Immature Phenotype Research Articles

Inflammation perturbs NK cell phenotype and function in chronic myeloid leukemia (CML)

Abstract Natural killer (NK) cells have the capacity to recognize and clear cancer cells by releasing cytotoxic granules. In fact, NK cells’ mature phenotype and abundance are associated with prolonged treatment-free remission in chronic myeloid leukemia (CML). However, NK cell anti-leukemic activity is suppressed during the disease, and the environmental triggers of this impairment are not fully characterized. Given the role of inflammatory cytokines in the progression of myeloid malignancies, we define their impact on NK cell function in a BCR-ABL1 +CML chimeric mouse model. Consistent with clinical observations, NK cells are reduced in counts in leukemic mice, display immature phenotype, low degranulation rate, and altered expression of activating and inhibitory receptors. Single-cell RNAseq analysis confirmed these observations at the transcriptional level, and revealed enrichment in genes and pathways associated with inflammatory cytokine response (e.g., GM-CSF, LTα/β, XCL1, IL-6st, IL-2Ra, SOCS1/2/3, CIS; IL-6/STAT3 and TNFa/NF-kB pathways) in CML-exposed NK cells. To validate the clinical relevance of our findings, we characterized NK cells from CML patients. As expected, NK cell frequencies are reduced in our cohort and display diminished K562-targeted degranulation. Mirroring mouse scRNAseq data, patient NK cells possess a pro-inflammatory gene signature with the activation of JAK-STAT, TNFa/NF-kB, and PI3K-Akt signaling pathways. As this is likely triggered by leukemic cytokines, we found that leukemic serum dampens ex vivo NK cell degranulation capacity. Thus, we speculate that NK cells are sensitive to CML-associated cytokines and that inflammation represents an optimal target for NK-boosting immunotherapies.

The miR-141/200c-STAT4 Axis Contributes to Leukemogenesis by Enhancing Cell Proliferation in T-PLL.

T-prolymphocytic leukemia (T-PLL) is a rare and mature T-cell malignancy with characteristic chemotherapy-refractory behavior and a poor prognosis. Molecular concepts of disease development have been restricted to protein-coding genes. Recent global microRNA (miR) expression profiles revealed miR-141-3p and miR-200c-3p (miR-141/200c) as two of the highest differentially expressed miRs in T-PLL cells versus healthy donor-derived T cells. Furthermore, miR-141/200c expression separates T-PLL cases into two subgroups with high and low expression, respectively. Evaluating the potential pro-oncogenic function of miR-141/200c deregulation, we discovered accelerated proliferation and reduced stress-induced cell death induction upon stable miR-141/200c overexpression in mature T-cell leukemia/lymphoma lines. We further characterized a miR-141/200c-specific transcriptome involving the altered expression of genes associated with enhanced cell cycle transition, impaired DNA damage responses, and augmented survival signaling pathways. Among those genes, we identified STAT4 as a potential miR-141/200c target. Low STAT4 expression (in the absence of miR-141/200c upregulation) was associated with an immature phenotype of primary T-PLL cells as well as with a shortened overall survival of T-PLL patients. Overall, we demonstrate an aberrant miR-141/200c-STAT4 axis, showing for the first time the potential pathogenetic implications of a miR cluster, as well as of STAT4, in the leukemogenesis of this orphan disease.

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes

BackgroundAlthough human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking.MethodsiPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs).ResultsWe showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction.ConclusionsOverexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Abstract 1851: Mechanisms underlying focused ultrasound-induced immunogenicity in melanoma

Abstract Advanced melanoma is an aggressive skin cancer characterized by low survival rates. The generation of a robust anti-tumor response relies on dendritic cells (DCs) to process tumor antigens, migrate to draining lymph nodes, and activate T-cells. Within the tumor microenvironment, immunosuppressive factors impede DC differentiation and activation, and the subsequent immature DC phenotype is a major mechanism underlying the ineffective T-cell response in melanoma patients. We have shown that low-intensity focused ultrasound (LOFU), a novel therapy that delivers mechanical and thermal stresses locally at the tumor site, may enhance DC function in a B16 murine melanoma model. We hypothesize that LOFU enhances the dendritic cell-mediated anti-tumor response by inducing changes in tumor cell and dendritic cell autophagy and increasing pro-immunogenic factors in the TME. Autophagy is a lysosome-dependent process of cellular content degradation that has previously been implicated in the regulation of multivesicular body formation/degradation, and thus in the secretion of extracellular vesicles. Autophagy may subsequently modify the cargo of tumor-derived extracellular vesicles, such as exosomes, in a pro-immunogenic manner. We have found that LOFU treatment is associated with an increase in autophagic flux in melanoma cells and have identified at least 82 proteins within melanoma-derived extracellular vesicles that are significantly enriched or reduced with LOFU treatment, with enriched proteins partaking in essential processes including antigen processing, intracellular trafficking, and cellular metabolism. DCs pulsed with vesicles from LOFU-treated melanoma cells have shown enhanced activation of both CD4+ and CD8+ T-cell activation, suggesting that the LOFU-induced increase in tumor cell autophagy and modification of vesicular cargo may promote a DC-activated anti-tumor antigen activation response. Characterization of other mechanisms by which LOFU may enhance DC function, such as direct changes in DC autophagy, is critical to elucidate DC-mediated pathways involved in the anti-tumor response. Further understanding of DC involvement in the anti-tumor response will provide novel targets for the development of immunotherapies to improve survival and quality of life in advanced melanoma patients. Citation Format: Sandra Pelka, Sanmay Bandyopadhyay, Sanjay Pandey, Chandan Guha, Fernando Macian. Mechanisms underlying focused ultrasound-induced immunogenicity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1851.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Exvivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1β condition murine and human MSCs invitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

Increased levels of immature and activated low density granulocytes and altered degradation of neutrophil extracellular traps in granulomatosis with polyangiitis.

Granulomatosis with Polyangiitis (GPA) is a small vessel vasculitis typically associated with release of neutrophil extracellular traps (NETs) by activated neutrophils. In this study, we further aimed to investigate the contributions of neutrophils and NETs to the complex disease pathogenesis. We characterized the phenotype of neutrophils and their capacity to induce NETs. In addition, the level of circulating NETs, determined by neutrophil elastase/DNA complexes, and the capacity of patient sera to degrade NETs were investigated from blood samples of 12 GPA patients, 21 patients with systemic lupus erythematosus (SLE) and 21 healthy donors (HD). We found that GPA patients had significantly increased levels of low-density granulocytes (LDGs) compared to HD, which displayed an activated and more immature phenotype. While the propensity of normal-density granulocytes to release NETs and the levels of circulating NETs were not significantly different from HD, patient sera from GPA patients degraded NETs less effectively, which weakly correlated with markers of disease activity. In conclusion, increased levels of immature and activated LDGs and altered degradation of circulating NETs may contribute to pathogenesis of GPA, potentially by providing a source of autoantigens that trigger or further enhance autoimmune responses.

Single-cell analysis of human nasal mucosal IgE antibody secreting cells reveals a newly minted phenotype

Immunoglobulin (Ig) E is central to the pathogenesis of allergic conditions, including allergic fungal rhinosinusitis. However, little is known about IgE antibody secreting cells (ASCs). We performed single-cell RNA sequencing from cluster of differentiation (CD)19+ and CD19- ASCs of nasal polyps from patients with allergic fungal rhinosinusitis (n=3). Nasal polyps were highly enriched in CD19+ ASCs. Class-switched IgG and IgA ASCs were dominant (95.8%), whereas IgE ASCs were rare (2%) and found only in the CD19+ compartment. Through Ig gene repertoire analysis, IgE ASCs shared clones with IgD-CD27- "double-negative" B cells, IgD+CD27+ unswitched memory B cells, and IgD-CD27+ switched memory B cells, suggesting ontogeny from both IgD+ and memory B cells. Transcriptionally, mucosal IgE ASCs upregulate pathways related to antigen presentation, chemotaxis, B cell receptor stimulation, and survival compared with non-IgE ASCs. Additionally, IgE ASCs have a higher expression of genes encoding lysosomal-associated protein transmembrane 5 (LAPTM5) and CD23, as well as upregulation of CD74 (receptor for macrophage inhibitory factor), store-operated Calcium entry-associated regulatory factor (SARAF), and B cell activating factor receptor (BAFFR), which resemble an early minted ASC phenotype. Overall, these findings reinforce the paradigm that human ex vivo mucosal IgE ASCs have a more immature plasma cell phenotype than other class-switched mucosal ASCs and suggest unique functional roles for mucosal IgE ASCs in concert with Ig secretion.

Recombinant Domain of Flagellin Promotes In Vitro a Chemotactic Inflammatory Profile in Human Immune Cells Independently of a Dendritic Cell Phenotype

Flagellin is the major component of the flagellum in gram-positive and -negative bacteria and is also the ligand for the Toll-like receptor 5 (TLR5). The activation of TLR5 promotes the expression of proinflammatory cytokines and chemokines and the subsequent activation of T cells. This study evaluated a recombinant domain from the amino-terminus D1 domain (rND1) of flagellin from Vibrio anguillarum, a fish pathogen, as an immunomodulator in human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs). We demonstrated that rND1 induced an upregulation of proinflammatory cytokines in PBMCs, characterized at the transcriptional level by an expression peak of 220-fold for IL-1β, 20-fold for IL-8, and 65-fold for TNF-α. In addition, at the protein level, 29 cytokines and chemokines were evaluated in the supernatant and were correlated with a chemotactic signature. MoDCs treated with rND1 showed low levels of co-stimulatory and HLA-DR molecules and kept an immature phenotype with a decreased phagocytosis of dextran. We probed that rND1 from a non-human pathogen promotes modulation in human cells, and it may be considered for further studies in adjuvant therapies based on pathogen-associated patterns (PAMPs).

Immunomodulatory effect of granulocyte colony-stimulating factor in repeated implantation failures in embryo transfer programs

The objective: to determine the effectiveness of intrauterine administration of granulocyte colony-stimulating factor depending on endometrium immune phenotype in patients with repeated implantation failures in embryo transfer programs.Material and methods. Endometrial biopsy during the implantation window in an artificial cycle before and (in some patients) after intrauterine administration of granulocyte colony-stimulating factor (G-CSF) was performed in 42 patients with repeated implantation failures and gestational carriers (20 women) as a control group. Flow cytometry was used to count classes and subclasses of endometrial lymphocytes in endometrial tissue samples.Results. The population of endometrial immune cells in patients with repeated implantation failures and fertile women was compared. Pregnancy and live birth rates depending on endometrium immune phenotype in patients with repeated implantation failures after intrauterine administration of granulocyte colony-stimulating factor in embryo transfer program were determined and the effect of intrauterine administration of granulocyte colony-stimulating factor on endometrial immune phenotype was evaluated.High expression of HLA-DR and CD16 on uterine natural killers is reliably associated with successful implantation after intrauterine administration of granulocyte colony-stimulating factor in an embryo transfer program. It was established that in the group with an immature immune endometrial phenotype the frequency of pregnancy (53.8 %) and the frequency of live births (53.8 %) were twice higher compared to the rest of the patients with other variants or the absence of changes in the immune profile (pregnancy rate and frequency live birth rate – 26.9 %).Conclusions. One-third of patients with repeated implantation failures in genetically tested embryos transfer programs have a unique immune status of endometrium characterized by high expression of HLA-DR and CD16 on uterine natural killers. Intrauterine administration of granulocyte colony-stimulating factor leads to decrease of HLA-DR and CD16 expression on uterine natural killers and promotes successful implantation in embryo transfer program in this group of patients.

Simulated Microgravity Disrupts Nuclear Factor κB Signaling and Impairs Murine Dendritic Cell Phenotype and Function.

During spaceflights, astronauts face different forms of stress (e.g., socio-environmental and gravity stresses) that impact physiological functions and particularly the immune system. In this context, little is known about the effect of such stress on dendritic cells (DCs). First, we showed that hypergravity, but not chronic ultra-mild stress, a socio-environmental stress, induced a less mature phenotype characterized by a decreased expression of MHCII and co-stimulatory molecules. Next, using the random positioning machine (RPM), we studied the direct effects of simulated microgravity on either splenic DCs or Flt-3L-differentiated bone marrow dendritic cells (BMDCs). Simulated microgravity was found to reduce the BM-conventional DC (cDC) and splenic cDC activation/maturation phenotype. Consistent with this, BMDCs displayed a decreased production of pro-inflammatory cytokines when exposed to microgravity compared to the normogravity condition. The induction of a more immature phenotype in microgravity than in control DCs correlated with an alteration of the NFκB signaling pathway. Since the DC phenotype is closely linked to their function, we studied the effects of microgravity on DCs and found that microgravity impaired their ability to induce naïve CD4 T cell survival, proliferation, and polarization. Thus, a deregulation of DC function is likely to induce immune deregulation, which could explain the reduced efficiency of astronauts' immune response.

STAT3 promotes a youthful epigenetic state in articular chondrocytes.

Epigenetic mechanisms guiding articular cartilage regeneration and age-related disease such as osteoarthritis (OA) are poorly understood. STAT3 is a critical age-patterned transcription factor highly active in fetal and OA chondrocytes, but the context-specific role of STAT3 in regulating the epigenome of cartilage cells remain elusive. In this study, DNA methylation profiling was performed across human chondrocyte ontogeny to build an epigenetic clock and establish an association between CpG methylation and human chondrocyte age. Exposure of adult chondrocytes to a small molecule STAT3 agonist decreased DNA methylation, while genetic ablation of STAT3 in fetal chondrocytes induced global hypermethylation. CUT&RUN assay and subsequent transcriptional validation revealed DNA methyltransferase 3 beta (DNMT3B) as one of the putative STAT3 targets in chondrocyte development and OA. Functional assessment of human OA chondrocytes showed the acquisition of progenitor-like immature phenotype by a significant subset of cells. Finally, conditional deletion of Stat3 in cartilage cells increased DNMT3B expression in articular chondrocytes in the knee joint in vivo and resulted in a more prominent OA progression in a post-traumatic OA (PTOA) mouse model induced by destabilization of the medial meniscus (DMM). Taken together these data reveal a novel role for STAT3 in regulating DNA methylation in cartilage development and disease. Our findings also suggest that elevated levels of active STAT3 in OA chondrocytes may indicate an intrinsic attempt of the tissue to regenerate by promoting a progenitor-like phenotype. However, it is likely that chronic activation of this pathway, induced by IL-6 cytokines, is detrimental and leads to tissue degeneration.

Alternative Chemotherapies: Angiotensin-Converting Enzyme Inhibitors Reduce Myeloid-Derived Suppressor Cells to Benefit Older Patients with Colorectal Cancer.

Older individuals are more likely to develop solid cancers, but at the same time are more sensitive to the side effects of chemotherapy. In addition, older adults are more likely to present with chronic diseases (comorbidities) and immunosenescence that may decrease immunosurveillance against cancer. Clinical outcomes for the older patient with cancer are different from the younger patient and require different research and treatment approaches. Thus, alternative therapeutic approaches tailored specifically to the older patients are required. Colorectal cancer (CRC) has a high incidence in older individuals and is the third leading cause of cancer death globally. Anti-hypertensives are used by a large proportion of older patients and some studies have pointed to a positive impact of angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB) on CRC outcomes. As we have previously shown in a mouse model, lung metastases express ACE and contain many infiltrating myeloid-derived suppressor cells (MDSC); particularly high levels of MDSC are also present in the blood of older patients with CRC and other cancers, and are associated with disease severity. In this Commentary, we hypothesize that one mechanism responsible for the positive impact of ACEi or ARB on the outcome of CRC is the modulation of myeloid cells contributing to their maturation to non-suppressive neutrophils/monocytes and diverting them away from retaining an immature MDSC phenotype.

Secretomes derived from osteogenically differentiated jaw periosteal cells inhibit phenotypic and functional maturation of CD14+ monocyte-derived dendritic cells.

The jaw periosteal tissue is generally recognized as a suitable source for the isolation of mesenchymal stem cells (MSCs). In previous studies we showed evidence that two- and three-dimensionally cultured jaw periosteum-derived MSCs (JPCs) are able to induce a more immature phenotype of dendritic cells (DCs). To further expand our knowledge of JPCs' immunoregulative function, we investigated the effects of JPC secretomes derived from undifferentiated (CO) or osteogenically differentiated cells (treated with or without dexamethasone: OB+/-D) on CD14+ monocyte-derived DCs (MoDCs). We detected a remarkably reduced formation of MoDC homotypic clusters under the influence of secretomes from osteogenically induced JPCs. Further, significantly decreased numbers of CD83+ cells, up-regulated CD209 and down-regulated CD80, CD86 and CD197 expression levels were detected on the surface of MoDCs. Whereas secretomes from JPCs osteogenically stimulated with dexamethasone significantly enhanced FITC-dextran uptake capacity of MoDCs, the increase by secretomes of JPCs treated without dexamethasone did not reach significance. The analysis of mixed lymphocyte reactions revealed that OB+/-D secretomes were able to significantly reduce the numbers of proliferating CD14- peripheral blood mononuclear cells (PBMCs) and of proliferating CD4+ T cells. The OB-D secretome significantly promoted the expansion of regulatory CD25+ T cells. Regarding gene expression of MoDCs, remarkably up-regulated mRNA expression of CD209, HLA-DRA, CSF3, IL10 and IL8 was detected when DCs were cultured in the presence of OB+/-D secretomes. At the same time, secretomes seemed to have an impact in the down-regulation of IFNγ and IL12B gene expression. At protein level, OB+/-D secretomes significantly up-regulated IL-10 and IDO (indoleamine-pyrrole 2,3-dioxygenase) levels whereas IL-12/IL-23p40 levels were down-regulated in supernatants of MoDCs when cultured under the presence of OB+/-D secretomes. Taken together, while secretomes from untreated JPCs had only little effects on the process of maturation of MoDCs, secretomes derived from osteogenically induced JPCs were able to inhibit the phenotypic and functional maturation of MoDCs.

Therapeutic Targeting of MERTK and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL. The early T-precursor (ETP-ALL) subset is characterized by an immature T-cell phenotype, chemoresistance, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in half of T-ALLs, particularly those with an immature T-cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is essential for ETP-ALL cell survival. Here, we show that MERTK and BCL-2 mRNA and protein are preferentially expressed in ETP-ALL patient samples. The dual MERTK/FLT3 inhibitor MRX-2843 decreased MERTK activation and downstream signaling, inhibited cell expansion, and induced cell death in ETP-ALL cell lines. Further, 54% (21/39) of primary T-ALL patient samples were sensitive to MERTK inhibition. Treatment with MRX-2843 significantly reduced leukemia burden and prolonged survival in cell-line-derived T-ALL and ETP-ALL xenograft models. In a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 markedly reduced peripheral blood leukemia and spleen weight compared to vehicle-treated mice and prolonged survival. MRX-2843 also synergized with venetoclax to provide enhanced anti-leukemia activity in ETP-ALL cell cultures, with a dose ratio of 1:20 MRX-2843:venetoclax providing optimal synergy. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL and provide rationale for clinical development. MRX-2843 monotherapy is currently being tested in patients with relapsed leukemia (NCT04872478). Further, our data indicate that combined MERTK and BCL-2 inhibition may be particularly effective for treatment of ETP-ALL.

Oral commensal bacterial flora is responsible for peripheral differentiation of neutrophils in the oral mucosa in the steady state

ObjectivesCommensal bacteria in the host body play a fundamental role in the differentiation and maintenance of the immune system. Studies on intestinal immunity have revealed that, under steady-state conditions, microflora have an important role in the maintenance of health. However, the role of oral commensal bacteria on the oral immune system is still unclear. Here, we clarify the interactions between commensal bacteria and the oral mucosal immune system under steady-state conditions. MethodsWe used germ-free mice that had never been exposed to bacteria and conventional mice grown with normal bacterial flora. Oral cells were isolated from the oral mucosa, stained with specific antibodies, and analyzed by flow cytometry. For the detection of myeloperoxidase and intracellular cytokines, oral cells were stimulated with N-formyl-methionine-leucyl-phenylalanine and phorbol 12-myristate 13-acetate/ionomycin, respectively. ResultsWe found that the oral mucosa harbored more neutrophils in germ-free mice than in conventional mice. However, the majority of neutrophils in the germ-free oral mucosa exhibited an immature phenotype. Other immune cells, including macrophages, T cells, and B cells, in the oral mucosa of germ-free mice showed similar differentiation to those in conventional mice. These results indicate that in the steady-state oral mucosa, the normal commensal flora promote the peripheral differentiation of neutrophils. ConclusionsThe presence of commensal flora is critical for the development of adequate immune system in the oral mucosa.

Accelerated maturation of ARPE‐19 cells for the translational assessment of gene therapy

AbstractPurpose: Retinal pigment epithelium (RPE) modulates the exchange of metabolite products to and from the retina, and are paramount to maintain retinal function. The human RPE cell‐line ARPE‐19 is commercially available and widely used as an alternative to primary RPE. However, these cells undergo epithelial‐mesenchymal transition (EMT) losing many features of primary RPE such as polarity, pigmentation, and expression of RPE signature markers. We aimed to determine whether a combination of RPE‐specific laminin (LN) coating and media supplementation with nicotinamide (NAM) could improve ARPE‐19 expression of genes and proteins, along with morphological phenotype resembling mature RPE to improve assessment of RPE‐specific gene therapy strategies.Methods: ARPE‐19 cells were propagated on tissue culture plastic with or without NAM supplementation and/or coated with human recombinant LN‐521. Cell morphology was evaluated by phase microscopy. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno associated virus (AAV)1 or AAV2, VMD2‐driven GFP, were assessed at 2‐ and 4‐weeks post‐plating in the different culturing condition with low multiplicity of infection.Results: The combination of LN‐521 coating with NAM supplementation promoted cytoskeletal and tight junction proteins reorganization, and expression of differentiation markers such as VMD2, RPE65 and PDGFRB. In addition, the combination reduced the expression of the EMT markers, TNFα and TGF‐β1; and NCAM1, an immature phenotype RPE marker. Both AAV1‐ and AAV2‐VMD2‐GFP expression was detected from 2‐weeks and improved at 4‐weeks post‐plating.Conclusions: Our study demonstrates that the combination of NAM with LN‐521 accelerated the kinetics of expression of mature RPE signature genes, reduced expression of EMT genes, accompanied with improved epithelial cell morphology and cytoskeletal organization, bringing the dedifferentiated ARPE‐19 closer to their in vivo phenotype. Moreover, viral‐mediated RPE‐specific gene expression demonstrates the amenability of the combinatorial cultures for translational assessment of RPE gene therapies.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated with Expansion and Maturation of NK Cells

ABSTRACT Background. Hodgkin Lymphoma (HL) Hodgkin Lymphoma (HL) is a B-Cell neoplasia characterized by a high degree of response to chemotherapy and an overall favourable outcome in responding patients. Despite the efficacy of first line therapy, however, about 30% of patients affected by HL are either refractory or eventually relapse (R/R) after first or second line therapy followed by autologous hemopoietic stem cell transplantation (ASCT). Immune checkpoint inhibitors (CI) have demonstrated clinical activity in HL patients relapsing after ASCT, although only 20% complete response (CR) rate are usually observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Aims. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI) and to investigate the lymphocyte subpopulation involved in the mechanism of response to CI in HL. Results. Seventeen patients with relapse/refractory (R/R) HL (median age 29 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 12 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 5 responding patients received ALI alone, as a control cohort. Most patients showed CMV seropositivity at the time of enrolment, without signs of active infection (positive anti-CMV IgG, negative IgM and undetectable CMV DNA). No adverse events were recorded, specifically, no immune-related toxicity was observed and none of the patients showed CMV reactivation. All patients receiving ALI + CI (treated patients) achieved negative PET scan CR and 11/12 are alive and disease-free after a median follow-up of 33 months. The only relapse occured 23 months after last ALI. Six of the responding patients underwent subsequent allogeneic SCT in a CR status and 1 patients are currently waiting for transplantation. Two of the patients in the control arm relapsed after a median of 6 months (4-8 months) Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients (Fig.1). In detail, different patterns of NK cell development could be identified. Indeed, starting from one month after transplantation, in the group of patients undergoing ALI plus nivolumab, a relevant fraction of NK cells displayed a mature phenotype characterized by a CD56dimCD16bright expression (Fig. 1). On the contrary, in control patients, NK cells were characterized by a more immature phenotype (high frequencies of CD56brightCD16neg/dim NK cells) even at late time points (three months) after transplantation (Fig. 1). This event occurred in all the treated (ALI + nivolumab) patients analyzed (Fig. 1). Finally, PB from patients undergoing ALI + nivolumab was enriched in unconventional CD56dimCD16dim NK cells (Fig. 1). One month after transplantation, CD94/NKG2A+ KIR- NK cells were largely predominant in all patients, whereas CD94/NKG2A- KIR+ NK cells were present in low proportions. An increase of CD94/NKG2A- KIR+ subset occurred primarily in treated patients starting from t2. At t3, the significant increase of CD94/NKG2A- KIR+ NK cells occurred in the treated group but not in the control group . This event occurred in all the treated (ALI + nivolumab) patients analyzed, regardless of their serological status for HCMV, but was more evident in treated patients who were seropositive for HCMV. Remarkably, PB from HCMV seropositive patients undergoing ALI + nivolumab became enriched over time in differentiated NK cells characterized by high expression of NKG2C receptor (the activating counterpart of NKG2A), as compared to HCMV+ control patients. This subset represents the so-called "adaptive" NK cell population that usually increases during HCMV infection/reactivation. In this case, however, this event occurred in the absence of HCMV reactivation, and only in HCMV seropositive patients treated with ALI + nivolumab. Conclusions. Our data show anti-tumour activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favour the expansion of the "adaptive" NK cell compartment, especially in patients with CMV seropositivity, in the absence of CMV reactivation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Proteomic Interrogation of FLT3-ITD Leukemia Compartment Revealsdistinct Leukemia Cell Populations Resistant to Quizartinib, Decitabine and Venetoclax

Triplet therapy (TT) combining Quizartinib(Quiz), Decitabine(Dec) and Venetoclax(Ven) is highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates (Yilmaz M, ASCO 2022).Signaling mutations, particularly those involving RAS/MAPK pathway, have emerged as key contributors to primary and secondary resistance to TT. Lack or loss of response to TT is not limited to presence or emergence of clones with signaling mutations and disease relapse occurs in FLT3-ITD AML patients with a variety of other mutations. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by therapy pressure, and aid in identifying proteomic correlates of response and therapeutic vulnerabilities. To this end, we performed comprehensive single-cell proteomic analysis of serially collected samples (n:162) from patients treated with TT (n:14) and doublet (n:10, DEC+Quiz) therapy, using CyTOF and interrogated leukemia phenotypic profiles, apoptotic networks, signaling pathways, differentiation states and trajectories. First, we performed unsupervised analyses to identify leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner, interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features that may correlate with clinical response and assessed whether distinct mutation profiles are associated with specific proteomic profiles that may confer resistance to therapy. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators across leukemia landscapes and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC), generally situated in close proximity to leukemia cells in UMAPs, lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells (Figure 1). Due to their contrasting apoptosis regulatory networks, we observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Of note, leukemia cell compartments harbor distinct differentiation hierarchies, the frequencies of which varied across patients. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered and reshaped the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutation diversified the leukemia landscape into a diverse continuum of differentiation states. Remarkably, we observed that RAS/MAPK mutations in FLT3-ITD AML overcame differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells(Figure1). Importantly, we observed that transitional and CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs preferentially persisted at D28, had active signaling pathways and expressed CD36, which indicates distinct metabolic programs could ensure survival under therapy pressure and also had lower levels of BCL2.The presence of CD34+ leukemia cells at D28, adapted under therapy pressure with persistent signaling at D28, both in CD34+ leukemia or MCs, was indicative of poor clinical outcomes. Conclusion: Single-cell proteomic assessment of FLT3-ITD AML treated with Quiz/Dec/Ven (TT) identifies a unique mode of resistance and proteomic features of surviving cells at D28, and elucidates how RAS/MAPK mutations diversify the leukemia proteomic landscape and modulate the architectural organization which eventually gives rise to emerging clones resistant to therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omic Single-Cell Sequencing Reveals Genetic and Immunophenotypic Clonal Selection in Patients With FLT3-Mutated AML Treated With Gilteritinib/Venetoclax

Background: The combination of the FLT3 inhibitor gilteritinib with the BCL-2 inhibitor venetoclax (VenGilt) is highly efficacious in relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML). In a recent phase 1b trial, VenGilt demonstrated a modified complete response rate of 75% (Daver et al, J Clin Oncol. 2022; published online). However, many patients (pts) relapsed after VenGilt. Proposed resistance mechanisms include RAS pathway mutations (Ven and Gilt) and monocytic immunophenotype (Ven). Using single cell (SC) multi-omic profiling, we sought to identify drivers of resistance to this combination. Methods: We analyzed 35 serial samples from 12 pts with R/R AML treated with VenGilt on the phase 1b trial (NCT03625505). We performed high-throughput SC DNA sequencing (Mission Bio Tapestri) on all samples and concurrent multi-omic SC cell-surface immunophenotyping on 18 samples. Results: We analyzed 107,528 cells (median 8960 cells/pt) from 12 pts with 2-5 serial timepoints. Eight pts had previously received tyrosine kinase inhibitors (TKI). Nine pts had clinical responses to VenGilt with a median relapse-free time of 8.3 mo. Of these 9 pts, 3 proceeded to transplant. Each pt had ≥1 genetically distinct clone identified (median 4.5, range 2-6). Fourteen FLT3mut clones were detected, with 2 pts having 2 separate FLT3mut clones. In all 12 pts, the FLT3mut clones were reduced with VenGilt. In 7 pts with a total of 9 clones, the FLT3mut population decreased to an undetectable level. Three pts had FLT3-IDH1/2 co-mutated clones, and the double mutant clones were reduced to undetectable levels in all 3. One pt developed a dominant IDH2mut clone at relapse without a FLT3 co-mutation. Nine pts demonstrated an initial decrease in non-FLT3mut clones, including clones with DNMT3A, NPM1, IDH1/2, TET2, EZH2, WT1, and NRAS mutations, suggesting some activity of VenGilt against non-FLT3mut populations. In 3 pts, all clones aside from those associated with clonal hematopoiesis of indeterminant potential (CHIP) were undetectable at follow-up. Five pts had ≥1 pre-existing NRAS- or KRAS-mutated clone. In 4/5 pts, the RASmutclone expanded on therapy and was dominant at relapse in 3/4 pts, with the fourth pt having a dominant IDH2mut clone. All 4 pts with expanding RASmutclones had prior TKI, while the pt whose RASmutclone decreased did not. Pre-existing RAS populations were small (0.001-18.1% of total cells) and were undetectable by bulk sequencing in 2/4 cases. RAS was not co-mutated with FLT3 in the same single cell in any cases. In 2 pts, FLT3mut clones present before VenGilt initially decreased but subsequently expanded on therapy. No de novo mutations within resistant/expanding FLT3mut clones were detected. We demonstrated immunophenotypic evolution for individual genotypes, including both FLT3mut and FLT3WT clones. For 5 pts with serial multi-omic SC samples, increased CD11b and CD13 expression was observed over time on a single cell basis in ≥1 genotype within individuals, suggesting increased monocytic-like features. These features were observed among 4 RASmut, 2 FLT3mut, and 1 TET2mut clones. At baseline, these 5 pts had heterogenous immunophenotypic profiles, with genetic clones occupying monocytic, immature, and mixed phenotypes; however, all 5 pts demonstrated ≥1 clone with increased monocytic immunophenotypic features after VenGilt treatment. Conclusion: Multi-omic SC sequencing enabled direct measurement of genetic and immunophenotypic clonal architecture and evolution with VenGilt treatment. VenGilt was highly effective against FLT3-mutated clones, even in the presence of co-mutations. Genetic resistance was associated with outgrowth of pre-existing subclones, primarily RAS mutations, rather than acquistion of de novo mutations. This differs from prior studies of Gilt monotherapy, in which both outgrowth of parallel clones and secondary mutations within the FLT3-mutated clone were associated with resistance. These data suggest the addition of Ven may suppress secondary resistance mutations, possibly due to enhanced apoptosis and cytotoxicity against FLT3-mutated cells. Multiple clones, predominatly those with a RAS mutation, developed monocytic features under VenGilt pressure. It is unknown whether monocytic immunophenotype is an independent resistance mechanism or a bystander to other mechanisms, such as increased MCL1 or RAS pathway gene expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Transcription Factor PU.1 Controls Alternative Splicing of Cflip By Binding to SRSF2/10 Splice Factors in AML Therapy Responses

cFLIP (CFLAR) is an inactive homolog of caspase 8 that functions downstream of death-receptor mediated apoptosis. CFLAR gene is alternatively spliced and exists in three isoforms: a long isoform (cFLIPL), which partially inhibits CASP8 activity, and two short forms (cFLIPR and cFLIPS) that entirely block caspase 8 activity. Moreover, cFLIPL is crucial for macrophage differentiation. Therefore, the ratio between cFLIPL and cFLIPS/R may regulate myeloid differentiation. The transcription factor PU.1 (SPI1) is necessary for myeloid differentiation as well as myeloid cell survival. PU.1 is expressed at low levels in hematopoietic progenitors and increasing levels of PU.1 promote macrophage and granulocyte differentiation. Additionally, aberrant low PU.1 expression contributes to an immature myeloid phenotype, e.g. seen in acute myeloid leukemia (AML). Interestingly, two studies indicated that high PU.1 protein levels were associated with alternative splicing in a murine erythroleukemia model by either direct binding to splice factors or RNA. Based on these observations we hypothesized that PU.1 controls alternative splicing of cFLIP during myeloid differentiation. In line with above hypothesis, differentiating primary CD34+ hematopoietic progenitors into granulocytes and monocytes or AML cells into granulocytes shifted cFLIP splicing in favor of cFLIPL paralleled by increased PU.1 mRNA and protein levels. Next, we assessed the direct impact of PU.1 protein on splicing during granulocytic differentiation by knocking down PU.1 in NB4 acute promyelocytic leukemia (APL) and MOLM13 AML cells. Silencing PU.1 in NB4 and MOLM13 cells decreased cell death and differentiation during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation. Moreover, knocking down PU.1 reduced cFLIPL/S protein and mRNA ratios during ATRA treatment. Interestingly, increasing the cFLIPL/S ratio by knocking down specifically cFLIPS or overexpressing cFLIPL reduced MOLM13 cell viability during ATRA treatment. Since the canonical function of CFLAR isoforms is the inhibition of extrinsic apoptosis, we next tested if increased cFLIPS levels found in AML cells depleted for PU.1 would show reduced sensitivity to TRAIL treatment. Indeed, TRAIL-induced extrinsic apoptosis via TRAIL receptor was significantly reduced in PU.1 depleted MOLM-13 AML cells. To investigate if PU.1 is directly involved in CFLAR splicing, we analyzed the CFLAR pre-mRNA sequence encompassing the alternative splicing site for PU.1 and splice factor binding motifs. We identified one putative PU.1 and SRSF2 as well as SRSF10 splice factor binding sites in the CFLAR pre-mRNA sequence. Using RNA immunoprecipitation, PU.1 pull-down and proximity ligation assays, we showed that all three proteins bind the CFLAR mRNA and physically interact with each other. Lastly, we asked if PU.1 additionally influences cFLIP alternative splicing via regulating splice factor transcription. Knocking down PU.1 in MOLM13 cells resulted in significantly reduced SRSF2 and SRSF10 mRNA and protein levels. We confirmed PU.1 binding to their promoter using ChIP assays. Accordingly, these splice factors showed altered mRNA expression when comparing mature granulocytes to primary AML cells. Since silencing SRSF2 and SRSF10 switched the CFLARL/S isoform ratio towards the long isoform, we suggest that PU.1-mediated induction of these splice factores represents a negative feedback loop. In conclusion, we provide evidence that PU.1 directly regulates alternative splicing of CFLAR during ATRA-mediated neutrophil differentiation and TRAIL-induced cell death in human AML cells. We propose a model where PU.1 binds to CFLAR pre-mRNA and blocks SRSF2 and SRSF10-mediated splicing ultimatively leading to exon 7 skipping and predominant expression of cFLIPL.