HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. METHODS: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. RESULTS: These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. CONCLUSIONS: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

A subset of HLA-DP molecules serve as ligands for the natural cytotoxicity receptor NKp44.

Natural killer (NK) cells can recognize virus-infected and stressed cells1 using activating and inhibitory receptors, many of which interact with HLA class I. Although early studies also suggested a functional impact of HLA class II on NK cell activity2,3, the NK cell receptors that specifically recognize HLA class II molecules have never been identified. We investigated whether two major families of NK cell receptors, killer-cell immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs), contained receptors that bound to HLA class II, and identified a direct interaction between the NK cell receptor NKp44 and a subset of HLA-DP molecules, including HLA-DP401, one of the most frequent class II allotypes in white populations4. Using NKp44ζ+ reporter cells and primary human NKp44+ NK cells, we demonstrated that interactions between NKp44 and HLA-DP401 trigger functional NK cell responses. This interaction between a subset of HLA-DP molecules and NKp44 implicates HLA class II as a component of the innate immune response, much like HLA class I. It also provides a potential mechanism for the described associations between HLA-DP subtypes and several disease outcomes, including hepatitis B virus infection5-7, graft-versus-host disease8 and inflammatory bowel disease9,10.

The clinical impact of humoral immunity in pediatric renal transplantation.

The development of anti-donor humoral responses after transplantation associates with higher risks for acute rejection and 1-year graft survival in adults, but the influence of humoral immunity on transplant outcomes in children is not well understood. Here, we studied the evolution of humoral immunity in low-risk pediatric patients during the first 2 years after renal transplantation. Using data from 130 pediatric renal transplant patients randomized to steroid-free (SF) or steroid-based (SB) immunosuppression in the NIH-SNSO1 trial, we correlated the presence of serum anti-HLA antibodies to donor HLA antigens (donor-specific antibodies) and serum MHC class 1-related chain A (MICA) antibody with both clinical outcomes and histology identified on protocol biopsies at 0, 6, 12, and 24 months. We detected de novo antibodies after transplant in 24% (23% of SF group and 25% of SB group), most often after the first year. Overall, 22% developed anti-HLA antibodies, of which 6% were donor-specific antibodies, and 6% developed anti-MICA antibody. Presence of these antibodies de novo associated with significantly higher risks for acute rejection (P=0.02), chronic graft injury (P=0.02), and decline in graft function (P=0.02). In summary, antibodies to HLA and MICA antigens appear in approximately 25% of unsensitized pediatric patients, placing them at greater risk for acute and chronic rejection with accelerated loss of graft function. Avoiding steroids does not seem to modify this incidence. Whether serial assessments of these antibodies after transplant could guide individual tailoring of immunosuppression requires additional study.

Beyond histology: lowering human leukocyte antigen antibody to improve renal allograft survival in acute rejection.

BACKGROUND: The common endpoint in the treatment of antibody-mediated rejection (AMR) is functional reversal (creatinine levels). Reduction of human leukocyte antigen (HLA) antibody strength is not commonly considered as an essential endpoint for AMR resolution. The purpose of this study was to determine whether reduction in HLA antibody intensity in patients with histologic AMR reversal influences long-term renal allograft survival. METHODS: Renal allograft recipients were included if he or she had a biopsy diagnosis of AMR (between August 2000 and October 2008) and serial evaluation for HLA antibodies prebiopsy and postbiopsy. Antibody reduction was defined as mean fluorescence intensity decrease more than 50% in highest intensity antibody after AMR therapy and the absence of new antibody formation. Patients were treated with plasmapheresis, thymoglobulin/OKT3, and corticosteroids. Survival analysis was performed using STATA/MP v10 (College Station, TX). RESULTS: Twenty-eight patients were analyzed. Antibody reduction failed to occur in 22 of 28 cases. Baseline characteristics were similar between groups. Antibody nonresponders had significantly shorter allograft survival time (61.4 months) compared with antibody responders (no failures) (P=0.04, log-rank test). CONCLUSIONS: In conclusion, failure to significantly reduce antibody levels and prevent new formation was strongly predictive of allograft loss. This observation suggests that the therapeutic intervention that reduces antibody production may prolong graft survival in transplantation.

Epitopes of HLA-A, B, C, DR, DQ, DP and MICA antigens.

This chapter presents lists of HLA epitopes that have been defined to date. It also presents examples of reactions of mAb and eluted allosera with the class I, class II and MICAsingle antigen beads. To date, we have identified 110 class I epitopes, of which 47 were defined by mAbs and 63 by alloantibodies that were eluted from rHLA class I single antigen cell lines. We listed 34 epitopes shared by the HLA-A locus antigens, 44 epitopes shared by HLA-B locus antigens, 4 epitopes shared by HLA-C locus antigens, 20 inter-locus epitopes shared by HLA-A-B locus antigens, 5 inter-locus epitopes shared by HLA-B-C locus antigens and 3 inter-locus epitopes shared by HLA-A-B-C locus antigens. Sixty HLA-DR epitopes have been defined mostly by one amino acid (aa) residue on the HLA-DR beta chain. Eighteen HLA-DQ epitopes have been defined on the HLA-DQB chain and on the HLA-DQA chain of the HLA-DQ antigens. A few DQ epitopes were defined by one aa residue. However, most can be defined by several alternative combinations of aa residues. DQA and DP epitopes--few in number at this time--were identified. Only seven MICA epitopes have been defined to date. All epitopes can be defined by an exclusive amino acid.

HLA class II DQ epitopes.

1. DQ epitopes were defined by studying the distinct reaction profiles of 17 allosera and 35 DQ-specific mouse monoclonal antibodies to HLA class II single antigen beads coated with purified recombinant DQ molecules. 2. DQ molecules are heterodimers with a polymorphic alpha chain and beta chain. Sixteen of the defined epitopes correlated to various DQB specificities and one defined epitope correlated to DQA1*0201. 3. Among the 16 DQB epitopes, 7 sites were recognized by both alloantibodies and DQ monoclonal antibodies, 5 sites were identified by DQ monoclonal antibodies and 4 sites by alloantibodies. The only unequivocal DQA1*0201 epitope found was identified by an alloantibody from a patient with a failed kidney graft. 4. Of the 17 DQ epitopes, 10 sites have a very restricted possible choice involving one to three amino acid(s) to form the unique sites; while the other 7 sites (6 DQB sites and 1 DQA site) have multiple potential sites. 5. The amino acid residues for all of 16 DQB epitope sites are unique and not shared with DQA, DR or DP antigens; while the DQA1*0201 epitope site is not shared with DQB, DR or DP antigens. 6. Except epitope #2008, all the other 16 epitopes consist of unique amino acids that appear to be exposed on the surface of DQ molecules as viewed from the 3-D model. 7. The existence of antibodies to multiple epitopes on the same HLA molecule invites molecular epitope analysis of serum reaction profiles. A single antigen cannot be considered to be a single epitope.

Detection of HLA and MICA antibodies before kidney graft failure.

390 serum samples taken from a series of 28 patients who lost their grafts were retrospectively investigated for antibodies by single antigen HLA Class I&II and MICA Luminex beads. In 20 patients with immunological failure, 10 patients had DSA, 16 had epitope-related NDSA, and 16 had NDSA or MICA antibodies. In 16 which increased antibodies leading up to the graft failure, DSA were found in 7 patients. However, in 9 patients who similarly rejected their grafts, 6 were epitope-related DSA and 3 were NDSA. We were unable to establish why NDSA might be associated with the failure, but this was clearly the case in these patients. Of the 25 patients whose grafts were rejected, 23 (92%) produced antibody prior to failure. Our results strongly suggest that antibody monitoring is important as a means of detecting chronic rejection before a rise in SCr. Early detection of antibody would allow for appropriate and timely interventions to save a graft.