Crystal structures of N-myristoylated lipopeptide-bound HLA class I complexes indicate reorganization of B-pocket architecture upon ligand binding.

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.

High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D.

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2ß and CDR3α. This allowed CDR3ß variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.

HLA Class I Downregulation by HIV-1 Variants from Subtype C Transmission Pairs.

HIV-1 downregulates human leukocyte antigen A (HLA-A) and HLA-B from the surface of infected cells primarily to evade CD8 T cell recognition. HLA-C was thought to remain on the cell surface and bind inhibitory killer immunoglobulin-like receptors, preventing natural killer (NK) cell-mediated suppression. However, a recent study found HIV-1 primary viruses have the capacity to downregulate HLA-C. The goal of this study was to assess the heterogeneity of HLA-A, HLA-B, and HLA-C downregulation among full-length primary viruses from six chronically infected and six newly infected individuals from transmission pairs and to determine whether transmitted/founder variants exhibit common HLA class I downregulation characteristics. We measured HLA-A, HLA-B, HLA-C, and total HLA class I downregulation by flow cytometry of primary CD4 T cells infected with 40 infectious molecular clones. Primary viruses mediated a range of HLA class I downregulation capacities (1.3- to 6.1-fold) which could differ significantly between transmission pairs. Downregulation of HLA-C surface expression on infected cells correlated with susceptibility to in vitro NK cell suppression of virus release. Despite this, transmitted/founder variants did not share a downregulation signature and instead were more similar to the quasispecies of matched donor partners. These data indicate that a range of viral abilities to downregulate HLA-A, HLA-B, and HLA-C exist within and between individuals that can have functional consequences on immune recognition.IMPORTANCE Subtype C HIV-1 is the predominant subtype involved in heterosexual transmission in sub-Saharan Africa. Authentic subtype C viruses that contain natural sequence variations throughout the genome often are not used in experimental systems due to technical constraints and sample availability. In this study, authentic full-length subtype C viruses, including transmitted/founder viruses, were examined for the ability to disrupt surface expression of HLA class I molecules, which are central to both adaptive and innate immune responses to viral infections. We found that the HLA class I downregulation capacity of primary viruses varied, and HLA-C downregulation capacity impacted viral suppression by natural killer cells. Transmitted viruses were not distinct in the capacity for HLA class I downregulation or natural killer cell evasion. These results enrich our understanding of the phenotypic variation existing among natural HIV-1 viruses and how that might impact the ability of the immune system to recognize infected cells in acute and chronic infection.

Stability and Expression Levels of HLA-C on the Cell Membrane Modulate HIV-1 Infectivity.

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/ß2 microglobulin [ß2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to ß2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to ß2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes ß2 microglobulin/peptide; the other conformation is not bound to ß2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from ß2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

The molecular basis for peptide repertoire selection in the human leucocyte antigen (HLA) C*06:02 molecule.

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.

Uncovering the peptide-binding specificities of HLA-C: a general strategy to determine the specificity of any MHC class I molecule.

MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8(+) T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database.

The immunodominant influenza A virus M158-66 cytotoxic T lymphocyte epitope exhibits degenerate class I major histocompatibility complex restriction in humans.

UNLABELLED: Cytotoxic T lymphocytes recognizing conserved peptide epitopes are crucial for protection against influenza A virus (IAV) infection. The CD8 T cell response against the M158-66 (GILGFVFTL) matrix protein epitope is immunodominant when restricted by HLA-A*02, a major histocompatibility complex (MHC) molecule expressed by approximately half of the human population. Here we report that the GILGFVFTL peptide is restricted by multiple HLA-C*08 alleles as well. We observed that M158-66 was able to elicit cytotoxic T lymphocyte (CTL) responses in both HLA-A*02- and HLA-C*08-positive individuals and that GILGFVFTL-specific CTLs in individuals expressing both restriction elements were distinct and not cross-reactive. The crystal structure of GILGFVFTL-HLA-C*08:01 was solved at 1.84 Å, and comparison with the known GILGFVFTL-HLA-A*02:01 structure revealed that the antigen bound both complexes in near-identical conformations, accommodated by binding pockets shaped from shared as well as unique residues. This discovery of degenerate peptide presentation by both HLA-A and HLA-C allelic variants eliciting unique CTL responses to IAV infection contributes fundamental knowledge with important implications for vaccine development strategies. IMPORTANCE: The presentation of influenza A virus peptides to elicit immunity is thought to be narrowly restricted, with a single peptide presented by a specific HLA molecule. In this study, we show that the same influenza A virus peptide can be more broadly presented by both HLA-A and HLA-C molecules. This discovery may help to explain the differences in immunity to influenza A virus between individuals and populations and may also aid in the design of vaccines.

Peptide-induced HLA-E expression in human PBMCs is dependent on peptide sequence and the HLA-E genotype.

Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA-E polymorphism is related to HLA-E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA-E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA-E*01:01 or HLA-E*01:03, by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide-induced HLA-E surface expression levels were higher in PBMC from HLA-E*01:03 homozygous subjects as compared to PBMC from HLA-E*01:01 homozygous subjects. HLA-E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA-E surface expression seemed to be not only dependent on the HLA-E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA-E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide-induced HLA-E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA-E expression is regulated and underlines a new role for extracellular peptides in inducing HLA-E translation, which may represent a defense mechanism against lytic viral infections and necrosis.

Amino acid substitution at peptide-binding pockets of HLA class I molecules increases risk of severe acute GVHD and mortality.

HLA disparity has a negative impact on the outcomes of hematopoietic cell transplantation (HCT). We studied the independent impact of amino acid substitution (AAS) at peptide-binding positions 9, 99, 116, and 156, and killer immunoglobulin-like receptor binding position 77 of HLA-A, B, or C, on the risks for grade 3-4 acute graft-versus-host disease (GVHD), chronic GVHD, treatment-related mortality (TRM), relapse, and overall survival. In multivariate analysis, a mismatch at HLA-C position 116 was associated with increased risk for severe acute GVHD (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.15-1.82, P = .0016). Mismatch at HLA-C position 99 was associated with increased transplant-related mortality (HR = 1.37, 95% CI = 1.1-1.69, P = .0038). Mismatch at HLA-B position 9 was associated with increased chronic GVHD (HR = 2.28, 95% CI = 1.36-3.82, P = .0018). No AAS were significantly associated with outcome at HLA-A. Specific AAS pair combinations with a frequency >30 were tested for association with HCT outcomes. Cysteine to tyrosine substitution at position 99 of HLA-C was associated with increased TRM (HR = 1.78, 95% = CI 1.27-2.51, P = .0009). These results demonstrate that donor-recipient mismatch for certain peptide-binding residues of the HLA class I molecule is associated with increased risk for acute and chronic GVHD and death.

HLA-B may be more protective against HIV-1 than HLA-A because it resists negative regulatory factor (Nef) mediated down-regulation.

Human leukocyte antigen HLA-B alleles have better protective activity against HIV-1 than HLA-A alleles, possibly due to differences in HLA-restricted HIV-1-specific CD8+ cytotoxic T lymphocyte (CTL) function, but the mechanism is unknown. HIV-1 negative regulatory factor (Nef) mediates down-regulation of surface expression of class I HLA (HLA-I) and may therefore impair immune recognition by CTL. Because of sequence differences in the cytoplasmic domains, HLA-A and -B are down-regulated by Nef but HLA-C and -E are not affected. However, the latter are expressed at low levels and are not of major importance in the CTL responses to HIV-1. Here, we compared the role of the cytoplasmic domains of HLA-A and -B in Nef-mediated escape from CTL. We found HLA-B cytoplasmic domains were more resistant to Nef-mediated down-regulation than HLA-A cytoplasmic domains and demonstrated that these differences affect CTL recognition of virus-infected cells in vitro. We propose that the relative resistance to Nef-mediated down-regulation by the cytoplasmic domains of HLA-B compared with HLA-A contributes to the better control of HIV-1 infection associated with HLA-B-restricted CTLs.

A comprehensive analysis of constitutive naturally processed and presented HLA-C*04:01 (Cw4)-specific peptides.

The human B lymphoblastoid cell line C1R is widely regarded as human leukocyte antigen-A (HLA-A)/HLA-B negative and is therefore frequently exploited as a recipient cell line to study HLA class I functions. However, the normal levels of HLA-C*04:01 often hamper the investigation of introduced HLA class I allomorphs, which is particularly evident in sensitive applications such as mass spectrometry. Here we describe the comprehensive analysis of endogenous HLA-C*04:01 ligands expressed on the surface of C1R cells to (i) define a large sequence dataset of HLA-C*04:01 ligands, to (ii) refine the HLA-C*04:01 peptide-binding motif and (iii) to provide a resource that allows discrimination between peptides bound to introduced HLA class I subtypes and to the endogenous HLA-C*04:01 molecules.

Description of two new HLA-C alleles: C*08:63 and C*14:44.

Here, we present two new HLA allelic variants at C locus: HLA-C*08:63 and HLA-C*14:44 detected by sequence-based typing. In both cases, a single-nucleotide mutation in exon 3 is responsible for a change in aminoacid translation. The extremely high polymorphism of human leucocyte antigen (HLA) system in human genome is responsible for the capability to recognize different antigens, including non-self-MHC (Major Histocompatibility Complex) molecules. This very high polymorphism and the improving accuracy of genomic HLA typing methods lead to an exponential increasing of known HLA alleles. Here, we describe the characterization of two new HLA-C alleles identified by sequence-based typing (SBT): HLA-C*08:63 and HLA-C*14:44.

The new HLA-C variant HLA-C*05:26 is likely to be structurally identical to the C*05:01P alleles.

The novel allele HLA-C*05:26 differs from HLA-C*05:01 by the non-synonymous amino acid exchange Gly16Ser.

The novel HLA-C*12:92 allele is characterized by one amino acid exchange located in the T-cell receptor binding region of the alpha 2 domain.

HLA-C*12:92 contains one amino acid exchange in the T-cell receptor binding region.

Molecular architecture of the MHC I peptide-loading complex: one tapasin molecule is essential and sufficient for antigen processing.

The loading of antigen-derived peptides onto MHC class I molecules for presentation to cytotoxic T cells is a key process in adaptive immune defense. Loading of MHC I is achieved by a sophisticated machinery, the peptide-loading complex (PLC), which is organized around the transporter associated with antigen processing (TAP) with the help of several auxiliary proteins. As an essential adapter protein recruiting MHC I molecules to TAP, tapasin catalyzes peptide loading of MHC I. However, the exact stoichiometry and basic molecular architecture of TAP and tapasin within the PLC remains elusive. Here, we demonstrate that two tapasin molecules are assembled in the PLC, with one tapasin bound to each TAP subunit. However, one tapasin molecule bound either to TAP1 or TAP2 is sufficient for efficient MHC I antigen presentation. By specifically blocking the interaction between tapasin-MHC I complexes and the translocation complex TAP, the MHC I surface expression is impaired to the same extent as with soluble tapasin. Thus, the proximity of the peptide supplier TAP to the acceptor MHC I is crucial for antigen processing. In summary, the human PLC consists maximally of 2× tapasin-ERp57/MHC I per TAP complex, but one tapasin-ERp57/MHC I in the PLC is essential and sufficient for antigen processing.

Allele-specific amplification of the complete HLA-C gene from genomic DNA - a novel Cw4 allele (C*04:71) with a Cw1 motif in the peptide-binding site.

To determine the complete sequence of a newly identified human leukocyte antigen (HLA)-C allele, we designed a method where the full genomic sequence of HLA-C*04 was amplified in isolation from the patient second HLA-C allele in a single polymerase chain reaction (PCR), using primers spanning its 5'- and 3'-untranslated regions. The new allele, officially designated HLA-C*04:71, differs from HLA-C*04:01:01:01 by two single-nucleotide polymorphisms: one determines substitution of phenylalanine for serine 9 at the B pocket of the peptide-binding site; the second substitution is a new polymorphism in intron 5. Phe-9 is characteristic of Cw1 alleles and its presence in C*04:71 most likely affects its peptide-binding repertoire. The principle we have used for C*04:71 isolation could be adapted for unambiguous sequence-based HLA-C typing of selected samples in a clinical setting.

T cells discriminate between groups C1 and C2 HLA-C.

Dimorphic amino acids at positions 77 and 80 delineate HLA-C allotypes into two groups, C1 and C2, which associate with disease through interactions with C1 and C2-specific natural killer cell receptors. How the C1/C2 dimorphism affects T cell recognition is unknown. Using HLA-C allotypes that differ only by the C1/C2-defining residues, we found that KRAS-G12D neoantigen-specific T cell receptors (TCRs) discriminated between C1 and C2 presenting the same KRAS-G12D peptides. Structural and functional experiments, and immunopeptidomics analysis revealed that Ser77 in C1 and Asn77 in C2 influence amino acid preference near the peptide C-terminus (pΩ), including the pΩ-1 position, in which C1 favors small and C2 prefers large residues. This resulted in weaker TCR affinity for KRAS-G12D-bound C2-HLA-C despite conserved TCR contacts. Thus, the C1/C2 dimorphism on its own impacts peptide presentation and HLA-C-restricted T cell responses, with implications in disease, including adoptive T cell therapy targeting KRAS-G12D-induced cancers.

HLA-C*03:93, a novel HLA-C*03 allele identified by sequence-based typing.

The new allele C*03:93 showed one nucleotide difference with C*03:04:01 at codon 140 (GCT/ACT).

Genetic variation and hitchhiking between structurally polymorphic Alu insertions and HLA-A, -B, and -C alleles and other retroelements within the MHC class I region.

We investigated structurally polymorphic Alu insertions (POALINs) at five loci in the major histocompatibility complex (MHC) class I genomic region to determine their allele and haplotype frequencies and associations with the human leukocyte antigen (HLA)-A, -B, and -C genes in three populations, the Australian Caucasians, Japanese, and African Americans. The POALINs varied in allelic frequency between 0% and 42.3% with significant differences between populations at three of the five loci. The linkage disequilibrium (LD) between Alu insertions and the HLA-A, -B, or -C alleles and previously published polymorphic retroelements (four SVA and human endogenous retrovirus type 9 (HERVK9) loci) within the class I region of the MHC were calculated in pairwise analyses of haplotypes to show strong allelic associations and possible crossing-over events between some loci. Each POALIN was in significant LD with a variety of HLA-A, -B, or -C two-digit alleles probably as a result of hitchhiking. The POALINs helped to further stratify the HLA-A:B:C haplotypes into different POALIN:HLA-A:B:C haplotype frequencies. Of the multilocus haplotype analyses, the seven- and eight-locus haplotypes showed the largest number of differences between the populations, and fewer matched haplotypes between populations that ranged, for example, from 49% for HLA-B:HLA-A haplotypes to 7% for AluMICB:HLA-B:HLA-C:AluTF:AluHJ:HLA-A:AluHG:AluTF haplotypes in the Japanese. This comparative study of multilocus POALINs in the HLA class I region of three ethnic populations shows that POALINs alone or together with the HLA class I alleles and other retroelements are informative ancestral markers for assessing the interrelationship of HLA class I haplotype lineages, LD, and genetic diversity within the same and/or different populations.

HLA mismatch combinations associated with decreased risk of relapse: implications for the molecular mechanism.

The finding that the risk of relapse in hematologic malignancy decreases after allogeneic hematopoietic stem cell transplantation (HSCT) has lead to the concept of a graft-versus-leukemia (GVL) effect. However, this beneficial effect is considered to be frequently offset by graft-versus-host disease (GVHD). Thus, improving HSCT outcomes by separating GVL from GVHD is a key clinical issue. This cohort study registered 4643 patients with hematologic malignancies who received transplants from unrelated donors. Six major human leukocyte antigen (HLA) loci were retrospectively genotyped. We identified 4 HLA-Cw and 6 HLA-DPB1 mismatch combinations responsible for a decreased risk of relapse; of these, 8 of 10 combinations were different from those responsible for severe acute GVHD, including all 6 of the HLA-DPB1 combinations. Pairs with these combinations of HLA-DPB1 were associated with a significantly better overall survival than were completely matched pairs. Moreover, several amino acid substitutions on specific positions responsible for a decreased risk of relapse were identified in HLA-Cw, but not in HLA-DPB1. These findings might be crucial to elucidating the mechanism of the decreased risk of relapse on the basis of HLA molecule. Donor selection made in consideration of these results might allow the separation of GVL from acute GVHD, especially in HLA-DPB1 mismatch combinations.