Engineered Interleukin-15 Autocrine Signaling Invigorates Anti-CD123 CAR-NK Cells

Introduction : Acute Myeloid Leukemia (AML) is an aggressive neoplastic disorder with poor outcomes in children and adults. NK cell adoptive transfer is an anti-cancer immunotherapy that has promise for AML treatment. We aimed to improve NK cell anti-tumor efficacy with expression of a Chimeric Antigen Receptor (CAR) on the cell surface. Our CAR consists of an extracellular single-chain variable fragment targeting the AML-associated antigen CD123 (IL3Rα) and intracellular domains derived from 2B4 and TCRζ. We sought to improve the persistence and long-term functionality of our CAR-NKs by introducing transgenic interleukin-15 (IL15). Methods: CD3-depleted PBMCs were first activated with lethally irradiated feeder cells, then transduced with transiently produced replication incompetent γ-retrovirus (αCD123.2B4.ζ, αCD123.2B4.ζ-IRES-sIL15, sIL15-IRES-mOrange) on day 4 of culture. CAR expression was measured on day 8 using FACS. Secretion of IL15 was verified with ELISA. Cytotoxicity was measured using ffLuc expressing target cells and bioluminescence (BL) measurement. In serial stimulation assays, target cells were repleted daily to maintain a 1:1 effector:target ratio. Immunophenotype and cell counts were assessed by FACS. Transcriptomic analysis (RNAseq) was performed on RNA derived from NK cells purified on D10. Xenograft modeling was performed using NSG mice engrafted with MV-4-11.ffLuc or MOLM-13.ffLuc AML cell lines. Mice were treated with NK cells on D4 or D4-7-10. Untreated mice served as controls. Tumor growth was serially tracked in vivo using BL imaging. NK cell persistence and expansion were measured in peripheral blood. Results: The 2B4.ζ CAR was well expressed on the surface of transduced NK cells (median transduction efficiency 95%, range 85-97%, n=3). 2B4.ζ CAR-NK treatment prolonged survival of AML engrafted mice when compared to treatment with unmodified NKs (median survival: 63 vs 55 days; n=8 mice; p=0.014). Serial peripheral blood analysis revealed a steady decline in circulating NK cells, which were undetectable in all cohorts within 21 days. NK cells were then engineered for constitutive secretion of IL15, with and without CAR expression. 2B4.ζ/sIL15 CAR-NKs had the most potent 24h-cytotoxicity against CD123+ targets (Fig. 1). After a 10-day chronic stimulation with MV-4-11, 2B4.ζ/sIL15- and sIL15-NKs expanded (x1.2 and x5.9 respectively), while NK cells without sIL15 decreased in number. In this assay, only 2B4.ζ/sIL15 CAR-NKs exhibited sustained tumor killing. Transcriptomic analysis after 10 days of serial stimulation showed sample clustering dependent on IL15 secretion. Differential gene expression analysis (DESeq2) identified upregulation of genes associated with cell cycle progression, apoptosis regulation, chemokine signaling, and NK cell mediated cytotoxicity in NK cells secreting IL15 compared to those without. In multiparameter flow cytometric analysis, 2B4.ζ/sIL15 CAR-NKs had a higher percentage of NK cells populating clusters defined by higher surface expression of NK cell activating receptors (NKp30, NKG2D, LFA-1) compared to 2B4.ζ and unmodified NK cells. In our MV-4-11 xenograft model, NKs armed with secreted IL15 expanded in vivo and had improved persistence. A single dose (D4) of 2B4.ζ/sIL15 CAR-NKs demonstrated an initial antitumor response, equivalent to that seen following 3 doses (D4-7-10) of 2B4.ζ CAR-NKs. However, mice treated with IL15-secreting NKs had short survival (Fig. 2). Compared to control mice, peripheral blood analysis showed increasing systemic hIL15 and higher levels of hTNFα. In our more aggressive MOLM-13 xenograft model, both single dose 2B4.ζ/sIL15 CAR-NK and multiple dose 2B4.ζ CAR-NK treatment prolonged survival compared to treatment with unmodified NKs. (27 and 26 vs 20 days; n=5 mice; p<0.01; Fig. 2). Conclusion: 2B4.ζ CAR-NKs have limited antitumor efficacy and short persistence in vivo. NK cells armored with secreted IL15 have enhanced anti-AML cytotoxicity and in vitro persistence. Introduction of IL15 secretion confers a distinctly activated phenotype that is maintained during chronic antigen stimulation. Constitutive local IL15 secretion improves in vivo NK cell persistence but may cause lethal toxicity when employed against AML. These results warrant further study and should impact the development of CAR-NK clinical products for patients with AML. Figure 1 Figure 1. Disclosures Ho: Rodeo Therapeutics/Amgen: Patents & Royalties; Exelixis: Consultancy; Sanofi: Research Funding. Bonifant: Kiadis Pharma: Research Funding; BMS: Research Funding; Merck, Sharpe, Dohme: Research Funding.

Irs1S57X Heterozygous Mutant Mice Display Normal Hematopoiesis and Phenotypic Features, While Homozygous Knockout Exhibit High Fetal or Postnatal Lethality

Introduction: Pharmacological inhibition of insulin receptor substrate 1 (IRS1) protein has been demonstrated to promote antineoplastic effects in hematological disorders, including myeloproliferative neoplasms, chronic myeloid leukemia and acute lymphoblastic leukemia. However, the role of IRS1 in normal hematopoiesis has not yet been elucidated. In this scenario, using a murine Irs1 knockout model represents an interesting tool to evaluate the role of this gene in normal hematopoiesis. B6.129S2-Irs1smla mouse carries a spontaneous nonsense mutation in serine 57 (Irs1S57X), which in homozygosis produces a knockout animal for Irs1, characterized by a small size. Throughout the development of the study, we noticed that the successive mating between Irs1smla heterozygous animals did not result in homozygous mice among the offspring. In view of these findings, the present study aimed to compare the hematological parameters of wild type and heterozygous mice for the Irs1S57X mutation and to describe the fetal lethality of homozygous mice. Methods: Protocols for mouse experiments were approved by the Animal Ethics Committee of the Institution. B6.129S2-Irs1smla/J mice (Jackson Laboratory, stock number: 007240) from a pure C57BL/6 background were mated to produce homozygous animals. For peripheral blood analysis, mice were evaluated monthly at different age ranges (8-10; 12-14; 16-18; 20-22 weeks of age). Samples were collected in heparin and submitted to automated blood cell count. The hematological parameters evaluated included white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT) and platelets. For fetal lethality evaluation, mating was performed between heterozygous mice. Females were followed up daily to check for the presence of a vaginal plug. The time that the plug was identified was considered as 0.5 days post coitus (dpc) or D+0.5. After the plug was found, the females were accommodated separately until the date of euthanasia. Euthanasia of the females was performed on gestational days D+9.5 (n=1), D+12.5 (n=1), D+15.5 (n=2) and D+18.5 (n=2). The fetuses were carefully separated from the maternal material to avoid contamination. Genotyping was performed by Sanger sequencing. Results: After 28 intercrossing events, a total of 101 heterozygotes, 46 wild type mice and 1 homozygous mouse were obtained, with an average of 4.9 (± 1.6) born alive per female. The absence of homozygous knockout adult mice precluded the inclusion of this genotype in the statistical analysis. The single Irs1 knockout mouse that survived to weaning was euthanized at 9 weeks of age for analysis. The Irs1-deficient mouse was male and characterized by low body weight (15.64 grams); 6.8x109/LWBC; 9.79x1012/L RBC, 16.4 g/dl HGB; 48.5% HCT and 311x109/Lplatelets. Wild type animals and heterozygotes were followed up for four months from 8 weeks of age, being weighed and submitted to peripheral blood analysis monthly. Among wild type animals (n= 7 females and 4 males) and heterozygotes (n= 7 females and 12 males) there were no significant differences in body weight and hematological parameters of peripheral blood, at all age ranges evaluated (all p>0.05). Irs1S57X mutation, in homozygosis, promotes fetal or postnatal lethality. Progeny genotyping demonstrated that homozygous fetuses developed until gestational day 18.5. Full-term deliveries (n=2 females) resulted in 17 pups, including 11 born alive and 6 miscarried/dead immediately after birth; the stillborn were exclusively homozygous. Conclusions: Irs1S57X mutation, in heterozygous state, does not result in phenotypic alterations in weight and hematological parameters. In this study, spontaneous truncated homozygosis mutation Irs1S57X, from a pure C57BL/6 genetic background, resulted in high fetal or postnatal lethality. High mortality was also described in a knockout model for SerpinB2, which spontaneously acquired the Irs1 S57X mutation, resulting in the total loss of Irs1 protein expression (WESTRICK et al., 2010). Irs1-deficient models generated by targeted disruption do not exhibit significative reduction in survival (ARAKI et al., 1994; TAMEMOTO et al., 1994). These data suggest that the elevated mortality observed in this study may not directly be related to Irs1 deficiency. Based on these findings, further hematologic studies of B6.129S2-Irs1smla/J mice should be focused on fetal hematopoiesis. Disclosures Traina: AbbVie: Other: Principal Investigator for Protocol Number M15-656 ; University of São Paulo at Ribeirão Preto Medical School: Current Employment.

Immune asynchrony in COVID-19 pathogenesis and potential immunotherapies

The outbreak of coronavirus disease 2019 (COVID-19) is an unprecedented global health crisis. Tissue and peripheral blood analysis indicate profound, aberrant myeloid cell activation, cytokine storm, and lymphopenia, with unknown immunopathological mechanisms. Spatiotemporal control of the quality and quantity of the antiviral immune responses involves synchronized cellular and molecular cascades and cross-talk between innate and adaptive immunity. Dysregulated responses in immunity, such as at the stages of immune sensing, alarming, polarization, and resolution, may contribute to disease pathology. Herein, we approach SARS-CoV-2 through an immunomodulatory lens, discussing possible mechanisms of the asynchronized antiviral immune response and proposing potential therapeutic strategies to correct the dysregulation.

Analysis of red blood cell parameters in dogs with various stages of degenerative mitral valve disease

IntroductionAlthough peripheral blood analysis has become increasingly automated, microscopy is the only available method for the diagnosis of anisocytosis and poikilocytosis. The aims of the study were to compare RBC volume data obtained with two different analysers and by manual assessment of smears and to compare this data between dogs in various stages of heart failure secondary to degenerative mitral valvular (DMV) disease. The impact of diuretic administration on RBC morphology was also assessed.Material and MethodsSixty-eight dogs, 56 in different stages of DMV disease and 12 as healthy controls, were studied. Impedance and flow cytometry haematological analyses were performed for each animal. Additionally, two smears were prepared for manual analysis. RBC structure, staining, and size differences were recorded.ResultsThere were no significant differences between the blood morphological parameters assessed using haematological analysers nor between dogs receiving diuretic treatment and those not treated. Based on the manual smear, significantly higher erythrocyte anisocytosis was observed in the dogs with symptomatic DMV disease than in the control group.ConclusionHaematological analysers based on impedance and flow cytometry provide reliable and comparable morphological results in dogs with heart failure. However, microscopic assessment of blood smears is a more reliable tool to detect erythrocyte anisocytosis.

An analysis of adaptive reactions in healthy subjects who have prolonged contact with tuberculosis patients

Prolonged contact of healthy subjects with Mycobacterium tuberculosis can change their blood formula and immune status, thus reflecting adaptive reactions to constant antigenic load. The peripheral blood analysis of health care workers (HCWs) in a tuberculosis hospital demonstrates changes in cell populations which prevent development of tuberculosis, in particular, CD4+ Т cells and CD3+ Т cells. It is shown that the number of the memory CD4+ Т cells specific to M.tuberculosis antigens which produce interferon gamma depends on the duration of work contact with tuberculosis patients. The HCWs blood characteristics usage as a control for tuberculosis patients is discussed.

Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1–targeted therapy in lung cancer patients

Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients’ responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non–small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1–targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients’ responses to PD-1–targeted therapies.

Podocalyxin Negatively Regulates Stress Myelopoiesis, Directly Binds Rap-1A and Modulates Its G-CSF-Induced Activity

Podocalyxin (PodxL) is a CD34 family member previously identified to mark hematopoietic stem cells (HSCs) and other progenitor cells. Previously, we discovered PodxL as a potent erythropoietin (EPO) response gene and demonstrated to promote egression of immature reticulocytes from bone marrow into circulation. PodxL is upregulated in several cancers, including myeloid and lymphoid leukemia. Herein, we aim to define the functional role of PodxL in hematopoiesis - specifically myelopoiesis - by employing conditional PodxL knock out (KO) mouse models. Hematopoietic-specific deletion was achieved using Cre mice with a Vav1 driver and myeloid-specific deletion was achieved with Lyzm2 - Cre driver. We confirmed the deletion of exons 3-7 at the gene, transcript and protein levels using PCR, RT-qPCR and western blotting, respectively. Peripheral blood analysis revealed no difference in blood cell lineages for either KO mouse strain. At steady state, colony forming unit-granulocyte/macrophage (CFU-GM) assay also showed no difference between the KO strains and wild type. In order to examine the functional role of PodxL during stress myelopoiesis, PodxL-/- ; Vav1-Cre mice were treated with 5-Fluorouracil (5FU), a chemotherapeutic agent induces myeloablation. Notably, during rebound of neutrophils, the PodxL-/- ; Vav1-Cre mice showed a sharp increase in neutrophil counts at day 12.5, which at later time points reverted to normal levels comparable to wild type mice. Previously, our in silico analyses combined with outcomes from truncated EpoR knock-in alleles had revealed that PodxL is a potential STAT5 transcriptional target. Here, we tested if G-CSF induces PodxL expression in hematopoietic progenitors. In vivo, G-CSF significantly induced PodxL expression four fold. We then tested the role of PodxL in G-CSF induced neutrophil formation in vivo. Both KO strains (Podxl-/-;Vav1-Cre and Podxl-/-;Lyzm2-Cre) and wild type were treated with G-CSF (125ug/kg/day) for 5 days. Peripheral blood analysis revealed increased neutrophil and monocyte levels in the PodxL-/-;Vav1-Cremice. In order to then determine a possible role of PodxL at the progenitor level, CFU-GM assays were performed. PodxL-/- ; lyzm2-Cre mice had increased colony forming capabilities but there was no difference in PodxL-/-;Vav1-Cre mice compared to wild type. Our results imply that PodxL is playing a negative regulatory role in stress myelopoiesis. Interestingly, the deletion of PodxL in hematopoietic progenitors (Vav1-Cre) resulted in enhanced migration of neutrophils, whereas deletion of PodxL in myeloid compartment (Lyzm2-Cre) resulted in decreased neutrophil migration. This may be in part due to a compensatory effect by CD34 in the hematopoietic compartment. To dissect the molecular mechanism of PodxL during stress myelopoiesis, upon in vivo G-CSF treatment, bone marrow derived hematopoietic progenitors were isolated and PodxL protein was immunoprecipitated. LC-MS/MS proteomic analysis was performed to identify the interacting partners with PodxL. Rap-1A, a small GTPase and member of the RAS family, was among the top interacting proteins. Rap-1A has been shown to promote adhesion and migration of myeloid cells. The association of PodxL with Rap-1A was further confirmed in hematopoietic progenitors by immunoprecipitation and western blotting. To determine if the interaction of PodxL directly regulates Rap-1A activity, a GTP-Rap-1A activity assay was performed in response to G-CSF, GM-CSF and IL-3. Rap-1A activity was significantly elevated in hematopoietic progenitors upon G-CSF treatment in PodxL-/-:Vav1-Cre mice compared to wild type, followed by IL3; however, GM-CSF did not affect Rap-1A activity. In conclusion, our results indicate an important functional role for PodxL in stress myelopoiesis, a function likely mediated via Rap-1A. A complete understanding of the PodxL/Rap-1A axis may reveal important molecular insights into G-CSF-induced mobilization of neutrophils and provide mechanistic understanding into the pathological role of PodxL in aggressive cancers, including leukemia, which in turn may facilitate identification of novel therapeutic targets in PodxL associated cancers. Disclosures No relevant conflicts of interest to declare.

Peripheral Blood Analysis By Flow Cytometry for the Diagnosis of Type I Chronic Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a clonal disorder with a diagnostic challenge encountered frequently in routine hematology consultations, especially when few dysplastic features in bone marrow and/or no cytogenetics or molecular alterations are detected. Thus, easily applicable parameters in peripheral blood analysis are warranted to aid diagnosis. We studied the utility of flow cytometry analysis of peripheral blood (PB) for the diagnosis of CMML type I. PB samples from 16 CMML type I, age- matched normal (n=10) and reactive monocytosis (>1 x109/L) cases (n=9) were studied. A large panel of monoclonal antibodies was used ( FITC:CD36, CD35, CD15 and CD4, CD16,PCP5.5:CD34, CD163, CD13, PC7: CD117, CD38, CD2,CD7, APC: CD300, CD33, CD11B , CD11C ,CD56 , APCH7: CD14 , HLADR V450, CD64 PE and CD45 OC515) in an 8-color combination, including a backbone of 4 colors in all tubes. At least 1x 10(6) events were acquired by FACSCanto II (BD Biosciences, San Jose, USA). The data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytes were selected on the automated population separator plot and confirmed by the expression of CD64 and HLADR. Lymphocytes were used as the internal control. As expected, CMML type 1 patients showed higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (p=0.001). CD64bright and CD163 expression were equally valid to identify monocytic population in all three groups (correlation r2 = 1). The majority of the monocytic population belonged to the late stages of maturation, being CD64+CD35+CD14+CD300+ in CMML as well as reactive and normal cases (p=0.14). CMML and reactive cases showed significantly lower levels of early stages of monocytic maturation (CD64+CD35-/+ CD14-CD300-) than normal controls ( p=0.04). The majority of type I CMML (69%) had more than 95% "classical" monocytes (CD14+CD16-) in the monocytic compartment, whereas only 22% of reactive cases and none of the normal cases had such condition (p=0.000). Statistically significant differences among the three groups were also found for "intermediate" (CD14+CD16+) and "non-classical" (CD14lowCD16+) monocytic populations, with the normal cases having higher percentage of "non classical" monocytes, and reactive cases higher "intermediate" (CD14+CD16+) monocytes (12% vs. 5% vs. 1%; -p <0.000-, and 2.6% vs. 3.7% vs. 1.4%; -p=0.01- for "non classical" and "intermediate" monocytes in normal, reactive and CMML cases, respectively). As for the aberrant antigenic expression, CD56 was most frequently expressed in monocytes of CMML (75% CMML with >20% CD56 positivity) followed by CD2 (31%) and CD7 (6%). CD56 could also be detected in reactive (22%) and normal (7%) cases, whereas CD2 and CD7 were always negative. Though useful, CD56 expression alone could overestimate the diagnosis of CMML type I whereas the utility of CD7 expression was doubtful due to its low frequency in CMMLs. When the pattern of distribution of "classical", "intermediate" and "non classical" monocytes together with the aberrant expression of CD56 and CD2 were considered, only CMML cases met both criteria (>95% of "classical" monocytes among the monocytic compartment in PB, and having an aberrant expression of CD56 or CD2 in >20% of monocytic cells), excluding all the reactive monocytosis and normal cases, although not all CMML (62%) met both criteria. Concerning the antigens analyzed on the monocytes, only CD163 and CD11b had a higher expression in CMML than in reactive or normal cases (p=0.01 & p=0.05). CD64, CD15, CD4, and CD45 were similarly expressed in CMML and reactive, but higher than in normal cases (p<0.03). CD35 was highest (p=0.01) and HLA-DR (p=0.01) and CD33 (p=0.02) lowest in reactive cases, compared to CMML or normal cases. No significant difference was found for the remaining antigens. In spite of these findings, in our opinion, its prospective utility to diagnose CMML or to distinguish it from reactive monocitosis was doubtful. Flow cytometry analysis can be useful to diagnose CMML type I in the routine laboratory by employing a few easily applicable parameters in PB (the pattern of distribution of "classical", "intermediate" and "non classical" monocytes together with the aberrant expression of CD56 or CD2), distinguishing it from reactive monocytosis, and helping to identify patients in whom other studies, as bone marrow assessment, should be performed. Disclosures Diez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Puig:The Binding Site: Consultancy; Janssen: Consultancy.

Analysis of the Expression and Polymorphism ofAPOE, HSP, BDNF,andGRIN2BGenes Associated with the Neurodegeneration Process in the Pathogenesis of Primary Open Angle Glaucoma

Glaucoma is characterized by optic neuropathy of the RGC or retinal nerve fiber. The aim of this study was to evaluate a relationship between the neurodegenerative genes’ polymorphisms of theAPOE(rs449647),BDNF(rs2030324),GRIN2B(rs3764028), andHSP70-1(rs1043618) and the occurrence risk of POAG and to investigate its effect on allele-specific gene expression. Genomic DNA was extracted from peripheral blood. Analysis of the genes’ polymorphisms was performed using PCR-RFLP. The level of mRNA expression was determined by QRT-PCR. We showed a statistically significant association ofBDNFandAPOEgenes’ polymorphisms with a risk of POAG occurrence. There was a statistically significant association of the rs2030324 polymorphism with progression of POAG based on cup disc ratio value and rs1043618 polymorphism based on nerve fiber index and rim area. Furthermore, we found that meanHSP70-1mRNA expression was significantly lower in the case of individuals with the G/G genotype than in the case of minor allele carriers, that is, G/C and C/C. We also found thatBDNFandHSP70-1expression level are associated with the progression of POAG based on rim area value. In conclusion, our results suggest thatBDNF,APOE, andHSP70-1genes might be associated with a risk of POAG occurrence in the Polish population.