Peripheral Blood Natural Killer Cells Research Articles

ПОДАВЛЕНИЕ ПРОДУКЦИИ ПРОВОСПАЛИТЕЛЬНЫХ ЦИТОКИНОВ В ЕСТЕСТВЕННЫХ КИЛЛЕРАХ КРОВИ ПАЦИЕНТОВ С ХРОНИЧЕСКОЙ ОБСТРУКТИВНОЙ БОЛЕЗНЬЮ ЛЕГКИХ ПОД ВЛИЯНИЕМ НОРТРИПТИЛИНА

Aim. To determine the ability of nortriptyline, alone and in combination with budesonide, to inhibit the production of pro-inflammatory cytokines in the peripheral blood natural killer (NK) cells of patients with chronic obstructive pulmonary disease (COPD) and to establish the molecular mechanisms of the combined action of these drugs. Material and methods. Blood cells from COPD patients (n=21) were incubated with budesonide, nortriptyline, or their combination. Cytokine production, expression of glucocorticoid receptor β (GRβ), histone deacetylase 2 (HDAC2), and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in NK cells were analyzed by flow cytometry. Results. The combination of nortriptyline (10 μM) and budesonide (10 nM) reduced the synthesis of interleukin 4 (IL-4), IL-8, interferon γ, tumor necrosis factor α as well as the expression of GRβ and p-p38 MAPK in NK cells; it also increased the expression of HDAC2 more significantly than budesonide alone. Conclusions. Nortriptyline enhances the anti-inflammatory effect of budesonide by altering the expression of HDAC2, GRβ, p-p38 MAPK in peripheral blood NK cells of patients with COPD. The data obtained indicate the advisability of combining nortriptyline with budesonide for the treatment of COPD.

O2-5 - Expression of cell adhesion molecules on peripheral blood NK cells in recurrent pregnancy losses

O2-5 - Expression of cell adhesion molecules on peripheral blood NK cells in recurrent pregnancy losses

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Bioactive compounds anthocyanins as a factor in the nutritional recovery of the body's adaptive potential after intense physical activity in the experiment: assessment of immunological and hematological indicators of adaptation

Restoring the adaptive potential of an athlete is of paramount importance not only for the implementation of his training and competitive activities, but also for maintaining health. One of the leading place in complex recovery programs in sports is given to full-fledged optimal nutrition, which provides for meeting the body's requirements not only in energy, macro- and micronutrients, but also in minor bioactive compounds. The use of anthocyanin-containing products is a promising strategy for the normalization of metabolic and immune disorders that develop as a result of intense physical and neuro-emotional stress not only in athletes, but also in other groups of people exposed to these factors, including military personnel undergoing training in conditions close to combat. This determines the relevance of this study. The aim of the research was to study the effect of an anthocyanin-enriched diet on hematological profile and cellular immunity in rats after intense physical activity. Material and methods. The experiment was carried out for 4 weeks on 4 groups of male Wistar rats with an initial body weight of ~300 g. The motor activity of the animals of the 1st (control) and 2nd groups was limited by the standard keeping animals in the vivarium, while physically active rats of the 3rd and 4th groups received additional physical activity - training on a treadmill. Before the end of the experiment, the animals of 3rd and 4th groups were given debilitating physical activity on a treadmill (until the rats refused to continue the exercise). Rats of all 4 groups received a standard semi-synthetic diet, water ad libitum. Animals in 2nd and 4th groups were additionally fed blueberry and blackcurrant extract (30% anthocyanins) as part of the diet at a daily dose of 15 mg anthocyanins/kg body weight. Hematological parameters were determined on a Coulter ACT TM 5 diff OV hematological analyzer. Expression of CD45R, CD3, CD4, CD8a, CD161 receptors on rat peripheral blood lymphocytes was determined by direct immunofluorescent staining of whole blood cells using a panel of monoclonal antibodies conjugated with fluorescent dyes: APC, FITC, PE. The measurements were carried out on an FC-500 flow cytometer. Results. Intense physical activity in rats of the 3rd group did not lead to a significant change in erythrocyte parameters compared with the control group. Enrichment of the diet with blueberry and black currant extract (the 2nd and the 4th groups) provided a significant (p<0.05) increase in blood content of hemoglobin (Hb) (150.7±0.9 and 154.4±2.0 vs 145.4±0.9 g/l in control), hematocrit (44.95±0.21 and 46.18±0.64 vs 43.78±0.32%) and the average content of Hb in erythrocytes (18.00±0.20 and 18.03±0.24 vs 17.35±0.24 pg). The absolute content of leukocytes and other cellular elements of the leukocyte formula, as well as leukocyte indices in rats of the experimental groups didn't significantly differ from those of the control rats, which confirms the absence of an inflammatory process. Intense physical activity and anthocyanin enrichment of the diet didn't have a significant effect on rat platelet parameters. Enrichment of the diet of rats of the 4th group with blueberry and black currant extract led to the activation of cellular immunity, as evidenced by a significant (p<0.01) increase in the percentage (from the total content of T-lymphocytes) of T-helpers (70.13 ±1.34 vs 63.75±0.99%) and a decrease in the relative content of cytotoxic T-lymphocytes (28.65±1.38 vs 34.71±0.95%) in comparison with those in rats of the 3rd group and at the level of the trend (р<0.1) - from the 1st group indexes (66.87±1.20 and 31.87±1.26%, accordingly). Intense physical activity led to a decrease in immunoregulatory index in rats of the 3rd group (1.86±0.07) compared with the control (2.13±0.12) (p<0.1), and in animals of the 4th group this indicator was significantly higher (2.50±0.14, p<0.05). In animals of the 3rd group a statistically significant (p<0.05) decrease in the relative content of NK cells in peripheral blood was found compared to the control. Enrichment of the diet of physically active rats with blueberry and black currant extract led to a significant (p<0.05) increase in the percentage of NK cells compared to this indicator in rats of the 3rd group (4.87±0.75 vs 2.08±0.18%) and had no significant difference with the indicator in rats of the control group (4.32±0.98%). Conclusion. The enrichment of the rats' diet with blueberry and blackcurrant extract containing a daily dose of 15 mg of anthocyanins per kg of body weight provides an increase in blood Hb content, hematocrit and the average content Hb in erythrocytes. It has been established that intense physical activity induces the cellular immunity suppression. The activating effect of anthocyanins on adaptive cellular immunity and NK cells, which are lymphocytes of innate immunity, was revealed. The data obtained indicate the effectiveness of the use of bioactive compounds (anthocyanins) to increase the adaptive potential of the organism.

Maternal cerium nitrate exposure induces developmental immunotoxicity in BALB/c mouse offspring

With the increasing application of cerium and rare-earth elements (REEs), cerium exposure is becoming more widespread. However, there remains a paucity of evidence on developmental immunotoxicity of cerium. This study was designed to examine the developmental immunotoxicity of gestational and postnatal exposure to cerium nitrate (CN) in BALB/C mouse offspring. Dams were given CN by oral gavage at 0, 0.002, 0.02 and 0.2 mg/kg from gestation day 5 (GD5) to postnatal day 21 (PND 21). On PND 21, the highest dose of CN significantly suppressed the NK cell cytotoxicity, and reduced the proportions of NK cells in peripheral blood and spleen of both female and male pups, however, the proportions of monocytes in peripheral blood and macrophages in spleen only increased in female pups. For adaptive immunity, on PND 21, the suppression of T/B lymphocyte proliferation, humoral and cellular immune responses (number of splenic plaque-forming cells, PFC, and delayed-type hypersensitivity, DTH) were observed in both female and male pup mice exposed to 0.2 mg/kg CN. However, the fall of proportions of T/B lymphocytes in peripheral blood (PB), spleen and mesenteric lymph node (MLN) only found in female pups at 0.2 mg/kg on PND 21. Most indications recovered to normal after 3-week cessation of CN exposure, except the reduction of DTH and PFC. From the findings in this study, the lowest-observed-adverse-effect level (LOAEL) of CN for developmental immunotoxicity was estimated to be 0.2 mg/kg bw per day.

Efficiency of intravenous therapy with Intralipid fat emulsion in patients with early reproductive loss

BACKGROUND: Immune mechanisms play a leading role in the pathogenesis of early reproductive loss. Modern methods of regulating disorders of immune homeostasis to overcome infertility and recurrent miscarriage include immune-efferent therapies such as immunocytotherapy, plasmapheresis, and intravenous administration of immunoglobulins and fat emulsions. Intralipid is the gold standard of the first generation parenteral fat emulsion that reduces the cytotoxicity of NK cells. In the literature, there are opposite data on the effectiveness of Intralipid fat emulsion in repeated implantation failure. This may be due to indications for its appointment and the choice of criteria for evaluating the effectiveness of this therapy.&#x0D; AIM: The aim of this study was to evaluate the effect of Intralipid therapy on the onset of pregnancy in patients with a history of early reproductive loss.&#x0D; MATERIALS AND METHODS: This prospective cohort study was conducted in Saint Petersburg, Russia in 20182021 and included 97 patients with a history of early reproductive loss (recurrent pregnancy loss or repeated In Vitro Fertilization failures). Patients were randomized into two study groups. The first group consisted of 41 women with Intralipid therapy, and the second group comprised 56 women without this therapy. Patients of the first group received 200 ml of Intralipid 20% (intravenous fat emulsion) once a month for three consecutive months before pregnancy. Inclusion criteria were ages 18 to 40 years, two cases of reproductive failure (pregnancy loss up to 12 weeks and / or unsuccessful In Vitro Fertilization attempts), and normal spouse karyotypes. Exclusion criteria were miscarriage due to fetal chromosomal abnormalities diagnosed by curettage and histopathological examination of products of conception from miscarriages, anatomical anomalies in the development of the genital apparatus, abnormal spouse karyotypes, and soy protein allergy. We evaluated anamnesis data and immunological parameters (functional activity of NK cells in peripheral blood before and after treatment). The efficacy of therapy was judged by the onset of pregnancy.&#x0D; RESULTS: The Mann Whitney U-test showed the difference in the levels of NKT cells before and after treatment with fat emulsions (p = 0.0076), this parameter decreasing 1.846 times compared to control. Clinical pregnancy in patients treated with Intralipid occurred twice as often compared to patients who did not receive fat emulsion therapy: 59.3% (n = 19) vs. 27.6% (n = 13), respectively (p = 0.0048).&#x0D; CONCLUSIONS: Intralipid fat emulsion increases pregnancy rates by decreasing NKT cells in women with early reproductive loss.

More severe lung lesions in smoker patients with active pulmonary tuberculosis were associated with peripheral NK cell subsets

BackgroundBoth pulmonary tuberculosis (PTB) and cigarette smoke (CS) exposure may lead to lung damage. The potential impact of CS exposure on tuberculosis-associated lung damage and the disturbance of immune cells and mediators involved, need to be further elucidated. MethodsWe firstly evaluated the chest X-ray (CXR) scores of a retrospective cohort of male patients with active PTB, followed for 6 months, and compared the scores between smoker (≥10 pack-years) and non-smoker patients. In a cross-sectional study, we measured the peripheral blood NK cell subsets and plasma inflammatory cytokines in male smoker and non-smoker patients with active PTB before anti-tuberculosis therapy, and the proportions of NK cell subsets and the levels of cytokines were analyzed for correlation with the CXR scores. ResultsIn the retrospective cohort, male smoker patients with active PTB showed a higher CXR score, characterized by more cavitary lesions, enlarged lymph nodes and emphysema, as compared to non-smokers. The cross-sectional study revealed that the CXR score in smoker patients was correlated inversely with the percentages of blood CD107a+, NKP46+, and TIGIT+ NK cells. ConclusionIn patients with active PTB, CS exposure was associated with more severe lung lesions, which were correlated with peripheral NK cell subsets.

Decidual natural killer cells and trophoblast cells: cellular, humoral and molecular mechanisms of interaction

Natural killer cells (NK cells) represent a group of lymphocytes of innate immunity. In addition to NK cells of peripheral blood, tissue-resident populations are described. NK cells of the decidual envelope (decidual NK cells) represent one of the local NK cell populations. Decidual NK cells differ in phenotype and function from peripheral blood NK cells. These cells have, mainly, regulatory functions. At the same time they retain the ability to perform cytotoxic effects. In the uterus, NK cells are located closely to the cells of fetal origin, i.e., trophoblast cells, which differentiate from the outer layer of the invading blastocyst. The purpose of the review article was to analyze the literature data on the studies of the molecular interactions between NK cells and trophoblast cells, as well as potential means of regulating these interactions. The review presents currently available data on receptor-mediated effects (due to adhesion molecules and cytotoxic receptors) and distant interactions (involving cytokines, chemokines and growth factors secreted by the both cell types) between NK population and trophoblast cells. The receptors regulating contacts of NK cells and trophoblast cells with extracellular matrix are also considered. The review provides information on activation of signaling pathways in NK cells and trophoblast cells resulting from their interaction with each other and components of the extracellular matrix. Currently, the molecular mechanisms regulating the NK cell functions and their interaction with trophoblast cells have not been studied sufficiently. The authors attempted to consider molecular regulation of the functional activity of NK cells mediated by the molecular complex of RNA polymerase II. We also describe participation of cyclin-dependent CDK8/19 kinases which comprise a part of the mediator complex which provides functioning of immune cells. The data on the participation of CDK8/19 in regulation of intracellular signaling pathways, as well as influence of CDK8/19 on the NK cell functions, are considered. Summarizing the data presented in the literature, one may emphasize that there is an extensive mutual influence of NK cells and trophoblast cells in decidual lining of uterus during pregnancy, thus leading to a changes in phenotype and functions of these cells. Experimental studies are required on the contribution of molecular mechanisms involved in transcription and translation processes to the biology of NK cells, and their role in maintaining interactions between NK cells and trophoblast cells, including the pathways involving CDK8/19.

Diversity of Circulating NKT Cells in Defense against Carbapenem-Resistant Klebsiella Pneumoniae Infection.

Nosocomial infection caused by carbapenem-resistant Klebsiella pneumonia (CRKP) infection has become a global public health problem. Human NK and NKT cells in peripheral immune responses are recognized as occupying a critical role in anti-bacterial immunity. Through performed scRNA-seq on serial peripheral blood samples from 3 patients with CRKP undergoing colonization, infection, and recovery conditions, we were able to described the immune responses of NK and NKT cells during CRKP infection and identified a mechanism that could contribute to CRKP clearance. The central player of CRKP infection process appears to be the NKT subset and CD56hiNKT subset which maintained immune competence during CRKP colonization. With time, CRKP leads to the loss of NK and CD160hiNKT cells in peripheral blood, resulting in suppressed immune responses and increased susceptibility to opportunistic infection. In summary, our study identified a possible mechanism for the CRKP invasion and to decipher the clues behind the host immune response that influences CRKP infection pathogenesis.

A combination of telomerase inhibition and NK cell therapy increased breast cancer cell line apoptosis

Triple-negative breast cancer (TNBC) is a subtype of breast tumor with the highest breast cancer stem cells (BCSCs) content and resistance to conventional treatment. Due to the immunosuppressive tumor microenvironment and immunogenicity of breast cancer cells, the use of immune cells, especially natural killer cells (NK) in the treatment of solid tumors, including breast cancer, has been unsatisfactory. Therefore, identifying novel therapies is requisite for breast cancer treatment. Furthermore, the combination of cancer therapies is an effective strategy to improve therapeutic effectiveness. In this study, we inhibited telomerase (hTERT) with BIBR1532, in stimulating NK cell cytotoxicity against breast cancer cells. The MDA-MB-231 cell line was cured with IC50 level of BIBR1532 for 24 h. Afterward, cells were washed with PBS and were co-cultured with peripheral blood NK cell for 5h. Finally, we assessed the impact of telomerase inhibition on the cytotoxicity of NK cells and apoptosis of breast cancer. Also, the expression of hTERT and apoptotic-related genes were evaluated. The data revealed that inhibition of telomerase increases NK cell cytotoxicity against breast cancer. Furthermore, telomerase inhibition and NK cell synergistically enhanced cell death in breast cancer cells by suppressing hTERT, upregulation of bax, and bad expression. In conclusion, telomerase suppression makes breast cancer cells more sensitive to NK cell therapy. Consequently, the combination of telomerase inhibition and NK cells can be useful in the treatment of breast cancer cells.

Expression of Tregs and IL-35 in Peripheral Blood of Patients with Newly Diagnosed Acute Myeloid Leukemia

To explore the expression of regulatory T cells (Tregs) and interleukin-35 (IL-35) in peripheral blood of patients with newly diagnosed acute myeloid leukemia (AML). Peripheral blood samples were collected from 33 newly diagnosed AML patients and 40 healthy volunteers. The levels of innate and adaptive immune cells and Tregs were detected by flow cytometry. Plasma IL-35 level was detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of P35 and EBI3 mRNA were detected by RT-PCR. The correlation between the levels of Tregs and IL-35 was analyzed. The proportion of CD4+T cells, CD8+T cells, B cells and NK cells in peripheral blood of newly diagnosed AML patients were significantly lower than those of control group (P<0.05). The levels of Tregs, IL-35, and the expression of P35 mRNA and EBI3 mRNA were significantly higher than that of healthy control group (P<0.01). The Tregs level was positively correlated with plasma IL-35 level (r=0.668,P<0.01). AML patients have a deficiency in immune function, which is characterized by the high levels of Tregs and IL-35 in peripheral blood, and it might be a new target for the treatment of AML.

Co-administration of tacrolimus and low molecular weight heparin in patients with a history of implantation failure and elevated peripheral blood natural killer cell proportion.

To investigate the therapeutic effect of co-administration of tacrolimus (TAC) and low-molecular-weight heparin (LMWH) or LMWH only on pregnancy outcomes in the female with a history of implantation failure and elevated peripheral blood natural killer (pNK) cell proportion in frozen-thawed embryo transfer cycles. To evaluate the pregnancy parameters for 249 patients with ≥2 implantation failures and pNK cell proportion ≥12% by analyzing a retrospective observational cohort study. Sixty patients had received the co-administration TAC and LMWH (TAC & LMWH group), 85 others had only taken LMWH (LWMH group), and the rest did not take any particular drugs (control group). The experimental finding indicated that the TAC & LMWH group and the LMWH group showed higher clinical pregnancy rates than the control group (p < 0.05), and TAC & LMWH group had a much higher live birth rate. According to the binary logistic regression analysis, the combination of TAC and LMWH was conducive to clinical pregnancy and live birth rate and reduced the possibility of miscarriage. It would not affect the result of spontaneous abortion and live birth, although the LMWH was only beneficial to clinical pregnancy. In addition, these findings were similar for these three groups' obstetrical and neonatal outcomes. The combination of TAC and LMWH can improve clinical pregnancy and live birth rates and reduce the risk of spontaneous miscarriage in patients with a history of implantation failure and elevated pNK ratio. LMWH is also beneficial to clinical pregnancy.

Senescence-associated secretory proteins induced in lung adenocarcinoma by extended treatment with dexamethasone enhance migration and activation of lymphocytes.

There is a need to improve response rates of immunotherapies in lung adenocarcinoma (AC). Extended (7-14days) treatment of high glucocorticoid receptor (GR) expressing lung AC cells with dexamethasone (Dex) induces an irreversible senescence phenotype through chronic induction of p27. As the senescence-associated secretory phenotype (SASP) may have either tumor supporting or antitumor immunomodulatory effects, it was interest to examine the effects of Dex-induced senescence of lung AC cells on immune cells. Dex-induced senescence resulted in sustained production of CCL2, CCL4, CXCL1 and CXCL2, both in vitro and in vivo. After Dex withdrawal, secretion of these chemokines by the senescent cells attracted peripheral blood monocytes, T-cells, and NK cells. Following treatment with Dex-induced SASP protein(s), the peripheral blood lymphocytes exhibited higher cell count and tumor cytolytic activity along with enhanced Ki67 and perforin expression in T and NK cells. This cytolytic activity was partially attributed to NKG2D, which was upregulated in NK cells by SASP while its ligand MICA/B was upregulated in the senescent cells. Enhanced infiltrations of T and NK cells were observed in human lung AC xenografts in humanized NSG mice, following treatment with Dex. The findings substantiate the idea that induction of irreversible senescence in high-GR expressing subpopulations of lung AC tumors using Dex pretreatment enhances tumor immune infiltration and may subsequently improve the clinical outcome of current immunotherapies.

Bispecific CS1-BCMA CAR-T Cells Are Clinically Active in Relapsed or Refractory Multiple Myeloma: An Updated Clinical Study

Background Bispecific CAR-T cell therapy has been proposed to mitigate some limitations of single-targeted CAR-T cells. CS1 plays a vital role in myeloma pathogenesis, and is also highly expressed on tumor cells in almost all patients (pts) with multiple myeloma (MM). Therefore, we aim to augment BCMA targeting with CS1. Bispecific CS1-BCMA CAR-T cells are effective in targeting MM cells in preclinical studies (Biomedicines 2021). Here we report the outcomes of 16 pts with refractory or relapsed (RR) MM in our phase I clinical trial (NCT04662099). Methods CS1-BCMA bispecific CAR contains a murine anti-CS1 scFv (clone 7A8D5) and a murine anti-BCMA scFv (clone 4C8) in tandem, and a 4-1BB costimulatory domain. The enrolled pts must have received at least 2 prior lines of therapy, and previous BCMA- or CS1-targeted immunotherapies were allowed. Pts were subjected to lymphodepleting regimens with cyclophosphamide (250 mg/m2, d-5 to d-3) and fludarabine (30 mg/m2, d-5 to d-3) daily prior to the CAR-T infusion (d0). Planned dose levels were 0.75x106, 1.5x106, and 3.0x106 CAR+T cells/kg, and repeated infusions were allowed. Primary objectives were incidence of adverse events (AEs). Cytokine release syndrome (CRS) and neurotoxicity were graded using the ASTCT criteria, and other AEs using CTCAE v5.0. Secondary objectives were overall response rate (ORR), overall survival (OS), duration of response (DOR), and progress-free survival (PFS). Response was assessed per the IMWG criteria 2016. Other objectives included in vivo kinetics of CAR-T cells, changes of soluble BCMA, immune assessment of infused CAR-T cells and patients’ lymphoid and immunosuppressive phenotype before and after infusion. Results As of July 31, 2022, 16 patients received the infusion of CS1-BCMA CAR-T cells and were included in the final analysis. Nine pts (56%) carried cytogenetic abnormalities, and 3 pts had extramedullary diseases (EMD) alone without detectable MM cells in bone marrow (BM). BCMA and CS1 were highly expressed on MM cells in BM by flow cytometry (mean BCMA+76.7% and CS1+ 96.5%) and EMD by trichromatic immunofluorescence. Nine pts (56%) were refractory, and 7 pts (44%) were relapsed and refractory. The median prior lines of treatment were 5.4 (range 2-10). All pts (100%) were exposed to bortezomib, 6 (38%) to ixazomib and 1 (6%) to cafilzomib. 13 pts (81%) received lenalidomide, 6 (38%) received thalidomide and 5 (31%) received pomadomide. 7 pts (44%) received autologous transplantation, 6 (38%) were exposed to daratumumab, 2 (13%) received selinexor and 2 (13%) had previous BCMA-targeted CAR-T therapy. Six pts (38%) experienced CRS, with median duration of 4 days (range 4-7). Grade 3 CRS occurred once and lasted for 5 days. Neurotoxicity was not observed. The ORR was 81% (13/16) for all patients, including 6 stringent complete, 3 very good partial, and 4 partial responses (Fig.1A). The ORR was 100% (13/13) for patients with MM cells in BM. Among them, 7 pts (54%) maintained ongoing response, 2 pts progressed after response, 1 withdraw from the study and 3 were lost due to poor compliance. Pt 16 had received prior anti-BCMA CAR-T treatment and achieved a partial response after CS1-BCMA CAR-T infusion. Although CAR-T cells infiltrated into the tumor tissue, response was not observed in the 3 pts with EMD alone (Fig.1B). Soluble BCMA decreased remarkably in peripheral blood (PB) and BM after infusion. At a median follow-up of 290 days, the median OS, PFS and DOR weren't reached, and OS, PFS and DOR at 12-month were 83.9%, 55.2% and 68.8%, respectively. On day 14 after infusion, CAR-T cells peaked at 138245 copies/ug DNA in PB (n=16) and 86654 copies/ug DNA in BM (n=13) by droplet digital PCR; CAR-T cells peaked at 493 CAR+T cells/ul PB (n=16) and 312 CAR+T cells/ul BM (n=13) by flow cytometry. CAR was detectable in vivo at a median of 6 months. Because CS1 is expressed on T and NK cells, we systematically evaluated the immune characteristics (differentiation, Fas, LAG3, PD1, TIM3, CS1) of infused products, absolute quantification and CS1 expression on T and NK cells in PB as well as myeloid derived suppressor cells, regulatory B and T cells in BM and PB. Further data will be released in the meeting. Conclusions Our study demonstrates bispecific CS1-BCMA CAR-T cells are clinical active with a good safety profile in heavily pretreated pts with MM, even after BCMA-targeted CAR-T cell therapy. Further studies are needed for pts with EMD alone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dynamic of Circulating DNAM-1+ Monocytes and NK Cells in Patients with STEMI Following Primary Percutaneous Coronary Intervention.

Although the role of inflammation and adverse cardiac remodeling in myocardial infarction (MI) have been extensively explored, gaps in knowledge on the complex interaction between these processes still exist. Data suggest that DNAX accessory molecule-1 (DNAM-1), an activating receptor implicated in NK cell education, may be involved in cardiac remodeling following coronary artery occlusion. In the present study, we aimed to explore the dynamic of DNAM-1+ monocytes and NK cells in peripheral blood in the early phase following reperfusion in patients with ST-elevation MI (STEMI). The study enrolled 49 patients older than 18 years of age diagnosed with STEMI, referred to primary percutaneous coronary intervention (pPCI). Blood samples were obtained at three distinct points (at admission, 3 h, and 24 h after pPCI) and analyzed using flow cytometry. The number of circulating DNAM-1+ monocytes (CD16++ and CD14++) and CD56dimCD16++NK cells was significantly reduced 3 h after pPCI and subsequently returned to initial levels 24 h after procedure (p = 0.003, p &lt; 0.001, and p = 0.002, respectively). Notably, such dynamic was dependent on age of patients. A positive correlation between high sensitivity troponin I levels and number of CD16++DNAM-1+ monocytes in peripheral blood 3 h after pPCI was observed (r = 0.431, p = 0.003). In conclusion, in the present study we delineated the post-reperfusion dynamic of DNAM-1-expresing leukocytes. Additionally, we demonstrated that the number of CD16++ DNAM-1+ monocytes correlate with the extent of myocardial injury.

B7-H3 Trike Enhances Killing of Myeloid Derived Suppressor Cells in Multiple Myeloma

Multiple myeloma (MM) is sensitive to Natural Killer (NK) cell-mediated killing but develops methods to attenuate NK cell function. One method to enhance NK cell specificity against myeloma is antibody dependent cellular cytotoxicity through the CD16 receptor. We developed a tri-specific killer engager (TriKE®) with a camelid single-domain antibody (sdAb) fragment against CD16 (on NK cells) as well as a linker region containing IL-15, to enhance NK cell survival. Delivery of IL-15 by TriKE supports NK cell proliferation while avoiding off-target effects on T cells. The second sdAb targets B7-H3 (CD276), a tumor antigen that is expressed on myeloma and is associated with decreased progression free survival. B7-H3 is differentially expressed on myeloma tumor lines MM1S, RPMI-8226, U266, and H929 but all four MM lines are significantly killed by peripheral blood NK cells in the presence of 3nM B7-H3 TriKE compared to NK cells alone. Bone marrow aspirates from 5 MM patients (3 newly diagnosed and 2 relapsed) were analyzed by flow cytometry and showed 58-96% expression of B7-H3 on CD138+ plasma cells. The bone marrow biopsy aspirates also showed monocytic myeloid derived suppressor cells (Mo-MDSC, CD14+, CD11b+) that expressed B7-H3 (38-87%) as well as the damage associated molecular pattern (DAMP) S100A8/9. In comparison, CD14+ cells from the peripheral blood of healthy donors expressed very low levels of B7-H3 or S100A8/9 (<5%). Mo-MDSC are important to the pathogenesis of MM because they support MM growth and contribute to suppression of NK in the tumor microenvironment. B7-H3 is highly expressed on mature osteoclasts and MDSC can develop into osteoclasts in mouse models of MM and breast cancer. We saw that Mo-MDSC expressing B7-H3 also co-expressed RANK, a receptor necessary for signaling osteoclastic development. We developed Mo-MDSC from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF or co-culture with MM cell lines at 1:1000 ratio. Both types of MDSC suppressed NK proliferation. Peripheral blood derived MDSC expressed >80% B7-H3 and RANK. We separated B7-H3 positive and negative populations by flow cytometry and attempted to develop osteoclasts with M-CSF and RANK-L over 14 days. Tartrate resistant acid phosphatase (TRAP) staining was performed but both groups were negative and exhibited no phenotypic changes by light microscopy. We then isolated CD14+ cells from the bone marrow of an MM patient who was in complete remission (CR) after autologous stem cell transplant but had a distinct population of B7-H3+, RANK+, S100A8/9+ Mo-MDSC in her surveillance bone marrow aspirate. After fourteen days of culture with M-CSF and RANK-L, TRAP staining was positive, and cells had grown larger, consistent with osteoclastic differentiation (Figure 1A). We tested the ability of B7-H3 TriKE to eliminate MDSC by NK cells, therefore diminishing there immune suppression. Cytotoxicity assays were conducted over 48 hours with NK cells with or without B7-H3 TriKE. Killing was significantly enhanced by B7-H3 TriKE (Figure 1B). Since MDSC are known to suppress NK cell function, we assessed metabolic fitness in NK cells with MDSC at 1:1 ratio for 48 hours with or without 3nM B7-H3 TriKE and found that B7-H3 TriKE improved both the oxygen consumption and extracellular acidification rates. These data show that B7-H3 TriKE enhances NK cell killing of MM through direct targeting and eliminates suppressive Mo-MDSC. B7-H3+ Mo-MDSC can differentiate into osteoclasts and persist even in patients who are in CR, possibly contributing to relapse. B7-H3 TriKE may be particularly useful for patients with bone lesions or as consolidative therapy. Commercial manufacturing of B7-H3 TriKE (GTB-5550) has begun, and a phase I trial is expected in late 2023. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel First-in-Class Drug ONC201 As a Post-Transplant Maintenance for AML and MDS: A Phase I Trial in Progress

Background: Relapses of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic hematopoietic cell transplantation represent an area of significant unmet clinical need and a research priority identified by the National Cancer Institute. For patients with high-risk AML and MDS, post-transplant relapses remain the principal cause of mortality. The role of chemotherapy agents outside of FLT3 inhibitors or TKIs, and post-transplant cellular therapy to prevent relapse of leukemia are not well established. However, chemotherapy agents are frequently utilized off-label in clinical practice, thus signifying an urgent need to develop effective therapeutic strategies. ONC201 is a novel first-in-class small molecule imipridone, that antagonizes G protein-coupled receptor, DRD2 resulting in AKT/ERK inactivation. ONC201 is also an allosteric agonist of mitochondrial caseinolytic protease P that activates an integrated stress responses. In preclinical studies, leukemia cells demonstrate sensitivity to ONC201 regardless of their genetic and mutation profiles. Leukemia cells with genetic and mutation changes such as TP53 mutation and complex karyotype that confer resistance to conventional therapy are sensitive to ONC201. ONC201 is also toxic to leukemia stem cells while sparing normal bone marrow cells [Sci Signal. 2016 Feb 16;9(415):ra17]. Several clinical trials in solid and hematologic malignancies have demonstrated that oral ONC201 has a low toxicity profile and is well tolerated. The profile of ONC201 is well suited for further development to prevent post-transplant relapses of AML and MDS. Here, we report the design of the first trial to use ONC201 as post-transplant maintenance (ClinicalTrials.gov ID: NCT03932643). Study Design and Methods: The study will follow a 3+3 phase 1 trial design. The primary objective is to determine the rate of dose-limiting toxicities (DLT) during the first cycle and grade ≥3 toxicities during the first 3 cycles. Key inclusion criteria include adult recipients of allogeneic hematopoietic stem cell transplant within 6-20 weeks for a history of high-risk AML or MDS; <5% bone marrow blasts; Karnofsky Performance Status of ≥70; absolute neutrophil count (ANC) greater than 1000/μL, and platelet count ³50,000/µL. Key exclusion criteria include a history of acute graft-versus-host disease (GVHD) grade III/IV; initiation of any new systemic immunosuppressive agent for GVHD within 4 weeks of enrollment, current use of prednisone at a dose of ≥0.25 mg/kg/day; uncontrolled serious infection; active ischemic heart disease, heart failure or arrhythmia; severe chronic obstructive pulmonary disease; resting O2 saturation <90%; aspartate transaminase, alanine transaminase or bilirubin >2 times the upper limit of normal; and creatinine clearance <30 mL/min. Patients will receive oral weekly ONC201 at a starting dose of 250 mg and dose escalation by 125 mg up to a maximum dose of 625 mg weekly, in the absence of DLT. Patients will be monitored for toxicities (using Common Terminology Criteria for Adverse Events, CTCAE version 5.0), quality of life (QOL) (Functional Assessment of Cancer Therapy-Bone Marrow Transplant, FACT-BMT), changes in functional status (KPS, instrumental activities of daily living and short physical performance battery), rates of disease relapse and mortality (See Study Schema). We will enroll a total of 20 patients; the first 6-9 patients are expected to receive escalating doses of ONC 201, and the remaining patients will receive the maximum tolerated doses. Clinical studies in solid malignancies have indicated that ONC201 can expand and activate NK cells in peripheral blood, or recruit CD8+ T-cells to the tumor. Hence, the study will assess expansion and activation of NK and T cell populations in peripheral blood and utilize transcriptomic analyses of NK and T cells to explore the mechanisms behind the immunologic changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NK Cells Suppress Hyperfunction of CTLs Via ATM-Mediated NKG2D/NKG2DLs Activation in Severe Aplastic Anemia

Object: The activating receptor NKG2D on the surface of NK cells in peripheral blood of patients with severe aplastic anemia (SAA) was significantly increased, and NK cells may be activated during the pathogenesis of SAA. However, the immunomodulatory effect of NK cells on CTL cells remains unclear. ATM protein is a serine/threonine protein kinase mainly involved in cell cycle regulation, DNA damage recognition and repair. Activation of ATM may be the key to regulating the expression of NKG2DLs in T cells stimulated by superantigens. By detecting the changes of NKG2DLs in CTL cells of SAA patients under the regulation of ATM, we explored the immunoregulatory effect of NK cells on autologous CTL cells through the signaling axis NKG2D/NKG2DLs. Methods: We used flow cytometry, RT-qPCR and Western Blot to detect the expression of NKG2DLs (MICA, ULBP1, ULBP3) in CTL cells of untreated SAA patients, remission SAA patients and healthy controls under the intervention of CD3/CD28 antibody and ATM agonist (chloroquine phosphate). NK and CTL cells from SAA patients were co-cultured in vitro, and the function of CTL under the regulation of ATM was detected. Results: Under the intervention of CD3/CD28 antibody and chloroquine phosphate, the level of MICA in CTL cells detected by flow cytometry in untreated SAA patients was higher than that in convalescent SAA patients and healthy controls (Fig.A). RT-qPCR detection of mRNA levels of NKG2DLs (MICA, ULBP1 and ULBP3) in CTL cells of untreated SAA patients was also higher than that of remission SAA patients and healthy controls (Fig.B). When ATM agonist (chloroquine phosphate), ATM inhibitor (KU55933), and chloroquine phosphate + KU55933 were used to intervene T cells, Western Blot detected the correlation between ATM phosphorylation and NKG2DLs (MICA, ULBP1) expression(Fig.D). We co-cultured CTL cells from SAA patients with different interventions in vitro with autologous NK cells. The expression of IFN-γ in CTL cells in the ATM agonist group was significantly lower than that in the ATM inhibition group, and the apoptosis rate was higher than that in the ATM inhibition group and the control group (Fig.C). There was no significant difference in the expression of perforin and granzyme B. In addition, NK cell apoptosis did not differ among the groups.The expression levels of free MICA and ULBP3 in the co-culture supernatant were detected by ELISA, and the ATM agonist group was higher than the inhibition group(p<0.05). Conclusion: Activation of ATM increases the expression of NKG2DLs in CTL cells of SAA patients, which promotes the inhibitory effect of NK cells on CTL cells. Our study further confirms that ATM can regulate the relationship between NK cells and CTL cells in SAA disease, and inhibit the hyperactive state of CTL cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preliminary Dose-Escalation Results from a Phase 1/2 Study of GEN3014 (HexaBody®-CD38) in Patients (pts) with Relapsed or Refractory Multiple Myeloma (RRMM)

Background: CD38-targeted antibodies (Abs) are foundational treatments (Tx) for pts with newly diagnosed or RRMM. GEN3014 (HexaBody®-CD38) is a novel human IgG1 anti-CD38 monoclonal Ab (mAb) with an E430G hexamerization-enhancing mutation that facilitates highly efficient complement-dependent cytotoxicity (CDC). GEN3014 showed preclinical tumor cell killing through highly potent CDC (more efficient than daratumumab) and Ab-dependent cellular cytotoxicity and phagocytosis. We present preliminary data from dose escalation in the first-in-human, phase 1/2 trial of GEN3014 in pts with RRMM (NCT04824794). Methods: Adults with RRMM were enrolled if they had ≥3 prior lines of Tx including a proteasome inhibitor (PI) and an immunomodulatory imide drug (IMiD), were double refractory to a PI and IMiD, or had ≥2 prior lines of Tx if 1 combined a PI and IMiD. GEN3014 was tested at 6 dose levels ranging from 0.2 to 24 mg/kg and administered intravenously in 28-d cycles (first dose split equally between D1 and D2 of cycle 1, then full doses QW through cycle 2; Q2W, cycles 3-6; Q4W, cycles ≥7). Primary objectives were to determine the recommended phase 2 dose (RP2D) and maximum tolerated dose. Other endpoints included pharmacokinetic (PK) and pharmacodynamic (PD) characterization. PD biomarker analysis included immunophenotyping and quantification of complement factors and cytokines in peripheral blood. Results: As of July 10, 2022, 24 pts with RRMM were treated (median age, 65 y; range, 45-84). Pts were heavily pretreated with a median of 7 prior lines of Tx (range, 3-13). Most pts (67%) had prior exposure to a daratumumab- or isatuximab-containing regimen, with 8 pts naive to anti-CD38 mAb. GEN3014 was well tolerated; the most common Tx-emergent AEs were infusion-related reactions (IRRs; 75.0%), neutropenia (62.5%), anemia (33.3%), diarrhea (33.3%), pyrexia (25.0%), and thrombocytopenia (25.0%). IRRs were mostly low grade (G; 62.5% G1-2, 12.5% G3, no G4), occurred mainly during the first infusion, and were manageable. There were no Tx-related deaths or dose-limiting toxicities observed at doses up to 16 mg/kg, the RP2D. Disease progression led to discontinuation in 80% of pts. Among 19 response-evaluable pts (5 naive to anti-CD38 mAb, 14 refractory to anti-CD38 mAb), preliminary antitumor activity was seen. In pts naive to anti-CD38 mAb, 2 pts achieved very good partial response (1 each at 4 and 24 mg/kg); 1 achieved minimal response (MR; 16 mg/kg). In pts refractory to anti-CD38 mAb, 2 pts achieved MR (1 each at 8 and 16 mg/kg). Five pts remained on Tx (4 naive and 1 refractory to anti-CD38 mAb). Updated data will be presented. Peak GEN3014 concentrations at the end of infusion increased roughly proportionally with dose. The interval area under the concentration-time curve showed a more than proportional increase with doses ≤8 mg/kg but a proportional or less than proportional increase at doses >8 mg/kg. These results suggest target-mediated drug disposition at lower doses and a high degree of target saturation at >8 mg/kg. Trough concentrations increased with weekly administration, most notably in pts responding to Tx, indicating target cell depletion over time. GEN3014 Tx was associated with a rapid, sustained decrease in peripheral blood natural killer cells at all doses in all pts (mean 92.4% ± 9.2% peak reduction from baseline [BL], n=21). T cells transiently decreased after administration of first doses ≥4 mg/kg, followed by expansion (≥100% increase from BL), particularly in pts naive to anti-CD38 mAb. GEN3014 induced transient reduction in complement component C2 (mean 58% ± 18% peak reduction from BL, n=18) and total complement lytic activity (CH50; mean 48% ± 24% peak reduction from BL for ≥8 mg/kg, n=11) at all evaluable doses, suggesting CDC. Complement parameters rapidly returned to BL, indicating that Tx does not exhaust complement. Plasma cytokine (IL-2, IL-6, IL-8, IL-10, IFNγ, and TNFα) levels generally remained low after Tx. Conclusions: Dose-escalation results show GEN3014 had a tolerable safety profile and clinical activity in pts with RRMM, including pts with prior anti-CD38 mAb. The PK profile is characterized by target-mediated drug disposition at lower doses and signs of saturation at doses >8 mg/kg. Biomarker analyses confirm biological activity in pts with RRMM at all evaluated doses. The expansion part of the trial evaluating the 16 mg/kg RP2D is ongoing.