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Heterozygosity for Hemoglobin (Hb) S, sickle cell trait (SCT), affects over 40 million people and confers resistance to severe infection by Plasmodium falciparum. Homozygosity for HbS, or compound heterozygosity with certain other alleles of Hb, affects over 4 million individuals and causes sickle cell disease (SCD). Hemolytic anaemia is a prominent feature of SCD and is mainly extravascular, mediated by hepatic and splenic macrophages. No ligands for this process have been identified. As many macrophage phagocytic receptors recognise carbohydrates, we surveyed surface glycan expression by sickle cells using a panel of 8 lectins and flow cytometry. Most glycans were similar to those of healthy red blood cells (RBC), except much higher expression of terminal mannose. We investigated the structural basis for these residues using glycomic mass spectroscopy, which showed them to be N-linked high (Man5-9GlcNAc2) mannoses, a surprising conclusion as these are usually intermediates in the formation of complex glycans and not displayed on cell surfaces. High resolution microscopy revealed the mannose residues to be carried in discrete microdomains on the surfaces of sickle cells. These structures were absent on the surfaces of healthy RBC, instead being present in the membrane skeleton under the cell surface. Lectin blots and immunoprecipitation showed the mannoses to co-migrate predominantly with spectrin. We showed these mannose-bearing structures were able to stimulate phagocytosis of RBC by using a peripheral blood derived macrophage uptake assay. Sickle RBC were taken up at high rates compared to healthy RBC and this could be inhibited by congeners of mannose. We identified the importance of a cognate ligand (CD206: the mannose receptor) using blocking antibodies and knockdown of CD206 expression using siRNA. The in vivo and pathogenic relevance of mannose exposure was investigated by taking advantage of the heterogeneity of hemolysis in SCD. RBC with SCD (n=94), SCT (n=57) and healthy individuals (n=54) were assayed for mannose exposure by flow cytometry. SCT and healthy RBC showed no mannose exposure but high levels were found on HbSS RBC (p Identification of a ligand pair mediating rapid clearance of sickle cells raised the possibility that they also mediate enhanced clearance of SCT RBC infected by malarial parasites. Indeed, P. falciparum cultures induced mannose expression at the pigmented trophozoite and schizont stages in infected HbAA RBCs, at levels corresponding to mild hemolysis in SCD. Mannose expression in infected HbAS RBCs was even higher, with levels corresponding to severe hemolysis in SCD. Infection with P. falciparum and selection for HbS arose only recently in human evolution, raising the question of what the physiological triggers for this mechanism are. Infection with malarial parasites causes oxidative stress. We therefore subjected healthy RBC to copper sulphate, which resulted in surface mannose exposure as well as uptake by macrophages. Oxidized SCT RBC displayed more mannose than oxidized healthy RBC. Thus, we have identified a new cell surface 'eat me' signalling mechanism that allows inspecting macrophages to engage with the rigid membrane skeleton and phagocytose the mannose displaying cell. The mechanism is stimulated by HbS: when present in high concentrations, the mechanism is activated constitutively, resulting in sickle cell anaemia. Heterozygosity for HbS is insufficient by itself to trigger mannose exposure. However, the mechanism is primed so that oxidative stress associated with infection by P. falciparum causes greater mannose display, increased parasitized cell clearance and protection against severe malaria. These findings should allow the design of inhibitors of sickle cell haemolysis and inducers of protection against malaria. Disclosures Cao: University of Aberdeen: Patents & Royalties. Barker:University of Aberdeen: Patents & Royalties. Vickers:University of Aberdeen: Patents & Royalties; GSK: Equity Ownership.

The K blood group remains an important target in hemolytic disease of the newborn (HDN), with no immune prophylaxis available. The aim was to characterize the Th response to K as a key step in designing specific immunotherapy and understanding the immunogenicity of the Ag. PBMCs from K-negative women who had anti-K Abs after incompatible pregnancy, and PBMCs from unimmunized controls, were screened for proliferative responses to peptide panels spanning the K or k single amino acid polymorphism. A dominant K peptide with the polymorphism at the C terminus elicited proliferation in 90% of alloimmunized women, and it was confirmed that responding cells expressed helper CD3+CD4+ and “memory” CD45RO+ phenotypes, and were MHC class II restricted. A relatively high prevalence of background peptide responses independent of alloimmunization may contribute to K immunogenicity. First, cross-reactive environmental Ag(s) pre-prime Kell-reactive Th cells, and, second, the K substitution disrupts an N-glycosylation motif, allowing the exposed amino acid chain to stimulate a Th repertoire that is unconstrained by self-tolerance in K-negative individuals. The dominant K peptide was effective in inducing linked suppression in HLA-transgenic mice and can now be taken forward for immunotherapy to prevent HDN because of anti-K responses.

The disposal of unwanted or dying cells is a key biological process driven by the display of 'eat me' signals that are recognised by phagocytes. Although these markers for uptake are known to include certain lipids and proteins, the role of glycans, which are abundant on cell surfaces, remains poorly characterized. Here, an unbiased glycomic survey by mass spectroscopic analyses of glycosidase (PNGase F) digested human red blood cell (RBC) membranes identified novel N-linked high mannose structures, which are sequestered inside healthy cells with spectrin, the major protein of the internal membrane skeleton, but exteriorised when cells are damaged as a dominant signal for uptake by macrophages. A panel of lectin probes was used to demonstrate that the mannose species were available as discrete patches on the surface of RBC that had been stressed by oxidation, but undetectable on unpermeabilized untreated cells. High mannoses co-localized with alpha-spectrin on lectin/Western blots and this signal was degraded by prior incubation with glycosidases. Super resolution microscopy showed co-localisation of mannose with spectrin in membrane protrusions of oxidized RBC. Co-localization of spectrin with N-linked mannose and exteriorisation on effete cells were also observed in nucleated cells. The mannose displayed on oxidized RBC represents a novel 'eat-me' signal for human cells, since congeners of mannose inhibited uptake by human monocyte-derived macrophages. The mannose receptor (CD206) was identified as an important receptor in this system by the use of blocking antibody and siRNA mediated knock-down. The mechanisms underlying the hemolysis characteristic of sickle cell disease (SCD) remain to be fully defined. We therefore investigated the contribution of this new pathway. RBC from patients with SCD demonstrated remarkably high levels of surface mannose, and super resolution microscopy showed the expression again to be limited to discrete patches and co-localized with spectrin in a disrupted membrane skeletal structure. The importance of mannose exposure to uptake of sickle cells by macrophages was confirmed by inhibition by congeners of mannose and blocking antibody to CD206. The prevalence of SCD is high because of selective pressure caused by the resistance of subjects with sickle cell trait to malarial parasites, which are mainly cleared by splenic macrophages. We therefore investigated whether RBC from subjects with sickle cell trait expressed surface mannose. We show that such RBC express modestly raised levels (p We also investigated whether the degree of mannose exposure correlated with peripheral blood count parameters. The Pearson correlation coefficients of log transformed surface mannose expression with hemoglobin levels, red cell counts and reticulocyte counts were -0.83, -0.84 and 0.73 respectively (all p values In summary, our results reveal a previously undescribed cryptic mannose exteriorization pathway, which mediates disposal of oxidatively damaged cells. The pathway is constitutively activated in SCD, where it mediates haemolysis and the degree of activation correlates well with clinical phenotypes of SCD and sickle cell trait. It thus represents a new mechanism for possible future therapeutic intervention. Disclosures Vickers: University of Aberdeen: Patents & Royalties: About to apply for patent. Barker: University of Aberdeen: Employment, Patents & Royalties: About to apply for patent. Cao: University of Aberdeen: Patents & Royalties: About to apply for patent.

Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune diseases directed against constituents of the blood, including hemolytic anemia (AIHA). We hypothesized that CLL cells predispose to hematologic autoimmunity by acting as aberrant antigen-presenting cells (APCs). Initially, it was confirmed that all studied patients with AIHA secondary to CLL harbored activated helper T (TH) cells specific for epitopes on the dominant red blood cell (RBC) autoantigens in primary AIHA, the Rh proteins. Rh-specific TH cells were also detected in a number of patients with CLL who, although they did not have AIHA, had low levels of anti-RBC antibody in their sera. Fractionation of putative APC populations from the peripheral blood of patients by negative selection showed that CD5+ CLL cells are the most effective cell type in processing and presenting purified Rh protein to autoreactive TH cells. This ability was confirmed using positively selected CD5+ CLL cells. Thus, our study provides the first evidence for malignant cells driving an autoimmune response by acting as aberrant APCs.

Regulatory T cells have been shown to control animal models of immune-mediated pathology by inhibitory cytokine production, but little is known about such cells in human disease. Here we characterize regulatory T-cell responses specific for a human red blood cell autoantigen in patients with warm-type autoimmune hemolytic anemia. Peripheral blood mononuclear cells from patients with autoimmune hemolytic anemia were found either to proliferate and produce interferon-γ or to secrete the regulatory cytokine interleukin 10 when stimulated in vitro with a major red blood cell autoantigen, the RhD protein. Flow cytometric analysis confirmed that the majority of the responding cells were of the CD4+phenotype. Serial results from individual patients demonstrated that this bias toward proliferative or interleukin-10 responses was unstable over time and could reverse in subsequent samples. Epitope mapping studies identified peptides from the sequence of the autoantigen that preferentially induced interleukin-10 production, rather than proliferation, and demonstrated that many contain naturally processed epitopes. Responses to such peptides suppressed T-cell proliferation against the RhD protein, an inhibition that was mediated largely by interleukin 10 and dependent on cytotonic T lymphocyte–associated antigen (CTLA-4) costimulation. Antigenic peptides with the ability to stimulate specific regulatory cells may represent a new class of therapeutic agents for immune-mediated disease.

Although considerable effort has been devoted to characterizing alloantibodies specific for the Rhesus D (RhD) blood group antigen, virtually nothing is known about the helper response that drives their production. Therefore, the aim of this study was to map alloreactive T-cell epitopes on the RhD protein. Peripheral blood mononuclear cells (PBMCs) were obtained from 22 RhD-negative volunteers in whom anti-D alloantibodies had developed after deliberate immunization or RhD-incompatible pregnancy. The PBMCs were stimulated with a panel of up to 68 overlapping synthetic 15-mer peptides spanning the complete sequence of the RhD protein. One or more peptides elicited proliferative responses by PBMCs from all 22 of the alloimmune volunteers but from only 2 of 8 alloantibody-negative control donors. Proliferation of PBMCs from the alloimmune donors was mediated by major histocompatibility complex class II–restricted T cells expressing the CD45RO marker of previous activation or memory. The number of peptides that induced proliferative responses was unrelated to either the frequency of, or time since, exposure to RhD-positive red blood cells, but it correlated strongly (Rs = 0.75;P

We have shown previously that the Rhesus (Rh) polypeptides are the commonest targets for pathogenic anti-red blood cell (RBC) autoantibodies in patients with autoimmune hemolytic anemia (AIHA). The aim of the current work was to determine whether activated T cells from such patients also mount recall responses to epitopes on these proteins. Two panels of overlapping 15-mer peptides, corresponding to the sequences of the 30-kD Rh proteins associated with expression of the D and Cc/Ee blood group antigens, were synthesized and screened for the ability to stimulate the in vitro proliferation of mononuclear cells from the peripheral blood or spleen of nine AIHA cases. Culture conditions were chosen that favor recall proliferation by previously activated T cells, rather than primary responses. In seven of the patients, including all four cases with autoantibody to the Rh proteins, two or more peptides elicited proliferation, but cells from eight of nine patients with other anemias and seven of nine healthy donors failed to respond to the panels. Multiple peptides were also stimulatory in two positive control donors who had been alloimmunized with Rh D-positive RBCs. Six different profiles of peptides elicited responses in the AIHA patients, and this variation may reflect the different HLA types in the group. Stimulatory peptides were identified throughout domains shared between, or specific to, each of the related 30-kD Rh proteins, but T cells that responded to nonconserved regions did not cross-react with the alternative sequences. Anti-major histocompatibility complex class II antibodies blocked the responses and depletion experiments confirmed that the proliferating mononuclear cells were T cells. Notably, splenic T cells that proliferated against multiple Rh peptides also responded to intact RBCs. We propose that pathogenic autoantibody production in many cases of AIHA is driven by the activation of T-helper cells specific for previously cryptic epitopes on the Rh proteins.

Background: The Kell blood group system contains over 25 antigens, with the K1 antigen being recognised as the most clinically important antigen after the ABO and RhD. K1 and its antithetical antigen K2 differ by a polymorphism at aa193, resulting in the substitution of Met for Thr in the K2 antigen. Our focus is on the role of helper T (Th) cells in the alloresponse to K1, which, occurs as a result of antigen mismatch during transfusion or pregnancy. In particular, the aim was to map the helper epitopes spanning the K1 polymorphism and to characterise the responding Th cells. Methods: To map the T-helper cell epitopes responsible for driving the alloresponse K1, peripheral blood mononuclear cells (PBMC) were isolated from K1 negative donors who had anti-K1 alloantibodies and stimulated with panels of overlapping 15-mer peptides, “walking” one amino acid at a time, down the sequences containing either the K1 Met, or K2 Thr polymorphism. The peptides were screened for the ability to stimulate recall proliferative responses and Th cytokine production, and the responding cells phenotyped by flow cytometric analysis. Results: PBMC from over 90% of alloimmune donors tested exhibited significant proliferation in response to the panel of K1 Met peptides, particularly those with the polymorphism at or near the C-terminus. Most notably, K1 peptide 1m(179–193), was found to induce significant proliferative responses from over 80% of K1 alloimmunised donors tested; proliferation was not associated with any particular HLA Class II type. There was no clear helper subset bias in the responses to the stimulatory peptides, with the balance between production of the respective Th1 and Th2 cytokines interferon-γ and interleukin-4 varying between donors. Flow cytometry confirmed that the proliferating cells were of the helper CD3+CD4+ phenotype and depletion experiments demonstrated that they were derived from the previously activated “memory” CD45RO+ population. Interestingly, a high background of proliferative responses was observed in response to the control K2 peptides by Th cells from both alloimmunised and unimmunised control donors. In vivo the K2 antigen is N-glycosylated at aa position 191, whereas the synthetic K2 peptide panel tested in vitro were not, raising the possibility that the presence or absence of the sugar residues affects Th cell recognition. The K1 substitution disrupts the glycosylation motif, and so such an effect would be relevant only for the K2 sequences. To test the possible role of glycosylation in Th responses to K2 peptides, PBMC were stimulated with either the original un-glycosylated version of peptide 1t (K2(179–193)), or a glycosylated version of the same peptide (WRISGKWTSLNF-Asn(AcNH-b-Glc)-RT-amide). Glycosylation resulted in abrogation of the Th cell proliferative responses previously observed to the original peptide 1t (K2(179–193)). Conclusion: A dominant alloreactive Th cell epitope has been mapped to K1 peptide 1m (K1(179–193)). Sugar residues attached to the gylosylation motif formed by the K2 substitution are important for Th recognition, and thus for self-tolerance to the corresponding peptides. Steric hindrance, preventing T cell receptor contact with the relevant amino acid residues, is the most likely mechanism, and, in the absence of sugar moieties on synthetic K2 peptides, these are effectively recognised as “foreign” by a Th repertoire that has been educated in the context of exposure to the gylosylated versions in vivo. We speculate that the immunogenicity of K1 may be attributed, at least in part, to the failure of peptides derived from the glycosylated K2 sequence to be recognised by Th cells during the establishment of self-tolerance in vivo, thereby leading to the survival of a larger pool of Th cells responsive to epitopes spanning the amino acid backbone at the polymorphic site. The identified dominant epitope is a candidate to be taken forward for inducing therapeutic tolerance, to prevent or switch off anti-K1 responses.

Regulatory T (Treg) cells contribute to immune evasion by malignancies. To investigate their importance in non-Hodgkin’s lymphoma (NHL), we enumerated Treg cells in peripheral blood mononuclear cells (PBMC) and involved tissues from 30 newly diagnosed patients. CD25+FoxP3+CD127lowCD4+ Treg cells were increased markedly in PBMC (median=20.4% CD4 T cells, n=20) versus healthy controls (median=3.2%, n=13; p