Diverse patterns of T-cell response against multiple newly identified human Y chromosome-encoded minor histocompatibility epitopes

Abstract

Purpose: Donor T cells respond to minor histocompatibility antigens (mHA), resulting in both graft-versus-host disease and graft versus leukemia after allogeneic hematopoietic stem cell transplantation. Because relatively few mHAs are known, we developed a new approach to predict and subsequently validate candidate mHA.

Experimental design: We developed an algorithm based on genetic disparities between Y chromosome-encoded and X chromosome-encoded proteins and known requirements for binding to HLA class I molecules to predict Y chromosome-derived, HLA A*0201-restricted peptides (HY) and ranked peptides based on potential immunogenicity. We evaluated T-cell responses to 41 candidate peptides in 28 male recipients with female donors (FM), 22 male recipients with male donors (MM), and 26 normal individuals. All patients and donors were HLA A*0201 positive.

Results: Thirteen peptides derived from five proteins elicited significantly greater T-cell responses in FM patients compared with MM patients and in normal females compared with normal males. Six peptides were more immunogenic than the only previously known HLA A*0201-restricted Y-encoded mHA. Twenty-seven of 28 FM patients responded to at least one HY peptide, but despite a common Y chromosome mismatch and expression of HLA A*0201, each patient responded to a unique set of peptides.

Conclusions: Novel HLA A*0201-restricted HY epitopes can be predicted and validated in patients after allogeneic hematopoietic stem cell transplantation. Highly diverse patterns of T-cell response against these epitopes have been identified. Prospective monitoring of responses to large panels of immunogenic peptides can facilitate the identification of clinically relevant targets of graft-versus-host disease and graft versus leukemia.

Figure 1

T cell responses to 13 immunogenic HY peptides in FM patients, MM patients, normal females (NF) and normal males (NM). 13 HY peptides are listed in the same order on the X axis in patient and control groups. The Y axis represents the number of spots/200,000 cells in ELISPOT assay for each peptide after subtraction of values for negative control peptides (HIV or HCV). Values >20 spots/200,000 cells are considered positive. Open symbols represent the previously known HLA A*0201 restricted HY.

Figure 2

No correlation between intensity and frequency of T cell response to individual HY peptides in FM patients. The percentage of FM patients (n=28) with a positive response to each HY peptide (n=13) is shown on the X axis. The Y axis illustrates the magnitude of response to each HY peptide (spots/200,000 cells) in the same individuals. The open square represents the previously reported HY (JARID1D311), which elicited a relatively strong response but this was only detected in 33.3% of FM patients.

Figure 3

Patterns of response to 13 immunogenic HY peptides among 28 FM patients. Quantitative T cell responses to each HY peptide measured by ELISPOT are clustered using dChip software (http://biosun1.harvard.edu/complab/dchip). Each row represents one of the 13 immunogenic HY peptides and each column represents a single FM patient. The intensity of ELISPOT reactivity for each peptide is represented on a color scale. A blank represents a missing data point; light blue represents a negative response; positive responses are in red and scaled as shown. Three patient clusters (A, B, C) with similar responses emerged from this analysis.

Figure 4

Persistence of T cell response to HY peptides in FM patients and temporal association with clinical acute GVHD. Panel 4a depicts all HY peptide responses in 28 FM patients. The X axis represent years from HSCT to sample collection. The Y axis represents the number of spots/200,000 cells after subtraction of appropriate negative control. Values >20 spots/200,000 cells are considered positive. Responses can be detected as long as 5 years post transplant. Open triangles represent responses against the known HLA A*0201 restricted HY peptide. Panel 4b shows the prospective monitoring of three additional HLA A*0201 FM patients for 6-9 months following transplant. Samples were obtained monthly at regular follow-up visits. Patient A developed acute GVHD at approximately 3 months post-transplant. Patient B developed a rash at day 52 that responded to treatment. He subsequently developed grade 3 acute GVHD at 94 days post transplant. Patient C did not develop GVHD. ALL relapse was suspected at day 137 when a mediastinal mass was detected and immune suppression was rapidly tapered. Bone marrow relapse was confirmed at day 211. All three patients developed significant ELISPOT responses post transplant but to different HY peptides. HY responses were no longer detected after high dose steroids or other therapy was initiated.