Minor histocompatibility antigen DBY elicits a coordinated B and T cell response after allogeneic stem cell transplantation

Abstract

We examined the immune response to DBY, a model H-Y minor histocompatibility antigen (mHA) in a male patient with chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplant from a human histocompatibility leukocyte antigen (HLA)-identical female sibling. Patient peripheral blood mononuclear cells were screened for reactivity against a panel of 93 peptides representing the entire amino acid sequence of DBY. This epitope screen revealed a high frequency CD4(+) T cell response to a single DBY peptide that persisted from 8 to 21 mo after transplant. A CD4(+) T cell clone displaying the same reactivity was established from posttransplant patient cells and used to characterize the T cell epitope as a 19-mer peptide starting at position 30 in the DBY sequence and restricted by HLA-DRB1*1501. Remarkably, the corresponding X homologue peptide was also recognized by donor T cells. Moreover, the T cell clone responded equally to mature HLA-DRB1*1501 male and female dendritic cells, indicating that both DBY and DBX peptides were endogenously processed. After transplant, the patient also developed antibodies that were specific for recombinant DBY protein and did not react with DBX. This antibody response was mapped to two DBY peptides beginning at positions 118 and 536. Corresponding DBX peptides were not recognized. These studies provide the first demonstration of a coordinated B and T cell immune response to an H-Y antigen after allogeneic transplant. The specificity for recipient male cells was mediated by the B cell response and not by donor T cells. This dual DBX/DBY antigen is the first mHA to be identified in the context of chronic GVHD.

Figure 1.

DBY elicits a sustained T cell response after allogeneic HSCT. (A) IFN-γ secretion ELISPOT assay was performed using unstimulated PBMCs collected 15 mo after HSCT and 93 overlapping DBY peptides distributed into 12 pools. Results are presented as number of spot-forming cells/10⁶ PBMCs. (B) The eight peptides included in pool-1 were tested individually using the same cells and according to the same procedure. (C) Patient PBMCs collected at various times before and after HSCT were tested by ELISPOT assay for reactivity to the DBY antigenic peptide. Time 0 corresponds to a sample collected before HSCT.

Figure 2.

The CD4⁺ T cell response to DBY antigen is restricted by HLA-DRB1*1501 molecules. (A) CD4⁺ T cell clone, 2F9, was stimulated with autologous EBV-immortalized B cells pulsed with DBY peptide (DBY_30–48) or another irrelevant DBY peptide. Anti–HLA class II mAb (9.49) and anti–HLA-DR mAb (G46.6) blocked antigen recognition, whereas anti–HLA class I (W6/32) or anti–HLA-DQ (SPVL.3) mAb did not. Results represent quantitative measurement of IFN-γ secretion in the coculture supernatant obtained by ELISA. (B) Clone 2F9 was stimulated with different DBY_30–48–pulsed EBV B cells (LCL) sharing the expression of either HLA-DRB1*0301 or HLA-DB1*1501 with the patient cells. After overnight incubation, IFN-γ secretion was measured in the coculture supernatant.

Figure 3.

DBX and DBY epitopes are both recognized by patient T cells. (A) 2F9 T cells were stimulated with autologous EBV B cells pulsed with different concentrations of DBX and DBY peptides. IFN-γ secretion was measured in the coculture supernatant after overnight incubation. The optimal 19-mer DBX and DBY peptides are shown in • and ○, respectively. Shorter, 14-mer suboptimal epitopes are also shown (▪ and □). Disparate residues between DBX and DBY versions of the peptides are represented with bold underlined characters. Peptide concentrations are expressed in microgram/milliliter. (B) IFN-γ secretion ELISPOT assays were performed using unstimulated PBMCs collected 8 and 21 mo after HSCT and both DBX_30–48 and DBY_30–48 peptides. (C) Clone 2F9 cytolytic activity was tested against EBV B cells pulsed with either DBX_30–48 (▪), DBY_30–48 (•), or an irrelevant peptide, DBY_536–552 (▴).

Figure 4.

T cell clone 2F9 recognizes DBX and DBY antigens endogenously processed and presented by mature DCs. (A) Female and male HLA-DRB1*1501 DCs (10⁴/well) were incubated for 24 h with medium alone or poly-(I)-poly-(C) to induce maturation. T cell clone 2F9 (10⁴/well) was then added to the wells in the presence of IL-2. After 18 h of incubation, secretion of IFN-γ was determined in cocultures using ELISA. (B) Clone 2F9 cytolytic activity toward DBY_30–48–pulsed ⁵¹Cr-labeled EBV B cells (LCL) at an E/T ratio of 1:1 was inhibited by the addition of mature unlabeled HLA-DRB1*1501 female or male DCs. Three ratios of unlabeled/labeled targets were tested in this experiment.

Figure 5.

DBY antigen elicits an antibody response after HSCT. IgG antibodies to recombinant DBY protein were detected by ELISA in patient serum samples collected at various times after allogeneic HSCT. Results are presented as serum titrations. Nonspecific reactivity to a control recombinant protein (HIV p24) was subtracted from each value.

Figure 6.

The antibody response after HSCT is specific for DBY antigen. DBX and DBY recombinant proteins were separated by SDS-PAGE electrophoresis, transferred on nitrocellulose membrane, and immunoblotted with either an anti-V5 mAb (left) or patient serum collected 21 mo after HSCT (right). Degradation products generating distinct patterns for DBX and DBY are noticeable in the figure.

Figure 7.

The antibody response to DBY continues to evolve 21 mo after HSCT. (A) Patient serum collected 16 mo after transplant was used to map the DBY-specific antibody response. The serum was diluted at 1:10 and tested by ELISA for reactivity with each of the 93 overlapping DBY peptides. (B) The same experiment was repeated using identical procedures with serum collected 21 mo after HSCT.

Figure 8.

Antibody responses after HSCT are specific for DBY antigen. Patient serum collected 21 mo after HSCT was tested in ELISA for reactivity with two DBY B cell epitopes, the corresponding DBX peptides, and both DBY and DBX T cell epitopes. Peptide sequences are shown with the X-Y–disparate residues noted with bold underlined characters.