Identification, immunomodulatory activity, and immunogenicity of the major helper T-cell epitope on the K blood group antigen.

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.

Combination peptide immunotherapy suppresses antibody and helper T-cell responses to the RhD protein in HLA-transgenic mice.

The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (P=0.008, Mann-Whitney rank sum test) or subcutaneously (P=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.

Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance.

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.

Different forms of helper tolerance to carcinoembryonic antigen: ignorance and regulation.

PURPOSE: Understanding the mechanisms of immune tolerance to tumor-associated antigens (TAA) is an important step in the design of cancer immunotherapy. The aim was to determine how T helper (Th) cell tolerance is mediated for a prototypic TAA, carcinoembryonic antigen (CEA). EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells from 50 healthy volunteers were stimulated with CEA, and the type and fine specificity of any Th cell responses were identified. The inhibitory effects of T regulatory (Tr) populations were determined by depleting "natural" CD25(+) Tr cells or neutralizing cytokine produced by the "induced" Tr form. RESULTS: Proliferative Th cell responses were consistently induced by CEA in 22 of 50 individuals. Responding cells were drawn from the CD45RA(+) "naive" or quiescent population. Depleting the CD25(+) fraction did not enhance CEA responsiveness. However, CEA elicited secretion of the Tr cytokine interleukin-10 (IL-10) in 23 of 50 donors, including 20 of 22 where no proliferation was induced. Neutralizing IL-10 revealed previously unseen proliferation to CEA by CD45RO(+) "memory" Th cells. Epitope maps revealed differences in the fine specificities of Th cells capable of proliferating or secreting IL-10. CONCLUSIONS: There are at least two major forms of CEA tolerance in different individuals. One is "ignorance," a failure of specific Th cells to respond to antigen presented in vivo. The other, seen when ignorance is lost, is mediated by IL-10-secreting Tr cells that recognize CEA. TAA tolerance, for example to colorectal carcinoma cells expressing CEA, may be overcome by peptide vaccines that exploit the differences in epitopes recognized by effector and Tr responses.