The biological significance of HLA-DP gene variation in haematopoietic cell transplantation

Authors


Dr Effie W. Petersdorf, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, D4–100, Seattle, WA 98109–1024, USA. E-mail: epetersd@fhcrc.org

Abstract

Although it has been over 25 years since HLA-DP was mapped to the major histocompatibility complex (MHC), its biological functions remain ill-defined. We sought to test the hypothesis that HLA-DP functions in a manner similar to that of other class II genes by measuring the risk of clinically severe grades III–IV acute graft-vs.-host disease (GVHD) associated with recipient HLA-DP disparity after haematopoietic cell transplantation. HLA-DPB1 exon 2 was sequenced in 205 patients who underwent transplantation from HLA-A, -B, -C, -DRB1 and -DQB1 allele-matched unrelated donors. HLA-DPB1 mismatched recipients experienced a significantly increased risk of acute GVHD compared with HLA-DP-identical transplants. Patients who were mismatched for a single HLA-DPB1 allele had an odds ratio (OR) of 1·0 (0·5, 2·2; P = 0·99) and patients who were mismatched for two alleles had an OR of 2·2 (1·0, 4·9; P = 0·06) for developing acute GVHD. Compared with matched and single-allele mismatched transplants, patients who were mismatched for two DPB1 alleles had an OR of 2·2 (1·2, 4·1; P = 0·01). HLA-DP plays an important role in the alloimmune response. A threshold effect of multiple HLA-DP disparities is evident in determining the risk of acute GVHD after haematopoietic cell transplantation from unrelated donors.

In 1980, a third locus within the major histocompatibility complex (MHC) class II region was identified that was capable of stimulating proliferative and cytotoxic responses in primed T cells (Shaw et al, 1980; Termijtelen et al, 1983). The locus was eventually named HLA-DP in 1984 following the Ninth International Histocompatibility Workshop (Bodmer et al, 1984). HLA-DP maps to 6p21.3 approximately 3 Mb centromeric to HLA-A (Rhodes & Trowsdale, 1999) and is comprised of a highly polymorphic B gene (DPB1) and an A gene (DPA1) that exhibits limited polymorphism (Bodmer et al, 1999). Studies of racially diverse populations have confirmed strong linkage disequilibrium between HLA-DPA1 and DPB1, but weak disequilibrium between HLA-DP and HLA-DR-HLA-DQ (Begovich et al, 1992; Spurkland & Vartdal, 1992; Sage et al, 1994; Hurley et al, 2000).

Originally, six HLA-DP allospecificities were defined using primed lymphocyte typing (PLT) reagents in a secondary mixed lymphocyte culture (MLC) (Shaw et al, 1980; Termijtelen et al, 1983; Wank & Schendel, 1984). Monoclonal antibodies specific for HLA-DP beta epitopes have been developed and provide a serological definition of HLA-DP alloantigens (Marshall et al, 1998). The failure of HLA-DP to stimulate consistent responses in primary (6 d) MLCs has limited its utility as an in vitro assay to evaluate HLA-DP disparity between donors and recipients (Farrell et al, 1988; Olerup et al, 1990). It was not until the advent of DNA-based methods that a precise and informative means for typing the unique alleles of HLA-DPB1 became available (Bugawan et al, 1990; Versluis et al, 1993). Application of DNA-based methods to the study of human populations has uncovered a substantial diversity of HLA-DP, with more than 89 new alleles described (Bodmer et al, 1999).

Insight into the role of HLA-DP in the immune response has been provided by disease association studies. HLA-DP may serve as a marker for susceptibility to arthritis, multiple sclerosis, hard metal lung disease and coeliac disease, indicating a potential role for HLA-DP in antigen presentation and HLA-DP-restricted T-cell responses (Begovich et al, 1989; Bugawan et al, 1989; Richeldi et al, 1993; Potolicchio et al, 1997; Yu et al, 1998). In renal transplantation, the clinical importance of disparity for HLA-DP between recipients and their donors has not been entirely defined (Rosenberg et al, 1992; Mytilineos et al, 1997). To date, the biological role of HLA-DP in haematopoietic stem cell transplantation has been inconclusive because of the relatively small numbers of informative HLA-DP-matched transplants, the complexity of multigene interactions and other risk factors that can affect clinical outcome after stem cell transplantation (Pawelec et al, 1986; al-Daccak et al, 1990; Kato et al, 1991; Petersdorf et al, 1993, 1998; Sasazuki et al, 1998; Varney et al, 1999).

Patients and methods

Study population Between May 10, 1985, and August 8, 1999, 572 patients received a marrow transplant at our centre from unrelated donors for the treatment of chronic myeloid leukaemia (CML) in chronic phase, accelerated phase or blast phase in remission. In an effort to reduce the effects of additive HLA disparities and of known risk factors for GVHD and survival (Hansen et al, 1998; Petersdorf et al, 1998), we restricted the current analysis to consecutive transplants who satisfied the following criteria (n = 205): (i) donors and recipients were allele-matched for graft-vs.-host recognition at HLA-A, -B, -C, -DRB1 and -DQB1; (ii) patients were prepared for transplantation with the use of a myeloablative regimen consisting of cyclophosphamide and total body irradiation; (iii) patients received T-replete marrow grafts using cyclosporin and methotrexate for GVHD prophylaxis; and (iv) DNA was available for retrospective DPB1 sequencing for both the recipient and the donor.

HLA typing HLA-A, -B and -C alleles were sequenced using direct automated fluorescence sequencing methods as described (Petersdorf et al, 1998). HLA-DRB1 and -DQB1 alleles were typed using sequence-specific oligonucleotide probe-based methods (Petersdorf et al, 1996). HLA-DQA1 and HLA-DPA1 were not given consideration in this study owing to the strong positive linkage disequilibrium with DQB1 and DPB1 respectively (Begovich et al, 1992; Spurkland & Vartdal, 1992; Sage et al, 1994; Hurley et al, 2000). Typing for HLA-DPB1 alleles in the current study was performed using direct automated fluorescence sequencing. HLA-DPB1 genes were amplified using 100–200 ng of genomic DNA, 0·75 U of AmpliTaq DNA Polymerase, and either 22·5 μl of HLA-DPB1 reverse polymerase chain reaction (PCR) ready mix (kind gift from Dr Malcolm McGinnis, Applied Biosystems) or buffer containing 10 mmol/l Tris-HCl (pH 8·3), 50 mmol/l KCl, 1·5 mmol/l MgCl2, 1·5 μl of 2·5 mmol/l dATP, dCTP, dGTP and dTTP, and 25 pmol of each primer DPB5 and DPB3 (Rozemuller et al, 1996). Initial denaturation at 98°C for 2 min was followed by 32 cycles of denaturation at 96°C for 45 s, annealing at 65°C for 1 min and extension at 72°C for 2 min. The final extension at 72°C for 10 min was followed by 4°C hold.

Amplified products were purified using Multiscreen-PCR filter plates according to manufacturer's protocols (Millipore, Bedford, MA, USA) with the following modification: PCR products were eluted with 75 μl of ddH2O and concentrated to 5–10 μl volume using Speed Vac Plus SC110A Concentrator (Savant Instruments, Holbrook, NY, USA). Purified PCR products were analysed using a direct fluorescence sequencing method as previously described (Petersdorf et al, 1999). The primer DPB5F (Rozemuller et al, 1996) was used to sequence exon 2 of the DPB1 gene in the 5′ orientation.

Sequencing data were manually reviewed, and HLA-A, -B and -C alleles were assigned by using alignments with known sequences. HLA-DPB1 sequence data were analysed using MatchTools PPC program (Applied Biosystems, Foster City, CA, USA) and by manual review.

When more than one possible combination of two DPB1 alleles could have resulted in the same chromatogram, allele-specific amplification was performed as follows: primer pair GSAP107C/DPB3 for heterozygous combinations DPB1*0401,0402/2301,5101; DPB5/GSAP205C for heterozygous combinations DPB1*02012,0401/0402,3301, DPB1*02012,2001/0402,0601, DPB1*02012,3501/0402,0901, DPB1*0301,1001/0901,2501/1401,3701, DPB1*0401,0601/ 20011,3301 and DPB1*0401,0901/3301,3501; primer pair DPB5/GSAP226C for heterozygous combinations DPB1*02012,1401/4501,4601, DPB1*02012,0301/0402, 2901/2501,4601, DPB1*02012,0901/1001,4601, DPB1* 0301, 0601/20011,2901, DPB1*0301,1601/0801,2001, DPB1*0301,1701/0601,3501/0901,2001 and DPB1*0202, 1001/4801,5401; and primer pair DPB5/GSAP256G for DPB1*0401,0501/2401,6301. Direct fluorescence sequencing was performed using primers DPB5F or DPB3F. Primer sequences have previously been described in detail (Versluis et al, 1995; Rozemuller et al, 1996). One new allele-specific primer DP03/26 (5′cagagaattacgt gtaccagttac3′, located at exon 2 position 11–34) was designed to resolve two heterozygous combinations DPB1*01011,0301/DPB1*26012,5001 and DPB1*01011, 20011/DPB1*2701,5001.

Transplant procedure All patients were prepared for transplantation with intravenous cyclophosphamide (60 mg/kg recipient body weight) administered on each of two successive days followed by total body irradiation (900–1575 cGy determined by phase of disease) from dual opposing 60Co sources. Prophylaxis for cytomegalovirus (CMV), fungal and Pneumocystis carinii infection was administered as described (Hansen et al, 1998). All protocols and consent forms were reviewed and approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center. The severity of acute GVHD was assessed according to criteria previously described (Petersdorf et al, 1998).

Statistical methods The major objective of this retrospective study was to test the hypothesis that HLA-DPB1 disparity increases the risk of clinically significant grades III–IV acute GVHD after unrelated donor stem cell transplantation for CML. Mismatching at HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 was defined as the presence of recipient alleles not shared by the donor (recipient disparity). The probability of grades III–IV GVHD was estimated among patients mismatched for 0, 1 and 2 HLA-DPB1 alleles, and odds ratios were obtained using logistic regression. In addition, the distribution of grades of GVHD was compared among groups using the Kruskal–Wallis test. The hazard of mortality and appropriate hazard ratios were estimated using proportional hazards regression. As HLA-DPB1 was not considered during donor selection, potential imbalances in non-HLA risk factors between groups defined by number of mismatched HLA-DPB1 alleles should have occurred only through chance. To account for this possibility, however, multivariable regression models were fitted by considering non-HLA risk factors previously shown to be associated with increased GVHD and mortality. Estimates of the probability of survival were obtained using the method of Kaplan and Meier (1958), and the probability of GVHD was summarized using cumulative incidence estimates (Gooley et al, 1999), in which death without GVHD was regarded as a competing risk for GVHD. Global tests were conducted to examine the difference in distribution of GVHD grades (using the Kruskal–Wallis test), probability of severe GVHD (score test) and the hazard of mortality (score test). If the global tests yielded significant or suggestive differences, selected pairwise comparisons were made. Two-sided P-values resulting from regression models appropriate to these comparisons were derived from the Wald test and no adjustments were made for multiple comparisons.

Results

Demographic characteristics

Patient and donor characteristics are displayed in Table I for the three groups defined by the number of disparate recipient alleles (0, 1 and 2 DPB1 mismatched alleles for graft-vs.-host immunological recognition). All three groups were balanced for known GVHD risk factors (Hansen et al, 1998).

Table I.  Transplant characteristics of the CML study population. Known GVHD and mortality risk factors are given for the 0, 1 and 2 DPB1 allele-mismatched groups.
 Number of mismatched HLA-DPB1 recipient alleles
0 (n = 49)1 (n = 90)2 (n = 66)
  1. *Body weight index, actual body weight divided by ideal body weight × 100% (Hansen et al, 1998).

  2.   †Marrow cell dose, × 106 nucleated cells/kg recipient body weight.

  3.   ‡FLU/GCV, fluconazole/ganciclovir (Hansen et al, 1998).

  4.   §Disease phase, CP (chronic phase); AP (accelerated phase); CP2 (second chronic phase).

  5.   CMV, cytomegalovirus.

Body weight index*Median 1·09Median 1·10Median: 1·08
Range: 0·93–1·54Range: 0·81–1·77Range: 0·82–1·58
Years from diagnosis to transplantMedian: 1·10Median: 1·25Median: 1·14
Range: 0·46–9·50Range: 0·23–8·60Range: 0·34–17·70
Patient ageMedian: 36Median: 38·5Median: 35
Range: 22–55Range: 6–54Range: 14–54
Marrow cell doseMedian: 24·76Median: 24·16Median: 25·48
Range: 1·52–168·72Range: 1·21–75·47Range: 8·51–257·41
Transplant date (% after July 1991)42 (86%)67 (74%)50 (76%)
Donor parity
 Male donor33 (67%)60 (67%)45 (68%)
 Parous female8 (16%)21 (23%)14 (21%)
 Nulliparous female6 (12%)8 (9%)5 (8%)
 Unknown female2 (4%)1 (1%)2 (3%)
CMV positivity
 Patient23 (47%)40 (44%)28 (42%)
 Donor12 (24%)20 (22%)20 (30%)
FLU/GCV available40 (82%)67 (74%)50 (76%)
Disease phase§
 CP38 (78%)64 (71%)46 (70%)
 AP11 (22%)20 (22%)16 (24%)
 CP206 (7%)4 (6%)
Follow-up (years) among survivorsMedian: 3·1Median: 4·0Median: 5·6
Range: 1·0–13·1Range: 1·0–14·0Range: 0·8–11·2

HLA-DPB1 typing and matching

To assess the role of HLA-DP mismatching in the development of clinically severe grades III–IV acute GVHD and in mortality after haematopoietic stem cell transplantation, we sequenced exon 2 of DPB1 in 205 patients and their unrelated donors using direct automated methods. Both DPB1 alleles could be assigned directly from the interpretation of the sequencing chromatogram in 289 (70%) of the 410 samples. For the remaining 121 samples (30%), allele-specific amplification followed by direct sequencing of the PCR product was performed to identify the two DPB1 sequences. Twenty-two of the 86 known DPB1 alleles were detected in our study population. Of the 820 DPB1 alleles typed in the 410 samples, HLA-DPB1*0401 was the most frequent (44%), followed by DPB1* 0201 (12%), DPB1*0402 (10%), DPB1*0301 (10%) and DPB1*0101 (7%). Three hundred and fifteen samples (77%) were HLA-DP heterozygous and 95 samples (23%) were homozygous. The homozygous pair DPB1*0401,0401 was the most frequently assigned genotype (19%), followed by heterozygous pairs DPB1* 0201,0401 (12%), DPB1*0401,0402 (9%), DPB1*0301,0401 (8%) and DPB1*0101,0401 (7%). We discovered one novel DPB1 allele among the 410 samples (unpublished observations).

Engraftment

All but two patients who survived for more than 28 d after the transplant had engraftment, as indicated by recovery of neutrophil counts above 0·5 × 109/l. Both patients were mismatched for a single DPB1 allele. One patient died on d 40. The second patient received a second transplant on d 54 and is alive at last contact on d 1955.

Acute GVHD

Acute GVHD data were available for 200 of the 205 patients. Of these 200 patients, 49 (25%) were matched, 90 (45%) were mismatched for one DPB1 allele and 61 (31%) were mismatched for two DPB1 alleles. Of the 200 HLA-A, -B, -C, -DRB1 and -DQB1 allele-matched transplants, 32 developed grades 0–I GVHD, 168 developed grades II–IV GVHD and 64 developed grades III–IV GVHD. The distribution of GVHD grades across the three groups was significantly different (global P = 0·05). The presence of a single HLA-DPB1 disparity did not affect the severity of GVHD, but the presence of two HLA-DPB1 disparities was associated with more severe GVHD than in patients with no disparity (Table II). Clinically significant grades III–IV GVHD occurred in 13 out of 49 (27%) DPB1-matched, 24 out of 90 (27%) one-DPB1 allele-mismatched and 27 out of 61 (44%) two-DPB1 allele-mismatched recipients (Fig 1, Table II). Univariate analysis of the association between HLA-DPB1 mismatching and the development of grades III–IV acute GVHD led to a global P-value = 0·05, and selected pairwise comparisons yielded an odds ratio (OR) of 1·0 (95% confidence interval: 0·5, 2·2; P = 0·99) for one-allele mismatch and an OR of 2·2 (1·0, 4·9; P = 0·06) for two-allele mismatches compared with DPB1-matched patients.

Table II.  Number of transplants developing grades 0, I, II, III or IV acute GVHD according to the number of HLA-DPB1 allele disparities in the recipient. Global P = 0·05.
 Number of recipient DPB1 disparities
GVHD Grade012
  1. Zero disparities vs. one disparity, P = 0·77 (unadjusted for multiple comparisons). Zero disparities vs. two disparities, P = 0·04 (unadjusted for multiple comparisons).

07103
I354
II265127
III112124
IV233
Figure 1.

Probability of developing grades III–IV acute GVHD according to 0, 1 or 2 DPB1-mismatched alleles.

Multivariable regression models were developed to examine the possible confounding effects of other GVHD risk factors (Table I). None of these risk factors had a marked effect on the magnitude of the association between DPB1 disparity and the probability of grades III–IV acute GVHD or the distribution of grades of GVHD. The adjusted OR for single-allele mismatched compared with matched pairs remained 1·0, and the adjusted OR for two-allele mismatched compared with matched pairs remained 2·1–2·2. These results indicate that a single HLA-DPB1 disparity was well-tolerated among patients matched for HLA-class I, DRB1 and DQB1, but two DPB1 disparities conferred a significant risk of severe GVHD. These data strongly suggest that the total number of HLA disparities is an important factor in determining acute GVHD risk after transplantation.

Survival

Survival was evaluated in the entire population of 205 CML transplants. Death occurred in 14 out of 49 (29%) HLA-DPB1-matched patients, 29 out of 90 (32%) one HLA-DPB1 allele-mismatched patients, and 27 out of 66 (41%) two HLA-DPB1 allele-mismatched patients. The Kaplan–Meier probabilities of survival at 3 years post transplant in these three groups were 73% (95% confidence interval: 61%, 86%), 69% (59%, 79%) and 59% (46%, 71%) respectively (Fig 2). In a univariate model, there was little suggestion of a difference in the hazards of mortality between the three groups of patients (global P = 0·38). In a multivariable model that adjusted for stage of disease (chronic phase vs. accelerated phase vs. second chronic phase), time interval from diagnosis to transplant (continuous variable), patient age (continuous variable) and the availability of fluconazole/ganciclovir peri-transplant, the hazards among groups did not differ significantly (P = 0·59). These results indicate that two-DPB1 allele mismatches conferred a substantially increased risk of severe acute GVHD, but mismatching did not have as significant an effect on survival.

Figure 2.

Kaplan–Meier probability of survival according to 0, 1 or 2 DPB1-mismatched alleles.

Discussion

We previously published a study of 129 patients who underwent marrow transplantation from HLA-A and -B serologically matched, and -DRB1 and -DQB1 allele-matched unrelated donors for the treatment of a variety of haematological disorders (Petersdorf et al, 1993). Application of sequence-specific oligonucleotide probe-based methods to type DPB1 alleles yielded an 80% mismatch and 20% match rate. The 10% difference in GVHD risk observed between the HLA-DP-matched and -mismatched groups was not statistically significant.

New information on the extent of class I allele disparity among phenotypically matched donors and recipients, and on the role of class I gene disparity in transplant outcome, has since become available (Petersdorf et al, 1998). A more complete understanding of the function of class I genes together with changes in the study design and a much larger transplant experience have now enabled the elucidation of the role of HLA-DP disparity in unrelated stem cell transplantation. Disparity for both class I and II has a significant effect on GVHD risk and mortality (Petersdorf et al, 1998; Sasazuki et al, 1998). By restricting our study population to patients who were matched for HLA-A, -B, -C, -DRB1 and -DQB1 alleles, we eliminated any contribution from these genes to the clinical end-points. The sequencing-based typing method was more complete than the probe-based method used previously, allowing us to define the matched and mismatched groups more accurately. Finally, in order to limit the potential contribution of other risk factors for GVHD, we confined the current study to good-risk CML patients (Hansen et al, 1998).

Increased acute GVHD risk in the two DPB1 allele-mismatched group compared with the matched and single allele-mismatched groups was consistent with a threshold effect for T-cell activation; two HLA-DP disparities may activate more T-cell clones than a single disparity. The influence of additive disparities is compatible with our previous observation that the presence of two or more HLA-DRB1 and/or DQB1 allele mismatches increased GVHD risk (Petersdorf et al, 1998). These results indicate that the total number of disparities may be as important a risk factor as the nature of the disparity. The results from the current study are also consistent with historical data from haploidentical transplants showing that the number of mismatched HLA loci has a profound effect on the risk of GVHD (Anasetti, 1999). Although GVHD risk was significantly affected by the number of mismatched DPB1 alleles, the differences in survival between the 0, 1 and 2 allele mismatches were not as large. The power to detect a clinically meaningful difference in survival was limited even in this relatively large homogeneous patient population.

Single DPB1 mismatches did not have a detectable effect on acute GVHD risk. In a previous study of unrelated donor transplants who were characterized for all HLA genes with the exception of DPB1, a single class II DRB1 or DQB1 allele mismatch conferred a significantly increased risk of grades III–IV acute GVHD (Petersdorf et al, 1998). The new information from the current DPB1 study implies that most of the historical ‘single class II mismatched’ pairs actually had DPB1 differences in addition to those at DRB1 and DQB1. Hence, the risk of GVHD previously observed for ‘single’ DRB1 and DQB1 mismatches may have resulted from combinations of DRB1, DQB1 and DPB1 disparities. The current analysis tested the hypothesis that HLA-DPB1 disparity increases acute GVHD risk by studying transplant pairs who were matched at all other HLA loci. As such, we did not have the opportunity to address the potential multilocus effects of mismatching for combinations of class I and class II disparities. Effects of multilocus mismatching may be additive and can be evaluated only in a large patient population without restriction to match status at these loci.

The relatively weak degree of linkage disequilibrium between HLA-DP and HLA-DR presents a clinical challenge in the identification of well-matched unrelated volunteer donors for transplantation. In this study, only 25% of HLA-A-, -B-, -C-, -DRB1- and -DQB1-matched patients were DPB1-matched with their donors. In order to address whether prospective DPB1 matching is feasible, we have initiated a prospective study of all patients referred to our centre for unrelated donor searches for CML. This pilot study will measure the extent to which DPB1-matched donors can be identified, the total number of donors required to be tested per patient in order to identify at least one matched donor, and the time required to type and match for HLA-DP. In this way, the biologically important information that HLA-DP functions as a transplantation determinant by initiating GVHD can now be applied to improve the selection of unrelated donors for the overall optimization of haematopoietic cell transplantation as curative therapy for bone marrow malignancies.

Acknowledgments

We thank Dr Malcolm McGinnis and Applied Biosystems for the kind donation of HLA-DP sequencing reagents. This work was supported by grants CA18029, CA72978, CA15704 and AI33484 from the National Institutes of Health.

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