Biology of Blood and Marrow Transplantation
Volume 14, Issue 1 , Pages 75-87, January 2008

Donor Killer Immunoglobulin-Like Receptor (KIR) Genotype-Patient Cognate KIR Ligand Combination and Antithymocyte Globulin Preadministration Are Critical Factors in Outcome of HLA-C-KIR Ligand-Mismatched T Cell–Replete Unrelated Bone Marrow Transplantation

  • Toshio Yabe

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • Corresponding Author InformationCorrespondence and reprint requests: Toshio Yabe, PhD, Japanese Red Cross Tokyo Metropolitan Blood Center, 2-1-67 Tatsumi, Koto-ku, Tokyo 135-8639, Japan; Tel: +81-3-5534-7546; Fax: +81-3-5534-7547.
    • ,
  • Keitaro Matsuo

      Affiliations

    • Aichi Cancer Center Research Institute, Division of Epidemiology and Prevention, Nagoya, Japan
    • ,
  • Kouyuki Hirayasu

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • University of Tokyo, Department of Human Genetics, Tokyo, Japan
    • ,
  • Koichi Kashiwase

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • ,
  • Sumiyo Kawamura-Ishii

      Affiliations

    • Japanese Red Cross Central Blood Institute, Tokyo, Japan
    • ,
  • Hidenori Tanaka

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • ,
  • Atsuko Ogawa

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • ,
  • Minoko Takanashi

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • ,
  • Masahiro Satake

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • ,
  • Kazunori Nakajima

      Affiliations

    • Japanese Red Cross Tokyo Metropolitan Blood Center, Tokyo, Japan
    • ,
  • Katsushi Tokunaga

      Affiliations

    • University of Tokyo, Department of Human Genetics, Tokyo, Japan
    • ,
  • Hidetoshi Inoko

      Affiliations

    • Tokai University School of Medicine, Division of Molecular Science, Isehara, Japan
    • ,
  • Hiroo Saji

      Affiliations

    • HLA Laboratory, NPO, Kyoto, Japan
    • ,
  • Seishi Ogawa

      Affiliations

    • Tokyo University Hospital, Tokyo, Japan
    • ,
  • Takeo Juji

      Affiliations

    • Japanese Red Cross Central Blood Institute, Tokyo, Japan
    • ,
  • Takehiko Sasazuki

      Affiliations

    • International Medical Center of Japan, Tokyo, Japan
    • ,
  • Yoshihisa Kodera

      Affiliations

    • Japanese Red Cross Nagoya First Hospital, Nagoya, Japan
    • ,
  • Yasuo Morishima

      Affiliations

    • Aichi Cancer Center Hospital, Department of Hematology and Cell Therapy, Nagoya, Japan
    • ,
  • Japan Marrow Donor Program

Received 18 July 2007; accepted 21 September 2007.

Article Outline

Abstract 

We previously reported the potent adverse effects of killer immunoglobulin-like receptor (KIR) ligand mismatch (KIR-L-MM) on the outcome of T cell–replete unrelated hematopoietic stem cell transplantation (UR-HSCT) through the Japan Marrow Donor Program. Other UR-HSCT studies have yielded inconsistent results. To address this discrepancy, we evaluated candidate factors contributing to the effects of KIR-L-MM on transplantation outcomes in retrospectively selected hematologic malignancy cases with uniform graft-versus-host disease (GVHD) prophylaxis (n = 1489). KIR-L-MM in the graft-versus-host direction (KIR-L-MM-G) was associated with a higher incidence of acute GVHD (aGVHD; P < .002) and a lower overall survival (OS; P < .0001) only without the preadministration of antithymocyte globulin (ATG). Furthermore, in KIR-L-MM-G, the donor KIR2DS2 gene with the patient cognate C1 ligand was associated with a higher incidence of aGVHD (P = .012). Multivariate analysis by Cox proportional hazard models suggested that donor 2DS2 and ATG preadministration were critical factors in grade III-IV aGVHD (hazard ratio = 1.96; 95% confidence interval = 1.01-3.80; P = .045, and hazard ratio = 0.56; 95% confidence interval = 0.31-0.99; P = .047, respectively). These results indicate that the adverse effects of KIR-L-MM-G depend on combination of donor-activating KIR genotype-patient cognate KIR ligand type and no ATG preadministration, thereby suggesting the importance of these factors in UR-HSCT and in leukemia treatment using natural killer (NK) cell alloreactivity.

 

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Introduction 

Natural killer (NK) cell alloreactivity plays an important role in hematopoietic stem cell transplantation (HSCT,) and its therapeutic use in leukemia treatment has been considered because of its possible graft-versus-leukemia (GVL) effect [1]. The beneficial effects of NK cell receptor killer immunoglobulin-like receptor (KIR) ligand incompatibility between patient and donor in the HLA-mismatched related hematopoietic stem cell transplantation (R-HSCT) has been reported 2, 3. These effects in unrelated hematopoietic stem cell transplantation (UR-HSCT) have been controversial, however [4]. We recently reported the potent adverse effects of HLA-C-KIR ligand incompatibility (ligand–ligand analysis) in unrelated T cell–replete HLA-A, -B, and -DR serologically matched bone marrow transplantation without preadministration of antithymocyte globulin (ATG) (n = 1790) through the Japan Marrow Donor Program (JMDP) [5]. Other UR-HSCT studies have documented either adverse or beneficial effects of KIR ligand incompatibility on transplantation outcome 6, 7, 8, 9, 10, 11, 12, 13, 14.

Candidate factors possibly accounting for this discrepancy include T cell depletion, KIR genotype of patients and donors, sample size, ethnicity, number and source of stem cells, ATG preadministration, graft-versus-host disease (GVHD) prophylaxis, and diseases. Associations between KIR genotype and clinical outcome have been reported in both related HLA-identical transplantation 15, 16, 17, 18, 19, 20, 21, 22 and unrelated transplantation settings 10, 23, 24, 25, 26, 27. However, the contribution of the KIR genotype to KIR ligand compatibility has not yet been well defined. Preadministration of ATG in the conditioning regimen both reduces stem cell rejection by host lymphocytes and prevents GVHD by donor-derived lymphocytes, as the drug remains in the patient's blood for several weeks after transplantation and affects residual donor mature lymphocyte activity and reconstitution of the lymphocyte repertoire from donor stem cells [28]. Earlier UR-HSCT studies have demonstrated the need for ATG administration to gain the beneficial effect of NK cell alloreactivity [7], whereas an adverse effect of KIR-L-MM has been reported in both a non-ATG preadministration study [6] and ATG preadministration studies 8, 9; however, a direct comparison of the ATG-administration and ATG-nonadministration groups in a single large cohort has never been performed. Such a study is desirable for a precise evaluation of the effect of ATG on KIR-L-MM.

In this study, patients with hematologic malignancy cases who received uniform GVHD prophylaxis were retrospectively selected from patients undergoing unrelated bone marrow transplantation through the JMDP. All cases were HLA-A, -B, and -DR serologically matched (ie, including HLA-A, -B, and -DR allele-mismatched pairs as well as HLA-Bw4 and HLA-A3 and -A11 KIR ligand matched and HLA-C-KIR ligand matched and mismatched pairs) and mostly uniform with regard to ethnicity and transplantation regimens 29, 30. In these cases, the effects of KIR genotype, KIR ligand compatibility, and ATG administration status on transplantation outcomes were analyzed to resolve the discrepant findings regarding the effects of KIR-L-MM.

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Patients and Methods 

Patient and Cohort Selection Criteria 

A cohort (n = 1489) was selected from among patients undergoing unrelated bone marrow transplantation between 1993 and 2000 through the JMDP. Characteristics of the patients and donors are summarized in Table 1. A source of hematopoietic stem cells of all transplantations were from T cell–replete and HLA-A, -B, and -DR serologically matched bone marrow. Patients with hematologic malignancies, including 401 cases of acute myelogenous leukemia (AML), 438 cases of acute lymphoblastic leukemia (ALL), 451 cases of chronic myelogenous leukemia (CML), 137 cases of myelodysplastic syndrome (MDS), and 62 cases of malignant lymphoma (non-Hodgkin lymphoma) were analyzed. GVHD prophylaxis other than the combination of cyclosporine and short-term methotexate (the most common treatment reported in the JMDP [68.1%]) was excluded. Ninety-four patients with preadministered ATG were included and analyzed separately or together with the nonadministered cases. Standard risk for relapse was defined as the status of first complete remission (CR) of AML or ALL, first chronic phase (CP) of CML at transplantation, or refractory anemia (RA) in MDS. High risk was defined as a more advanced status than standard risk in AML, ALL, CML, and MDS. All patients were preconditioned with a myeloablative regimen, and 1204 patients received total body irradiation (TBI)-containing regimens, whereas 285 received non–TBI-containing regimens. The final clinical survey of these patients was performed as of June 1, 2005. The mean and range for clinical follow-up were 2914 days and 1639-4597 days, respectively. A part of the subject population (leukemia treated with cyclosporine and short-term methotexate; n = 1210) was overlapped with that reported in our previous study [5]. Written informed consent was obtained from all patients and donors, and the study design was approved by the institutional review boards of the Japanese Red Cross Tokyo Metropolitan Blood Center, the Aichi Cancer Center, and the JMDP.

Table 1. Patient characteristics and matching of HLA allele between patient and donor
C-mismatch
All patientsC-matchKIR-L-MM-GKIR-L-M
Analyzed number1489101381395
AML4012861798
ALL43830624108
CML45129625130
MDS137821441
Malignant lymphoma6243118
Patient age26272527
Donor age (90 high risk)34353534
Sex match5756.659.357.5
TBI80.9817980.8
Status of leukemia (% high risk)55.153.57056
HLA-allele mismatch, %
A18.514.228.427.6
B9.13.525.920.1
C320100100
DRB118.915.532.124.8
DQB12218.728.429.1
DPB171.374.882.776.7
ATG+94561127
ATG−139595770368
Donor KIR 2DS2 analyzed233838070
Patient–donor 16 KIR type analyzed187705562

HLA and KIR Ligand Typing and Compatibility Characterization of Patient–Donor Pairs 

HLA-A, -B, -C, -DR, -DQ, and -DP alleles of all patients and donors were retrospectively determined by DNA typing as described previously [5]. For analysis of GVHD and leukemia relapse, HLA allele mismatch among donor–patient pairs was defined as the patient's alleles not being shared by the donor. KIR ligand specificity of the HLA-C antigen was determined according to the amino acid residues of the HLA-C allele. C1 ligand specificity consists of Asn 80 (Cw1, w3, w7, w8, and others); C2 specificity consists of Lys 80 (Cw2, w4, w5, w6, and others). In the cohort (patients and donors, n = 2978), the numbers of C1C1, C1C2, and C2C2 were 2555 (85.8%), 399 (13.4%), and 24 (0.81%), respectively. HLA-C mismatched pairs (n = 476) were divided into KIR ligand mismatch in the GVH direction (KIR-L-MM-G) (n = 81) and KIR ligand match in the GVH direction (KIR-L-M) (n = 395). KIR-L-MM-G was defined as the donor's KIR ligand for HLA-C not being shared by the patient's ligand. KIR-L-M included ligand match and ligand mismatch in the host-versus-graft (HVG) rejection direction. The combinations of KIR ligands in KIR-L-MM-G were as follows: C1C1 (patient)–C1C2 (donor), 78 (96.2%); C2C2–C1C2, 1 (1.2%); C1C1–C2C2, 2 (2.5%); and C2C2–C1C1, 0.

KIR Genotyping and Profile Analysis 

KIR genotyping was performed using genomic DNA from patient and donor, and the presence of the 16 KIR genes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, and 3DP1) was determined by the polymerase chain reaction sequence-specific primer (PCR-SSP) method [31] with minor modifications [32]. Pairs of all of KIR-L-MM-G (n = 81) cases and also KIR-L-M from HLA-C mismatch cases were selected, and HLA-C–matched cases were randomly selected as controls for the comparison. From the 260 pairs analyzed, all 16 KIR types of both patients and donors were successfully obtained in only 187 pairs, because of either insufficient quantity or quality of DNA. These data were used for evaluating KIR gene frequency and performing statistical analyses (Table 1). For the KIR-L-MM-G donor 2DS2 analysis, 46 cases, in which donor 2DS2 status was obtained, were added (for a total of 233 cases). KIR haplotype A is defined as carrying a single activating KIR gene, 2DS4; KIR haplotype B has additional activating KIR genes [33].

Definition of Transplantation-Related Events 

The occurrence of acute GVHD (aGVHD) was evaluated according to grading criteria in patients who survived for more than 8 days after transplantation, as described previously [30].

Statistical Analysis 

Statistical analysis was performed as described previously [5]. All analyses were conducted using STATA version 8.2 (STATA Corp, College Station, TX). Overall survival (OS) rate was assessed using the Kaplan-Meier product limit method. Cumulative incidence of aGVHD and leukemia relapse were assessed as described previously [5] to eliminate the effects of competing risks. The competing events regarding aGVHD and relapse were defined as death without aGVHD and death in remission (treatment related mortality), respectively. For each endpoint, a log-rank test was applied to assess the impact of the factor of interest. Multivariate analysis by Cox proportional hazard models was applied to assess the impact of KIR ligand compatibility, donor KIR genotype, and ATG administration along with potential confounders. Confounders considered were HLA-A, -B, -DR, -DQ, and -DP matching (GVH direction), sex (donor–patient pairs), patient age (linear), donor age (linear), type of disease, risk of leukemia relapse (standard and high, leukemia only analyzed), number of cells transplanted (linear), and preconditioning (TBI vs non-TBI). The number of nucleated cells before the manipulation of bone marrow was replaced with the number of cells transplanted. P values < .05 were considered statistically significant. Adjustment of P values for multiple comparison was done because of an a priori hypothesis that activating KIR would interact with the cognate ligand and transduce a stimulatory signal only when the inhibitory signal was inactive.

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Results 

Adverse Effects of KIR Ligand Incompatibility 

We first confirmed the effects of KIR-L-MM in the newly selected cohort in this study (Table 1). The cumulative incidence of aGVHD and OS are shown in Figure 1. KIR-L-MM-G showed a significantly higher incidence of grade III-IV aGVHD (41.1%; 95% confidence interval [CI] = 29.5%-51.9%) compared with KIR-L-M in HLA-C–mismatched patients (29.7%; 95% CI = 25.2%-34.3%; P = .032). A similar trend was seen in grade II-IV aGVHD (data not shown). In addition, in HLA-C–mismatched patients, KIR-L-MM-G had a lower 5-year OS rate (23.2%; 95% CI = 14.6%-32.9%) than KIR-L-M (41.8%; 95% CI = 36.9%-46.7%; P < .0001). Multivariate analysis (Table 2, group A [n = 1489]) also demonstrated the strong adverse effects of KIR-L-MM-G in HLA-C mismatch on aGVHD (grade III-IV GVHD: hazard rate [HR] = 3.08, P < .001; grade II-IV GVHD: HR = 1.76, P = .001) and on OS (HR = 1.93; P < .001), but not on relapse (HR = 1.27; P = .40). Allele mismatches of HLA-A, -B, -DR, -DQ, and -DP loci of the patient and donor were considered confounders in the analysis; consequently, the observed KIR-L-MM-G effects in HLA-C mismatch were adjusted for other HLA disparities. These adverse effects of KIR-L-MM-G on aGVHD and OS were consistent with those found in our previous study [5]. Consequently, we further analyzed the factors responsible for the effects of KIR-L-MM-G on transplantation outcome using this cohort.

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  • Figure 1 

    Effects of KIR ligand mismatch on transplantation outcome. Cumulative incidence of acute GVHD (grade III-IV) (A) and overall survival (B) by matching of KIR ligands in the GVHD direction. The directions of HLA-C mismatching were GVH and/or HVG. All patients were analyzed. The solid line represents HLA-C match (CM), the thin dotted line represents HLA-C mismatch KIR ligand match in the GVHD direction (C-MM KIR-L-M), and the thick dotted line represents HLA-C mismatch KIR ligand mismatch in the GVH direction (C-MM KIR-L-MM-G). The log-rank test was applied between CMM KIR-L-MM-G and CMM KIR-L-M.

Table 2. Multivariate analysis of the effects of KIR ligand matching, donor KIR genotype, and ATG preadministration
aGVHD 3-4aGVHD 2-4RelapseOS
GroupConfoundersSubject numberHR (95%CI)P valueHR (95%CI)P valueHR (95%CI)P valueHR (95%CI)P value
AHLA-C (HCX) matched10131.00(reference) 1.00(reference) 1.00(reference) 1.00(reference)
HLA-C-MM (HCX) and KIR-L-MM-G813.08(2.05-4.62)<.0011.76(1.28-2.43).0011.27(0.73-2.22).4031.93(1.47-2.53)<.001
HLA-C-MM (HCX) and KIR-L-M3952.00(1.54-2.61)<.0011.47(1.23-1.77)<.0010.58(0.41-0.81).0011.17(0.99-1.37).065
ATG (yes vs no)94 vs 13950.56(0.31-0.99).0470.63(0.43-0.93).0191.01(0.60-1.71).9571.23(0.92-1.65).158
BKIR-L (MM-G vs M)80 vs 701.36(0.76-2.44).3041.32(0.81-2.15).2582.16(0.89-5.24).0871.60(1.05-2.44).027
Donor KIR genotype (2DS2 + vs −)28 vs 1221.96(1.01-3.80).0451.62(0.92-2.85).0950.78(0.24-2.47).6661.04(0.62-1.74).889

Adjusted for HLA-A, -B, -DR –DQ, DP(GVH direction), age, donor age, donor-recipient sex pattern, disease, TBI, and risk.

Group A: all pairs, n=1489; group B: HLA-C-mismatched and donor 2DS2-typed n=150.

KIR Genotypes and Profiles of Patients and Donors 

The selected patients and donors were analyzed using the PCR-SSP method for genotyping 16 different KIR genes. Data for 187 pairs were obtained, including 55 cases of KIR-L-MM-G and 62 cases of KIR-L-M in HLA-C mismatch and 70 cases of HLA-C match (Table 1). Table 3 shows the frequency of each KIR gene and the KIR profiles of patients and donors, demonstrating no significant differences between the patients and donors. The frequency of each KIR was similar to that of the healthy Japanese population 32, 33, 34. Nearly half of the patients had only haplotype A.

Table 3. KIR genotpye analysis of patient and donor of the cohort (n=374)
PatientDonorKIR number∗∗
HaplotypeProfile2DL12DL22DL32DL42DL52DS12DS22DS32DS42DS53DL13DL23DL33DS12DP13DP1NumberFrequencyNumberFrequencyInhibitoryActivatingTotal
A#1+−++−−−−+−+++−++950.51920.49617
B#2+−++++−−++++++++240.13350.197411
B#3++++−−+−+−+++−++130.0790.05729
B#4+−++++−++−++++++120.0640.027411
B#5+−++++−−−+−+++++80.0480.04639
B#6+−++−−−−−−+++−++60.0350.03606
B#7++++++++++++++++60.0350.038614
B#8+−++++−−−+++++++40.0230.027310
B#9+++++++++−++++++40.0230.028513
B#10++++++++−+−+++++20.0120.017512
Others ∗ 130.07210.11
Patient0.990.161.001.000.380.370.170.170.880.250.941.001.000.351.001.00
FrequencyDonor0.990.140.981.000.410.390.160.140.870.320.931.000.990.370.991.00
Total0.990.150.991.000.390.380.160.150.870.280.931.001.000.360.991.00

∗ Combined profiles < 1% frequency; ∗∗ Not include pseudo-gene.

Donor KIR2DS2 Exacerbated aGVHD in KIR-L-MM-G 

To statistically evaluate the possible involvement of KIR genotype in the adverse effects of KIR-L-MM-G, we investigated the particular combinatory effects of donor KIR genotype and patient cognate KIR ligand type (receptor-ligand analysis). For the inhibitory KIR, we attempted to examine the combination of a particular ligand in the patient and absence of the cognate inhibitory KIR gene in the donor. But with regard to HLA-C-KIR ligand specificity, almost all individuals were positive for both C1 and C2 inhibitory KIRs (2DL2 and/or 2DL3, and 2DL1, respectively; Table 3); therefore, there was no mismatch between patient KIR ligand and donor inhibitory KIR genotype combination or vice versa. In contrast, activating KIR genotypes were quite variable among individuals, and mismatch (ie, reactive) combinations of activating KIR with its presumed ligand (2DS1 with C2 and 2DS2 with C1, respectively) were present.

For activating KIR, the combination of a particular ligand in the patient and presence of the cognate-activating KIR but absence of the corresponding inhibitory KIR in the donor was selected and analyzed. This choice was based on dominance of the inhibitory signal over the cognate-activating signal 35, 36. The corresponding activating donor KIR genotypes to patients C1C1 and C2C2 in KIR-L-MM-G were 2DS2 and 2DS1, respectively. However, the frequency of C2C2 in the JMDP cases was too low (only 1 case in this study) to permit statistical evaluation. As shown in Figure 2, donor 2DS2-positive cases in KIR-L-MM-G had a significantly higher incidence of aGVHD (grade III-IV GVHD, 70.9% [95% CI = 40.0%-87.9%]; grade II-IV GVHD, 78.6% [95% CI = 47.2%-92.5%]) compared with the donor 2DS2-negative cases (grade III-IV GVHD, 33.6% [95% CI = 22.0%-45.7%]; grade II-IV GVHD, 54.4% [95% CI = 40.8%-66.1%]; P = .012 and .029, respectively). This was not true for KIR-L-M cases, however. These results suggest that the adverse effects of KIR-L-MM-G depend on combinations of the donor-activating KIR genotype and cognate patient ligand C1.

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  • Figure 2 

    Effects of donor KIR2DS2 in KIR ligand mismatch on transplantation outcome: Cumulative incidence of aGVHD, relapse, and overall survival with presence or absence of donor KIR2DS2 gene in HLA-C–mismatched patients. Grade III-IV GVHD (A and E), grade 2-4 GVHD (B and F), relapse (C and G), and overall survival (D and H) with KIR-L-MM-G (A-D) or KIR-L-M (E-H) cases were analyzed. The solid line represents donor KIR2DS2-positive; the dotted line, donor KIR2DS2-negative.

To explore the possibility of the neighboring activating KIR loci being the primary factor in outcomes because of possible linkage disequilibrium, we next investigated the associations between other KIR genotypes and transplantation outcomes. No other activating KIR, but inhibitory 2DL2 (located adjacent to and tightly linked with 2DS2) showed a significant association with the incidence of aGVHD (data not shown). No significant associations between donor 2DS2 with relapse or OS in KIR-L-MM-G could be observed (Figures 2C and D, respectively).

Multivariate analysis (Table 2; group B [n = 150]) demonstrated that the donor 2DS2 was a possible risk factor for grade III-IV aGVHD in HLA-C–mismatched cases (HR = 1.96; P = .045). The same trend was observed for grade II-IV GVHD (HR = 1.62; P = .095). We also tested the currently proposed model for the KIR genotype effects on HSCT outcomes (donor KIR gene numbers 10, 16, 18, 26, 37, comparison of KIR genotype and profile between patient and donor [receptor–receptor analysis] 17, 19, 25, compatibility score [24], ligand homozygosity in patients 21, 27, 38, 39, and “missing ligand” effect 3, 11, 13, 40, 41), and found no significant associations in this cohort (data not shown).

ATG Preadministration Ameliorates the Adverse Effects of KIR-L-MM-G on aGVHD and OS 

In our previous study [5], the incidence of aGVHD was high in KIR-L-MM-G, where all cases did not involve ATG administration in the conditioning regimen, which is common in the JMDP cases. In the present study, we included rare ATG-administered cases (n = 94) in the analysis and evaluated the effects of ATG administration on KIR-L-MM-G. We found no significant differences in most of the parameters between the ATG-administered and non–ATG-administered groups, except for patient average age (18 years vs 27 years). Multivariate analysis (Table 2; group A [n = 1489]) indicated that ATG administration was a risk-reducing factor for severe aGVHD (grade III-IV GVHD: HR = 0.56; P = .047; grade II-IV GVHD: HR = 0.63, P = .019), whereas no significant effects on relapse or OS could be seen.

The cumulative incidence of aGVHD was assessed separately in the non–ATG-administered and ATG-administered groups (Figures 3A and 3D, respectively). In the non–ATG-administered group, the incidence of grade III-IV GVHD was significantly higher in KIR-L-MM-G than in KIR-L-M (47.7% [95% CI = 35.2%-59.2%] vs 29.4% [95% CI=24.8%-34.1%]; P = .0014), as found in our previous study [5]. In contrast, no grade III-IV aGVHD was observed in KIR-L-MM-G cases in the ATG-administered group (2 cases of grade 2, 2 cases of grade 1, and 7 cases of grade 0), and the preventive effects of KIR-L-MM-G on severe aGVHD were significant (P = .042) although only a small number were analyzed (n = 38). We analyzed the effects of 2DS2 in the non–ATG-administered cases. In KIR-L-MM-G, the incidence of grade III-IV aGVHD was significantly higher in the donor 2DS2-positive cases (n = 15) than in the donor 2DS2-negative cases (n = 54) (76.4% [95% CI = 43.5-91.7%] vs 40.1% [95% CI = 26.5%-53.2%]; P = .048), suggesting that the adverse effects of donor 2DS2 are independent of ATG administration. In ATG-administered cases, no grade III-IV aGVHD was observed in donor 2DS2-negative KIR-L-MM-G (n = 15); in 1 donor 2DS2-positive KIR-L-MM-G case, the patient failed engraftment but showed no aGVHD, and died on day 35. Therefore, we could not statistically evaluate the effect of ATG on the 2DS2-positive cases.

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  • Figure 3 

    Effects of ATG preadministration in KIR ligand mismatch on transplantation outcome: Cumulative incidence of aGVHD, relapse, and overall survival of patients not receiving ATG (A-C) and those receiving ATG (D-F). The solid line represents HLA-C match (C-M), the thick dotted line represents HLA-C mismatch KIR ligand match in the GVHD direction (C-MM KIR-L-M), and the thin dotted line represents HLA-C mismatch KIR ligand mismatch in the GVH direction (C-MM KIR-L-MM-G). The log-rank test was applied between CMM KIR-L-MM-G and CMM KIR-L-M.

As shown in Figure 3B, in non–ATG-administered cases, the cumulative incidence of relapse was higher in KIR-L-MM-G than in KIR-L-M (16.1% [95% CI = 8.6%-25.8%] vs 11.9% [95% CI = 8.9%-15.3%]; P = .046), which was seen mainly in ALL (data not shown), as was found in our previous study [5]. In contrast, no significant increase in relapse was obtained in ATG-administered cases (P = .082) (Figure 3E). As in our previous study [5], in non–ATG-administered cases, overall survival rate was significantly lower in KIR-L-MM-G than in KIR-L-M (21.0% [95% CI = 12.2%-31.3%] vs 42.0% [95% CI =36.8%-47.0%]; P < .0001) (Figure 3C). On the other hand, in ATG-administered cases, no significant difference was observed between KIR-L-MM-G and KIR-L-M (36.4% [95% CI = 11.2%-62.7%] vs 39.5% [95% CI = 21.2%-57.3%]; P = .79) (Figure 3F), suggesting that ATG preadministration in the conditioning regimen abolished the adverse effect of KIR-L-MM-G on survival.

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Discussion 

In the present study, we identified donor KIR genotype–patient KIR ligand combination and no ATG preadministration as critical factors for the adverse effects of KIR-L-MM-G on transplantation outcomes in the JMDP. The cases analyzed in this study were all HLA-A, -B, and -DR serologically matched; thus, we were able to evaluate the HLA-C ligand compatibility effects, because the HLA-Bw4 and HLA-A3 and -A11 KIR ligands were all matched. Other groups included mostly Bw4 ligand mismatch cases in KIR-L-MM-G analysis 2, 6, 7, 10, 11, 12, 14, 21, 42. The Bw4 (patient) -3DL1 (or -3DS1) (donor) combinatory effect also may affect transplantation outcome.

In the KIR-L-MM-G combination, the patient lacks the donor's KIR ligand. In this situation, donor NK cells may react with the patient cells according to the “missing self” model [43]. Previous KIR ligand compatibility data, together with the present data, confirm that the KIR-L-MM-G has potent adverse effects on UR-HSCT. In most KIR-L-MM-G cases in the JMDP, the donor and patient ligand types are C1C2 and C1C1, respectively, suggesting that C1C2 donor NK cells (and/or some T cells) respond to C1C1 patient cells. In this case, donor NK cells lack the inhibitory KIR for C1 (2DL2 and 2DL3) in terms of genotype or phenotype, or both. As shown in the present results, almost all JMDP donors examined posessed an inhibitory KIR gene for C1 (2DL3). The subpopulation of donor NK cells thus appears to lack cell surface expression of the C1-inhibitory KIR molecule, despite the presence of the genes. This is explained by the “at least one inhibitory receptor expression” model [44], in which each NK cell must express 1 inhibitory receptor for the self–major histocompatibility complex (MHC) class I to avoid autoreactivity, but expression of other receptors is “stochastic.” Consequently, NK cell subpopulations lacking the C1-inhibitory KIR (2DL2 and 2DL3) but having the C2-inhibitory KIR (2DL1) would react with C1C1 (C2-lacking) patient cells. Therefore, the donor inhibitory KIR repertoire at the expression level, not at the genomic level, appears to influence outcome in the JMDP. The importance of the inhibitory KIR expression repertoire and functional analysis of donor NK cells has been discussed previously 3, 45.

With a lack of inhibitory KIR signals, NK cells respond to target cells through activation signals from activating receptors. 2DS1 and 2DS2 are assigned to recognize C2 and C1, respectively, but other activating KIR ligand specificities (2DS3-5 and 3DS1) are unidentified [35]. Therefore, we were able to evaluate only these 2 KIRs for combinatory effects with their ligands. As described in Results, a higher incidence of severe aGVHD was observed in the 2DS2-positive donors in the KIR-L-MM-G cases, but not in the KIR-L-M cases. This suggests that 2DS2-positive lymphocytes (NK cells and/or some T cells) react with cognate ligand (C1)-positive cells and exacerbate aGVHD. Recently, La Nasa et al.[27] reported that the patient KIR ligand homozygosity, but not donor KIR genotype, is predictive for the outcome of HLA-matched UR-HCT in patients with beta-thalassemia. Their cases were all KIR ligand-matched transplantation and the donor-activating KIR–patient cognate ligand combination had no significant effect on the outcomes. Their results are consistent with our findings indicating that the donor 2DS2–patient C1 combination of ligand-matched pairs has no effect on any outcomes (Figure 2E-H). This is in accordance with the notion that an activating KIR works only when the patient has the cognate ligand and that the donor inhibitory KIR does not function (Fig. 4). Chewning et al [36] reported that KIR 2DS1-positive NK cells recognized C2-expressing target cells and showed alloreactivity in vitro supporting the concept of this model.

  • View full-size image.
  • Figure 4 

    Model of interaction between activating KIR and cognate KIR ligand. Donor-activating KIR transduces an activating signal on recognition of the cognate KIR ligand of the patient cell in KIR-L-MM-G case (B). The activating signal is canceled by an inhibitory signal from inhibitory KIR, which recognizes the KIR ligand of the patient cell in KIR-L-M case (D).

Although adverse impacts of donor 2DS2 on transplantation outcome have been documented previously 15, 18, 23, the present study is the first report on the adverse effects of the 2DS2–cognate ligand C1 combination on aGVHD incidence. Because we had an a priori hypothesis, we did not apply adjustment of P-value in our analysis; however, our results must be interpreted with caution. KIR-L-MM-G is infrequent in the JMDP (only 81 of 1489 cases in the present study), and the frequency of 2DS2 is low in Japan 32, 33, and confirmation in other independent cohorts from different populations will support our findings. Combinatory effects of 2DS2 and cognate ligand C1 also have been reported in disease susceptibility studies, including studies of type I diabetes mellitus [46], ulcerative colitis [47], rheumatoid vasculitis [48], and tuberculosis [49]. Furthermore, extensive genetic analysis of KIR and HLA genotypes of various ethnic populations have demonstrated a strong negative correlation of activating KIR and its putative ligand combination including 2DS2–C1, suggesting coevolution of the activating receptor–ligand loci [50]. Taken together with our data, these clinical and population genetic studies suggest a direct receptor–ligand interaction between 2DS2 and C1; however, binding studies using soluble 2DS2 molecules have shown no or a very weak binding to C1 molecules or C1-transfected cells, challenging the notion of C1 as a 2DS2 ligand 51, 52, 53. Recombinant 2DS1 also showed very low or no affinity to C2 [54]. This disparity may be linked to differences in the nature of ligand binding between inhibiting and activating receptors. One possible factor is class I-binding peptides. The peptide-dependent binding with class I-binding receptors is recognized in most of the inhibitory receptors 53, 55, 56, 57, 58 and also has been suggested in activating KIR 53, 58, 59. The peptide repertoire that allows strong KIR binding might be more restricted in activating KIR cases than in inhibitory ones. Alternatively, activating KIR–ligand binding may be somehow strengthened under stress conditions, such as transplantation or viral infection. Epstein Barr virus–transformed C1-positive cells were found to be stained slightly by recombinant 2DS2 tetramers [53]. A mutation study found that only 1 amino acid substitution in 2DS2 increased its level of binding to C1 to that of inhibitory 2DL2, suggesting that a very fine conformational microstructure change controls KIR binding specificity [60].

Inhibitory 2DL2 also showed a significant association with the incidence of severe aGVHD. This may be secondary to the 2DS2–C1 association [50]; alternatively, donor 2DL2-positive NK cells might have a different effect than 2DL3-positive NK cells on acute GVHD incidence, because the binding affinity to C1 is higher in 2DL2 than in 2DL3 [60]. Other groups have analyzed activating KIR gene number and outcome and have reported both beneficial and adverse associations 10, 16, 26, 37. We did not find such quantitative KIR loci effects in this JMDP cohort (data not shown); KIR genotype variation among various ethnic groups may be responsible for these differences.

Preadministration of ATG to a patient is also a critical factor in attenuating the adverse effects of KIR-L-MM-G on transplantation outcome. Our findings demonstrate that KIR-L-MM-G had potent adverse effects (higher aGVHD incidence and lower OS) without ATG administration, and that ATG administration in the conditioning regimen ameliorated most of these adverse effects. Although the average patient age in the ATG-administered group was about 10 years younger than that in the non–ATG-administered group in this study, multivariate analysis including age as a confounder also identified the ATG effect as an independent factor for incidence of aGVHD (see Table 2). To the best of our knowledge, this is the first direct comparison UR-HSCT study on the effects of ATG preadministration under the same transplantation regimen with similar genetic backgrounds. Because far fewer ATG-administrated cases than non–ATG-administered cases are included in the JMDP (an imbalance that could bias statistical results), further evaluation of large numbers of ATG-preadministered cases in different ethnic populations are needed.

Administration of ATG extensively depletes patient and donor T cells, thus strongly inhibiting the responses of alloreactive T cells. Because the JMDP cases are all unmanipulated T cell–replete marrow, donor alloreactive T cell response may be very strong, which would obscure some of the NK cell beneficial effects [61]. In KIR-L-MM-G without ATG preadministration, alloreactive NK cells were activated by 2DS2–C1 interaction without inhibitory KIR signals and may have augmented alloreactive donor T cell responses, resulting in increased aGVHD incidence and mortality. Alternatively, KIR-positive T cells may have been responsible for inducing aGVHD. In contrast, with ATG preadministration, donor T cells are largely depleted, and the beneficial effects of NK cell alloreactivity on aGVHD incidence may become prominent. Too few ATG-treated cases were analyzed (n = 11) to allow confirmation of the preventive effects of KIR-L-MM-G on acute GVHD, but the results are consistent with those for the HLA haplo-mismatched, ATG-preadministered R-HSCT [2]. In mouse GVHD models, alloreactive NK cells prevented donor alloreactive T cell stimulation and suppressed aGHVD by lysing donor antigen-presenting cells [2]. These mechanisms might explain the preventive effects of KIR-L-MM-G on the incidence of aGVHD. NK cell reconstitution after transplantation might be influenced by ATG treatment as well as by KIR ligand and KIR genotype variability 39, 62, 63. Our data suggest that the KIR-L-MM-G combination must be avoided in JMDP transplantation unless ATG is used in the conditioning regimen.

Another possible factor is mismatch combination dissimilarity resulting from genetic variability in HLA and KIR in populations with different ethnic backgrounds. There are allele frequency differences in HLA-C among human populations in terms of the HLA-C KIR ligand [50]. Because the C1 ligand type is dominant in the Japanese population (allele frequency 0.92), KIR-L-MM-G is relatively rare (5%) compared with the incidence in White populations. Furthermore, in the KIR-L-MM-G, the C1C1 (patient)–C1C2 (donor) combination is common (95%) [5]. Therefore, we could focus on the KIR ligand incompatibility and the 2DS2 effects on the C1-homozygous patients in this study. In contrast, the White population more frequently exhibits the C2 type [50]. Consequently, the KIR-L-MM-G frequency is higher in Caucasian than Japanese and might include C2C2 (patient)–C1C2 (donor), C2C2–C1C1, and C1C1–C2C2 combinations, in addition to the C1C1–C1C2 combination. Therefore, not only the C1C1 (patient)–2DS2 (donor) combination, but also the C2C2 (patient)–2DS1 (donor) combination, might contribute considerably to the effects of KIR-L-MM-G in White [36]. The inhibitory capacity of C1 is reportedly weaker than that of C2 [64], and the binding strength of inhibitory KIR to the ligand HLA-C is different as well (2DL1 > 2DL2 > 2DL3) [60]. There may be more variability in inhibitory pathways in White populations; indeed, several groups have reported that the transplantation outcomes vary between C1-homozyous and C2-homozygous patients 15, 19, 38, 39. KIR genotype also shows ethnic variability 33, 50; Japanese have a markedly high frequency of the A haplotype and a very low frequency of 2DS2 (16% in the JMDP, compared with a frequency of > 40% in most Caucasian and African populations). One potential factor not examined in the present study is KIR allelic polymorphism. Yawata et al. [34] have shown that allelic polymorphism modulates the level and frequency of KIR3D expression, as well as its inhibitory capacity. These allelic differences might influence outcomes even though HLA-A- and B-KIR ligand specificities were the same in donors and recipients in the present study.

Here we found that the combination of donor-activating KIR genotype–patient cognate KIR ligand type and ATG administration in the conditioning regimen were critical factors in the adverse effects of KIR-L-MM-G on transplantation outcome. Alloreactivity of NK cells may be either beneficial or adverse depending on the above factors. However, other important parameters also may contribute to transplantation outcome. Further large-scale international collaborative studies, including a variety of ethnic populations and statistical comparisons under uniform regimens, are needed to gain further insight into the effects of NK cell alloreactivity on transplantation and to guide the development of cell therapy using alloreactive NK cells for leukemia and other diseases.

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Acknowledgments 

The authors thank the staff members of the transplantation center, the donor centers, and the JMDP office. They also thank Drs Kenji Tadokoro, Hiroshi Furukawa, and Nobuyo and Makoto Yawata for useful suggestions and discussion, and Ms. Mutsuko Minemoto and Mr. Takahiro Ichihara for technical assistance. This work was supported in part by a Health and Labor Science Research Grant from the Ministry of Health, Labor and Welfare of Japan (Research on Human Genome, Tissue Engineering), a grant from Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Grant-in Aid B (15390309) from the Japan Society for the Promotion of Science, and a Grant of Third-Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor and Welfare, Japan. The following institutions participated in and registered patients in the present study: Hokkaido University Hospital, Sapporo University Hospital, Sapporo Hokuyu Hospital, Japanese Red Cross Asahikawa Hospital, Asahikawa Medical College Hospital, Hirosaki University Hospital, Iwate Medical University Hospital, Tohoku University Hospital, Yamagata University Hospital, Akita University Hospital, Fukushima Medical College, Toranomon Hospital, National Cancer Center Central Hospital, National Center for Child Health and Development, Institute of Medical Science at the University of Tokyo, Toho University Hospital, Omori Hospital, Tokyo Metropolitan Komagome Hospital, Nihon University Hospital, Itabashi Hospital, Jikei University Hospital, Keio University Hospital, Tokyo Medical College Hospital, Tokyo Medical and Dental University Hospital, Tokyo University Hospital, Yokohama City University Hospital, Kanagawa Children's Medical Center, Kanagawa Cancer Center, Tokai University Hospital, St. Marianna University Hospital, Chiba University Hospital, Chiba Children's Hospital, Kameda General Hospital, Saitama Children's Medical Center, Saitama Cancer Center Hospital, Saitama Medical School Hospital, Ibaraki Children's Hospital, Jichi Medical School Hospital, Tsukuba University Hospital, Dokkyo University Hospital, Saiseikai Maebashi Hospital, Gunma University Hospital, Niigata University Hospital, Niigata Cancer Center Hospital, Shinshu University Hospital, Hamamatsu University Hospital, Hamamatsu Medical Center, Shizuoka General Hospital, Shizuoka Children's Hospital, Japanese Red Cross Nagoya First Hospital, Nagoya Daini Red Cross Hospital, Meitetsu Hospital, Nagoya University Hospital, Nagoya Ekisaikai Hospital, Nagoya Medical Center, Aichi Cancer Center Hospital, Aichi Medical University Hospital, Nagoya City University Hospital, Showa Hospital, Anjo Kousei Hospital, Fujita Health University Hospital, Mie University Hospital, Yamada Red Cross Hospital, Kanazawa University Hospital, Kanazawa Medical University Hospital, Toyama Prefectural Central Hospital, Fukui Medical School Hospital, Shiga University of Medical Science, Center for Adult Disease in Osaka, Kinki University Hospital, Osaka University Hospital, Osaka City University Hospital, Osaka Medical Center and Research Institute for Maternal and Child Health, Matsushita Memorial Hospital, Hyogo College of Medicine Hospital, Hyogo Medical Center for Adults, Kobe City General Hospital, Kobe University Hospital, Kyoto University Hospital, Kyoto Prefectural University of Medicine Hospital, Kyoto City Hospital, Kansai Medical University Hospital, Tenri Hospital, Nara Medical University Hospital, Tottori University Hospital, Hiroshima Red Cross Hospital and Atomic-Bomb Survivors Hospital, Yamaguchi University Hospital, Ehime Prefectural Central Hospital, Okayama Medical Center, Kurashiki Central Hospital, Kyushu University Hospital, Harasanshin General Hospital, Hamanomachi General Hospital, National Kyushu Cancer Center, St. Mary's Hospital, Kokura Memorial Hospital, Nagasaki University Hospital, Kumamoto Medical Center, Oita Medical University Hospital, Imamura Hospital, and Kagoshima University Hospital.

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PII: S1083-8791(07)00489-2

doi:10.1016/j.bbmt.2007.09.012

Biology of Blood and Marrow Transplantation
Volume 14, Issue 1 , Pages 75-87, January 2008

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