T-Cell Monitoring for the Recently Expanded Repertoire of HLA Class I-Restricted Minor Histocompatibility Antigens Using Barcoded Peptide-MHC Multimers
Authors: Fuchs Kyra J. , Göransson Marcus , Kester Michel G.D. , Ettienne Natasja W. , Meent Marian van de , de Jong Rob C.M. , Koster Eva A.S. , Halkes Constantijn J.M. , Scheeren Ferenc A. , Heemskerk Mirjam H.M. , Van Balen Peter , Falkenburg J.H. Frederik , Hadrup Sine R. , Griffioen Marieke 
Published: December 2024

Allogeneic stem cell transplantation provides a curative treatment option for patients with hematological malignancies. After HLA-matched allogeneic stem cell transplantation, donor-derived T cells recognize minor histocompatibility antigens (MiHAs), which are polymorphic peptides presented on patient cells by HLA surface molecules. MiHAs are not presented on donor cells due to differences in single nucleotide polymorphisms between patient and donor. Donor T cells targeting MiHAs that are broadly expressed on hematopoietic as well as non-hematopoietic tissues may induce desired Graft-versus-Leukemia (GvL) reactivity as well as undesired Graft-versus-Host Disease (GvHD), while T cells for MiHAs with restricted or preferential expression on either hematopoietic cells or non-hematopoietic tissues may skew immune responses more towards a selective GvL effect or GvHD, respectively. Besides tissue expression, the overall strength of GvL and GvHD is also determined by the T-cell frequencies that are induced against MiHAs after allogeneic stem cell transplantation.

Recently, we discovered a large repertoire of new MiHAs in common HLA class I alleles by genome-wide association screening.1,2 T-cell clones were isolated from 39 patients who responded to donor lymphocyte infusion after T cell-depleted HLA-matched allogeneic stem cell transplantation. T-cell clones recognizing unknown MiHAs were tested for reactivity against 191 EBV-B cell lines of the 1000 Genomes Project to identify the target antigens. By discovering 89 new MiHAs, the repertoire of HLA class I-restricted MiHAs was expanded to 159 total antigens. The data showed that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients, demonstrating that the dominant repertoire of MiHAs in common HLA class I alleles has been largely discovered and available to screen patient cohorts to investigate the kinetics of MiHA-specific T-cell responses after allogeneic stem cell transplantation.

Here, the expanded repertoire of MiHAs was used to screen patients for specific T cells with barcoded peptide-MHC multimers. Barcoded peptide-MHC multimers were produced for 255 MiHA peptides and 103 viral peptides. The 255 MiHA peptides included 126 autosomal MiHAs and 5 H-Y antigens, as well as various length variants for MiHAs and peptides for allelic variants. All peptides were validated to form stable peptide-MHC complexes and tested for potential background staining on peripheral blood samples from healthy individuals. In total, 227 MiHA peptides and 93 viral peptides passed the quality controls and were selected for T-cell monitoring. In 16 patients who responded to donor lymphocyte infusion after T cell-depleted HLA-matched allogeneic stem cell transplantation, variable T-cell frequencies up to 30.5% of CD8+ T cells were measured against 49 MiHAs. High T-cell frequencies above 1% were measured in 12 patients against 15 mismatched MiHAs with peak responses detected 6-8 weeks after donor lymphocyte infusion and at onset of GvHD. The 12 patients included nine of ten patients with severe GvHD, two of three patients with limited GvHD, and one of three patients without GvHD. The data demonstrated that T-cell frequencies were particularly high in patients with GvHD, and that T-cell responses were directed against MiHAs with variable tissue distribution in all patient groups.

In conclusion, we explored whether barcoded peptide-MHC multimers are a feasible method to detect and follow MiHA-specific T-cell responses after allogeneic stem cell transplantation. We demonstrated that the technique can be used to measure and monitor patients for MiHA-specific T-cell frequencies during treatment to investigate the kinetics of immune responses and their impact on development of GvL and GvHD after allogeneic stem cell transplantation.

1. Fuchs KJ, Honders MW, van der Meijden ED, et al. Optimized Whole Genome Association Scanning for Discovery of HLA Class I-Restricted Minor Histocompatibility Antigens. Front Immunol. 2020;11:659.

2. Fuchs KJ, van de Meent M, Honders MW, et al. Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens. Blood 2024; 143(18):1856-1872.