C1 Epitope Research Articles

Is the Development of Ascites in Alcoholic Liver Patients Influenced by Specific KIR/HLA Gene Profiles?

Decompensated cirrhosis is the most common cause of ascites due to hemodynamic and renal alteration by continuous fluid leakage from the hepatic sinusoids and splanchnic capillaries into the interstitial space. Then, fluid leakage exceeds lymphatic return, leading to progressive fluid accumulation directly into the peritoneal cavity. Alcohol consumption is one of the main risks of developing alcoholic cirrhosis (AC), but not all AC patients develop ascites. Avoiding the development of ascites is crucial, given that it deteriorates prognosis and increases the patient mortality patient. The innate immune system plays a crucial role in cirrhosis through natural killer cells, which are abundant in the liver. The aim of this study was to analyze the KIR/HLA-C genetic profile in AC patients with and without ascites to understand this pathology and find predictive clinical susceptibility biomarkers that can help to establish risks and prevent the development of ascites in AC patients. A total of 281 AC patients with and without ascites were analyzed and compared with 319 healthy controls. Genomic DNA was extracted from peripheral blood in all groups. A PCR-SSO assay was performed for KIR/HLA genotyping analysis. A total of 16 activating and inhibitor KIR genes and their corresponding known ligands, epitopes of HLA-C, and their genotypes were analyzed. According to our analysis, C1 epitopes were statistically significantly decreased in AC patients with and without ascites. When comparing AC patients with ascites and healthy controls, a significant decrease in C1 epitope frequency was also observed. A statistically significant decrease was also found when comparing the C1C2 genotype in AC patients without ascites with controls. In conclusion, the absence of KIR2DL2 and KIR3DL1 genes may be a predisposing factor for the development of ascites in AC patients. The KIR2DS2/KIR2DL2 may could be involved in grade I ascites development, and the presence of the C1+ epitope and the homozygous C2C2 genotype may be protective genetic factors against ascites development in AC patients.

Escaping Ability of Human-Porcine Hybrid Factor VIII from Anti-Factor VIII Inhibitors with an A2 or C2 Epitope

Porcine (p) factor (F)VIII has limited cross-reactivity to inhibitors against human (h) FVIII (Lollar P, JBC 1991). Plasma-derived or recombinant pFVIII agents, therefore, have been used as therapeutics for uncontrollable bleedings in congenital or acquired hemophilia A (HA). Alternatively, most of FVIII inhibitors bind to the epitopes located in the A2, C2 and/or A3-C1 domains of FVIII (Scandella D, Blood 1989, Fulcher C, PNAS 1985), and monospecific inhibitors for domains other than A2 and C2 are infrequent (Prescott R, Blood 1997). In the present study, based on a hypothesis that human-porcine hybrid (hp)FVIII could escape the FVIII-inhibitory effects by inhibitors, we converted A2 domain and/or C2 domain of hFVIII into corresponding sequence of pFVIII, respectively, and investigated the escaping ability of these mutants from inhibitors. Three mutants were created using stable BHK cell line by converting hFVIII A2 domain and/or C2 domain into corresponding sequence of pFVIII, termed by hp(A2), hp(C2), and hp(A2/C2), respectively. Specific activity assessed by a one-stage clotting assay of hp(A2), hp(C2) and hp(A2/C2) was 0.75, 0.94 and 0.60 relative to hFVIII respectively. First, the escaping ability of these mutants from commercial anti-FVIII A2 or C2 monoclonal antibody (mAb) was investigated. Each of two different anti-hFVIII A2 mAbs and two different anti-C2 mAbs was spiked into FVIII-deficient plasma (FVIIId), respectively at the Bethesda titer of 2.7-4.4 BU/mL, followed by spiking hFVIII or hpFVIII at the concentration of 1 nM each. Incubation of each sample at 37 °C was initiated and FVIII activity was determined (FVIII:C) sequentially, calculating the FVIII:C at the fold of initial (no addition of inhibitor). In anti-A2 mAbs, FVIII:C of hFVIII and hp(C2) were immediately attenuated, resulting in suppression under 20% of initial level after 30 min, however, hp(A2) and hp(A2/C2) were not suppressed at all. In contrast, with anti-C2 mAbs, FVIII:C in hFVIII and hp(A2) was attenuated, whilst hp(C2) and hp(A2/C2) maintained their FVIII:C. Next, similar experiment was conducted with the purified IgG from the HA patients with inhibitor whose inhibitor was confirmed to recognize A2 only or both A2/C2 domains by Western blotting. Four patients with anti-A2 and anti-A2/C2 inhibitors (2 patients each) were investigated. Mutants converted certain domain(s) could escape inhibitory effect of purified IgG recognizing the corresponding domain(s), although partial attenuation exhibited even in the corresponding mutants. To further investigation, thrombin generation assay (TGA) and rotation thromboelastometry (ROTEM) were used. In TGA, purified IgG from patient with anti-A2 or anti-A2/C2 inhibitor was added to FVIIId, followed by spiking hFVIII or hpFVIII at the concentration of 1 nM. In the absence of IgG, peak thrombin of FVIIId was 128.0 nM, and then by adding hFVIII, hp(A2), hp(C2) and hp(A2/C2), the parameter enhanced to 228.8, 208.6, 207.3, and 219.9 nM, respectively. In the presence of anti-A2 inhibitor (5.0 BU/mL), peak thrombin of hp(A2) and hp(A2/C2) was not attenuated (203.1 and 257.1 nM), whilst those of hFVIII and hp(C2) was suppressed equally to FVIIId (137.3 and 134.5 nM respectively). As for anti-A2/C2 inhibitor (2.8 BU/mL), only hp(A2/C2) was not attenuated, whereas the others were suppressed comparable to FVIIId . Whole bloods collected from severe HA with anti-A2 inhibitor (3.1 BU/mL) were mixed with hFVIII or hpFVIII at the concentration of 2 nM, respectively, and were measured by ROTEM. Without any FVIII spiking, clot time (CT) was 7,151 sec and clot formation time (CFT) was undetectable. The addition of hp(A2) and hp(A2/C2) shortened CT/CFT to 1,525/536 sec and 1,987/690 sec, respectively, whilst hFVIII or hp(C2) was not effective (4,257/2,270 and 3,200/1,022 sec, respectively). On the other hand, purified IgG from patients with anti-A2/C2 inhibitor was added to whole blood from another severe HA without inhibitor. Under this condition (4.7 BU/mL), only hp(A2/C2) addition shortened CT/CFT from 6,482/2,163 to 1,724/503 sec, whereas the addition of the other FVIII resulted in limited effect to CT/CFT (2,703-4,796/551-2,024). We concluded that hybrid FVIII converted into porcine A2 and/or C2 domain sequences were able to escape corresponding inhibitors and that hpFVIII could be effective therapeutically for HA patients with inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Association of KIR Genes and MHC Class I Ligands with Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic illness that is associated with immune dysregulation. NK cell function has previously been associated with AD. NK cells directly interact with polymorphic HLA class I ligand variants using killer cell Ig-like receptors (KIRs). The purpose of this study was to identify potential associations between NK cell function and AD by evaluating variation in the presence of KIR genes as well as KIR gene interactions with the appropriate HLA class I KIR-specific ligands. Human DNA from the genetics of AD case-control study was used to genotype HLA class I KIR-specific ligands and the presence of KIR genes. In the full cohort, an increased risk of AD was noted for KIR2DL5 (1.51 [1.13, 2.01]), KIR2DS5 (1.72 [1.26, 2.34]), and KIR2DS1 (1.41 [1.04, 1.91]). Individuals with KIR2DS5 or KIR2DS1 and the HLA-C*C2 epitope were at an increased risk of AD (1.74 [1.21, 2.51] and 1.48 [1.04, 2.12], respectively). The HLA-B*-21T (TT) leader sequence increased the risk of AD across ethnicity. African Americans with KIR2DL2, KIR2DS1, KIR2DL5, and KIR2DS5 are more likely to have AD, and the risk increased for KIR2DS1 and KIR2DS5 in the presence of appropriate HLA-C C2 epitope. The risk of AD also increased for individuals with the HLA-B*-21T leader sequence. Future studies should focus on KIR gene allelic variation as well as consider cell-based measurements of KIR and the associated HLA class I epitopes.

Epitope Mapping of Inhibitor Patient Plasmas during Immune Tolerance Induction

Background The most significant complication in the management of patients with severe hemophilia A continues to be the development of anti-factor VIII antibodies. The standard of care for inhibitor eradication is immune tolerance induction (ITI). ITI refers to frequent and prolonged exposure to fVIII, often at high doses, as a method to induce tolerance. Patients with severe hemophilia A typically develop a polyclonal B-cell response to fVIII, with antibodies to the A2 and C2 domains being the most prevalent. We have previously identified 5 non-overlapping B cell epitopes with unique characteristics in the A2 domain, and 3 in the C2 domain. Specifically within the A2 and C2 domain we have shown that epitope is a better predictor of response to treatment than the Bethesda titer. Using monoclonal antibodies (MAbs) with known epitopes, we tracked the change in A2 and C2 epitope response over time in 9 high-titer inhibitor patients during ITI.Methods B-cell epitopes were mapped using a previously published competition ELISA. For each epitope tested, a single well characterized biotinylated anti-fVIII MAb was competed against antibodies present in patient plasma. FVIII deficient plasma was used as a no antibody control. In the A2 domain, 4 of the 5 non-overlapping epitopes were tested (groups A, B, D, and E). All three non-overlapping epitopes in the C2 domain were tested (A, B, and C). Absorbance was measured at 405 nm and ELISA titers were calculated using an O.D. value of 0.3. Samples were considered positive if there was at least a 20% decrease in the ELISA titer ratio between patient and control plasma.Results We tested 85 plasma samples from 9 patients, with each individual patient contributing between 4-13 samples over the course of ITI. At the point of highest titer, patients had a mean of 4.4 of 7 tested epitopes. The most frequently detected epitopes were 2-76 (group A) and 4F4 (group B) in the A2 domain and 2-117 (group C) in the C2 domain. All patients (9 of 9) had antibodies to these epitopes at one or more time points during ITI. The 1D4 (group E) and 2-54 (group D) epitopes within the A2 domain and the I54 (group A) and 1B5 (group B) epitopes within the C2 domain were less frequent but still prominent, with 8 of 9 patients having the I54 epitope, 7 of 9 having the 1D4 epitope, and 6 of 9 having the 2-54 and 1B5 epitopes.Eighty of the 85 patient samples tested were found to have a positive Bethesda titer, indicating inhibitory antibodies were present. Higher inhibitory titers correlated with more epitopes detected. Of the 80 inhibitory samples, 64% of them showed antibodies to the 2-76 epitope, 59% to the 4F4 epitope, and 53% of them showed antibodies to the 2-117 epitope. The less frequent 1D4, 2-54, I54, and 1B5 epitopes were seen in 28%, 18%, 23%, and 16% respectively. The number of epitopes and the titers for those epitopes detected increased or decreased with the Bethesda titer over time. However, the most prevalent epitopes remained dominant even at times of relapse.Conclusion A broad antibody spectrum was seen in patients with hemophilia A and inhbitors. Multiple different epitopes within the A2 and C2 were present. Given our previous studies that showed epitope was more important than titer in the pathogenicity of these antibodies, tracking epitopes over time may provide valuable knowledge regarding response to therapy and may direct the clinician to adjust ITI strategy. Ultimately, knowledge of the dominant epitope that persists or recurs after ITI may be a target for individualized treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Polyfunctional Tier 2-Neutralizing Antibodies Cloned following HIV-1 Env Macaque Immunization Mirror Native Antibodies in a Human Donor.

Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C-infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 μg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2-neutralizing V3 Abs and demonstrates partial reproduction of the human donor's humoral immune response through nonhuman primate vaccination.

KIR B donors improve the outcome for AML patients given reduced intensity conditioning and unrelated donor transplantation

AbstractNatural killer (NK) cell recognition and killing of target cells are enhanced when inhibitory killer immunoglobulin-like receptors (KIR) are unable to engage their cognate HLA class I ligands. The genes of the KIR locus are organized into either KIR B haplotypes, containing 1 or more activating KIR genes or KIR A haplotypes, which lack those genes. Analysis of unrelated donor (URD) hematopoietic cell transplants (HCT), given to acute myeloid leukemia (AML) patients between 1988 and 2009, showed that KIR B haplotype donors were associated with better outcomes, primarily from relapse protection. Most of these transplants involved marrow grafts, fully myeloablative (MAC) preparative regimens, and significant HLA mismatch. Because the practice of HCT continues to evolve, with increasing use of reduced intensity conditioning (RIC), peripheral blood stem cell grafts, and better HLA match, we evaluated the impact of URD KIR genotype on HCT outcome for AML in the modern era (2010-2016). This analysis combined data from a prospective trial testing URD selection based on KIR genotypes (n = 243) with that from a larger contemporaneous cohort of transplants (n = 2419). We found that KIR B haplotype donors conferred a significantly reduced risk of leukemia relapse and improved disease-free survival after RIC, but not MAC HCT. All genes defining KIR B haplotypes were associated with relapse protection, which was significant only in transplant recipients expressing the C1 epitope of HLA-C. In the context of current HCT practice using RIC, selection of KIR B donors could reduce relapse and improve overall outcome for AML patients receiving an allogeneic HCT.

Differences in MHC-B diversity and KIR epitopes in two populations of wild chimpanzees

The major histocompatibility complex (MHC) class I genes play a critical role within the immune system, both by the presentation of antigens from intracellular pathogens to immunocompetent cells and by the interaction with killer cell immunoglobulin-like receptors (KIR) on natural killer cells (NK cells). Genes of the MHC are highly diverse, and MHC variation can have effects on the immune functionality of individuals; hence, comparisons of MHC diversity among closely related phylogenetic taxa may give insight into the factors responsible for the shaping of its diversity. The four geographically separated chimpanzee subspecies differ in their overall genetic diversity, have different population histories, and are confronted with different pathogens in their natural habitat, all of which may affect MHC class I DNA sequence diversity. Here, we compare the MHC-B exon two DNA sequence diversity from 24 wild western and 46 wild eastern chimpanzees using necropsy and noninvasively collected fecal samples, respectively. We found a higher MHC-B exon two nucleotide diversity, in our western than eastern chimpanzees. The inclusion of previously published MHC-B exon two data from other western and eastern chimpanzees supported this finding. In addition, our results confirm and extend the finding of a very low C1 epitope frequency at eastern chimpanzee MHC-B molecules, which likely affects the ability of these molecules to interact with NK cells. While the understanding of the differing pathogen environments encountered by disparate populations of a species is a challenging endeavor, these findings highlight the potential for these pathogens to selectively shape immune system variation.

A Novel Injury Site-Targeted Complement Inhibitor Which Protects against Lung Transplant Ischemia Reperfusion Injury

Purpose Self-reactive natural Abs initiate complement activation and injury following ischemia and reperfusion of certain tissues, but their role in post-ischemic lung injury is unknown. Here, we investigated the expression of two previously identified post-ischemic neoepitopes, namely a modified annexin IV epitope recognized by B4 IgM mAb, and an epitope consisting of a subset of phospholipids recognized by C2 IgM mAb, and additionally determined their role in lung transplant ischemia reperfusion injury (IRI). Methods Lungs from C57Bl/6 mice were orthotopically transplanted into Rag1-/- (antibody-deficient) mice reconstituted with either B4 and C2 mAbs. Results Rag1-/- mice were protected from lung IRI, but reconstitution with either B4 or C2 natural IgM mAbs restored lung IRI. The C2 mAb was more effective than the B4 mAb at restoring injury, as determined by histological and micro-CT analysis. Based on this information, we developed and characterized a therapeutic strategy that exploited the post-ischemia recognition system of natural antibodies. We constructed a single-chain antibody (scFv) derived from C2 mAb and linked it to Crry, an inhibitor of C3 activation (C2scFv-Crry). In models of syngeneic (B6-B6) and allogeneic (Balb/c-B6) lung transplant models in which recipients contain a full natural antibody repertoire, a single post-transplant injection of C2scFv-Crry blocked the binding of endogenous IgM to the graft, inhibited complement activation, and significantly reduced graft inflammation and injury as determined by histology, μCT, and immunhistochemical analysis. The severity of IRI is associated with the onset of acute rejection. Conclusion We identified pathophysiologically important epitopes expressed within the lung after lung ischemia and reperfusion post transplantation. We further describe a novel translatable strategy for complement inhibition that is specifically targeted to the transplanted lung, and that has several advantages over currently available approaches that rely on systemic complement inhibition. Significantly, the C2 epitope has been identified in both mouse and man, increasing the translational relevance of the approach.

Two to Tango: Co-evolution of Hominid Natural Killer Cell Receptors and MHC.

Natural killer (NK) cells have diverse roles in hominid immunity and reproduction. Modulating these functions are the interactions between major histocompatibility complex (MHC) class I molecules that are ligands for two NK cell surface receptor types. Diverse killer cell immunoglobulin-like receptors (KIR) bind specific motifs encoded within the polymorphic MHC class I cell surface glycoproteins, while, in more conserved interactions, CD94:NKG2A receptors recognize MHC-E with bound peptides derived from MHC class I leader sequences. The hominid lineage presents a choreographed co-evolution of KIR with their MHC class I ligands. MHC-A, -B, and -C are present in all great apes with species-specific haplotypic variation in gene content. The Bw4 epitope recognized by lineage II KIR is restricted to MHC-B but also present on some gorilla and human MHC-A. Common to great apes, but rare in humans, are MHC-B possessing a C1 epitope recognized by lineage III KIR. MHC-C arose from duplication of MHC-B and is fixed in all great apes except orangutan, where it exists on approximately 50% of haplotypes and all allotypes are C1-bearing. Recent study showed that gorillas possess yet another intermediate MHC organization compared to humans. Like orangutans, but unlike the Pan-Homo species, duplication of MHC-B occurred. However, MHC-C is fixed, and the MHC-C C2 epitope (absent in orangutans) emerges. The evolution of MHC-C drove expansion of its cognate lineage III KIR. Recently, position −21 of the MHC-B leader sequence has been shown to be critical in determining NK cell educational outcome. In humans, methionine (−21M) results in CD94:NKG2A-focused education whereas threonine (−21T) produces KIR-focused education. This is another dynamic position among hominids. Orangutans have exclusively −21M, consistent with their intermediate stage in lineage III KIR-focused evolution. Gorillas have both −21M and −21T, like humans, but they are unequally encoded by their duplicated B genes. Chimpanzees have near-fixed −21T, indicative of KIR-focused NK education. Harmonious with this observation, chimpanzee KIR exhibit strong binding and, compared to humans, smaller differences between binding levels of activating and inhibitory KIR. Consistent between these MHC-NK cell receptor systems over the course of hominid evolution is the evolution of polymorphism favoring the more novel and dynamic KIR system.

Discovery of gorilla MHC-C expressing C1 ligand for KIR

In comparison to humans and chimpanzees, gorillas show low diversity at MHC class I genes (Gogo), as reflected by an overall reduced level of allelic variation as well as the absence of a functionally important sequence motif that interacts with killer cell immunoglobulin-like receptors (KIR). Here, we use recently generated large-scale genomic sequence data for a reassessment of allelic diversity at Gogo-C, the gorilla orthologue of HLA-C. Through the combination of long-range amplifications and long-read sequencing technology, we obtained, among the 35 gorillas reanalyzed, three novel full-length genomic sequences including a coding region sequence that has not been previously described. The newly identified Gogo-C*03:01 allele has a divergent recombinant structure that sets it apart from other Gogo-C alleles. Domain-by-domain phylogenetic analysis shows that Gogo-C*03:01 has segments in common with Gogo-B*07, the additional B-like gene that is present on some gorilla MHC haplotypes. Identified in ~ 50% of the gorillas analyzed, the Gogo-C*03:01 allele exclusively encodes the C1 epitope among Gogo-C allotypes, indicating its important function in controlling natural killer cell (NK cell) responses via KIR. We further explored the hypothesis whether gorillas experienced a selective sweep which may have resulted in a general reduction of the gorilla MHC class I repertoire. Our results provide little support for a selective sweep but rather suggest that the overall low Gogo class I diversity can be best explained by drastic demographic changes gorillas experienced in the ancient and recent past.

OR15 Hierarchy of HLA types in provoking antibody production following kidney transplantation

Aim HLA antigens are diverse in cell surface expression, peptide presentation, binding immune receptors, and in eliciting immune responses. Here we examined the ability of distinct HLA types in inducing antibody (Ab) production following kidney transplantation (Ktx). Methods A cohort of 308 candidates (44% female) waiting for 2nd Ktx was studied. Prior to 1st Ktx, all were negative for HLA Abs by One Lambda single antigen bead (OL-SAB) assay. HLA Abs were measured in pre-2nd tx sera using OL-SAB. The strength (mean of MFIs) and frequency of each Abs in pre-2nd tx sera of 308 candidates were determined and plotted. Results A positive linear correlation was observed between Ab strengths to frequency with an r2 = 0.6 and p DQ8 > DQ7 > B76 > DR9 > DQ4 > B45 > DQ6 > B57 > DQ2 > A1 > DQ5 > DR7 > B57 > B58 > A24 > B49 > DR53 > A2 > B82 > B44 > A96 > A23 > A11 > A68. All HLA-Cw antigens and a subset of DPB antigens (DP15, DP13, DP5, DP1, DP23, DP4) induce Ab production at low levels and low frequency. Within Cw types, the most poorly immunogenic represents to be those carrying C1 epitope that binds to KIR2DL2/L3. The moderate inducer includes most HLA-B, HLA-A and DR antigens, and the hierarchy from the strongest inducer within this group is B7 > A10 > B5 > DR52 > DR51 CREG. The B5 CREG was diverse and observed to have four outliers from the linear fit. The strongest to weakest to trigger Abs in B5 CREG is ordered as follows: B76 > B63 > B73 > B62 > B75 > B78 > B46. Conclusions Our findings categorize 121 HLA types into three hierarchical sets based on their ability in eliciting Ab production following Ktx – strongly immunogenic, moderately immunogenic, and poorly immunogenic. The strongly immunogenic HLA types should be considered for matching in Ktx, while the poorly immunogenic HLA antigens are tolerable.

Missing HLA C group 1 ligand in patients with AML and MDS is associated with reduced risk of relapse and better survival after allogeneic stem cell transplantation with fludarabine and treosulfan reduced toxicity conditioning.

Reduced-toxicity conditioning with fludarabine and treosulfan is a dose-intensive regimen with enhanced anti-leukemia effect and acceptable toxicity in AML/MDS. HLA-C regulates natural-killer (NK) cell function by inhibiting Killer immunoglobulin-like receptors (KIR) and is divided into C1 and C2 epitopes. The missing-ligand theory suggests that missing recipient KIR ligands drives NK-alloreactivity after SCT, in the absence of HLA-mismatch by activating unlicensed donor NK cells. We analyzed SCT outcomes in 203 patients with AML/MDS, median age 58 years, given SCT from matched-siblings (n = 97) or matched-unrelated donors (n = 106), using two treosulfan doses (total 36 or 42 g/m2 ). 34% expressed one HLA-C group 1 allele (C1C1), 19% one HLA-C group 2 allele (C2C2), and 48% both KIR ligands (C1C2). Median follow-up was 48 months. 5-year relapse, nonrelapse mortality (NRM) and leukemia-free survival (LFS) rates were 38%, 27%, and 36%, respectively. Relapse rates were 43%, 45%, and 26% in patients expressing C1C1, C1C2, and C2C2 ligands, respectively (P = .03). Multivariate-analysis identified chemo-refractory disease (HR 3.1, P = .003), poor cytogenetics (HR 1.7, P = .08), female donor to male recipient (HR 0.4, P = .01) and C2C2 ligands (HR 0.4, P = .04) as independent factors predicting relapse. HLA-C ligands were not associated with GVHD or NRM. LFS was 33%, 30%, and 46%, respectively (P = .07). Chemorefractory disease (HR 3.1, P = .0004) and C2C2 group ligand (HR 0.6, P = .06) independently predicted LFS. Treosulfan dose did not predict any SCT outcome. In conclusion, missing HLA-C group 1 ligand is associated with reduced relapse risk, similar NRM and improved LFS, after HLA-matched SCT with treosulfan conditioning in AML/MDS.

Missing or altered self: human NK cell receptors that recognize HLA-C.

Natural killer (NK) cells are fast-acting and versatile lymphocytes that are critical effectors of innate immunity, adaptive immunity, and placental development. Controlling NK cell function are the interactions between killer-cell immunoglobulin-like receptors (KIRs) and their HLA-A, HLA-B and HLA-C ligands. Due to the extensive polymorphism of both KIR and HLA class I, these interactions are highly diversified and specific combinations correlate with protection or susceptibility to a range of infectious, autoimmune, and reproductive disorders. Evolutionary, genetic, and functional studies are consistent with the interactions between KIR and HLA-C being the dominant control mechanism of human NK cells. In addition to their recognition of the C1 and C2 epitopes, increasing evidence points to KIR having a previously unrecognized selectivity for the peptide presented by HLA-C. This selectivity appears to be a conserved feature of activating KIR and may partly explain the slow progress made in identifying their HLA class I ligands. The peptide selectivity of KIR allows NK cells to respond, not only to changes in the surface expression of HLA-C, but also to the more subtle changes in the HLA-C peptidome, such as occur during viral infection and malignant transformation. Here, we review recent advances in understanding of human-specific KIR evolution and how the inhibitory and activating HLA-C receptors allow NK cells to respond to healthy cells, diseased cells, and the semi-allogeneic cells of the fetus.

KIR2DS5 allotypes that recognize the C2 epitope of HLA-C are common among Africans and absent from Europeans.

IntroductionKIR2DS5 is an activating human NK cell receptor of lineage III KIR. These include both inhibitory KIR2DL1, 2 and 3 and activating KIR2DS1 that recognize either the C1 or C2 epitope of HLA‐C. In Europeans KIR2DS5 is essentially monomorphic, with KIR2DS5*002 being predominant. Pioneering investigations showed that KIR2DS5*002 has activating potential, but cannot recognize HLA‐A, ‐B, or ‐C. Subsequent studies have shown that KIR2DS5 is highly polymorphic in Africans, and that KIR2DS5*006 protects pregnant Ugandan women from preeclampsia. Because inhibitory C2‐specific KIR2DL1 correlates with preeclampsia, whereas activating C2‐specific KIR2DS1 protects, this association pointed to KIR2DS5*006 being an activating C2‐specific receptor. To test this hypothesis we made KIR‐Fc fusion proteins from all ten KIR2DS5 allotypes and tested their binding to a representative set of HLA‐A, ‐B and ‐C allotypes.ResultsSix African‐specific KIR2DS5 bound to C2+HLA‐C but not to other HLA class I. Their avidity for C2 is ∼20% that of C2‐specific KIR2DL1 and ∼40% that of C2‐specific KIR2DS1. Among the African C2 receptors is KIR2DS5*006, which protected a cohort of pregnant Ugandans from pre‐eclampsia. Three African KIR2DS5 allotypes and KIR2DS5*002, bound no HLA‐A, ‐B or ‐C. As a group the C2‐binding KIR2DS5 allotypes protect against pre‐eclampsia compared to the non‐binding KIR2DS5 allotypes. Natural substitutions that contribute to loss or reduction of C2 receptor function are at positions 127, 158, and 176 in the D2 domain.Conclusions KIR2DS5*005 has the KIR2DS5 consensus sequence, is the only allele found at both centromeric and telomeric locations of KIR2DS5, and is likely the common ancestor of all KIR2DS5 alleles. That KIR2DS5*005 has C2 receptor activity, points to KIR2DS5*002, and other allotypes lacking C2 receptor function, being products of attenuation, a characteristic feature of most KIR B haplotype genes. Alleles encoding attenuated and active KIR2DS5 are present in both centromeric and telomeric locations.

Anti-citrullinated protein antibodies cause arthritis by cross-reactivity to joint cartilage.

Today, it is known that autoimmune diseases start a long time before clinical symptoms appear. Anti-citrullinated protein antibodies (ACPAs) appear many years before the clinical onset of rheumatoid arthritis (RA). However, it is still unclear if and how ACPAs are arthritogenic. To better understand the molecular basis of pathogenicity of ACPAs, we investigated autoantibodies reactive against the C1 epitope of collagen type II (CII) and its citrullinated variants. We found that these antibodies are commonly occurring in RA. A mAb (ACC1) against citrullinated C1 was found to cross-react with several noncitrullinated epitopes on native CII, causing proteoglycan depletion of cartilage and severe arthritis in mice. Structural studies by X-ray crystallography showed that such recognition is governed by a shared structural motif "RG-TG" within all the epitopes, including electrostatic potential-controlled citrulline specificity. Overall, we have demonstrated a molecular mechanism that explains how ACPAs trigger arthritis.

The Intergenic Recombinant HLA-B∗46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands

SummaryHLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

Two Orangutan Species Have Evolved Different KIR Alleles and Haplotypes.

The immune and reproductive functions of human NK cells are regulated by interactions of the C1 and C2 epitopes of HLA-C with C1-specific and C2-specific lineage III killer cell Ig-like receptors (KIR). This rapidly evolving and diverse system of ligands and receptors is restricted to humans and great apes. In this context, the orangutan has particular relevance because it represents an evolutionary intermediate, one having the C1 epitope and corresponding KIR but lacking the C2 epitope. Through a combination of direct sequencing, KIR genotyping, and data mining from the Great Ape Genome Project, we characterized the KIR alleles and haplotypes for panels of 10 Bornean orangutans and 19 Sumatran orangutans. The orangutan KIR haplotypes have between 5 and 10 KIR genes. The seven orangutan lineage III KIR genes all locate to the centromeric region of the KIR locus, whereas their human counterparts also populate the telomeric region. One lineage III KIR gene is Bornean specific, one is Sumatran specific, and five are shared. Of 12 KIR gene-content haplotypes, 5 are Bornean specific, 5 are Sumatran specific, and 2 are shared. The haplotypes have different combinations of genes encoding activating and inhibitory C1 receptors that can be of higher or lower affinity. All haplotypes encode an inhibitory C1 receptor, but only some haplotypes encode an activating C1 receptor. Of 130 KIR alleles, 55 are Bornean specific, 65 are Sumatran specific, and 10 are shared.

Activating KIR2DS4 Is Expressed by Uterine NK Cells and Contributes to Successful Pregnancy.

Tissue-specific NK cells are abundant in the pregnant uterus and interact with invading placental trophoblast cells that transform the maternal arteries to increase the fetoplacental blood supply. Genetic case-control studies have implicated killer cell Ig-like receptor (KIR) genes and their HLA ligands in pregnancy disorders characterized by failure of trophoblast arterial transformation. Activating KIR2DS1 or KIR2DS5 (when located in the centromeric region as in Africans) lower the risk of disorders when there is a fetal HLA-C allele carrying a C2 epitope. In this study, we investigated another activating KIR, KIR2DS4, and provide genetic evidence for a similar effect when carried with KIR2DS1. KIR2DS4 is expressed by ∼45% of uterine NK (uNK) cells. Similarly to KIR2DS1, triggering of KIR2DS4 on uNK cells led to secretion of GM-CSF and other chemokines, known to promote placental trophoblast invasion. Additionally, XCL1 and CCL1, identified in a screen of 120 different cytokines, were consistently secreted upon activation of KIR2DS4 on uNK cells. Inhibitory KIR2DL5A, carried in linkage disequilibrium with KIR2DS1, is expressed by peripheral blood NK cells but not by uNK cells, highlighting the unique phenotype of uNK cells compared with peripheral blood NK cells. That KIR2DS4, KIR2DS1, and some alleles of KIR2DS5 contribute to successful pregnancy suggests that activation of uNK cells by KIR binding to HLA-C is a generic mechanism promoting trophoblast invasion into the decidua.

P095 HLA class I variation in Iranian Lur and Kurd populations: High allele and haplotype diversity with an abundance of KIR ligands

HLA-A, -B and -C alleles of 285 individuals, representing three Iranian Lur populations and one Iranian Kurd population were sequenced completely, yielding HLA class I genotypes at high resolution and filling four fields of the official HLA nomenclature. Each population has 87–99 alleles, evenly distributed between the three HLA class I genes, 145 alleles being identified in total. These alleles were already known, named and deposited in the HLA database. The alleles form 316 different HLA A-B-C haplotypes, with each population having between 80 and 112 haplotypes. The four Iranian populations form a related group that is distinguished from other populations, including other Iranians. All four KIR ligands – the A3/11, Bw4, C1 and C2 epitopes – are well represented, particularly Bw4, which is carried by three high-frequency allotypes: HLA-A∗24:02, HLA-A∗32:01 and HLA-B∗51:01. In the Lur and Kurd populations, between 82% and 94% of individuals have the Bw4 epitope, the ligand for KIR3DL1. HLA-B∗51:01 is of Neandertal origin and associated with Behcet’s disease, also known as the Silk Road disease. The Lordegan Lur have the highest frequency of HLA-B∗51:01 in the world. This allele is present on 46 Lur and Kurd haplotypes. Present at lower frequency is HLA-B∗51:08, which is also associated with Behcet’s disease. In the four Iranian populations, 31 haplotypes encode both Bw4+HLA-A and Bw4+HLA-B, a dual combination of Bw4 epitopes that is relatively rare in other populations, worldwide. This study both demonstrates and emphasizes the value of studying HLA class I polymorphism at highest resolution in anthropologically well-defined populations.

OR3 The strength of positive linear correlations between donor-specific HLA antibodies and flow-cytometry crossmatch is locus-dependent

To determine correlation between the mean fluorescence intensity (MFI) of donor specific antibodies (DSA) and the median channel shift (MCS) of the flow crossmatch (FXM). HLA antibodies (Ab) in DTT-treated sera were analyzed using One Lambda (OL) single antigen beads (SAB). T cell FXM (51 pronase & 120 untreated) with single class I DSA and B cell FXM (41 pronase & 101 untreated) with single class II DSA were analyzed. MCS values were plotted against the MFI of the DSA and Pearson’s correlations (R2 were assessed. Positive correlations between T-FXM and DSA to HLA-A (R2 = 0.86) or HLA-B (R2 = 0.83) were strong, but low for Cw DSA (R2 = 0.28) [Figure]. B-FXM and DSA to DR (R2 = 0.54) and DQ (R2 = 0.55) had medium degree of +ve correlations while DP (R2 = 0.48) correlation was low. Pronase enhanced MCS but did not improve XM-DSA correlations. DSA with MFI < 2K to a single HLA-A/B or to a single DR/DQ did not cause +ve T or B FXM respectively. For Cw, +ve T-FXM was not observed when DSA was <8K MFI. Similarly, +ve B-FXM was not observed for DP when DSA MFI was <10K. XM reactivity of Cw Ab can be serum dependent. Pronase T-FXM between 3 Cw4+ donors and 2 sera with comparable Ab profiles (Ab to KIR2DL1-binding C2 epitope) on both OL and Immucor SAB panels produced conflicting results. Serum 1 (Cw4 MFI = 8081) was +ve with all donors (MCS = 78, 109, 155) while serum 2 (Cw4 MFI=9610) yielded all T-FXM -ve (MCS ⩽ 10). Sera can be false +ve with OL Cw beads, i.e., −ve with Immucor SAB and −ve T-FXM. Some displayed 10–20K MFI reactivity to certain Cw beads (Cw1, 12, 15) and others reacted to all Cw beads except to self. Continuing correlation studies between DSA and XM are critical for the precise virtual XM prediction. Standalone Cw Ab require additional scrutiny including screening by another reagent and T-FXM. Compared to HLA-A/B/DR/DQ DSAs, 4 or 5 times more Ab are required to yield +ve XM by Cw and DP DSAs respectively, and thus elevating MFI cutoff for Cw and DP unacceptable antigen listing will draw an enhanced donor offer for broadly sensitized patients.Download : Download high-res image (262KB)Download : Download full-size image