anti-MICA Antibodies Research Articles

The impact of Anti-HLA and Anti-MICA specific antibodies on allograft function of kidney trans-plant

Objective To explore the impact of anti-HLA and MICA antibodies on kidney graft func-tion by dynamic monitoring for two years. Methods The 135 patients to be subject to renal allngraft were de-teeted for specificity of anti-HLA and anti-MICA antibodies by flow PRATM beads. Among them 33 patients ac-cepted two-year dynamic monitoring. Also the IRA and MICA genotyping was done by using PR-SSOP,and do-nor specific antibody (DSA) and non-donor specific antibody (NDSA) were identified. Simultaneously,the ser-um ereatinine levels and clinical data were analyzed. Results The 46 patients were positive for antibody before transplantation in 135 patients (34. 1%). Among the 33 patients subject to dynamic monitoring,20 patients were negative for anti-HLA and anti-MICA antibodies before and after transplantation:7 patients had anti-MICA anti-body:2 patients were positive for both anti-HLA and anti-MICA antibodies:one patient was positive for anti-HLA antibody,and the remaining 3 patients developed de novo antibody at 6th month after transplantation (9.1%). Two patients negative for antibodies before transplantation had de novo antibodies: A33, A31 (one case), and DQ7,DRI7,DR18 (one case) at the 6th month after transplantation,then specific antibodies were DR12, and DRII at first to second years after transplantation. One patient had de novo MICA27 ,MICA02 antibody and HLA-B41 ,A32 antibedies one year after transplantation. There was significant difference in the Scr levels in 33 patients between positive and negative antibody groups (P ＜ 0. 05). The Scr levels were most significandy in-creased in anti-MICA antibody group (P ＜ 0.01). Conclusion Whether there is HLA and MICA antibodies before transpLantation,dynamic monitoring of the changes in HLA and MICA specific antibodies after transplan-tation is of great importance in predicting rejection and guiding clinieal therapy to prevent the occurrence and development of renal allograft function deficiency. Key words: Anti-HLA antibody; Anti-MICA antibody; Kidney transplantation

Approaches To Anti-HLA Antibody Profiles In Patients Waiting For Renal Re-Transplantation

This review focuses on the mechanisms of anti-HLA antibody development in patients waiting for a renal re-transplantation. Pre- existence or post transplant de novo production of these antibodies have been found to contribute to renal allograft failure in years. In addition to the detrimental effect of donor specific and non-donor specific anti-HLA antibodies on graft survival, many researchers emphasized the effect of non-HLA antibodies (e.g.; anti-MICA antibodies), auto-antibodies (e.g.; anti-vimentin antibodies), allo-nephrectomy and combined liver-kidney transplantations on rejection processes. Beyond all these, a newly developed computer algorithm, 'HLAMatchmaker' has changed the concept of transplantation possibility in highly sensitized patients.

Identification of Epitopes and Immunodominant Regions on the MICA Protein Defined by Alloantibodies From Kidney Transplant Patients

Several reports showed a contribution of anti-MICA (major histocompatibility complex class I chain-related molecule A) antibodies (Abs) to the development of acute and chronic rejection. Identification of the epitopes to which the Abs bind may help to determine immunoreactive regions essential for the major histocompatibility complex compatibility between donor and recipients, leading to the best outcome of the transplant. Sera from 284 kidney transplant patients were screened for anti-MICA Abs by Luminex assay. MICA allele typing of the recipients was determined. The epitopes of MICA were mapped by screening a synthesized library of overlapping peptides from the extracellular domains of the protein against the sera from kidney transplant patients with anti-MICA Abs. Antibodies against MICA were detected in 50 of 284 patients (17.6%) and correlated with the development of acute rejection. Nine antigenic regions were immunoreactive with anti-MICA Abs in the sera samples. Four of these continuous epitopes mapped to polymorphic amino acids (aa). Five antigenic regions were shared epitopes found in all the MICA alleles. The polymorphic residues, 173 (E/K), 175 (S/G), and 181 (R/T), had determined allele-specific epitopes (reactivity patterns 1 and 2). In contrast, the aa 208Y and 213T were implicated in the cross-reactivity among alleles. The presence of anti-MICA Abs could be an important marker for diagnosis because of their contribution to the outcome of the graft, regardless of presence of anti-HLA Abs. Additionally, the identification of epitopes revealed the in vivo antigens of the transplant and is spurring the development of new matching strategies to reduce the incidence of acute and chronic rejection.

Anti-MICA Antibodies Are Related to Adverse Outcome in Heart Transplant Recipients

The clinical relevance of the post-transplant presence of anti-major histocompatibility complex class I chain-related A (MICA) antibodies as a marker for chronic graft failure in heart transplantation was examined using post-transplant sera from 159 heart transplant recipients. Mean follow-up after transplantation was 7 +/- 4.9 years. The sera were screened by Luminex (Luminex Corp, Austin, TX) for MICA antibodies. Samples that tested positive were confirmed using a Luminex MICA single-antigen bead assay. The antigen specificity of the detected antibodies was identified. Outcome parameters were survival, cardiac allograft vasculopathy (CAV), and cellular rejection. We retrospectively selected 159 patients: 107 with 0 or 1 rejection and 52 with 2 or more acute rejection episodes, of whom 36 (22.6%) had a positive screen for anti-MICA antibodies. In 19 of 36 samples, specific anti-MICA antibodies were confirmed by single antigen assay. The presence of post-transplant specified anti-MICA antibodies in patients' sera was associated with acute rejection (63.2% vs 28.6%, p < 0.01) and CAV (78.9% vs 32.8%, p < 0.01). Multivariate analysis identified anti-MICA positivity as an independent risk factor for the development of CAV. The results indicate that anti-MICA antibodies may be related to adverse outcome after heart transplantation. Post-transplantation monitoring of anti-MICA antibodies could identify patients with an increased risk for acute rejection and vasculopathy.

Identifying compartment-specific non-HLA targets after renal transplantation by integrating transcriptome and “antibodyome” measures

We have conducted an integrative genomics analysis of serological responses to non-HLA targets after renal transplantation, with the aim of identifying the tissue specificity and types of immunogenic non-HLA antigenic targets after transplantation. Posttransplant antibody responses were measured by paired comparative analysis of pretransplant and posttransplant serum samples from 18 pediatric renal transplant recipients, measured against 5,056 unique protein targets on the ProtoArray platform. The specificity of antibody responses were measured against gene expression levels specific to the kidney, and 2 other randomly selected organs (heart and pancreas), by integrated genomics, employing the mapping of transcription and ProtoArray platform measures, using AILUN. The likelihood of posttransplant non-HLA targets being recognized preferentially in any of 7 microdissected kidney compartments was also examined. In addition to HLA targets, non-HLA immune responses, including anti-MICA antibodies, were detected against kidney compartment-specific antigens, with highest posttransplant recognition for renal pelvis and cortex specific antigens. The compartment specificity of selected antibodies was confirmed by IHC. In conclusion, this study provides an immunogenic and anatomic roadmap of the most likely non-HLA antigens that can generate serological responses after renal transplantation. Correlation of the most significant non-HLA antibody responses with transplant health and dysfunction are currently underway.

Structurally based epitope analysis of major histocompatibility complex class I–related chain A (MICA) antibody specificity patterns

Recent studies have suggested a clinical significance to the detection of anti–major histocompatibility complex class I–related chain A (MICA) antibodies in transplantation. We have developed an eplet-based version of the HLAMatchmaker algorithm to assess the epitope specificity of these antibodies. Molecular viewing of the MICA structure and the determination of amino acid sequence differences between MICA alleles has yielded a repertoire of 38 potentially immunogenic MICA eplets. These eplets are based on the functional epitope structure that considers a configuration of amino acids within a 3-Ångstrom radius of an antibody-accessible polymorphic residue on the molecular surface. In this study MICA-reactive sera were screened in Luminex assays with single MICA allele panels and analyzed with HLAMatchmaker. We identified reactivity patterns that correspond to eplet-specific antibodies to identify of unacceptable MICA mismatches including those alleles that have not been tested with the serum. In conclusion, HLAMatchmaker is a useful algorithm to analyze the reactivity patterns of anti-MICA antibodies and the determination of MICA mismatch acceptability at the structural level.

Production and characterization of monoclonal antibodies against major histocompatibility complex class I chain‐related gene A

Major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand for the activating immunoreceptor natural killer group 2D (NKG2D), is expressed on stressed cells such as tumor cells. Study of expression of this molecule on tumor cells and patients' sera is useful to define patients' stages leading to proper selection of therapy. In this study, mouse anti-MICA monoclonal antibodies (mAbs) were produced by DNA immunization using a gene gun. Screening of anti-MICA-producing mouse and hybridomas were performed by immunoblot and cell enzyme-linked immunosorbent assay (ELISA) against MICA-positive HeLa and -negative Me1386 cell lines. MAbs were characterized against MICA-positive and -negative cell lines by immunoblot, cell ELISA and flow cytometry. The mAbs were also characterized for locus and allele specificities of MICA and MHC class I chain-related gene B (MICB) as well as for their ability to stain formalin-fixed paraffin-embedded tissues by immunohistochemistry. Although all mouse immune sera were positive with MICA-positive cells by both immunoblot and cell ELISA methods, some hybridomas were positive only with one method. The mAbs had diverse specificities to detect MICA and MICB and different abilities to stain formalin-fixed paraffin-embedded tissues. Thus, DNA immunization by gene gun is an effective method to generate immune mice for the production of mAbs with a variety of specificities against native and denatured forms of MIC proteins.

17-OR: Analysis of anti-MICA antibody reactivity patterns

17-OR: Analysis of anti-MICA antibody reactivity patterns

Renal Transplantation

It has been known for many years that patients with well-matched kidney transplants, even those who are matched at HLA-A, -B, and -DR, do not have indefinite allograft survival, and some kidneys fail as a result of rejection. In an attempt to understand the mechanism for rejection with graft loss in the face of excellent HLA matching, Zou et al. took pretransplantation sera from 1910 kidney transplant recipients and tested these sera for antibodies to MHC class I–related chain A (MICA) antigens. These antigens are expressed on the surface of epithelial cells, endothelial cells, fibroblasts, and monocytes. The MICA antigens are ligands of an activating receptor on natural killer cells and CD8+ T lymphocytes. Genes for these antigens are located close to the HLA-A and -B loci. ### Findings. Antibodies against these MICA antigens were detected in 11.4% of the 1910 patients. More important, the presence of these antibodies was associated with inferior graft survival compared with transplant centers without such antibodies (88 versus 93%; P = 0.01). In patients with good HLA matching (zero or one antigen mismatch of HLA-A plus HLA-B plus HLA-DR), sensitization to MICA was associated with even worse graft survival (83 versus 95% in individuals without such antibodies; P = 0.002). ### Commentary. This study provides a mechanism by which some allografts can be rejected, despite excellent matching and without anti-HLA antibodies. An editorial accompanying this article suggested that anti-MICA antibodies might be surrogate markers for a fundamental mechanism that mediates graft rejection, specifically, natural killer cell alloreactivity, which is due to these antigens’ binding to an activating ligand (1). It is possible that exploring this further may lead to better screening and cross-matching and further understanding of the role that these antigens may play in development of chronic allograft nephropathy.

The NKG2D expression on CD8+ T cells and the efficacy of polysaccharide K (PSK) in gastric cancer

3065 Some studies suggest that the immunoreceptor NKG2D expression on CD8+ T cells is down-regulated and this reduction may be involved in immune evasion in cancer patients. The present study was designed to investigate NKG2D expression on CD8+ T lymphocytes and its relationship to immune evasion in gastric cancer patients. NKG2D expression on both circulating and tumor-infiltrating CD8+ T cells was evaluated by multicolor flow cytometry. Soluble MHC class I chain-related gene A (MICA) in the sera was quantitated by ELISA. Transwell experiments were carried out to determine the effect of cancer cells on NKG2D expression. NKG2D expression on circulating CD8+ T cells was down-regulated and significantly correlated with IFN-gamma production in gastric cancer patients (r = 0.68; P = 0.007). NKG2D expression was closely related to undifferentiated cancer (P = 0.021) as was the depth of invasion (P = 0.012). There was no difference in soluble MICA between gastric cancer patients and normal controls. NKG2D expression on CD8+ T cells was remarkably reduced in the tissue of gastric cancer compared with peripheral blood (P = 0.046). Complete removal of tumor by surgery restored NKG2D expression on CD8+ T cells (P = 0.0049). Transwell experiments showed that this down-regulation was induced by direct contact between cancer cells and CD8+ T cells and that soluble factors did not affect the NKG2D expression. This phenomenon was blocked by the addition of anti-MICA antibodies. Furthermore, the NKG2D expression down-regulated by tumor cells was dose-dependently recovered by administration of non-specific immunopotentiator, polysaccharide K (PSK). Therefore, decreased NKG2D expression may be one of the key mechanisms responsible for immune evasion by tumors in gastric cancer. The NKG2D expression may be a promising biomarker for monitoring the efficacy of PSK. No significant financial relationships to disclose.

MHC class I chain-related protein A antibodies and shedding are associated with the progression of multiple myeloma.

Monoclonal gammopathy of undetermined significance (MGUS) is a common disorder of aging and a precursor lesion to full-blown multiple myeloma (MM). The mechanisms underlying the progression from MGUS to MM are incompletely understood but include the suppression of innate and adaptive antitumor immunity. Here, we demonstrate that NKG2D, an activating receptor on natural killer (NK) cells, CD8(+) T lymphocytes, and MHC class I chain-related protein A (MICA), an NKG2D ligand induced in malignant plasma cells through DNA damage, contribute to the pathogenesis of MGUS and MM. MICA expression is increased on plasma cells from MGUS patients compared with normal donors, whereas MM patients display intermediate MICA levels and a high expression of ERp5, a protein disulfide isomerase linked to MICA shedding (sMICA). MM, but not MGUS, patients harbor circulating sMICA, which triggers the down-regulation of NKG2D and impaired lymphocyte cytotoxicity. In contrast, MGUS, but not MM, patients generate high-titer anti-MICA antibodies that antagonize the suppressive effects of sMICA and stimulate dendritic cell cross-presentation of malignant plasma cells. Bortezomib, a proteasome inhibitor with anti-MM clinical efficacy, activates the DNA damage response to augment MICA expression in some MM cells, thereby enhancing their opsonization by anti-MICA antibodies. Together, these findings reveal that the alterations in the NKG2D pathway are associated with the progression from MGUS to MM and raise the possibility that anti-MICA monoclonal antibodies might prove therapeutic for these disorders.

On the utility of screening for anti-MICA antibodies before kidney transplantation

On the utility of screening for anti-MICA antibodies before kidney transplantation

Kidney-Transplant Rejection and Anti-MICA Antibodies

Kidney-Transplant Rejection and Anti-MICA Antibodies

Anti-MICA antibodies might contribute to kidney allograft loss

Anti-MICA antibodies might contribute to kidney allograft loss

MICA Polymorphism Identified by Whole Genome Array Constitutes a Disease Predisposition Factor in T-Cell Large Granular Lymphocyte Leukemia.

Large granular lymphocyte leukemia (LGL) is a disease of semiautonomous proliferation of cytotoxic T-cells (CTL) often accompanied by immune cytopenias, particularly neutropenia. LGL related cytopenias have been attributed to LGL cellular cytotoxicity or proapoptotic cytokines rather than intrinsic properties of the neutrophils. The association of LGL with autoimmunity suggests that genetic predisposition may contribute to disease pathogenesis. We studied 69 patients with LGL leukemia using a case-control approach; control populations included ethnically matched healthy individuals (N=82) and disease controls of aplastic anemia (N=48) and kidney transplant recipients (N=48). Initially, we applied the Illumina 12K non-synonymous SNP array to a subcohort of 36 LGL patients and 54 healthy controls (training set). Results were subjected to independent hypothesis-generating biostatistical algorithms. First, Exemplar automated analysis determined disease prediction based on independent χ2 analysis for each SNP. As expected, no SNP in this underpowered study reached Bonferroni corrected statistical significance, but our analysis allowed for ranking based on p-value. Second, Random Forests, a nonparametric tree method was applied, whereby all SNP information was calculated multivariately to predict disease. In a non-Mendelian inherited disease, this method more closely reflects the biology of complex polygenic traits; remarkably, those SNP identified by Random Forest were among the highest ranking SNP by Exemplar. Our initial hypothesis-generating set identified 1 SNP in unknown gene C8orf31 and 4 SNP within the MHC class I related-chain A (MICA) gene. We focused on MICA, a non-peptide presenting, tightly regulated stress response HLA molecule that could play a role in pathogenesis of neutropenia in LGL. To further substantiate our finding, the initial training set results were subjected to technical validation; fidelity was rechecked by PCR genotyping with 93% concordance. Biological validation was determined by confirmation in an independent test set consisting of 33 LGL patients and additional 28 controls. As only limited numbers of SNP were tested, there was no need for α-error adjustment. MICA SNP rs1063635 was found to have the most predictive value in both the training set (PPV=56%, NPV=89%) and test set (PPV=64%, NPV=86%). Overall, the control frequency of this SNP in homozygous form was 12% vs 60% in LGL (p<.01, OR=9.1). MICA alleles have been implicated in autoimmune diseases and malignancies. Although this SNP may not define a particular MICA genotype, it is possible that it is in linkage disequilibrium with genotype-defining polymorphisms. To study the functional consequences of our findings, flow cytometric analysis using anti-MICA antibodies was performed, which identified higher expression of MICA in neutrophils from patients as compared to controls (p=.04). MICA overexpression decreased after immunosuppressive therapy (p<.01). While the mechanism of MICA induction is unknown, we stipulate that the presence of MICA alleles leads to a persistent stimulatory signal in LGL predisposing to clonal outgrowth. In sum, our findings suggest that MICA polymorphisms may represent a predisposition factor in LGL and/or LGL-associated neutropenia.

Antibodies against MICA Antigens and Kidney-Transplant Rejection

Good HLA-A, HLA-B, and HLA-DR matches do not guarantee rejection-free renal transplantation. Some kidney transplants fail despite such matches, suggesting that other antigens might be targets for rejection. Major-histocompatibility-complex (MHC) class I-related chain A (MICA) antigens are polymorphic and can elicit antibody production. We sought to determine whether an immune response to MICA antigens might play a role in the failure of kidney allografts. Pretransplantation serum samples from 1910 recipients of kidney transplants from deceased donors were tested for anti-MICA antibodies with an assay in which single MICA antigens were attached to polystyrene microspheres. Antibodies against MICA alleles were detected in 217 of the 1910 patients (11.4%). The presence of MICA antibodies was associated with renal-allograft rejection. The mean (+/-SE) 1-year graft-survival rate was 88.3+/-2.2% among recipients with anti-MICA antibodies as compared with 93.0+/-0.6% among recipients without anti-MICA antibodies (P=0.01). Among recipients of first kidney transplants, the survival rate was even lower among MICA antibody-positive patients (87.8+/-2.4%) than among MICA antibody-negative recipients (93.5+/-0.6%, P=0.005). In addition, the association of MICA sensitization with reduced graft survival was more evident in kidney-transplant recipients with good HLA matching: among 326 recipients who received well-matched kidneys (0 or 1 HLA-A plus HLA-B plus HLA-DR mismatch), sensitization against MICA was associated with poorer allograft survival (83.2+/-5.8% among those with anti-MICA antibodies vs. 95.1+/-1.3% among those without such antibodies, P=0.002). Presensitization of kidney-transplant recipients against MICA antigens is associated with an increased frequency of graft loss and might contribute to allograft loss among recipients who are well matched for HLA.

24-P: Pre transplant anti MICA antibodies and long term follow up in deceased donor kidney transplants

24-P: Pre transplant anti MICA antibodies and long term follow up in deceased donor kidney transplants

The Relationship of Anti-MICA Antibodies and MICA Expression with Heart Allograft Rejection

The role of MICA antibodies in acute heart allograft rejection was examined utilizing 190 pre- and post-transplant serum samples from 44 patients collected during the first year after transplantation. MICA antibodies were detected by CDC test on recombinant cell lines and by the newly developed Luminex MICA antibody detection assay. Additionally, MICA expression was analyzed by 'real time' RT-PCR and by immunohistochemistry in 10 endomyocardial biopsies. Only two subjects had HLA antibodies post-transplant. Nevertheless, MICA antibodies were found in a significant number of subjects. The prevalence of MICA antibodies was significantly higher among those with severe acute rejection (AR) than in those without rejection (60.7% vs. 14.3%, p = 0.0038 by CDC; 55.5% vs. 5.7%, p = 0.0020 by Luminex). In most cases, the appearance of MICA antibodies post-transplant precedes AR. Following transplantation, MICA up-regulation correlated with histological evidence of severe rejection. Monitoring for MICA antibodies post-transplant may be useful to establish new risk factors for acute rejection.

Monitoring of Anti-HLA and Anti–Major Histocompatibility Complex Class I Related Chain A Antibodies in Living Related Renal Donor Transplantation

This study was undertaken with the aim to analyze the clinical relevance of posttransplant anti-HLA and anti–major histocompatibility complex class I related chain A (MICA) antibodies in response to living related donor renal transplantation. A total of 185 consecutive post–renal transplant recipient serum samples were analyzed for the detection of anti-HLA and MICA antibodies using enzyme-linked immunosolvent assay techniques. Patients carrying both anti-HLA as well as anti-MICA antibodies (MICA +/HLA +) were the worst affected, showing significantly poorer graft survival compared with the MICA-/HLA-negative group (17% vs 89%, χ 2 = 19.63, P = .000). Similarly, patients with only MICA antibodies or those with only HLA antibodies also had significantly lower graft survival ( P = .035 and P = .001, respectively) as compared to the nonsensitized group. The study illustrated that posttransplant monitoring antibodies to both MICA as well as HLA could be good predictors of renal allograft failure.

Decreased NKG2D Expression on CD8+ T Cell Is Involved in Immune Evasion in Patients with Gastric Cancer

Some studies suggest that the immunoreceptor NKG2D expression on CD8(+) T cells is down-regulated and this reduction may be involved in immune evasion in cancer patients. The present study was designed to investigate NKG2D expression on CD8(+) T lymphocytes and its relationship to immune evasion in gastric cancer patients. NKG2D expression on both circulating and tumor-infiltrating CD8(+) T cells was evaluated by multicolor flow cytometry. Soluble MHC class I chain-related gene A (MICA) in the sera was quantitated by ELISA. Transwell experiments were carried out to determine the effect of cancer cells on NKG2D expression. NKG2D expression on circulating CD8(+) T cells was down-regulated and significantly correlated with IFN-gamma production in gastric cancer patients (r = 0.68; P = 0.007). NKG2D expression was closely related to undifferentiated cancer (P = 0.021) as was the depth of invasion (P = 0.012). There was no difference in soluble MICA between gastric cancer patients and normal controls. NKG2D expression on CD8(+) T cells was remarkably reduced in the tissue of gastric cancer compared with peripheral blood (P = 0.046). Complete removal of tumor by surgery restored NKG2D expression on CD8(+) T cells (P = 0.0049). Transwell experiments showed that this down-regulation was induced by direct contact between cancer cells and CD8(+) T cells and that soluble factors did not affect the NKG2D expression. This phenomenon was blocked by the addition of anti-MICA antibodies. Decreased NKG2D expression may be one of the key mechanisms responsible for immune evasion by tumors in gastric cancer.