Human CD2 Research Articles

Abstract 6641: ATG-031, a first-in-class humanized anti-CD24 antibody, demonstrates potent in vivo efficacy and repolarizes tumor-associated macrophages in the TME

Abstract Background: Tumor microenvironment (TME) is shaped by not only cancer cells but also a variety of resident and infiltrating cells, including stromal fibroblasts, endothelial cells and immune cells like tumor-associated macrophages (TAM) and lymphocytes (Roghayyeh et al., 2020). Most TAMs are considered as the M2 subtype with pro-tumor/anti-inflammation effects. CD24 is a small, highly glycosylated cell adhesion protein and was reported to be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and acts as a novel “don’t eat me” target. CD24 is over-expressed on tumor cells and can help them avoid being engulfed by macrophages through its interaction with Siglec-10 expressed by TAM. ATG-031 is a humanized anti-CD24 monoclonal antibody that binds to CD24 with high affinity and specificity and blocks the interaction of CD24 with Siglec-10 on macrophages, enhancing macrophage-mediated phagocytosis of tumor cells. In this study, we evaluated the in vivo efficacy of ATG-031 and explored its pharmacodynamic effects. Methods: C57BL/6J mice were inoculated with wild-type (WT) MC38 cells on the left flank and MC38 cells that express human CD24 (MC38-hCD24) on the right flank. When the average tumor volume reached 120 to 140 mm3 for MC38-hCD24, mice were treated with vehicle control (PBS) or ATG-031 at 3 mg/kg intravenously twice a week for four doses. A group of mice inoculated with only MC38 tumor cells was set as the control and treated with ATG-031 by the same way. Tumor volume was measured twice a week to evaluate the efficacy of ATG-031. Tumor samples were isolated at the endpoint of this efficacy study, and a multiplex IHC staining was performed to evaluate the change of tumor-infiltrating immune cells in response to ATG-031. Results: ATG-031 treatment significantly inhibited the tumor growth of MC38-hCD24 on the right flank of mice with a tumor growth inhibition (TGI) of 60.52%. Interestingly, although the WT MC38 tumor on the left flank did not express the antigen, human CD24, ATG-031 still demonstrated efficacy in controlling the tumor growth with a TGI of 21.17%. Without the co-inoculation of MC38-hCD24, ATG-031 did not affect the WT MC38 tumor growth in the control group. Multiplex IHC staining revealed that the proportion of M1 macrophage increased in response to ATG-031 treatment, not only in MC38-hCD24 tumors but also in WT MC38 tumors on the left flank. Conclusions: ATG-031, a first-in-class anti-CD24 antibody, demonstrated potent anti-tumor activity in vivo, re-polarizing M2-like TAM to anti-tumor M1 subtype in the TME and induced systemic anti-tumor immunity. A clinical study to investigate the safety and efficacy of ATG-031 in cancer patients is being developed. Citation Format: Peng Chen, Min Deng, Yun Liu, Jiamei Luo, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-031, a first-in-class humanized anti-CD24 antibody, demonstrates potent in vivo efficacy and repolarizes tumor-associated macrophages in the TME. [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 6641.

A senolytic immunotoxin eliminates p16INK4a-positive T cells and ameliorates age-associated phenotypes of CD4+ T cells in a surface marker knock-in mouse

Senescent cells were recently shown to play a role in aging-related malfunctions and pathologies. This consensus has been facilitated by evidence from senolytic model mice capable of eliminating senescent cells in tissues using well-characterized senescent markers, such as p16INK4a (hereafter p16). However, since the incomplete or artificial gene expression regulatory regions of manipulated marker genes affect their cognate expression, it currently remains unclear whether these models accurately reflect physiological senescence. We herein describe a novel approach to eliminate p16-expressing cells from mice at any given point in time, generating a new type of knock-in model, p16hCD2 mice and a toxin-conjugated anti-human CD2 antibody (hCD2-SAP) as an inducer. p16hCD2 mice possess an intact Cdkn2a locus that includes a p16 coding region and human CD2 (hCD2) expression unit. We confirmed cognate p16-associated hCD2 expression in mouse embryonic fibroblasts (MEFs) and in several tissues, such as the spleen, liver, and skin. We detected chronological increases in the hCD2-positive population in T lymphocytes that occurred in a p16-dependent manner, which reflected physiological aging. We then confirmed the high sensitivity of hCD2-SAP to hCD2 and validated its efficacy to remove p16-positive cells, particularly in T lymphocytes. The multiple administration of hCD2-SAP for a prolonged p16-positive cell deficiency partially restored aging-related phenotypes in T lymphocytes, such as the contraction of the CD4+ naïve population and expansion of senescence-associated T cells. Our novel approach of targeting p16-positive senescent cells will provide novel insights into the mechanisms underlying physiological aging in vivo.

Single and Combinational Multiplex Base-Edited 'Universal' CAR T Cells in a Humanised Model of Primary CD7+CD33+ AML

Introduction Universal allogeneic donor CAR T cells are being developed as part of a 'deep conditioning' strategy against AML to secure disease clearance ahead of allogeneic stem cell transplantation (Allo-SCT). CD33 is widely expressed on AML, sometimes on the entire leukemia-associated populations, but is also present on healthy precursors; CD7 may be expressed on AML subpopulations and is present on normal T and NK cells, resulting in problematic fratricide effects during CAR T manufacture. Healthy donor derived CAR T cell banks generated using cytidine deamination base-editing and lentiviral transduction with CARs against both CD33 and CD7 circumvent such issues and could offer cell based anti-leukemic therapy alongside preparative effects ahead of allo-SCT. Here we investigated single and combinational BE-CAR T cells against primary human AML in vivo. Methods: Healthy donor lymphocytes were activated by anti-CD3/28 stimulation ahead of electroporation to deliver codon optimised base editor mRNA and guide RNAs against CD7, CD52 and TRBC, and lentiviral transduction to express either an anti-CD7 or anti-CD33 CAR. Magnetic bead depletion removed residual TCRab T cells, and disruption of CD7 ensured cells avoided fratricide. In vitro, antigen specific function was verified in cytotoxicity and cytokine release assays, and in vivo potency was confirmed against luciferase modified Molm14 and Kasumi lines in NSG mice. Responses against primary AML were investigated in a patient derived xenograft model (PDX) from a KIT mutated AML patient, established by serial transplantation, with full expression of CD33 (100% of blasts) and partial expression of CD7 (30% of blasts). Cells were injected into irradiated NSG mice (n=10 per group). On day 24 at a mean leukemic engraftment of 15% human CD45 positive cells in peripheral blood, mice were treated with either 10x106 BE-CAR33, BE-CAR7 or a combination of both or with anti-CD19-CAR-T cells (CAR19) as a control group (in total 20x106 T cells per mouse). AML engraftment, T cell persistence and expression of checkpoint markers were monitored by multicolour flow cytometric analysis. Results: On days 14 and 28 after CAR-T cell infusion, AML cells were readily detected in peripheral blood of control CAR19 animals (42.8% and 90% respectively) and the BE-CAR7 group (27.8% and 67.6%), but were notably reduced in the BE-CAR33 alone group (2.3% and 8.3%) and the BE-CAR33 and BE-CAR7 combination group (2.5% and 3.4%) (Figure 1A). Survival was significantly prolonged for BE-CAR33 and BE-CAR33/BE-CAR7 treated mice compared to the other groups (P<0.001) (Figure 1B). CD7+ AML cells were depleted to a mean of 1% and 4.5% in the BE-CAR33 group, to 0.6% and 0.7% in the BE-CAR33/BE-CAR7 and to 2.6% and 2.9% in the BE-CAR7 group, while CD7+ AML cells increased to 12% and 30% in the CAR19 group at day 14 and 28 after CAR-T cell infusion. Total CD2+CAR-T cell detection in peripheral blood ranged from 1.6 to 9.8% at day 14, 1 to 3.7% at day 28, and 0.8 to 2.7% at day 42, indicating durable persistence over the investigation period. Interestingly, PD-L1 on AML cells was present in all groups while checkpoint markers PD1, GAL9, TIM3, and LAG3 were significantly upregulated in BE-CAR33 cell treated mice, but not BE-CAR7 or CAR19 cell treated mice (P<0.001 for all comparisons). Conclusions: Lentiviral transduction and multiplexed base editing to remove TCR, CD52 and the shared AML/T lineage antigen CD7, enabled the generation of universal donor CAR T cells for combinational use. This data demonstrates robust activity of BE-CAR33 alone and in combination with BE-CAR7 against human CD7+CD33+AML in mice and highlights the importance of strategies that ensure complete targeting of leukemic populations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting C3d in Combination with Daratumumab:a Strategy to Enhance Therapeutic Potential in Multiple Myeloma

The monoclonal antibody (mAb) daratumumab (DARA) targeting CD38 can induce minimal residual disease negative remissions in patients with multiple myeloma (MM). However, majority of patients subsequently relapse or become refractory to treatment (RRMM). Rapid reduction of surface CD38 levels in MM cells following DARA infusion due to trogocytosis by monocytes and granulocytes leads to reduced complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC), effector functions that rely on antigen density. High expression of complement regulatory proteins, CD55 and CD59 on MM cell surface can also impede CDC activity. Therefore, modalities to overcome these limitations and improve clinical outcome for patients with RRMM are needed. Daratumumab is a strong activator of complement. While a subset of cells may be lysed by the membrane attack complex, all cells reacted with daratumumab are opsonized by C3d, the terminal component of C3. C3d remains covalently attached at the cell surface. Thus, the newly acquired membrane bound C3d constitutes a target for additional intervention, particularly when the cell surface CD38 is lost or low following treatment with DARA. Using phage display technology, we generated and characterized recombinant rabbit/human chimeric mAbs that selectively bind C3 fragments including C3d but not full length C3. We selected two high affinity anti-C3d mAbs (clones 5S8 and d301 with kD values 3.35 nM and 0.99 nM, respectively) that showed reactivity against both human and mouse C3d. To deposit C3d on myeloma cell lines (MM1-S, MM1-R, RPMI8226, NCI-H929, KMS-12-PE) without activating CDC, the cells were reacted with DARA in C5 depleted normal human serum. Both 5S8 and d301 bound to the C3d opsonized cells. Based on SPR analysis these two mAbs do not compete for C3d binding suggesting that they recognize distinct epitopes. In normal human serum, the anti-C3d mAbs enhanced killing by CDC of DARA-opsonized target cells compared to DARA alone (P<0.0001). The non-targeting IgG1 isotype control mAb trastuzumab (TRA) did not bind C3d or enhance DARA-mediated killing. To test the combination of DARA with anti-C3d mAbs in vivo, we used Balb/c-SCID mice bearing subcutaneous (s. c.) MM1-R tumors. First, using clones 5S8 and d301, we demonstrated in vivo binding of mouse C3d on MM1-R tumor cells harvested 24 h after a single intraperitoneal (i. p.) injection of DARA. Next, we challenged the mice s. c. with MM1-R cells and randomly divided them into the following treatment groups: 20 mg/kg DARA single agent (n=7), or combination treatments of 10 mg/kg of each of two mAbs: DARA + TRA (n=10), DARA + 5S8 (n=13), DARA + d301 (n=13). The mAbs were given via i. p. injections weekly starting on day 7 post tumor challenge and continued for 6 weeks. Palpable tumors were noticed in mice between days 7 and 14, and tumor growth was monitored by caliper measurements twice weekly. Mice were euthanized when tumors reached pre-defined limits. All the mice that received DARA + TRA showed progressive tumor growth with median survival of 38 days and all mice had to be euthanized by day 50. Doubling the dose of DARA to 20mg/kg slightly increased median survival to 43 days (P = 0.12 vs DARA+TRA). Two of 7 mice that received DARA alone (at 20 mg/kg) survived to day 175. Median survival for mice treated with DARA (10mg/kg) plus an anti-C3d mAb was 162 days (P = 0.006 for comparison to DARA 20mg/kg), and 13 of 26 (50%) mice in this cohort survived tumor-free beyond 200 days. There was no statistically significant difference between groups treated with 5S8 or d301 in combination with DARA (P = 0.23). All the surviving mice showed no evidence of tumor, weight loss or other morbidity indicating that they are essentially tumor-free. In summary, these results demonstrate that combination treatment of DARA with anti-C3d mAbs improves efficacy over single agent DARA leading to long-term survival in the MM.1R xenograft model. The combination of C3d targeting mAbs with DARA could improve clinical efficacy and overcome resistance to single agent therapy.

Population Pharmacokinetics (PPK) and Exposure-Response to Support Body Surface Area (BSA)-Based Dosing of Brentuximab Vedotin in Pediatric Patients with Advanced-Stage Newly-Diagnosed Hodgkin Lymphoma (HL)

Introduction Brentuximab vedotin is an antibody-drug conjugate (ADC), consisting of antibody cAC10 specific to human CD30 and the antimicrotubule agent monomethyl auristatin E (MMAE), conjugated by a protease-cleavable linker. PPK models were developed to identify factors impacting the pharmacokinetics of ADC and MMAE, and exposure-response analyses were performed to assess the risk-benefit profiles of brentuximab vedotin combined with doxorubicin, vinblastine, and dacarbazine (A+AVD) to support BSA-based dosing of brentuximab vedotin in pediatric patients with advanced-stage newly-diagnosed HL. Methods Data from two open-label phase 1/2 pediatric studies were included in the PPK analysis: study C25002 (NCT01492088), which was a dose-escalation study (brentuximab vedotin 1.4-1.8 mg/kg every 3 weeks) in patients with relapsed or refractory systemic anaplastic large cell lymphoma or HL, and study C25004 (NCT02979522) in patients with advanced-stage, newly-diagnosed HL (brentuximab vedotin 48 mg/m2 every 2 weeks [Q2W] +AVD; A 25 mg/m2, V 6 mg/m2, D 375 mg/m2). Samples for measuring ADC and MMAE concentrations and immunogenicity assessment were collected. Sources of brentuximab vedotin pharmacokinetic variability were quantified using nonlinear mixed-effects modeling. ADC exposures achieved by BSA-based dosing of brentuximab vedotin in pediatric patients were compared to those in adults who received weight-based dosing of brentuximab vedotin in the ECHELON-1 trial (NCT01712490). Exposure-response analyses included patients in study C25004 treated with brentuximab vedotin 48 mg/m2 Q2W in combination with AVD. Relationships between ADC/MMAE exposures, progression-free survival (PFS), and incidence of adverse events (AEs) were analyzed by logistic regression in patients receiving A+AVD. Results Data from 95 patients (6-17 years) informed pharmacokinetics models of ADC (linear 3-compartment with zero-order input and first-order elimination) and MMAE (2-compartment with first-order elimination and formation of MMAE from ADC). BSA (0.79-2.03 m2) and albumin level (23-51 g/L) were significant covariates for clearance of ADC and MMAE. Age, body weight, sex, and race were not identified as covariates for ADC and MMAE pharmacokinetics. Albumin concentrations did not result in clinically meaningful change in clearance of ADC and MMAE. BSA-based brentuximab vedotin dosing resulted in similar systemic exposures of ADC and MMAE across age groups (<12 [n=23], 12-16 [n=56], and >16 years [n=16]). In exposure-response analyses (n=59), steady-state ADC exposures (area under the curve [AUC]) with BSA-based dosing of brentuximab vedotin at 48 mg/m2 Q2W in pediatric patients were comparable to those with weight-based dosing of brentuximab vedotin at 1.2 mg/kg Q2W in adults in the ECHELON-1 trial (Figure). A significant increase (p<0.05) in the incidence of febrile neutropenia was related to increasing exposure of MMAE, consistent with what has been observed in adult patients. No apparent relationship was identified between ADC exposure and the incidence of peripheral neuropathy, grade ≥3 neutropenia, febrile neutropenia, or grade ≥3 treatment-emergent AEs, or between ADC/MMAE exposure and PFS. Conclusions PPK and exposure-response analyses support brentuximab vedotin 48 mg/m2 Q2W in combination with AVD as a treatment option for pediatric patients with advanced-stage newly-diagnosed HL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunophenotypic transformation in relapsed/refractory mantle cell lymphoma treated with human anti-CD5 chimeric antigen receptor T cells: A Case Report

Relapsed/refractory (R/R) mantle cell lymphoma (MCL) with primary drug resistance to Bruton tyrosine kinase inhibitor and mutated TP53 responds poorly to conventional treatments. Chimeric antigen receptor (CAR) T cell therapy has emerged as one of the most effective treatments for R/R B cell lymphoma. However, no reports of CD5 CAR T cell treatment for MCL have been reported. In this paper, we report a R/R MCL patient with primary drug resistance to BTK inhibitors and TP53 mutation enrolled in a human CD5 CAR T cell trial. Remission of the primary disease was observed half a month after CAR T cell infusion. However, ascites was observed 2 weeks later. Flow cytometry suggested disease progression and immunophenotypic transformation. CD5 in CAR T cells turned negative and the expression of CD38 was enhanced. The patient was treated with a combination of daratumumab and Gemox (gemcitabine + oxaliplatin), abdominal distension and pain were markedly reduced, and ascites disappeared. We report the first case of human CD5 CAR T cell treatment for a patient with R/R MCL, providing insight on treatment strategies for such patients.

Tooth Root Formation

The formation of the tooth crown implies dentin and pulp construction (including mantle dentin, odontoblasts, Höhl cells, and vascular, axons and pulp cells). The root formation involves acellular and cellular cementum, peripheral and central dentins. Vascular cells and nerves are located in the central part of the root, whereas the superficial layer (Hopewell-Smith and Tomes granular layers) are located at the periphery. In the central part of the pulp, fibroblasts and immunocompetent cells are identified as T-lymphocytes, B-lymphocytes, dendritic cells, NK cells. In human deciduous teeth, HLA-DR+, CD68+, and factor XIIIa are present, whereas T and B-lymphocytes form clusters. The Hertwig’s Epithelial Enamel Epithelium (HERS’s) is formed by two layers. The HERS’s cells dissociate, and cells, taking origin from the dental follicular sac and from the papilla, migrates. The OEE becomes periodontal ligament cells whereas the IEE becomes cementoblasts. The interconversion of epithelial cells into mesenchymal cells seems to be a crucial event in tooth root formation. Tooth eruption is associated to root lengthening. The temporary tooth is gradually destroyed (root cementoclasts), whereas the permanent coronal part of the tooth erupts and acquire it final shape and position.

FeMV is a cathepsin-dependent unique morbillivirus infecting the kidneys of domestic cats

Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.

Exosome biopotentiated hydrogel restores damaged skeletal muscle in a porcine model of stress urinary incontinence

Urinary incontinence afflicts up to 40% of adult women in the United States. Stress urinary incontinence (SUI) accounts for approximately one-third of these cases, precipitating ~200,000 surgical procedures annually. Continence is maintained through the interplay of sub-urethral support and urethral sphincter coaptation, particularly during activities that increase intra-abdominal pressure. Currently, surgical correction of SUI focuses on the re-establishment of sub-urethral support. However, mesh-based repairs are associated with foreign body reactions and poor localized tissue healing, which leads to mesh exposure, prompting the pursuit of technologies that restore external urethral sphincter function and limit surgical risk. The present work utilizes a human platelet-derived CD41a and CD9 expressing extracellular vesicle product (PEP) enriched for NF-κB and PD-L1 and derived to ensure the preservation of lipid bilayer for enhanced stability and compatibility with hydrogel-based sustained delivery approaches. In vitro, the application of PEP to skeletal muscle satellite cells in vitro drove proliferation and differentiation in an NF-κB-dependent fashion, with full inhibition of impact on exposure to resveratrol. PEP biopotentiation of collagen-1 and fibrin glue hydrogel achieved sustained exosome release at 37 °C, creating an ultrastructural “bead on a string” pattern on scanning electron microscopy. Initial testing in a rodent model of latissimus dorsi injury documented activation of skeletal muscle proliferation of healing. In a porcine model of stress urinary incontinence, delivery of PEP-biopotentiated collagen-1 induced functional restoration of the external urethral sphincter. The histological evaluation found that sustained PEP release was associated with new skeletal muscle formation and polarization of local macrophages towards the regenerative M2 phenotype. The results provided herein serve as the first description of PEP-based biopotentiation of hydrogels implemented to restore skeletal muscle function and may serve as a promising approach for the nonsurgical management of SUI.

SAM-based Genome Editing to Obtain Isogenic Cell Lines Overexpressing Human CD5 Protein

In vitro and in vivo experiments often require construction of convenient cell instruments to reliably assess the specificity of the molecules or therapeutic approaches being tested against the protein target of interest. Using model isogenic cell lines that differ only in the expression of the target protein represents an ideal solution to this problem. Cloning and efficient delivery of genetic cassettes encoding such proteins, particularly the large ones, is typically challenging, much as the knock-out of the respective genes. To tackle this issue, we adapted a CRISPR/ Cas9-based SAM (Synergistic Activation Mediator) platform, and successfully established four model isogenic cell line pairs (U343, HeLa, HT-1080 и HEp-2) overexpressing human CD5.

Anti-Human CD9 Fab Fragment Antibody Blocks the Extracellular Vesicle-Mediated Increase in Malignancy of Colon Cancer Cells.

Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand–receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies.

The extracellular sialidase NEU3 primes neutrophils.

Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have 4 sialidases and can be elevated in inflammation and fibrosis. In this report, we show that incubation of human neutrophils with the extracellular human sialidase NEU3, but not NEU1, NEU2 or NEU4, induces human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed human neutrophil markers CD11b, CD18, and CD66a to localize to the cell cortex, and decreases the localization of the unprimed human neutrophil markers CD43 and CD62-L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. Human neutrophils treated with NEU3 show a decrease in cortical levels of Sambucus nigra lectin staining and an increase in cortical levels of peanut agglutinin staining, indicating a NEU3-induced desialylation. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

A dual-responsive bio-amicable fluorophore for trace level recognition of Zn2+ and Cd2+: Prefatory diagnosis of neoplastic disease from urine and ALS from saliva

A fluorogenic and electrochemical chemosensor BHMMP has been synthesized for aqueous phase ppb level detection of Zn2+ and Cd2+. The chemosensor exhibited distinct CHEF-based turn-on fluorometric transformation upon the addition of Zn2+ and Cd2+ respectively. Distinct fluorogenic response of BHMMP in presence of urinary excreted Zn2+ and human salivary Cd2+ indorsed the chemosensor towards a prefatory diagnostic tool for urinary Neoplastic disease and salivary Amyotrophic Lateral Sclerosis (ALS). The bio-compatibility of BHMMP was validated through in-vitro detection of Zn2+ and Cd2+ distinctively in the MCF-7 cancer cell line. BHMMP exhibited a paper-strip-based distinct fluorogenic response upon the addition of a trace amount of Zn2+ and Cd2+. A lumino-readable ‘information storage system’ was developed which strengthened the on-field applicability of BHMMP. The sensing response was rejuvenated by the addition of EDTA2−, which showcased a design of a four-input-three-output ‘fishtail’ mimicking logic gate circuitry. Sensing phenomena were corroborated through, Photoluminescence, ESI-MS, cyclic voltammetry, 1H NMR, FTIR, and DLS along with an in-silico approach. Additionally, synthesized ‘H’ type organic nano aggregate out of BHMMP (BHMMP-ONA) exhibited F− scavenging efficiency in a wide range of contaminated water samples. Therefore, the multichannel applications of this probe are expected to find imminent effectiveness in the realm of point-of-care testing.

A Human CD68 Promoter-Driven Inducible Cre-Recombinase Mouse Line Allows Specific Targeting of Tissue Resident Macrophages.

Current genetic tools designed to target macrophages in vivo often target cells from all myeloid lineages. Therefore, we sought to generate a novel transgenic mouse which has a tamoxifen inducible Cre-recombinase under the control of the human CD68 promoter (hCD68-CreERT2). To test the efficiency and specificity of the of Cre-recombinase activity we crossed the hCD68-CreERT2 mice with a loxP-flanked STOP cassette red fluorescent protein variant (tdTomato) mouse. We established that orally dosing mice with 2 mg of tamoxifen for 5 consecutive days followed by a 5-day induction period resulted in robust expression of tdTomato in CD11b+ F4/80+ tissue resident macrophages. Using this induction protocol, we demonstrated tdTomato expression within peritoneal, liver and spleen macrophages and blood Ly6Clow monocytes. Importantly there was limited or no inducible tdTomato expression within other myeloid cells (neutrophils, monocytes, dendritic cells and eosinophils), T cells (CD4+ and CD8+) and B cells (CD19+). We also demonstrated that the level of tdTomato expression can be used as a marker to identify different populations of peritoneal and liver macrophages. We next assessed the longevity of tdTomato expression in peritoneal macrophages, liver and splenic macrophages and demonstrated high levels of tdTomato expression as long as 6 weeks after the last tamoxifen dose. Importantly, hCD68-CreERT2 expression is more restricted than that of LysM-Cre which has significant expression in major myeloid cell types (monocytes and neutrophils). To demonstrate the utility of this novel macrophage-specific Cre driver line we demonstrated tdTomato expression in recruited CD11b+CD64+F4/80+ monocyte-derived macrophages within the atherosclerotic lesions of AAV8-mPCSK9 treated mice, with limited expression in recruited neutrophils. In developing this new hCD68-CreERT2 mouse we have a tool that allows us to target tissue resident macrophages, with the advantage of not targeting other myeloid cells namely neutrophils and inflammatory monocytes.

Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows.

CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other's results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.

Surface Plasmon Resonance Immunosensor with Antibody-Functionalized Magnetoplasmonic Nanoparticles for Ultrasensitive Quantification of the CD5 Biomarker

A surface plasmon resonance (SPR) immunosensor signal amplification strategy based on antibody-functionalized gold-coated magnetic nanoparticles (mAuNPs) was developed for ultrasensitive and quantitative detection of the CD5 biomarker using an indirect sandwich immunoassay format. The gold surface of the SPR sensor disk and mAuNPs was modified with a self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA), and the coupling method using N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide was used to immobilize capture antibodies against human CD5 (anti-CD52A) and detection antibodies against human CD5 (anti-CD52B), respectively. The mAuNPs and anti-CD52B conjugates (mAuNPs–anti-CD52B) were separated by an external magnetic field and used to amplify the SPR signal after the formation of the anti-CD52A/CD5 immune complex on the SPR sensor disk. Compared to the direct CD5 detection with a limit of detection (LOD) of 1.04 nM and a limit of quantification (LOQ) of 3.47 nM, the proposed sandwich immunoassay utilizing mAuNPs–anti-CD52B significantly improved the LOD up to 8.31 fM and the LOQ up to 27.70 fM. In addition, it showed satisfactory performance in human blood serum (recovery of 1.04 pM CD5 was 109.62%). These results suggest that the proposed signal amplification strategy has superior properties and offers the potential to significantly increase the sensitivity of the analysis.

ScRNA-Seq analysis of human CD5+ innate-like B cells identifies AHR expression as a marker of human CD9+ IL-10+ BRegulatory cells

Abstract B lymphocytes are adaptive immune cells responsible for the production of antigen specific antibodies and the release of pro- and anti-inflammatory cytokines. A previous report from our laboratory found that human, primary CD5+ innate-like B cells (ILB) were preferentially sensitive to aryl hydrocarbon receptor (AHR)-mediated immune suppression compared to CD5− adaptive B cells. CD5+ ILB are a heterogenous population of B cells unified by their expression of CD5 and thought to encompass IgM memory B cells, human B1 B cells, marginal zone B cells, and regulatory B cells among others. Specifically, we demonstrated that CD5+ ILB were marked by elevated expression of programmed cell death protein-1 (PD-1) and its ligands. We also identified a putative role for AHR in the expression of PD-1, suggesting AHR may be important for B cell regulatory functions. Unlike TRegulatory cells, BRegulatory cells are heterogenous, unidentifiable by FoxP3 expression, and express a wide range of surface proteins. To elucidate AHR expression in human B cell populations, naïve, circulating CD19+ B cells were isolated directly from PBMC and enriched for CD5 expression. We then used single cell RNA-sequencing (scRNA-seq) to identify different B cell subsets found within human CD5+ ILB. We found that AHR expression was strongly correlated with expression of CD9 and IL10 as well as CYP1B1. Interestingly, CD9 is reported as a marker of IL-10 producing Bregs suggesting a role for AHR in Breg IL-10 production. These findings were confirmed directly with qRT-PCR and flow cytometry. (Supported by NIH grant P42ES004911) Supported by NIH grant P42ES004911

Role of Programmed Cell Death Protein-1 and Lymphocyte Specific Protein Tyrosine Kinase in the Aryl Hydrocarbon Receptor- Mediated Impairment of the IgM Response in Human CD5+ Innate-Like B Cells.

Innate-like B cells (ILBs) are a heterogeneous population B cells which participate in innate and adaptive immune responses. This diverse subset of B cells is characterized by the expression of CD5 and has been shown to secrete high levels of immunoglobulin M (IgM) in the absence of infection or vaccination. Further, CD5+ ILBs have been shown to express high basal levels of lymphocyte specific protein tyrosine kinase (LCK) and programmed cell death protein-1 (PD-1), which are particularly sensitive to stimulation by interferon gamma (IFNγ). Previous studies have demonstrated that activation of the aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, results in suppressed IgM responses and is dependent on LCK. A recent study showed that CD5+ ILBs are particularly sensitive to AHR activation as evidenced by a significant suppression of the IgM response compared to CD5- B cells, which were refractory. Therefore, the objective of this study was to further investigate the role of LCK and PD-1 signaling in AHR-mediated suppression of CD5+ ILBs. In addition, studies were conducted to establish whether IFNγ alters the levels of LCK and PD-1 in CD5+ ILBs. We found that AHR activation led to a significant upregulation of total LCK and PD-1 proteins in CD5+ ILBs, which correlated with suppression of IgM. Interestingly, treatment with recombinant IFNγ reduced LCK protein levels and reversed AHR-mediated IgM suppression in CD5+ ILBs in a similar manner as LCK inhibitors. Collectively, these results support a critical role for LCK and PD-1 in AHR-mediated suppression of the IgM response in human CD5+ ILBs.

Isolation and molecular characterization of circulating extracellular vesicles from blood of chronic Chagas disease patients

Extracellular vesicles (EVs) are lipid bilayer envelopes that encase several types of molecules. Their contents mostly reflect their cell origin and possible targets at other locations in the organism and can be modified in pathological conditions to interfere with intercellular communication, thus promoting disease establishment and development. These characteristics, in addition to their presence in virtually all body fluids, make such vesicles ideal for biomarker discovery in human diseases. Here, we describe the effect of different anticoagulants and the combination of two purification methods for isolation and characterization of circulating EVs from blood of chronic Chagas disease (CCD) patients. We illustrated this procedure by studying a population of patients with Chagas disease at the indeterminate chronic stage, in which the Trypanosoma cruzi is very scarce in circulation. EVs were harvested from blood collected without or with different anticoagulants. Protein and nanoparticle tracking analysis was used to measure EVs size and concentration. The EVs were purified by ultracentrifugation, followed by size-exclusion chromatography and characterized by chemiluminescent enzyme-linked immunosorbent assay and dot blot using antibodies that recognized parasite-derived EVs, such as hyperimmune sera, polyclonal and monoclonal antibodies against trans-sialidase and mucins. In parallel, antibodies against classical human EV markers CD9, CD63, CD81, and CD82, were also analyzed. The results showed that anticoagulants did not interfere with the analyzed parameters and circulating EVs from CCD patients contain T. cruzi antigens and classical human exosomal markers. Overall, our protocol is adequate for the isolation of the total circulating EVs and can serve as an important basis for further studies on biomarker discovery in Chagas' disease.

Identification of Scd5 as a functional regulator of visceral fat deposition and distribution

SummaryEctopic deposition of visceral adipose tissue (VAT) in abdomen is usually accompanied with systematic chaos of energy metabolism, a higher risk of cardiovascular diseases and type II diabetes. Here, we identified a previously unexplored gene Scd5 as a master regulator of fat distribution, which alone plays a significant role in determining the VAT accumulation. Firstly, zebrafish scd5 had the highest homology with human SCD5 compared to other SCDs in mouse and rat. We then observed that scd5-homozygous mutant zebrafish displayed a puffy, short and rounded apple-shaped figure. Whole-mount micro-CT scan showed that excessive VAT deposition and short spine are responsible for the abnormal body ratio. And the supplementation of ω3-polyunsaturated fatty acid (ω3-PUFA) in dietary significantly decreased VAT accumulation in scd5−/− zebrafish. Lastly, transcriptional analyses revealed that the Wnt, PPAR, C/EBP, and fat synthesis signaling pathways are significantly affected in the VAT of scd5−/− mutant and restored by ω3-PUFA.