Levels Of MICA Research Articles

Comparative Study of the Effects of Docosahexaenoic Acid, Linoleic Acid, and Taxol in Decreasing the Expression of miR-10b, and miR-20a OncomiRs, and their Target Major Histocompatibility Complex Class I Chain-Related Proteins A (MICA) in Triple-Negative Metastatic Breast Cancer Cell Line

Background: Molecular signatures of the effects of widely used omega-3 and omega-6 fatty acids as dietary supplements and even in cancer therapy have been studied. Of them, microRNAs, have been shown to undergo altered expression after treatment with docosahexaenoic acid (DHA) and linoleic acid (LA) in different cancer types. In this study, the expression level of two well-known onco-microRNAs (miR), miR-10b and miR-20a, and also major histocompatibility complex class I chain-related protein A (MICA) as the target for these oncomiRs were investigated after treatment with DHA, LA, and common therapeutic agent Taxol. Methods: MDA-MB-231 cells were cultured in a complete RPMI 1640 medium and an MTT assay was performed to determine IC50 of DHA, LA, and Taxol. Cells were treated by DHA (100µM), LA (50µM), and Taxol (0.3µM) alone or in different combinations. After RNA extraction and cDNA synthesis, the expression levels of miR-10b, miR-20a, and MICA were determined by quantitative real-time PCR. U6 and β-Actin were used as endogenous controls, respectively. Results: Our findings showed that DHA, LA, and Taxol, and their combinations led to the significantly decreased expression of miR-10b and miR-20a (all P<0.0001). The expression level of miR-10b and miR-20a significantly decreased when treated with the LA+TAX combination. MICA as the target of miR-10b and miR-20a was also down-regulated significantly (P<0.0001) especially when treated with DHA+LA+TAX. Conclusion: Useful effects of DHA, LA, and their combination in breast cancer (BC) could be interpreted in part by decreasing the expression level of oncomiRs. In the case of MICA have had some contrary. High expression of MICA/B caused an approving outcome concerning the relapse-free period in early BC patients, however; some studies have shown that higher MICA expression was found as a sign of poor prognosis in BC. The decreased expression of MICA in the present study suggests the potential role of DHA and LA should be used in dietary supplements of BC patients.

Tregs, gMDSCs, and levels of soluble MICA as prognostic biomarkers for combined NEO-201 and pembrolizumab.

2530 Background: The humanized IgG1 monoclonal antibody NEO-201 binds to Core 1 and/or extended Core 1 O-glycans expressed by several human solid and blood tumors, as well as neutrophils, and mediates killing of cancer cells, neutrophils, regulatory T cells (Tregs) and granulocytic myeloid-derived suppressor cells (gMDSCs) via ADCC and CDC. Resistance to PD-1/PD-L1 blockade may be due to the accumulation of Tregs and gMDSCs in the tumor microenvironment (TME). NEO-201 was proven to bind and reduce the amount of circulating Tregs in cancer patients. This supports the rationale of the ongoing phase II clinical trial (NCT03476681) evaluating the activity of NEO-201 with pembrolizumab in adults with solid tumors resistant to prior checkpoint inhibitors. Furthermore, elevated serum levels of soluble MICA (sMICA) have been correlated with decreased NK cell activity. Methods: Cancer patients were treated each cycle with 3 doses of NEO-201 1.5mg/kg every 2 weeks and pembrolizumab 400mg IV every 6 weeks and imaged for response every 2 cycles. PBMCs and serum from cancer patients pre- and at multiple time points post-treatment were used to evaluate the percentage of circulating gMDSCs and Tregs (flow cytometry) and sMICA levels (ELISA). Results: NEO-201 recognizes naïve Tregs (nTregs: CD3+/CD4+/CD45RA+/Foxp3low cells) but not effector Tregs (eTregs: CD3+/CD4+/CD45RA-/Foxp3high cells). NEO-201 also binds to gMDSCs, defined as HLA-DRneg/CD33+/CD15+/ CD14neg/CD66b+ cells. In patients with durable SD, a patient with HNSCC (SD >11 months) showed >50% reduction of nTregs and >90% of gMDSCs at C3D1 (beginning of cycle 3) compared to baseline. One patient with cervical cancer (SD >8 months) showed 40% reduction of nTregs at C3D1 compared to baseline, while gMDSCs trended down to baseline levels at C3D1 after initial increase. Conversely, we observed a general uptrend of nTregs and gMDSCs in patients with PD after treatment. Median serum levels of sMICA pre-treatment were 33-fold higher in patients with PD compared to patients with SD. Levels of sMICA remained elevated in patients with PD and low in patients with SD at all time points post-treatment. Conclusions: Depletion of circulating Tregs and gMDSCs may prevent their accumulation in the TME and enhance the efficacy of pembrolizumab in subjects with tumors resistant to checkpoint inhibitors. The decrease in circulating nTregs and gMDSCs after treatment with NEO-201 and pembrolizumab was associated with durable SD. High levels of sMICA can impair ADCC mediated by NEO-201, resulting in poor clinical response. Low levels of sMICA in patients with SD, together with the reduction of both circulating nTregs and gMDSCs, could be favorable prognostic markers for clinical benefit following treatment with NEO-201 and pembrolizumab. Ongoing enrollment in this clinical trial will validate these findings in larger cohorts. Clinical trial information: NCT03476681 .

Functional MICA Variants Are Differentially Associated with Immune-Mediated Inflammatory Diseases.

As the principal ligand for NKG2D, MICA elicits the recruitment of subsets of T cells and NK cells in innate immunity. MICA gene variants greatly impact the functionality and expression of MICA in humans. The current study evaluated whether MICA polymorphisms distinctively influence the pathogenesis of psoriasis (PSO), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) in Taiwanese subjects. The distributions of MICA alleles and levels of serum soluble NKG2D were compared between healthy controls and patients with PSO, RA, and SLE, respectively. The binding capacities and cell surface densities of MICA alleles were assessed by utilizing stable cell lines expressing four prominent Taiwanese MICA alleles. Our data revealed that MICA*010 was significantly associated with risks for PSO and RA (PFDR = 1.93 × 10-15 and 0.00112, respectively), while MICA*045 was significantly associated with predisposition to SLE (PFDR = 0.0002). On the other hand, MICA*002 was associated with protection against RA development (PFDR = 4.16 × 10-6), while MICA*009 was associated with a low risk for PSO (PFDR = 0.0058). MICA*002 exhibited the highest binding affinity for NKG2D compared to the other MICA alleles. Serum concentrations of soluble MICA were significantly elevated in SLE patients compared to healthy controls (p = 0.01). The lack of cell surface expression of the MICA*010 was caused by its entrapment in the endoplasmic reticulum. As a prevalent risk factor for PSO and RA, MICA*010 is deficient in cell surface expression and is unable to interact with NKG2D. Our study suggests that MICA alleles distinctively contribute to the pathogenesis of PSO, RA, and SLE in Taiwanese people.

Cellular immunotherapy combined with platinum-based chemotherapy prolongs survival for non-small cell lung cancer patients.

Platinum-based chemotherapy is the primary treatment option for advanced non-small cell lung cancer (NSCLC) patients without a driver gene mutation, but its efficacy is still modest. Through a potential synergistic effect, autologous cellular immunotherapy (CIT) composed of cytokine-induced killer (CIK, natural killer (NK), and T cells) might enhance it. NK cells exhibited in vitro cytotoxicity toward lung cancer cells (A549 cells) following platinum therapy. Using flow cytometry, the expression of MICA, MICB, DR4, DR5, CD112, and CD155 on lung cancer cells was assessed. In this retrospective cohort study, there were included 102 previously untreated stage IIIB/IV NSCLC patients ineligible for tyrosine kinase inhibitor (TKI) target therapy who received either chemotherapy alone (n = 75) or combination therapy (n = 27). The cytotoxicity of NK cells for A549 cells was increased obviously and a time-dependent enhancement of this effect was also observed. After platinum therapy, the levels of MICA, MICB, DR4, DR5, CD112, and CD155 on the surface of A549 cells were increased. In the combination group, the median PFS was 8.3 months, compared to 5.5 months in the control group (p = 0.042); the median overall survival was 18.00 months, compared to 13.67 months in the combined group (p = 0.003). The combination group had no obvious immune-related adverse effects. The combination of NK cells with platinum showed synergistic anticancer effects. Combining the two strategies increased survival with minor adverse effects. Incorporating CIT into conventional chemotherapy regimens may improve NSCLC treatment. However, additional evidence will require multicenter randomized controlled trials.

Suppression of CD56bright NK cells in breast cancer patients is associated with the PD-1 and TGF-βRII expression.

Natural killer (NK) cells, as professional cytotoxic cells, play a key role againstcancer intheearly and metastaticstages. Their functional defects are highly associated with the initiation or progression of breast cancer (BC). Here, we investigated the phenotypic characterization of NK cells in 26 newly diagnosed BC patients in comparison to 12 healthy counterparts. Expression of CXCR3 and PD-1, and also NKG2D, and TGF-βRII were studied on CD56dim and CD56bright NK cells from fresh peripheral blood (PB) samples using flow cytometry. The plasma levels of IFN-γ and soluble MIC-A levels were also assessed by ELISA. Both CD56dim and CD56bright NK subtypes showed lower CXCR3 and NKG2D expression in BC patients than healthy subjects. Furthermore, patients' CD56bright NK cells significantly showed higher expression levels of TGF-βRII and PD-1. Interestingly, increased concentration of MIC-A level in plasma of BC patients was associated with the higher TGF-βRII and PD-1 expression in all NK cells, while the plasma level of IFN-γ was associated with the lower TGF-βRII expression on CD56bright NK cells in these patients. Our results demonstrated phenotypically suppressed-NK cells, especially in the CD56bright subset of BC patients. It specifies their potential incompetence and outlines decrement of their anti-tumor activity, which could be interrelated with the tumor pathogenesis, TME immunosuppression, and so disease progression. The induction of compensatory mechanisms revives NK cells function and could be used in combination with the conventional treatments as a putative therapeutic approach for targeted immunotherapy.

NK Cells Suppress Hyperfunction of CTLs Via ATM-Mediated NKG2D/NKG2DLs Activation in Severe Aplastic Anemia

Object: The activating receptor NKG2D on the surface of NK cells in peripheral blood of patients with severe aplastic anemia (SAA) was significantly increased, and NK cells may be activated during the pathogenesis of SAA. However, the immunomodulatory effect of NK cells on CTL cells remains unclear. ATM protein is a serine/threonine protein kinase mainly involved in cell cycle regulation, DNA damage recognition and repair. Activation of ATM may be the key to regulating the expression of NKG2DLs in T cells stimulated by superantigens. By detecting the changes of NKG2DLs in CTL cells of SAA patients under the regulation of ATM, we explored the immunoregulatory effect of NK cells on autologous CTL cells through the signaling axis NKG2D/NKG2DLs. Methods: We used flow cytometry, RT-qPCR and Western Blot to detect the expression of NKG2DLs (MICA, ULBP1, ULBP3) in CTL cells of untreated SAA patients, remission SAA patients and healthy controls under the intervention of CD3/CD28 antibody and ATM agonist (chloroquine phosphate). NK and CTL cells from SAA patients were co-cultured in vitro, and the function of CTL under the regulation of ATM was detected. Results: Under the intervention of CD3/CD28 antibody and chloroquine phosphate, the level of MICA in CTL cells detected by flow cytometry in untreated SAA patients was higher than that in convalescent SAA patients and healthy controls (Fig.A). RT-qPCR detection of mRNA levels of NKG2DLs (MICA, ULBP1 and ULBP3) in CTL cells of untreated SAA patients was also higher than that of remission SAA patients and healthy controls (Fig.B). When ATM agonist (chloroquine phosphate), ATM inhibitor (KU55933), and chloroquine phosphate + KU55933 were used to intervene T cells, Western Blot detected the correlation between ATM phosphorylation and NKG2DLs (MICA, ULBP1) expression(Fig.D). We co-cultured CTL cells from SAA patients with different interventions in vitro with autologous NK cells. The expression of IFN-γ in CTL cells in the ATM agonist group was significantly lower than that in the ATM inhibition group, and the apoptosis rate was higher than that in the ATM inhibition group and the control group (Fig.C). There was no significant difference in the expression of perforin and granzyme B. In addition, NK cell apoptosis did not differ among the groups.The expression levels of free MICA and ULBP3 in the co-culture supernatant were detected by ELISA, and the ATM agonist group was higher than the inhibition group(p<0.05). Conclusion: Activation of ATM increases the expression of NKG2DLs in CTL cells of SAA patients, which promotes the inhibitory effect of NK cells on CTL cells. Our study further confirms that ATM can regulate the relationship between NK cells and CTL cells in SAA disease, and inhibit the hyperactive state of CTL cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CNSC-13. TUMOR INFILTRATION OF GAMMA-DELTA T CELLS IN GLIOBLASTOMA

Abstract Gamma-delta (γδ) T cells are innate immunity effector lymphocytes with known prominent anti-tumor reactivity against aggressive glioblastoma (GBM). However, therapeutic approaches have had limited success due to the protective blood-brain-barrier and the immunosuppressive GBM tumor microenvironment. In this study, we determined Vδ1 a Vδ2 γδ T cell populations in peripheral blood and paired tumor tissue samples in patients (n=40) following the resection and throughtout the therapy follow-up. Tumor samples were processed using enzymatic kits and gentleMACSTM Dissociator (Miltenyi Biotec Inc.) and tumor-infiltrating γδ T lymphocytes (TILs) were analyzed by flow cytometry.We found infiltration of both intratumoral CD3+ γδ T cell subsets in 68% tumor samples. We detected Vδ1 γδ T cells in the range 0-0.8% (median 0.26%). Majority of GBM patients presented the Vδ2 subset among TILs in the range 0-13.8% (median 1.5%). Functional studies showed prominent cytotoxicity of magnetically sorted Vδ1 a Vδ2 γδ T cells against GBM cell lines and more importantly against primary tumors. Detailed phenotypic profiling and single-cell sequencing of Vδ2 γδ T cells is currently underway. Next, we identified the EphA2 receptor as one of the targets for tumor-reactive Vδ1 γδ T cells. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of GBM killing mediated by Vδ1 γδ T cells. Furthermore, Luminex xMAP technology identified significantly elevated levels of stress ligand MICA and check-point inhibitor ligands PD-L1 (B7-H1, CD274) and B7-H3 (CD276) and galectin-9 in patients‘ plasma samples at diagnosis compared to age-matched controls.The patient’s clinical course and therapeutic protocols will be discussed.This study was supported by Ministry of Health, Czech Republic (grant NV19-05-00410 to AK) by Ministry of Health, Czech Republic-conceptual development of research organization (FNBr, 65269705). All rights reserved.

Association of MICA gene Exon-5 polymorphism in oral submucous fibrosis

The present study was conducted to explore the allele frequencies of MICA gene Exon-5 transmembrane and to measure the circulatory MICA levels in various histologic grades of patients with oral submucous fibrosis (OSF) compared to healthy individuals. We enrolled a total of 595 patients for this cross-sectional study and divided them into 2 groups: healthy controls (n=320) and patients with OSF (n=275). Further, patients with OSF were subdivided based on their histologic gradings. The genomic DNA was extracted followed by a polymerase chain reaction and genotyping using the ABI Prism DNA Sequencer (ThermoFisher Scientific, Inc., Waltham, MA, USA). Our study showed that the A5 allele of the MICA gene in the Exon-5 region conferred significant risk for patients with OSF. With reference to the histologic gradings of OSF, we found that the MICA gene conferred statistically significant risk among patients with grade III OSF. On the other hand, the A8 allele of MICA gene in the Exon-5 region conferred significant protection among the overall OSF cohort and in the grade III of histologic grade. Finally, the circulatory human MICA levels were found to have a stepwise increase from grade I toward grade III in patients with OSF. Our results suggested that the A5 allele in MICA might confer risk for the progression of OSF among the South Indian ethnic population.

Immune Evasion of Hepatoma Cancer Stem-Like Cells from Natural Killer Cells

Poor prognosis in liver cancer is due to its high frequency of intrahepatic metastasis. Cancer stem-like cells (CSLCs), which possess the properties of stemness, tumor initiation capability, and resistance to therapy, also exhibit metastatic potential. Immune surveillance plays an important role in the accomplishment of metastasis. Herein, the property of immune evasion in CSLCs was investigated. Sphere cells were induced as CSLCs using a sphere induction medium containing neural survival factor-1. The expression of genes involved in immune evasion was determined using RNA-sequencing for sphere and parental cells followed by validation using flow cytometric analysis and ELISA. Susceptibility to natural killer (NK) cell-mediated cytotoxicity was examined by a chromium release assay. A xenograft model using BALB/c nu/nu mice was used to assess tumor growth. Gene set enrichment analysis was performed for interpreting RNA sequencing. The cell surface expressions of PD-L1, PD-L2, and CEACAM1 were upregulated and those of ULBP1 and MICA/MICB were downregulated in SK-sphere, CSLCs derived from SK-HEP-1, compared with that in parental cells. Levels of soluble MICA were elevated in conditioned medium from SK-sphere. Expression of HLA class I was not downregulated in SK-sphere. The susceptibilities to NK cell-mediated killing and secreted perforin were significantly lower in both CSLCs derived from SK-HEP-1 and HLE than in parental cells. Tumors formed upon inoculation of SK-sphere in immunodeficient mice harboring NK cells were larger than those formed upon inoculation of parental cells. Human hepatoma cell line-derived CSLCs may possess immune evasion properties, especially from NK cell-mediated immunity.

Роль факторов иммуносупрессии в прогнозе эффективности клеточной иммунотерапии у пациентов с солидными опухолями

Production of different factors with wide immunosuppressive properties by tumor cells is one of the mechanisms that support tumor growth. We think that immunosuppressive action of tumor cells should be taken into account for cellular antitumor therapy optimization. Aim of the study – to evaluate the effectiveness of cellular immunotherapy factors (ISF) for response prediction in patients receiving tag7/PGRP-S gene modified tumor cells (GMV) or autologous dendritic cells vaccine (DCV) for melanoma (Mel), kidney cancer (RCC) and soft tissue sarcoma (STS). Material and methods. We have treated 204 patients with cellular therapy since 2001 till 2016 (88 – GMV, 1 16 – DCV). ISF (MICA, TGF-β, IL-10 и VEGF) were measured in tumor tissue cultures of 88 patients (71 – Mel, 6 – RCC, 11 – STS). Median age of patients was 51 year. Patient were divided as having insufficient response (IR) or sufficient response (SR) for the analysis purpose. Results. ISF concentration in cultures of patients with IR was higher than in patients with SR (р &lt; 0.05). SR could be predicted correctly in 74.4% using ISF. The most important factor was TGF-β1. In the Cox model TGF-β1 and MICA levels were significant predictors of overall survival for Mel patients (р &lt; 0.05). VEGF was the most important factor for time to progression prognosis. Spirman correlations showed moderate significant correlation between ISF (р &lt; 0.05). Conclusions. ISF concentration in cell cultures obtained from the patients with Mel, RCC or STS is important independent predictive factor for time to progression and overall survival. Our data supports rigid selection criteria based on biological properties of tumor cells for patients receiving cellular therapy for solid tumors

Prognostic Value of Serum MICA Levels as a Marker of Severity in COVID-19 Patients

ABSTRACT The COVID-19 global pandemic and high mortality rates necessitate the development of diagnostic and prognostic tools, as well as expanding testing capacity. Existing methods for detecting and characterizing SARS-CoV-2 infection are typically based on viral genome detection or measuring COVID-19-specific antibody levels. Despite their value, these methods are unable to predict disease outcomes in patients. Given the critical role of innate immune cells, particularly natural killer (NK) cells, in antiviral defense, this study sought to determine the prognostic value of serum secretory MHC class I polypeptide–related sequence A (sMICA) levels as an essential ligand for the NKG2D receptor, the master regulator of NK cell development and responsiveness. Serum MICA levels were measured by ELISA assay. Sera (n = 60) from SARS-CoV-2 positive patients were collected, and disease severity was determined using clinical criteria. The patient group included 30 patients with mild disease and 30 severely ill patients, as well as 30 healthy controls. Our findings revealed that serum MICA levels were significantly higher in patients than in controls, especially in cases with severe complications (P < .0001). Higher serum MICA levels may be associated with respiratory failure in COVID-19 and may serve as a marker of clinical severity in patients infected with SARS-CoV-2, particularly when clinical manifestations are insufficient to make a confident prediction.

AMR or CAV without HLA-DSA: Could MICA-DSA Be the Culprit?

<h3>Purpose</h3> The role of non-HLA antibodies, such as MICA and AT1R, in pediatric heart transplant (HTx) is not well delineated. While both may contribute to worse outcomes after HTx, regular surveillance monitoring and B-cell treatment is generally reserved for recipients with HLA-DSA. <h3>Methods</h3> Case report describing the development of non-HLA DSA in a patient who is s/p second HTx with concern for CAV in both allografts. <h3>Results</h3> We introduce a 16-yo male who received a HTx for familial DCM in 2007, at age 2yo. Prior to HTx he was mechanically supported with ECMO (10 dys) and a Berlin Heart (6 wks). PRA were negative and his retrospective cross match (XM) was invalid due to auto-reactivity. Immunosuppression (ISX) was maintained with tacrolimus and sirolimus. Three years (2010) after HTx <i>de novo</i> HLA-DSA (DQ8, DR53) were detected following a period of medical non-compliance; HLA-DSA gradually increased in strength to 20,000 MFI. While all surveillance biopsies since transplant remained without evidence of ACR or AMR, he required treatment for clinical rejection in 2011 (pulse steroids, IVIG) and 2015 (pulse steroids, ATG) and MMF was added to his ISX. He eventually developed micro-vascular CAV, presenting as heart block and diastolic dysfunction, and received a second HTx in 2016. His retrospective XM again showed autoreactivity; he had a negative virtual XM and never developed new HLA-DSA. A recent biopsy, prompted by mild diastolic dysfunction, revealed new endothelial swelling (ACR 1R, AMR 1H, C4D neg). Additional analysis showed high levels of donor-specific MICA, and AT1R antibodies; both remained high despite initiation of B-cell therapy (rituximab, bortezomib, IVIG). Further retrospective analysis revealed that the MICA antibodies were developed <i>de novo</i> against his first heart (targeting a common epitope present on both donors' MICA) alongside the HLA-DSA, while AT1R antibodies were already present before his first HTx. Thus, MICA-DSA may have contributed to accelerated CAV in his first allograft, and cause endothelial swelling and AMR in his second allograft. This may have been further exacerbated by the ongoing presence of AT1R, though their role in our patient's clinical course remains less clear. <h3>Conclusion</h3> Non-HLA DSA can be injurious to cardiac allografts, and the presence of AMR or CAV without HLA-DSA warrants further evaluation for MICA-DSA and AT1R antibodies.

A study of the association between long non coding RNA (ULBP1 and MICA/B) expression and preeclampsia in Egypt

BackgroundPreeclampsia is associated with excessive inflammation and increased serum monokines. Although preeclampsia pathogenesis has been connected to uterine natural killer (NK) cells, natural killer group 2, member D (NKG2D) is thought to have a role in immune response mediation. NKG2D ligands (NKG2DLs) are significantly expressed in preeclampsia, and the heterogeneity of NKG2DL expression is unclear. Aim of the studyIn current study we aimed to study the expression levels of ULBP1 and MICA/B genes in women with preeclampsia. Patients & methodsThe Medical Biochemistry and Molecular Biology Department, as well as the Gynecological Department, at Menofia University's Faculty of Medicine, conducted this research. There were 120 female in all, with 80 of them having preeclampsia (40 mild and 40 severe preeclampsia). There were 40 healthy pregnant women as control group. All were chosen from the Menofia University Hospital's, Gynecological and Obstetric Departments between May 2020 and May 2021. After all subjects have given their informed permission, full history taking, clinical examination were performed on all participants and laboratory examinations include: measurement of liver function tests (AST&ALT), renal function tests (urea, creatinine, and uric acid), complete blood count, assessment of protein in urine and calculation of gene expression of ULPB1, MICA, and MICB in placental tissue by real time q PCR method. ResultsThere was significant increase in ULPB1, MICA and MICB genes expression in cases versus control and there was significant higher level of ULPB1, MICA, and MICB in severe cases compared to mild cases. ConclusionFrom this study, it could be concluded that expression of ULPB1, MICA and MICB levels might be used as biomarkers for diagnosis and detection of severity of preeclampsia.

Low MICA Gene Expression Confers an Increased Risk of Graves' Disease: A Mendelian Randomization Study.

Background: Expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) plays a major role as a "danger signal" on stressed cells to promote removal of the latter by NKG2D-expressing cytotoxic lymphocytes. NKG2DL expression has been found in peripheral immune cells as well, such as in macrophages; however, the effect of this expression is yet to be determined. Methods: We determined instrumental variables (IVs; R2 <0.01 in linkage disequilibrium), explaining the major variance in major histocompatibility complex class I chain-related protein A (MICA) and B (MICB) gene expression levels from the expression-quantitative trait locus (eQTL) of NKG2DLs based on the RNA-seq analysis of peripheral blood mononuclear cells (PBMCs) from 381 Japanese. Simultaneously, the target outcomes were filtered by PheWAS from 58 health risks, using a community-based cohort study composed of 44,739 Japanese residents. Finally, we estimated the causal effect of gene expression levels on the outcomes using the Mendelian randomization approach. Results: We determined nine and four IVs, explaining 87.6% and 33.0% of MICA and MICB gene expression levels, respectively. In the association test, we identified 10 or 13 significant outcomes associated with the MICA or MICB eQTLs, respectively, as well as the causal effect of MICA expression on Graves' disease (GD) (p = 4.2 × 10-3; odds ratio per 1 S.D. difference in the expression: 0.983 [confidence interval: 0.971-0.995]), using the weighted median estimator, without significant pleiotropy (p > 0.05), and the results were consistent across the sensitivity analyses. Conclusions: Our study provide novel evidence associating NKG2DL expression with GD, an autoimmune thyroiditis; direction of the effect indicated the immunoregulatory role of MICA expression in PBMCs, suggesting the importance of further functional assays in inflammatory diseases.

Associations of high-resolution-typing-defined MICA and MICB polymorphisms, and the levels of soluble MICA and MICB with Oral Squamous Cell Carcinoma in Bulgarian patients.

Oral Squamous Cell Carcinoma (OSCC) is a malignancy characterized by an aggressive tumor growth and significant mortality. Clarifying mechanisms responsible for immunomodulation are among the main challenges for the development of personalized approaches for the management of patients with Oral Squamous Cell Carcinoma. The aim of the present study was to analyze the relevance of MICA and MICB to Oral Squamous Cell Carcinoma pathogenesis focusing on allele polymorphisms and the levels of soluble MICA and MICB molecules. 73 patients diagnosed with Oral Squamous Cell Carcinoma and 149 healthy controls from the Bulgarian population were included in the study. MICA and MICB polymorphism was analyzed at high-resolution level using Next-Generation Sequencing. Serum levels of soluble MICA and MICB molecules were measured by ELISA. Our results show significant protective association with MICB*002:01, while relatively rare alleles MICB*018, *019, and *020 were observed with statistically significant increased frequency in Oral Squamous Cell Carcinoma patients compared to controls. Additionally, a predisposing association was observed for MICA*008:01-MICB*019 haplotype. A correlation analysis between functionally relevant MICA polymorphisms and sMICA showed that homozygosity for MICA-A5.1 or 129Val in OSCC patients was associated with significantly higher serum levels of sMICA. This is the first study showing significant associations between MICB alleles and Oral Squamous Cell Carcinoma and suggesting the possible role of MICB in immunosurveillance in Oral Squamous Cell Carcinoma development. Observed correlations between the levels of soluble MICA molecules and functionally relevant polymorphisms might represent a further step toward a better understanding of molecular mechanisms of Oral Squamous Cell Carcinoma and developing strategies for therapeutic targeting harnessing effective immunosurveillance.

Analysis of Serum Vitamin D and Major Histocompatibility Complex Class I-Related Chain a (Mica) Levels in Patients With Breast Carcinoma

Introduction: Breast carcinoma is the most deadly cancer in women worldwide, with an incidence rateof 26 cases per 100,000 women in Indonesia. Treatment and prognosis is very dependent on the stage ofbreast carcinoma. Vitamin D can inhibit proliferation and invasive cancer cells, induction of differentiation,apoptosis and promotion of angiogenesis. Major Histocompatibility Complex class I - related chain A(MICA) plays a role in reducing the expression of NKG2D receptors in breast carcinomas so that they canbe targeted for treatment of breast carcinoma treatment. The aim of this study was to determine vitamin Dand MICA levels in serum patients with non-metastatic and metastatic breast carcinoma.Method: We examined 86 samples of breast carcinoma patients (44 non-metastatic samples and 42metastatic samples) with a range of 29 - 68 years old. Vitamin D levels were examined by the EnzymeLinked Flourescent Assay (ELFA) method while the MICA levels were examined by Enzyme LinkedImmunoabsorbant Assay (ELISA).Result: There were significant differences in mean vitamin D levels in non-metastatic and metastatic breastcarcinomas (29.88 ng/mlvs19.06 ng/ml; p &lt;0.01).Serum MICA levels in patients with non-metastatic breastcarcinoma were lower (333.52 pg/ml) than in metastatic breast carcinoma(333.52 pg/mlvs528.71 pg/ml; p&lt;0.01).A negative correlation was found between vitamin D levels andMICA in both groups (Pearson correlation, p: -0.58).Conclusion: Metastatic breast carcinoma had lower vitamin D levels and higher serum MICA levels thannon-metastatic breast carcinoma.

The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding

MICA/B proteins are expressed on the surface of various types of stressed cells, including cancer cells. Cytotoxic lymphocytes expressing natural killer group 2D (NKG2D) receptor recognize MICA/B and eliminate the cells. However, cancer cells evade such immune recognition by inducing proteolytic shedding of MICA/B proteins. Therefore, preventing the shedding of MICA/B proteins could enhance antitumor immunity. Here, by screening a protease inhibitor library, we found that the fatty-acid amide hydrolase (FAAH) inhibitor, URB597, suppresses the shedding of MICA/B. URB597 significantly reduced the soluble MICA level in culture medium and increased the MICA level on the surface of cancer cells. The effect was indirect, being mediated by increased expression of tissue inhibitor of metalloproteinases 3 (TIMP3). Knockdown of TIMP3 expression reversed the effect of URB597, confirming that TIMP3 is required for the MICA shedding inhibition by URB597. In contrast, FAAH overexpression reduced TIMP3 expression and the cell-surface MICA level and increased the soluble MICA level. These results suggest that inhibition of FAAH could prevent human cancer cell evasion of immune-mediated clearance.

AML-396: Development of a Relevant Cell Model for NKG2D CAR-T Efficiency Assessment

Context AML is a severe blood pathology with a complicated therapy strategy and high risk of relapse. Objective Different types of cancer cells exhibit various expression rates of NKG2D ligands. An effective cell model is a crucial stage for profound NKG2D CAR-T functional cell estimation. Design We have designed two NKG2D plasmid constructs for two types of CAR-T cells' production and consequent modification with costimulatory signals (4-1BB, CD 28, TIGIT, ICOS). By means of qPCR, we analyzed expression of main NKG2D receptor ligands: MICA, MICB, ULBP 1–6 in K562 (myelogenous leukemia), NB-4 (acute myeloid leukemia), and HeLa (cervical cancer) tumor cell lines. Prominent ligand expression within HeLa cells makes it a sensitive model for NKG2D CAR-T efficiency verification. For cell model creation, we designed shRNA constructs for knockdown of the mentioned NKG2D ligands in HeLa cells. Setting The first designed NKG2D vector includes a suicide iCasp9 sequence. The second vector contains EGFR with a signal sequence. For cloning these sequences and plasmid assembly, the lentiviral pLVT1 vector was used in both cases. The NKG2D-CAR gene sequence was adopted from the Baumeister et al. study and contains human cytoplasmic CD3ζ fused to the full-length human NKG2D gene (KLRK1). NKG2D-CD3ζ fusion was obtained after gene synthesis (Evrogen, Russia). Both newly designed plasmids contain DAP10 sequence for NKG2D signal transduction enhancement. shRNA sequences were designed for MICA, MICB, and ULBP 1–6 ligands and cloned into pLKO.1 vector (Addgene, Plasmid #10878). After lentiviral assembly, all designed shRNA was transduced into the HeLa cell line. Results We observed the levels of MICA, MICB, and ULBP3–6 to be the highest and upregulated in HeLa cells, compared to K562 and NB-4 (p Conclusions Due to patient-specific patterns of ligand expression and its influence on CAR-T cell therapy efficiency, we developed tumor cell line models allowing the assessment of NKG2D CAR-T efficiency in the case of different levels of ligands on tumor cell membranes.

ABCL-041: Soluble MIC-A and MIC-B, and NKG2D Expression in NK Cells, NKT Cells, and Gamma-Delta T Lymphocytes in Patients with De Novo Non-Hodgkin Lymphoma and Post-Chemotherapy

Context: The presence of soluble NKG2D receptor ligands in different types of cancer affects the cytotoxic functionality of NK cells by interfering with the receptor. Objective: Determine the levels of soluble MIC-A and MIC-B (sMICA, sMICB), the NKG2D receptor and the subpopulations of NK, NKT and Tγδ cells, in patients with de novo NHL and post-chemotherapy, and its relationship to treatment response. Design: Peripheral blood (SP) in EDTA from patients diagnosed with NHL was taken prior to the start of treatment and at least 3 months after chemotherapy. Healthy donors (SD) were included as controls. Setting: By flow cytometry using monoclonal antibodies lymphocyte subpopulations and NKG2D receptor expression were determined, and by ELISA, the levels of sMICA and sMICB. Patients or other participants: 50 patients with de novo NHL, indolent (N=17) and aggressive (N=33), from these 26 post-chemotherapy samples were obtained. Patients of both genders, over 18 years old, with informed consent, never treaty with chemotherapy and without contaminating viral disease. Interventions: There were not, only the sample taken described in setting. Main outcome measures: Before carrying out the project we hypothesized that the high levels of sMICA and sMICB in patients with NHL, as well as the low expression of the NKG2D receptor in SP of these patients are associated with poor response to treatment and poor prognosis in disease evolution. Results: Decrease (P Conclusions: The high level of sMIC in the serum of patients with NHL and the decrease of circulating dendritic, NK, helper (Th), and TCD4reg cells propose a possible mechanism for evasion of the antitumor response of NHL cells.

Abstract 6707: Inhibition of proteolytic cleavage of NKG2D ligands increases immune cell-mediated cell killing of tumor cells in vitro

Abstract Natural Killer Group 2, member D (NKG2D) is an activating receptor that is expressed on the key immune cells involved in the cytotoxic arm of the immune system. Ligands of NKG2D, such as MICA and MICB, are not normally expressed but are upregulated in response to stress, such as oxygen or nutrient deprivation, viral infection or oncogenic transformation. One mechanism that tumor cells, that express MICA and MICB, have adopted to evade the cytotoxic potential of NK and T cells, that express the NKG2D receptor, is to cleave MICA/B from the cell surface. Proteolytic cleavage of MICA/B occurs within the α-3 domain of MICA/B. In order to evaluate the therapeutic potential of blocking cleavage of MICA/B we have generated multiple antibodies that bind to the α-3 domain of MICA/B. The lead antibody, PDI-01, was found to bind to multiple recombinant human MICA alleles and human MICB and to recombinant MIC proteins derived from non-human primate sequences with low nM affinity. PDI-01 bound to target antigen on the surface of multiple cancer cell lines including HCT-116 colon carcinoma, HCC1534 lung carcinoma and PLC/PRF/5 liver cancer cell lines. Each of those tumor cell lines were positive for MICA and MICB expression. Low expression of other NKG2D ligands, known as UL-16 binding proteins 1-6 (ULBP 1-6), has been shown in those cell lines. PDI-01 was found to prevent cleavage of MICA from the surface of HCT-116, HCC1534 and PLC/PRF/5 cells via ELISA quantification of soluble MICA levels. Exposure of HCT-116, HCC1534 and PLC/PRF/5 tumor cell lines to PDI-01 increased surface MICA levels as measured by flow cytometry analysis. An increase in immune cell mediated killing of HCT-116, HCC1534 and PLC/PRF/5 cells was observed when tumor cells were incubated with NK-92 cells (NKG2D positive) in the presence of PDI-01. PDI-01 also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) against these three tumor cell lines. Co-culture of PBMCs, or purified NK cells, with PLC/PRF/5 cells in the presence of PDI-01 led to an increase in TNF-α, IFN-γ, CCL4, CCL5 and IL-6. PDI-01 is being advanced into clinical development and the Phase 1 program is scheduled to be initiated in the 2Q, 2020. Citation Format: Safak Yalcin, Vanessa Cicchini, Xin Du, Xin Wu, Tom Kaever, Neil Gibson. Inhibition of proteolytic cleavage of NKG2D ligands increases immune cell-mediated cell killing of tumor cells in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6707.