Ligand Expression Research Articles

WNT signaling contributes to the extrahepatic bile duct proliferative response to obstruction in mice.

Biliary obstruction and cholangiocyte hyperproliferation are important features of cholangiopathies affecting the large extrahepatic bile duct (EHBD). The mechanisms underlying obstruction-induced cholangiocyte proliferation in the EHBD remain poorly understood. Developmental pathways, including WNT signaling, are implicated in regulating injury responses in many tissues, including the liver. To investigate the contribution of WNT signaling to obstruction-induced cholangiocyte proliferation in the EHBD, we used complementary in vivo and in vitro models with pharmacologic interventions and transcriptomic analyses. To model obstruction, we used bile duct ligation (BDL) in mice. Human and mouse biliary organoids and mouse biliary explants were used to investigate the effects of WNT activation and inhibition in vitro. We observed an upregulation of WNT ligand expression associated with increased biliary proliferation following obstruction. Cholangiocytes were identified as both WNT ligand-expressing and WNT responsive cells. Inhibition of WNT signaling decreased cholangiocyte proliferation in vivo and in vitro, while activation increased proliferation. WNT effects on cholangiocyte proliferation were β-catenin-dependent, and we showed a direct effect of WNT7B on cholangiocyte growth. Our studies suggested that cholangiocyte-derived WNT ligands can activate WNT signaling to induce proliferation after obstructive injury. These findings implicated the WNT pathway in injury-induced cholangiocyte proliferation within the EHBD.

Transcriptionally distinct B cell profiles in systemic immune tissues and peritoneal cavity of Atlantic salmon (Salmo salar) infected with salmonid alphavirus subtype 3.

Teleost B cells producing neutralizing antibodies contribute to protection against salmonid alphavirus (SAV) infection, the etiological agent of pancreas disease, thereby reducing mortality and disease severity. Our previous studies show differences in B cell responses between the systemic immune tissues (head kidney (HK) and spleen) and the peritoneal cavity (PerC) after intraperitoneal SAV3 infection in Atlantic salmon (Salmo salar) where the response in PerC dominates at the late time points. By employing the same infection model, we aimed to further characterize these B cells. Immunophenotyping of teleost B cells is challenging due to limited availability of markers; however, RNA-seq opens an opportunity to explore differences in transcriptomic responses of these cells. Our analysis identified 334, 259 and 613 differentially expressed genes (DEGs) in Atlantic salmon IgM+IgD+ B cells from HK, spleen, and PerC, respectively, at 6 weeks post SAV3 infection. Of these, only 34 were common to all the three immune sites. Additionally, out of the top 100 genes with the highest fold change in expression, only four genes were common across B cells from the three sites. Functional enrichment analyses of DEGs using KEGG and GO databases demonstrated differences in enriched innate immune signaling and the cytokine-cytokine interaction pathways in B cells across the sites, with varying numbers of genes involved. Overall, these findings show the presence of transcriptionally distinct B cell subsets with innate immune functions in HK, spleen and PerC of SAV3-infected Atlantic salmon. Further, our data provide new insights into the immunoregulatory role of fish B cells through the differential expression of various cytokine ligands and receptors and will be a useful resource for further studies into B cell immune compartments.

Immuno-oncologic profiling by stage-dependent transcriptome and proteome analyses of spontaneously regressing canine cutaneous histiocytoma.

Canine cutaneous histiocytoma (CCH) is a tumor that originates from dermal Langerhans cells and affects particularly young dogs. The common spontaneous regression of CCH makes it an interesting model in comparative oncology research. Previous studies have indicated that anti-tumor immune responses may be involved, but details remain speculative to date. Here, we asked which specific immuno-oncological dynamics underlie spontaneous regression of CCH on mRNA and protein levels. QuantSeq 3' mRNA sequencing with functional over-representation analysis and an nCounter RNA hybridization assay were employed on 21 formalin-fixed, paraffin-embedded CCH samples representing three different tumor stages (dataset information: GSE261387-Immuno-Oncologic Profiling by Stage-Dependent Transcriptome and Proteome Analyses of Spontaneously Regressing Canine Cutaneous Histiocytoma-OmicsDI). Nine additional samples were subjected to matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Surprisingly, only minor stage-specific differences were found. When we investigated expression of B7 family ligands and CD28 family receptors holding co-stimulatory and -inhibitory functions, respectively, we found a higher abundance of CD80, CD86, CTLA4 and CD28, which may trigger a balanced activation of lymphocyte-mediated immune responses. CD80 and CD86 expressing cells were further quantified by in situ hybridization and compared with data from three cases of canine histiocytic sarcoma (HS), a malignant tumor variant originating from antigen-presenting interstitial dendritic cells. A stage-specific increase of CD80 expressing cells was recorded in CCH from the tumor bottom to the top, while CD86 was continuously and homogenously expressed at high levels. Overall expression of CD80 in CCH was similar to that in HS (73.3 ± 37.4% vs 62.1 ± 46.4%), while significantly more CD86 expressing tumor cells were found in CCH (94.7 ± 10.3%) when compared to HS (57.6 ± 11.0%). Our data suggest that major immuno-oncological pathways are not regulated during regression of CCH on the mRNA or protein levels as detectable by the methods used. Instead, our data provide further evidence supporting previous hypotheses towards a role of immune stimulatory B7 family ligands and CD28 family receptors in the regression of CCH.

Mll4 in Skeletal Muscle Fiber Maintains Muscle Stem Cells by Regulating Notch Ligands.

Muscle stem cells (MuSCs) undergo numerous state transitions throughout life, which are critical for supporting normal muscle growth and regeneration. Therefore, it is crucial to investigate the regulatory mechanisms governing the transition of MuSC states across different postnatal developmental stages. To assess if myofiber-expressed Mll4 contributes to the maintenance of MuSCs, we crossed MCK Cre/+ or HSA MerCreMer/+ mice to Mll4 f/f mice to generate myofiber-specific Mll4-deleted mice. Investigations were conducted using 8-week-old and 4-week-old MCK Cre/+ ;Mll4 f/f mice Investigations were conducted using 8-week-old and 4-week-old HSA Cre/+ ;Mll4 f/f mice were utilized. During postnatal myogenesis, Mll4 deleted muscles were observed with increased number of cycling MuSCs that proceeded to a differentiation state, leading to MuSC deprivation. This phenomenon occurred independently of gender. When Mll4 was ablated in adult muscles using the inducible method, adult MuSCs lost their quiescence and differentiated into myoblasts, also causing the depletion of MuSCs. Such roles of Mll4 in myofibers coincided with decreased expression levels of distinct Notch ligands: Jag1 and Dll1 in pubertal and Jag2 and Dll4 in adult muscles. Our study suggests that Mll4 is crucial for maintaining MuSCs in both pubertal and adult muscles, which may be accomplished through the modulation of distinct Notch ligand expressions in myofibers. These findings offer new insights into the role of myofiber-expressed Mll4 as a master regulator of MuSCs, highlighting its significance not only in developmental myogenesis but also in adult muscle, irrespective of sex.

The effect of c-Myc on regulating the immune-related ligands in Y subtype small cell lung cancer through histone deacetylase 1

Objective: To explore the effect and mechanism of c-Myc on regulating the expression of immune-related ligands in Y subtype small-cell lung cancer (SCLC) characterized by high expression of immune-related molecules. Methods: The Y subtype SCLC cell line H196 was randomly divided into the control group, c-Myc inhibitor 10058-F4 group, histone deacetylase 1 (HDAC1) inhibitor pyroxamide group, and 10058-F4 plus pyroxamide group. The co-culture system with NK-92MI cells was used to determine the effect of H196 cells on the function of natural killer (NK) cells. Western Blotting and co-immunoprecipitation assays were used to detect the effect of c-Myc on class Ⅰ HDAC, and flow cytometry was used to detect the regulatory effect of c-Mycon CD47, programmed cell death ligand 1 (PD-L1), and CD155, which are highly expressed immune checkpoints in Y subtype SCLC, and major histocompatibility complex classⅠ-related chains A and (MICA/B), which is a poorly expressed immune-activating ligand in SCLC, and the role of HDAC. Chromatin immunoprecipitation (ChIP) assay and real-time quantitative polymerase chain reaction (RT-qPCR) were used to determine the regulatory mechanism of c-Myc-HDAC1 on MICA/B expression. Results: Inhibition of c-Myc decreased the mortality of H196 cells in the co-culture system and down-regulated the expression of MICA/B. Compared with the NK+H196 group [(42.54±2.47)%], the proportion of cells killed by NK-92MI cells in the NK+H196+10058-F4 group was lower [(28.48±3.38)%, P＜0.001]. The mean fluorescence intensity (MFI) of MICA/B on the cells in the 10058-F4 group (36.40±0.82) was lower than that in the control group (91.23±8.60, P＜0.001). And c-Myc could bind to HDAC1, whose protein level was up-regulated by 10058-F4 while the mRNA level was not. Compared with the cells in the control group (90.10±4.91), the MFI of MICA/B on the cells in the pyroxamide group was significantly increased (145.70±5.86, P＜0.001), and the MFI of MICA/B on the cells in the 10058-F4+pyroxamide group (54.60±2.88) was significantly increased compared with the cells in the 10058-F4 group (35.97±1.60, P＜0.001). The percentage of MICA promoter gene fragments in the c-Myc antibody precipitation group (0.125±0.037) was significantly higher than that in the IgG group (0.000 8±0.000 3, P=0.004). MICB had a similar trend, suggesting that the c-Myc-HDAC1 complex could bind to the promoter region of MICA/B. The MFI of CD47 on the cells in the 10058-F4 group (60.07±0.21) was significantly lower than cells in the control group (70.27±1.37, P＜0.001), but the MFIs of PD-L1 (13.50±0.61) and CD155 (829.70±41.19) were significantly higher than those on the cells in the control group (9.23±0.94, P＜0.01; 496.00±4.36, P＜0.001, respectively). Conclusions: c-Myc may promote the expression of MICA/B and CD47 in Y subtype SCLC cells by binding and inhibiting HDAC1, while it may also be involved in inhibiting the expression of PD-L1 and CD155 in SCLC cells.

IL-1 receptor antagonist anakinra downregulates inflammatory cytokines during renal normothermic machine perfusion: Preliminary results.

The interleukin 1 (IL-1) cytokine group plays a key role in sterile inflammation and may be an important target for transplant-related renal injury. This study examined the effects of anakinra, a non-specific IL-1 receptor antagonist, administered during normothermic machine perfusion (NMP) of porcine kidneys. Paired porcine kidneys (n = 5 pairs) underwent 15 min of warm ischemia plus 2 h of static cold storage in ice. Kidneys were then perfused with autologous whole blood using an exvivo NMP platform. Kidneys were randomly allocated to receive anakinra or vehicle administered at the start of NMP. Cortical biopsies were collected at baseline before ischemic injury and at the end of NMP. Functional parameters were recorded and calculated, and inflammatory markers were measured by qPCR and ELISA techniques. During NMP, there were no statistically significant differences in renal blood flow, urine output, creatinine clearance or fractional excretion of sodium in the anakinra and control groups. The administration of anakinra significantly downregulated transcriptional expression of IL-6, Fas ligand and intercellular adhesion molecule 1 (p = 0.029, 0.029, 0.028, respectively). Anakinra, an IL-1 receptor blocker, successfully attenuated the downstream inflammatory and immune-mediated response within the kidney during NMP.

Therapeutic BCG vaccine protects against long COVID: The BATTLE randomized clinical trial.

Bacillus Calmette-Guérin (BCG) injected during the COVID-19 convalescence period was safe and enhanced recovery from anosmia and dysgeusia in the acute phase. To report the long-term results of the BATTLE trial, BCG vaccine in adults with mild COVID-19. Design: Double-blind, placebo-controlled, randomized (1:1) clinical trial. BCG intradermal vaccine and placebo. A total of 157 BCG and 142 placebo recipients participated in the 6-month follow-up, and 97 BCG and 95 placebo recipients participated in the 12-month follow-up. Long COVID symptoms and mechanistic analyses. BCG reduced hearing problems at 6 months (odds ratio [OR]=0.26) and sleeping, concentration, memory, and vision problems at 12 months (OR=0.45, 0.36, 0.38, and 0.36, respectively). Sensitivity analyses confirmed that long COVID-19 symptoms were reduced at the 6- and 12-month follow-ups (p=0.010 and 0.031, respectively). BCG's crossover interaction paradoxically increased hair loss in women and decreased it in men at 6 months (p=0.032). BCG immunomodulation is likely mediated through inhibition of Fas ligand expression in the blood and increased induction of IL6, IL10, interferon-induced transmembrane protein 3, and angiotensin-converting enzyme 2 in cultured human macrophages. Long-term follow-up of the BATTLE trial participants revealed that BCG protects against long COVID development if administered within the COVID-19 convalescence period. The response to BCG was subject-specific, including a paradoxical crossover interaction based on sex. Not tested for previous mycobacterial exposure; loss to follow-up, particularly at 12 months.

Abstract C037: Chemotherapy causes de novo induction of invasive breast cancer cells

Abstract Metastasis is the leading cause of breast cancer (BC) death, and tumor cells must migrate and invade to metastasize. BC cells that express the actin regulatory protein MenaINV have an enhanced ability to migrate and intravasate within the primary tumor, and extravasate at secondary sites. Though chemotherapy improves patient survival, treatment with paclitaxel, a chemotherapy used for BC, leads to upregulation of MenaINV and an increase in metastasis in mice. MenaINV expression can be induced in BC cells through Notch1 signaling with macrophages, which are often recruited to tumors in response to chemotherapy. MenaINV-expressing cells are also resistant to paclitaxel, begging the question of whether paclitaxel increases MenaINV by de novo induction or by selectively killing non-MenaINV-expressing cells. We hypothesized that paclitaxel causes de novo MenaINV induction by increasing macrophage-tumor cell Notch signaling. Understanding this pro-metastatic effect of chemotherapy is crucial to refining treatment strategies. MMTV-PyMT mice bearing spontaneous mammary tumors were used as a model of BC. Mice were treated with paclitaxel or vehicle every 5 days for a total of 3 treatments. To study the role of macrophages in the paclitaxel-mediated MenaINV increase, in addition to paclitaxel, one cohort of mice was co-administered the macrophage-depleting agent clodronate or vehicle. To study the role of Notch signaling, a second cohort of mice was co-administered the Notch inhibitor DAPT or vehicle. Immunofluorescence staining was used to evaluate tumors for: %MenaINV+ area, proportion of apoptotic cells, macrophage density, and macrophage expression of Notch ligands Jag1 and Jag2. Paclitaxel-treated tumors expressed significantly more MenaINV even when tumor cell death did not increase, indicating that chemotherapy increases MenaINV expression by induction. Both macrophages and Notch signaling were needed for this effect to occur. Interestingly, paclitaxel did not increase macrophage infiltration or macrophage expression of Notch ligands, suggesting that paclitaxel may increase MenaINV by acting on tumor cells to promote Notch signaling. Paclitaxel causes de novo MenaINV induction that is macrophage- and Notch-dependent. Studies aiming to further elucidate the mechanism behind paclitaxel-mediated MenaINV induction are ongoing. These results lay the groundwork for novel microenvironment-based therapies to alleviate the pro-metastatic effects of chemotherapy in BC. Citation Format: Madeline Friedman-DeLuca, George S. Karagiannis, Camille L. Duran, Luis Rivera Sanchez, Prachiben P. Patel, Suryansh Shukla, Nicole D. Barth, Ved P. Sharma, Michael Papanicolaou, John S. Condeelis, Maja H. Oktay, David Entenberg. Chemotherapy causes de novo induction of invasive breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C037.

CD34+ and CD34- MM cells show different immune-checkpoint molecule expression profiles: high expression of CD112 and CD137 ligand on CD34+ MM cells.

Despite the introduction of new drugs, multiple myeloma (MM) still remains incurable. We previously reported that CD34+ MM cells, which are clonogenic and self-renewing, are therapy-resistant and persist as a major component of minimal residual disease, expanding during relapse. To investigate the effects of immunotherapies such as immune-checkpoint inhibitors, CAR-T therapy, and bispecific antibodies on CD34+ MM cells, we analyzed immune profiles of both MM cells and T cells from MM patients using microarrays and flow cytometry. Ingenuity pathway analysis revealed 14 out of 289 canonical pathways were more active in CD34+ MM cells compared to CD34- cells, many of which were involved in inflammation and immune responses. Notably, PD-1 signaling-related genes were highly expressed in CD34+ MM cells. Among 10 immune-checkpoint molecules, CD34+ cells more frequently expressed CD112, CD137L, CD270, CD275, and GAL9 than CD34- cells in both newly diagnosed and relapsed/resistant patients. In addition, CD4+ and CD8+ T cells more frequently expressed TIGIT and CD137, suggesting that CD112/TIGIT and CD137L/CD137 interactions may suppress T-cell activity against CD34+ MM cells. Furthermore, our finding of higher FcRH5 expression on CD34+ MM cells is encouraging for future research into the efficacy of FcRH5-targeted therapy in MM.

EXTH-33. COMBATING TUMOR HETEROGENEITY IN GLIOBLASTOMA WITH A NOVEL CAR-BASED PLATFORM

Abstract Tumor heterogeneity is a major hindrance impeding success of antigen targeting immunotherapies, such as chimeric antigen receptor (CAR) T cells, in solid tumors. CAR T cells engineered to target EGFRvIII, IL13Rα2, and HER2 have mostly failed clinically to treat glioblastoma, because these antigens are typically only expressed on a subset of a tumor cells within a patient. Thus, finding a surface target that more universally characterizes tumor cells will likely be critical if antigen-specific immunotherapies are to succeed in glioblastoma and other solid tumors. Recently, we uncovered a mechanism whereby CD8 T cells can directly kill tumor cells independently of tumor-expressed major histocompatibility complex (MHC)-I. We found this mechanism to be dependent on NKG2D expression on T cells and NKG2D ligand (NKG2DL) expression on tumor cells. Interestingly, NKG2DL expression was not limited to MHC-I-deficient tumor cells, nor was it limited to gliomas, but NKG2DL expression characterized a variety of solid tumor types. Therefore, NKG2DL is potentially an ideal target for immune-based therapy. Here, we developed a bi-specific fusion protein comprised of the extracellular domain of NKG2D linked to an anti-CD3scFv. Thus, T cells will be redirected to target NKG2DL-expressing tumor cells. We have termed this fusion protein Bi-specific Engagers without Antigen Requirements (BEAR). To ensure local, intratumoral delivery of BEAR, we engineered an EGFRvIII-targeting CAR T cell to secrete BEAR (VIII CAR Engineered with BEAR; VIII “CARE-BEAR”). VIII CARE-BEAR demonstrated potent tumor kill in both in vitro and in vivo tumor heterogeneity models. Moreover, we engineered an EGFRwt-targeting (D2C7) CARE-BEAR to exemplify the potential utility of CARE-BEAR beyond glioblastoma.

Changes in soluble PD-L1 levels in patients with advanced non-small cell lung cancer

Abstract Soluble programmed death-1 ligand (sPD-L1) in the serum of non-small cell lung cancer (NSCLC) patients is a crucial factor in disease prognosis and an indicator for immunotherapy response. This study aimed to evaluate changes in sPD-L1 levels in advanced NSCLC patients. Between May 2018 and October 2022, 80 patients with advanced-stage NSCLC and 30 healthy volunteers participated in a prospective study. The findings revealed a significant rise in sPD-L1 concentration in the lung cancer group compared to the normal control group (1.08 vs. 0.42, p < 0.001). No apparent correlation was found between sPD-L1 concentration and membrane-bound programmed death-1 ligand (mPD-L1) expression. The optimal diagnostic threshold for sPD-L1 was set at 0.92 ng/mL, showing a sensitivity of 56.25% and specificity of 77.33%, with an area under the curve (AUC) of 0.743 (p = 0.001). Although increased sPD-L1 levels were prevalent in individuals with advanced age, prolonged disease duration, large tumor size, and finger clubbing, these associations did not reach statistical significance (p > 0.05). In conclusion, sPD-L1 levels significantly increased in advanced NSCLC patients compared to controls (p < 0.05), with no association observed with mPD-L1 (p = 0.304). The study suggests that sPD-L1 concentration can be a specific biomarker for guiding the diagnosis of advanced NSCLC, with a recommended cutoff value of 0.92 ng/mL, demonstrating a sensitivity of 56.25% and specificity of 77.33% (p = 0.001). Importantly, no apparent relationship was established between sPD-L1 levels and clinical or paraclinical characteristics in advanced NSCLC patients.

A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

Abstract T-cell activation is a multistep process requiring T-cell receptor engagement by peptide–MHC complexes (Signal 1) coupled with CD28-mediated costimulation (Signal 2). Tumors typically lack expression of CD28 ligands, so tumor-specific Signal 1 (e.g., neoepitope presentation) without costimulation may be ineffective or even induce T-cell anergy. We designed the bispecific antibody XmAb808 to co-engage the tumor-associated antigen B7-H3 with CD28 to promote T-cell costimulation within the tumor microenvironment. XmAb808 costimulation was measured by its ability to activate and expand T cells and enhance T cell–mediated cancer cell killing in cocultures of human peripheral blood mononuclear cells and cancer cells and in mice engrafted with human peripheral blood mononuclear cells and tumor xenografts. XmAb808 avidly bound cancer cells and stimulated IL2 and IFNγ secretion from T cells cocultured with cancer cells engineered to deliver Signal 1 to T cells via a surface-expressed anti-CD3 antibody. XmAb808 enhanced expression of the antiapoptotic factor Bcl-xL and CD25, promoting survival and IL2-dependent expansion of T cells coupled with increased T cell–mediated cytotoxicity in vitro. XmAb808 combined with an EpCAM×CD3 bispecific antibody to enhance target cell killing through IL2-dependent expansion of CD25+ T cells. This combination also suppressed pancreatic tumor xenograft growth in mice. Furthermore, XmAb808 combined with an anti–programmed cell death protein 1 antibody to suppress breast tumor xenograft growth in mice. XmAb808 as monotherapy and in combination with an anti–programmed cell death protein 1 antibody is currently in clinical development in patients with advanced solid tumors. Our results suggest that XmAb808 may also combine with tumor antigen–targeted anti-CD3 (Signal 1) T-cell engagers.

ICOSL deficiency promotes M1 polarization to alleviate liver fibrosis in Schistosomiasis Mice

The expression of inducible co-stimulator ligand (ICOSL) on macrophage (Mφ) implies their ability to interact with inducible co-stimulator (ICOS)-expressing T cells, thereby modulating immune responses within the liver microenvironment. This study aimed to elucidate the mechanism underlying ICOS/ICOSL signaling in the regulation of Mφ polarization during Schistosomiasis-induced liver fibrosis. To investigate this, ICOSL-knock out (KO) and wildtype (WT) C57BL/6 mice were infected with Schistosoma japonicum (S. japonicum) to examine the dynamic changes in Mφ phenotype and observe the pathology alterations in the liver. There was significantly decreased expression of ICOSL both in monocytes of cirrhosis patients and the liver tissue of mice infected with S. japonicum. Furthermore, ICOSL-KO mice exhibited reduced liver granuloma formation and fibrosis during S. japonicum infection. Simultaneously, Mφ in ICOSL-KO mice polarized towards M1-type and induced apoptosis of hepatic stellate cells (HSCs). Overall, the blockade of ICOSL signaling could promote M1 polarization, induce HSCs apoptosis, and ameliorate hepatic fibrosis, suggesting that ICOSL may serve as a potential biomarker for prognosis and therapeutic target for schistosomiasis-induced hepatic fibrosis.

A mechanism of human cytomegalovirus for establishing latency through inhibition of HCMV UL16 expression by hcmv-miR-US33-5p.

Human cytomegalovirus (HCMV) is the only beta herpesvirus that can encode microRNA (miRNA). As one of the 26 HCMV miRNAs, hcmv-miR-US33-5p has been reported to inhibit viral DNA synthesis and DNA replication via downregulation of the host gene syntaxin 3. Here, we tested the luciferase activity of 8 other putative target mRNAs of hcmv-miR-US33-5p, which were identified via hybrid PCR, 7 of which decreased following the over expression of hcmv-miRNA-US33-5p. A viral gene, HCMV UL16, was confirmed to be a direct target of hcmv-miR-US33-5p since both luciferase activity and protein levels were decreased by hcmv-miR-US33-5p overexpression. Moreover, by using a reconstituted virus with UL16 deletion, we found that UL16 is not an essential gene for the ability of hcmv-miR-US33-5p to downregulate DNA replication. UL16 plays a critical role in NK cell evasion by sequestering NKG2D ligands in the endoplasmic reticulum (ER) and reducing their cell surface expression. We demonstrated that over expression of hcmv-miR-US33-5p induced some NKG2D ligands independent of other HCMV genes and decreased the interaction between MICB and UL16. These stimulating NKG2D ligands can be recognized by NKG2D and subsequently activate NK cells. Our results provide insight into the mechanisms by which HCMV promotes latency through the inhibition of UL16 and the stimulation of NKG2D ligand expression by hcmv-miR-US33-5p.

Tertiary Lymphoid Structure in Dental Pulp: The Role in Combating Bacterial Infections.

Tertiary lymphoid structure (TLS) is associated with various pathologies, including those of cancers and chronic infections. Depending on the organ, multiple factors regulate the formation of TLS. However, the role of TLS in immune response and the molecules that drive its formation remain uncertain. The dental pulp, includes a few immune cells surrounded by rigid mineralized tissue, and opens to the outside through the apical foramen. Owing to this special organization, the dental pulp generates a directional immune response to bacterial infection. Considering this aspect, the dental pulp is an ideal model for comprehensively studying the TLS. In the present study, single-cell RNA sequencing of healthy and inflamed human dental pulp reveals known markers of TLS, including C-C motif chemokine ligand 19 (CCL19), lysosome-associated membrane glycoprotein 3 (LAMP3), CC chemokine receptor 7 (CCR7), and CD86, present in inflamed dental pulp. Compared with the healthy pulp, types and proportions of immune cells increase, along with enhanced cellular communication. Multiple immunofluorescence staining reveals that typical TLS emerges in dental pulp with pulpitis, consistent with the high expression of CC chemokine ligand 3 (CCL3), which may be a key driver of TLS formation. Moreover, TLS is also observed in a mouse model of pulpitis. These findings collectively offer insights into the formation and function of TLS in response to infection.

Effect of Hypoxia on Siglec-7 and Siglec-9 Receptors and Sialoglycan Ligands and Impact of Their Targeting on NK Cell Cytotoxicity.

Tumor microenvironmental hypoxia is an established hallmark of solid tumors. It significantly contributes to tumor aggressiveness and therapy resistance and has been reported to affect the balance of activating/inhibitory surface receptors' expression and activity on NK cells. In the current study, we investigated the impact of hypoxia on the surface expression of Siglec-7 and Siglec-9 (Sig-7/9) and their ligands in NK cells and tumor target cells. The functional consequence of Siglec blockage using nanoparticles specifically designed to target and block Sig-7/9 receptors on NK cell cytotoxicity was elucidated. CD56⁺ CD3- NK cells were isolated from PBMCs along with an NK-92 clone and used as effector cells, while MCF-7 and K562 served as target cells. All cells were incubated under normoxic or hypoxic conditions for 24 h. To assess Siglec-7 and Siglec-9 receptor expression, U937, NK-92, and primary NK cells were stained with PE-labeled antibodies against CD328 Siglec-7/9. Interactions between Siglec-7/9 and their sialylated ligands, along with their functional impact on NK cell activity, were evaluated using polymeric nanoparticles coated with a sialic acid mimetic. Immunological synapse formation and live-cell imaging were performed with a ZEISS LSM 800 with Airyscan at 10× magnification for 24 h. Our data indicate that hypoxia had no effect on the expression of Siglec-7/9 receptors by NK cells. In contrast, hypoxic stress resulted in an increase in Siglec-7 sialoglycan ligand expression by a sub-population of NK target cells. Using polymeric nanoparticles coated with a sialic acid mimetic that binds both Siglec-7 and -9 (Sig-7/9 NP), we demonstrated that incubation of these nanoparticles with NK cells resulted in increased immunological synapse formation, granzyme B accumulation, and killing of NK target cells. These studies indicate that hypoxic stress may have an impact on NK cell-based therapies and highlight the need to consider the hypoxic microenvironment for tumor-specific glycosylation. Our findings point to the role of Siglec-sialylated glycan interactions in hypoxic stress-induced NK cell dysfunction and recommend the potential integration of the manipulation of this axis through the targeting of Siglecs in future cancer immunotherapy strategies.

Cytochalasin H enhances sensitivity to gefitinib in non-small-cell lung cancer cells through inhibiting EGFR activation and PD-L1 expression

In our previous study, we have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove plant and found that CyH inhibited the proliferation of non-small cell lung cancer (NSCLC) cells. Recently, epidermal growth factor receptor (EGFR) activation and programmed cell death 1 ligand (PD-L1) expression have been demonstrated to mediate NSCLC resistance to gefitinib, first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI). Here, we further investigated the effect of CyH on EGFR activation, PD-L1 expression, and gefitinib sensitivity in NSCLC cell lines, A549 (wild-type EGFR), HCC827 (EGFR mutation), and NCI-H1975 (dual EGFR mutations and acquired gefitinib resistance) and animal model. Our results showed that CyH significantly inhibited EGFR activation and PD-L1 expression in NSCLC cells. Additionally, CyH dramatically promoted the inhibitory effect of gefitinib on the proliferation of A549 and HCC827 cells, and enhanced the sensitivity to gefitinib in NCI-H1975 cells. Moreover, CyH increased the inhibitory effect of gefitinib on EGFR activation and PD-L1 expression in HCC827 and NCI-H1975 cells. Animal experiments further demonstrated that CyH significantly promoted the inhibitory effect of gefitinib on the growth of NSCLC and the expression of Ki-67, p-EGFR, and PD-L1 in NCI-H1975 NSCLC xenograft tumors of nude mice. Furthermore, CyH inhibited the activation of JAK3/STAT signaling pathway. Taken together, our findings suggest that CyH promotes the sensitivity to gefitinib in NSCLC cells through the inhibition of EGFR activation and PD-L1 expression.

The hepcidin-ferroportin axis modulates liver endothelial cell BMP expression to influence iron homeostasis in mice

AbstractThe liver hormone hepcidin regulates systemic iron homeostasis to provide enough iron for vital processes while limiting toxicity. Hepcidin acts by degrading its receptor ferroportin (encoded by Slc40a1) to decrease iron export to plasma. Iron controls hepcidin production in part by inducing liver endothelial cells (LECs) to produce bone morphogenetic proteins (BMPs) that activate hepcidin transcription in hepatocytes. Here, we used in vitro and in vivo models to investigate whether ferroportin contributes to LEC intracellular iron content to modulate BMP expression and, thereby, hepcidin. Quantitative proteomics of LECs from mice fed different iron diets demonstrated an inverse relationship between dietary iron and endothelial ferroportin expression. Slc40a1 knockdown primary mouse LECs and endothelial Slc40a1 knockout mice exhibited increased LEC iron and BMP ligand expression. Endothelial Slc40a1 knockout mice also exhibited altered systemic iron homeostasis with decreased serum and total liver iron but preserved erythropoiesis. Although endothelial Slc40a1 knockout mice had similar hepcidin expression to control mice, hepcidin levels were inappropriately high relative to iron levels. Moreover, when iron levels were equalized with iron treatment, hepcidin levels were higher in endothelial Slc40a1 knockout mice than in controls. Finally, LEC ferroportin levels were inversely correlated with hepcidin levels in multiple mouse models, and treatment of hepcidin-deficient mice with mini-hepcidin decreased LEC ferroportin expression. Overall, these data show that LEC ferroportin modulates LEC iron and consequently BMP expression to influence hepcidin production. Furthermore, LEC ferroportin expression is regulated by hepcidin, demonstrating a bidirectional communication between LECs and hepatocytes to orchestrate systemic iron homeostasis.

H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

BackgroundABO blood group antigens (ABH antigens) are carbohydrate chains glycosylated on epithelial and red blood cells. Recent findings suggest reduced ABH expression in psoriasis and atopic dermatitis, a chronic inflammatory skin disease with retained scale. H antigen, a precursor for A and B antigens, is synthesized by fucosyltransferase 1 (FUT1). Desmosomes, critical for skin integrity, are known to require N-glycosylation for stability. We investigate the impact of H antigens, a specific type of glycosylation, on desmosomes in keratinocytes.MethodPrimary human keratinocytes were transfected with FUT1 siRNA or recombinant adenovirus for FUT1 overexpression. Cell adhesion and desmosome characteristics and their underlying mechanisms were analyzed.ResultThe knockdown of FUT1, responsible for H2 antigen expression in the skin, increased cell-cell adhesive strength and desmosome size in primary cultured keratinocytes without altering the overall desmosome structure. Desmosomal proteins, including desmogleins or plakophilin, were upregulated, suggesting enhanced desmosome assembly. Reduced H2 antigen expression via FUT1 knockdown led to increased keratinocyte differentiation, evidenced by elevated expression of differentiation markers. Epidermal growth factor receptor (EGFR) has been described to be associated with FUT1 and promotes cell migration and differentiation. The effects of FUT1 knockdown were recapitulated by an EGFR inhibitor concerning desmosomal proteins and cellular differentiation. Further investigation demonstrated that the FUT1 knockdown reduced EGFR signaling by lowering the levels of EGF ligands rather than directly regulating EGFR activity. Moreover, FUT1 overexpression reversed the effects observed in FUT1 knockdown, resulting in the downregulation of desmosomal proteins and differentiation markers while increasing both mRNA and protein levels of EGFR ligands.ConclusionThe expression level of FUT1 in the epidermis appears to influence cell-cell adhesion and keratinocyte differentiation status, at least partly through regulation of H2 antigen and EGFR ligand expression. These observations imply that the fucosylation of the H2 antigen by FUT1 could play a significant role in maintaining the molecular composition and regulation of desmosomes and suggest a possible involvement of the altered H2 antigen expression in skin diseases, such as psoriasis and atopic dermatitis.

Transcriptomic profiling of Schlemm’s canal cells reveals a lymphatic-biased identity and three major cell states

Schlemm’s canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm’s canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.