Biology of Blood and Marrow Transplantation
Volume 15, Issue 5 , Pages 580-588, May 2009

What Is the Role for Donor Natural Killer Cells after Nonmyeloablative Conditioning?

  • Frédéric Baron

      Affiliations

    • Giga Research, University of Liège, Liège, Belgium
    • ,
  • Effie W. Petersdorf

      Affiliations

    • Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington, Seattle, Washington
    • ,
  • Ted Gooley

      Affiliations

    • Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington, Seattle, Washington
    • ,
  • Brenda M. Sandmaier

      Affiliations

    • Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington, Seattle, Washington
    • ,
  • Mari Malkki

      Affiliations

    • Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington, Seattle, Washington
    • ,
  • Thomas R. Chauncey

      Affiliations

    • Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington, Seattle, Washington
    • Veterans Affairs Medical Center, Seattle, Washington
    • ,
  • David G. Maloney

      Affiliations

    • Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington, Seattle, Washington
    • ,
  • Rainer Storb

      Affiliations

    • Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington, Seattle, Washington
    • Corresponding Author InformationCorrespondence and reprint requests: Rainer Storb, MD, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, D1-100, PO Box 19024, Seattle, WA 98109-1024.

Received 19 November 2008; accepted 26 January 2009.

Article Outline

Abstract 

We investigated the impacts of the tempo of early (days 14, 28, and 42) donor T cell and natural killer (NK) cell engraftment, missing recipient killer cell immunoglobulin-like receptor (KIR) ligands, and numbers of donor inhibitory and activating KIR genes on hematopoietic cell transplantation (HCT) outcomes in 282 patients with hematologic malignancies given nonmyeloablative conditioning. Modeling chimerism levels as a continuous linear variable, we found that high early donor T cell chimerism was significantly associated with acute graft-versus-host disease (aGVHD) (P = .01), whereas high donor NK cell chimerism levels had no such association (P = .38). Conversely, high donor NK cell chimerism levels were significantly associated with low relapse risk (P = .0009), whereas no significant association was seen with high donor T cell chimerism (P = .10). The qualitative associations between donor T cell and NK cell chimerism levels and GVHD and relapse did not change after adjustment for the presence of recipient KIR ligands or numbers of donor inhibitory or activating KIR genes. Our data indicate that prompt engraftment of donor NK cells correlated with lessened risks of relapse, but not with GVHD, whereas the converse was true for T cells.

Key Words: Allogeneic hematopoietic cell transplantation, Nonmyeloablative, NK cells, Chimerism, KIR

 

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Introduction 

Allogeneic hematopoietic cell transplantation (HCT) after nommyeloablative conditioning relies nearly exclusively on graft-versus-tumor effects for tumor eradication 1, 2, 3, 4, 5, 6. Although it is generally accepted that graft-versus-tumor effects after HLA-identical HCT mainly result from T cells 7, 8, 9, observations by the Perugia group showed that after HLA-haploidentical HCT, graft-versus-host natural killer (NK) cell reactivity was associated not only with less relapse in patients with acute myelogenous (but not lymphoblastic) leukemia (AML), but also with less graft rejection and less acute graft-versus-host disease (aGVHD) [10]. These findings have sparked renewed interest in NK cells in the HCT setting.

NK cell activities are thought to be regulated by a quantitative balance between inhibitory signals mediated by inhibitory killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A, and by activating signals mediated by the natural cytotoxicity receptors (NCRs) NKG2D and DNAX accessory molecule-1 (DNAM-1, CD226) [11]. KIRs recognize allotypic determinants that are shared by different HLA class 1 alleles; KIR2/DL2 and KIR2/DL3 recognize HLA-C group 1 alleles, KIR2/DL1 recognizes HLA-C group 2, and KIR3/DL1 recognizes HLA-Bw4 alleles [11]. Conversely, CD94/NKG2A recognizes overall expression of HLA class 1 molecules on target cells through HLA-E. Although HLA and KIR genes are inherited independently, clonal analyses have demonstrated that in healthy individuals, each NK cell either expresses at least one inhibitory receptor for self HLA (either KIR or the nonspecific CD94/NKG2A receptor) or is developmentally immature 12, 13.

After transplantation, donor NK cells arising from hematopoietic stem cells (HSCs) regenerate the same KIR repertoire as the donor, [14] leading to a high frequency of graft-versus-host reactive NK cells in those recipients who lack ligands for donor NK cell KIRs [15]. Such alloreactive NK cells are detectable for only a few months after HCT [15]; thereafter, they become tolerant to the host, likely in part through expression of KIRs specific to the recipient HLA [16]. NK alloreactivity has been associated with graft-versus-tumor effects after both HLA-mismatched and HLA-matched HCT 10, 16, 17, 18, 19.

In previous work, we analyzed the correlations between the tempo of engraftment of various peripheral blood stem cell (PBSC) subpopulations and HCT outcomes in 120 patients with various hematologic malignancies who underwent HCT after nonmyeloablative conditioning [20]. We found a strong suggestion that rapid establishment of high levels of donor NK cell chimerism between days 14 and 100 after HCT predicted better progression-free survival (PFS) [20]. In the present study, we examined the impact of both early donor T cell and NK cell chimerism levels on transplantation outcomes in the context of recipient HLA ligand and donor KIR receptor genetic data in 282 patients with cancer who underwent HCT from either a related or an unrelated donor after a minimal-intensity conditioning regimen that included 2 Gy of total body irradiation (TBI).

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Patients and Methods 

Patients and Donors 

Data from 282 patients who underwent allogeneic HCT after nonmyeloablative conditioning at the Fred Hutchinson Cancer Research Center, University of Washington Medical Center, Children's Hospital and Regional Medical Center, and the Veterans Affairs Medical Center (all in Seattle) between March 1998 and November 2006 were included in this study. Patient characteristics are summarized in Table 1. Median patient age was 54 years (range, 5 to 74 years). Diagnoses included hematologic malignancies (n = 274) and solid tumors (n = 8). Patients were classified as having indolent or aggressive disease, as characterized previously [21]. A total of 152 patients received grafts from HLA-identical related donors, 1 from a 1 HLA-A antigen–mismatched related donor, 95 from HLA-A, -B, -C, -DR, and -DQ allele–matched unrelated donors (URDs), and 34 from 1 HLA class I allele– and/or 1 antigen-mismatched URDs. Comorbidities at the time of HCT were assessed using the HCT comorbidity index (HCT-CI) score [22]. Prospective research protocols were approved by the Fred Hutchinson Cancer Research Center's Institutional Review Board for the 4 participating institutions. Informed consent was obtained from all patients.

Table 1. Patient, Disease, and Transplantation Characteristics (n = 282)
CharacteristicValue
Median patient age, years (range)54 (5-74)
Recipient sex, male/female, n (%)182 (65)/100 (35)
Median donor age, years (range)45 (19-78)
Donor sex, male/female, n (%)147 (52)/135 (48)
Diagnosis, n (%)
Acute myelogenous leukemia63 (22.3)
Acute lymphoblastic leukemia8 (2.8)
Chronic myelogenous leukemia15 (5.3)
Chronic lymphocytic leukemia33 (11.7)
Myelodysplastic syndrome31 (11.0)
Multiple myeloma44 (15.6)
Non-Hodgkin lymphoma52 (18.4)
Hodgkin disease25 (8.9)
Waldenström macroglobulinemia3 (1.0)
Renal cell carcinoma7 (2.5)
Cervical cancer1 (0.4)
Disease risk, n (%)
Indolent∗123 (44)
Aggressive†159 (56)
Tandem autologous/allogeneic HCT, n (%)41 (14.5%)
Donor, n (%)
Related
HLA-identical152 (53.9)
One HLA-antigen mismatched1 (0.4)
Unrelated
10/10 HLA allele-matched95 (33.7)
One HLA-allele mismatched16 (5.7)
One HLA-antigen mismatched13 (4.6)
One HLA-antigen + 1 HLA allele mismatched5 (1.8)
Numbers of inhibitory genes on donor NK cell KIR, n (%)
Unknown30 (11)
26 (2)
391 (32)
4104 (37)
551 (18)
Number of activating genes on donor NK cell KIR, n (%)
Unknown53 (18.8)
177 (27.3)
246 (16.3)
321 (7.4)
437 (13.1)
533 (11.7)
615 (5.3)
Hematopoietic stem cell source, n (%)
G-PBMC271 (96)
Bone marrow11 (4)
Conditioning regimen, n (%)
2 Gy TBI54 (19)
2 Gy TBI + fludarabine228 (81)
Cell dose, × 106/kg recipient, median (range)
CD34+ cells7.8 (0.8-42.6)
T cells312 (16-934)
Sustained engraftment/graft rejection, n (%)264 (94)/18 (6)
Acute GVHD, n (%)
Grade 0/I113 (40.1)
Grade II127 (45.0)
Grade III31 (11.0)
Grade IV11 (3.9)
Chronic GVHD, n (%)
No135 (48)
Yes147 (52)
3-year overall survival (%)50
3-year progression-free survival (%)37

TBI indicates total body irradiation; GVHD, graft-versus-host disease; KIR, killer cell immunoglobulin-like receptor.

∗Defined as acute myelogenous leukemia in first complete remission, acute lymphoblastic leukemia in first complete remission, myelodysplastic syndrome–refractory anemia, chronic myelogenous leukemia in first chronic phase, chronic lymphoblastic leukemia, low-grade non-Hodgkin lymphoma, multiple myeloma in partial or complete remission, and Waldenstrom macroglobulinemia.

†All other diagnoses.

Treatment and Evaluation 

A total of 54 recipients of related grafts were conditioned with 2 Gy TBI alone [3], whereas the remaining 99 recipients of related grafts and all recipients of unrelated grafts also received fludarabine (Flu) 30 mg/m2/day on days −4, −3, and −2 before HCT 3, 4, 23, 24, 25. Donor granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood mononuclear cell (G-PBMC) grafts (n = 271) or bone marrow (BM) grafts (n = 11) were infused without processing on day 0. Postgrafting immunosuppression included mycophenolate mofetil (MMF) and cyclosporine (CsA) or tacrolimus in all patients, as described previously 3, 4, 23, 25, 26.

Grading and treatment of aGVHD and chronic GVHD (cGVHD) were performed according to established criteria [27]. Disease-dependent restaging after HCT occurred monthly for the first 3 months and then at 6 months, 1 year, and yearly thereafter. Persistent or progressive malignancies in the absence of GVHD were treated by rapid tapering and discontinuation of immunosuppression, and 24 patients received donor lymphocyte infusion (DLI) [28]. Eight additional patients with low or failing T cell chimerism received DLI 2 days after receiving pentostatin (4 g/m2) aimed at preventing graft rejection, as reported previously [29].

Relapse and progression were defined according to the criteria of Kahl et al. [6] and Rotta et al. [30]. Relapse was defined as recurrence of malignancy based on one or more of the following parameters: marrow morphology, flow cytometry, cytogenetic studies (including fluorescein in situ hybridization [FISH]), electrophoresis, immunofixation assays, polymerase chain reaction (PCR)-based assays for disease markers, and imaging results. Disease progression was defined as a ≥ 50% increase in disease burden or a 25% increase in any disease marker for patients with multiple myeloma (MM).

Chimerism Analyses 

The different peripheral blood subpopulations were sorted by 3-color flow cytometry using a Vantage SE cytometer (BD, San Jose, CA). Cell types were defined as follows: T cell, CD3+CD56- sidescatterlow; NK cell, CD56+CD3-CD14- sidescatterlow. Percentages of donor chimerism in the different blood cell populations were assessed using PCR-based analyses of polymorphic minisatellite or microsatellite regions (VNTR/STR) or FISH for X and Y chromosomes if patients and donors were sex-mismatched, as reported previously 3, 20.

HLA Typing, KIR Genotyping, and the Missing KIR Ligand Algorithm 

Sequence-specific oligonucleotide hybridization and/or sequencing-based typing methods were used to define exons 2 and 3 of HLA-A, -B, and -C alleles and exon 2 of -DRB1 and -DQB1 alleles in all unrelated donor–recipient pairs. Pairs with the same HLA-A, -B, -C, -DRB1, and -DQB1 alleles were defined as “10/10” allele matched; all other pairs were defined by the number of mismatched class I and/or class II alleles or antigens (Table 1). DNA for KIR genotyping was available for 264 of the 282 donors (94%). The presence or absence of 10 KIR genes (2DL1, 2DL2, 2DL3, 3DL1, 2DL5, 2DS2, 2DS3, 2DS4, 2DS4-22bp deletion, and 2DS5) was assessed using a commercial PCR–sequence-specific primers (SSP) kit (Invitrogen, Carlsbad, CA), following the manufacturer's protocol. Typing for the 2DS1 gene was performed using previously published PCR-SSP primers [31]. KIR pseudogenes (KIR2DP1 and 3DP1) were not typed. KIR framework genes (KIR2DL4, KIR3DL2, and KR3DL3) are present in all individuals [32] and thus were not genotyped. Recipient HLA-A, -B, and -C alleles and donor KIR2DL1, KIR2DL2/3, KIR3DL1, and KIR3DL2 loci were evaluated as follows: absence of recipient HLA-Bw4 epitopes in HLA-A and/or -B with the presence of donor KIR3DL1; absence of recipient Cw3 epitopes (C1 group) in the presence of donor KIR2DL2/3; absence of recipient Cw4 epitopes (C2 group) in the presence of donor KIR2DL1, and absence of recipient HLA-A3/A11 alleles/antigens in the presence of donor KIR3DL2 [33]. The total numbers of donor-activating KIR genes (2DS1, 2DS2, 2DS3, 2DS4, and 2DS5) and of donor-inhibitory KIR genes (2DL1, 2DL2, 2DL3, 3DL1, and 2DL5) were determined for each pair.

Statistical Analyses 

Potential associations between chimerism levels and pretransplantation/transplantation characteristics were determined using the generalized estimating equation method. The effects of the percentage of donor chimerism on the incidence of rejection and aGVHD were assessed using logistic regression, and the effects on PFS, relapse, and nonrelapse mortality (NRM) were assessed using Cox regression. The association between KIR genetics and outcome also was examined using Cox regression. In some cases, chimerism was modeled as a time-dependent covariate (within the first 42 days after HCT for relapse, NRM and PFS); in other cases, the effects of chimerism values at a specified timepoint on outcome (rejection and aGVHD) were examined. Analyses of associations of chimerism level and KIR genetics with relapse, NRM, and PFS were adjusted for indolent/aggressive disease, HCT-CI score, donor relationship, and previous autologous HCT. The effects of KIR genetics on the association between NK chimerism and outcome were assessed by including various models of KIR (described later) in the appropriate regression models. Spearman correlation coefficients were estimated to evaluate the correlations between donor cell subsets. P values from regression models were derived from the Wald test.

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Results 

Kinetics of Donor Engraftment 

Median donor T cell and NK cell chimerism levels were 66% (range, 1% to 100%) and 78% (range, 3% to 100%), respectively, on day 14, 79% (range, 1% to 100%) and 90% (range, 2% to 100%) on day 28, and 81% (range, 1% to 100%) and 88% (range, 1% to 100%) on day 42. The Pearson correlation coefficient between donor T cell and NK cell chimerism level was 0.60 on day 14, 0.76 on day 28, and 0.75 on day 42.

Factors Affecting Kinetics of Donor Engraftment 

Transplantation/Pretransplantation Factors 

High numbers of transplanted CD34+ cells correlated with high levels of donor T cell chimerism (P = .002) and NK cell chimerism (P = .004) (Table 2). Furthermore, whereas high numbers of transplanted T cells correlated closely with high levels of early donor T cell chimerism (P = .004), the association with NK cell chimerism was weaker (P = .06). Patients receiving bone marrow grafts had lower levels of donor T cell chimerism (P < .0001) and NK cell chimerism (P = .007) compared with those receiving G-PBMCs. Patients with lymphogenous malignancies had higher levels of donor T cell chimerism compared with those with myelogenous malignancies (P = .0006) or solid tumors (P = .007), although the associations between disease category and donor NK cell chimerism level did not reach statistical significance.

Table 2. Associations between Transplantation/Pretransplantation Factors and Donor T Cell and NK Cell Chimerism Level∗
FactorNK Cell ChimerismT Cell Chimerism
Number of CD34 cells transplantedP = .004 (greater number transplanted, higher chimerism levels)P = .002 (greater number transplanted, higher chimerism levels)
Number of T cells transplantedP = .06 (greater number transplanted, higher chimerism levels)P = .004 (greater number transplanted, higher chimerism levels)
Bone marrow (vs G-PBMC)P = .007 (bone marrow lower 30%)P < .0001 (bone marrow lower 43%)
URD (vs related donor)P = .49 (URD lower 2%)P = .45 (URD lower 2%)
Disease category
Lymphoid malignancies——
Myeloid malignanciesP = .23 (myeloid lower 4%)P = .0006 (myeloid lower 11%)
Solid tumorsP = .34 (solid lower 8%)P = .007 (solid lower 16%)

NK indicates natural killer; PBMC, peripheral blood mononuclear cells; URD, unrelated donor.

∗P values were obtained from generalized estimated equations, using chimerism values measured at days 14, 28, and 42 after HCT.

Associations between KIR/KIR ligand and kinetics of donor NK cell engraftment 

The absence of 1 or more recipient ligands for donor KIR did not have a statistically significant effect on average donor NK cell chimerism level. Specifically, the average donor NK cell chimerism level on days 14, 28, and 42 was 5.6% lower in patients with all ligands present compared with those missing 1 or more ligands (P = .23).

When the numbers of donor-inhibitory genes were modeled as continuous linear variables, an increasing number of genes was associated with decreased donor NK cell chimerism, but the correlation was not statistically significant (P = .11). Similarly, an increasing number of donor-activating genes was associated with lower chimerism levels, but this association also was not statistically significant (P = .10).

Associations between Tempo of Donor Cell Engraftment and HCT Outcome 

Graft rejection 

Eighteen of the 282 patients (6%) experienced graft rejection between 13 and 1123 days (median, 74 days) after HCT, with 4 rejections occurring despite administration of pentostatin followed by DLI. After excluding patients who received DLI to prevent graft rejection, data on T cell chimerism at day-14 were available for 7 graft rejectors and 170 nonrejectors, and similar data for NK cells were available for 6 rejectors and 142 nonrejectors. The mean day-14 T cell chimerism level was 14% in rejectors and 65% in nonrejectors (P < .0001), with corresponding day-14 NK cell chimerism levels of 34% and 73% (P < .0001). Of the 7 rejectors among patients with T cell chimerism levels available at day 14, 5 had values < 10%, 1 had a value between 10% and 50%, and 1 had a value between 50% and 75%. Of the 6 rejectors among patients with NK chimerism levels available on day 14, 2 had values < 10%, 3 had values between 10% and 50%, and 1 had a value between 75% and 90%. The percentages of patients who were missing at least 1 ligand for donor NK cell KIRs were similar in rejectors and nonrejectors (82% vs 90%).

GVHD 

Grade II, III, and IV aGVHD occurring beyond day 14 was diagnosed in 45%, 10%, and 4% of patients, respectively. With chimerism modeled as a categorical variable, the probability of grade II-IV aGVHD increased with increasing levels of donor T cells (P = .01; trend test), but not of NK cells (P = .38; trend test) (Table 3). When both day-14 donor T cell and NK cell chimerism levels were included in a model for grade II-IV aGVHD, the (nonstatistically significant) association for donor NK cell chimerism level became even less significant (P = .83; trend test), whereas the association for T cell chimerism remained statistically significant (P = .01; trend test). These observations remained qualitatively similar after adjustment for the presence or absence of 1 or more ligands for donor NK cell KIRs, as well as after adjustment for the number of activating or inhibitory genes (data not shown).

Table 3. Associations between Day-14 Donor Chimerism Levels and Acute GVHD
Patients with Acute GVHD/Patients at Risk, n (%)∗
% Donor Chimerism on Day 14T CellsNK Cells
0-5022/47 (47%)10/20 (50%)
51-7534/65 (52%)24/42 (57%)
76-9032/43 (74%)31/53 (58%)
91-1009/13 (69%)16/25 (64%)
Trend test†P = .01‡P = .38§

∗Patients with GVHD or death before day 14 were excluded from the analyses.

†P values adjusted for donor type (related vs unrelated).

‡P = .01 after adjusting for donor NK cell chimerism level.

§P = .83 after adjusting for donor T cell chimerism level.

Extensive cGVHD was present in 52% of the patients. When donor chimerism levels were modeled as average values for days 14, 28, and 42, no statistically significant association was found between NK cell level and cGVHD (P = .80; trend test with chimerism modeled as categorical variable), whereas a significant association was found between high T cell level and cGVHD (P = .05; trend test). The results were similar after adjustment for the presence or absence of 1 or more ligands for donor NK cell KIR, as well as after adjustment for the number of activating or inhibitory genes.

Taken together, these results suggest an association between GVHD and the tempo of donor T cell engraftment, but not NK cell engraftment.

Relapse, achievement of complete remission, NRM, and PFS 

With a median follow-up of 4 years for surviving patients, 107 patients relapsed/progressed between 4 and 1978 days (median, 166 days). When donor chimerism level was modeled as a continuous variable, no significant association between donor T cell chimerism level and relapse was found (P = .10), whereas high donor NK cell chimerism level was associated with decreased risk of relapse in time-dependent analyses (P = .0009; Table 4). The magnitude of the association was similar if the analysis was restricted to patients with a day-14 chimerism level of ≥ 25%. Furthermore, the associations between donor T cell and NK cell chimerism levels and relapse risk were qualitatively the same when the analysis was restricted to patients with hematologic malignancies. The conclusions also were qualitatively the same after adjustments for the presence or absence of 1 or more ligands for donor NK cell KIRs, as well as for the number of activating or inhibitory genes (data not shown). The risk of relapse was lower in patients who had all ligands for donor NK cell KIRs compared with those missing 1 or more ligands for donor NK cell KIRs, but the difference was not statistically significant (adjusted hazard ratio [HR] = 0.74; 95% confidence interval [CI] 0.39 to 1.41; P = .36) (Figure 1A). Table 5 gives the raw percentages of relapse according to the specific missing ligand [33]. A formal statistical analysis based on these specific ligands is not possible because of the large number of unique categories; the raw percentages in Table 5 do not suggest any striking associations, however.

Table 4. Associations between Donor Chimerism Level and Relapse
HR (95% CI)
% Donor Chimerism on Day 14T CellsNK Cells
0-5011
51-750.35 (0.17-0.72; P = .004)0.29 (0.10-0.82; P = .02)
76-900.48 (0.25-0.91; P = .03)0.29 (0.13-0.63; P = .002)
91-1000.50 (0.26-0.96; P = .04)0.20 (0.09-0.45; P = .0001)
Trend test∗P = .10P = .0009

∗P values obtained by the Cox model. Chimerism was modeled as a time-dependent covariate. All models were adjusted for donor relationship (unrelated vs related), aggressive versus indolent disease, HCT-CI, and previous autologous transplantation.

Table 5. Associations between Matching of Recipient HLA Class I Ligand to Donor NK Cell KIR and Proportion of Relapse
Patients, n (%)Relapses, n (%)
Recipient has all ligands for donor inhibitory KIR28 (9.9)11/28 (39)
Recipient misses one or more ligands for donor inhibitory KIR237 (84)91/237 (38)
Recipient A3 or 11−/donor 3DL2+67 (23.8)27/67 (40)
Recipient Bw4−/donor 3DL1+14 (5)5/14 (36)
Recipient C1−/donor 2DL2/3+7 (2.5)2/7 (29)
Recipient C2−/donor 2DL1+11 (3.9)2/11 (18)
Recipient A3/11− and Bw4−/donor 3DL2+ and 3DL1+11 (3.9)5/11 (45)
Recipient A3/11− and C1−/donor 3DL2+ and 2DL2/3+21 (7.4)7/21 (33)
Recipient A3/11− and C2−/donor 3DL2+ and 2DL1+43 (15.2)21/43 (49)
Recipient Bw4− and C1−/donor 3DL1+ and 2DL2/3+4 (1.4)1/4 (25)
Recipient Bw4− and C2−/donor 3DL1+ and 2DL1+26 (9.2)11/26 (42)
Recipient A3/11− and Bw4− and C1−/donor 3DL2+ and 3DL1+ and 2DL2/3+3 (1.1)1/3 (33)
Recipient A3/11− and Bw4− and C2−/donor 3DL2+ and 3DL1+ and 2DL2/3+30 (10.6)9/30 (30)
Unknown KIR genotype17 (6)5/17 (29)

A total of 172 patients had measurable disease at HCT, and 69 of them achieved complete remission (CR). The average CD3 donor chimerism level was 72% in those patients who achieved CR and 67% in those who did not (P = .23).

A total of 64 patients died of a nonrelapse-associated cause between 20 and 2466 days (median, 230 days) after HCT. No statistically significant associations were found between donor T cell (P = .67), or NK cell (P = .21) chimerism level and NRM when chimerism was modeled as a continuous linear variable. These results did not change after adjustments for both the presence or absence of 1 or more ligands for donor NK cell KIRs and the numbers of activating or inhibitory genes (data not shown).

When chimerism level was modeled as a continuous linear variable, high levels of both donor T cell (P = .01) and NK cell (P < .0001) chimerism were associated with better PFS. The results were similar after adjustment for the presence of 1 or more ligands for donor NK cell KIRs and the numbers of activating or inhibitory genes (data not shown). The risk of failure for PFS was higher in patients missing at least 1 ligand for donor NK cell KIRs compared with those not lacking such ligands, but the difference was not statistically significant (HR = 1.33; 95% CI = 0.81 to 2.20; P = .26).

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Discussion 

It has been commonly accepted that donor T cells play important roles in engraftment, GVHD, and graft-versus-tumor effects after HLA-matched HCT in patients with malignancies [9]. The removal of T cells from hematopoietic grafts by various T cell depletion techniques generally is associated with higher graft rejection rates, less GVHD, and more frequent relapse/progression of underlying malignancies compared with the use of unmodified grafts 34, 35. Consistent with these previous observations, our patients with high numbers of grafted T cells experienced rapid, high donor T cell chimerism, which in turn was associated with a lower rate of graft rejection, increased rates of aGVHD and cGVHD, and a slightly (but not statistically significant) decreased risk of relapse.

The role of donor NK cells in these transplantation settings remains far less clear. One of the earliest studies, of 175 patients receiving an HLA-matched related marrow graft after conventional conditioning, found relatively rapid recovery of NK cell function but no correlation between the tempo of recovery and the risk of GVHD, infection, or recurrence of malignancy [7]. Nonetheless, renewed interest in the role of NK cells in the HCT setting was rekindled by findings from investigators in Perugia, Italy, showing that after HLA-haploidentical HCT, graft-versus-host NK cell reactivity was associated with lower rates of graft rejection, aGVHD, and relapse in patients with acute myelogenous leukemia (AML) [10]. A more recent study by this group found similar incidences of aGVHD in patients with or without graft-versus-host NK cell reactivity, however [36].

The most robust predictor of donor versus recipient NK cell alloreactivity in the HCT setting has been a matter of extensive debate. Based on experimental observations, the Perugia investigators proposed that all mature NK cells express at least 1 inhibitory receptor for self HLA, and thus the presence or absence of functional KIRs can be detected by HLA genotype 10, 37. Based on this hypothesis, that group developed a simple algorithm, known as the KIR ligand incompatibility model, in which comparison between donor and recipient HLA class I genotype allows prediction of NK alloreactivity. Several other groups have retrospectively tested the KIR ligand incompatibility model in patients given grafts from HLA-mismatched URDs 17, 18, 38, 39. Although some of these studies found a lower risk of relapse in patients with KIR ligand incompatibility in the graft-versus-host direction 17, 18, others failed to identify such an association 38, 39, 40.

Given that HLA and KIR genes are encoded on chromosomes 6 and 19, respectively, they are inherited independently [41]. Consequently, 75% of HCTs with HLA-matched siblings and almost all HCTs with URDs are KIR genotype–mismatched [41]. These observations are the basis for the “missing KIR ligand model,” in which donor–recipient NK cell alloreactivity is predicted by analyses of donor KIR genotype and recipient HLA genotype 42, 43. Both HLA-matched and HLA-mismatched donor–recipient pairs may have missing ligands. In support of this model, one study found that early after transplantation, engrafted donor stem cells gave rise to a NK cell wave that expressed the same repertoire as the donor cells [14] and contained high frequencies of donor-versus-recipient alloreactive NK cells in HCTs in which the recipients lacked ligands for donor NK cell KIRs [15]. Several studies have evaluated the missing KIR ligand model in HLA-identical HCT, with differing results. Although some of the studies found a lower risk of relapse in patients missing 1 or more KIR ligands 16, 19, others failed to find such an association or even found a detrimental effect of missing KIR ligands 44, 45.

Given the strong association between rapid posttransplantation establishment of high donor NK cell chimerism levels and better PFS reported in the current study [20], along with previous data demonstrating the importance of KIR ligands in HLA-haploidentical transplantation [10], we explored whether immunogenetic factors possibly could provide additional information on the mechanisms underlying donor NK chimerism and, specifically, whether donor–recipient HLA and KIR genotype information could predict those patients who might have a higher probability of achieving robust donor NK chimerism. Interestingly, we found no association between missing 1 or more ligands for donor NK cells and risk of relapse. Furthermore, the qualitative association between prompt NK cell engraftment and less relapse did not differ after adjustment for number of activating or inhibitory donor KIR genes (which served as markers for KIR donor haplotype). Several significant characteristics of our study population might help explain this lack of association of genotype with outcome. Most importantly, the conditioning and GVHD prophylaxis regimens and the use of T cell–replete donor stem cells in our patients provided a setting that favored T cell reconstitution 46, 47 and differed greatly from the regimens used in the HLA-haploidentical transplantation setting [10]. Both the HLA and KIR genetic systems are highly polymorphic [48], and the organization of KIR genes on haplotypes is complex [33]; examination of the clinical impact of KIR ligands together with their cognate receptors resulted in very small numbers of donor–recipient pairs with each combination, and might have limited our ability to evaluate their clinical importance. Likewise, although we found a lower risk of relapse in those patients with all ligands for donor KIRs compared with those missing 1 or more ligands, along with a greater risk of failure to achieve progression-free survival in patients missing at least 1 ligand for donor KIRs compared with patients with all such ligands, these differences were not statistically significant. Examination of these questions in a larger transplantation experience will be important in the future. Analyses of recipient and donor KIR haplotypes are ongoing to investigate a possible association between KIR haplotype and HCT outcome after nonmyeloablative conditioning, as has been observed in the myeloablative setting 33, 49.

A previous study comparing KIR reconstitution in patients given T cell–depleted or unmanipulated grafts found that T cells in the graft altered HLA-C–binding KIR reconstitution, whereas, interestingly, reconstitution of KIR3/DL1, binding to Bw4, was less affected by the number of T cells in the grafts [50]. This finding prompted us to investigate whether missing ligands for donor Bw4 would impact HCT outcomes. Confirming the findings from analysis of all KIR ligands together, we found no impact of a missing ligand for KIR3/DLI on relapse risk and other HCT outcomes.

Our main finding in the present study is that prompt donor NK cell engraftment correlated with low risk of relapse. This association was not affected by donor type (related vs unrelated) or by disease category. Our results are in agreement with recent findings reported by Savani et al. 51, 52 that rapid versus slow NK cell recovery was associated with lower risk of relapse and better overall survival (OS) in patients with chronic myelogenous leukemia (CML) given T cell–depleted G-PBMCs after myeloablative conditioning 51, 52. Interestingly, in those studies, rapid NK cell recovery correlated not only with high numbers of transplanted CD34+ cells, but also with higher numbers of donor total (inhibitory and activating) KIR genes 51, 52. It might be argued that prompt NK cell engraftment after nonmyeloablative conditioning can serve as a marker of “good graft function” without implicating donor NK cells in graft-versus-tumor effects. But, this hypothesis is unlikely, because previous studies have failed to identify a correlation between the kinetics of granulocyte engraftment and risk of relapse 3, 20, 23. Furthermore, the association between high donor NK chimerism levels and low risk of relapse remained quantitatively similar when the analyses were restricted to patients with day-14 NK chimerism levels > 25%, this demonstrating that this association was not affected by patients with very poor engraftment (who were likely to have graft rejection and thus relapse). Importantly, prompt donor NK cell engraftment was not associated with higher incidence of aGVHD or cGVHD, suggesting that initiation of NK cell adoptive immunotherapy early after HCT could be a promising approach to separate graft-versus-tumor effects from GVHD.

In conclusion, we found that robust engraftment of donor NK cells correlated with low risk of graft rejection, low risk of relapse, and high PFS, but not with aGVHD. These associations did not depend on donor NK cell alloreactivity. The clinical importance of recipient ligand and donor KIR haplotypes on posttransplantation donor NK chimerism and HCT outcomes merits further study.

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Acknowledgments 

Financial disclosure: This work was supported in part by National Institutes of Health Grants CA78902, CA92058, HL36444, CA18029, CA49605, and CA15704. D.G.M. also was supported by a grant from the Gabrielle Rich Leukemia Foundation, and R.S. also received support from the Laura Landro Salomon Endowment Fund. FB is a research associate of the National Fund for Scientific Research (FNRS), Belgium. We thank Serina Gisburne, Sam Shin, Patrice Stroup, and Eustacia Zellmer for their excellent technical assistance; Mohamed Sorror, MD, for the HCT-CI scores; Gresford Thomas and Heather Hildebrant for data processing; research nurses Michelle Bouvier, Mary Hinds, and John Sedgwick for research assistance; all of the participating physicians, physicians' assistants, and clerical staff for their dedicated patient care; and Bonnie Larson, Helen Crawford, and Sue Carbonneau for help with manuscript preparation.

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 F.B. and E.W.P. contributed equally to this work.

 Financial disclosure: See Acknowledgments on page 587.

PII: S1083-8791(09)00063-9

doi:10.1016/j.bbmt.2009.01.018

Biology of Blood and Marrow Transplantation
Volume 15, Issue 5 , Pages 580-588, May 2009

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