD-AD) with that of the basic MNXD prescription (MNXD-BD) on the immunostimulating activity of MNXD. The immunopotentiation of MNXD, MNXD-BD, and MNXD-AD was evaluated using a cyclophosphamide (CTX)-immunosuppressed mouse model. Their influences on non-specific and specific immunity were evaluated using immune organ indexes, peripheral white blood cell (WBC) count, red blood cell (RBC) count, platelet count, phagocytosis, macrophage-secreted nitric oxide (NO) and cytokines, natural killer (NK) cytotoxic activity, lymphocyte proliferation, serum cytokines, splenic T-lymphocyte subpopulations, and quantitative hemolysis of sheep red blood cell (QHS SRBC) assays. MNXD, MNXD-BD, and MNXD-AD increased the spleen and thymus indexes, as well as the peripheral WBC, RBC, and platelet counts. They also promoted phagocytosis, NO and cytokine secretion from macrophages, NK cytotoxic activity, and lymphocyte proliferation, and also raised the CD4+ /CD8+ T-cell ratio, serum cytokine concentrations, and haemolysin formation in CTX-treated immunosuppressed mice. Compared with MNXD-BD and MNXD-AD, MNXD was superior in restoring the phagocytic index, concanavalin A (ConA)-induced T-lymphocyte proliferation, NO secretion from macrophages, and haemolysin formation, as well as the levels of interleukin 1 beta (IL-1β), and serum interleukin-2 (IL-2) and interferon gamma (INF-γ). MNXD, MNXD-BD, and MNXD-AD have excellent immunostimulating and myelosuppression-restoring activities on CTX-immunosuppressed mice. Among them, MNXD-AD might be an immunomodulator, which may happen to be in line with the clinical experience of Tibetan medicine physicians of using it to promote the efficacy of MNXD-BD.

Inhibitory KIR2DL2 Gene: Risk for Deep Endometriosis in Euro-descendants.

Endometriosis (EDT) is an inflammatory disease characterized by implantation/growth of endometrial tissue, glands, and/or stroma, outside the uterus. Reduced NK cell cytotoxic activity has been implicated in its pathogenesis, together with other immunologic alterations. We investigated the influence of KIR gene polymorphisms and their HLA ligand combinations in deep endometriosis (DE) susceptibility. One hundred sixty women with a histological diagnosis of DE and 202 control women without the disease, who underwent laparoscopy, were enrolled. The DE group was subdivided into initial (I/II; n = 60) and advanced stages (III/IV, n = 100). KIR and HLA class I gene polymorphisms were typed by PCR-SSP and sequence-based-typing (SBT), respectively. We observed a significant association of KIR2DL2, an inhibitory gene of B haplotype, conferring risk for DE in Euro-descendants. Positive associations of Bx haplotype and centromeric AB segments were also found. However, no association with KIR-HLA ligand combination was observed. Our data suggest KIR2DL2 gene to be a relevant factor favoring NK inhibition in DE in Euro-descendants, contributing to the defective NK cytotoxic activity and impaired clearance of ectopic endometrial cells in the disease.

Alterations in the innate immune system due to exhausting exercise in intensively trained rats

It is known that intensive physical activity alters the immune system’s functionality. However, the influence of the intensity and duration of exercise needs to be studied in more depth. We aimed to establish the changes in the innate immune response induced by two programmes of intensive training in rats compared to sedentary rats. A short training programme included 2 weeks of intensive training, ending with an exhaustion test (short training with exhaustion, S-TE). A second training programme comprised 5-week training including two exhaustion tests and three trainings per week. In this case, immune status was assessed before (T), immediately after (TE) and 24 h after (TE24) an additional final exhaustion test. Biomarkers such as phagocytic activity, macrophage cytokine and reactive oxygen species (ROS) production, and natural killer (NK) cell activity were quantified. S-TE was not enough to induce changes in the assessed innate immunity biomarkers. However, the second training was accompanied by a decrease in the phagocytic activity, changes in the pattern of cytokine secretion and ROS production by macrophages and reduced NK cell proportion but increased NK cytotoxic activity. In conclusion, a 5-week intense training programme, but not a shorter training, induced alterations in the innate immune system functionality.

Enumeration of peripheral blood NKp46 positive NK lymphocytes reflects NK cytotoxic activity in vitro

Natural killer (NK) cells are the predominant innate lymphocyte subsets that mediate anti-tumor and anti-viral responses. The monitoring of NK cells function is important in various physiological and pathological conditions. Different approaches have been used to directly or indirectly evaluate NK cells activities.The purpose of this study was to investigate the correlation between the number of NK cells and cytotoxic activity of NK cells and to determine whether NKp46+NK cells reflect NK cytotoxicity status.In our study, we retrospectively analyzed laboratory data on NK cytotoxicity and NK lymphocyte levels of 4896 infertile women which underwent routine immunology investigation after IVF failures. In healthy women, NKp46 expression was assessed on NK cells (n = 214) and cytotoxicity activity was evaluated with regard to NKp46 expression. We found that despite a significant correlation coefficient (n = 4689, r = 0.447), the correlation with cytotoxicity is maintained only within the zones with a low or high NK cells frequency. NK cells frequency has no significant prognostic value for their cytotoxicity – within the medium NK frequency zone the samples may have any cytotoxicity, both reduced and elevated.However, our data demonstrate that NKp46+NK cells frequency correlates with cytotoxicity activity even more significantly than the NK cells frequency (n = 214, r = 0.67 and r = 0.62, respectively) and has significant prognostic value for the abnormal NK cytotoxicity status indications, both low and increased.Our results further support an important role of NKp46 in NK cells killing and afford grounds for using the measurement of the NKp46+NK cells frequency as an alternative method for abnormal NK cytotoxicity status indication, which is responsive, simple and reliable.

Inhibition of the NKp44-PCNA Immune Checkpoint Using a mAb to PCNA.

mAb-based blocking of the immune checkpoints involving the CTLA4-B7 and PD1-PDL1 inhibitory axes enhance T-cell-based adaptive immune responses in patients with cancer. We show here that antitumor responses by natural killer (NK) cells can be enhanced by a checkpoint-blocking mAb, 14-25-9, which we developed against proliferating cell nuclear antigen (PCNA). PCNA is expressed on the surface of cancer cells and acts as an inhibitory ligand for the NK-cell receptor, NKp44-isoform1. We tested for cytoplasmic- and membrane-associated PCNA by FACS- and ImageStream-based staining of cell lines and IHC of human cancer formalin fixed, paraffin embedded tissues. The mAb, 14-25-9, inhibited binding of chimeric NKp44 receptor to PCNA and mostly stained the cytoplasm and membrane of tumor cells, whereas commercial antibody (clone PC10) stained nuclear PCNA. NK functions were measured using ELISA-based IFNγ secretion assays and FACS-based killing assays. The NK92-NKp44-1 cell line and primary human NK cells showed increased IFNγ release upon coincubation with mAb 14-25-9 and various solid tumor cell lines and leukemias. Treatment with 14-25-9 also increased NK cytotoxic activity. In vivo efficacy was evaluated on patient-derived xenografts (PDX)-bearing NSG mice. In PDX-bearing mice, intravenous administration of mAb 14-25-9 increased degranulation (CD107a expression) of intratumorally injected patient autologous or allogeneic NK cells, as well as inhibited tumor growth when treated long term. Our study describes a mAb against the NKp44-PCNA innate immune checkpoint that can enhance NK-cell antitumor activity both in vitro and in vivo.

Natural killer cell cytotoxicity is a predictor of outcome for patients with high risk myelodysplastic syndrome and oligoblastic acute myeloid leukemia treated with azacytidine

The aim of the present study was to identify biomarkers predictive of the outcome of patients with high-risk myelodysplastic syndrome and oligoblastic acute myeloid leukemia (AML) treated with 5-azacytidine (AZA). We prospectively examined the association between NK-cytotoxic activity, myeloid-derived suppressor cells (MDSCs), and T-regulatory cells (Tregs) on the overall survival (OS) of patients. Patients with NK-cytotoxicity above a critical threshold had a longer duration of response and survived longer than patients with severe impairment of NK-cytotoxicity. The numbers of MDSCs, and Tregs in the PB of patients after a short exposure to AZA were not different from normal donors. In conclusion, the results of our study suggest that the therapeutic activity of AZA is at least partly mediated by an immunomodulatory effect. To our knowledge, this is the first study reported so far, that shows a positive correlation between NK cytotoxicity and OS of AZA-treated patients.

Elucidating the Immune-Related Mechanisms by Which Probiotic Strain Lactobacillus casei BL23 Displays Anti-tumoral Properties.

We have recently described antitumor properties of Lactobacillus casei BL23 strain in both a mouse allograft model of human papilloma virus (HPV)-induced cancer and dimethylhydrazine-associated colorectal cancer. However, the mechanisms underlying these beneficial effects are still unknown. Interestingly, in vitro cellular models show that this bacterium is able to stimulate the production of high levels of IL-2. Because this cytokine has well-known antitumor properties, we decided to explore its role in the anti-cancer effects of BL23 using the HPV-induced cancer model. We found a negative correlation between IL-2 and tumor size confirming the necessity of IL-2 to protect from tumor development. Then, we blocked IL-2 synthesis using neutralizing monoclonal antibodies in mice that were challenged with lethal levels of tumor cells; this led to a significant reduction in the protective abilities of BL23. Next, we used a genetically modified strain of Lactococcus lactis to deliver exogenous IL-2 to the system, and in doing so, we were able to partially mimic the antitumor properties of BL23. Additionally, we showed the systemic role of T-cells in tumor protection through a negative correlation between tumor size and T-cells subpopulations and an increasement of BL23-specific local Foxp3 levels in tumor-bearing mice. Finally, we observed a negative correlation between tumor size and NK+ cells, but local recruitment of NK cells and cytotoxic activity appeared specific to BL23 treatment. Taken together, our data suggest that IL-2 signaling pathway plays an important role in the anti-tumoral effects of probiotic strain L. casei BL23. These results encourage further investigation in the use of probiotic strains for potential therapeutic applications to clinical practice, in particular for the treatment of colorectal cancer. Furthermore, our approach could be extended and applied to other potential beneficial microorganisms, such as gut microbiota, in order to better understand the crosstalk between microbes and the host.