Surface CD4 Research Articles

Abstract 2334: Reprogramming M2-like tumor associated macrophages using a natural compound, glycyrrhizin, as a potential therapeutic strategy against prostate cancer

Abstract Tumor Associated Macrophages (TAMs), copiously found in the Tumor Microenvironment (TME) are key players in creating an immunosuppressive environment, increasing angiogenesis and metastasis in prostate cancer (PCa). They are plastic in nature and usually exist as, M1 (anti-tumorigenic) or M2 (pro-tumorigenic) phenotype, with relative abundance of M2. In this study, we aimed to explore the phenotypic effects of TAMs on PCa and investigate the potential of glycyrrhizin, a natural compound, to reprogram M2 TAMs, as a feasible treatment approach against PCa. THP-1 monocytes and RAW 264.7 were used to study macrophage differentiation and glycyrrhizin-mediated repolarization. THP-1 cells were cultured in conditioned media (CM) of LNCaP and BPH-Cd to obtain PCa associated macrophages. Flow cytometric analysis revealed increase in expression of surface markers CD32, CD64, CD206 and CD163 confirming M1/M2 differentiation. qPCR analysis also demonstrated upregulation of CD80, CD86, CD68, TGF-β and IL10. M2 markers such as CD68, CD163, CCL2, CCL5 and TGF-β were expressed in BPH-Cd TAMs. LNCaP TAMs also co-expressed CD68 and CD163. In spheroid assay on BPH-Cd cells, treatment with BPH-Cd TAM resulted in abnormally shaped, enlarged spheroids. Furthermore, the CM enhanced migration of BPH-Cd cells in transwell migration assay and downregulated P21 in western blot analysis, thus confirming its tumor-promoting role. MTT assay performed prior to studying the polarization potential of glycyrrhizin showed an increase in viability of glycyrrhizin-treated THP-1 and RAW 264.7 and selectively promoted viability in M1. The M1-polarizing properties of glycyrrhizin was revealed in the qPCR analysis of glycyrrhizin-treated M0 macrophages. The reduction in the size of the spheroids and decreased migration of BPH-Cd upon treatment with glycyrrhizin-treated M0 CM confirmed the same. Differentiation media (DM) of glycyrrhizin-treated M2 TAMs resulted in smaller, disintegrated BPH-Cd spheroids indicating possible reprogramming effect. Interestingly, glycyrrhizin-treated M2 CM downregulated the gene expression of stemness-associated markers such as NANOG, SNAIL, OCT3/4 and COX2 with concurrent suppression of EMT marker, N-Cadherin in LNCaP cells. Cell cycle analysis on LNCaP revealed a decrease in the percentage of cells in S phase compared to glycyrrhizin-treated M2 CM, suggesting hindered proliferation. A notable observation to help understand the plausible mechanism of M2 TAM suppression was the decrease in Reactive Oxygen Species (ROS) levels in glycyrrhizin-treated M1 and M2 TAMs derived from THP-1 cells. The glycyrrhizin-mediated inhibition of ROS was also confirmed in RAW 264.7. In conclusion, our findings suggest that glycyrrhizin could potentially reprogram TAMs to intercept proliferation, metastasis and stemness characteristics in PCa. Citation Format: Amy Thomas, Gnanasekar Munirathinam. Reprogramming M2-like tumor associated macrophages using a natural compound, glycyrrhizin, as a potential therapeutic strategy against prostate cancer [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 2334.

Abstract 4123: Rhus verniciflua Stokes (RVS) inhibits PD-1 expression and induces anticancer effects via enhancing T-cell function

Abstract Rhus verniciflua Stokes (RVS) has traditionally been used for centuries as a therapeutic herb and food supplement in East Asian countries. Over the last decade, the anticancer effects of RVS have been demonstrated in various preclinical and clinical studies. RVS contains major flavonoids such as fustin, fisetin, and sulfuretin, which were known to have apoptotic or anti-proliferative activities in various cancer cell lines. In clinical retrospective studies, RVS administration exclusively suggested more favorable outcomes with prolonged overall survival in solid cancer patients. However, the effect of RVS on immuno-oncology, especially the functional properties of T cells and their phenotypes remain unclear. Dried RVS grown in Wonju (Korea) was extracted with sterile distilled water after removing the allergen (urushiol). Peripheral blood mononuclear cells (PBMCs) from breast cancer patients with HLA-A*02:01 were isolated using a vacutainer tube and cultured for T cell expansion in the presence of interleukin (IL)-2 for 14 days. Cells were enriched above 90% CD3+ T cells after 14 days of expansion. Red fluorescent protein (RFP)-expressing HLA-A*02:01-matched breast cancer cell lines (MCF7 and MDA-MB-231), were used to determine the anticancer activity of T cells in the presence or absence of RVS. For in vitro killing assays, MCF7 and MDA-MB-231 cell lines expressing RFP were co-cultured with T cells with or without RVS treatment for 48 h, and cell viability was measured by the MTT assay. The phenotypic characteristics of the T cells were profiled by staining with various T cell surface markers, including CD3, CD4, CD8, PD-1, and CTLA4. RVS toxicity in the target cells (MCF7 and MDA-MB-231) was not observed at RVS concentrations up to 250 ug/mL. The anticancer activity of T cells against breast cancer cells was significantly increased by adding both 10 ug/mL and 100ug/mL of RVS. T cells co-cultured with MCF7 and MDA-MB-231 cells in the presence of 100 ug/mL of RVS showed 20.6% increases in cytotoxicity in MCF7 cells and 36.2% in MDA-MB-231 cells compared to no RVS treatment. Interestingly, relative reductions in programmed death-1 (PD-1) were found in T cells co-cultured with target cells by adding RVS, even though there was no significant difference in the other markers observed. Our findings showed that RVS could improve the function of T cells against cancer cells in the tumor microenvironment, as interpreted by reduced PD-1 expression in T cells after RVS treatment. Therefore, the components of RVS are candidates for restoring T cells exhausted against cancer. Active compounds should be identified for clinical efficacy, and further studies are necessary for combination strategies with conventional cancer treatments, such as chemotherapy and radiotherapy. Citation Format: Seoyoung Kim, Young-Kwan Lee, Hyoun Jong Moon, Sunki Lim, Yujeong Gho, Wang Jun Lee, Sanghun Lee, Excelsisbio Inc.. Rhus verniciflua Stokes (RVS) inhibits PD-1 expression and induces anticancer effects via enhancing T-cell function. [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 4123.

Abstract 1751: Prognostic value of CD20/CD73 ratio in sensitive and relapsed/refractory diffuse large B-cell lymphoma cell lines

Abstract Purpose: Developing novel therapy strategies to treat relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) is critically needed. Previous studies in our group found despite dysregulation of apoptotic pathway in resistant DLBCL, upregulation in glycose metabolism and energetic ATP production were also found in r/r DLBCL. Therefore, decreasing or blockage ATP production may provide a novel method to overcome resistance in lymphoma. ATP/ADP is hydrolyzed into AMP by NTPDases, and AMP into adenosine and phosphate by ecto-5’-nucleotidase, known as CD73. CD73 is the rate limiting enzyme anchored the surface of tumor and immune cell. The expression level of CD73 on the tumor was reported significantly associated with inferior prognosis in variety of cancers. Moreover, expression level of CD73 on immune cell has been shown immunosuppression in multiple cancers. Targeting CD73 by monoclonal antibody or small inhibitor currently underwent multiple clinical trials. However, the role of CD73 in r/r DLBCL and the relationship between CD73 and CD20 have not been explored. Methods: Germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines generated in our lab were used. Surface expression of CD73 and CD20 were determined by staining anti-CD73 FITC and anti-CD20 Per-cy5.5 antibodies and running flow cytometry. The expression levels of CD73 and CD20 were detected by media fluorescence index (MFI). The CD20/CD73 ratio was calculated by CD73 MFI/CD20 MFI. Result: Previously our group found surface CD20 levels exhibited a linear correlation with rituximab induced cellular mediated cytotoxicity. The CD20 MFI of germinal center B-cell like, activated B-cell like lymphoma, Burkitt’s lymphoma and rituximab resistant cell lines were: DHL4 8652, DOHH2 6472, TMD8 3981, RL 2849, Raji 2011, U2932 604, RL 4RH 599 and Raji 4RH 576. The higher MFI of CD20, the more sensitive to rituximab treatment. Here we discovered that the surface level of CD73 had an inverse correlation with CD20 level in these cell lines. In detail, CD73 MFI were: DHL4 464, DOHH2 387, TMD8 363, RL 299, Raji 611, U2932 486, RL 4RH 631 and Raji 4RH 702. The CD20/CD73 ratio of cell lines from high to low were DOHH2, DHL4, TMD8, RL, Raji, U2932, Raji 4RH and RL 4RH, respectively. This CD20/CD73 ratio in each of cell lines was found more straight forward to represent the sensitivity of rituximab or chemo drug treatment in vitro condition. Conclusion: Our study was first time to reveal the association between CD20/CD73 ratio and immune-chemo response in lymphoma cell lines, especially in resistant lymphoma in vitro condition. This may give us prognostic value of CD20/CD73 ratio in predicting lymphoma response to immune/chemotherapy in clinical setting. Further study to find CD20 and CD73 association in the clinical setting and mechanism of action need to be warranted. Citation Format: Juan J. Gu, Cory Mavis, Jessica Wang, Taylor Mandeville, Francisco Hernandez-Ilizaliturri. Prognostic value of CD20/CD73 ratio in sensitive and relapsed/refractory diffuse large B-cell lymphoma cell lines [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 1751.

Adiponectin Enhances Fatty Acid Signaling in Human Taste Cells by Increasing Surface Expression of CD36.

Adiponectin, a key metabolic hormone, is secreted into the circulation by fat cells where it enhances insulin sensitivity and stimulates glucose and fatty acid metabolism. Adiponectin receptors are highly expressed in the taste system; however, their effects and mechanisms of action in the modulation of gustatory function remain unclear. We utilized an immortalized human fungiform taste cell line (HuFF) to investigate the effect of AdipoRon, an adiponectin receptor agonist, on fatty acid-induced calcium responses. We showed that the fat taste receptors (CD36 and GPR120) and taste signaling molecules (Gα-gust, PLCβ2, and TRPM5) were expressed in HuFF cells. Calcium imaging studies showed that linoleic acid induced a dose-dependent calcium response in HuFF cells, and it was significantly reduced by the antagonists of CD36, GPR120, PLCβ2, and TRPM5. AdipoRon administration enhanced HuFF cell responses to fatty acids but not to a mixture of sweet, bitter, and umami tastants. This enhancement was inhibited by an irreversible CD36 antagonist and by an AMPK inhibitor but was not affected by a GPR120 antagonist. AdipoRon increased the phosphorylation of AMPK and the translocation of CD36 to the cell surface, which was eliminated by blocking AMPK. These results indicate that AdipoRon acts to increase cell surface CD36 in HuFF cells to selectively enhance their responses to fatty acids. This, in turn, is consistent with the ability of adiponectin receptor activity to alter taste cues associated with dietary fat intake.

T-Cell Prolymphocytic Leukemia With t(X;14)(q28;q11.2): A Clinicopathologic Study of 15 Cases.

T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell leukemia usually characterized by inv(14)(q11.2q32)/t(14;14)(q11.2;q32). In this study, we aimed to investigate the clinicopathologic features and molecular profile of T-PLL associated with t(X;14)(q28;q11.2). The study group included 10 women and 5 men with a median age of 64 years. All 15 patients had a diagnosis of T-PLL with t(X;14)(q28;q11.2). All 15 patients had lymphocytosis at initial diagnosis. Morphologically, the leukemic cells had features of prolymphocytes in 11 patients, small cell variant in 3, and cerebriform variant in 1. All 15 patients had hypercellular bone marrow with an interstitial infiltrate in 12 (80%) cases. By flow cytometry, the leukemic cells were surface CD3+/CD5+/CD7+/CD26+/CD52+/TCR α/β+ in 15 (100%) cases, CD2+ in 14 (93%) cases, CD4+/CD8+ in 8 (53%) cases, CD4+/CD8- in 6 (40%) cases, and CD4-/CD8 + in 1 (7%) case. At the cytogenetic level, complex karyotypes with t(X;14)(q28;q11.2) were seen in all 15 patients assessed. Mutational analysis showed mutations of JAK3 in 5 of 6 and STAT5B p.N642H in 2 of 6 patients. Patients received variable treatments, including 12 with alemtuzumab. After a median follow-up of 17.2 months, 8 of 15 (53%) patients died. T-PLL with t(X;14)(q28;q11.2) frequently shows a complex karyotype and mutations involving JAK/STAT pathway, and it is an aggressive disease with a poor outcome.

Angioimmunoblastic T-Cell Lymphoma Diagnosed From Serous Effusion by Integration of Cytologic Features and Ancillary Studies.

To explore the approach to the diagnosis of malignant serous effusion (SE) caused by angioimmunoblastic T-cell lymphoma (AITL). The clinical, cytomorphologic, immunophenotypic, and molecular features of 6 patients were summarized. Clinically, SE caused by AITL was predominant in middle-aged and older male patients with multiple SEs and lymphadenopathy. Cytomorphology showed small to medium-sized, irregular lymphocytes with clear cytoplasm and mixed with various inflammatory cells and apoptosis. Hodgkin/Reed-Sternberg-like cells were detected in 2 of 6 cases. Furthermore, 2 patterns of cytomorphology were described for the first time. Flow cytometry revealed abnormal T-cell populations with loss of surface CD3 (3/4 cases) and CD7 (3/4 cases). In addition, B-cell populations lacking surface immunoglobulin (Ig) were identified in 2 of 4 cases. Immunocytochemical staining revealed expression of at least 2 T follicular helper markers. Epstein-Barr virus-encoded RNA (EBER)-positive cells were demonstrated in 4 of 5 cases. Clonal T-cell receptor γ chain rearrangement was detected in 6 cases, and 3 of them had concomitant clonal immunoglobulin gene rearrangement. Moreover, 2 cases revealed discrepant findings regarding IgH/Igκ rearrangements in cytohistologic correlation. This study broadens the morphologic spectrum of malignant SE caused by AITL and provides diagnostic criteria in routine practice.

Experimental Study on the Characteristic Changes of the Immunological Microenvironment at the Maternal-Fetal Interface in IVF-ET Pregnancy

To investigate the characteristic functional changes of the decidual natural killer (NK) cells and γδ T cells, two immunocytes in the decidua, at the maternal-fetal interface in in vitro fertilization-embryo transfer (IVF-ET) pregnancy. Decidual samples were collected from 12 women of natural pregnancy (NP) and 32 women of IVF-ET pregnancy, who were enrolled in the NP group and the IVF-ET group, respectively. Then part of the decidual samples were paraffin-embedded for HE staining and immunofluorescence staining, while the rest of the samples were digested and Percoll was used for isolating decidual immunocytes (DICs) by gradient centrifugation. Flow cytometry was used to determine the cell counts of decidual NK cells and γδ T cells and the expression levels of their surface activation markers, CD69 and NKG2D in the NP and the IVF-ET groups. In addition, the expression levels of IFN-γ, TNF-α, IL-17A, and IL-10, the intracellular cytokines, and granzyme B, perforin, and granulysin, the cytolytic granules, were measured. The characteristic changes in the relevant immunological indicators were compared and analyzed. HE staining of the tissue specimens showed that the typical structure of decidua was observed, and that lymphocytes were enriched in the decidua. Immunofluorescence staining showed that the percentage of decidual NK (dNK) cells in nucleated cells of the IVF-ET group was significantly lower than that of the NP group ( P<0.05). Flow cytometry analysis of DICs showed that, compared with those of the NP group, the percentage of dNK cells of the IVF-ET group was decreased ( P<0.05) and the expression levels of IL-10 and perforin were significantly decreased in the IVF-ET group ( P<0.05). However, there was no significant difference in the decidual γδ T (dγδT) cell count between the two groups. The expression of IL-10, IL-17A, and perforin was downregulated in the IVF-ET group ( P<0.05). There was no significant difference in the expression of IFN-γ, TNF-α, granzyme B, and granulysin, the cellular function indicators ( P>0.05). The dNK cell count and the secretion of some intracellular cytokines of dNK and dγδT cells of women of IVF-ET pregnancy decreased to some degree, which suggests that certain changes may have taken place in the immunological microenvironment at the maternal-fetal interface. The specific effect of these changes on pregnancy outcomes needs further investigation.

The association between altered intestinal microbiome, impaired systemic and ocular surface immunity, and impaired wound healing response after corneal alkaline-chemical injury in diabetic mice.

We aim to investigate the effect of sustained hyperglycemia on corneal epithelial wound healing, ocular surface and systemic immune response, and microbiome indices in diabetic mice compared to controls after alkaline chemical injury of the eye. Corneal alkaline injury was induced in the right eye of Ins2Akita (Akita) mice and wild-type mice. The groups were observed at baseline and subsequently days 0, 3, and 7 after injury. Corneal re-epithelialization was observed under slit lamp with fluorescein staining using a cobalt blue light filter. Enucleated cornea specimens were compared at baseline and after injury for changes in cornea thickness under hematoxylin and eosin staining. Tear cytokine and growth factor levels were measured using protein microarray assay and compared between groups and time points. Flow cytometry was conducted on peripheral blood and ocular surface samples to determine CD3+CD4+ cell count. Fecal samples were collected, and gut microbiota composition and diversity pattern were measured using shotgun sequencing. Akita mice had significantly delayed corneal wound healing compared to controls. This was associated with a reduction in tear levels of vascular endothelial growth factor A, angiopoietin 2, and insulin growth factor 1 on days 0, 3, and 7 after injury. Furthermore, there was a distinct lack of upregulation of peripheral blood and ocular surface CD3+CD4+ cell counts in response to injury in Akita mice compared to controls. This was associated with a reduction in intestinal microbiome diversity indices in Akita mice compared to controls after injury. Specifically, there was a lower abundance of Firmicutes bacterium M10-2 in Akita mice compared to controls after injury. In diabetic mice, impaired cornea wound healing was associated with an inability to mount systemic and local immune response to ocular chemical injury. Baseline and post-injury differences in intestinal microbial diversity and abundance patterns between diabetic mice and controls may potentially play a role in this altered response.

Engineering d-band states of (CuGa)xZn1-2xGa2S4 material for photocatalytic syngas production

The d-band states of catalytic materials participate in adsorbing reactive intermediate species and determine the catalytic behaviors in CO2 reduction reactions. However, surface d-band states relating to the photocatalytic CO2 reduction reactions behaviors are rarely concerned. Herein, a slightly amount of Cd2+ is decorated on the surface of (CuGa)xZn1-2xGa2S4 material (Cd2+/(CuGa)xZn1-2xGa2S4) to tune the surface d-band states for improved CO2 reduction reactions. The Cd2+/(CuGa)xZn1-2xGa2S4 is fabricated via the facile ions-exchange method to make that slightly Zn2+ is substituted by Cd2+. The Cd2+/(CuGa)xZn1-2xGa2S4 exhibits much enhanced photocatalytic activity in CO2 reduction reactions to produce CO and water splitting to produce H2. Physical characterizations show that the energy band structure is not changed obviously. Density functional theory reveals that Cd2+/(CuGa)xZn1-2xGa2S4 possesses a closer shift of d-band center to Fermi level than (CuGa)xZn1-2xGa2S4, suggesting easier adsorption of CO2 reduction reactive intermediates after Cd2+ decoration. Further calculations confirm that a relatively reduced adsorption Gibbs energy of reactive intermediates in CO2 reduction reaction is required on Zn atoms in Cd2+/(CuGa)xZn1-2xGa2S4 material, benefiting the photocatalytic CO2 reduction reactions. This work engineers surface d-band states by surface Cd2+ decoration, which gives an effective strategy to design highly efficient photocatalysts for syngas production.

The prohibitin complex regulates macrophage fatty acid composition, plasma membrane packing, and lipid raft-mediated inflammatory signaling

Prohibitins (PHB1 and PHB2) are ubiquitously expressed proteins which play critical roles in multiple biological processes, and together form the ring-like PHB complex found in phospholipid-rich cellular compartments including lipid rafts. Recent studies have implicated PHB1 as a mediator of fatty acid transport as well as a membrane scaffold mediating B lymphocyte and mast cell signal transduction. However, the specific role of PHBs in the macrophage have not been characterized, including their role in fatty acid uptake and lipid raft-mediated inflammatory signaling. We hypothesized that the PHB complex regulates macrophage inflammatory signaling through the formation of lipid rafts. To evaluate our hypothesis, RAW 264.7 macrophages were transduced with shRNA against PHB1, PHB2, or scrambled control (Scr), and then stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-α), which activate lipid raft-dependent receptor signaling (CD14/TLR4 and TNFR1, respectively). PHB1 knockdown was lethal, whereas PHB2 knockdown (PHB2kd), which also resulted in decreased PHB1 expression, led to attenuated nuclear factor-kappa-B (NF-κB) activation and subsequent cytokine and chemokine production. PHB2kd macrophages also had decreased cell surface TNFR1, CD14, TLR4, and lipid raft marker ganglioside GM1 at baseline and post-stimuli. Post-LPS, PHB2kd macrophages did not increase the concentration of cellular saturated, monounsaturated, and polyunsaturated fatty acids. This was accompanied by decreased lipid raft formation and modified plasma membrane molecular packing, further supporting the PHB complex's importance in lipid raft formation. Taken together, these data suggest a critical role for PHBs in regulating macrophage inflammatory signaling via maintenance of fatty acid composition and lipid raft structure. SummaryProhibitins are proteins found in phospholipid-rich cellular compartments, including lipid rafts, that play important roles in signaling, transcription, and multiple other cell functions. Macrophages are key cells in the innate immune response and the presence of membrane lipid rafts is integral to signal transduction, but the role of prohibitins in macrophage lipid rafts and associated signaling is unknown. To address this question, prohibitin knockdown macrophages were generated and responses to lipopolysaccharide and tumor necrosis factor-alpha, which act through lipid raft-dependent receptors, were analyzed. Prohibitin knockdown macrophages had significantly decreased cytokine and chemokine production, transcription factor activation, receptor expression, lipid raft assembly and membrane packing, and altered fatty acid remodeling. These data indicate a novel role for prohibitins in macrophage inflammatory signaling through regulation of fatty acid composition and lipid raft formation.

Intestinal and blood lymphograms as new diagnostic tests for celiac disease.

Accurate celiac disease (CD) diagnosis is still challenging for some specific patients or circumstances. Thus, much effort has been expended last decades focused on seronegative or low grade enteropathy CD and, especially, on enable early diagnosis of individuals on a gluten-free diet (GFD). We discuss here two diagnostic approaches based on immunophenotyping by flow cytometry that we expect to reduce the persistent low diagnostic rates and the common diagnostic delay. The intraepithelial lymphogram is based on determining the percentage of TCRγδ+ and surface CD3- lymphocytes in the intestinal epithelium. The concomitant increase in TCRγδ+ and decrease in surface CD3- intraepithelial lymphocytes has been termed the celiac lymphogram and has been proved to be discriminative in seronegative, low grade enteropathy and potential CD, as well as in most CD patients on a GFD. A blood lymphogram based on the analysis of activated gut-homing CD8+ T cells combined with a 3-day gluten challenge is also considered, which has shown high sensitivity and specificity to diagnose seropositive Marsh 1 and Marsh 3 CD in individuals following a GFD. In addition, flow cytometry can be extremely useful in cases of refractory CD type II to identify aberrant cells. Those approaches represent highly accurate methods for CD diagnosis, being simple, fast, highly reproducible and of easy implementation in clinical practice.

Airway and Systemic Prostaglandin E2 Association with COPD Symptoms and Macrophage Phenotype.

Polymorphisms and products of the cyclooxygenase (COX) pathway have been associated with the development of chronic obstructive pulmonary disease (COPD) and adverse outcomes. COX-produced prostaglandin E2 (PGE-2) may play a role in the inflammation observed in COPD, potentially through deleterious airway macrophage polarization. A better understanding of the role of PGE-2 in COPD morbidity may inform trials for therapeutics targeting the COX pathway or PGE-2. Urine and induced sputum were collected from former smokers with moderate-severe COPD. The major urinary metabolite of PGE-2 (PGE-M) was measured, and ELISA was performed on sputum supernatant for PGE-2 airway measurement. Airway macrophages underwent flow cytometry phenotyping (surface CD64, CD80, CD163, CD206, and intracellular IL-1β, TGF-β1). Health information was obtained the same day as the biologic sample collection. Exacerbations were collected at baseline and then monthly telephone calls. Among 30 former smokers with COPD (mean±SD age 66.4±8.88 years and forced expiratory volume in 1 second [FEV1] 62.4±8.37 percent predicted), a 1 pg/mL increase in sputum PGE-2 was associated with higher odds of experiencing at least one exacerbation in the prior 12 months (odds ratio 3.3; 95% confidence interval: 1.3 to15.0), worse respiratory symptoms and health status. PGE-M was not associated with exacerbations or symptoms. Neither airway PGE-2 nor urinary PGE-M was uniformly associated with an M1 or M2 polarization. Elevated levels of sputum PGE-2, rather than systemic PGE-2, is associated with increased respiratory symptoms and history of exacerbation among individuals with COPD. Additional studies focused on mechanism of action are warranted.

CD19 Is Internalized Together with IgM in Proportion to B Cell Receptor Stimulation and Is Modulated by Phosphatidylinositol 3-Kinase in Bone Marrow Immature B Cells.

Newly generated immature B cells that bind self-antigen with high avidity arrest in differentiation and undergo central tolerance via receptor editing and clonal deletion. These autoreactive immature B cells also express low surface levels of the coreceptor CD19, a key activator of the PI3K pathway. Signals emanating from both CD19 and PI3K are known to be critical for attenuating receptor editing and selecting immature B cells into the periphery. However, the mechanisms that modulate CD19 expression at this stage of B cell development have not yet been resolved. Using in vivo and in vitro models, we demonstrate that Cd19 de novo gene transcription and translation do not significantly contribute to the differences in CD19 surface expression in mouse autoreactive and nonautoreactive immature B cells. Instead, CD19 downregulation is induced by BCR stimulation in proportion to BCR engagement, and the remaining surface IgM and CD19 molecules promote intracellular PI3K-AKT activity in proportion to their level of expression. The internalized CD19 is degraded with IgM by the lysosome, but inhibiting lysosome-mediated protein degradation only slightly improves surface CD19. In fact, CD19 is restored only upon Ag removal. Our data also reveal that the PI3K-AKT pathway positively modulates CD19 surface expression in immature B cells via a mechanism that is independent of inhibition of FOXO1 and its role on Cd19 gene transcription while is dependent on mTORC1.

The effect of ox-LDL and platelets on macrophages, M2 macrophage polarization, and foam cell formation.

The accumulation of oxidized LDL (ox-LDL) in macrophages in association with platelet activity leads to the formation of foam cells, which play a key role in the pathophysiology of atherosclerosis and coronary artery diseases (CAD). Here, in this study, we aimed to investigate the simultaneous effect of ox-LDL and platelets on foam cell formation, as well as modification in cell markers. First, the U937, a human monocytic cell line, was cultured in RPMI-1640. Then, isolated platelets were co-cultured with the U937 and exposed to ox-LDL (80 µg/ml) to evaluate the impact of ox-LDL on foam cell formation using Oil red O (ORO) staining. Also, the expression of foam cells' surface markers and CD36, ABCA1, SR-B1, ACAT1, and LXRα genes, which are involved in macrophage metabolism and ox-LDL uptake, was measured by flow cytometry and real-time PCR, respectively. Our findings suggest that platelets promoted foam cell formation (ORO-positive cells), accompanied by a higher level of CD163+ M2 macrophages. Furthermore, the expression of CD36, ABCA1, SR-B1, ACAT1, and LXRα genes, which are implicated in cholesterol accumulation in macrophages, was significantly upregulated in the ox-LDL+ platelets group compared to the control (P < 0.05). Moreover, the up-regulation of CD36, ABCA1, and SR-B1 genes in the ox-LDL+ platelets group was more accentuated compared to the ox-LDL group (P < 0.05). Owing to the positive effector role of platelets in the formation of foam cells and CD163+ cells, it could be assumed that platelets play a dual role in the development of these cells.

Multiple human B cell subsets give rise to regulatory B cells

Multiple human B cell subsets give rise to regulatory B cells

Efficient removal and recovery of Cd2+ from aqueous solutions by capacitive deionization (CDI) method using biochars

Biochars are low-cost and sustainable materials for environmental technologies. In this work, we prepared three biochars using pomelo peel (P-BC), algae (A-BC), and corncob (C-BC) to recover Cd2+ from wastewater via capacitive deionisation (CDI). A-BC possesses the highest amount of mesopores and nitrogen functionality and attains the highest removal of Cd2+ via physical adsorption. For the electro-sorption capacity, C-BC and A-BC perform better capacitive removal of Cd2+ than P-BC due to the smaller charge-transfer and mass-transfer resistances. Also, this work investigated the impacts of surface morphology, cell voltage, NaCl, initial pH, and Cd2+ concentrations on Cd2+ capacitive removal and electrode regeneration performances. The results indicated that A-BC and C-BC may be prospective materials for Cd2+ removal from wastewater by CDI. However, the presence of competing cations at high concentrations may influence the removal of Cd2+ at a low level, requiring the modification of A-BC and C-BC in future work.

Unique roles of co-receptor-bound LCK in helper and cytotoxic T cells

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4–LCK and CD8–LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor–LCK interactions as promising targets for immunomodulation.

1044. Identification and Characterization of an Unconventional NK Subset in COVID-19

Abstract Background Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is associated with dysregulation in the innate immune response including NK cells. NK cells are integral in the innate immune response against viral infections. Canonical NK cells are classified as CD56dim CD16+ and CD56bright CD16-. An unconventional subset of CD56dim CD16- NK cells has previously been identified in COVID-19 that is not present in other viral infections. Here we characterize phenotypic changes in the NK cells of patients with severe COVID-19 as work towards determining the functional status of this unconventional subset. Methods Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from healthy donors (n=5) and patients with severe COVID-19 on Extra Corporeal Membrane Oxygenation (ECMO) (n=15). Primary NK cells were stimulated in vitro with plasma from patients with severe COVID-19 or healthy donors. Flow cytometry was used to phenotype the NK cells. A separate cohort of PBMC samples (n=7) from patients requiring hospitalization for COVID-19 underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) analysis. Results The CD56bright CD16- NK subset was expanded in PBMCs from patients with severe COVID-19 as compared to healthy controls. CITE-Seq demonstrated that NK cells without surface CD16 clustered separately based on transcriptional profiling and did express FCGR3A at the translational level. Stimulation with COVID-19 plasma recapitulated the loss of CD16 from primary human NK cells and led to increased activity of Caspase 3/7. Figure 1.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset in patients with severe COVID-19. a) Representative gating of NK cell subsets by Flow Cytometry in healthy and COVID-19 patient peripheral blood mononuclear cells (PBMCs). b) Percentage of total NK cells belonging to a particular cell subset compared between healthy donor samples (n=4) and COVID-19 patient samples (n=8). Data points represent an individual patient sample. Error bars represent the standard deviation of the mean. Differences between groups was analyzed using a two tailed t-test. *: p&amp;lt; 0.05, ns: not significant Figure 2.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset after stimulation with COVID-19 plasma in vitro a) Representative gating of NK cell subsets by Flow Cytometry analysis in healthy donor NK cells stimulated by healthy plasma and COVID-19 patient plasma. b)Relative change in percentage of total NK cells belonging to a particular cell subset compared between healthy donor plasma (n=6)and COVID-19 patient plasma (n=15) stimulation conditions. Error bars represent the standard deviation of the mean and the difference between groups was analyzed using a two-tailed T-test. **: p&amp;lt; 0.01, ***: p&amp;lt; 0.001, ns: not significant. Conclusion We demonstrate and characterize a nonclassical population of CD56dim CD16- NK cells that are present in patients with severe COVID-19 and replicate this phenotype in vitro. Reproduction of this in vivo phenotype in an in vitro system will allow for additional studies on the functional state of NK cell subsets in COVID-19. The presence of this NK cell population may reflect a dysregulated innate immune response and immunopathogenesis of COVID-19. Disclosures All Authors: No reported disclosures.

Transport Form and Pathway from the Intestine to the Peripheral Tissues and the Intestinal Absorption and Metabolism Properties of Oleamide.

This study aimed to obtain information on the transport form and pathway from the intestine to the peripheral tissues and on the intestinal absorption and metabolism properties of oleamide (cis-9-octadecenamide). Oleamide was primarily transported via the portal vein. Density gradient centrifugation indicated that plasma oleamide was enriched in the fractions containing albumin in the portal and peripheral blood. Oleamide formed a complex with albumin in an endothermic reaction (apparent Kd = 4.4 μM). The CD36 inhibitor inhibited the oleamide uptake into the intestinal epithelial Caco-2 cells, and oleamide decreased the cell surface CD36 level. The fatty acid amide hydrolase (FAAH) inhibitor increased the transepithelial transport of oleamide across Caco-2 cells and the plasma oleamide concentration in mice intragastrically administered with oleamide. These results indicate that oleamide is transported primarily via the portal vein as a complex with albumin. Furthermore, we suggest that oleamide is taken up via CD36 in the small intestine and degraded intracellularly by FAAH.

Different Types of Chronic Inflammation Engender Distinctive Immunosenescent Profiles in Affected Patients.

Immunosenescence encompasses a spectrum of lymphocyte phenotypic alterations. The aim of the study was to evaluate immunosenescent effect of two different forms of chronic inflammation, Systemic Lupus Erythematosous (SLE), a systemic autoimmune disease, and End-Stage Kidney Disease (ESKD), a chronic inflammatory disorder. Certain lymphocyte surface molecules, including CD31, CD45RA, CCR7, CD28, CD57, for T, and IgD, CD27 for B lymphocytes, were analyzed by flow cytometry in 30 SLE and 53 ESKD patients on hemodialysis (HD), and results were compared to 31 healthy controls (HC) of similar age, gender, and nationality. Significant Lymphopenia was evident in both SLE and ESKD-HD patients, compared to HC, affecting B cells 75.4 (14.4−520.8), 97 (32−341), and 214 (84−576) cells/μL, respectively, p < 0.0001, and CD4 cells 651.2 (71.1−1478.2), 713 (234−1509), and 986 (344−1591) cells/μL, respectively, p < 0.0001. The allocation of B cell subpopulations was remarkably different between SLE and ESKD-HD patients. SLE showed a clear shift to senescence (CD19IgD-CD27−) cells, compared to ESKD-HD and HC, 11.75 (10)% vs. 8 (6) vs. 8.1 (10), respectively. Regarding T lymphocytes, Central Memory CD8 cells predominated in both SLE and ESKD-HD patients compared to HC, 53 (50)%, 52 (63), and 24 (64)%, respectively, while ESKD-HD but not SLE patients also had increased expression of CD4CD28− and CD8CD28− cells. In conclusion, both diseases are followed by significant lymphopenia; however, the senescent phenomenon affects the B lymphocyte compartment in SLE patients and T lymphocytes in ESKD-HD patients.