Mature NK Cells Research Articles

Modified Hemocyanins from Rapana thomasiana and Helix aspersa Exhibit Strong Antitumor Activity in the B16F10 Mouse Melanoma Model.

Melanoma is one of the most common tumors worldwide, and new approaches and antitumor drugs for therapy are being investigated. Among the promising biomolecules of natural origin for antitumor research are gastropodan hemocyanins-highly immunogenic multimeric glycoproteins used as antitumor agents and components of therapeutic vaccines in human and mouse cancer models. A murine melanoma model established in C57BL/6 mice of the B16F10 cell line was used to study anticancer modified oxidized hemocyanins (Ox-Hcs) that were administered to experimental animals (100 μg/mouse) under different regimens: mild, intensive, and with sensitization. The solid tumor growth, antitumor response, cell infiltration in tumors, and survival were assessed using flow cytometry, ELISA, and cytotoxicity assays. Therapy with Ox-RtH or Ox-HaH resulted in the generation of enhanced specific immune response (increased levels of tumor-infiltrated mature NK cells (CD27+CD11b+) in sensitized groups and of macrophages in the intensively immunized animals) and tumor suppression. Beneficial effects such as delayed tumor incidence and growth as well as prolonged survival of tumor-bearing animals have been observed. High levels of melanoma-specific CTLs that mediate cytotoxic effects on tumor cells; tumor-infiltrating IgM antibodies expected to enhance antibody-dependent cellular cytotoxicity; type M1 macrophages, which stimulate the Th1 response and cytotoxic cells; and proinflammatory cytokines, were also observed after Ox-Hcs administration. The modified Hcs showed strong antitumor properties in different administration regimens in a murine model of melanoma with potential for future application in humans.

Clinical and genetic profile of Chinese patients with indolent natural killer-cell lymphoproliferative disorder of the gastrointestinal tract

Indolent natural killer cell lymphoproliferative disorder of the gastrointestinal tract (iNKLPD-GI) is an uncommon, recently recognized lymphoid proliferation of mature NK cells primarily manifesting in the GI tract. Unlike NK/T lymphoma, iNKLPD-GI exhibits a rather indolent clinical course, underscoring the need for cautious management to prevent unnecessary interventions. However, clinical and molecular features of this entity have not been thoroughly understood. This study aimed to add more information to the current knowledge of this disease. Seven patients with iNKLPD-GI were included in our study. Clinical data included initial symptoms, endoscopic manifestations, pathological features, and therapies. Besides, next-generation sequencing was arranged to explore the underlying genetic mechanism of this disease. In our study, iNKLPD-GI in the urinary bladder was first identified. Edema of extremities (3, 42.8 %) was the most prevalent onset symptom which was reported for the first time. Pathological and immunohistological features were found to display the phenotype of NK cells. Unlike extranodal NK/T cell lymphoma, Epstein-Barr virus-encoded small RNA (EBER) were negative in all patients. Moreover, we found that two patients harbored JAK3 mutation. Apart from JAK3 K563_C565del previously reported in the literature, we discovered new JAK3 mutation sites. Other mutations including BRAF, KRAS, and SH2B3 were also identified. In conclusion, iNKLPD-GI was an indolent atypical NK-cell proliferation with diverse clinical characteristics. “Watch and wait” therapy was preferable to intense chemotherapy. Recurrent JAK3 mutation may be the underlying mechanism responsible for the neoplastic nature of the disease and may serve as a potential target for patients with severe symptoms.

Recombinant Human Interleukin-2 Corrects NK Cell Phenotype and Functional Activity in Patients with Post-COVID Syndrome

Post-COVID syndrome develops in 10–20% of people who have recovered from COVID-19 and it is characterized by impaired function of the nervous, cardiovascular, and immune systems. Previously, it was found that patients who recovered from infection with the SARS-CoV-2 virus had a decrease in the number and functional activity of NK cells. The aim of the study was to assess the effectiveness of recombinant human IL-2 (rhIL-2) administered to correct NK cell phenotype and functional activity in patients with post-COVID syndrome. Patients were examined after 3 months for acute COVID-19 of varying severity. The phenotype of the peripheral blood NK cells was studied by flow cytometry. It was found that disturbances in the cell subset composition in patients with post-COVID syndrome were characterized by low levels of mature (p = 0.001) and cytotoxic NK cells (p = 0.013), with increased release of immature NK cells (p = 0.023). Functional deficiency of NK cells in post-COVID syndrome was characterized by lowered cytotoxic activity due to the decreased count of CD57+ (p = 0.001) and CD8+ (p 0.001) NK cells. In the treatment of patients with post-COVID syndrome with recombinant IL-2, peripheral blood NK cell count and functional potential were restored. In general, the effectiveness of using rhIL-2 in treatment of post-COVID syndrome has been proven in patients with low levels of NK cells.

Immune signature of patients with cardiovascular disease predicts increased risk for a severe course of COVID-19.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection can lead to life-threatening clinical manifestations. Patients with cardiovascular disease (CVD) are at higher risk for severe courses of COVID-19. So far, however, there are hardly any strategies for predicting the course of SARS-CoV-2 infection in CVD patients at hospital admission. Thus, we investigated whether this prediction is achievable by prospectively analysing the blood immunophenotype of 94 nonvaccinated participants, including uninfected and acutely SARS-CoV-2-infected CVD patients and healthy donors, using a 36-colour spectral flow cytometry panel. Unsupervised data analysis revealed little differences between healthy donors and CVD patients, whereas the distribution of the cell populations changed dramatically in SARS-CoV-2-infected CVD patients. The latter had more mature NK cells, activated monocyte subsets, central memory CD4+ T cells, and plasmablasts but fewer dendritic cells, CD16+ monocytes, innate lymphoid cells, and CD8+ T-cell subsets. Moreover, we identified an immune signature characterised by CD161+ T cells, intermediate effector CD8+ T cells, and natural killer T (NKT) cells that is predictive for CVD patients with a severe course of COVID-19. Thus, intensified immunophenotype analyses can help identify patients at risk of severe COVID-19 at hospital admission, improving clinical outcomes through specific treatment.

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2+ influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca ++ influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2+ flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70 . Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

Disrupting Smad3 potentiates immunostimulatory function of NK cells against lung carcinoma by promoting GM-CSF production

Through Smad3-dependent signalings, transforming growth factor-β (TGF-β) suppresses the development, maturation, cytokine productions and cytolytic functions of NK cells in cancer. Silencing Smad3 remarkably restores the cytotoxicity of NK-92 against cancer in TGF-β-rich microenvironment, but its effects on the immunoregulatory functions of NK cells remain obscure. In this study, we identified Smad3 functioned as a transcriptional repressor for CSF2 (GM-CSF) in NK cells. Therefore, disrupting Smad3 largely mitigated TGF-β-mediated suppression on GM-CSF production by NK cells. Furthermore, silencing GM-CSF in Smad3 knockout NK cells substantially impaired their anti-lung carcinoma effects. In-depth study demonstrated that NK-derived GM-CSF strengthened T cell immune responses by stimulating dendritic cell differentiation and M1 macrophage polarization. Meanwhile, NK-derived GM-CSF promoted the survival of neutrophils, which in turn facilitated the terminal maturation of NK cells, and subsequently boosted NK-cell mediated cytotoxicity against lung carcinoma. Thus, Smad3-silenced NK-92 (NK-92-S3KD) may serve as a promising immunoadjuvant therapy with clinical translational value given its robust cytotoxicity against malignant cells and immunostimulatory functions to reinforce the therapeutic effects of other immunotherapies.

Impact of immune system humanization on atherosclerosis in dyslipidemic immunocompromised mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Cariplo Foundation. PRIN (Italian Ministry of University and Research) Background and aim Given the key role of immune response during atherosclerosis and the therapeutic interest on biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and test therapeutic approaches for atherosclerosis is continuously increasing. Here we provide an immune and metabolic characterization of an innovative immunodeficient mouse humanized with human hematopoietic cells on an atheroprone background. Methods LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse (TKO-LDLR KO mice) recipient of human hematopoietic stem cells (hCD34+). Results TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet, TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia, steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next we tested the impact of immune system humanization on atherosclerosis. TKO-LDLR KO pups received a low-dose irradiation (200 cGy) and thereafter 2 x 10^5 hCD34+ were injected in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells. The humanization with hCD34+ cells worsens atherosclerosis development as compared to non-humanized TKO-LDLR KO mice. Conclusion We have generated and characterized the humanized dyslipidemic TKO-LDLR KO mouse. This mouse model presents human B and T cells and represents and important toll to investigate the impact of human adaptive immune cell pharmacological modulation in the context of atherosclerosis.

Quercetin promotes the proportion and maturation of NK cells by binding to MYH9 and improves cognitive functions in aged mice

BackgroundQuercetin is a flavonol compound widely distributed in plants that possesses diverse biological properties, including antioxidative, anti-inflammatory, anticancer, neuroprotective and senescent cell-clearing activities. It has been shown to effectively alleviate neurodegenerative diseases and enhance cognitive functions in various models. The immune system has been implicated in the regulation of brain function and cognitive abilities. However, it remains unclear whether quercetin enhances cognitive functions by interacting with the immune system.ResultsIn this study, middle-aged female mice were administered quercetin via tail vein injection. Quercetin increased the proportion of NK cells, without affecting T or B cells, and improved cognitive performance. Depletion of NK cells significantly reduces cognitive ability in mice. RNA-seq analysis revealed that quercetin modulated the RNA profile of hippocampal tissues in aging animals towards a more youthful state. In vitro, quercetin significantly inhibited the differentiation of Lin−CD117+ hematopoietic stem cells into NK cells. Furthermore, quercetin promoted the proportion and maturation of NK cells by binding to the MYH9 protein.ConclusionsIn summary, our findings suggest that quercetin promotes the proportion and maturation of NK cells by binding to the MYH9 protein, thereby improving cognitive performance in middle-aged mice.Graphical abstract

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma

IntroductionNK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. “suicide mechanisms” regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. ObjectivesWe sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. MethodsPrimary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. ResultsAZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. ConclusionsmTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Abstract 2778: Influence of the differentiation strategy on the phenotypic and genotypic features of iPSC derived NK cells

Abstract The iPSC-derived NK (iNK) cells offer new perspectives to produce large-scale of homogeneous immunotherapeutic cellular products and open the way towards manufacturing off-the-shelf cancer immunotherapies. Numerous strategies and protocols of differentiation of iPSCs toward NK lineage have been published in the last decade. Nevertheless, the embryological development of the NK cells and the impact of the differentiation strategy on the activity of the NK cells are still not clearly understood. Furthermore, very few studies have compared different NK differentiation strategies from iPSC. this study, we compared two previously reported protocols for NK cell generation from the same iPSC line, one feeder-based and the other feeder-free. In the first protocol, 3D hematopoietic differentiation was followed by lymphoid differentiation, using an OP9 DLL4 expressing feeder cell line. The second protocol employed a clinical-grade approach, utilizing the “spin embryoid body” method to generate primitive hematopoietic progenitors. Subsequently, these progenitors were differentiated towards the NK lineage in a feeder-free environment. Hematopoietic progenitors produced by these protocols have distinct phenotypes, suggesting variations in their maturity levels. Furthermore, the protocols have different kinetics of differentiation revealed by CD56 and CD7 phenotypes. Although, both protocols were able to efficiently generate functional and mature NK cells, characterized by the expression of activating and maturity ligands but with a distinct transcriptomic profile. iNK cells generated using feeders exhibit a more pronounced inflammatory profile with the expression of hallmark NFKB and STAT3 signaling pathways. In contrast, feeder-free iNK cells have a more proliferative profile as evidenced by increased expression of cell cycle factors such as E2F or Myc. Feeder-based iNK cells exhibit increased expression of transcription factors involved in NK cell differentiation, such as GATA2, STAT1, and IRF1. These transcriptional variances are reinforced by superior functionality observed in feeder-based NK cells compared to feeder-free NK cells, evidenced by increased degranulation (p-value < 0.0001) and cytotoxicity (p-value = 0.0004) at a 1:2 (effector-to-target) ratio against the K562 cell line. Our findings indicate that, despite the advantages of the feeder-free strategy, the feeder-based protocol remains more efficient and produces iPSC-derived NK cells with an enhanced cytotoxic profile in vitro. Our comprehensive transcriptional analysis offers significant insights into the biological and functional properties of iNK cells. This information has the potential to oncover novel mechanisms for NK differentiation strategies from iPSCs. Citation Format: Matthias Huyghe, Christophe Desterke, Jusuf Imeri, Nathan Belliard, Ali G. Turhan, Annelise Bennaceur Griscelli, Frank Griscelli. Influence of the differentiation strategy on the phenotypic and genotypic features of iPSC derived NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2778.

Long-Term Follow-Up of Abatacept, Post-Transplantation Cyclophosphamide, and Sirolimus-Based Haploidentical Transplantation in Younger Patients with Nonmalignant Diseases

BackgroundHaploidentical (Haplo) HCT for nonmalignant diseases (NMD) faces inherent challenges of both alloreactivity and graft failure. Building upon promising results from pilot studies, where abatacept was combined with post-transplantation cyclophosphamide (PTCy) and sirolimus (AbaCyS) in younger NMD patients undergoing haplo-HCT, we present the long-term outcomes of this protocol. Study DesignOn the back of uniform disease-specific conditioning regimens containing ATG (4.5 mg/kg) from day-9 to -7, GVHD prophylaxis (AbaCyS) consisted of abatacept administered on days 0, +5, +20, +35, and monthly until 180 days with PTCy and sirolimus. The patients were followed up with longitudinal assessment of immune reconstitution, growth and reproductive development and quality of life (QoL) analysis. ResultsAmongst 40 patients (aplastic anemia-24, hemoglobinopathies-14, and primary immunodeficiencies-2) with a median age of 10 years (range, 2-25), 95% achieved sustained engraftment. Post-transplant Hemophagocytic Syndrome was detected in 3 patients, leading to graft failure in 2 cases. The incidence of aGVHD was 2.6%, while that of cGVHD was 14.3%. CMV, adenovirus and EBV infections were observed in 45%, 5% and 0% respectively. Non-relapse mortality, overall survival, EFS and GEFS rates were 5%, 95% 90% and 82%, respectively at a median follow-up of 4.6 years. Absence of cGVHD correlated with younger patient age and early sustained recovery of Tregs and mature NK cells, which in turn was associated with improved QoL and lack of late infections. ConclusionsAbaCyS protocol is associated with excellent long-term survival with attenuation of both early and late alloreactivity in over 80% of younger patients undergoing Haplo-HCT for NMD. The study sheds light on predispositions to cGVHD and its impact on QoL, warranting further optimization of this approach.

The immunological landscape of peripheral blood in glioblastoma patients and immunological consequences of age and dexamethasone treatment.

Glioblastomas manipulate the immune system both locally and systemically, yet, glioblastoma-associated changes in peripheral blood immune composition are poorly studied. Age and dexamethasone administration in glioblastoma patients have been hypothesized to limit the effectiveness of immunotherapy, but their effects remain unclear. We compared peripheral blood immune composition in patients with different types of brain tumor to determine the influence of age, dexamethasone treatment, and tumor volume. High-dimensional mass cytometry was used to characterise peripheral blood mononuclear cells of 169 patients with glioblastoma, lower grade astrocytoma, metastases and meningioma. We used blood from medically-refractory epilepsy patients and healthy controls as control groups. Immune phenotyping was performed using FlowSOM and t-SNE analysis in R followed by supervised annotation of the resulting clusters. We conducted multiple linear regression analysis between intracranial pathology and cell type abundance, corrected for clinical variables. We tested correlations between cell type abundance and survival with Cox-regression analyses. Glioblastoma patients had significantly fewer naive CD4+ T cells, but higher percentages of mature NK cells than controls. Decreases of naive CD8+ T cells and alternative monocytes and an increase of memory B cells in glioblastoma patients were influenced by age and dexamethasone treatment, and only memory B cells by tumor volume. Progression free survival was associated with percentages of CD4+ regulatory T cells and double negative T cells. High-dimensional mass cytometry of peripheral blood in patients with different types of intracranial tumor provides insight into the relation between intracranial pathology and peripheral immune status. Wide immunosuppression associated with age and pre-operative dexamethasone treatment provide further evidence for their deleterious effects on treatment with immunotherapy.

Primary Cutaneous Follicle Center Lymphoma in an Adult African American Male

Primary cutaneous B-Cell Lymphomas (PCBCL) is a form of a lymphoproliferative neoplasm consisting of proliferation localized to the skin. Cutaneous lymphomas originate from mature T-lymphocytes, B-lymphocytes, or NK cells. The incidence of PCBCL is significantly lower in patients with Skin of Color (SoC) and is quite rare among African American (AA) individuals, compared to their Caucasian counterparts. The clinical course of PCFCL in SoC is not widely studied or reported in literature due to the low incidence in the AA population. We present a case of PCBCL in an AA individual along with a brief review of the clinical presentation, pathology, and treatment options of the condition. According to the U.S. Census Bureau, approximately half of the U.S population will have SoC by 2050; by adding our case of PCFCL to the body of literature, we hope to promote awareness of the presentation and course of this cutaneous disorder in SoC patients.

The Impact of the Peptide Drug Conjugate Melflufen on the Myeloma Tumour Microenvironment

Introduction Melphalan flufenamide (melflufen), is a peptide-drug conjugate that delivers alkylating agents in tumors upon cleavage by aminopeptidases, and recently approved by the EMA for the treatment of relapsed/refractory multiple myeloma (MM). Although melflufen's MoA on tumor cells had been extensively studied, little is known about the drug impact on the tumor microenvironment. By applying single cell, multi-parametric, flow cytometry-based analysis to bone marrow mononuclear cells (BM-MNC) from MM patient samples treated ex vivo, we investigated the effect of melflufen on immune cell populations to assess for potential immune modulatory effects and to identify potential immune-based biomarkers of response. Methods Viably frozen BM-MNC from 26 samples of patients diagnosed with MM were used for multi-parametric flow cytometry-based drug sensitivity and resistance testing evaluation to melflufen after written informed consent following approved protocols in compliance with the Declaration of Helsinki. Drug sensitivity was assessed using five different concentrations of melflufen ranging between 0.5-5000 nM and cells incubated with drug or DMSO control for 72 hrs incubation. For analysis, we used antibody panels allowing us to identify and phenotype T and B lymphocytes, NK, progenitor, and malignant plasma cells (PC). Based on drug sensitivity scores (DSS) of gated PCs from drug treated and controls samples, we divided the samples into two groups - high sensitivity (HS, Q3 of PC-DSS) and low sensitivity (LS, Q1 of PC-DSS). Samples with no PCs detected (n=11) were excluded from PC subset analysis. Results The MM samples were divided based on the sensitivity of the PC population to melflufen with DSS > 21.25 defining a highly sensitive group and DSS < 15.9 defining a less sensitive group. We found that HS samples have higher abundance of mature NK and iNKT cells, although significance was low, likely due to the small cohort size. In addition, we discovered that effector memory (EM) and terminal effector (TE) T cells have higher sensitivity to melflufen, compared to naïve and central memory (CM) phenotypes (Fig. 1). More mature NK cells (Stage 5 and 6) also showed higher sensitivity than NK cells of earlier stages of maturation. Importantly, we observed that in many of the tested samples, progenitor-like CD34+CD38- cells either were not sensitive to the cytotoxic effect of the drug, or even proliferated in higher drug concentrations (Fig. 2). Interestingly, HS samples had significantly higher baseline abundance of this cell population (p<0.05). At the same time, response profiles of healthy B and malignant PCs were similar. Finally, in our tests, PCs in samples with trisomy 11 abnormality showed lower sensitivity to melflufen (p<0.05). Conclusion In a previous study, we found that effector-like cells could be associated with better response to melflufen. In this study, we observed the same trends and confirm our earlier findings demonstrating an association of NK and NKT cells with higher sensitivity to melflufen. Moreover, we identified differential response of CD4, CD8 and NK cell populations comprising the tumor microenvironment to melflufen suggesting that in addition to direct tumor cell killing, melflufen may have modulating effects on different tumor associated immune cell populations. Finally, we observed proliferation of CD34+CD38- progenitor cells, which might be relevant for lymphocyte proliferation and function, and additionally supporting potential modulating effect. Further analyses are being done to validate these findings.

Immune system humanization in dyslipidemic Rag2-KO/IL2rg-KO/CD47-KO/LDL-R KO mice controls atherosclerosis

Abstract Background and aim Given the key role of the immune response during atherosclerosis and the therapeutic interest of biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and to test therapeutic approaches for atherosclerosis is continuously increasing. Here we describe the characteristics of an innovative immunodeficient mouse humanized with hCD34+ cells on an atheroprone background. Methods LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse recipient TKO-LDLR KO of human hematopoietic stem cells (hCD34+). Results TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet for 8 weeks, both male and females TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia (total cholesterol 870.9 and 890.1 mg/dL male and females respectively), steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next we tested the impact of immune system humanization on atherosclerosis. TKO-LDLR KO pups received a low-dose irradiation (150-200 cGy) and thereafter 1,5-2 x 10^5 hCD34+ were injected with in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells. The humanization with hCD34+ cells affected atherosclerosis development as compared to non-humanized TKO-LDLR KO mice. Conclusion We have generated and characterized for the first time a humanized dyslipidemic TKO-LDLR KO mice. This mouse model presents human B and T cells and represent and important toll to investigate the impact of biologics targeted toward human targets in the context of atherosclerosis.

Expansion, Persistence, and Characteristics of Autologous, Bhv-1100 Armored Memory-like NK Cells Infused Prior to Autologous Stem Cell Transplant in MRD+ Multiple Myeloma Patients: A First-in-Human Trial

Introduction/Background Autologous stem cell transplant (ASCT) improves MRD negativity and prolongs progression-free survival in patients with multiple myeloma (MM) in their first or second remission following induction chemotherapy. MM NK cells are dysfunctional, negatively impacting outcomes. BHV-1100, a novel Antibody Recruiting Molecule ( ARM), binds to CD38 and recruits NK cells for antibody-dependent cell cytotoxicity (ADCC) without inducing fratricide. Allogeneic, cytokine induced memory-like (CIML) NK cells effectively treat myeloid disorders, however, it is not known if autologous CIML NK cells, when coated with BHV-1100, would further improve ASCT outcomes in MM. Methods We designed a first-in-human study of autologous CIML NK cells coated ex-vivo with BHV-1100 for MRD+, MM patients undergoing ASCT following first or second remission. NK cells were isolated from non-mobilized leukapheresis on day -1 (prior to melphalan for HCT) using CD3 depletion followed by CD56 positive selection with Miltenyi's CliniMACS. The NK cells were incubated overnight (12-16 hours) with IL-12 (10ng/ml), IL-15 (100ng/ml), and IL-18 (50ng/ml) to induce CIML differentiation, washed and subsequently coated with BHV-1100 for one hour prior to infusion. The product was infused fresh on D0 after standard melphalan 200 mg/m2 myeloablative conditioning and followed by stem cell infusion. Low dose IL2 (1 mIU/m 2) was administered SQ starting on D+1, QOD for a total of 7 doses. Results This is an ongoing trial (NCT04634435) with a median follow-up of 191 days. We are herein reporting data on the in vivo expansion and functional characterization of ARMored CIML NK for the first 5 patients. CIML NK cells were manufactured with a 100% success rate and infused at a target dose of 5-10x10 6 cells/kg-body weight, 24 hours after 200 mg/m 2 melphalan administration. Patients received between 3.9-6.0x10 6/Kg stem cells. Engraftment based on recovery of neutrophil count occurred on D+12-D+14. There was a 3-fold expansion of NK cells in the peripheral blood from D+7 (from 12% to 42%) to D+28 that persisted until D+60 (25% total PBMC, Fig. 1A). Most expanded NK cells were CD56 dim, CD16 high, KIR high and CD57 high. CD57 and KIR expression increased over time from D+7 to D+60, whereas NKG2A expression decreased, indicating the expansion of mature, activated, and cytotoxic NK cells. Regulatory T cells increased by D+7 (3% vs 15% total PBMC) and returned to baseline after D+14 most likely reflecting the effect of IL-2 treatment. The functional capacity of the infused product was tested in vitro against MOLP8 MM cell line. The BHV-1100 ARMored CIML NK cells showed increased IFN (53% vs 48% at 0H and 53% vs 44% at 24H) and CD107a (Fig.1B) (26% vs 13% at 0H and 34% vs 15% at 24H) production compared to untreated CIML NK cells and the product was stable for 24 hours. Conclusion Autologous, BHV-1100 ARMored CIML NK cells have enhanced anti-MM activity as well as expand and persist in vivo peaking at D+28 after infusion. This represents an innovative approach to boost autologous cancer immunosurveillance in the context of ASCT. Aside from anticipated infusion reactions, no severe/unexpected adverse events were noted; longer follow-up is required to assess safety and efficacy.

Low levels of CD26 on certain cellular subtypes of donor harvest is associated with better clinical outcomes post allogeneic stem cell transplantation through regulation of NF-κB pathway and pro-inflammatory cytokines

BackgroundWe had previously reported significant association of immunoectoenzyme CD26 expression on donor harvest with acute Graft-versus-Host-Disease (aGVHD) in allogeneic stem cell transplantation (ASCT) patients. The current study was aimed at analysing CD26 signaling pathway molecules and understanding their impact on immune reconstitution and clinical outcomes post-ASCT. Subjects and MethodologyThe study cohort included 26 transplant donors/patients who underwent reduced intensity (n = 21), myeloablative (n = 4) and non-myeloablative (n = 1) ASCT for hematological malignancies. Donors were matched related donors (n = 19) and haploidentical donors (n = 7). Surface expression of CD26, CD73 and ADA, and various immune cell subtypes were assessed by multicolour-flow cytometry. Soluble CD26 (sCD26) and cytokine levels were measured in plasma samples by ELISA and Multiplex Luminex assay, respectively. Immune cells from healthy individuals were stimulated with phytohemagglutinin (PHA) in the presence or absence of CD26 inhibitor. Effect of CD26 inhibition on NF-κB localization in PHA stimulated cells was analysed by immunofluorescence and confocal microscopy. Pro-inflammatory cytokines from the culture supernatants were detected with Cytometric bead array flow cytometry. Association of all measured markers with clinical outcomes was evaluated using appropriate statistical tests. ResultsCD26 surface expression on PBSC donor harvest cells showed increased risk of chronic GVHD (cGVHD, p = 0.055). Amongst the various immune cell subtypes, decreased B cells in harvest showed significant association with aGVHD (p = 0.022) whereas increased myeloid dendritic cells and CD3+T cells at Day100 in peripheral blood of transplant recipients correlated with cGVHD (p = 0.046) and aGVHD (p = 0.035), respectively. Further, high sCD26 in transplant recipients at Day100 exhibited association with reduced event-free survival (EFS) (p = 0.011). Higher CD26 expression on more & less mature NK cells, naïve & post-switched memory B cells and Treg cells in the donor harvest (p < 0.05) led to lower EFS in transplant recipients. Mechanistically, CD26 inhibitor caused dose-dependent reduction in CD26 enzyme activity and in pro-inflammatory cytokine production in post mitogen-stimulated T cell cultures. ConclusionOur study has implicated that lower CD26 expression on immune cell subtypes of the donor stem cell harvest is associated with reduced risk of GVHD and better survival. The underlying mechanism was found to be through NF-κB pathway and pro-inflammatory cytokines. Based on these observations, chemically designed or natural resources-based CD26 inhibitors can be explored further in clinical trials for improving ASCT outcomes.

Peripheral unspecified T-cell lymphoma with damage to the heart and large vessels of the mediastinum

Peripheral unspecified T-cell lymphoma not otherwise specified (PTCL NOS) is a malignant neoplasm that develops from mature T-lymphocytes and NK cells. PTCL NOS is a rare malignant neoplasm that occurs most often in men, predominantly over 60 years of age. This type of lymphoma accounts for about 15% of all non-Hodgkin’s lymphomas. The paper describes a clinical case of PTCL NOS , which was difficult to diagnose.

The aryl hydrocarbon receptor maintains antitumor activity of liver resident natural killer cells after partial hepatectomy in C57BL/6J mice.

Liver-resident natural killer (lr-NK) cells are distinct from conventional NK cells and exhibit higher cytotoxicity against hepatoma via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the mechanism by which partial hepatectomy (PH) significantly suppresses TRAIL expression in lr-NK cells remains unclear. This study aimed to investigate the PH influence on the function and characteristics of liver-resident NK (lr-NK) cells using a PH mouse model. Here, we report that PH alters the differentiation pattern of NK cells in the liver, and an aryl hydrocarbon receptor (AhR) molecule is involved in these changes. Treatment with the AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) inhibited the maturation of NK cells. FICZ increased the immature subtype proportion of NK cells with high TRAIL activity and decreased the mature subtype of NK cells with low TRAIL activity. Consequently, FICZ increased the expression of TRAIL and cytotoxic activity of NK cells in the liver, and this effect was confirmed even after hepatectomy. The participation of AhR promoted FoxO1 expression in the mTOR signaling pathway involved in the maturation of NK cells, resulting in TRAIL expression. Our findings provide direct in-vivo evidence that partial hepatectomy affects lrNK cell activity through NK cell differentiation in the liver. Perioperative therapies using an AhR agonist to improve NK cell function may reduce the recurrence of hepatocellular carcinoma after hepatectomy.

In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.

Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%. In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months. At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001). This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.