Identification of T-Cell Epitopes on the Rhesus Polypeptides in Autoimmune Hemolytic Anemia

Abstract

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.

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat‐shock protein 60

“…By contrast, the proliferative response of PBMC from many RA patients to hsp 60 peaked earlier than those from normal individuals. Since the peak proliferative response of human peripheral blood T cells to recall antigens is earlier than that to non‐recall antigens 24,25,29 this result suggests that PBMC from some RA patients are primed against hsp 60. It is envisaged that this priming to hsp 60 in vivo occurs during the disease process where increased expression of hsp 60 in the inflamed joint leads to antigen processing and presentation of normally cryptic hsp 60 epitopes to the immune system.…”

Characterization of the alloreactive helper T‐cell response to the platelet membrane glycoprotein IIIa (integrin‐β3) in human platelet antigen‐1a alloimmunized human platelet antigen‐1b1b women

“…Third, the culture conditions were designed to minimize the primary responses to allopeptides that may be seen in unimmunized donors. As previously validated, 20,26,28 this was achieved by performing the proliferation assays in microtiter plates over a short time course, conditions designed to favor recall proliferation by primed T cells, rather than primary responses of naïve T cells. And finally, the responding T cells exhibited helper function as well as phenotype, because anti‐HPA‐1a was generated after prolonged culture of PBMNC‐containing T cells that had proliferated after stimulation with the immunodominant peptides.…”

“…Two panels of 15‐mer peptides were synthesized corresponding to the sequences of the HPA‐1 polymorphic region, with either the Leu 33 or Pro 33 at each possible position (Department of Biochemistry, University of Bristol, UK; Table 1). To ensure purity, each panel was synthesized by F‐moc chemistry as reported previously 26 . The peptides were reconstituted to a concentration of 2.0 mg per mL and used for stimulation of PBMNCs at 20 µg per mL in culture.…”

“…As previously described, 20,26 assays were carried out in culture conditions designed to favor proliferation by previously activated T cells rather than primary responses. PBMNCs were cultured at a concentration of 1.25 × 10 6 cells per mL in alpha modification of Eagle's medium (Sigma Diagnostics) supplemented with 5 percent AB serum.…”

“…Results were presented either as the mean counts per minute ± SD of the triplicate samples or as a stimulation index (SI) expressing the ratio of mean counts per minute in stimulated versus unstimulated control cultures. An SI of greater than 3 was interpreted as representing a positive response 26 …”