Clinical impact of natural killer cell reconstitution after allogeneic hematopoietic transplantation

Although the optimal donor for allogeneic hematopoietic stem cell transplantation is a human leukocyte antigen (HLA)-matched sibling, 75% of patients do not have a match and alternatives are matched unrelated volunteers, unrelated umbilical cord blood units, and full HLA-haplotype-mismatched family members. This review will focus on the open issues of allogeneic hematopoietic transplantation and on the benefits of natural killer (NK) cell alloreactivity and its underlying mechanisms. Donor-versus-recipient NK cell alloreactivity derives from a mismatch between inhibitory receptors for self major histocompatibility complex (MHC) class I molecules on donor NK clones and the MHC class I ligands on recipient cells. These NK clones sense the missing expression of the self MHC class I allele on the allogeneic targets (“missing self”) and mediate alloreactions. Here, we review the translation of NK cell allorecognition into the clinical practice of allogeneic hematopoietic transplantation and discuss how it has opened innovative perspectives in the cure of leukemia.     

Role of KIRs and KIR ligands in hematopoietic transplantation

This review focuses on recent research demonstrating the role alloreactive natural killer (NK) cells play in adoptive immunotherapy of leukemia in allogeneic hematopoietic transplantation. For patients with hematologic malignancies and an indication to allogeneic hematopoietic transplantation who do not have a matched sibling donor, unrelated donor, or cord blood transplants are almost always available (as long as the patient's ethnicity is represented in the donor registries). However, up to one half of patients relapse and do not make it to transplant during the time required for the donor search, completion of donor HLA typing, bone marrow harvest, and shipment. Donor-versus-recipient NK cell alloreactivity is effected by a functional repertoire of NK cells which express inhibitory Killer-Cell Immunoglobulin-like Receptor(s) (KIR) for self class I ligand(s), sense missing expression of donor KIR ligand(s) in the recipient and mediate alloreactions. It improves outcomes of HLA haplotype-mismatched ('haploidentical') transplants by controlling acute myeloid leukemia relapse without causing graft-versus-host disease. It is hoped the dramatic improvements afforded by the discovery of the role of NK cell alloreactivity will extend the use of haploidentical transplants, as the donors are, unlike the unrelated, immediately available family members.     

The beneficial role of inhibitory KIR genes of HLA class I NK epitopes in haploidentically mismatched stem cell allografts may be masked by residual donor-alloreactive T cells causing GVHD

HLA allele mismatches will provoke T-cell alloreactivity after allogeneic stem cell transplantation. As donors and recipients are usually HLA matched, the public HLA epitopes that are recognized by natural killer (NK) cells (NK epitopes) are rarely mismatched, and therefore there is rarely potential for NK alloreactivity arising from the absence of ligands for inhibitory killer immunoglobulin-like receptors (KIR). Transplants using related donors sharing only one haplotype (haploidentical donors) represent a setting in which NK epitopes are often mismatched, thus resulting in the potential for NK alloreactivity. We have analyzed engraftment, acute graft vs host disease (GVHD), leukemia relapse, and survival in 62 haploidentical transplants in relationship with potential NK alloreactivity, inhibitory, and activating KIR genes of class I HLA NK epitopes. Potential NK alloreactivity in the rejection direction was not associated with any outcome variable. Potential NK alloreactivity in the GVHD direction was associated with an increased incidence of severe GVHD and poorer patient survival but not with non-engraftment nor leukemia relapse. A higher number of activating KIR receptors in the genome of the donor was associated with a higher prevalence of GVHD. These results suggest that lack of extensive T-cell depletion in haploidentical transplantation is associated with high GVHD rates and diminishes the benefits of NK-cell alloreactivity.     

Immunogenomics of hematopo?etic stem cell transplantation.

Recipients of allogeneic hematopo?etic stem cell transplantation (HSCT) incur the risk of graft-versus-host disease (GVHD) even when the donor is a sibling who shares the major histocompatibility antigens. Therefore, even the perfect HLA match does not represent the optimal genetic match between donors and recipients in HSCT. In addition to the HLA complex other genetic systems operate and affect the outcome of HSCT. These include minor histocompatibility systems (inducing bona fide allogeneic responses) as well as a series of functional polymorphisms in cytokines and chemokines and receptors genes. Among the items affecting the outcome of HSCT the incidence and severity of infections have an important impact. Polymorphisms of genes controlling both arms of the immune responses to pathogens (innate vs. cognate) are strong candidates for susceptibility factors to infection in allogeneic transplantation. These include the MHC alleles (HLA class I, class II, MIC) CD1, Toll and TLR genes MBP, MPO genes.). In addition to the NK alloreactivity induced by HLA class I epitopes mismatching (a common situation in HSCT) variations in the genotype of the KIR genes may also be encountered between the donor and the recipient leading to potentially harmful or beneficial combinations. An integrated knowledge of the role and hierarchy of the most important genetic factors (MHC and non-MHC) will provide the rationale for a comprehensive matching in HSCT. This short review provides a panorama of this strategic issue for further development of HSCT.     

KIR matching in hematopoietic stem cell transplantation

Although the key role of MHC-restricted T lymphocytes in hematopoietic stem cell transplantation (HSCT) has been known for a long time, recent data have focused on complementary or alternative effector cell populations, and in particular on NK cells. Spontaneously generated NK cell alloreactivity from stem cell grafts involves specific interactions between NK receptors, including killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands. The combined effects of HLA and KIR polymorphic genes might explain discrepancies in the impact of donor-recipient matching observed in HSCT.     

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.     

Immunogenomics of hematopoietic stem cell transplantation

Recipients of allogeneic hematopoietic stem cell transplantation (HSCT) incur the risk of graft-versus-host disease even when the donor is a sibling who shares the Major Histocompatibility Antigens. Therefore, even the perfect HLA match does not represent the optimal genetic match between donors and recipients in HSCT. In addition to the HLA complex other genetic systems operate and affect the outcome of HSCT. These include minor histocompatibility systems (Martin P. Applicability of matching for minor histocompatibility antigens in human bone marrow transplantation. In: Roopenian DC, Simpson E, editors. Minor histocompatibility antigens: From the laboratory to the clinic. Georgetown: Landis Bioscience; 2000. p. 97-103) (inducing bona fide allogeneic responses) as well as a series of functional polymorphisms in cytokines and chemokines and receptors genes (Transplantation 1997;64:553). Among the items affecting the outcome of HSCT the incidence and severity of infections have an important impact. Polymorphisms of genes controlling both arms of the immune responses to pathogens (innate versus cognate) are strong candidates for susceptibility factors to infection in allogeneic transplantation. These include the MHC alleles (HLA class I, class II, MIC) CD1, Toll and TLR genes MBP, MPO genes, ...). In addition to the NK alloreactivity induced by HLA class I epitopes mismatching (a common situation in HSCT) variations in the genotype of the KIR genes (Tissue Antigens 2001;57:358) may also be encountered between the donor and the recipient leading to potentially harmful or beneficial combinations. An integrated knowledge of the role and hierarchy of the most important genetic factors (MHC and non-MHC) will provide the rationale for a comprehensive matching in HSCT (Curr Opin Hematol 3 (1996) 416). This short review provides a panorama of this strategic issue for further development of HSCT.     

异基因外周血造血干细胞移植治疗白血病*

背景：异基因外周血造血干细胞移植是治疗白血病的有效手段。 目的：比较血缘与非血缘供者异基因外周血造血干细胞移植治疗白血病的造血重建、免疫重建、感染、移植物抗宿主病及疗效。 方法：选择接受异基因外周血造血干细胞移植治疗的白血病患者45例,其中30例患者接受血缘供者造血干细胞移植(血缘组),15例患者接受非血缘供者造血干细胞移植(非血缘组)。 结果与结论：①造血重建：血缘组白细胞和血小板重建时间均快于非血缘组(P < 0.05)。在移植后30~40 d植活证据指标测定提示异体造血干细胞在受者体内完全植活。②T细胞重建：两组移植后各时间点T细胞重建差异无显著性意义。③感染发生率：两组移植后早期感染发生率,急、慢性移植物抗宿主病发生率差异无显著性意义(P > 0.05)。④白血病复发：两组移植后复发率差异无显著性意义(P > 0.05)。⑤无病生存：两组移植后2年无病生存率差异无显著性意义(P > 0.05)。表明血缘供者异基因外周血造血干细胞移植后的造血重建较非血缘供者迅速,但两者间移植后T细胞重建、感染发生率、移植物抗宿主病及无病生存并无差异。      

Patients benefit from the addition of KIR repertoire data to the donor selection procedure for unrelated haematopoietic stem cell transplantation

Killer cell immunoglobulin-like receptors (KIRs) expressed on donor natural killer (NK) cells are important for induction of NK cell alloreactivity in haematopoietic stem cell transplantation (HSCT). Current criteria in the selection procedure of an unrelated donor do not account for this potential NK alloresponse. In this study the KIR gene repertoire of 21 HSCT patients and all their potential, unrelated donors (N=64) has been identified by the sequence-specific priming (SSP) procedure. KIR genotype characteristics are correlated with HLA and clinical data. These data show that for 16 cases an HLA compatible alternative donor was available. Among those 16 were 8 donors with a favourable predicted NK alloreactivity directed against the leukaemic cells. In conclusion, it is feasible and clinically relevant to add the KIR repertoire to the unrelated donor selection procedure.     

Allorecognition by murine natural killer cells: lysis of T-lymphoblasts and rejection of bone-marrow grafts

Summary: Natural killer (NK) cells of inbred mice reject allogeneic bone-marrow cells, and NK cells of F1 hybrid mice can reject parental bone-marrow cells (hybrid resistance). In some cases these patterns of rejection can be mimicked in vitro by utilizing IL-2 cultured NK effector cells and allogeneic or parental T-lymphoblasts as target cells. Lysis of allogeneic parental targets in vitro can be explained on the basis of the missing self hypothesis. Subsets of NK cells that bear non-overlapping MHC class I inhibitory receptors belonging to the Ly49 family lyse allogeneic targets because they do not express self class I molectiles of the NK cell donor. Parental strain targets are lysed because they do not express all of the self class I antigens of the Fl hybrid, and hence fail to deliver inhibitory signals to all subsets of Fl NK cells. The expression of Ly49 receptors on NK cells is regulated by liost MHC to ensure maximal sensitivity to alterations in self class I molecules and to prevent autoreactivity. In many instances, however, the rejection of allogeneic bone marrow cells in vivo cannot be readily explained by the missing self hypothesis. In these instances, it appears that rejection is initiated by class 1 MHC receptors on NK ceils Out recognize allogeneic class I molecules as non-self, and activate rather than inhibit NK cell function.     

Taking license with natural killer cell maturation and repertoire development

Summary:  Combining population analysis with in-depth analysis of selected individuals, the tolerance of human natural killer (NK) cells to autologous major histocompatibility complex (MHC) class I and potential reactivity to allogeneic MHC class I have been studied. Analysis of NK cell clones in long-term culture and peripheral blood NK cells after short-term culture (20–24 h) shows that NK cell tolerance is determined by interactions of autologous MHC class I with CD94:NKG2A and inhibitory killer cell immunoglobulin-like receptors (KIRs). Alloreactivity is predicted whenever the donor of the allogeneic target lacks a cognate MHC class I–KIR, ligand–receptor pair that is present in the NK cell donor. In the human population, there is a wide variation in the NK cell repertoire of KIRs and CD94:NKG2A expression. Variation is principally due to KIR gene variation and polymorphism, with a smaller effect due to MHC class I. The presence of MHC class I increases the frequency of NK cells expressing the cognate KIR, an effect that is diminished by the presence of other cognate–ligand pairs. The minor influence of MHC class I on the KIR repertoire indicates that NK cell development is an efficient process in which the expression of inhibitory MHC class I receptors at the final stages ensures that functionally active human NK cells are self-tolerant.     

Relevance of KIR gene polymorphisms in bone marrow transplantation outcome

Natural Killer (NK) cells may be involved both in allogeneic bone marrow transplantation (BMT) rejection and graft-versus-host disease (GVHD). The physiologic functions of NK cells appear to be regulated by diverse non-inhibitory and inhibitory receptors including the killer cell immunoglobulin-like receptors (KIR). Although human leukocyte antigen (HLA) epitope mismatches are well-known causes of NK alloreactivity, the role of KIR genes in transplantation remains to be further investigated. In this study, we have evaluated whether KIR genotype differences between donors and recipients of HLA identical (related and unrelated) compared with HLA non-identical unrelated BMT, had an impact on transplantation outcome. Our results show that 5 of 15 KIR genes were always identical in donors and recipients and most variations were observed in the number and specificity of noninhibitory KIR genes. Based on the presence or absence of particular KIR genes, 70 different genotypes were obtained from all individuals. According to the donor or recipient KIR genotype, different combination patterns were described. Interestingly, when the recipient KIR genotype was "included" in the donor KIR genotype, 100% (11/11 pairs) of unrelated BMT developed GVHD compared with 60% (18/30) in all other combinations (p = 0.012). In contrast, no GVHD was observed in related BMT when the recipient KIR genotype was "included" in the donor KIR genotype (p = 0.0001). In conclusion, our results reveal a great diversity for KIR genotypes in donors and recipients of BMT and that the risk of GVHD was maximum in unrelated BMT when the recipient KIR genotype was "included" in the donor KIR genotype.     

Single Infusion of Donor Mononuclear Early Apoptotic Cells as Prophylaxis for Graft-versus-Host Disease in Myeloablative HLA-Matched Allogeneic Bone Marrow Transplantation: A Phase I/IIa Clinical Trial

Because of its potent immunomodulatory effect, an infusion of donor mononuclear early apoptotic cells (ApoCell) was tested in addition to cyclosporine and methotrexate as prophylaxis for acute graft-versus-host disease (GVHD) after HLA-matched myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) from a related donor. In a phase I/IIa clinical trial, we treated 13 patients (median age, 37 years; range, 20 to 59 years) with hematologic malignancies: 7 patients with acute lymphoblastic leukemia, 5 patients with acute myeloid leukemia, and 1 patient with chronic myeloid leukemia, who received conventional myeloablative conditioning, with 35, 70, 140, or 210 × 10⁶ cell/kg of donor ApoCell, on day -1 of transplantation. Engraftment was successful in all patients with median time to neutrophil recovery of 13 days (range, 11 to 19), and platelet recovery of 15 days (range, 11 to 59). Serious adverse effects were reported on 10 occasions in the trial, all of which were considered unrelated (n = 7) or unlikely to be related (n = 3) to ApoCell infusion. The nonrelapse mortality at day 100 and 180 after transplantation was 7.7% and the overall survival at 100 and 180 days after transplantation was 92% and 85%, respectively. All ApoCell preparations showed an in vitro significant tolerogenic effect upon interaction with dendritic cells. The overall incidence of acute grades II to IV GVHD was 23%, whereas among those receiving the 2 higher doses (n = 6), the rate was 0%. These results suggest that a single infusion of donor ApoCell in HLA-matched allogeneic HSCT is a safe and potentially effective prophylaxis for acute GVHD occurring after myeloablative conditioning. No dose limiting toxicity was observed. (Clinicaltrials.gov no. NCT00524784).     

Status of donor-recipient HLA class I ligands and not the KIR genotype is predictive for the outcome of unrelated hematopoietic stem cell transplantation in beta-thalassemia patients.

Several studies have investigated the role played by killer immunoglobulin-like receptors (KIRs) and their ligands on the outcome of hematopoietic stem cell transplantation (HSCT) in patients affected by oncohematologic diseases. However, the interpretation of the results of these studies is considerably hampered by the heterogeneity of the diseases, disease status at transplantation, and the different protocols employed for both conditioning and graft-versus-host disease (GVHD) prophylaxis. To better define the role of KIRs in HSCT, we studied KIR genotypes and HLA class I ligands in a homogeneous group of 45 thalassemia patients transplanted with bone marrow cells from an HLA-identical, unrelated donor. Patients that were heterozygotes for HLA-Cw groups 1 (HLA-Cw(Asn80)) and 2 (HLA-Cw(Lys80)) had a higher risk of developing acute GVHD than C1/C1 or C2/C2 homozygotes (relative risk [RR] = 8.75; 95% confidence interval [CI]: 1.63-46.76; P = .007). Vice versa, all patients who experienced primary/secondary graft failure were C1/C1 or C2/C2 homozygotes (RR = 20.45; 95% CI = 1.08-384.24; P = .009). Moreover, the presence of the HLA-A11 antigen conferred protection against GVHD (0% versus 35%, P = .02). Our results suggest that C1/C2 heterozygosity, may favor the development of donor alloreactivity and thereby increase the risk of GVHD. Conversely, C1/C1 and C2/C2 homozygosity seems to reduce the risk of GVHD but may increase the incidence of graft rejection. These data may be helpful in tailoring the intensity of GVHD prophylaxis and conditioning regimens in thalassemia patients receiving HSCT from an HLA-identical volunteer donor.     

KIR受配体模式在治疗急性淋巴细胞白血病中的研究

目的 研究在无关供体造血干细胞移植中急性淋巴细胞白血病(ALL)的杀伤细胞免疫球蛋白样受体(KIR)受配体模式对自然杀伤(NK)细胞的异源反应性活性预示造血干细胞移植的影响.方法 采用基因测序和序列特异性引物聚合酶链反应(PCR-SSP)的方法,对中国造血干细胞捐献者资料库中提供的23对HLA全相合供受者进行KIR及HLA高分辨基因分型;流式缃胞术动态随访CD158分子表达水平;患者均为ALL.结果 23对供受者中17例供者KIR2D12/L3有相应的患者配体HLACw1、3、7、8、12、14;6例供者KIR2DL1有相应的患者配体HLA-Cw6、15;16例供者KIR3DL1有相应的患者配体HLA-Bw4;12例供者3DL2有相应的患者配体HLA-A11.23对供受者中有19对接受了造血干细胞移植,供受者KIR基因完全相同或宿主抗移植物(HVG)方向移植相关死亡率高,分别为33.3%和40.0%;移植物抗宿主(GVH)方向移植卡甘关死亡率低,为12.5%.供受者在GVH方向时,移植物抗宿主病(GVHD)发生率高(50.0%)且有多种激活性(aKIR)的组合;而HVG方向GVHD发生率低(20.0%).19对供受者有5对均为KIR基因A单体型,其中2对供受者为KIR2DS4*001/002亚型,移植后死亡;3对供受者KIR2DS4为KIR2DS4*003-007亚型,1年后无病生存.移植后随访无GVHD发生时,CD158a的表达逐渐下降;有GVHD发生时,CD158a的表达逐渐增高;移植后早期受者NK细胞百分比为(23.4±3.8)%,高于正常人水平[(2.04±0.58)%,P<0.05],差异有统计学意义.结论 供者的KIR2DL1、KIR3DL1是引起NK细胞异源反应活性的重要抑制性KIR.KIR受配体模式不仅能预示无关供体异基因造血于细胞移植的预后,更能帮助临床提高ALL异基凶造血干细胞移植的总生存率及无病生存率,降低移植后相关死亡率和防止白血病复发.     

CD3+/CD19+ Depleted Matched and Mismatched Unrelated Donor Hematopoietic Stem Cell Transplant with Targeted T Cell Addback Is Associated with Excellent Outcomes in Pediatric Patients with Nonmalignant Hematologic Disorders

Unrelated donor hematopoietic stem cell transplantation (HSCT) is increasingly being used to cure nonmalignant hematologic diseases (NMHD) in patients who lack HLA matched related donors. Both graft rejection and graft-versus-host disease (GVHD) remain major barriers to safe and effective transplant for these patients requiring unrelated donors. Partial T cell depletion combined with peripheral stem cell transplantation (pTCD-PSCT) has the potential advantages of providing a high stem cell dose to facilitate rapid engraftment, maintaining cells that may facilitate engraftment, and decreasing GVHD risk compared with T cell–replete HSCT. Here, we report a single-institution, retrospective experience of unrelated donor pTCD-PSCT for pediatric patients with NMHD. From 2014 to 2017, 12 pediatric patients with transfusion-dependent NMHD underwent matched unrelated donor (MUD) or mismatched unrelated donor (MMUD) pTCD HSCT in our center using disease-specific conditioning. Donor PSCs underwent CD3⁺ T cell and CD19⁺ B cell depletion using CliniMACS, followed by a targeted addback of 1?×?10⁵ CD3⁺ T cells/kg to the graft before infusion. All 12 patients demonstrated rapid trilinear engraftment. At a median follow-up of 740days (range, 279 to 1466), all patients were alive with over 92% total peripheral blood donor chimerism and without transfusion dependence or recurrence of their underlying hematologic disease. Immune reconstitution was rapid and comparable with T cell–replete HSCT. No patients developed severe acute GVHD (grades III to IV) or chronic extensive GVHD, and all patients had discontinued systemic immune suppression. Viral reactivations were common, but no patient developed symptoms of life-threatening infectious disease. Our data indicate that MUD and MMUD pTCD-PSCTs are safe and effective approaches that enable rapid engraftment and immune reconstitution, prevent severe GVHD, and expand availability of HSCT to any patients with NMHD who have closely MUDs.     

KIR-ligand mismatch in allogeneic hematopoietic stem cell transplantation

KIR-ligand mismatch in the graft-versus-host (GVH) direction between donor and recipient in allogeneic stem cell transplantation (ASCT) may trigger NK cell alloreactivity. Such KIR-ligand mismatch has been associated with improved survival after haploidentical ASCT for acute myeloid leukemia (AML). Its role in unrelated ASCT has been more controversial. In an analysis of 190 unrelated ASCTs for hematological malignancies, we observed that KIR-ligand mismatch was associated with increased transplantation related mortality (TRM) and decreased overall survival. The increased TRM was a consequence of a higher rate of infections. Thus, the presence of alloreactive NK cells can potentially interfere with effective immunity to infections in the early post-transplantation period. Here, we review the discrepancies in published reports on KIR-ligand mismatch in unrelated ASCT.     

NK细胞在小鼠异基因骨髓移植中的作用

目的:研究自然杀伤(NK)细胞在异基因骨髓移植中对移植物抗宿主病(GVHD)、移植排斥、骨髓植入及造血重建的影响。方法:以近交系小鼠C57/6j(H-2^b)为供鼠、BALB/c(H-2^d)为受鼠,在移植物中增加供者的外周T细胞和/或NK细胞进行异基因骨髓移植,用流式细胞仪检测受鼠的CD₃₄^＋细胞计数和H-2K^b＋细胞表达水平,血细胞自动分析仪检测外周血白细胞计数,并结合临床表现和病理检查,比较不同移植组的存活率、GVHD、植入水平及造血重建等。结果:增加NK细胞组的小鼠存活率显著大于不增加NK细胞组,小鼠出现GVHD的数量少、程度轻,外周血白细胞及骨髓CD₃₄^＋细胞恢复快、H-2K^b＋细胞表达水平高。结论:NK细胞抑制小鼠异基因骨髓移植中的GVHD和移植排斥,促进骨髓植入及造血重建。     

Real-time in vivo cellular imaging of graft-versus-host disease and its reaction to immunomodulatory reagents

Visualizing the in vivo dynamics of individual donor cells after allogeneic hematopoietic stem cell transplantation (HSCT) will enable deeper understanding of the process of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). In this study, using non-invasive in vivo fluorescence imaging of the ear pinna, we successfully visualized green fluorescent protein (GFP) donor cells at the single cell level in the skin. This imaging model enabled visualization of the movement of GFP cells into blood vessels in real time after allogeneic HSCT. At day 1, a few donor cells were detected, and the movement of donor cells in blood vessels was readily observed at day 4. Early donor cell infiltration into non-lymphoid tissue was increased by treatment with croton oil, as an inflammatory reagent. Treatment with dexamethasone, as an anti-inflammatory reagent, suppressed donor cell infiltration. The in vivo cellular fluorescence imaging model described here is a very useful tool for monitoring individual donor cells in real-time and for exploring immunomodulatory reagents for allogeneic HSCT, as well as for understanding the mechanism of GVHD.     

Donor Selection for Killer Immunoglobulin-like Receptors B Haplotype of the Centromeric Motifs Can Improve the Outcome after HLA-Identical Sibling Hematopoietic Stem Cell Transplantation

After hematopoietic stem cell transplantation (HSCT), natural killer (NK) cell alloreactivity in HLA cells of recipients is regulated by killer immunoglobulin-like receptors (KIRs) on donor NK cells. The effect of KIRs on HSCT outcomes is controversial, particularly in those undergoing HLA-identical sibling HSCT. In this study, effects of KIR and HLA genotypes on the HSCT outcome were investigated in a 5-year retrospective study comprising 219 patient-donor pairs undergoing HLA-identical sibling HSCT for myeloid and lymphoid malignancies. We found that 39.7% (87 of 219) of these pairs, which were KIR mismatched, had better overall survival (OS) and reduced grade III to IV acute graft-versus-host disease (aGVHD), especially in acute myeloid leukemia (AML) patients. Bx1 donor KIR genotype with haplotype B on a telomeric region was a risk factor for the OS and relapse-free survival (RFS). Donor centromeric (c) and telomeric (t) KIR haplotype analysis showed that donor KIR cB-tA/tB was associated with improved OS and RFS compared with cA-tA or cA-tB. Furthermore, donor KIR B haplotype of the centromeric motifs (Cen-B) was an independent beneficial factor in improving OS and RFS and in protecting from relapse after HSCT. In AML patients, the occurrence of aGVHD was significantly lower in HLA-C1 group compared with that in HLA-C2 group, although such effect was not observed in patients with acute lymphoblastic leukemia or chronic myelogenous leukemia. Our results suggest that KIR could impact outcome and donor KIR haplotype with Cen-B confer significant survival benefits to HLA-identical sibling HSCT.