Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2012 Sep 27;19(2):274–282. doi: 10.1016/j.bbmt.2012.09.008

Association of disparities in known minor histocompatibility antigens with relapse-free survival and graft-versus-host-disease after allogeneic stem cell transplantation

Willemijn Hobo 1,*, Kelly Broen 1,*, Walter JFM van der Velden 2, Annelies Greupink-Draaisma 1, Niken Adisty 1, Yannick Wouters 2, Michel Kester 3, Hanny Fredrix 1, Joop H Jansen 1, Bert van der Reijden 1, JH Frederik Falkenburg 3, Theo de Witte 4, Frank Preijers 1, Ton Schattenberg 2, Ton Feuth 5, Nicole M Blijlevens 2, Nicolaas Schaap 2, Harry Dolstra 1
PMCID: PMC3553241  NIHMSID: NIHMS411012  PMID: 23022467

Abstract

Allogeneic stem cell transplantation (allo-SCT) can induce remission in patients with hematological malignancies due to graft-versus-tumor (GVT) responses. This immune-mediated anti-tumor effect, however, is often accompanied by detrimental graft-versus-host disease (GVHD). Both GVT and GVHD are mediated by minor histocompatibility antigen (MiHA)-specific T cells recognizing peptide products from polymorphic genes that differ between recipient and donor. In this study, we evaluated whether mismatches in a panel of seventeen MiHA are associated with clinical outcome after partial T cell-depleted allo-SCT. Comprehensive statistical analysis revealed that DNA mismatches for one or more autosomal-encoded MiHA was associated with increased relapse-free survival in sibling transplants, (P =0.04), particularly in patients suffering from multiple myeloma (P =0.02). Moreover, mismatches for the ubiquitous Y chromosome-derived MiHA resulted in a higher incidence of acute GVHD (grade 3–4; P =0.004), while autosomal MiHA mismatches, ubiquitous or restricted to hematopoietic cells, were not associated with severe GVHD. Finally, we demonstrated considerable differences between MiHA in their capability to induce in vivo T cell responses using dual-color tetramer analysis of peripheral blood samples collected post-SCT. Importantly, detection of MiHA-specific T cell responses was associated with improved relapse-free survival in sibling transplants (P =0.01). Our findings provide a rationale to further boost GVT immunity towards autosomal MiHA with a hematopoietic restriction to improve outcome after HLA-matched allo-SCT.

Keywords: minor histocompatibility antigen, CD8+ T cells, stem cell transplantation, GVHD, graft-versus-tumor

Introduction

Allogeneic stem cell transplantation (allo-SCT) combined with donor lymphocyte infusion (DLI) is a potent treatment for patients with hematological malignancies.1;2 Many clinical and experimental studies in human leukocyte antigen (HLA)-identical allo-SCT provide evidence that both the potentially curative graft-versus-tumor (GVT) effect and graft-versus-host disease (GVHD) develop as a result of donor T cell responses directed against disparate minor histocompatibility antigens (MiHA).3–5 These MiHA are polymorphic HLA-bound peptides derived from cellular proteins that can induce powerful alloreactive T cell responses. It has been demonstrated that emergence of MiHA-specific T cells precedes clinical remission in patients treated with DLI.4;6;7 While various MiHA, including Y chromosome-encoded MiHA, are expressed ubiquitously, increasing numbers of autosomal-encoded MiHA are being identified that are exclusively expressed by hematopoietic cells and their malignant counterparts.8–10 The molecular identification of these GVHD- and GVT-associated MiHA has made it possible to study the clinical impact of MiHA mismatches and their specific T cell responses post-transplantation.

Several studies in HLA-matched allo-SCT have reported an association between mismatches in MiHA and clinical outcome. Mismatches in individual MiHA, including HA1, HA2 and HA8, have been associated with increased GVHD occurrence and lower relapse rates11–13, but other studies could not confirm these results14–16. Furthermore, previous studies have predominantly investigated cohorts of HLA-matched non-T cell-depleted transplants, and found only an increase in chronic GVHD (cGVHD) and reduced relapse rate upon HY MiHA disparity.17;18 Moreover, investigation of the role of MiHA incompatibility in transplant outcome is hampered by the requirements to restrict studies to specific HLA types and low frequencies of particular MiHA alleles. Recently however, it was reported that HLA-A2+ chronic myeloid leukemia (CML) patients who developed acute GVHD (aGVHD) showed an improved overall survival (OS) and relapse-free survival (RFS) when receiving a transplant from a HA1-mismatched donor.19

In this study, we performed a retrospective analysis on the impact of a panel of seventeen immunogenic MiHA mismatches in a relatively large cohort of patients who received partial T cell-depleted allo-SCT. In sibling transplants, mismatches in one or more of the studied autosomal-encoded MiHA resulted in an improved RFS (P =0.04), especially in multiple myeloma (MM) patients (P =0.02). In contrast, no significant association between autosomal MiHA mismatches and acute or chronic GVHD was observed, whereas the occurrence of a HY disparity led to more grade 3–4 aGVHD (P =0.004). Finally, this report describes for the first time the potential of disparate MiHA to induce productive T cell responses post-transplantation. Tetramer analysis revealed that the ability of different MiHA to mount specific CD8+ T cell responses post-transplant varies strongly (0% – 60%). More importantly, presence of MiHA-specific T cell immunity was associated with improved RFS, without inducing severe aGVHD or cGVHD. Together, these data provide rationale for further boosting of GVT immunity towards autosomal hematopoietic-restricted MiHA to improve relapse-free survival after HLA-matched allo-SCT.

Patients, materials and methods

Patients and donors

Three hundred and twenty-seven (N=327) adult SCT recipients and their donors were included in this study. They were selected from the total transplant cohort of our centre, treated between 1995 and 2010 with HLA-matched partial T cell-depleted allo-SCT for a hematological malignancy. HLA was typed using sequence-specific PCR. In sibling transplants, patients were transplanted with a HLA-identical sibling donor, and in matched unrelated transplants (MUD), patients were transplanted with a 8 to 10 out of 10 HLA-matched voluntary donor, not considering HLA-DP. Only patient-donor couples with the HLA types HLA-A1, -A2, -A3, -A24, -B7, -B8 or -B44 were included, because the selected set of MiHA was restricted to these HLA types. Furthermore, couples were selected based on the availability of both patient and donor material. The characteristics of patients, donors and SCT procedures are shown in Table 1. Patients and donors had given their informed consent to the prospective collection of data and samples for investigational use, which was approved by the Radboud University Nijmegen Medical Centre (RUNMC) Institutional Review Board.

Table 1.

Recipient, donor, and SCT characteristics (N=327)

Characteristic
Recipient age, years, mean (range) 46 (18–67)
Donor age, years, mean (range) 46 (11–71)
Recipient gender male, no (%) 195 (60%)
Donor gender male, no (%) 191 (58%)
Gender combination, no (%)
 - Male patient/female donor 76 (23%)
 - Other 251 (77%)
Donor relation, no (%)
 - Matched sibling donor 264 (81%)
 - Matched unrelated donor 63 (19%)
Disease category, no (%)
 - AML/MDS 118 (36%)
 - ALL 34 (10%)
 - CML 53 (16%)
 - NHL/CLL 67 (20%)
 - MM 55 (17%)
Stem cell source
 - Mobilized peripheral blood, no (%) 175 (54%)
  - CD34 ×106/kg, median (range) 5.6 (1.3–13.8)
  - CD3 ×106/kg, median (range) 0.5 (0.04–1.7)
 - Bone marrow, no (%) 152 (46%)
  - CD34 ×106/kg, median (range) 1.9 (0.5–6.5)
  - CD3 ×106/kg, median (range) 0.7 (0.5–1.1)
SCT date, no (%)
 - 1995–1999 98 (30%)
 - 2000–2004 83 (25%)
 - 2005–2010 146 (45%)
Conditioning regimens, no (%)
Myeloablative:
 - (Ida)-Cy-Bus 18 (5.5%)
 - (Ida)-Cy-TBI 182 (55.5%)
 - Cy-ATG-Bus 8 (2.5%)
 - Cy-ATG-TBI 39 (12%)
Non-myeloablative:
 - Flu-Cy 50 (15.5%)
 - Flu-Cy-ATG 16 (5%)
 - TBI alone 14 (4%)
GVHD prophylaxis, no (%)
 - Cyclosporine alone 305 (93%)
 - Cyclosporine/MMF 13 (4%)
 - None 9 (3%)
Disease status, no (%)
 - Early 193 (59%)
 - Intermediate 81 (25%)
 - Advanced 53 (16%)
Diagnosis before SCT, no (%)
 - ≤ 1 year 205 (62.5%)
 - > 1 year 122 (37.5%)
CMV status, no (%)
 - Negative/negative 76 (23%)
 - Other combination 229 (70%)
 - Missing 22 (7%)
Gratwohl score, no (%)
 - Score 0 30 (9%)
 - Score 1 183 (56%)
 - Score 2 93 (28.5%)
 - Score 3 21 (6.5%)
Open in a new tab

Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphatic leukemia; CML, chronic myeloid leukemia; NHL, non-Hodgkin lymphoma; CLL, chronic lymphatic leukemia; MM, Multiple Myeloma; SCT, stem cell transplantation; Ida, idarubicin; Cy, Cyclophosphamide; Bus, busulphan; TBI, total body irradiation; ATG, anti-thymocyte globulin; Flu, fludarabine; MMF, mycophenolate mofetil; GVHD, graft-versus-host disease; CMV, cytomegalovirus.

Treatment protocol

All patients were treated according to protocols described previously.20–22 Myeloablative conditioning regimens consisted of cyclophosphamide (60 mg/kg for 2 days) in combination with either total body irradiation (TBI; 4.5 Gy for 2 days) or busulfan (4 mg/kg for 4 days). Idarubicin (42 mg/m2 in 48 hours intravenously) was often added to reduce the risk of relapse in the setting of partial T cell-depleted SCT.23 Non-myeloablative conditioning regimens consisted mainly of cyclophosphamide (1200 mg/m2 for 4 days) in combination with fludarabine (30 mg/m2 for 4 days), sometimes only TBI (2 Gy). Patients receiving a MUD-graft received anti-thymocyte globulin (ATG) (2 mg/kg for 4 days). After conditioning, patients received a partial T cell-depleted graft derived either from bone marrow (BM) or mobilized peripheral blood stem cells (PBSC). The median number of CD34+ stem cells in the graft was 1.9×106 cells/kg (range 0.5–6.5) for BM and 5.6×106 cells/kg (range 1.3–13.8) for PBSC, and the median number of CD3+ T cells in BM grafts was 0.7×106 T cells/kg and in PBSC grafts 0.5×106 T cells (Table 1). GVHD prophylaxis consisted of cyclosporine A (CsA) only in almost all patients, and was dosed 1.5 mg/kg bidaily (bid) intravenously for the first two weeks, and thereafter 1 mg/kg bid intravenously or 2.5–3 mg/kg bid orally. Tapering of CsA was started in the absence of GVHD after two months, and stopped at three months. Several patients received prophylactic or therapeutic DLI following transplantation. Prophylactic DLI was restricted to patients who had stopped CsA for at least three months, and had not developed aGVHD grade ≥2 or cGVHD.

Definition of outcome variables

Acute GVHD was graded according to the criteria of Przepiorka et al.24 and cGVHD was classified according to the revised Seattle criteria of Lee et al.25. Patient risk scores for outcome were determined according to the Gratwohl score.26 OS, RFS, and non-relapse mortality (NRM) were defined according to the standard criteria proposed by the EBMT.

MiHA genotyping using the KASPar system

HLA-matched SCT donor-recipient pairs were genotyped for a panel of 17 MiHA with the KASPar assay system (KBioscience, Hoddesdon, UK), which is a fluorescence-based competitive allele-specific PCR using non-labeled primers. Details of this method can be found at http://www.kbioscience.co.uk.

Tetramer staining and validation by T cell culture

PE- and APC-labeled MiHA tetramers were produced as described previously.27 Tetramer stainings were performed directly on cryopreserved peripheral blood mononuclear cells (PBMC) after thawing, and 7 days after ex vivo re-stimulation with the appropriate MiHA peptide.28 For this, PBMC were stimulated once with MiHA peptide-pulsed (10 μM) Epstein-Barr virus lymphoblastoid cell lines (EBV-LCL) on day 0. MiHA peptides were loaded on a corresponding EBV-LCL stably transduced with either HLA-A2, -A3, or -B7. For additional HLA types (i.e. HLA-A24 and HLA-B8) matching healthy donor EBV-LCL were used. After initial ex vivo re-stimulation, 100 IU/ml IL-2 (Chiron, Emeryville, CA) and 10 ng/ml IL-15 (Immunotools) was added at day 2 and 5. For tetramer staining, ~1×106 cells were stained with 0.2 μg tetramer for 15 min at room temperature. The stability of all tetramers was verified using HPLC analysis in combination with a HLA-binding assay (MHC-ELISA or MHC-bead assay).29;30 In addition, the functional reactivity of all tetramers, except HEATR, was confirmed by staining CTL clones specific for the corresponding epitope, and subsequent flow cytometrical analysis. After tetramer staining, cells were washed with PBS/0.5% bovine serum albumin (BSA; Sigma, St Louis, MO, USA) and labeled with AlexaFluor700-conjugated CD8 (Invitrogen) in combination with FITC-conjugated CD4, CD14, CD16 and CD19 (Beckman Coulter) for 30 min at 4°C. Finally, cells were washed and resuspended in PBS/0.5% BSA containing 0.2 μM Sytox Blue (Invitrogen) to allow dead-cell exclusion. Data acquisition was performed on a Cyan-ADP analyzer (Beckman Coulter) and analyzed using Kaluza 1.1 software (Beckman Coulter). CD8+ T cells were defined as viable Sytox Blue-negative, single-cell lymphocytes, CD8-positive and FITC (CD4, CD14, CD16, CD19) negative cells. Within the CD8+ T cell population, cells positive for both tetramers (APC and PE) were quantified (Supplementary Figure 1). Patients were classified as having a positive tetramer response, when MiHA-specific CD8+ T cells (≥0.01% tetramer+ cells within the CD8+ T cell population) were found either directly after thawing, and/or 7 days after peptide ex vivo re-stimulation using peripheral blood samples obtained during the effector or memory phase of the immune response.

Statistical analysis

The outcome variables aGVHD and cGVHD, relapse, RFS, OS, and NRM after allo-SCT were analyzed in relation to MiHA disparity. Associations with RFS and OS were analyzed using Kaplan Meier curves and log-rank tests. Statistical differences in the RFS incidences at certain time points were analyzed using Kaplan Meier point estimates and their associated errors. Associations with cumulative incidences of aGVHD, cGVHD, relapse, and NRM were estimated respecting the presence of competing risks using the Gray test. As competing risks we considered death within 100 days from other toxicities or relapse for aGVHD, death after 100 days from other toxicities or relapse for cGVHD, NRM for relapse, and death from relapse for NRM.

Furthermore, in case a P-value was ≤0.20 in univariable analyses, Cox regression analyses (for the endpoints RFS and OS) and Fine and Gray regression analyses (for the endpoints aGVHD, cGVHD, relapse, and NRM) were used to adjust for the following confounding risk factors: patient age, stem cell source, year of transplant, conditioning regimen, diagnosis-subgroup, CMV seropositivity of either recipient and/or donor, and aGVHD grade 2–4. Analyses were performed using SAS 8.2 software and the cmprsk package of open source language R version 2.6.2. (www.r-project.org). P-values <0.05 were considered statistically significant. MiHA mismatch parameters were defined as disparate HY (HY MiHA restricted to HLA-A2, -B7 or -B8), disparate HA1, and disparate autosomal MiHA (mismatched for one or more MiHA listed in Table 2, excluding for HY).

Table 2.

MiHA disparity rate

MiHA HLA restriction Peptide sequence Reference NA Number of disparate pairs (rates)B
HA3 A1 VTEPGTAQY 40 82/87 3 (3.7%)
HA1 A2 VLHDDLLEA 41 195/197 32 (16.4%)
HA2 A2 YIGEVLVSV 42;43 195/197 6 (3.1%)
HA8 A2 RTLDKVLEV 44 194/197 22 (11.3%)
HY A2 FIDSYICQV 9 197/197 42 (21.3)
ADIR A2 SVAPALALAFPA 45 195/197 30 (15.4%)
HwA11 A2 CIPPDSLLFPA 46 195/197 6 (3.1%)
SP110 A3 SLPRGTSTPK 47 92/93 7 (7.6%)
PANE1 A3 RVWDLPGVLK 48 92/93 5 (5.4%)
ACC1 A24 DYLQYVLQI 49 54/54 6 (11.1%)
ACC2 B44 KEFEDDIINW 49 64/65 16 (25.0%)
LRH1 B7 TPNQRQNVC 35 97/98 19 (19.6%)
HY B7 SPSVDKARAEL 8 98/98 26 (26.5%)
ECGF B7 RPHAIRRPLAL 50 97/98 5 (5.2%)
ZAPHIR B7 IPRDSWWVEL 51 97/98 12 (12.4%)
HY B8 LPHNMTDL 52 67/67 14 (20.9%)
HEATR B8 ISKERAEAL 53 64/67 8 (12.5%)
Open in a new tab
A

N = number of patient donor couples typed, within the total number of couples presenting the correct HLA molecule.

B

The number of disparate pairs and disparity rates of a particular MiHA were determined within all typed couples presenting the correct HLA-molecule.

Results

Clinical outcome parameters after HLA-matched allo-SCT

We analyzed the clinical outcome after partial T cell-depleted allo-SCT in patients (N=327) suffering from a hematological malignancy. The median follow up of patients alive at last follow up was 7.1 years (range 0.5–17), and 1.1 years (range 0.05–12.6) in those who died. In Figure 1A–B, the survival curves for OS (5 year: 68%) and RFS (5 year: 44%) are depicted for the complete cohort. NRM after 5 years was 15.6% (i.e. 43.4% of all deaths, Figure 1C) and 40.4% of the patients experienced relapse. Acute GVHD occurred in 19.6% of patients and was severe (grade 3–4) in only 6.5%. Of all evaluable patients, 22.7% developed limited cGVHD and 14.2% extensive cGVHD.

Figure 1. Clinical outcome parameters after partial T cell-depleted allo-SCT.

Figure 1

Open in a new tab

Within the complete cohort of allo-SCT recipients, (A) overall survival, (B) relapse-free survival and (C) the cumulative incidence of non-relapse mortality (NRM) was analyzed using Kaplan Meier analysis. Allo-SCT, allogeneic stem cell transplantation.

MiHA allele frequency and phenotype disparity rate

To determine the MiHA allele frequency and disparity rates, recipient and donor DNA was typed. Paired couples were only genotyped when the appropriate HLA-molecule was expressed to which the MiHA is restricted, leading to actual immunogenic disparity rates. HY.A2, HY.B7 or HY.B8 disparity was scored when a female-donor-male-recipient (FDMR) transplantation was performed. The highest disparity rates, within the restricting HLA type, were observed for all Y chromosome-derived MiHA (20.9%-26.5%), as well as for ACC2 (25.0%), LRH1 (19.6%) and HA1 (16.4%). Overall, 60 (18.2%) and 137 (41.6%) of the 327 pairs were disparate for Y-chromosome or autosomal chromosome encoded MiHA, respectively (Table 2).

HY disparity is associated with increased aGVHD

In HY mismatched patients a significantly higher grade 3–4 aGVHD incidence of 19% at day 100 after SCT was observed than in HY matched patients (4%, univariable analysis P <0.001) when respecting for the presence of competing risks. Multivariable analysis confirmed this association for HY incompatibility with the increased risk to develop aGVHD grade 3–4 (HR 4.1, 95%CI: 1.6–10.3, P <0.001 Figure 2A). Notably, none of the non-sex linked MiHA mismatched categories (HA1 or autosomal MiHA mismatched) showed an association with acute or chronic GVHD (Figure 2A–B), possibly due to the hematopoietic-restricted tissue expression pattern of the majority of the autosomal MiHA tested.

Figure 2. Occurrence of GVHD following MiHA-mismatched transplantation.

Figure 2

Open in a new tab

Within the complete cohort, the incidence of (A) aGVHD grade 3–4 and (B) limited/extensive cGVHD was analyzed using Fine and Gray competing risk regression models. Groups were categorized based on MiHA disparity in mismatched and matched at the DNA level. GVHD, graft-versus-host-disease; MiHA, minor histocompatibility antigen.

Autosomal MiHA disparity is associated with improved relapse-free survival in sibling transplants

Statistical analysis revealed that mismatches in the studied MiHA did not have an impact on relapse-free survival in the complete transplant cohort (Figure 3A). Because recipients transplanted with a MUD graft might have a HLA-DP mismatch, as well as a higher rate of unknown MiHA mismatches, we separately analyzed the effect of disparities in known MiHA on clinical outcome in the sibling cohort (N=264). Interestingly, recipients transplanted with an autosomal MiHA-mismatched sibling graft showed significantly better RFS in multivariable analysis (HR 0.68, 95%CI: 0.48–0.98, P =0.04; Figure 3B, Table 3). This beneficial effect of autosomal MiHA disparity on RFS could be attributed to both a trend in improved NRM (HR 0.59, 95%CI: 0.29–1.25, P =0.19) and less relapse (HR 0.77, 95%CI: 0.50–1.11, P =0.14) as assessed in multivariable Fine and Gray competing risk analyses. Furthermore, also in sibling allo-SCT HY disparity was associated with a higher incidence of grade 3–4 aGVHD (HR 4.2, 95%CI: 1.6–10.9, P =0.004, Table 3). Analysis of clinical outcome in recipients of a MUD graft (N=61) showed no significant associations of MiHA disparity with neither RFS (Figure 3C) nor aGVHD (data not shown). Taken together, these data indicate that recipients of a HLA-identical sibling donor stem cell graft that is mismatched for the studied autosomal MiHA may induce beneficial GVT immunity post-SCT.

Figure 3. Autosomal MiHA disparity is associated with increased relapse-free survival after transplantation with a sibling graft.

Figure 3

Open in a new tab

Relapse-free survival was analyzed for (A) the complete cohort of recipients versus patients transplanted with (B) a sibling graft or (C) an unrelated graft using the log-rank test. Groups were categorized based on autosomal MiHA disparity in mismatched (black line) and matched (grey line) at the DNA level. Allo-SCT, allogeneic stem cell transplantation; N, number of patients within the group; MiHA, minor histocompatibility antigen. When significant, P-values of multivariable analyses are given.

Table 3.

Univariable analysis of patient characteristics and clinical outcome within the SIB cohort.

Outcome parameter MiHA disparity Univariable analysis Multivariable analysis
HR (95% CI) P-value Confounding risk factors HR (95% CI) P-value
aGVHD gr 3–4 HY 4.80 (2.00–11.20) <0.001 Patient age, diagnosis, year of transplantation, stem cell source, conditioning regimen 4.20 (1.60–10.90) 0.004
MiHA 1.10 (0.50–2.50) 0.74 NA NA NA
cGVHD lim/ext HY 1.30 (0.70–2.20) 0.44 NA NA NA
MiHA 1.01 (0.63–1.67) 0.96 NA NA NA
Time to relapse HY 0.58 (0.35–0.98) 0.04 Diagnosis, year of transplantation, stem cell source, conditioning regimen 0.65 (0.40–1.08) 0.095
MiHA 0.77 (0.53–1.11) 0.16 Diagnosis, year of transplantation, stem cell source, conditioning regimen 0.77 (0.50–1.11) 0.14
RFS HY 0.75 (0.49–1.16) 0.20 Patient age, diagnosis, year of transplantation, stem cell source, conditioning regimen 0.81 (0.52–1.27) 0.35
MiHA 0.73 (0.52–1.03) 0.07 Patient age, diagnosis, year of transplantation, stem cell source, conditioning regimen 0.68 (0.48–0.98) 0.04
OS HY 0.90 (0.52–1.55) 0.70 NA NA NA
MiHA 0.88 (0.56–1.35) 0.56 NA NA NA
NRM HY 1.30 (0.70–2.70) 0.43 NA NA NA
MiHA 0.59 (0.29–1.25) 0.13 Patient age, year of transplantation, CMV seropositive, aGVHD grade 2–4 0.59 (0.29–1.25) 0.19
Open in a new tab

Abbreviations: aGVHD, acute graft-versus-host-disease; cGVHD, chronic graft-versus-host-disease; RFS, relapse-free survival; OS, overall survival; NRM, non-relapse mortality; CMV, cytomegalovirus; CI, confidence interval; MiHA, minor histocompatibility antigen; NA, not applicable. Associations with OS and RFS were analyzed using Kaplan Meier curves and log-rank tests. Associations with cumulative incidences of aGVHD, cGVHD, relapse and NRM were estimated respecting the presence of competing risks using the Gray test. Furthermore, in case a P-value was ≤0.20 in univariable analyses, Cox regression analyses (for the endpoints OS and RFS) and Fine and Gray regression analyses (for the endpoints aGVHD, cGVHD, relapse and NRM) were used to adjust for known confounding risk factors.

MM patients show improved RFS upon transplantation with a MiHA-mismatched sibling graft

Interestingly, our multivariable analysis including the correction for diagnosis subgroups revealed an association between autosomal MiHA disparity and improved RFS in HLA-matched sibling transplantation, which indicates a diagnosis independent effect. To study which patients benefit the most from a disparity in the studied MiHA, we separately analyzed the influence of MiHA mismatches on clinical outcome in different diagnosis subgroups. Notably, we were only able to confirm the significant correlation between the occurrence of a disparate autosomal MiHA and clinical outcome in the MM subgroup. Of the 22 mismatched MM patients, 15 patients were mismatched for 1 MiHA, 6 patients for 2 MiHA and 1 patient for 4 MiHA. Importantly, transplanted MM patients showed improved RFS when at least one autosomal MiHA mismatch was present (HR 0.41, 95%CI: 0.19–0.89, P =0.02, Figure 4A). In addition, patients with an autosomal MiHA mismatch developed significantly less relapse as observed in univariable Fine and Grey competing risk analysis (HR 0.46, 95%CI: 0.21–1.00, P =0.049, Figure 4B). The occurrence of a disparate MiHA was also correlated with an increase in limited cGVHD (univariable analysis P =0.03, Figure 4C). Notably, the effects on clinical outcome parameters were not a result of differences in disease status between MiHA matched and mismatched patients (Figure 4D). Interestingly, 22.6% of the MiHA mismatched MM patients had a HA1 disparity (Figure 4E). HA1 disparity was previously reported to be associated with improved RFS in CML patients who developed aGVHD.19 Altogether, these data suggest that mismatches in the studied MiHA induce improved graft-versus-myeloma immunity following partial T cell-depleted sibling allo-SCT.

Figure 4. Multiple myeloma patients transplanted with a related MiHA-mismatched graft show improved relapse-free survival.

Figure 4

Open in a new tab

Multiple myeloma patients transplanted with a sibling graft were grouped based on autosomal MiHA disparity in mismatched and matched at the DNA level. Between these subgroups differences in (A) relapse-free survival and (B) relapse were analyzed using the log-rank test and Fine and Gray competing risk regression model, respectively. N = number of patients within the group. P-values of univariable analyses are given. (C) The incidence of cGVHD and (D) disease status were analyzed using the Fisher Exact test. (E) Within the total of mismatched MiHA, the relative contribution of each MiHA is depicted. Allo-SCT, allogeneic stem cell transplantation; cGVHD, chronic graft-versus-host-disease; Lim, limited; Ext, extensive; Int, intermediate disease; Adv, advanced disease; n.s., not significant; MiHA, minor histocompatibility antigen.

Ability to induce tetramer+ MiHA-specific T cell responses varies among MiHA

Although genetic MiHA incompatibility shows significant differences on clinical outcome post-SCT, this does not necessarily mean that corresponding T cell responses actually occur. Therefore, we investigated the potential of 15 of the 17 studied MiHA to induce productive MiHA-specific T cell responses in vivo. For this, we analyzed recipient PBMC material obtained at the median of 9 months (range 2–70 months) after allo-SCT using a dual-color MiHA-multimer approach. Patients were classified as having a positive tetramer response, when MiHA-specific CD8+ T cells were detected directly after thawing and/or after one ex vivo peptide re-stimulation. Although we were unable to analyze all patients at the same time interval, we believe that a positive detection of MiHA tetramer+ CD8+ T cells at variable time points reflects either an ongoing effector immune response, or a sustained effector-memory response after immune contraction. In Figure 5A, representative tetramer screenings of three different allo-SCT recipients are depicted. Already in freshly thawed samples low numbers of MiHA-specific CD8+ T cells were observed in 27 out of 40 (67.5%) MiHA T cell responsive patients. In addition, in 13 out of 40 (32.5%) responsive patients, tetramer+ T cells were detectable after 1 week stimulation with MiHA peptide-pulsed EBV-LCL. Notably, when examining the whole cohort of MiHA-mismatched recipients, certain MiHA-specific T cell responses were observed more frequently than others (Figure 5B). Especially, disparity for HA1, HA2, PANE1, LRH1, ACC1 and the HY-chromosome encoded antigens HY.A2 and HY.B7 resulted in MiHA-specific CD8+ T cell responses in 25–60% of the MiHA-mismatched patients. HA8-, SP110- and ZAPHIR-specific CD8+ T cells were found in 10–20%. In contrast, no productive CD8+ T cell responses against ADIR, HwA11, ECGF, HEATR and HY.B8 were observed, despite genetic disparity. These results indicate that certain MiHA appear relatively more productive in inducing MiHA-specific CD8+ T cell responses after partial T cell-depleted allo-SCT than others.

Figure 5. Detection of tetramer+ MiHA-specific CD8+ T cell responses is associated with improved RFS after allo-SCT.

Figure 5

Open in a new tab

Recipient PBMC samples obtained after allo-SCT were analyzed for presence of MiHA-specific CD8+ T cells using the dual-color MiHA-multimer flow cytometry assay. Patients were classified as having a positive tetramer response, when MiHA-specific CD8+ T cells were found directly after thawing, or 7 days after stimulation with peptide-loaded EBV-LCL. (A) The number in the dot plots indicates the percentage of MiHA-specific cells positive for both tetramers (PE and APC) within the CD8+, CD4−, CD14−, CD16−, CD19− T cell population. Three representative examples are shown. (B) For each disparate MiHA, the number of tetramer+ responses (white bars) within the total number of screenings (grey bars) is depicted for the complete cohort of MiHA-mismatched recipients. Percentages indicate the relative number of productive responses. (C–D) Relapse-free survival was analyzed for (C) the complete cohort of recipients versus patients transplanted with (D) a sibling graft using the log-rank test. Groups were categorized based on detection of MiHA-specific T cell responses (black line) vs. no MiHA-specific T cell responses or no mismatching for any of the studied MiHA (grey line) after allo-SCT. Allo-SCT, allogeneic stem cell transplantation; N, number of patients within the group; MiHA, minor histocompatibility antigen. Statistical differences in RFS incidences were analyzed using Kaplan Meier point estimates and their associated errors, univariable P-values are given.

Finally, we examined whether presence of a MiHA-specific T cell response, including those targeting autosomal MiHA (N=25 in the complete cohort, and N=20 in the SIB cohort) or HY (N=15 in both the complete cohort and the SIB cohort), was associated with improved outcome post-transplant. When analyzing the whole RFS curves no significant differences were observed (complete cohort: HR 0.82, 95%CI: 0.52–1.30, P =0.39; SIB cohort: HR 0.73, 95%CI: 0.44–1.22, P =0.23). Nevertheless in the first years after allo-SCT, the RFS of patients with and without a MiHA-specific T cell response clearly differs. Therefore, the RFS incidences at 3 years post-transplant were compared and we found that detection of MiHA-specific T cell responses was associated with improved RFS in both the complete cohort (RFS incidence of 69% vs. 51%, univariable analysis P =0.03, Figure 5C), as well as in the sibling transplants (RFS incidence of 73% vs. 52%, univariable analysis P =0.01, Figure 5D). Notably, this association with improved RFS at 3 years after allo-SCT can be mainly attributed to less relapse in patients with a MiHA-specific T cell response (complete cohort: relapse incidence of 26% vs. 39%, P =0.09; SIB cohort: relapse incidence of 24% vs. 40%, P =0.047), and to a smaller extent to improved NRM (complete cohort: NRM incidence of 5% vs. 10%, P =0.26; SIB cohort: NRM incidence of 3% vs. 9%, P =0.10) as assessed in univariable Fine and Gray competing risk analyses. Despite inclusion of T cell responses against ubiquitously expressed MiHA, including HA8 and HY (i.e. 17 out of 40 or 35 in the complete and the SIB cohort respectively), the incidence of neither grade 3–4 aGVHD (P >0.7) nor limited/extensive cGVHD was affected (P >0.2, univariable Fine and Grey competing risk analysis). To conclude, these results indicate that productive MiHA-specific T cell responses contribute to the beneficial GVT immunity following partial T cell-depleted allo-SCT.

Discussion

As the dominant target antigens in HLA-matched allo-SCT, MiHA play a pivotal role in both GVT responses, as well as GVHD. Precise understanding of involved MiHA-specific T cell responses may not only lead to a better prediction of clinical outcome in allo-SCT recipients, but also provide a rationale for the selection of the most potent MiHA in post-transplant immunotherapy. Interestingly, our statistical analysis of immunogenic MiHA disparity rates in sibling transplants revealed that DNA mismatches in autosomal-encoded MiHA are associated with improved clinical outcome. In particular, MM patients showed a lower incidence of relapse and increased RFS when transplanted with a MiHA-mismatched sibling transplant. In addition, we demonstrated considerable variance in the relative immunogenicity of different MiHA in inducing productive T cell responses post-transplantation. Most importantly, presence of these MiHA-specific T cell responses was associated with improved GVT immunity after partial T-cell depleted allo-SCT.

Characteristics of all recipients and their corresponding donors were analyzed for clinical outcome parameters (Table 3). Importantly, we observed that mismatched autosomal MiHA, including those with a ubiquitous expression pattern, were not correlated with higher incidences of severe acute or chronic GVHD after partial T cell-depleted SCT. In accordance with previous reports, only the HY MiHA was associated with increased frequency of grade 3–4 aGVHD. Similar findings were previously reported by Gratwohl et al.18 and Stern et al.31 who used female-to-male alloreactivity as a model for MiHA HY mismatches. However, not only MiHA play an important role in the FDMR transplants, also non-inherited maternal/paternal antigens (NIMA and NIPA) are involved. In non-T cell-depleted haploidentical sibling NIMA-mismatched allo-SCT, lower aGVHD rates were observed than in NIPA-mismatched recipients.32 In our current study, we have no information on the NIMA or NIPA status of the transplant couples and cannot exclude the influence of these antigens on clinical outcome after partial T cell-depleted allo-SCT. Moreover, unknown MiHA not present in our panel and other general genetic disparities might be important as well. Therefore, we analyzed patients transplanted with a sibling donor separately, thereby circumventing the higher likelihood of unknown polymorphic differences between recipients and donors. Analysis showed again a role for a disparate HY MiHA, and although more cases of severe aGVHD (grade 3–4) were observed, this gender mismatch showed a trend towards a lower incidence of relapse (P =0.095; Table 3), which was previously reported by Gratwohl et al.18.

Our findings indicate that a disparity in at least one known autosomal MiHA was associated with higher RFS, without increasing aGVHD incidence or severity after partial T cell-depleted allo-SCT. This contribution of autosomal MiHA mismatches to improved RFS in the sibling transplant setting becomes even more apparent when compared with patients transplanted with a MUD graft. In the MUD cohort, effects of mismatched MiHA on RFS could not be observed, which might be attributed to the smaller size of this cohort. Furthermore, these MUD patients receive ATG treatment20, which results in an additional in vivo T cell depletion, thereby reducing the chance of inducing tumor-reactive MiHA-specific T cell responses.

Besides the role of alloreactive T cells in the GVT response, the underlying malignancy of the recipients might also be important. We found that autosomal MiHA disparity in sibling transplants was associated with improved RFS in multivariable analysis, which included the correction for diagnosis subgroups, suggesting a diagnosis independent effect. However, we could only confirm the observed significant correlation between the occurrence of disparate autosomal MiHA and improved RFS in the MM subgroup. The reason why we did not observe similar effects in the other diagnosis-subgroups could be related to differences in the immune susceptibility of the various malignancies and the number of patients included in this study. Furthermore, partial T cell-depleted SCT results in a low incidence of acute and chronic GVHD, which might have downgraded the clinical impact of MiHA mismatches. When focusing on patients suffering from MM within the sibling cohort, an evident role for disparate MiHA on both relapse incidence, as well as RFS was observed independent of disease status. Moreover, the occurrence of disparate MiHA was also associated with a higher incidence of limited cGVHD. These results show that especially MM patients can benefit from a MiHA-driven GVT effect in the presence of an acceptable degree of cGVHD. It is well known that MM is an immunogenic tumor and that patients can respond well to DLI.33;34 This phenomenon could be the result of multiple factors, likely including high antigen presentation, good susceptibility to killing, and possibly a good window for immune recognition and killing due to the relatively slow tumor growth. Furthermore, post-transplant treatment with immunomodulatory drugs such as lenalidomide has a promoting impact on GVT immunity.

Overall, we observed low incidence of aGVHD (6.5% grade 3–4) in our partial T cell-depleted setting, even when ubiquitous expressed MiHA mismatches were present. This indicates that there is a potential safe clinical application for transplant mismatching of certain strongly immunogenic MiHA in allo-SCT. Previously, the MiHA HA1, HA2, LRH1 and ACC1 have been implicated to selectively induce a GVT effect without GVHD.7;35–37 We show that they are also potent in inducing MiHA-specific T cell responses in vivo, adding to the promise of these MiHA not only in transplant mismatching but also in vaccination or adoptive T cell strategies. However, timing of the tetramer-based analysis of post-transplant PBMC samples is crucial in detecting MiHA-specific T cell responses, and our study had a rather wide time frame of sampling after allo-SCT, which probably resulted in underscoring the frequency of positive responses. In addition, some of the MiHA-specific T cell responses could be masked (subdominant) by other (un)known MiHA. Moreover, the underlying malignancy might skew the MiHA-specific T cell repertoire towards a particular hematopoietic compartment such as the BM, which may prevent the detection of MiHA-specific T cells in peripheral blood. Therefore, absence of tetramer+ T cells during analysis does not necessarily mean a complete lack of MiHA-specific T cells in vivo. Nevertheless, we observed that presence of MiHA-specific T cell responses resulted in improved RFS at 3 years after allo-SCT despite heterogeneity of the cohort. However, after 5 years the RFS curves of patients with and without MiHA-specific immunity no longer differ. As the group size is relatively limited at start and becomes even smaller in time due to positive events or censoring of patients, the data is likely less reliable at late time-points. Furthermore, due to the heterogeneity of the patient group skewing towards late-relapsing, less immunogenic, malignancies likely occurs over time. It could also well be that the patients that develop late relapses have impaired MiHA-specific T cell immunity due to immune escape mechanisms exploited by surviving tumor cells, like the PD-1/PD-L1 and BTLA/HVEM co-inhibitory pathways as we have reported previously38;39. Due to this negative signaling T cells can become exhausted in time, and patients may lose the advantage of having MiHA-specific immunity post-transplant.

Importantly, occurrence of in vivo MiHA-specific T cell responses, including those recognizing ubiquitously expressed MiHA, including the HY antigens, was not associated with an increased incidence of severe acute or chronic GVHD. Unfortunately, the group of patients having MiHA-specific T cell immunity was too small to focus on diagnosis subgroups or perform multivariable analyses. Therefore, these observations should be confirmed in a larger and more homogenous cohort of allo-SCT recipients with longer follow-up.

In conclusion, this study shows that the occurrence of MiHA mismatches is associated with improved clinical outcome after partial T cell-depleted HLA-matched allo-SCT, particularly in MM patients but likely also in other hematological malignancies. The observed positive effects might be attributed to MiHA-specific CD8+ T cells inducing GVT responses. However, not all MiHA seem to have the same potential to induce MiHA-specific T cells as was shown by tetramer analysis of PBMC samples collected post-transplant. By further studying the MiHA that are most productive in this respect, we can select hematopoietic-restricted MiHA as safe and potent target antigens in allo-SCT in order to prevent or treat tumor recurrences by post-transplant immunotherapeutic strategies.

Supplementary Material

01. Supplemental Figure 1. Gating strategy for detection of tetramer+ MiHA-specific T cell responses.

Recipient PBMC samples, obtained after allo-SCT or DLI were stained with MiHA-multimers in PE and APC, CD8-AlexaFluor 700, CD4-, CD14-, CD16- and CD19-FITC and Sytox Blue. Subsequently, cell populations were analyzed by flow cytometry. Sytox Blue- single cells were selected and gated on CD8+FITC- lymphocytes. Within this population the percentage of cells positive for both tetramers was determined.

Acknowledgments

We would like to thank Dr. Arnold van der Meer for providing us with DNA of transplant couples, Rob Woestenenk for assistance in flow cytometry, and all hematologists of our transplant center for providing patient material. Theo de Witte is a member of the advisory board of Novartis, Celgene and Clavis. The remaining authors have no competing financial interests to declare. This work was supported by grant NIH RO1CA118880 and a grant from the Dutch Cancer Society (KWF 2008-4018).

Footnotes

Conflict of interest disclosure

Theo de Witte is a member of the advisory board of Novartis, Celgene and Clavis. The remaining authors declare no competing financial interests.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Appelbaum FR. Haematopoietic cell transplantation as immunotherapy. Nature. 2001;411:385–389. doi: 10.1038/35077251. [DOI] [PubMed] [Google Scholar]
  • 2.Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood. 1990;75:555–562. [PubMed] [Google Scholar]
  • 3.Bleakley M, Riddell SR. Molecules and mechanisms of the graft-versus-leukaemia effect. Nat Rev Cancer. 2004;4:371–380. doi: 10.1038/nrc1365. [DOI] [PubMed] [Google Scholar]
  • 4.Goulmy E. Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy. Immunol Rev. 1997;157:125–140. doi: 10.1111/j.1600-065x.1997.tb00978.x. [DOI] [PubMed] [Google Scholar]
  • 5.Riddell SR, Berger C, Murata M, Randolph S, Warren EH. The graft versus leukemia response after allogeneic hematopoietic stem cell transplantation. Blood Rev. 2003;17:153–162. doi: 10.1016/s0268-960x(03)00007-9. [DOI] [PubMed] [Google Scholar]
  • 6.Marijt WA, Heemskerk MH, Kloosterboer FM, et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. Proc Natl Acad Sci U S A. 2003;100:2742–2747. doi: 10.1073/pnas.0530192100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kircher B, Stevanovic S, Urbanek M, et al. Induction of HA-1-specific cytotoxic T-cell clones parallels the therapeutic effect of donor lymphocyte infusion. Br J Haematol. 2002;117:935–939. doi: 10.1046/j.1365-2141.2002.03536.x. [DOI] [PubMed] [Google Scholar]
  • 8.Wang W, Meadows LR, den Haan JM, et al. Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein. Science. 1995;269:1588–1590. doi: 10.1126/science.7667640. [DOI] [PubMed] [Google Scholar]
  • 9.Meadows L, Wang W, den Haan JM, et al. The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity. 1997;6:273–281. doi: 10.1016/s1074-7613(00)80330-1. [DOI] [PubMed] [Google Scholar]
  • 10.Ivanov R, Aarts T, Hol S, et al. Identification of a 40S ribosomal protein S4-derived H-Y epitope able to elicit a lymphoblast-specific cytotoxic T lymphocyte response. Clin Cancer Res. 2005;11:1694–1703. doi: 10.1158/1078-0432.CCR-04-1772. [DOI] [PubMed] [Google Scholar]
  • 11.Goulmy E, Schipper R, Pool J, et al. Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation. N Engl J Med. 1996;334:281–285. doi: 10.1056/NEJM199602013340501. [DOI] [PubMed] [Google Scholar]
  • 12.Akatsuka Y, Warren EH, Gooley TA, et al. Disparity for a newly identified minor histocompatibility antigen, HA-8, correlates with acute graft-versus-host disease after haematopoietic stem cell transplantation from an HLA-identical sibling. Br J Haematol. 2003;123:671–675. doi: 10.1046/j.1365-2141.2003.04676.x. [DOI] [PubMed] [Google Scholar]
  • 13.Perez-Garcia A, De la Camara R, Torres A, Gonzalez M, Jimenez A, Gallardo D. Minor histocompatibility antigen HA-8 mismatch and clinical outcome after HLA-identical sibling donor allogeneic stem cell transplantation. Haematologica. 2005;90:1723–1724. [PubMed] [Google Scholar]
  • 14.Lin MT, Gooley T, Hansen JA, et al. Absence of statistically significant correlation between disparity for the minor histocompatibility antigen-HA-1 and outcome after allogeneic hematopoietic cell transplantation. Blood. 2001;98:3172–3173. doi: 10.1182/blood.v98.10.3172. [DOI] [PubMed] [Google Scholar]
  • 15.Spellman S, Warden MB, Haagenson M, et al. Effects of mismatching for minor histocompatibility antigens on clinical outcomes in HLA-matched, unrelated hematopoietic stem cell transplants. Biol Blood Marrow Transplant. 2009;15:856–863. doi: 10.1016/j.bbmt.2009.03.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Heinemann FM, Ferencik S, Ottinger HD, Beelen DW, Grosse-Wilde H. Impact of disparity of minor histocompatibility antigens HA-1, CD31, and CD49b in hematopoietic stem cell transplantation of patients with chronic myeloid leukemia with sibling and unrelated donors. Transplantation. 2004;77:1103–1106. doi: 10.1097/01.tp.0000120175.25116.cb. [DOI] [PubMed] [Google Scholar]
  • 17.Markiewicz M, Siekiera U, Karolczyk A, et al. Immunogenic disparities of 11 minor histocompatibility antigens (mHAs) in HLA-matched unrelated allogeneic hematopoietic SCT. Bone Marrow Transplant. 2009;43:293–300. doi: 10.1038/bmt.2008.326. [DOI] [PubMed] [Google Scholar]
  • 18.Gratwohl A, Hermans J, Niederwieser D, van Biezen A, van Houwelingen HC, Apperley J. Female donors influence transplant-related mortality and relapse incidence in male recipients of sibling blood and marrow transplants. Hematol J. 2001;2:363–370. doi: 10.1038/sj.thj.6200117. [DOI] [PubMed] [Google Scholar]
  • 19.Mutis T, Brand R, Gallardo D, van Biezen A, Niederwieser D, Goulmy E. Graft-versus-host driven graft-versus-leukemia effect of minor histocompatibility antigen HA-1 in chronic myeloid leukemia patients. Leukemia. 2010;24:1388–1392. doi: 10.1038/leu.2010.115. [DOI] [PubMed] [Google Scholar]
  • 20.Schattenberg A, van der Meer A, Preijers F, et al. Addition of ATG to the conditioning regimen is a major determinant for outcome after transplantation with partially lymphocyte-depleted grafts from voluntary unrelated donors. Bone Marrow Transplant. 2004;33:1115–1121. doi: 10.1038/sj.bmt.1704490. [DOI] [PubMed] [Google Scholar]
  • 21.van der Velden WJFM, Blijlevens NM, Maas FM, et al. NOD2 polymorphisms predict severe acute graft-versus-host and treatment-related mortality in T-cell-depleted haematopoietic stem cell transplantation. Bone Marrow Transplant. 2009;44:243–248. doi: 10.1038/bmt.2009.21. [DOI] [PubMed] [Google Scholar]
  • 22.Mandigers CM, Raemaekers JM, Schattenberg AV, et al. Allogeneic bone marrow transplantation with T-cell-depleted marrow grafts for patients with poor-risk relapsed low-grade non-Hodgkin’s lymphoma. Br J Haematol. 1998;100:198–206. doi: 10.1046/j.1365-2141.1998.00539.x. [DOI] [PubMed] [Google Scholar]
  • 23.Schaap N, Schattenberg A, Bar B, et al. Outcome of transplantation for standard-risk leukaemia with grafts depleted of lymphocytes after conditioning with an intensified regimen. Br J Haematol. 1997;98:750–759. doi: 10.1046/j.1365-2141.1997.d01-3499.x. [DOI] [PubMed] [Google Scholar]
  • 24.Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995;15:825–828. [PubMed] [Google Scholar]
  • 25.Lee SJ, Vogelsang G, Flowers ME. Chronic graft-versus-host disease. Biol Blood Marrow Transplant. 2003;9:215–233. doi: 10.1053/bbmt.2003.50026. [DOI] [PubMed] [Google Scholar]
  • 26.Gratwohl A, Stern M, Brand R, et al. Risk score for outcome after allogeneic hematopoietic stem cell transplantation: a retrospective analysis. Cancer. 2009;115:4715–4726. doi: 10.1002/cncr.24531. [DOI] [PubMed] [Google Scholar]
  • 27.Burrows SR, Kienzle N, Winterhalter A, Bharadwaj M, Altman JD, Brooks A. Peptide-MHC class I tetrameric complexes display exquisite ligand specificity. J Immunol. 2000;165:6229–6234. doi: 10.4049/jimmunol.165.11.6229. [DOI] [PubMed] [Google Scholar]
  • 28.Broen K, Greupink-Draaisma A, Woestenenk R, Schaap N, Brickner AG, Dolstra H. Concurrent detection of circulating minor histocompatibility antigen-specific CD8 T cells in SCT recipients by combinatorial encoding MHC multimers. PLoS One. 2011;6:e21266. doi: 10.1371/journal.pone.0021266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Rodenko B, Toebes M, Hadrup SR, et al. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protoc. 2006;1:1120–1132. doi: 10.1038/nprot.2006.121. [DOI] [PubMed] [Google Scholar]
  • 30.Eijsink C, Kester MG, Franke ME, et al. Rapid assessment of the antigenic integrity of tetrameric HLA complexes by human monoclonal HLA antibodies. J Immunol Methods. 2006;315:153–161. doi: 10.1016/j.jim.2006.07.020. [DOI] [PubMed] [Google Scholar]
  • 31.Stern M, Brand R, de Witte T, et al. Female-versus-male alloreactivity as a model for minor histocompatibility antigens in hematopoietic stem cell transplantation. Am J Transplant. 2008;8:2149–2157. doi: 10.1111/j.1600-6143.2008.02374.x. [DOI] [PubMed] [Google Scholar]
  • 32.van Rood JJ, Loberiza FR, Jr, Zhang MJ, et al. Effect of tolerance to noninherited maternal antigens on the occurrence of graft-versus-host disease after bone marrow transplantation from a parent or an HLA-haploidentical sibling. Blood. 2002;99:1572–1577. doi: 10.1182/blood.v99.5.1572. [DOI] [PubMed] [Google Scholar]
  • 33.Lokhorst HM, Schattenberg A, Cornelissen JJ, Thomas LL, Verdonck LF. Donor leukocyte infusions are effective in relapsed multiple myeloma after allogeneic bone marrow transplantation. Blood. 1997;90:4206–4211. [PubMed] [Google Scholar]
  • 34.Alyea E, Weller E, Schlossman R, et al. T-cell--depleted allogeneic bone marrow transplantation followed by donor lymphocyte infusion in patients with multiple myeloma: induction of graft-versus-myeloma effect. Blood. 2001;98:934–939. doi: 10.1182/blood.v98.4.934. [DOI] [PubMed] [Google Scholar]
  • 35.de Rijke B, van Horssen-Zoetbrood A, Beekman JM, et al. A frameshift polymorphism in P2X5 elicits an allogeneic cytotoxic T lymphocyte response associated with remission of chronic myeloid leukemia. J Clin Invest. 2005;115:3506–3516. doi: 10.1172/JCI24832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Hambach L, Vermeij M, Buser A, Aghai Z, van der Kwast T, Goulmy E. Targeting a single mismatched minor histocompatibility antigen with tumor-restricted expression eradicates human solid tumors. Blood. 2008;112:1844–1852. doi: 10.1182/blood-2007-11-125492. [DOI] [PubMed] [Google Scholar]
  • 37.Overes IM, Fredrix H, Kester MG, et al. Efficient activation of LRH-1-specific CD8+ T-cell responses from transplanted leukemia patients by stimulation with P2X5 mRNA-electroporated dendritic cells. J Immunother. 2009;32:539–551. doi: 10.1097/CJI.0b013e3181987c22. [DOI] [PubMed] [Google Scholar]
  • 38.Norde WJ, Maas F, Hobo W, et al. PD-1/PD-L1 interactions contribute to functional T-cell impairment in patients who relapse with cancer after allogeneic stem cell transplantation. Cancer Res. 2011;71:5111–5122. doi: 10.1158/0008-5472.CAN-11-0108. [DOI] [PubMed] [Google Scholar]
  • 39.Hobo W, Norde WJ, Schaap N, et al. B and T Lymphocyte Attenuator Mediates Inhibition of Tumor-Reactive CD8+ T Cells in Patients After Allogeneic Stem Cell Transplantation. J Immunol. 2012;189:39–49. doi: 10.4049/jimmunol.1102807. [DOI] [PubMed] [Google Scholar]
  • 40.Spierings E, Brickner AG, Caldwell JA, et al. The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein. Blood. 2003;102:621–629. doi: 10.1182/blood-2003-01-0260. [DOI] [PubMed] [Google Scholar]
  • 41.den Haan JM, Meadows LM, Wang W, et al. The minor histocompatibility antigen HA-1: a diallelic gene with a single amino acid polymorphism. Science. 1998;279:1054–1057. doi: 10.1126/science.279.5353.1054. [DOI] [PubMed] [Google Scholar]
  • 42.Pierce RA, Field ED, Mutis T, et al. The HA-2 minor histocompatibility antigen is derived from a diallelic gene encoding a novel human class I myosin protein. J Immunol. 2001;167:3223–3230. doi: 10.4049/jimmunol.167.6.3223. [DOI] [PubMed] [Google Scholar]
  • 43.den Haan JM, Sherman NE, Blokland E, et al. Identification of a graft versus host disease-associated human minor histocompatibility antigen. Science. 1995;268:1476–1480. doi: 10.1126/science.7539551. [DOI] [PubMed] [Google Scholar]
  • 44.Brickner AG, Warren EH, Caldwell JA, et al. The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing. J Exp Med. 2001;193:195–206. doi: 10.1084/jem.193.2.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.van Bergen CA, Kester MG, Jedema I, et al. Multiple myeloma-reactive T cells recognize an activation-induced minor histocompatibility antigen encoded by the ATP-dependent interferon-responsive (ADIR) gene. Blood. 2007;109:4089–4096. doi: 10.1182/blood-2006-08-043935. [DOI] [PubMed] [Google Scholar]
  • 46.Tykodi SS, Fujii N, Vigneron N, et al. C19orf48 encodes a minor histocompatibility antigen recognized by CD8+ cytotoxic T cells from renal cell carcinoma patients. Clin Cancer Res. 2008;14:5260–5269. doi: 10.1158/1078-0432.CCR-08-0028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Warren EH, Vigneron NJ, Gavin MA, et al. An antigen produced by splicing of noncontiguous peptides in the reverse order. Science. 2006;313:1444–1447. doi: 10.1126/science.1130660. [DOI] [PubMed] [Google Scholar]
  • 48.Brickner AG, Evans AM, Mito JK, et al. The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL. Blood. 2006;107:3779–3786. doi: 10.1182/blood-2005-08-3501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Akatsuka Y, Nishida T, Kondo E, et al. Identification of a polymorphic gene, BCL2A1, encoding two novel hematopoietic lineage-specific minor histocompatibility antigens. J Exp Med. 2003;197:1489–1500. doi: 10.1084/jem.20021925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Slager EH, Honders MW, van der Meijden ED, et al. Identification of the angiogenic endothelial-cell growth factor-1/thymidine phosphorylase as a potential target for immunotherapy of cancer. Blood. 2006;107:4954–4960. doi: 10.1182/blood-2005-09-3883. [DOI] [PubMed] [Google Scholar]
  • 51.Broen K, Levenga H, Vos J, et al. A polymorphism in the splice donor site of ZNF419 results in the novel renal cell carcinoma-associated minor histocompatibility antigen ZAPHIR. PLoS One. 2011;6:e21699. doi: 10.1371/journal.pone.0021699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Warren EH, Gavin MA, Simpson E, et al. The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen. J Immunol. 2000;164:2807–2814. doi: 10.4049/jimmunol.164.5.2807. [DOI] [PubMed] [Google Scholar]
  • 53.Bleakley M, Otterud BE, Richardt JL, et al. Leukemia-associated minor histocompatibility antigen discovery using T-cell clones isolated by in vitro stimulation of naive CD8+ T cells. Blood. 2010;115:4923–4933. doi: 10.1182/blood-2009-12-260539. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplemental Figure 1. Gating strategy for detection of tetramer+ MiHA-specific T cell responses.

Recipient PBMC samples, obtained after allo-SCT or DLI were stained with MiHA-multimers in PE and APC, CD8-AlexaFluor 700, CD4-, CD14-, CD16- and CD19-FITC and Sytox Blue. Subsequently, cell populations were analyzed by flow cytometry. Sytox Blue- single cells were selected and gated on CD8+FITC- lymphocytes. Within this population the percentage of cells positive for both tetramers was determined.

NIHMS411012-supplement-01.jpg (2.5MB, jpg)

RESOURCES