A Soluble NK-CAR Mediates the Specific Cytotoxicity of NK Cells toward the Target CD20+ Lymphoma Cells.

The structures of chimeric antigen receptors (CARs) currently designed for natural killer (NK) cells are mostly based on knowledge gained about CAR-T cells. Although these CAR-NK cells have shown promising effects, there are still many limitations to their application. In this study, we designed a soluble NK-CAR since the membrane protein NKG2D expressed by NK cells can directly trigger NK cell cytotoxicity by binding with the ligand MICA. This CAR is composed of three segments: the extracellular domain of MICA, an anti-CD20 single-chain variable fragment (anti-CD20 ScFv), and a human IgG Fc component. The nucleotide sequence of the soluble NK-CAR was cloned into a eukaryotic expression vector and expressed in suspension HEK293 cells, and the recombinant NK-CAR protein was then purified in a Staphylococcus aureus protein A column. The novel NK-CAR exhibited bifunctional activity, recognizing both the CD20 antigen of target cells and the NKG2D receptor of NKL cells. The NK-CAR activated the NKG2D receptor signaling pathway, causing NKL cells to express CD107a and secrete interferon-gamma. The soluble NK-CAR mediated the NKL cell killing of CD20+ Daudi cells in vitro, with a 1 µg/mL concentration inducing the maximum killing effect. Moreover, 51.7% (p < 0.01) of Daudi cells were killed at the effector-to-target ratio of 10:1. In the presence of recombinant rMICA and NKG2D-Ig proteins, this killing effect was reduced to 30% (P < 0.01) owing to competitive interference. Our results highlight the clinical application potential of this novel immunotherapy for killing target tumor cells.

Posttransplant-Alloantibodies Against MICA Antigens Associated With Decreased Long-Term Allograft Survival of Kidney Transplant Recipients.

BACKGROUND: Previous evidence showed that antibodies against major histocompatibility complex class I-related chain A (MICA) could lead to antibody-mediated rejection in kidney transplantation in case where the patients had no alloantibodies against HLA. However, the effects of posttransplant anti-MICA antibodies on long-term renal allograft survival and function remained unsettled. We tested the posttransplant anti-MICA antibodies in 150 kidney transplant patients. The aim of this study was to compare the long-term graft survival and function between patients who were MICA positive and those who were negative. METHODS: The posttransplant serum samples from 150 patients receiving kidney transplantation in our center from 2012 to 2013 were tested for MICA antibodies and HLA antibodies by Luminex single antigen array technology. Graft survival and function were followed up for a mean time of 74.2 months. The research was conducted in accordance with the Helsinki Congress and the Declaration of Istanbul. RESULTS: Of the 150 patients, 38 (25.3%) were sensitized against MICA after transplantation. The anti-MICA antibodies-positive (anti-MICA+) group had a worse long-term renal allograft survival than that of anti-MICA-negative (anti-MICA-) group (P = .029), even when stratified by posttransplant HLA sensitization status or donor source. Anti-MICA antibodies also had a detrimental impact on renal allograft function, but only at 1 year posttransplantation (estimated glomerular filtration rates at 1 year: anti-MICA+ 66.6 mL/min/1.73 m2 vs anti-MICA- 78.7 mL/min/1.73 m2; P = .023). CONCLUSION: Posttransplant anti-MICA antibodies were associated with decreased long-term renal allograft survival and short-term renal allograft function.

Proteome-wide analysis of lysine ß-hydroxybutyrylation in the myocardium of diabetic rat model with cardiomyopathy.

Lysine ß-hydroxybutyrylation (kbhb), a novel modification of lysine residues with the ß-hydroxybuty group, is associated with ketone metabolism in numerous species. However, its potential role in diabetes, especially in diabetic cardiomyopathy (DCM), remains largely unexplored. In this study, using affinity enrichment and liquid chromatography-mass spectrometry (LC-MS/MS) method, we quantitatively analyze the kbhb residues on heart tissues of a DCM model rat. A total of 3,520 kbhb sites in 1,089 proteins were identified in this study. Further analysis showed that 336 kbhb sites in 143 proteins were differentially expressed between the heart tissues of DCM and wild-type rats. Among them, 284 kbhb sites in 96 proteins were upregulated, while 52 kbhb sites in 47 proteins were downregulated. Bioinformatic analysis of the proteomic results revealed that these kbhb-modified proteins were widely distributed in various components and involved in a wide range of cellular functions and biological processes (BPs). Functional analysis showed that the kbhb-modified proteins were involved in the tricarboxylic acid cycle, oxidative phosphorylation, and propanoate metabolism. Our findings demonstrated how kbhb is related to many metabolic pathways and is mainly involved in energy metabolism. These results provide the first global investigation of the kbhb profile in DCM progression and can be an essential resource to explore DCM's pathogenesis further.

Genomic characterization of MICA gene using multiple next generation sequencing platforms: A validation study.

We have developed a protocol regarding the genomic characterization of the MICA gene by next generation sequencing (NGS). The amplicon includes the full length of the gene and is about 13 kb. A total of 156 samples were included in the study. Ninety-seven of these samples were previously characterized at MICA by legacy methods (Sanger or sequence specific oligonucleotide) and were used to evaluate the accuracy, precision, specificity, and sensitivity of the assay. An additional 59 DNA samples of unknown ethnicity volunteers from the United States were only genotyped by NGS. Samples were chosen to contain a diverse set of alleles. Our NGS approach included a first round of sequencing on the Illumina MiSeq platform and a second round of sequencing on the MinION platform by Oxford Nanopore Technology (ONT), on selected samples for the purpose of either characterizing new alleles or setting phase among multiple polymorphisms to resolve ambiguities or generate complete sequence for alleles that were only partially reported in the IMGT/HLA database. Complete consensus sequences were generated for every allele sequenced with ONT, extending from the 5' untranslated region (UTR) to the 3' UTR of the MICA gene. Thirty-two MICA sequences were submitted to the IMGT/HLA database including either new alleles or filling up the gaps (exonic, intronic and/or UTRs) of already reported alleles. Some of the challenges associated with the characterization of these samples are discussed.

Tumor-Derived Soluble MICA Obstructs the NKG2D Pathway to Restrain NK Cytotoxicity.

The natural killer group 2D (NKG2D) receptor and its ligands play important roles in immune surveillance. In this study, we observed that the average serum soluble MICA (sMICA) concentration of 174 hepatocellular carcinoma (HCC) patients was significantly higher than that in 80 healthy subjects (602.17 ± 338.15 vs. 72.26 ± 87.88 pg/ml, t = 3.107, P=0.002). The levels of serum sMICA in 44 HCC patients with initial levels above 400 pg/ml declined significantly after surgical removal of the liver cancer tissue (P<0.001). Moreover, the mean survival time of HCC patients who had sMICA above 400 pg/ml was significantly shorter than that HCC patients with lower sMICA levels (P<0.001). Using the reporter cell line (NKG2D-2B4) in which activation of the NKG2D receptor pathway results in GFP expression based on the stimulation of immobilized rMICA, we showed that the number of GFP-expressing cells decreased sharply in presence of sMICA. Upon adding sMICA, the release of cytokines IFN-γ, TNF-α, and IL-8 by NK cell line (NKL) under stimulation of immobilized rMICA was blocked. Using MICA-expressing cells as the target cells, we observed that about 80% of target cells were killed by NKL at E:T of 10:1, but in presence of sMICAhigh serum of HCC patients, the dead target cells were reduced to 30.8%. Compared in presence of sMICAlow serum from HCC patients, there were 63.7% of target cells dead (p=0.043). Thus, our data suggested that sMICA obstructs the activation of NKG2D pathway to protect tumor cells from NK cell-mediated cytotoxicity.

LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration.

Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML.

NK cell-mediated anti-leukemia cytotoxicity is enhanced using a NKG2D ligand MICA and anti-CD20 scfv chimeric protein.

NK cells are important innate cytotoxic lymphocytes that have potential in treatment of leukemia. Engagement of NKG2D receptor on NK cells enhances the target cytotoxicity. Here, we produced a fusion protein consisting of the extracellular domain of the NKG2D ligand MICA and the anti-CD20 single-chain variable fragment (scfv). This recombinant protein is capable of binding both NK cells and CD20+ tumor cells. Using a human NKG2D reporter cell system we developed, we showed that this fusion protein could decorate CD20+ tumor cells with MICA extracellular domain and activate NK through NKG2D. We further demonstrated that this protein could specifically induce the ability of a NK cell line (NKL) and primary NK cells to lyse CD20+ leukemia cells. Moreover, we found that downregulation of surface HLA class I expression in the target cells improved NKL-mediated killing. Our results demonstrated that this recombinant protein specifically lyses leukemia cells by NK cells, which may lead to development of a novel strategy for treating leukemia and other tumors.

Polymorphism of keratin 1 associates with systemic lupus erythematosus and systemic sclerosis in a south Chinese population.

Both systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) diseases are related to the genetic and environmental factors, causing damage to the skin. The mutations of keratin 1 gene (KRT1) were reported to associate with skin diseases. The single-nucleotide polymorphism (SNP, rs14024) and the indel polymorphism (cds-indel, rs267607656), consisting mostly of the common haplotypes and could be used for genotyping of KRT1. We used the PCR with sequence specific primers (PCR-SSP) to determine the genotype of KRT1 in 164 SLE, 99 SSc patients, and 418 healthy controls. The results showed that the mutant with G at SNP rs14024 was associated with the high risk to SLE (p = 6.48×10-5) and SSc (p = 8.75×10-5), while the deletion allele at rs267607656 was associated with the low risk to SSc (p = 4.89×10-4) comparing to the normal controls. Haplogenotype, Del-/MU+ was associated with high susceptibility to SLE (OR = 1.87, p = 0.001) and SSc (OR = 2.29, p = 2.34×10-4). In contrast, the Haplogenotype Del+/MU- was associated with resistance to SLE (OR = 0.35, p = 6.24×10-5) and SSc (OR = 0.34, p = 0.001). This study demonstrates that the variations in KRT1 and the specific polymorphism of KRT1 in this Chinese Han population are associated with autoimmune diseases SLE and SSc. Typing KRT1 might be helpful to identify SLE and SSc patients.

Analysis of Sera of Recipients with Allograft Rejection Indicates That Keratin 1 Is the Target of Anti-Endothelial Antibodies.

Anti-endothelial cell antibodies (AECAs) are usually directed against the surface antigens on the vascular endothelial cells. Clinical studies suggest a pathogenic role for nonhuman leukocyte antigen in antibody-mediated rejection; however, the antigens on the donor vascular endothelium that serve as the first-line targets for an immune response during allograft rejection have not been fully identified. Here, we used immunoprecipitation and mass spectrometry to identify antigens from the sera of kidney transplant recipients who were experiencing antibody-mediated rejection. Keratin 1 (KRT1) was identified as a novel antigenic target expressed on endothelial cells. To validate our finding, we produced recombinant proteins representing the three most common alleles of KRT1. The serum used for immunoprecipitation showed a strong reaction to KRT1 recombinants in western blot and ELISA. In the kidney transplant cohort, more AECA-positive recipients than AECA-negative recipients had KRT1 antibodies (32.2% versus 11.9%, p = 0.002). Sera from 255 renal recipients were tested by ELISA. Of the 77 recipients with deteriorating graft function (serum creatinine > 120 µmol/L), 23 had anti-KRT1 antibodies. KRT1-IgG positivity was, therefore, associated with a higher risk of kidney transplant rejection (29.9% (23/77) versus 16.9% (30/178), p = 0.0187). A better understanding of this antigenic target will improve long-term allograft survival.

Cytokine response to Hantaan virus infection in patients with hemorrhagic fever with renal syndrome.

Hantaan virus (HTNV) infection of the human body causes a severe acute infectious disease known as hemorrhagic fever renal syndrome (HFRS). The aim of this study was to correlate patient cytokine profiles to HFRS severity. In this study, we discuss the clinical significance of evaluating HFRS treatment outcomes using cytokine information. The levels of 18 cytokines were quantitatively determined in three groups: 34 HTNV IgM+ cases, 63 HTNV IgM- negative cases, and 78 healthy volunteers. The level of 14 serum cytokines were higher in the patient group than that in the healthy control group. In the 34 HTNV IgM+ patient sera, a set of 27 cytokines was further assessed. The cytokines of TNF-ß, IL-1ra, and IL-6 were detected at higher level in the IgM+ group than that in the IgM- group. The deterioration of HFRS was accompanied with multiple cytokines increased, such as IL-1ra, IL-12p70, IL-10, IP-10, IL-17, IL-2, and IL-6. Our data indicate that serum cytokine levels are associated with the progression of HFRS.

Acute Antibody-Mediated Rejection in Presence of MICA-DSA and Successful Renal Re-Transplant with Negative-MICA Virtual Crossmatch.

The presence of donor-specific alloantibodies (DSAs) against the MICA antigen results in high risk for antibody-mediated rejection (AMR) of a transplanted kidney, especially in patients receiving a re-transplant. We describe the incidence of acute C4d+ AMR in a patient who had received a first kidney transplant with a zero HLA antigen mismatch. Retrospective analysis of post-transplant T and B cell crossmatches were negative, but a high level of MICA alloantibody was detected in sera collected both before and after transplant. The DSA against the first allograft mismatched MICA*018 was in the recipient. Flow cytometry and cytotoxicity tests with five samples of freshly isolated human umbilical vein endothelial cells demonstrated the alloantibody nature of patient's MICA-DSA. Prior to the second transplant, a MICA virtual crossmatch and T and B cell crossmatches were used to identify a suitable donor. The patient received a second kidney transplant, and allograft was functioning well at one-year follow-up. Our study indicates that MICA virtual crossmatch is important in selection of a kidney donor if the recipient has been sensitized with MICA antigens.