Donor Killer Cell Immunoglobulin-like Receptor Research Articles

Donor KIR genotype based outcome prediction after allogeneic stem cell transplantation: no land in sight.

Optimizing natural killer (NK) cell alloreactivity could further improve outcome after allogeneic hematopoietic cell transplantation (alloHCT). The donor's Killer-cell Immunoglobulin-like Receptor (KIR) genotype may provide important information in this regard. In the past decade, different models have been proposed aiming at maximizing NK cell activation by activating KIR-ligand interactions or minimizing inhibitory KIR-ligand interactions. Alternative classifications intended predicting outcome after alloHCT by donor KIR-haplotypes. In the present study, we aimed at validating proposed models and exploring more classification approaches. To this end, we analyzed samples stored at the Collaborative Biobank from HLA-compatible unrelated stem cell donors who had donated for patients with acute myeloid leukemia (AML) or myelodysplastic neoplasm (MDS) and whose outcome data had been reported to EBMT or CIBMTR. The donor KIR genotype was determined by high resolution amplicon-based next generation sequencing. We analyzed data from 5,017 transplants. The median patient age at alloHCT was 56 years. Patients were transplanted for AML between 2013 and 2018. Donor-recipient pairs were matched for HLA-A, -B, -C, -DRB1, and -DQB1 (79%) or had single HLA mismatches. Myeloablative conditioning was given to 56% of patients. Fifty-two percent of patients received anti-thymocyte-globulin-based graft-versus-host disease prophylaxis, 32% calcineurin-inhibitor-based prophylaxis, and 7% post-transplant cyclophosphamide-based prophylaxis. We tested several previously reported classifications in multivariable regression analyses but could not confirm outcome associations. Exploratory analyses in 1,939 patients (39%) who were transplanted from donors with homozygous centromeric (cen) or telomeric (tel) A or B motifs, showed that the donor cen B/B-tel A/A diplotype was associated with a trend to better event-free survival (HR 0.84, p=.08) and reduced risk of non-relapse mortality (NRM) (HR 0.65, p=.01). When we further dissected the contribution of B subtypes, we found that only the cen B01/B01-telA/A diplotype was associated with a reduced risk of relapse (HR 0.40, p=.04) while all subtype combinations contributed to a reduced risk of NRM. This exploratory finding has to be validated in an independent data set. In summary, the existing body of evidence is not (yet) consistent enough to recommend use of donor KIR genotype information for donor selection in routine clinical practice.

Gene and genotype frequencies of immunoglobulin-like natural killer cell receptors in the population of Samara region

Background. The using of killer-cell immunoglobulin-like receptor (KIR) composition data is of increasing interest in clinical practice to select an optimal donor for allogeneic hematopoietic stem cell transplantation for treatment of hematologic malignancies to reduce graft versus host disease and the risk of relapse. It is also of interest to study the frequencies of KIR genes and genotypes in different populations. For the Russian Federation, KIR gene and genotype frequencies have been described for only a few relatively small samples and have not been fully studied. The study of KIR gene and genotype frequencies has not been conducted for the Samara region population to date.Aim. To study the frequencies of KIR genes and genotypes in the population of Samara region and to compare the data with previously described Russian populations.Materials and methods. To study the frequencies of KIR genes and genotypes, molecular genetic typing of 142 CBUs from the public cord blood bank of the Samara Regional Medical Centre “Dynasty” was performed. Molecular genetic typing of KIR genes was performed by polymerase chain reaction with sequence-specific primers with subsequent visualization of products in agarose gel. 16 KIR genes and pseudogenes were analyzed: 2DL1, 2DL2, 2DL3, 3DL1, 3DL2, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DS1, 2DP1, 3DL3, 2DL4, 2DL5, 3DP1. KIR gene frequencies were determined by direct counting. Genotypes were determined using Allele Frequencies database. A determination B-content group was performed using the Donor KIR B-content group calculator. Statistical analysis was performed using the χ2 test.Results. The highest frequency of KIR inhibitory genes was found for KIR2DL1 (98.6 %), KIR3DL1 (98.6 %), KIR2DL3 (96.5 %), KIR2DL5 (46.5 %), and KIR2DL2 (34.5 %). The most frequent among the activating receptor genes was KIR2DS4 (89.4 %), the frequencies of other KIR activating genes were KIR2DS2 – 45.1 %, KIR2DS1 – 35.9 %, KIR2DS3 33.8 %, and KIR2DS5 – 26.1 %. Comparative analysis of KIR gene frequencies in the population of Samara region and other Russian populations revealed certain differences. Significant differences in the frequencies of occurrence were found for KIR2DL3, KIR2DS4, KIR2DL2, KIR2DL5, KIR2DS3, KIR2DS5, as well as KIR2DP1 and KIR3DP1. Examination of 142 samples revealed 45 different genotypes: AA genotypes were detected in 30 % and Bx genotypes in 70 % of cases. AA genotype ID195 with a frequency of 5.6 % was detected, which has not been previously described in Russian populations. Among the Samara region population sample, only 3.5 % had the “best” status, 20.4 % had the “better” status, and 76.1 % had the “neutral” status of the B-content.Conclusion. The results obtained in this sample on the frequencies of KIR genes and genotypes differ from the previously published data for the Russian Federation. Of interest is the finding of a greater diversity of genotypes among a rather small study group, the detection of an atypical ID195 genotype, and the difference in the representation of B-content groups. The analysis of KIR genotypes in the population of Samara region can be used in the selection of optimal CBU and hematopoietic cell/bone marrow donors in addition to HLA typing. Studying the frequency distribution of KIR and HLA genes and genotypes can play a role in the study of fundamental aspects of human immunology and population genetics.

A retrospective analysis to evaluate if KIR B haplotype donors associate with a reduced risk of relapse in patients with haematological malignancies following haploidentical transplantation at the Blood and Bone Marrow Transplant Unit at Hammersmith Hospital ICHNHST.

Relapse is a major cause of treatment failure in haploidentical haematopoietic progenitor cell transplant (HPCT) with PTCy. Natural killer cells suppress graft versus host disease and mediate the graft versus leukaemia effect, driven by killer cell immunoglobulin-like receptors (KIRs). Emerging research suggests that donor KIR genotype may influence graft outcome in haploidentical transplants with varying impacts between patient cohorts. This study investigates whether donors with greater KIR B motifs associate with outcomes such as greater relapse-free survival (RFS), overall survival (OS), nonrelapse mortality (NRM), acute graft versus host disease (GvHD) and infection. The study cohort included 98 haploidentical donor-recipient (D/R) pairs (myeloablative n = 37, RIC n = 61) with various haematological malignancies, receiving primary T-cell replete haploidentical HSCT with PTCγ. Following KIR SSO genotyping, donors are categorised into neutral (n = 63) or better and best (n = 35), based on KIR B motif content. Kaplan-Meier and Cox regression survival functions are performed to investigate associations with outcomes. Our results show that the better and best category has significantly poorer RFS (p = 0.013; hazard ratio [HR] 3.16, 95% CI 1.21-8.24: p = 0.018). The greater risk of relapse associated with poorer OS (p = 0.011; HR 2.24, 95% CI 1.18-4.24: p = 0.01) in the better and best category. The competing KIR receptor-ligand and missing licensing proof models failed to predict transplant outcomes. Here, we show neutral donors associate with favourable outcomes in T-cell replete haplo-HPCT with PTCγ after categorisation using the KIR B content model, due to the increased risk of relapse associated with the use of better and best donors.

Refining eligibility criteria of unit selection for myeloablative cord blood transplantation in acute leukemia: Real-world experience of a referral center.

The algorithm for cord blood (CB) unit selection is still somewhat ambiguous. We retrospectively analyzed 620 cases of acute leukemia between 2015 and 2020, who were treated with myeloablative single-unit umbilical CB transplantation (UCBT). We found that, when human leukocyte antigen (HLA) mismatch was ≤3/10, CD34+ cell dosage<0.83 × 105/kg-considerably lower than prevalent guidelines-was permissible without affecting survival. Moreover, synergy between donor killer-cell immunoglobulin-like receptors (KIR) haplotypes-B and donor-recipient HLA-C mismatch protected against relapse-related mortality. We submit that minimum required CD34+ cell dosage can possibly be relaxed to broaden access to UCBT, and donor KIR genotyping should be considered during unit selection.

Donor NK and T Cells in the Periphery of Lung Transplant Recipients Contain High Frequencies of Killer Cell Immunoglobulin-Like Receptor-Positive Subsets

IntroductionFor end-stage lung diseases, double lung transplantation (DLTx) is the ultimate curative treatment option. However, acute and chronic rejection and chronic dysfunction are major limitations in thoracic transplantation medicine. Thus, a better understanding of the contribution of immune responses early after DLTx is urgently needed. Passenger cells, derived from donor lungs and migrating into the recipient periphery, are comprised primarily by NK and T cells. Here, we aimed at characterizing the expression of killer cell immunoglobulin-like receptors (KIR) on donor and recipient NK and T cells in recipient blood after DLTx. Furthermore, we investigated the functional status and capacity of donor vs. recipient NK cells.MethodsPeripheral blood samples of 51 DLTx recipients were analyzed pre Tx and at T0, T24 and 3wk post Tx for the presence of HLA-mismatched donor NK and T cells, their KIR repertoire as well as activation status using flow cytometry.ResultsWithin the first 3 weeks after DLTx, donor NK and T cells were detected in all patients with a peak at T0. An increase of the KIR2DL/S1-positive subset was found within the donor NK cell repertoire. Moreover, donor NK cells showed significantly higher frequencies of KIR2DL/S1-positive cells (p<0.01) 3wk post DLTx compared to recipient NK cells. This effect was also observed in donor KIR+ T cells 3wk after DLTx with higher proportions of KIR2DL/S1 (p<0.05) and KIR3DL/S1 (p<0.01) positive T cells. Higher activation levels of donor NK and T cells (p<0.001) were detected compared to recipient cells via CD25 expression as well as a higher degranulation capacity upon activation by K562 target cells.ConclusionHigher frequencies of donor NK and T cells expressing KIR compared to recipient NK and T cells argue for their origin in the lung as a part of a highly specialized immunocompetent compartment. Despite KIR expression, higher activation levels of donor NK and T cells in the periphery of recipients suggest their pre-activation during the ex situ phase. Taken together, donor NK and T cells are likely to have a regulatory effect in the balance between tolerance and rejection and, hence, graft survival after DLTx.

CT-163: Role of KIR Genotype in Haploidentical Stem Cell Transplant

Context Natural killer cells (NK) play a major role in defense against viruses and graft-versus-tumor effect. NK functions are regulated by killer-cell-immunoglobulin-like-receptor (KIRs) and their ligands (HLA class I molecules). In haploidentical stem cell transplantation (Haplo-SCT), donor KIR genotype is not routinely considered when choosing an “ideal donor”. Objective Evaluate the relevance of KIR genotype in a series of patients with hematologic malignancies submitted to Haplo-SCT. Design Retrospective cohort study. Setting Single-center study in a private referral hospital. Patients or other participants Donors and patients over 18 years old submitted to Haplo-SCT with post-transplantation cyclophosphamide as graft-versus-host disease (GVHD) prophylaxis, from 2015-2020. KIR genotype was studied in donors. Patients whose donors' blood samples were insufficient to determine KIR genotype were excluded. Main outcome measures We analyzed the impact of the KIR genotype on overall survival (OS), relapse-free survival (RFS), event-free survival (EFS), and GVHD. The variables include: patient ligands (C1×/C2, Bw4, and HLA 3-11), donor KIR haplotype, B content, KIR inhibitor mismatches, presence of KIR2DS1 or KIR3DS1, activating KIRs matches, mismatching in KIR ligands, and the iKIR score. Secondary outcomes included the impact of KIR2DS2, KIR2DS4, KIR2DL2 mismatch, and KIR3DL2 on cytomegalovirus (CMV) reactivation. Results Thirty patients and donors were included, with a median follow-up of 532 days. Median age of patients and donors were 45 years and 37 years, respectively. The main diagnosis was acute myeloid leukemia (40%). The cohort's 2-year OS was 63%. Patients with C1/x ligands (70%, n=21) presented a higher 1-year EFS than patients with C2/2 (75% vs 23% p=0.015). KIR2DL2 mismatch showed an improvement in 1-year OS (84% vs 20%, p Conclusions KIR genotyping can provide useful information to select the donor for Haplo-SCT. Larger cohort studies are needed to confirm these findings.

Impact of donor KIRs and recipient KIR/HLA class I combinations on GVHD in patients with acute leukemia after HLA-matched sibling HSCT

In addition to T cells, NK cells can also participate in the outcome of hematopoietic stem cell transplantation (HSCT) mainly through the interaction between donor killer cell immunoglobulin-like receptors (KIRs) and recipient human leukocyte antigen (HLA) class I molecules. There is a risk of GVHD other than leukemia relapse after allogeneic HSCT that activation of donor NK cells in the absence of appropriate inhibitory ligands will be one of the reasons. To investigate the impact of donor KIRs and recipient KIR/HLA class I combinations on GVHD and leukemia relapse in patients with acute leukemia after HSCT, 100 patients with acute leukemia who received HSCT from their HLA-matched siblings were included in this study. Genotypes of 16 KIR genes and two 2DS4 variants (full length and deleted alleles), along with HLA-A/B genotypes, were determined by PCR-SSP. HLA-C genotyping was done with the SSO-Luminex method. Chimerism analysis was done using 16 short tandem repeats (STRs) to detect early leukemia relapse. Acute (a)GVHD occurred in 38 patients, and 16 of them died during the study. None of the recipients showed any sign of leukemia relapse after HSCT. Full donor chimerism was observed in all tested patients during the first year after HSCT. Our results also indicated an increased risk of aGVHD in AA recipients with the C2/Cx, Bw4+ (or A-Bw4+) or HLA-A3−/A11− genotypes who received HSCT from Bx donors. Our results showed that donor selection based on donor-recipient KIR genotypes and recipient HLA class I status can improve the outcome of HSCT.

Presence of donor-encoded centromeric KIR B content increases the risk of infectious mortality in recipients of myeloablative, T-cell deplete, HLA-matched HCT to treat AML.

The reported influence of donor Killer-cell Immunoglobulin-like Receptor (KIR) genes on the outcomes of haematopoietic cell transplantation (HCT) are contradictory, in part due to diversity of disease, donor sources, era and conditioning regimens within and between different studies. Here, we describe the results of a retrospective clinical analysis establishing the effect of donor KIR motifs on the outcomes of 119 HLA-matched, unrelated donor HCT for adult acute myeloid leukaemia (AML) using myeloablative conditioning (MAC) in a predominantly T-cell deplete (TCD) cohort. We observed that HCT involving donors with at least one KIR B haplotype were more likely to result in non-relapse mortality (NRM) than HCT involving donors with two KIR A haplotypes (p = 0.019). Upon separation of KIR haplotypes into their centromeric (Cen) and telomeric (Tel) motif structures, we demonstrated that the Cen-B motif was largely responsible for this effect (p = 0.001). When the cause of NRM was investigated further, infection was the dominant cause of death (p = 0.006). No evidence correlating donor KIR B haplotype with relapse risk was observed. The results from this analysis confirm previous findings in the unrelated, TCD, MAC transplant setting and imply a protective role for donor-encoded Cen-A motifs against infection in allogeneic HCT recipients.

Donor KIR2DS1-Mediated Decreased Relapse and Improved Survival Depending on Remission Status at HLA-Haploidentical Transplantation with Post-Transplantation Cyclophosphamide

HLA-haploidentical allogeneic hematopoietic cell transplantation (allo-HCT) using post-transplantation cyclophosphamide (PT/Cy-haplo) is becoming the standard of care for patients without an HLA-matched related or unrelated donor. PT/Cy-haplo can give more patients the opportunity to undergo allo-HCT, because most patients have multiple available HLA-haploidentical related donor candidates. The optimal donor selection algorithm in the PT/Cy-haplo setting has not yet been established, however. To contribute to the establishment of a donor selection formula based on disease status and killer-cell immunoglobulin-like receptor (KIR) genotype, we retrospectively analyzed 91 patients who underwent PT/Cy-haplo at our institution. In both patients and donors, HLA allele genotyping was performed for HLA-A, -B, -C, and -DRB1, and 16 KIR genes were genotyped. Patients in complete remission (CR) who underwent PT/Cy-haplo from a KIR2DS1-positive donor had a significantly lower cumulative incidence of relapse (CIR) than those who underwent PT/Cy-haplo from a KIR2DS1-negative donor (1-year CIR: 0% versus 32.6%, P=.037; 2-year CIR: 9.2% versus 42%, P=.037). Moreover, PT/Cy-haplo from a KIR2DS1-positive donor was significantly associated with improved overall survival (OS) (1-year OS: 91.7% versus 58.7%, P=.010; 2-year OS: 83% versus 34%, P=.010). In contrast, in non-CR individuals, PT/Cy-haplo from KIR2DS1-positive donors did not significantly improve CIR or OS (1-year CIR: 56.5% versus 64.7%, P=.973; 2-year CIR: not reached versus 64.7%, Pnot evaluable; 1-year OS: 25.4% versus 20.6%, P=.418; 2-year OS: 5.1% versus 20.6%, P=.418). In addition, lower infused CD34+ cell dose, female-to-male transplantation, and acute myelogenous leukemia were significantly associated with increased risk of relapse and mortality. This study demonstrates that graft-versus-leukemia/tumor effects were exerted through donor KIR2DS1 at PT/Cy-haplo when patients have low tumor burdens. It would be worth examining the inclusion of donor KIR genotyping and disease status assessment in establishing optimal donor selection criteria for PT/Cy-haplo.

Donor and host coexpressing KIR ligands promote NK education after allogeneic hematopoietic stem cell transplantation

AbstractThe rate and extent of natural killer (NK)–cell education after hematopoietic cell transplantation correlates with leukemia control. To study the effect of donor and host HLA on NK-cell reconstitution, single killer-cell immunoglobulin-like receptor (KIR)+ NK cells (exhibiting KIR2DL1, KIR2DL2/KIR2DL3, or KIR3DL1 as their sole receptor) were grouped into 4 groups based on the interaction between donor/host HLA and donor inhibitory KIR in 2 cohorts (n = 114 and n = 276, respectively). On days 90 to 180 after transplantation, the absolute number and responsiveness against K562 cells (CD107a or interferon-γ expression) of single-KIR+ NK cells were higher in pairs where donor and host HLA both expressed ligands for donor inhibitory KIRs than in pairs where 1 or both of the donor and recipient HLA lacked at least 1 KIR ligand. NK-cell responsiveness was tuned commensurate with the number of inhibitory receptors from the donor. When both donor and host expressed the 3 major KIR ligands (HLA-C1, HLA-C2, and HLA-Bw4), NK cells expressing 3 inhibitory receptors (KIR2DL1/2DL3/3DL1) reached the maximum responsiveness against K562 cells compared with those NK cells expressing only 1 or 2 inhibitory receptors. When donor and host HLA both expressed all ligands for donor inhibitory KIRs, patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) showed the lowest recurrence rate after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). In conclusion, this study demonstrates that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells achieve better functional education and contribute to least relapse among patients. This observation study was registered at www.clinicaltrials.gov as #NCT02978274.

Impact of Donor KIR and HLA Genotypes on Clinical Outcomes According to Pre-Transplant Remission Status after HLA-Haploidentical Transplantation with Post-Transplantation Cyclophosphamide

Impact of Donor KIR and HLA Genotypes on Clinical Outcomes According to Pre-Transplant Remission Status after HLA-Haploidentical Transplantation with Post-Transplantation Cyclophosphamide

Impact of KIR/HLA Incompatibilities after Posttransplant Cyclophosphamide Based T Cell-Replete Haploidentical Hematopoietic Stem Cell Transplantation

Introduction: Posttransplantation cyclophosphamide (PTCy) based T cell-replete haploidentical (haplo) hematopoietic stem cell transplantation (HSCT) is a valid option for patients with indication for allogeneic HSCT without a human leucocyte antigen (HLA) matched donor. However, selection criteria to determine the optimal among several available haplo donors are still a matter of debate. Especially, the impact of killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen (HLA) incompatibilities (inc) in the setting of PTCy T cell-replete haplo HSCT is unclear. PTCy has been reported to eliminate most mature donor NK cells infused with the graft, including single KIR+ NK cells, thereby blunting NK cell alloreactivity in this setting (Russo et al., Blood 2018). Willem et al. (J Immunol 2019) reported (i) a significant loss of KIR2DL2/3+ NK cells at day +30 in patients with inhibitory KIR/HLA incompatibility (inc.) suggesting that PTCy might target responsive KIR NK cells and (ii) a correlation of genetic KIR2DL/HLA inc. with less relapse, but more graft-versus-host-disease (GvHD). Similarly, NK alloreactivity defined as KIR receptor-ligand mismatch or group B KIR haplotype with the presence of KIR2DS2 has been correlated with improved survival (Salomon et al., BBMT 2018). Aims of our study were to evaluate the impact of (i) HLA/KIR inc, (ii) donor KIR genotype and (iii) HLA-DP mismatch status on survival and incidence of relapse, acute and chronic GvHD in our homogeneously treated, independent patient cohort. Patients and methods: We retrospectively analyzed the outcome of 51 consecutively transplanted patients (AML/MDS (n=28/5), ALL (n=9), HD (n=2), NHL (n=5), CML (n=1), PMF (n=1)) receiving a PTCy based T cell-replete haplo HSCT between 01/2011-12/2018. All patients received a myeloablative conditioning regimen (fludarabine/total body irradiation (FTBI) or thiotepa/busulfan/fludarabine (TBF)) with unmanipulated bone marrow (98%) as the preferred graft (median CD34+ cells: 3.02 x 106/kg (range, 1.50-6.90) and median CD3+ T cells: 3.54 x 107/kg (range 1.52-43.74)). GvHD prophylaxis with ciclosporin A started on day 0, mycophenolate-mofetil on day +1, PTCy was applied on day +3 and +5. Results: Patient, donor and transplant characteristics as detailed in table 1 were well balanced between the inh. KIR/HLA inc. group (n=29) vs. no inh. KIR/HLA inc. group (n=22) with the exception of the median donor age (41.7 (range, 23.4-73.7) vs. 33.6 years (range, 19.0-56.2), resp. All patients engrafted. At day +28 (range, 20-29; n=26) CD3+ cells were 88.5/nL (range, 3-665), CD3+CD4+ cells 22.5/nL (range, 0-277.0), CD3+CD8+ cells 117.0/nL (range, 7-478), CD19+ cells 1.0/nL (range, 0-12), CD56bright cells 74.4/nL (range11.1-93.4), CD56dim cells 25.5/nL (range, 6.4-88.9) measured by flow cytometry and without differences between the inh. KIR/HLA inc. group vs. no inh. KIR/HLA inc. group. Cytomegalovirus (CMV) reactivation occurred in 73.3% of patients at risk and median time of occurrence was 32 days (range, 12-97) without difference between groups. Median follow-up for surviving patients was 26.1 months (range, 2.8-92.8) and we found no significant differences in 2-year overall survival (OS; 65.3±10.3 vs. 89.6±7.0, p=0.311), 2-year relapse-free survival (RFS; 66.0±9.4 vs 77.8±10.2, p=0.235), GvHD- and relapse-free survival (GRFS; 48.4±9.8 vs 60.5±12.0, p=0.182) as well as cumulative incidence (CI) of relapse (23.3% vs 16.2%, p= 0.283), acute GvHD grade 2-4 (27.6% vs 31.8, p=0.563), moderate-severe chronic GvHD (22.2% vs. 9.9%, p=0.227) and NRM (16.3% vs 5.3%, p=0.283) between the inh. KIR/HLA inc. group vs. no inh. KIR/HLA inc. group. This was also the case for donor KIR genotype AA vs AB (n=46; 2-y OS: 74.9±13.0% vs. 73.0±9.9%, p=0.844; 2-y RFS: 60.0±14.8% vs 74.5±8.4%, p=0.645) and HLA-DP-identical/permissive mismatch (MM) vs non permissive MM (n=45; 2-y OS: 70.7±10.0% vs 72.7±13.4%, p=0.945; 2-y RFS: 73.2±8.2% vs 63.6.0±14.5%, p=0.798) Conclusion: Our outcome data support the hypothesis of PTCy eliminating mature donor NK cells infused with the graft and thereby reducing the impact of alloreactivity in this setting. However, our patient number is quite small and the findings need to be validated in larger cohorts and preferably prospective studies. Disclosures Lindner: Celegene, Sanofi, Neovii: Honoraria, Research Funding. Berg:Riemser Pharma GmbH: Consultancy, Honoraria; Incyte, Abbvie, Astellas, Alexion and Celgene: Other: travel support. Bug:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene Neovii: Other: travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Hexal: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Sanofi: Other: travel grants. Schwaeble:Uniqure BV: Research Funding. Ullrich:CellGenix: Honoraria, Research Funding; Novartis: Research Funding.

Role of killer cell immunoglobulin-like receptor (KIR)-ligand interactions to prevent relapse in patients (pts) receiving matched unrelated stem cell transplant (SCT) for acute myeloid leukemia (AML).

7049 Background: NK cells play a pivotal role in cancer immune vigilance. NK cell function is regulated by a balance between activating and inhibitory signals derived from various receptor-ligand interactions. Methods: 390 AML pts received 10/10 unrelated donor SCT. KIR genotype was performed by PCR with sequence specific primers. Hazard ratio (HR) for 2-year relapse was calculated using Fine-Gray regression and adjusted for disease risk index, remission status, pre-SCT MRD, conditioning regimen and presence of HLA-DP mismatch. KIR-ligand (K-L) match was defined as the presence a given KIR in the donor and the presence of its reported ligand in the patient (ex. 2DL1 and HLA-C2). KIRs that have no known ligands were not included in this analysis. The Table shows pt characteristics and K-L matches. Results: There was no correlation between the number of inhibitory or activating KIRs or KIR haplotype (A or B) and the probability of relapse after SCT. However, donor KIRs had a dramatic effect on relapse when they were considered together with the presence of the corresponding ligand in the pt. The 210 pts who had ≥3 inhibitory K-L matches had a significantly higher probability of relapse (HR=1.748, CI=1.147-2.667, p=0.009) than the remaining 180 pts. Similarly, the 96 pts who had at least one known activating K-L match had a lower probability of relapse (HR=0.581, CI=0.345-0.978, p=0.04). When we considered inhibitory and activating K-L matches together, we found that the 168 pts who had ≥3 inhibitory and no activating K-L matches had a significantly higher probability of relapse (HR 2.001, CI=1.376-2.908, p&lt;0.001) than the 222 remaining pts. Conclusions: Donor-pt KIR-ligand matching should be taken into account when choosing unrelated donors for AML pts. [Table: see text]

Donor KIR Genotype Impacts on Clinical Outcome after T Cell–Depleted HLA Matched Related Allogeneic Transplantation for High-Risk Pediatric Leukemia Patients

Preliminary data suggest that ex vivo T cell-depleted matched related donor (MRD) hematopoietic stem cell transplantation (HSCT) is feasible and confers a clinically beneficial reduction in graft-versus-host disease. Classically, T cell-depleted grafts were associated with reduction of the graft-versus-leukemia (GVL) effect because of delayed T cell immune reconstitution. However, natural killer (NK) cell alloreactivity is also critical for an early GVL effect as well as for lymphocyte immune reconstitution. Here, we study the role of NK cells in MRD T cell-depleted HSCT, which is still poorly understood. Given that MRD ligands for inhibitory killer cell immunoglobulin-like receptors (KIRs) are matched, we focused on activating KIR receptors. We retrospectively analyzed KIR genotyping in patients and MRDs in 40 ex vivo T cell-depleted pediatric HSCTs. The log-rank test and Cox proportional risk test were performed to correlate genotype with clinical outcome (relapse rate, disease-free survival, and overall survival) and immune reconstitution. The statistical analysis revealed poorer overall survival when donors have a KIR-B content score of ≥2, a best/better subtype, or present the KIR2DS1 gene. The patient's relapse rate was higher when donors present the KIR2DL5A gene, as well as a poorer probability of disease-free survival when the donor is classified with a best/better subtype. Regarding immune reconstitution, donor KIR haplotype A or the presence of inhibitory KIR genes promote best recovery of T lymphocytes, whereas donor KIR haplotype B or the presence of activating KIR genes confer better expansion of NK cells. These findings suggest that the selection of MRDs with an inhibitory KIR phenotype improve T cell expansion as well as the clinical outcome after pediatric ex vivo T cell-depleted HSCT.

OR8 Killer cell immunoglobulin-like receptor genotype BX1 and centromeric gene-content motifs influences survival after 3-models of allogeneic hematopoietic stem cell transplantation

Aim To investigate the role of donor killer cell immunoglobulin-like receptor (KIR) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to develop a donor selection strategy for Chinese population. Methods In this 5-year retrospective study, we performed KIR genotyping on a total of 626 donor and recipient pairs by PCR-SSP. All patients underwent HSCT, comprising 219 HLA-identical sibling HSCT, 210 HLA-identical unrelated donor (URD) HSCT, and 197 HLA haplo-identical HSCT, for treatment of myeloid and lymphoid leukemia. Results Donor KIR genotypes and KIR gene motifs influenced transplantation outcomes for AML/MDS but not ALL/NHL patients in 3 models of transplants. In sibling HSCT for AML/MDS patients (n = 128), KIR mismatch between patient and donor had a better overall survival (OS; 87.3% versus 69.3%; P = 0.027) and reduced grade III to IV acute graft-versus-host disease (aGVHD; 4.6% versus 13.6%; P = 0.029). However, the effect was not observed in URD (n = 122) and haplo-identical HSCT (n = 108) for AML/MDS patients. Multivariate analysis indicated that centromeric B motifs contributed to improved survival in all models of transplants: (1) In sibling HSCT, donors’ haplotype cB-tA/tB were associated with improved OS compared with cA-tB (96.6% versus 71.2%; P = 0.021) or cA-tA (96.6% versus 72.1%; P = 0.017); (2) In URD-HSCT, donors with Cen -B were associated with a significantly improved OS (HR = 0.256; 95% CI, 0.084 to 0.774; P = 0.016) and RFS (HR = 0.252; 95% CI, 0.084 to 0.758; P = 0.014); (3) In haplo-identical HSCT, the same effect of Cen -B was observed on OS (HR, 0.335; 95% CI, 0.164 to 0.667; P = 0.002), RFS (HR, 0.331; 95% CI, 0.167 to 0.657; P = 0.002), and NRM (HR, 0.286; 95% CI, 0.120 to 0.683; P = 0.005). Regarding the KIR genotypes, compared with other donor KIR B/x genotypes: (1) In sibling HSCT, genotype Bx1 was a risk factor for lower OS (HR, 8.667; 95% CI, 2.321 to 32.37; P = 0.001) and RFS (HR, 9.207; 95% CI, 2.631 to 32.22; P = 0.001); (2) The same negative effect of Bx1 was also found in URD-HSCT (OS: P = 0.047; RFS: P = 0.041) and haplo-identical HSCT (OS: P = 0.002; RFS: P = 0.002). Conclusions Avoiding the selection of Bx1 donors is strongly advisable for AML/MDS patients undergoing both related and unrelated HSCT whereas the presence of the Cen -B motifs was rather recommended in donor selection.

Killer Immunoglobulin-Like Receptor Allele Determination Using Next-Generation Sequencing Technology.

The impact of natural killer (NK) cell alloreactivity on hematopoietic stem cell transplantation (HSCT) outcome is still debated due to the complexity of graft parameters, HLA class I environment, the nature of killer cell immunoglobulin-like receptor (KIR)/KIR ligand genetic combinations studied, and KIR+ NK cell repertoire size. KIR genes are known to be polymorphic in terms of gene content, copy number variation, and number of alleles. These allelic polymorphisms may impact both the phenotype and function of KIR+ NK cells. We, therefore, speculate that polymorphisms may alter donor KIR+ NK cell phenotype/function thus modulating post-HSCT KIR+ NK cell alloreactivity. To investigate KIR allele polymorphisms of all KIR genes, we developed a next-generation sequencing (NGS) technology on a MiSeq platform. To ensure the reliability and specificity of our method, genomic DNA from well-characterized cell lines were used; high-resolution KIR typing results obtained were then compared to those previously reported. Two different bioinformatic pipelines were used allowing the attribution of sequencing reads to specific KIR genes and the assignment of KIR alleles for each KIR gene. Our results demonstrated successful long-range KIR gene amplifications of all reference samples using intergenic KIR primers. The alignment of reads to the human genome reference (hg19) using BiRD pipeline or visualization of data using Profiler software demonstrated that all KIR genes were completely sequenced with a sufficient read depth (mean 317× for all loci) and a high percentage of mapping (mean 93% for all loci). Comparison of high-resolution KIR typing obtained to those published data using exome capture resulted in a reported concordance rate of 95% for centromeric and telomeric KIR genes. Overall, our results suggest that NGS can be used to investigate the broad KIR allelic polymorphism. Hence, these data improve our knowledge, not only on KIR+ NK cell alloreactivity in HSCT but also on the role of KIR+ NK cell populations in control of viral infections and diseases.

Analysis of the Impact of KIR B Haplotype Donors on Outcomes after Haploidentical (HI) and Matched Related (MR) Hematopoietic Stem Cell Transplantation (HSCT)

Introduction:Natural killer (NK) cell alloreactivity has been associated with graft versus leukemia effects in HSCT. These effects are in part mediated through killer cell immunoglobulin-like receptors (KIR) on the surface of NK cells. The absence of inhibition of NK cells via inhibitory KIR in KIR ligand mismatched T-cell depleted HI HSCT, and the use of unrelated donors possessing activating KIR haplotypes (KIR-B) have been associated with improved overall survival (OS) due to relapse protection. Inhibitory KIR ligand mismatch in the setting of T-cell replete HI HSCT has not consistently been shown to be beneficial, and data regarding the impact of using KIR-B donors in T-replete HI and MR HSCT is limited. In this preliminary retrospective review, we compared outcomes between patients with KIR-B donors versus KIR A haplotype (KIR-A) donors after HI and MR HSCT to help inform donor selection.Methods:All patients underwent HSCT on our 2-step approach in which after conditioning, a fixed T cell dose (2 x 108/kg) is administered (step 1), followed 2-3 days later by cyclophosphamide (CY) for bidirectional tolerization. Two days after CY, patients receive a CD 34-selected donor product (step 2). To be included in the analysis, patients had to be alive for at least 1 month post HSCT and be without evidence of graft rejection. Recipients were divided into 2 groups based on whether they were transplanted from a KIR-B (possessing one or more non-framework activating KIR genes) versus a KIR-A donor (genotypically negative for non-framework activating KIR genes). The relationship between the use of a KIR-B donor and the end points of OS, relapse, grades II-IV acute graft versus host disease (GVHD), chronic GVHD and non-relapse mortality (NRM) was examined. Probability of OS was estimated by Kaplan-Meier (KM) method with log-rank analysis and multivariate analyses using Cox proportional hazards. Donor and recipient age and gender, HCT-CI score, Revised Disease Risk Index, presence of any inhibitory KIR ligand mismatch between donor and recipient and CMV reactivation after HSCT were considered as confounding variables. Cumulative incidence functions with competing risk analyses were used to estimate the probabilities of relapse, NRM, acute and chronic GVHD.Results:The study group consisted of 128 consecutive patients with myeloid malignancies (AML=91 MDS=27, CML=1, MPD=9) undergoing HI [n=84] or MR [n=44] HSCT. The median recipient age and follow-up of the study group was 58 (range 19-78) years and 40.6 (range: 1.07-117.27) months respectively. Only 13 (10.2%) patients had grade III or higher acute GVHD and only 7 (5.5%) patients developed severe, chronic GVHD (score of 3) respectively. Seventy-four (58%) and 54 (42%) of recipients had KIR-B versus KIR-A donors respectively. Fifty-five (74.3%) KIR-B donors possessed HLA C1 or C1/2 alleles in the presence of KIR2L2/3/2DS2. On multivariate Cox regression analysis, OS (HR, 0.579; 95% CI, 0.337-0.995; P= 0.048) was significantly higher in patients who received HSCT from KIR-B donors. On competing risk analysis, relapse (HR, 0.756; P=0.403) and NRM (HR, 0.466; P=0.128) were lower in patients who had KIR haplotype B donors though not statistically significant. In contrast, the risk of grades II to IV acute GVHD (with death as a competing event) was higher in patients with KIR-B donors (HR, 1.972; 95% CI, 1.009-3.852; P=0.047). Similarly transplants from KIR-B donors were associated with a higher risk of chronic GVHD (HR, 2.89; P=0.053).Discussion:Our results show that within the context of the 2-step approach, the use of a KIR-B donor is associated with improved OS and an increased risk of acute and chronic GVHD in patients with myeloid neoplasms receiving HI and MR HSCT. While not reaching significance, the results for relapse and NRM suggest that the OS benefit in patients with KIR-B donors was derived from a combination of lower relapse rates and NRM that was not adversely impacted by GVHD which was mostly lower grade in this population. Our findings support the concept of KIR-B donors as more alloreactive than KIR-A donors, specifically those possessing a C1-C1/2/2DL2/3/2DS2 repertoire, the primary type possessed by KIR-B donors in this analysis. The data argues for the continued use of KIR genotyping at our institution and analyses of HSCT outcomes based on donor KIR repertoire and other donor characteristics to optimize donor selection in HI and MR HSCT. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Influence of KIR Genes and HLA Class I Ligands on Overall and Event-Free Survivals after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myelogenous Leukemia

Acute myelogenous leukemia (AML) is the most sensitive to natural killer (NK)-cell reactivity among blood malignancies treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Function of NK cells is regulated by their inhibitory and activating surface receptors, including killer-cell immunoglobulin-like receptors (KIRs). KIRs with specificity to different HLA class I-encoded ligands seem to play a major role in allorecognition: HLA-C allotypes with asparagine at position 80 (HLA-C1ligands) are recognized by KIR2DL2/3, HLA-C allotypes with lysine at position 80 (HLA-C2 ligands) are recognized by KIR2DL1and KIR2DS1, and HLA-B allotypes with a polymorphic sequence motif at position 77-83 (Bw4 ligands) are recognized by KIR3DL1.There are numerous but sometimes contradictory data about the role of KIR genes and genes of their HLA ligands in outcomes after HLA-identical related and HLA-matched unrelated HCST in patients with AML.The goal of our prospective study was to evaluate the influence of KIR genes and HLA class Iligands on overall survival (OS) and event-free survival (EFS) after allo-HSCT in adult patients with AML.35 patients with AML (median age 36 years, range 19-60) treated with allogeneic HSCT in our transplant center from HLA-identical related (n=19) and HLA-matched (10/10) unrelated (n=16) donors were included in the study. Median follow-up was 18 months (range, 5-56). The pre-transplantation risk category included standard or high risk. Patients in complete remission 1 (CR1) were categorized as standard risk (n=22), whereas patients in > CR1 were considered as high risk (n=13). In case of HLA-identical related HSCT donor KIR genotyping was performed simultaneously with HLA-typing. In case of HLA-matched unrelated HSCT donor KIR genotyping was performed in time of confirmatory HLA-testing or in day of HSCT. KIR genotyping was done using a PCR-SSP kit (Olerup, Sweden). Overall survival (OS) and event-free survival (EFS) were calculated by Kaplan-Meier method, and compared with log-rank test. OS was defined as survival without lethal event from any cause, EFS was defined as survival in complete remission without lethal event from any cause.The 3-year estimated OS and EFS in all patients were 66 and 43%, respectively. 10 patients died (29%), relapse (hematological and/or molecular) was diagnosed in 18 patients (51%). The pre-transplantation risk category was the main factor affecting OS and EFS after HSCT. The probability of the 3-year OS and EFS for standard-risk patients was 86 and 57%, respectively. Nobody of our patients with high risk lived more than18 months. Conditioning regimen, graft source and donor/patient gender combination had no significant effect on OS and EFS. The patients with unrelated HLA-matched donors had better (not significantly) EFS in comparison with recipients of related HLA-identical grafts (p=0.12).The influence of KIR and HLA- ligand genes on EFS after allo-HSCT was investigated in patients with standard risk. We did not find the immunogenetic factors (presence of B- haplotypes in donors, donor KIR B content, presence of centromeric or telomeric B- motifs in donors, presence of missing HLA ligand for any inhibitory KIR of donor), which significantly affected EFS, but we found two obvious tendencies. The patients with HLA-C1/C1 homozygosity had a tendency to improved EFS compared with patients having either HLA-C1/C2 or HLA-C2/C2 ligands (p=0.09). There was a tendency to better EFS in HLA-C1/x recipients of KIR2DS1 -positive allografts (p=0.09).Our data suggest that KIRs and their HLA class I ligands play role in the graft versus leukemia effect in AML. It seems that HLA-C1 homozygosity improves EFS in patients after allo-HSCT, and KIR2DS 1-positive donors are preferable for HLA-C1/x patients. DisclosuresNo relevant conflicts of interest to declare.

Negative Impact of Unidirectional Host-versus-Graft Killer Cell Immunoglobulin-like Receptor Ligand Mismatch on Transplantation Outcomes after Unmanipulated Haploidentical Peripheral Blood Stem Cell Transplantation for Acute Myeloid Leukemia.

This study explored the influence of mismatched inhibitory killer cell immunoglobulin-like receptor (KIR) ligands on the outcome of haploidentical transplantation using T cell-replete, granulocyte colony-stimulating factor-mobilized peripheral blood stem cells in adult patients with acute myeloid leukemia (AML). Three groups were examined: unidirectional graft-versus-host KIR ligand mismatched (GVH-KIR-MM; n= 33), bidirectional KIR ligand matched (KIR-M; n= 41), and unidirectional host-versus-graft KIR ligand mismatched (HVG-KIR-MM; n= 26). All recipients were treated with the same conditioning regimen (800 cGy total body irradiation, fludarabine, busulfan, and antithymocyte globulin). After a median follow-up of 26 months, the 2-year cumulative incidence of relapse was significantly higher in HVG-KIR-MM (40.3% ± 10.3%) versus others (18.9% ± 4.8%, P= .044). In the standard-risk group, the 2-year disease-free survival (DFS) was significantly lower in HVG-KIR-MM (51.8% ± 11.2%) compared with GVH-KIR-MM (88% ± 8.1%, P= .025). Multivariate analysis showed that HVG-KIR-MM was significantly associated with higher relapse (hazard ratio [HR], 10.7; P= .002) and lower DFS (HR, 3.4; P= .012). Subgroup analysis revealed increased DFS with higher doses of CD3(+)CD8(+) and CD3(-)CD56(+) grafts in GVH-KIR-MM (90.9% ± 8.7%, P= .006); there was no such effect in the other groups. Although our conclusions are limited by the absence of donor KIR genotype data, our study suggests unidirectional KIR ligand incompatibility in the host-versus-graft vector has a detrimental effect on T cell-replete haploidentical transplantation outcomes in adult patients with AML.

Genes of killer cell immunoglobulin-like receptors and their HLA ligands after allogeneic hematopoietic stem cell transplantation in myeloid leukemia patients

To study the impact of the genes of donor killer cell immunoglobulin-like receptors (KIR) and HLA-KIR ligands on overall (OS) and event-free survival (EFS) rates in patients with myeloid leukemia after transplantation with allogeneic hematopoietic stem cells (allo-HSCT) from HLA-identical related and HLA-compatible unrelated donors. The investigation enrolled 29 patients who had undergone allo-HSCT from KIR-genotyped donors at the Department of Bone Marrow Transplantation, Hematology Research Center (see symbol) in 2010-2013. OS and EFS rates after allo-HSCT were calculated using the Kaplan-Meier method. The main predictor of recurrence and survival in patients after allo-HSCT was a recurrence-risk group the patient belonged to before transplantation. The standard-risk group patients whose donors had telomeric gene-content motifs of KIR-B haplotypes had higher EFS rates than those whose donors lacked these genes. The standard-risk patients homozygous for HLA-1 alleles (i.e. without HLA-C2 ligand) tended to have higher EFS rates, so did the patients without HLA-Bw4 ligand. The donors having telomeric gene-content motifs of KIR-B haplotypes are more preferred for allo-HSCT for patients with myeloid leukemia as the presence of donor telomeric KIR-B genes increases EFS rates in standard-risk patients.