CD3ε Expression Research Articles

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.

CD3ε of a pan T cell marker involved in mouse Aspergillus fumigatus keratitis.

To explore whether CD3ε is involved in the adaptive immunity of Aspergillus fumigatus (A. fumigatus) keratitis in mice and the role of innate and adaptive immunity in it. Mice models of A. fumigatus keratitis were established by intra-stromal injection and corneal epithelial scratching. Subconjunctival injections of natamycin, wedelolactone, LOX-1 inhibitor (poly I) or Dectin-1 inhibitor (laminarin) were used to treat mice with A. fumigatus keratitis. Mice were pretreated by intraperitoneal injection of anti-mouse CD3ε. We observed the corneal infection of mice under the slit lamp microscope and made a clinical score. The protein expression of CD3ε and interleukin-10 (IL-10) was determined by Western blotting. With the disease progresses, the degree of corneal opacity and edema augmented. In the intra-stromal injection models, CD3ε protein expression began to increase significantly on the 2nd day. However, in the scraping epithelial method models, CD3ε only began to increase on the 3rd day. After natamycin treatment, the degree of corneal inflammation in mice was significantly attenuated on the 3rd day. After wedelolactone treatment, the severity of keratitis worsened. And the amount of CD3ε protein was also reduced, compared with the control group. By inhibiting LOX-1 and Dectin-1, there was no significant difference in CD3ε production compared with the control group. After inhibiting CD3ε, corneal ulcer area and clinical score increased, and IL-10 expression was downregulated. As a pan T cell marker, CD3ε participate in the adaptive immunity of A. fumigatus keratitis in mice. In our mice models, the corneas will enter the adaptive immune stage faster. By regulating IL-10, CD3ε exerts anti-inflammatory and repairs effects in the adaptive immune stage.

Extranodal NK/T-Cell Lymphoma Predominantly Composed of Anaplastic Cells: A Frequently Misdiagnosed and Highly Aggressive Variant.

Extranodal NK/T-cell lymphoma (ENKTL) is a non-Hodgkin lymphoma associated with the Epstein-Barr virus that primarily affects individuals in East Asia and indigenous populations in Central and South America. Morphologically, ENKTL typically consists of medium-sized cells or a combination of small and large cells. This report presents 10 cases characterized by predominantly anaplastic cells with diffuse expression of CD30, resembling anaplastic lymphoma kinase-negative anaplastic large cell lymphoma (ALK-negative ALCL) and demonstrating highly aggressive behavior. The cohort included 9 males and 1 female, ranging in age from 29 to 65 years (median age: 47y). Eight patients presented with nasal disease, while 2 had non-nasal disease. Five patients had stage I/II disease, and the remaining 5 had stage III/IV disease. Morphologically, necrosis was observed in 9 cases, angiocentric-angiodestructive growth in 3 cases, and pseudoepitheliomatous hyperplasia in 2 cases. Anaplastic cells predominated in all cases, with some displaying eccentric, horseshoe-shaped, or kidney-shaped nuclei (referred to as "Hallmark" cells). The morphology profile was monomorphic in 3 cases and polymorphic in 7 cases. Immunohistochemically, all cases tested positive for cytotoxic granule markers (TIA1 and granzymeB) and Epstein-Barr virus-encoded RNA. Cytoplasmic expression of CD3ε and CD56 was observed in 9 of 10 cases. Interestingly, most cases (7 of 8) exhibited variable expression of MuM1, ranging from 10% to 90%. All cases showed diffuse positivity for CD30 but were negative for ALK, resulting in 3 cases being initially misdiagnosed as ALK-negative ALCL. Compared with nonanaplastic cases, anaplastic cells predominant ENKTL had a significantly higher frequency of "B" symptoms, bone marrow involvement, hemophagocytic lymphohistiocytosis, and higher Ki67 proliferative index. These findings provide valuable information for pathologists, expanding their understanding of the cytologic spectrum of ENKTL. This rare variant of ENKTL, characterized by the predominance of anaplastic cells and diffuse CD30 expression, exhibits high aggressiveness and should be differentiated from ALK-negative ALCL. Awareness of this uncommon variant is crucial in preventing misdiagnosis and ensuring the timely initiation of therapy.

HIgD-Fc-Ig fusion protein regulates T cell functions by inhibiting TCR signaling pathway in adjuvant arthritis rats.

This study aimed to investigate the effect of hIgD-Fc-Ig on TCR-Lck-Erk activated by IgD in adjuvant arthritis (AA) rats. Wistar rats were divided into the normal, AA model, hIgD-Fc-Ig (1mg/kg, 3mg/kg and 9mg/kg) and Etanercept (3mg/kg) groups. The overall index of AA rats was measured every 3days. The pathologic examination of knee joints and the proliferation of the spleen and thymus of AA rats were detected by H&E staining and CCK-8. The blood flow signal of knee joints of experimental rats was examined by US. The articular bone injury was detected by X-ray. The changes in PBMCs and spleen T cell subsets were detected by flow cytometry. The expression of CD3ε, p-Lck, p-Zap70, Ras, and p-Erk in rat spleens was detected by immunofluorescence and WB. Rat spleen T cells or Jurkat cells treated by IgD to observe the effect of hIgD-Fc-Ig on TCR and its downstream protein expression. The results showed that hIgD-Fc-Ig had a therapeutic effect on AA rats by reducing the secondary inflammation, improving pathological changes. hIgD-Fc-Ig can reduce the ratio of Th cells of PBMCs of AA rats, the ratio of Th, Th1, Th17 cells and increase the ratio of Th2, Treg cells of AA rat spleens. hIgD-Fc-Ig could down-regulate the expression of CD3ε, p-Lck, p-Zap70, Ras, p-Erk in vivo or in vitro. In conclusion, hIgD-Fc-Ig could alleviate the symptoms of AA rats and regulate T cells through TCR-Lck-Erk signaling pathway and maybe a new promising biological agent for RA.

Immunohistochemical analysis of the thymus in newborn and adult yaks (Bos grunniens).

The thymus is the site of development and maturation of functional T lymphocytes and is critically important to the immune system. The purpose of this study was to examine the expression of markers of T lymphocytes, macrophages, dendritic cells, B lymphocytes and plasmocytes in the yak thymus. Twenty healthy male yaks were divided into newborn (2-4 weeks old, n = 10) and adult (3-4 years old, n = 10) group. qRT-PCR was used to evaluate the mRNA expression level of the main markers of the studied cell types. Immunohistochemistry was used to detect the distribution of CD3+ T lymphocytes, CD68+ macrophages, SIRPα+ dendritic cells, CD79α+ B lymphocytes, IgA and IgG+ plasmocytes. Within the same age group, the mRNA expression of CD3ε was highest (P < 0.05), followed by that of CD68, SIRPα, CD79α, IgG and IgA. Furthermore, CD3ε, CD68, and SIRPα mRNA expression levels were higher in newborn yaks than in the adult ones (P < 0.05), whereas those of CD79α, IgA, and IgG were higher in adults (P < 0.05). Immunohistochemical results showed localization of CD3+ T lymphocytes in the thymic cortex and medulla. CD68+ macrophages, SIRPα+ dendritic cells, CD79α+ B lymphocytes, IgA+ and IgG+ plasmocytes were mainly observed in the cortico-medullary region and medulla. In the same age group, the frequency of CD3+ T lymphocytes was higher than that of CD68+ macrophages and SIRPα+ dendritic cells (P < 0.05), followed by those of CD79α+ B lymphocytes and IgA+ and IgG+ plasmocytes. No significant difference was observed between B lymphocyte and plasmocyte frequencies in the yak thymus in both age groups (P > 0.05). The frequency of CD3+, CD68+ and SIRPα+ cells decreased from newborns to adults (P < 0.05). However, the frequencies of CD79α+, IgA+ and IgG+ cells increased from newborn to adult yaks (P < 0.05). The thymus of newborn yaks is well-developed, with higher numbers of T lymphocytes, macrophages, and dendritic cells than those in the adult thymus. However, higher frequencies of plasmocytes and B lymphocytes were detected in the adult thymus, suggesting that adults may better resist infections through humoral immunity as this organ undergoes involution. Furthermore, there was no significant difference in the number of IgA and IgG plasmocytes, which differs from what is observed in rodents and humans. This difference might be related to the fact that yaks live in low-oxygen plateaus.

CD3 engagement as a new strategy for allogeneic “off-the-shelf” T cell therapy

Allogeneic “off-the-shelf” (OTS) chimeric antigen receptor T cells (CAR-T cells) hold promise for more accessible CAR-T therapy. Here, we report a novel and simple way to make allogeneic OTS T cells targeting cancer. By engineering T cells with a bispecific T cell engager (BiTE), both TCRαβ and CD3ε expression on the T cell surface are dramatically reduced. BiTE-engineered T (BiTE-T) cells show reduced reaction to TCR stimulation in vitro and have low risk of graft-versus-host disease (GvHD) in vivo. BiTE-T cells down-regulated CD3ε/TCRαβ on bystander T cells by releasing BiTEs. BiTE-T cells produce much fewer cytokines and are comparable to CAR-T cells on anti-cancer efficacy in xenograft mouse models with pre-existing HLA-mismatched T cells. Co-expressing co-stimulatory factors or T cell-promoting cytokines enhanced BiTE-T cells. Our study suggests CD3ε engagement could be a new strategy for allogeneic T cell therapy worthy of further evaluation.

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8β, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rβ, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation.

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.

The potential target of double negative T cells in cancer immunotherapy.

e15180 Background: Double negative T cell (DNT) is defined by the expression of CD3 (or TCRαβ) with the absence of CD4, CD8 and iNKT cell markers. DNTs account for 1-3% of the total peripheral lymphocytes. Although healthy donor cells derived allogeneic DNTs were reported to have anti-tumor effects in several cancer models, the function of DNTs in tumor microenvironment is not well understood. In this study, we aim to characterize DNTs in cancer tissues and lymph nodes of colorectal cancer patients including their immunological functions. Methods: A total of 14 colorectal cancer tissues and regional lymph nodes were collected and examined for the proportion and the immunological functions of DNTs by FACS analysis. Results: DNTs, which were defined in this study as TCRαβ positive with CD4-, CD8- and CD56- T cells, were detected in colorectal cancer tissues. DNT cells account for more than 5% of TCRαβ-positive populations in 12 cases and out of which 7 cases were more than 20% observed in this study. In all of these 7 cases, DNT cells were also found to be higher in the tumor tissues than in non-metastatic lymph nodes. In addition to colorectal cancer, DNTs were found to be more than 5% of TCRαβ-positive populations in cancer tissues in 3 out of the 6 cases of ovarian and uterine cancer cases.To further evaluate the potential immunological functions of DNTs, expression of cell membrane protein markers were evaluated. CD3ε expression was down regulated in almost all of the DNTs. The CD107a expression of DNTs were higher than that of CD8+ T cells in tumor tissues. When co-culturing DNTs with tumor cells, CD107a expression of DNTs was significantly upregulated. Notably, the intracellular production of perforin and granzyme B were lower than CD8+ T cells. Fas-L expression were found to be higher on DNT cells compared to other T cell populations, suggesting DNT might possess cytotoxic activity in the tumor microenvironment. It is known that upregulation of Fas expression is caused by T cell activation and subsequently induces T cell apoptosis. The upregulation of Fas expression was also observed in DNT cells from non-metastatic lymph nodes. In contrast, Fas expression of DNT cells in tumor tissues was observed to be lower than CD4+ or CD8+ T cells, suggesting that DNT could escape from apoptosis. This downregulation of Fas expression was speculated to be one of the reasons for the accumulation of DNTs in tumor tissues. Conclusions: Although the function of DNTs was still not well elucidated, the accumulation of DNT cells in tumor microenvironment suggests DNTs could be a potential target for cancer immunotherapy.

CD3ε is involved in host immune response against challenges in Nile tilapia (Orechromis niloticus)

CD3 is an important membrane molecule of mature T cells. TCR-CD3 complex plays important roles in the activation and signal transduction of T cells. In this study, the CD3ε (OnCD3ε) gene was cloned from Nile tilapia (Oreochromis niloticus), and its tissue distribution was detected by qPCR. The expression changes in CD3ε were analysed after three challenges (S. agalactiae, A. hydrophila and poly (I:C)) in vivo and in vitro. The open reading frame (ORF) of OnCD3ε contains 531 bp, encoding 176 amino acids, which found that OnCD3ε consists of conserved amino acid residues and motifs (cysteine residues, CXXC motif, immunoreceptor tyrosine-based activation motifs and PxPDY). In addition, tissue distribution displayed that OnCD3ε has the highest expression in thymus and significant expression level in mucosal immune-related tissues (intestine and gills). In vivo, all three challenges could significantly up-regulate the expression of CD3ε in the head kidney and spleen. For further in vitro assays, after poly (I:C) stimulation, OnCD3ε appeared significant up-regulation at 3 hr post stimulation, and two pathogenic bacteria also induce significant up-regulation of OnCD3ε in spleen leucocytes (12 hr). These results suggested that OnCD3ε was likely to get involved in host immune response against pathogenic challenges.

Peculiar Expression of CD3-Epsilon in Kidney of Ginbuna Crucian Carp.

TCR/CD3 complex is composed of the disulfide-linked TCR-αβ heterodimer that recognizes the antigen as a peptide presented by the MHC, and non-covalently paired CD3γε- and δε-chains together with disulfide-linked ζ-chain homodimers. The CD3 chains play key roles in T cell development and T cell activation. In the present study, we found nor or extremely lower expression of CD3ε in head- and trunk-kidney lymphocytes by flow cytometric analysis, while CD3ε was expressed at the normal level in lymphocytes from thymus, spleen, intestine, gill, and peripheral blood. Furthermore, CD4-1+ and CD8α+ T cells from kidney express Zap-70, but not CD3ε, while the T cells from other tissues express both Zap-70 and CD3ε, although expression of CD3ε was low. Quantitative analysis of mRNA expression revealed that the expression level of T cell-related genes including tcrb, cd3ε, zap-70, and lck in CD4-1+ and CD8α+ T cells was not different between kidney and spleen. Western blot analysis showed that CD3ε band was detected in the cell lysates of spleen but not kidney. To be interested, CD3ε-positive cells greatly increased after 24 h in in vitro culture of kidney leukocytes. Furthermore, expression of CD3ε in both transferred kidney and spleen leukocytes was not detected or very low in kidney, while both leukocytes expressed CD3ε at normal level in spleen when kidney and spleen leukocytes were injected into the isogeneic recipient. Lower expression of CD3ε was also found in kidney T lymphocytes of goldfish and carp. These results indicate that kidney lymphocytes express no or lower level of CD3ε protein in the kidney, although the mRNA of the gene was expressed. Here, we discuss this phenomenon from the point of function of kidney as reservoir for T lymphocytes in teleost, which lacks lymph node and bone marrow.

CD3ε Expression Defines Functionally Distinct Subsets of Vδ1 T Cells in Patients With Human Immunodeficiency Virus Infection.

Human γδ T cells expressing the Vδ1 T cell receptor (TCR) recognize self and microbial antigens and stress-inducible molecules in a major histocompatibility complex-unrestricted manner and are an important source of innate interleukin (IL)-17. Vδ1 T cells are expanded in the circulation and intestines of patients with human immunodeficiency virus (HIV) infection. In this study, we show that patients with HIV have elevated frequencies, but not absolute numbers, of circulating Vδ1 T cells compared to control subjects. This increase was most striking in the patients with Candida albicans co-infection. Using flow cytometry and confocal microscopy, we identify two populations of Vδ1 T cells, based on low and high expression of the ε chain of the CD3 protein complex responsible for transducing TCR-mediated signals (denoted CD3εlo and CD3εhi Vδ1 T cells). Both Vδ1 T cell populations expressed the CD3 ζ-chain, also used for TCR signaling. Using lines of Vδ1 T cells generated from healthy donors, we show that CD3ε can be transiently downregulated by activation but that its expression is restored over time in culture in the presence of exogenous IL-2. Compared to CD3εhi Vδ1 T cells, CD3εlo Vδ1 T cells more frequently expressed terminally differentiated phenotypes and the negative regulator of T cell activation, programmed death-1 (PD-1), but not lymphocyte-activation gene 3, and upon stimulation in vitro, only the CD3εhi subset of Vδ1 T cells, produced IL-17. Thus, while HIV can infect and kill IL-17-producing CD4+ T cells, Vδ1 T cells are another source of IL-17, but many of them exist in a state of exhaustion, mediated either by the induction of PD-1 or by downregulation of CD3ε expression.

Novel Immunotherapy Options for Extranodal NK/T-Cell Lymphoma.

Extranodal NK/T-cell lymphoma (ENKTCL) is a highly aggressive mature NK/T-cell neoplasm marked by NK-cell phenotypic expression of CD3ε and CD56. While the disease is reported worldwide, there is a significant geographic variation with its highest incidence in East Asian countries possibly related to the frequent early childhood exposure of Epstein–Barr virus (EBV) and specific ethnic–genetical background, which contributes to the tumorigenesis. Historically, anthracycline-based chemotherapy such as CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone) was used, but resulted in poor outcomes. This is due in part to intrinsic ENKTCL resistance to anthracycline caused by high expression levels of P-glycoprotein. The recent application of combined modality therapy with concurrent or sequential radiation therapy for early stage disease, along with non-anthracycline-based chemotherapy regimens consisting of drugs independent of P-glycoprotein have significantly improved clinical outcomes. Particularly, this neoplasm shows high sensitivity to l-asparaginase as NK-cells lack asparagine synthase activity. Even still, outcomes of patients with advanced stage disease or those with relapsed/recurrent disease are dismal with overall survival of generally a few months. Thus, novel therapies are needed for this population. Clinical activity of targeted antibodies along with antibody–drug conjugates, such as daratumumab (naked anti-CD38 antibody) and brentuximab vedotin (anti-CD30 antibody conjugated with auristatin E), have been reported. Further promising data have been shown with checkpoint inhibitors as high levels of programmed death-ligand 1 expression are observed in ENKTCL due to EBV-driven overexpression of the latent membrane proteins [latent membrane protein 1 (LMP1) and LMP2] with activation of the NF-κB/MAPK pathways. Initial case series with programmed death 1 inhibitors showed an overall response rate of 100% in seven relapsed patients including five with a complete response (CR). Furthermore, cellular immunotherapy with engineered cytotoxic T lymphocytes targeted against LMP1 and LMP2 have shown encouraging results with durable CRs as either maintenance therapy after initial induction chemotherapy or in the relapsed/refractory setting. In this paper, we review this exciting field of novel immunotherapy options against ENKTCL that hopefully will change the treatment paradigm in this deadly disease.

Relative Target Affinities of T-Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model.

Anti-HER2/CD3, a T-cell-dependent bispecific antibody (TDB) construct, induces T-cell-mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell-targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ε expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell-rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell-containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776-85. ©2018 AACR.

Herpesviruses possess conserved proteins for interaction with Nedd4 family ubiquitin E3 ligases

Nedd4 is a family of ubiquitin E3 ligases that regulate numerous cellular processes. In this report, we showed that alpha- and beta-herpesviruses have membrane proteins that regulate the function of the Nedd4 family members. Although the homology search score was quite low, UL56 of herpes simplex virus type 1 and 2, ORF0 of varicella-zoster virus, UL42 of human cytomegalovirus, and U24 of human herpesvirus 6A, 6B, and 7 all possess at least one PPxY (PY) motif in their cytoplasmic domain, and are able to bind with Itch, a member of the Nedd4 family. These viral proteins altered the localization of Itch and decreased Itch expression in co-expressing cells. In addition, these viral proteins reduced the production of retrovirus vectors through the regulation of the Nedd4 family of proteins. U24, but not the other proteins, effectively reduced CD3ε expression on the T cell surface. These viral molecules are thought to contribute to the specific function of each virus through the regulation of Nedd4 family activity.

Indoleamine 2,3-dioxygenase (IDO1), PD-L1, and overall survival (OS) of patients diagnosed with esophageal cancer.

50 Background: Immune checkpoint inhibition of PD-L1 is emerging as an important therapeutic target for patients with advanced esophageal cancer. However, response rates to therapy remain low. IDO1 is a rate-limiting immunosuppressive enzyme that has emerged as an important immunotherapeutic target in human cancer. The role, expression pattern, and relevance of IDO1 in esophageal cancer are currently unknown. Here, we utilize gene expression analysis of the cancer genome atlas and quantitative immunohistochemistry (IHC) to understand whether IDO1 contributes to a poor esophageal cancer patient prognosis. Methods: mRNA expression was assessed using Hi-RNA sequencing in an esophageal squamous cell carcinoma (SCC) cohort of 87 patients and an adenocarcinoma (AC) cohort of 97 patients. Survival data was obtained from the Cancer Genome Atlas. Patient survival was analyzed by the Kaplan-Meier Method. IHC for a second cohort of 93 cases of esophageal SCC were stained for IDO1, PD-L1, and CD3ε, followed by light microscopic immunoscoring analysis. Correlation between markers was analyzed using Fisher’s exact test. Results: The median OS for high versus low IDO1 mRNA levels was 15.9 months vs 41.5 months, respectively (p =0.02) in the SCC cohort. The median OS was 20.1 months and 58.6 months in the high vs low IDO1 mRNA levels, respectively (p = 0.036) in the esophageal AC cohort. High co-expression for IDO1 and PD-L1 vs low co-expression of these markers, demonstrated a median OS of 15.1 months and 41.5 months, respectively, in the SCC cohort, and 13.7 months and 41.5 months, respectively, in the AC cohort. IHC for IDO1 SCC showed a significant correlation with PD-L1 (p = &lt; 0.0001) and CD3ε (p = &lt; 0.0001). PD-L1 expression also significantly correlated with CD3ε expression (p = &lt; 0.0001). Conclusions: Esophageal cancer with high IDO1 and PD-L1 levels is associated with significantly decreased survival. The expression of IDO1 and PD-L1 is significantly enhanced by the coincident intratumoral increase of T cells. These data suggest that combinatorial approaches for combination therapies that simultaneously inhibit IDO1 and PD-(L)1 may enhance T-cell mediated control of esophageal cancer in patients.

Abstract P139: Dual Angiotensin Receptor and Neprilysin Inhibition Improves Renal Hemodynamics and Reduces Kidney Damage in Diabetic Rats

Dual blockade with an Angiotensin Receptor/Neprilysin Inhibitor (ARNI) reduces proteinuria and glomerulosclerosis in diabetic TGR(mREN2)27 rats (i.e., rats displaying angiotensin II-dependent hypertension) more strongly than single AR blockade (ARB), despite a similar effect on blood pressure. Here we investigated whether this is due to improved renal hemodynamics and/or suppression of renal inflammation. TGR(mREN2)27 rats were made diabetic with streptozotocin for 12 weeks, and treated with placebo (n=10), valsartan (ARB; n=8) or valsartan/sacubitril (ARNI; n=8) from week 9-12. Blood pressure was measured by telemetry. Effective renal plasma flow (eRPF) and glomerular filtration rate (GFR) were assessed by quantifying para-aminohippuric acid and inulin clearances. Renal inflammation was quantified by qPCR (CD68 and CD3ε expression, representative for macrophages and T cells, respectively) and fluorescent activated cell sorting (FACS) analysis. ARNI and ARB lowered blood pressure identically, while only ARNI reduced albuminuria. Severe, chronic ischemia and globally sclerotic glomeruli occurred less frequently in kidneys of ARNI-treated animals vs. ARB-treated animals and controls. ARNI, but not ARB, increased eRPF, and a similar trend was observed for GFR. No treatment affected filtration fraction. ARNI decreased CD68 mRNA expression in both renal cortex and medulla, while ARB increased CD68 as well as CD3ε expression in renal medulla. FACS analysis revealed no differences between the treatment groups in the immune-cell fractions that had infiltrated the kidney. In conclusion, improved renal hemodynamics, combined with reduced macrophage infiltration, may underlie the stronger beneficial effects of ARNI on albuminuria and renal histology in diabetic TGR(mREN2)27 rats versus ARB.

OP.5A.07] IMPROVED RENAL HEMODYNAMICS REDUCE ALBUMINURIA AND RENAL MACROPHAGE INFILTRATION IN DIABETIC RATS TREATED WITH DUAL ANGIOTENSIN RECEPTOR AND NEPRILYSIN INHIBITION

Objective: Dual blockade with an Angiotensin Receptor/Neprilysin Inhibitor (ARNI) reduces proteinuria and glomerulosclerosis in diabetic TGR(mREN2)27 rats (i.e., rats displaying angiotensin II-dependent hypertension) more strongly than single AR blockade (ARB), despite a similar effect on blood pressure. Here we investigated whether this is due to improved renal hemodynamics and/or suppression of renal inflammation. Design and method: TGR(mREN2)27 rats were made diabetic with streptozotocin for 12 weeks, and treated with placebo (n = 10), valsartan (ARB; n = 8) or valsartan/sacubitril (ARNI; n = 8) from week 9–12. Blood pressure was measured by telemetry. Effective renal plasma flow (eRPF) and glomerular filtration rate (GFR) were assessed by quantifying para-aminohippuric acid and inulin clearances. Renal inflammation was quantified by qPCR (CD68 and CD3ε expression, representative for macrophages and T cells, respectively) and fluorescent activated cell sorting (FACS) analysis. Results: ARNI and ARB lowered blood pressure identically, while only ARNI reduced albuminuria. Severe, chronic ischemia and globally sclerotic glomeruli occurred less frequently in kidneys of ARNI-treated animals vs. ARB-treated animals and controls. ARNI, but not ARB, increased eRPF, and a similar trend was observed for GFR. No treatment affected filtration fraction. ARNI decreased CD68 mRNA expression in both renal cortex and medulla, while ARB increased CD68 as well as CD3ε expression in renal medulla. FACS analysis revealed no differences between the treatment groups in the immune-cell fractions that had infiltrated the kidney. Conclusions: Improved renal hemodynamics, combined with reduced macrophage infiltration, may underlie the stronger beneficial effects of ARNI on albuminuria and renal histology in diabetic TGR(mREN2)27 rats versus ARB.

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247

Primary T-cell immunodeficiency with functional revertant somatic mosaicism in CD247

Inhibition of reactive astrocytosis in established experimental autoimmune encephalomyelitis favors infiltration by myeloid cells over T cells and enhances severity of disease

Reactive astrocytosis, involving activation, hypertrophy, and proliferation of astrocytes, is a characteristic response to inflammation or injury of the central nervous system. We have investigated whether inhibition of reactive astrocytosis influences established experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We made use of transgenic mice, which express herpes simplex virus-derived thymidine kinase under control of a glial fibrillary acidic protein promotor (GFAP HSV-TK mice). Treatment of these mice with ganciclovir leads to inhibition of reactive astrocytosis. When GFAP HSV-TK mice were treated for seven days following onset of EAE with ganciclovir, disease severity increased. Although aquaporin-4 staining on astrocyte endfeet at the glia limitans remained equally detectable, GFAP immunoreactivity and mRNA expression in CNS were reduced by this treatment. Ganciclovir-treated GFAP HSV-TK mice with EAE had a 78% increase in the total number of infiltrating myeloid cells (mainly macrophages), whereas we did not find an increase in infiltrating T cells, using quantitative flow cytometry. Per cell expression of mRNA for the macrophage-associated molecules TNFα, MMP-12 and TIMP-1 was elevated in spinal cord of GFAP HSV-TK mice treated with ganciclovir. Relative expression of CD3ε was downregulated, and expression levels of IFNγ, IL-4, IL-10, IL-17, and Foxp3 were not significantly changed. mRNA expression of CCL2 was upregulated, and CXL10 was downregulated. Thus, inhibition of reactive astrocytosis after initiation of EAE leads to increased macrophage, but not T cell, infiltration, and enhanced severity of EAE. This emphasizes the role of astrocytes in controlling leukocyte infiltration in neuroinflammation.