Monocyte Markers Research Articles

P-438. T-cell Immune Exhaustion and the Atherogenic Index of Plasma in People Living with HIV on Antiretroviral Therapy

Abstract Background The atherogenic index of plasma (AIP), an index that correlates with atherosclerosis, has been reported to be abnormal among people with human immunodeficiency virus (PWH). Few subclinical markers of atherosclerosis were linked to T-cell immune exhaustion. However, no study has explored the relationship of AIP to the different immune exhaustion markers. We compared the AIP and immunologic parameters of PWH on antiretroviral therapy (ART) to that of people without HIV (PWoH). Correlation of different clinical, laboratory and immunologic parameters with AIP in PWH were investigated.Table 1.Demographic and clinical characteristics of study participants (N=100) Methods This is a cross-sectional study conducted from March 2018 to February 2019 in a treatment hub in Manila, Philippines. We recruited 50 virally suppressed PWH on ART and 50 age- and sex- matched PWoH. Demographic and clinical data were recorded. Fasting blood samples were collected for complete blood count, blood glucose, and lipid profile. Peripheral blood monocytes were isolated and analyzed for immune exhaustion markers in T-cells and monocytes (LAG-3, PD-1, TIGIT+, TIM-3) via flow cytometry. AIP was computed as log (trigylyceride/high density lipoprotein-c). Abnormal AIP was defined as values in the medium (0.1-0.24) and high-risk ( >0.24) categories. Pearson correlation was performed to determine linear correlation between clinical, laboratory and immunologic factors and AIP.Table 2.Laboratory and immunologic parameters of study participants (N=100) Results Majority of the participants were male (96%) with mean age of 31. Among PWH, the mean recent CD4+ count is at 515 cells/mm3. Median ART exposure is 4 years. Majority were on efavirenz based ART (41, 82%). Few were on protease inhibitors (6, 12%). All PWH had abnormal AIP. There is no difference in AIP between PWH and PWoH (0.5; 0.3 vs 0.62; 0.3, p 0.5). Recent CD4+ (R 0.32, p 0.02), nadir Cd4+ (R 0.31, p 0.03) and zenith CD4+ (R 0.30, p 0.04) were positively correlated with AIP in PWH. Only dual expression of PD-1 TIGIT+ CD8+ T-cells showed positive correlation with AIP (R 0.20, p 0.04).Figure 1.Correlation of AIP levels with CD4+ count Conclusion AIP in PWH is comparable to that of PWoH. The correlation of AIP to PD-1 TIGIT+ CD8+ T-cells supports the role of T-cell immune exhaustion in the pathogenesis of dyslipidemia and atherosclerosis. Strategies to decrease immune exhaustion can possibly prevent or delay metabolic complications in PWH. Correlation of AIP levels with frequency expression of PD-1 TIGIT+ on CD8+ T-cells Disclosures All Authors: No reported disclosures

BMP6 participates in the molecular mechanisms involved in APAP hepatotoxicity.

Given the lack of accurate diagnostic methods of acetaminophen (APAP)-induced acute liver failure (ALF), the search for new biomarkers for its diagnosis is an urgent need. The aim of this study was to evaluate the role of bone morphogenetic protein 6 (BMP6) in APAP-induced ALF progression and its potential value as a biomarker of ALF. Hepatic and circulating BMP6 expression was assessed in APAP-treated mice and in serum samples from patients with APAP overdose. In addition, BMP6 expression and release was evaluated in hepatocytes after APAP exposure. BMP6 gene was silenced in Huh7 cells prior to APAP treatment and the culture medium (CM) was added to THP1 cells to evaluate the paracrine effects of hepatocyte BMP6 on APAP toxicity. Hepatic and serum BMP6 levels were increased in mice after APAP-induced ALF. In addition, a positive correlation was observed between circulating BMP6 and ALT activity in patients exposed to APAP overdose. Moreover, hepatocytes expressed and released BMP6 to the CM after APAP treatment. Indeed, the CM from APAP-treated Huh7 cells upregulated M1 and M2 markers in THP1 monocytes. The CM from BMP6-silenced Huh7, which was depleted of BMP6, reduced the expression of M2 markers in THP1 cells. In fact, expression of M2 markers was increased in THP1 cells exposed to BMP6. This study reveals that hepatic BMP6 expression is increased in APAP-induced acute liver injury, positioning it as a potential new biomarker of liver damage severity. Moreover, our data indicate that BMP6 might play a role in the hepatocyte-macrophage crosstalk during APAP-induced hepatotoxicity.

Liver GPBAR1 Associates With Immune Dysfunction in Primary Sclerosing Cholangitis and Its Activation Attenuates Cholestasis in Abcb4-/- Mice.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterised by progressive biliary inflammation and fibrosis, leading to liver cirrhosis and cholangiocarcinoma. GPBAR1 (TGR5) is a G protein-coupled receptor for secondary bile acids. In this study, we have examined the therapeutic potential of BAR501, a selective GPBAR1 agonist in a PSC model. Single-cell analysis of healthy human liver samples and gene expression analysis of PSC liver samples were conducted. Invitro studies on a human cholangiocyte cell line (NHC), U937 and human hepatic stellate cells (hSteCs) were performed. Additionally, Abcb4-/- mice were treated with BAR501 for 12-24 weeks. Single-cell analysis demonstrated that GPBAR1 is expressed by macrophages, NK cells, sinusoidal cells and to a lesser extent by cholangiocytes. Total liver expression of GPBAR1 increases in PSC patients compared to that in healthy controls and positively correlates with markers for monocytes and NK cells and cytokeratin 19. Invitro treatment of NHCs with BAR501 reversed the acquisition of a pro-inflammatory phenotype and the downregulation of GPBAR1 expression promoted by LPS in an NF-κB-dependent manner. Treating Abcb4-/- mice reduced bile duct inflammation and liver fibrosis and prevented the downregulation of GPBAR1 expression. Treating mice with BAR501 also modulated the bile acid pool composition and reduced the dysbiosis-associated gut permeability, and intestinal and systemic inflammation. Exvivo experiments using conditioned media from BAR501-treated cholangiocytes mitigated the activation of macrophages. Our study provides evidence for the therapeutic potential of selective GPBAR1 agonists in intestinal inflammation-associated cholestasis, warranting the evaluation of BAR501 in PSC patients.

B Lymphocyte-A Prognostic Indicator in Post-Acute Pancreatitis Diabetes Mellitus.

To determine the value of lymphocyte subsets and granulocyte/monocyte surface markers in predicting the risk of post-acute pancreatitis diabetes (PPDM-A). This study included 308 in patients with acute pancreatitis (AP). The markers of granulocytes and monocytes and lymphocyte subsets were detected by flow cytometry, and the fluorescence intensity, absolute count and percentage were obtained. Based on the occurrence of diabetes after AP, patients were divided into two groups: PPDM-A and PPNG-A (post-acute pancreatitis with normal glucose). Correlations between granulocyte and monocyte surface markers and lymphocyte subsets were analyzed. Binary logistic regression was used to analyze the potential influencing factors of PPDM-A. Compared with patients with PPNG-A, patients with PPDM-A tend to be younger (p < 0.001) and have a higher proportion of fatty liver, recurrent pancreatitis, and hyperlipidemic pancreatitis. The results of linear regression showed that B% was negatively correlated with MFI of HLA-DR on monocytes (R2 = 0.145, p < 0.001), B% was positively correlated with CD10-NEUT% (R2 = 0.291, p < 0.001), and MFI of HLA-DR on monocytes was negatively correlated with CD10-NEUT% (R2 = 0.457, p < 0.001). Multivariate logistic regression analysis revealed that age, serous effusion, fatty liver, recurrent pancreatitis, and B% were independent risk factors for the occurrence of PPDM-A. Our study has first confirmed the correlation between PPDM-A and lymphocyte subsets and CD10-NEUT%. Furthermore we indicated that age, fatty liver, serous effusion, recurrent AP, and B% were independent risk factors for PPDM-A. The mechanism of granulocyte and monocyte surface markers and B lymphocytes on PPDM-A is worthy of study. This would help clarify the pathogenesis of PPDM-A at the cellular level and potentially provide new strategies for immunotherapy and even disease prevention.

A study on the level of serum neopterin a new novel biomarker in acute myocardial infarction

Introduction: Acute myocardial infarction (AMI) is one of the most common diseases, and it is one of the important medical emergencies admitted in the hospitals. It is characterized by the presence of clinical features like sudden onset of severe chest pain with dysponea, palpitation and profuse sweating leading to cardiac arrythmias, cardiogenic shock, cardiac failure and death if not treated early(2). Among the biomarkers for diagnosis and prognosis in AMI, the Neopterin, a pteridine derivative is considered to be one of the new novel bio-markers which is found to be elevated in patients with AMI. Neopterin was first isolated from larvae of bee in 1963(3). Eventually Neopterin was identified as the fluorescent component that was elevated in the urine of mice with Ehrlich ascites tumor (4). Estimation of Serum levels of Neopterin is a new novel biomarker in AMI and seems to be a prognostic marker for MACE in AMI. Aim: To elucidate the role of Neopterin levels and its short term prognostic significance in AMI. Methods: Neopterin is an activation marker for monocytes / macrophages and its circulating levels in the Serum is considered to be one of the prognostic markers in the treatment of AMI.

The Tumor Metabolite 5'-Deoxy-5'Methylthioadenosine (MTA) Inhibits Maturation and T Cell-Stimulating Capacity of Dendritic Cells.

Metabolite accumulation in the tumor microenvironment fosters immune evasion and limits the efficiency of immunotherapeutic approaches. Methylthioadenosine phosphorylase (MTAP), which catalyzes the degradation of 5'-deoxy-5'methylthioadenosine (MTA), is downregulated in many cancer entities. Consequently, MTA accumulates in the microenvironment of MTAP-deficient tumors, where it is known to inhibit tumor-infiltrating T cells and NK cells. However, the impact of MTA on other intra-tumoral immune cells has not yet been fully elucidated. To study the effects of MTA on dendritic cells (DCs), human monocytes were maturated into DCs with (MTA-DC) or without MTA (co-DC) and analyzed for activation, differentiation, and T cell-stimulating capacity. MTA altered the cytokine secretion profile of monocytes and impaired their maturation into dendritic cells. MTA-DCs produced less IL-12 and showed a more immature-like phenotype characterized by decreased expression of the co-stimulatory molecules CD80, CD83, and CD86 and increased expression of the monocyte markers CD14 and CD16. Consequently, MTA reduced the capability of DCs to stimulate T cells. Mechanistically, the MTA-induced effects on monocytes and DCs were mediated by a mechanism beyond adenosine receptor signaling. These results provide new insights into how altered polyamine metabolism impairs the maturation of monocyte-derived DCs and impacts the crosstalk between T and dendritic cells.

Epstein Barr Virus (EBV) Latent Membrane Protein 1 (LMP-1) Regulates Functional Markers in Intermediate and Non-Classical Monocytes.

Background: The Epstein-Barr virus (EBV) infects more than 90 percent of the human population. In pediatric patients, the innate immune response against EBV primary infection plays a key role. Monocytes and macrophages can have distinct functions depending on the microenvironment surrounding them. At least three monocyte subpopulations can be differentiated depending on membrane protein expression: classical (C, CD14++CD16-), intermediate (I, CD14++CD16+), and non-classical (NC, CD14+CD16++). They also modulate T and B lymphocyte activation/inhibition through the expression of costimulatory molecules such as CD80, CD86, and PD-L1. Yet, little is known about monocytes' role in EBV infection. Methods: Peripheral blood and tonsil biopsies of EBV primary infected (PI) patients, healthy carriers (HCs), and patients undergoing reactivation (R) were studied. Results: Classical monocytes prevailed in all infectious statuses. Tonsillar CD163 positively correlated with CD163 expression in NC monocytes in HCs. PD-L1+ cells in the tonsil positively correlated with PD-L1 expression in NC monocytes. LMP-1 viral latent protein presented a positive correlation with PD-L1, CD163, and CD206 expression in the NC subpopulation. Conclusions: Our results evidence the predominant role of I and NC monocytes' response against EBV infection. Furthermore, the viral oncoprotein LMP-1 could be involved in the expression of regulatory proteins in I and NC monocytes.

CD14loCD301b+ macrophages gathering as a proangiogenic marker in adipose tissues.

The role of the monocyte marker CD14 in the regulation of obesity is increasingly recognized. Our observations indicated that Cd14-/- mice exhibited a leaner body shape compared to their wild type (WT) counterparts. And the loss of CD14 alleviated high-fat diet (HFD)-induced obesity in mice. In human subjects, CD14 level was tested to be positively correlated with overweight and obesity. However, the relationship between CD14 and the development of obesity remains only partially understood. To investigate the underlying mechanisms, adipose tissues (AT) from Cd14-/- and WT mice were subjected to deep RNA sequencing. Gene Ontology enrichment analysis revealed a significant enhancement of angiogenesis-related function in the Cd14-/- epWAT compared to WT counterpart, which was accompanied by an upregulation of Cd301b. Subsequent assays confirmed the enhanced angiogenesis and more accumulation of CD301b+ macrophages in Cd14-/- epWAT. Because Igf1 expression has been suggested to be associated with Cd301b expression through pseudotime analysis, we found it was IGF-1 secreted from Cd14-/- macrophages that mediated the angiogenesis enhancement. Collectively, our findings indicate that the accumulation of CD14loCD301b+ macrophages may serve as a proangiogenic marker in adipose tissues, providing novel insights into the relationship between CD14 and obesity development.

Diabetic Glycation of Human Serum Albumin Affects Its Immunogenicity.

Advanced glycation end-products (AGEs) are products of a non-enzymatic reaction between amino acids and reducing sugars. Glycated human serum albumin (HSA) increases in diabetics as a consequence of elevated blood glucose levels and glycating metabolites like methylglyoxal (MGO). The impact of different types of glycation on the immunomodulatory properties of HSA is poorly understood and is studied here. HSA was glycated with D-glucose, MGO, or glyoxylic acid (CML). Glycation-related biochemical changes were characterized using various biochemical methods. The binding of differentially glycated HSA to AGE receptors was determined with inhibition ELISAs, and the impact on inflammatory markers in macrophage cell line THP-1 and adherent monocytes isolated from human peripheral blood mononuclear cells (PBMCs) was studied. All glycation methods led to unique AGE profiles and had a distinct impact on protein structure. Glycation resulted in increased binding of HSA to the AGE receptors, with MGO modification showing the highest binding, followed by glucose and, lastly, CML. Additionally, modification of HSA with MGO led to the increased expression of pro-inflammatory markers in THP-1 macrophages and enhanced phosphorylation of NF-κB p65. The same pattern, although less prominent, was observed for HSA glycated with glucose and CML, respectively. An increase in pro-inflammatory markers was also observed in PBMC-derived monocytes exposed to all glycated forms of HSA, although HSA-CML led to a significantly higher inflammatory response. In conclusion, the type of HSA glycation impacts immune functional readouts with potential relevance for diabetes.

IMMU-59. AN IMMUNOSUPPRESSIVE PROGRAM ENCOMPASSING MULTIPLE IMMUNE CELL TYPES IN GBM TYPIFIED BY NOVEL MARKER LRRC25 ASSOCIATED WITH TYROBP SIGNALING

Abstract Immunosuppression in glioblastoma is a powerful impediment to effective immunotherapy. We used maximal tumor sampling and genome-wide coexpression analysis of RNA sequencing, on 69 samples from 8 patients, to identify a module of 270 genes marked by LRRC25, determined by top fidelity score, that covaries with many immunosuppressive molecules. An immunosuppressive module was independently derived from an expanded cohort of 319 samples from 28 GBM also marked by LRRC25 by top fidelity score. Within the gene module, we observed strong co-variation of markers for monocytes, neutrophils, and NK cells with LRRC25. In published single-cell RNA-seq from glioma, we determined that LRRC25 is expressed by neutrophils and myeloid cells while absent from NK-cells. LRRC25 is also constitutively expressed in monocytes and neutrophils while being upregulated by microglia and DCs in the tumor core and necrotic regions. In addition to covariation with multiple cell-type specific markers we also discovered that LRRC25 expression is tightly associated with several members of the TYROBP causal signaling network in microglia. Thus, LRRC25 may play a novel role in TYROBP signaling which may, in turn, represent a novel mechanism of NK cell and neutrophil immunosuppression in glioma. We confirmed LRRC25 RNA expression in patient samples by RNAscope co-stained with an anti-CD163 antibody indicating myeloid expression, as well as tumor cell expression determined by histopathology. Although LRRC25 is known to mediate IFN and NFKb signaling in myeloid cells in-vitro, its function in glioma remains understudied. While significant efforts have focused on mechanisms of myeloid to T-cell immunosuppression, less focus has been given to mechanisms of myeloid to NK-cell and neutrophil immunosuppression, despite the fact they exist at levels comparable to T-cells in the tumor microenvironment. This work will be followed up with antibody validation as well as genetic manipulation of LRRC25 in-vitro and in-vivo.

Non-classical monocyte levels correlate negatively with HIV-associated cerebral small vessel disease and cognitive performance.

Despite antiretroviral treatment (cART), aging people living with HIV (PWH) are more susceptible to neurocognitive impairment (NCI) probably due to synergistic/additive contribution of traditional cerebrovascular risk factors. Specifically, transmigration of inflammatory CD16+ monocytes through the altered blood brain barrier (BBB) may exacerbate cerebral small vessel disease (CSVD), a known cause of vascular cognitive impairment. PWH on cART (n=108) and age, sex, and Reynold's cardiovascular risk score-matched uninfected individuals (PWoH, n=111) were enrolled. This is a longitudinal observational study but only cross-sectional data from entry visit are reported. Neuropsychological testing and brain magnetic resonance imaging (MRI) were performed. CSVD was diagnosed by Fazekas score ≥1. Flow cytometric analyses of fresh whole blood were conducted to evaluate circulating levels of monocyte subsets (classical, intermediate, and non-classical) and markers of monocyte activation (CCR2, CD40, PSGL-1, TNFR2 and tissue factor). ELISAs were used to measure sCD14, ICAM, and Osteoprotegerin. Two-way analysis of variance (ANOVA), and linear regression models were performed to study the effects of HIV status, CSVD status, and their interaction to outcome variables such as cognitive score. Two-sample t-tests and correlation analyses were performed between and within PWoH with CSVD and PWH with CSVD participants. PWH with CSVD (n=81) had significantly lower total cognitive scores, higher levels of NCMs and soluble CD14 and intracellular adhesion molecule 1 (ICAM-1) as compared to PWoH with CSVD group (n=68). sCD14 and ICAM1 were positively correlated with each other indicating that monocyte and endothelial activation are associated with each other. Cognition was negatively correlated with NCMs, especially in the PWH with CSVD group. Among other blood biomarkers measured, osteoprotegerin levels showed mild negative correlation with cognitive performance in individuals with CSVD irrespective of HIV status. Elevated levels of NCMs may contribute to neuroinflammation, CSVD and subsequent cognitive impairment. This finding is of particular relevance in aging PWH as both HIV and aging are associated with increased levels of NCMs. NCMs may serve as a potential biomarker to address these comorbidities. Further longitudinal studies are needed to evaluate whether changes in NCM levels are associated with changes in CSVD burden and cognitive impairment.

Abstract P183: Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus Induce Inflammation and Salt Sensitivity of Blood Pressure in Humanized Mice

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease (CVD), even in normotensive people. Systemic lupus erythematosus (SLE) is also a risk factor for CVD and hypertension. SLE and CVD are on the rise, and it is not known if this is attributable to the increased dietary salt in the modern diet and SSBP. Isolevuglandins (IsoLGs) adduct self-proteins forming neoantigens in antigen-presenting cells (APC), activate T cells, and produce cytokines that promote sodium retention, kidney damage, SLE, and SSBP. We hypothesized the adoptive transfer of peripheral blood mononuclear cells (PBMCs) from patients with SLE into immunodeficient mice (NSG/MHCI/MHCII-/-) would increase inflammation and SSBP after high salt feeding. In vitro studies were performed using PBMCs from patients with SLE and treated with and without high salt, and myeloid cell activation and cytokine expression were measured with flow cytometry. We humanized mice with PBMCs from patients with SLE (n=7) and controls without autoimmune disease (n=7) in animal studies. The mice were fed a 4% high-salt diet for two weeks. Blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography. A high salt diet significantly increased systolic blood pressure in lupus mice compared to the controls (133.0 vs.116.4 mmHg, p=0.001). APC infiltration (macrophages, monocytes, dendritic cells), proinflammatory markers, and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. In lupus mice, classical monocyte and proinflammatory markers were significantly elevated in the spleen lymphocytes, and proinflammatory markers were increased in the kidney, spleen, and aorta in lupus mice. These mice also exhibited mesenteric artery vascular dysfunction (p&lt;0.05). Lupus mice tended to excrete more urinary albumin than the controls (25.89 vs. 17.24 µg/ml p=0.142), suggesting kidney injury. High salt treatment in vitro increased intermediate and nonclassical monocytes with increased CD86, IsoLG, and TNF-alpha expression compared to normal salt-treated cells. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt, increase SSBP and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular function; and worsen albuminuria in immunodeficient mice.

Cardiovascular risk in ANCA-associated vasculitis: Monocyte phenotyping reveals distinctive signatures between serological subsets

Background and aimsAnti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) is associated with an increased cardiovascular risk, particularly the myeloperoxidase AAV serotype (MPO-AAV). Distinct alterations in monocyte phenotypes may cause accelerated atherosclerotic disease in AAV. MethodsA cohort including 43 AAV patients and 19 healthy controls was included for downstream analyses. Extensive phenotyping of monocytes and monocyte-derived macrophages was performed using bulk RNA-sequencing and flow cytometry. An in vitro transendothelial migration assay reflecting intrinsic adhesive and migratory capacities of monocytes was employed. Subsequent sub-analyses were performed to investigate differences between serological subtypes. ResultsMonocyte subset analysis showed increased classical monocytes during active disease, whereas non-classical monocytes were decreased compared to healthy controls (HC). RNA-sequencing revealed upregulation of distinct inflammatory pathways and lipid metabolism-related markers in monocytes of active AAV patients. No differences were detected in the intrinsic monocyte adhesion and migration capacity. Compared to proteinase-3(PR3)-AAV, monocytes of MPO-AAV patients in remission expressed genes related to inflammation, coagulation, platelet-binding and interferon signalling, whereas the expression of chemokine receptors indicative of acute inflammation and monocyte extravasation (i.e., CCR2 and CCR5) was increased in monocytes of PR3-AAV patients. During active disease, PR3-AAV was linked with elevated serum CRP and increased platelet counts compared to MPO-AAV. ConclusionsThese findings highlight changes in monocyte subset composition and activation, but not in the intrinsic migration capacity of AAV monocytes. MPO-AAV monocytes are associated with sustained upregulation of inflammatory genes, whereas PR3-AAV monocytes exhibit chemokine receptor upregulation. These molecular changes may play a role in elevating cardiovascular risk as well as in the underlying pathophysiology of AAV.

Macrophages of multiple hematopoietic origins reside in the developing prostate.

Tissue-resident macrophages contribute to the organogenesis of many tissues. Growth of the prostate is regulated by androgens during puberty, yet androgens are considered immune suppressive. In this study, we characterized the localization, androgen receptor expression and hematopoietic origin of prostate macrophages, and transiently ablated macrophages during postnatal prostate organogenesis in the mouse. We show that myeloid cells were abundant in the prostate during puberty. However, nuclear androgen receptor expression was not detected in most macrophages. We found Cx3cr1, a marker for macrophages, monocytes and dendritic cells, expressed in interstitial macrophages surrounding the prostate and associated with nerve fibers. Furthermore, we provide evidence for the co-existence of embryonic origin, self-renewing, tissue-resident macrophages and recruited macrophages of bone-marrow monocyte origin in the prostate during puberty. Our findings suggest that prostate macrophages promote neural patterning and may shed further light on our understanding of the role of the innate immune system in prostate pathology in response to inflammation and in cancer.

Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma

AbstractHematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies, being particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we characterized 48 patients treated with B-cell maturation antigen (BCMA) CAR-T cells to understand kinetics of cytopenia, identify predictive factors, and determine potential mechanisms underlying these toxicities. We observed that overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, being still present after 1 year in 4 and 3 patients, respectively. Baseline cytopenia and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms underlying cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPC differentiation, promoting more immature phenotypes, which could be prevented with a combination of interferon γ, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). These results suggest that CAR-T activation induces HSPC maturation arrest through paracrine effects and provides potential treatments to mitigate the severity of this toxicity.

Plac8-ERK pathway modulation of monocyte function in sepsis

Sepsis, a life-threatening condition caused by infection, is characterized by the dysregulation of immune responses and activation of monocytes. Plac8, a protein, has been implicated in various inflammatory conditions. This study aimed to investigate the effect of Plac8 upregulation on monocyte proliferation and activation in sepsis patients. Peripheral blood samples were collected from healthy individuals and sepsis patients. Monocytes were stimulated with lipopolysaccharide (LPS) to create an in vitro sepsis model, while a murine sepsis model was established using cecal ligation and puncture (CLP). The levels of monocyte markers, proliferation index (PI), and pro-inflammatory cytokines were assessed using flow cytometry and qPCR, respectively. Plac8 and phosphorylated ERK protein levels were determined by western blot, and TNF-α, IL-6, and IL-10 levels were quantified using ELISA. The CCK-8 assay was used to evaluate PBMC proliferation and activation. The results showed that Plac8 was highly expressed in sepsis models, promoting the survival, proliferation, and activation of monocytes. Plac8 upregulation activated the ERK pathway, leading to increased phosphorylation of ERK protein and elevated levels of CD14, CD16, TNF-α, IL-6, Plac8, and IL-10. In sepsis mice, Plac8 overexpression similarly activated the ERK pathway and promoted the survival, proliferation, and activation of monocytes. In conclusion, the upregulation of Plac8 enhances the activation of the ERK pathway and promotes monocyte proliferation and activation in sepsis patients.

Impact of monocytic differentiation on acute myeloid leukemia patients treated with venetoclax and hypomethylating agents.

Although the combination of venetoclax (VEN) and hypomethylating agents (HMAs) results in impressive efficacy in acute myeloid leukemia (AML), there is still a subset of patients who are refractory. We investigated the outcomes of AML patients with monocytic differentiation who were treated with frontline VEN/HMA. A total of 155 patients with newly diagnosed AML treated with frontline VEN/HMA were enrolled in the study. Monocyte-like AML was identified by flow cytometry with typical expression of monocytic markers, and M5 was identified according to French, American, and British category. We compared the outcomes of patients with different characteristics. The rate of complete remission (CR) and CR with incomplete recovery of blood counts (CRi), progression-free survival (PFS), and overall survival (OS) in monocyte-like AML were inferior to those in nonmonocyte-like AML (CR/CRi rates, 26.7% vs. 80.0%, p < 0.001; median PFS, 2.1 vs. 8.8 months, p < 0.001; median OS, 9.2 vs. 19 months, p = 0.013). CR/CRi rate in M5 was lower than that in non-M5 (60.7% vs. 75.5%, p = 0.049). Multivariate analyses showed that monocyte-like AML was associated with lower odds of CR/CRi and higher risk of progression. Our study suggested that newly diagnosed AML with a monocytic immunophenotype had a poor prognosis with VEN/HMA treatment.

#1323 Kidney health in children with X-linked hypophosphatemia: lessons from the prospective multicenter study in Germany and Switzerland

Abstract Background and Aims X-linked hypophosphatemia (XLH) is the most common genetic cause of hypophosphatemia. Mutations in the PHEX gene cause elevated circulating levels of fibroblast growth factor 23 (FGF23), phosphaturia, rickets and osteomalacia. Conventional treatment with phosphate salts and active vitamin D is associated with nephrocalcinosis and preclinical studies in mice have shown that a chronic high phosphate load causes proximal tubular injury and fibrosis. There is no detailed analysis on kidney health in XLH patients yet. Method We conduct a prospective observational multicenter study in Germany and Switzerland to evaluate kidney health in pediatric XLH patients. Patients receive conventional therapy or burosumab, a fully humanized anti-FGF23 antibody. Clinical and biochemical data, blood and urine samples are collected annually. Results To date, 137 patients (80 girls, mean age 10.5 years) from 40 centers are included. 22% of patients have been treated conventionally for 7.5 years, 78% of patients have received burosumab for 2.3 years with 3.7 years of conventional therapy beforehand. Children with XLH show increased systolic blood pressure and body mass index (BMI), and overweight (BMI &amp;gt;90th percentile) and hypertension (systolic blood pressure &amp;gt;95th percentile) were each found in 20% of patients. A reduced eGFR (&amp;lt;90 ml/min/1.73 m2) and/or nephrocalcinosis was noted in 7% and 28% of patients, respectively. Microalbuminuria is twice as prevalent in children with XLH as in healthy children (14% vs 7%). Urinary lithogenic substances (calcium, oxalate, glycolate) are increased in 1.8-22.8%, and citrate is decreased in 12.3% of XLH patients. Tubular injury markers neutrophil gelatinase-associated lipocalin (NGAL) and Dickkopf-3 (DKK3) are elevated in urine of children with XLH, Chitinase 3-like 1 (CHl3L1) is comparable to children with chronic kidney disease, while kidney injury molecule-1 (Kim-1), indicating proximal tubule cell injury, is within a normal range. The renal inflammation marker monocyte chemoattractant protein-1 (MCP-1) is elevated in children with XLH, and epidermal growth factor (EGF), a marker of tubular cell repair, is decreased compared to healthy adults. Conclusion Pediatric XLH patients on conventional or burosumab therapy show enhanced systolic blood pressure slightly associated with elevated BMI, and significant renal comorbidity, i.e. reduced eGFR, nephrocalcinosis, increased urinary lithogenic substances, elevated urinary markers of glomerular and tubular injury and inflammation. The impact of conventional versus burosumab treatment are still being investigated.

Addition of CD14 improves discrimination of lymphocytes in the TBNK phenotyping panel.

Peripheral blood lymphocyte phenotyping panels typically include CD45 for discrimination of the lymphocyte population, and fluorophore-conjugated monoclonal antibodies to identify T, B, and Natural Killer (NK) cells. While CD45 combined with side scatter is generally sufficient to clearly distinguish lymphocytes from monocytes in the majority of peripheral blood samples, it is challenging to accurately gate lymphocytes in samples from patients with monocytosis or significant lymphopenia, or from very young infants. Addition of a monocyte marker to lymphocyte phenotyping panels for monocyte exclusion has previously been evaluated for improved discrimination of lymphocytes, albeit largely in healthy donor adult samples. Here we evaluate the effect of the addition of CD14 to a standard lymphocyte phenotyping panel on total lymphocyte, T, B, and NK cell percentages in a predominantly pediatric population of patients under evaluation chiefly for immunodeficiency, immune-depletion, or immune reconstitution. Addition of CD14 to the standard lymphocyte phenotyping improved discrimination of lymphocytes from monocytes, resulted in decreased NK cell percentages, likely because CD16+ and/or CD56+ monocytes were included in the CD56+CD16+ NK cell gate with conventional gating, and although less significant, resulted in an increased percentage of B cells, since relatively larger B cells were likely gated out by more restrictive light scatter gating used with the conventional gating approach. The change in NK and B cell percentages were more pronounced in samples from patients below a year of age, and in patients who were relatively lymphopenic. These data suggest that addition of CD14 to conventional lymphocyte phenotyping panels that utilize CD45 versus side scatter gating results in significant improvement in the accuracy of lymphocyte gating, and accurate quantification of NK and B cells particularly in samples from infants and lymphopenic individuals.

The Role of Invariant Natural Killer T (iNKT) Cells in Early Responses to Influenza A Virus (IAV)

Abstract iNKT cells are a CD1d-restricted innate-like lineage of ⍺ßT cells that are prevalent in mucosal tissues, where they influence early immune responses. iNKT cells differentiate in the thymus into—iNKT1, iNKT2, and iNKT17 analogous to Th1, Th2, and Th17 cells. We have shown in ET-2;CD4cre mice, that sustained E protein activity results in changes in iNKT subset composition, with a shift from iNKT1s towards iNKT2/NKT17 cells and other subsets. To understand the physiological relevance of these changes, we tested the response of ET2 mice to IAV. ET2 mice showed less weight loss, reduction in lung-infected areas, decreased neutrophil infiltration, lower activation markers in inflammatory monocytes, reduced levels of CCL2 and interferon-responsive genes in ET2. We observed increased levels of IFNλ in infected ET2 mice vs. elevated IFNα in WT mice 24 hours post-infection. scRNA-seq analysis of sorted iNKT from WT and ET2 mice showed that ET2 profiles are closer to conventional naïve T cells, and that, after infection, their response is marked by overexpression of Ccr7, -Lselectin, IL1R1. WT response is predominantly type 1. Gene Ontology pathway analysis of infected WT show enrichment of cytokine production and immune cell activation pathways, while pathways enriched in infected ET-2 are associated with adhesion and repair. These experiments suggest that early type 1 responses driven by NKT cells during IAV infection can be deleterious, intensifying inflammation, and neutrophilia.