MICA Alleles Research Articles

Distributions of MICA and MICB Alleles Typed by Amplicon-Based Next-Generation Sequencing in South Koreans.

Major histocompatibility complex class I chain-related genes A and B (MICA and MICB) play a role as ligands in activating the NKG2D receptor expressed in natural killer cells, γδ T-cells and αβ CD8 T-cells and have been defined in human diseases and haematopoietic stem cell transplantation (HSCT). MICA and MICB alleles were genotyped at the three-field level by amplicon-based next-generation sequencing (NGS) using a MiSeqDx system and compared with the results from previous studies in healthy South Korean donors. Exons 2-5 of MICA and exons 2-4 of MICB were amplified using a multiplex polymerase chain reaction (PCR). Sequence reads of ≥ 51 depth counts were consistently obtained for each sample exon, and target exons were determined to match reference sequences contained in the IPD-IMGT/HLA database. MICA and MICB alleles were tested using exon combinations. The program was designed to recognise specific sequences and discriminate between the MICA*008:01:01/*027 alleles. A total of 22 alleles were found in MICA and MICB. We observed 1 HLA-C ~ HLA-B ~ MICA ~ MICB ~ HLA-DRB1 haplotype with significant linkage disequilibrium between alleles at all neighbouring HLA loci. These results are consistent with previous microarray results. Genotyping of MICA and MICB was possible using 11-loci HLA genes. We updated the distribution of MICA and MICB based on three-field allele and haplotype frequencies containing linkage disequilibrium in South Koreans using amplicon-based NGS. These data suggest that high-resolution MICA and MICB typing data obtained using NGS may aid in performing HSCT and disease association studies.

Accurate multi-population imputation of MICA, MICB, HLA-E, HLA-F and HLA-G alleles from genome SNP data.

In addition to the classical HLA genes, the major histocompatibility complex (MHC) harbors a high number of other polymorphic genes with less established roles in disease associations and transplantation matching. To facilitate studies of the non-classical and non-HLA genes in large patient and biobank cohorts, we trained imputation models for MICA, MICB, HLA-E, HLA-F and HLA-G alleles on genome SNP array data. We show, using both population-specific and multi-population 1000 Genomes references, that the alleles of these genes can be accurately imputed for screening and research purposes. The best imputation model for MICA, MICB, HLA-E, -F and -G achieved a mean accuracy of 99.3% (min, max: 98.6, 99.9). Furthermore, validation of the 1000 Genomes exome short-read sequencing-based allele calling against a clinical-grade reference data showed an average accuracy of 99.8%, testifying for the quality of the 1000 Genomes data as an imputation reference. We also fitted the models for Infinium Global Screening Array (GSA, Illumina, Inc.) and Axiom Precision Medicine Research Array (PMRA, Thermo Fisher Scientific Inc.) SNP content, with mean accuracies of 99.1% (97.2, 100) and 98.9% (97.4, 100), respectively.

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.

MICA and NKG2D gene polymorphisms influence graft survival, and response to therapy in kidney transplantation.

Antibody-mediated rejection is a significant cause of kidney transplant failure. Recent studies have shown that the MHC class I MICA gene influences the transplantation outcome. However, the role of the primary MICA receptor, NKG2D, has yet to be explored. We aimed to investigate the correlation between recipient/donor MICA allele matching and NKG2D genotype with the risk of antibody-mediated rejection and their potential clinical effects and implications for organ maintenance therapy. Of the 524 patients who underwent transplantation, 387 were eligible for the study. Complete MICA allele and two functional polymorphisms of NKG2D (rs1049174C>G and rs2255336G>A) were analyzed in 148 transplanted patients and 146 controls. Increased recipient/donor MICA allele mismatches correlate with an elevated risk of antibody-mediated rejection (X2 = 6.95; Log-rank=0.031). Notably, the rs1049174[GG] genotype contributes to a significantly increased risk of antibody-mediated rejection (X2 = 13.44; Log-rank=0.001 and X 2 = 0.34; Log-rank=0.84). The combined effect of two MICA allele mismatches and rs1049174[GG] genotype shows the highest risk (X2 = 23.21; Log-rank<0.001). Most importantly, patients with rs1049174[GG] and rs2255336[AA] genotypes may respond less to mTOR inhibitor immunosuppressive therapy than Calcineurin inhibitors (rs1049174[GG]; P=0.035; and rs2255336[AA]; P=0.002). Recipient/donor MICA allele mismatches and specific NKG2D variants, as well as their combinations, influence kidney transplant outcomes, providing insights for personalized treatment and enhancing graft survival.

Mutation hot spots in MICA/MICB extracellular domains

MICA and MICB are non-classical MHC molecules that indicate cellular stress. They act as ligands for NKG2D receptors found on NK cells, thereby triggering a cytotoxic response against damaged, infected, or transformed cells. The production of soluble forms of MICA/MICB occurs via the cleavage of their extracellular domains (ECDs). The expression of MICA/MICB molecules in tumor sections or the levels of their soluble forms in blood have potential as diagnostic tools for cancer. They can predict important clinical outcomes for cancer patients, such as overall and recurrence-free survival. However, their extensive molecular polymorphism complicates the development of monoclonal antibodies (mAbs) for diagnostic use. Therefore, the diagnostic value of mAb-based assays may vary depending on the frequencies of allelic variants in local human populations. We examined the ECD amino acid sequences of more than 280 MICA and 50 MICB allelic variants. Additionally, we identified 172 and 58 single nucleotide polymorphisms (SNPs) located in the coding regions of the respective genes and resulting in amino acid replacements. The most frequent amino acid replacements (&gt; 10%) in the ECD occur at 11 and 4 sites of MICA and MICB, respectively. We found that the frequencies of SNPs in the identified hot spots strongly correlate with each other in different human populations, despite the diversity of allelic variant frequencies. The functional role of only one site is known. The replacement of valine with methionine at position 152 enhances the affinity of MICA to NKG2D receptor. As the hot spots are dispersed throughout the entire ECD sequences, they may play a role other than modulating affinity with the NKG2D receptor interaction. We recommend that Ag sets used to validate anti-MICA/MICB mAbs meet two criteria. First, they should include both MICA and MICB alleles, as these genes have highly similar sequences. Second, the alleles should cover the variability observed in the identified hot spots.

Cross-Dressing of Multiple Myeloma Cells Mediated by Extracellular Vesicles Conveying MIC and ULBP Ligands Promotes NK Cell Killing.

Natural Killer (NK) cells are innate cytotoxic lymphoid cells that play a crucial role in cancer immunosurveillance. NKG2D is an activating receptor that binds to MIC and ULBP molecules typically induced on damaged, transformed, or infected cells. The secretion of NKG2D ligands (NKG2DLs) through protease-mediated cleavage or in an extracellular vesicle (EV) is a mode to control their cell surface expression and a mechanism used by cancer cells to evade NKG2D-mediated immunosurveillance. EVs are emerging as important players in mediating cell-to-cell communication due to their ability to transfer biological material to acceptor cells. Herein, we investigated the spreading of NKG2DLs of both MIC and ULBP molecules through the EV-mediated cross-dressing on multiple myeloma (MM) cells. We focused our attention on two MICA allelic variants, namely MICA*008 and MICA*019, representing the prototype of short and long MICA alleles, respectively, and on ULBP-1, ULBP-2, and ULBP-3. Our findings demonstrate that both ULBP and MICA ligands can be acquired from tumor cells through EVs enhancing NK cell recognition and killing. Moreover, besides MICA, EVs expressing ULBP-1 but not ULBP-2 and 3 were detected in bone marrow aspirates derived from a cohort of MM patients. Our findings shed light on the role of EV-associated MICA allelic variants and ULBP molecules in the modulation of NKG2D-mediated NK cell immunosurveillance in the tumor microenvironment. Moreover, the EV-mediated transfer of NKG2DLs could suggest novel therapeutic approaches based on the usage of engineered nanoparticles aimed at increasing cancer cell immunogenicity.

MICA and MICB allele assortment in Finland.

Genetic variation in the MICA and MICB genes located within the major histocompatibility complex region has been reported to be associated with transplantation outcome and susceptibility to autoimmune diseases and infections. Only limited data of polymorphism in these genes in different populations are available. We here report allelic variation at 2-field resolution and the haplotypes of the MICA and MICB genes in Finland (n= 1032 individuals), a north European population with historical bottleneck and founder effects. Altogether 24 MICA and 18 MICB alleles were found, forming 70 estimated MICA-MICB haplotypes. As compared to other populations frequency differences were found, for example, MICA*010:01 was found to be at an allele frequency of 0.133 in Finland which is higher than in other European populations (0.021-0.077), but close to Asian populations (0.151-0.220). Three novel alleles with amino acid change are described. The results demonstrate a relatively high level of polymorphism and population differences in MICA and MICB allele distribution.

Analysis of the association between MICA gene polymorphisms and schizophrenia.

The major histocompatibility complex (MHC) has been implicated in schizophrenia. This study aimed to explore the correlation between the major histocompatibility complex class I polypeptide-related sequence A (MICA) polymorphisms and schizophrenia. A total of 220 Han schizophrenia patients, 47 Han healthy controls, 155 Li schizophrenia patients, and 48 Li controls were selected from Hainan Province, China. The diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria. Sequencing-based-typing (PCR-SBT) technology was used for MICA allele typing, and the correlation analyses of MICA gene polymorphism and schizophrenia were performed. In the Han group, the three allele frequencies of MICA*002:01, MICA*A4, and MICA*A9 in the schizophrenia group were significantly higher than those in the healthy control group, and the differences were statistically significant (pc < 0.05; pc values were 0.024, 0.030, and 0.031, respectively). Yet, there was no difference in the MICA gene between the schizophrenia group and the healthy controls group in the Li population. We found MICA*002:01, MICA*A4, and MICA*A9 may be susceptibility alleles for schizophrenia in the Han population, while the MICA allele polymorphism in the Li population is not associated with schizophrenia in Chinese.

Genetic polymorphism associated with cervical cancer: a systematic review

Introduction. Cervical cancer (CC) is one of the most common cancers in women. The CC etiological agent is the high-risk oncogenic human papillomavirus. In the meantime, not all women infected with this virus can develop cancer, thus suggesting that there is genetic predisposition to CC.The aim of the study was to analyze information about single nucleotide polymorphisms associated with the CC risk.Materials and methods. The performed search was focused on genome-wide association studies (GWAS) and meta-analyses conducted over the last 10 years and addressing the genetic risk of CC in the Caucasian population.Results. The most significant associations with CC were found in the following single nucleotide polymorphisms. Based on the GWAS data, they involve risk alleles rs138446575-T (OR = 2.39) TTC34; rs73728618-T (OR = 1.48) HLA-DQA1; rs3130196-C (OR = 1.4) HLA-DPB1; rs2516448-T (OR = 1.39 and 1.44) MICA and protective alleles rs9271898-A (OR = 0.64) and 9272143-C (OR = 0.65) between HLA-DRB1 and HLA-DQA1, rs55986091-A HLADQB1 (OR = 0.66). Based on the meta-analysis data, they involve genotype rs4646903-СС (OR = 4.65) CYP1A1 and protective alleles rs1801133-T (OR = 0.77) MTHFR, rs2333227-AA (OR = 0.57) MPO.Conclusion. The obtained data are critically important for development of laboratory techniques and reagent kits allowing for a personalized approach to identification of risk groups, which could benefit from compulsory vaccination and screening for pre-cancers of the cervix.

Abstract 3506: CLN-619, a clinical-stage MICA/MICB-specific IgG1 antibody, restores the MICA/MICB-NKG2D axis to promote NK-mediated tumor cell lysis

Abstract Natural killer (NK) cells play a central role in the immune surveillance of cancer. NK cells express multiple inhibitory and activating surface receptors, including the activating receptor, NKG2D. Engagement of NKG2D by the stress-induced ligands MICA/MICB stimulates NK cell activation and subsequent target cell lysis. While MICA/MICB expression is restricted in normal tissues, expression is strongly induced upon viral infection, genotoxic stress or malignant transformation. In cancer patients, the tumor microenvironment (TME) facilitates evasion of immune surveillance by NK cells. One mechanism by which tumor cells can escape NKG2D-mediated lysis is via proteolytic release of MICA/MICB from the cell surface due to proteases present in the TME. High concentrations of shed MICA have been observed in serum from patients across multiple tumor types and correlate with poor survival. In mouse models, expression of MICA on tumor cells delayed tumor growth, while the absence of NKG2D accelerated tumor growth. Given the established role of the MICA/MICB-NKG2D axis in the immune surveillance of tumor cells, a therapeutic agent that effectively restores this axis is a compelling approach to harness the immune system for treating cancer. CLN-619 is a humanized anti-MICA/MICB monoclonal IgG1 antibody being developed for the treatment of multiple cancer indications. The antibody was selected based on its high-affinity binding to MICA/MICB, potent in vitro activity and robust anti-tumor activity in xenograft models. MICA/MICB is highly polymorphic, with &amp;gt;150 MICA and 47 MICB alleles in humans, and expression level, binding affinity to NKG2D, and degree of MICA/MICB shedding is thought to be allele-dependent. CLN-619 demonstrated high affinity binding to all common allelic variants of MICA, including the most prevalent MICA allele, MICA*008, and the canonical allelic variant of MICB. CLN-619 was able to prevent proteolytic cleavage of MICA/MICB as evidenced by the reduction of shed MICA/MICB in cell culture supernatants and the corresponding increase in surface MICA/MICB on cancer cells. In addition to the ability to restore the MICA/MICB-NKG2D axis, CLN-619 enhanced the binding of MICA to NKG2D on NK cells. This was enabled by both FcγR engagement on NK cells as well as an intrinsic enhancement of MICA binding to NKG2D. Finally, CLN-619 was shown to elicit potent ADCC of MICA/MICB expressing tumor cells. Consistent with the multimodal mechanisms of action described above, CLN-619 treatment resulted in robust anti-tumor activity at low doses in mice bearing MICA/MICB-expressing human tumor xenografts. Given the pan-cancer expression of MICA/MICB in both solid and hematological malignancies, CLN-619 is expected to have broad anti-tumor activity. CLN-619 is currently being investigated in a Phase 1 clinical trial for the treatment of patients with advanced solid tumors. Citation Format: Kerry A. Whalen, Naveen K. Mehta, Safak Yalcin, Kristan Meetze, Neil W. Gibson, Jennifer S. Michaelson, Patrick A. Baeuerle. CLN-619, a clinical-stage MICA/MICB-specific IgG1 antibody, restores the MICA/MICB-NKG2D axis to promote NK-mediated tumor cell lysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3506.

Single-Nucleotide Polymorphisms in MICA and MICB Genes Could Play a Role in the Outcome in AML Patients after HSCT

NKG2D and its ligands, MICA and MICB, are known as the key regulators of NK cells. NK cells are the first reconstituted cells after the allogeneic hematopoietic stem cell transplantation (HSCT); therefore, it is crucial to understand their role in HSCT outcome. In the presented study, we investigated the single amino acid changes across the exons 2–4 of MICA and MICB genes, and point mutations within the NKG2D gene, which defines the type of NKG2D haploblock (HNK/LNK) in the donors (n = 124), as well as in patients with acute myeloid leukemia (n = 78). In our cohort, we found that graft from a donor with at least one MICA allele containing glycine at position 14 (MICA-14Gly) is significantly associated with deterioration of a patient’s overall survival (OS) (p < 0.05). We also observed a negative effect of MICB-58 (Lys → Glu) polymorphism on relapse-free survival (RFS), although it was not statistically significant in multivariate analysis (p = 0.069). To our knowledge, this is the first work describing the role of MICA-14 and MICB-58 polymorphisms on HSCT outcome.

Establishment of HLA class I and MICA/B null HEK-293T panel expressing single MICA alleles to detect anti-MICA antibodies

Pre- and post-transplantation anti-MICA antibody detection development are associated with an increased rejection risk and low graft survival. We previously generated HLA class I null HEK-293T using CRISPR/Cas9, while MICA and MICB genes were removed in this study. A panel of 11 cell lines expressing single MICA alleles was established. Anti-MICA antibody in the sera of kidney transplant patients was determined using flow cytometric method (FCM) and the Luminex method. In the 44 positive sera, the maximum FCM value was 2879 MFI compared to 28,135 MFI of Luminex method. Eleven sera (25%) were determined as positive by FCM and 32 sera (72%) were positive by the Luminex method. The sum of total MICA antigens, MICA*002, *004, *009, *019, and *027 correlation showed a statistically significant between the two methods (P = 0.0412, P = 0.0476, P = 0.0019, P = 0.0098, P = 0.0467, and P = 0.0049). These results demonstrated that HEK-293T-based engineered cell lines expressing single MICA alleles were suitable for measuring specific antibodies against MICA antigens in the sera of transplant patients. Studies of antibodies to MICA antigens may help to understand responses in vivo and increase clinical relevance at the cellular level such as complement-dependent cytotoxicity.

Prevalence and impact of HLA and MICA allele mismatching on donor-specific antibodies induction in kidney transplant rejection.

Donor-recipient antigen mismatching for anti-human leucocyte antigen (HLA) and MICA is one of the risk factors for antibody induction leading to graft rejection. Our aim was to analyze the incidence and specificity of the different DSAs developing and to investigate the impact of HLA and MICA allele mismatches on antibody production in kidney transplant patients experiencing antibody-mediated rejection (AMR). We retrospectively reviewed 253 consecutive recipients of kidney transplant who were diagnosed as experiencing AMR. Our results showed that around 27% of our patients were positive for DSAs over a median follow-up period of 24 months. Antibody to HLA-DQ7 was the most prevalent DSA detected. The allele mismatch number was significantly lower for DQ loci than -A and -B loci (DQ vs. A, p < .001; DQ vs. B, p=.002). Considering each HLA antigen, the incidence rate of DQ-DSA [41.9 (32.92-51.46; 95%CI)] was much higher than the rate observed for DSA directed to -A, -DR and -B loci. Half of the recipients in the DQ-DSA-only group, and the DQ-DSA together with non-DQ group, had MFI > 5000. Only one case developed de novo MICA-DSA (MICA002). Our study indicates that mismatching for HLA and MICA alleles leads to the development of HLA and MICA antibodies in some kidney transplant recipients. We have also demonstrated that DSA to the DQ locus is the most prevalent in kidney transplant patients with AMR. Thus, matching the DQ locus in kidney allocation algorithms may reduce post-transplant development of DSA.

Nghiên cứu xác định đa hình đơn Nucleotide RS2596542 của gen mica ở bệnh nhân ung thư vòm họng

MICA is an antigen that is commonly expressed on cell surface of tumoRS. MICA had a low expression level in normal cells but increased in pathological/cancer cells. The goal of the study was to determine the association of SNP RS2596542 with the risk of nasopharyngeal cancer (NPC). 144 biopsy samples diagnosed with NPC and 135 blood samples from healthy people were collected. Genotypes and alleles of RS2596542 MICA were determined by Realtime PCR genotyping. The genotype rate of MICA at RS2596542 in patients with NPC was 34.72% CC; 44.44% CT and 20.84% TT. The genotype of TT and T allele in the disease group was significantly higher than the control group (p=0.01*; OR=2.47; 95%CI: 1.22-4.97) and (p=0.01*; OR=1.51; 95%CI: 1.07-2.13). The T-allele at SNP RS2596542 of the MICA gene increases the risk of NPC and suggests that this could be used as a biomarker for NPC screening in Viet Nam. Keywords: MICA, RS2596542, nasopharyngeal cancer, NPC, Single Nucleotide Polymorphism, SNPs, Realtime PCR

Distribution of MICA alleles and haplotypes associated with HLA-B in Greek population

IntroductionThe Major Histocompatibility Complex Class I-related chain A gene (MICA) is a highly polymorphic functional gene located close to the HLA-B locus. Certain MICA alleles have been related to inflammatory and autoimmune diseases while MICA antibodies have been implicated in organ allograft rejection or graft-versus-host disease (GVHD). AimThe aim of this study was to identify the frequencies of MICA alleles and MICA ~ HLA-B haplotypes in the Greek population since, as far as we know, these data are still limited. MethodsDNA was obtained from 277 unrelated healthy Greek individuals of Caucasian origin, volunteer donors of blood stem cells. HLA-B* and MICA* genotyping was performed by reverse PCR-SSOP. ResultsA total of 18 MICA alleles were defined in the present study. The five most frequent alleles in the Greek population were MICA*008 (24.6%), MICA*009 (22.36%), MICA*018 (16.03%), MICA*002 (8.02%) and MICA*004 (7.17%) which altogether account for 77.8% of all alleles. The most common MICA ~ HLA-B haplotypes were MICA*018 ~ B*18 (12.5%) and MICA*009 ~ B*51(11.5%). ConclusionsThe five most frequent MICA alleles in the Greek population were *008, *009, *018, *002, *004. In other Caucasian populations, two of these alleles (*008, and *004) were observed in similar frequencies. MICA*002 was observed less frequently (8.02%) in the Greek population compared to other Caucasian groups (frequencies > 15%). Also, MICA*009 and MICA*018 had elevated frequencies (above 15%) whereas in other Caucasian populations they were found around 10% or less. These data may be important for the elucidation of the role that MICA polymorphisms play in organ and stem cell transplantation and to identify the relation of certain MICA with susceptibility to specific diseases.

High-throughput genotyping of HLA-G, HLA-F, MICA, and MICB and analysis of frequency distributions in healthy blood donors from Catalonia.

Similarly to HLA class I molecules, certain non-classical HLA class I genes and MHC class I polypeptide-related sequences A and B (MICA and MICB) act as ligands for KIR and NKG2D natural killer receptors. Although these genes are less polymorphic than HLA class I, few studies have analyzed their association with diseases. Information on allele frequencies in healthy donors is needed to map their distribution worldwide. This study is the first to analyze high-resolution HLA-G, HLA-F, MICA, and MICB allele frequencies using a novel high-throughput next generation-sequencing method. We analyzed DNA samples from 96 unrelated blood donors resident in Catalonia, Spain, and registered in the Barcelona Blood and Tissue Bank. Using the first two fields of the HLA nomenclature, we detected six HLA-G and two HLA-F alleles. The most frequent alleles were HLA-G*01:01 (77.08%) and HLA-F*01:01(84.90%). When the four fields were analyzed, we detected 16 and 10 alleles, respectively. Nineteen alleles were detected for MICA and 10 for MICB. The most frequent alleles in these cases were MICA*008:01 (16.15%) and MICB*005:02 (46.84%). All frequencies were in Hardy Weinberg equilibrium except MICA. We also estimated maximum-likelihood haplotype frequencies and calculated corresponding linkage disequilibrium (LD) values and found that few allele pairs were in disequilibrium. Strong LD between MICA and HLA-B (using data from a previous study) was observed. Our findings will be useful for guiding further research evaluating the functional role of these genes in different diseases and populations.

Association of MICA and HLA‐B alleles with leprosy in two endemic populations in Brazil

Leprosy is a prevalent disease in Brazil, which ranks as the country with the second highest number of cases in the world. The disease manifests in a spectrum of forms, and genetic differences in the host can help to elucidate the immunopathogenesis. For a better understanding of MICA association with leprosy, we performed a case-control and a family-based study in two endemic populations in Brazil. MICA and HLA-B alleles were evaluated in 409 leprosy patients and in 419 healthy contacts by PCR-SSOP-Luminex-based technology. In the familial study, analysis of 46 families was completed by direct sequencing of all exons and 3'/5'untranslated regions, using the Ilumina MiSeq platform. All data were collected between 2006 and 2009. Statistical analysis was performed using the Chi-square or Fisher's exact test together with a multivariate analysis. Family-based association was assessed by transmission disequilibrium test (TDT) software FBAT 2.0.4. We found associations between the haplotype MICA*002-HLA-B*35 with leprosy in both the per se and the multibacillary (MB) forms when compared to healthy contacts. The MICA allele *008 was associated with the clinical forms of paucibacillary (PB). Additionally, MICA*029 was associated with the clinical forms of MB. The association of MICA*029 allele (MICA-A4 variant) with the susceptibility to the MB form suggests this variant for the transmembrane domain of the MICA molecule may be a risk factor for leprosy. Two MICA and nine HLA-B variants were found associated with leprosy per se in the Colônia do Prata population. Linkage disequilibrium analysis revealed perfect linkage disequilibrium (LD) between HLA-B markers rs2596498 and rs2507992, and high LD (R2 =.92) between these and the marker rs2442718. This familial study demonstrates that MICA association signals are not independent from those observed for HLA-B. Our findings contribute the knowledge pool of the immunogenetics of Hansen's disease and reveals a new association of the MICA*029 allele.

A Chimeric Antigen Receptor That Binds to a Conserved Site on MICA.

The NKG2D ligand MHC class I chain-related protein A (MICA) is expressed on many varieties of malignant cells but is absent from most normal tissues, and thus represents a potential target for chimeric Ag receptor (CAR) T cell-based therapeutics. However, there are more than 100 alleles of MICA, so the ability to target a conserved site is needed for a therapy to be used in most patients. In this study, we describe a fully human anti-MICA CAR created by fusing the single-chain fragment variable B2 to the full length DAP10 protein and the traditional CD3ζ signaling domain. Human T cells expressing the B2 CAR killed MICA-positive tumor cells, produced IFN-γ when in contact with MICA-positive tumor cells or plate-bound MICA protein, and inhibited PANC-1 growth in a mouse xenograft model. To localize B2's epitope on MICA, we used novel computational methods to model potential binding modes and to design mutational variants of MICA testing these hypotheses. Flow cytometry using a commercial anti-MICA/MICB Ab indicated that the variant proteins were expressed at high levels on transduced P815 cell lines. One variant protein (R38S/K40T/K57E) showed reduced staining with a B2-IgG1 fusion protein compared with controls and did not induce IFN-γ production by human T cells expressing the B2 CAR. These results show antitumor activity of MICA-specific CAR T cells and indicate an essential role for a conserved site in the exposed loop involving aa 38-57 of MICA. This study describes a novel MICA-specific CAR and discusses its potential use as a cancer therapeutic.

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.

MICA ∗012:01 Allele Facilitates the Metastasis of KRAS-Mutant Colorectal Cancer.

Major histocompatibility complex (HLA) class I chain-related protein A (MICA) regulates immune surveillance through activation of NKG2D (natural killer group 2D) receptor. However, the genetic association, potential function, and predictive ability of MICA alleles with colorectal cancer (CRC) prognosis remain undefined. In this study, we characterized MICA alleles in tissue samples from 104 patients with CRC and 536 healthy controls and carried out genetic association studies by molecular and clinical CRC phenotypes. Preliminary sequence analysis revealed that MICA ∗009:01 or ∗049 alleles were significantly decreased in patients with CRC (p = 0.0049), and further stratification analysis indicated that MICA ∗012:01 allele was associated with patients with CRC and carrying KRAS codon 12 mutation (p = 0.027). The functional consequences of MICA alleles were examined via transfected CRC cell lines which showed that overexpression of MICA ∗012:01 enhanced the proliferation, invasion, and metastatic phenotype of CRC. Preliminary analysis of disease-free survival time in patients with and without MICA ∗012:01 suggest this allele may be predictive for poor prognosis of patients with KRAS codon 12 mutated CRC, as no somatic mutation of MICA gene was detected in CRC tumors compared to paracancerous tissues. Our study indicates that MICA ∗012:01 allele is associated with KRAS-mutated CRC, facilitates CRC invasion and metastasis, and possibly reduces the survival of patients with KRAS-mutated CRC.