Allogeneic transplantation of peripheral blood stem cell grafts results in a massive decrease of primitive hematopoietic progenitor frequencies in reconstituted bone marrows

Abstract

The success of allogeneic hematopoietic stem cell transplantation (alloSCT) is indicated by the reconstitution of the peripheral blood system of patients after alloSCT and the engraftment of hematopoietic stem and progenitor cells (HSPCs) into their bone marrow (BM). The number of CD34+ cells is commonly used as surrogate for the content of hematopoietic stem cells in the grafts. During the last decade, several antigens (including CD133, CD45RA, CD38, and CD10) were identified allowing discrimination of different HSPC subpopulations within the human CD34+ cell compartment. Although such studies increased our understanding of early human hematopoiesis tremendously, hardly any study dissected the CD34+ compartment in the alloSCT setting. Consequently, we comprehensively analyzed the CD34+ compartment in G-CSF-stimulated peripheral blood stem cell grafts of allogeneic donors, in BM samples of the respective recipients 4 weeks after alloSCT, and in BM samples of healthy donors. We demonstrate that alloSCT is associated with a dramatic shift from primitive to more mature HSPC types. Upon investigating whether the composition of engrafted CD34+ cells has any impact on the incidence and severity of graft-versus-host disease, we did not find any correlation. However, more detailed analyses of the CD34+ compartment may elucidate associations with other transplantation-related complications.

Introduction

Allogeneic hematopoietic stem cell transplantation (alloSCT) offers a curative therapy option for a variety of hematological disorders. A sufficient number of transfused hematopoietic stem and progenitor cells (HSPCs) are required for a successful alloSCT. The fraction of CD34+ cells is commonly viewed as a surrogate for the content of hematopoietic stem cells (HSCs) in a graft [1]. However, the CD34+ cell fraction is heterogeneous and contains a variety of different HSPCs, which were reported to influence engraftment kinetics after alloSCT [2].

The classical model of hematopoiesis subdivides progeny of multipotent hematopoietic progenitors (MPP) into common lymphoid progenitors (CLP) and common myeloid progenitors (CMP), the precursors of all lymphoid or myeloid cells, respectively [3, 4]. This dichotomous model, however, was challenged by the identification of lymphoid progenitors, which retain partial myeloid potential [5,6,7]. By considering CD133 expression to discriminate specific functional progenitor populations, we were able to redefine the hematological taxonomy. We demonstrated that HSPCs with long-term in vitro and in vivo potential reside in the CD34+CD133+ cell fraction, which is lineage negative (lin) [8, 9]. This cell fraction can be discriminated by means of CD45RA into CD34+CD133+CD45RA cells, which showed all lymphoid and myeloid including erythroid potentials, thus fulfilling the criteria of MPP [8, 10, 11], and into CD34+CD133+CD45RA+ cells that have lost erythroid, megakaryocytic as well as basophil and eosinophilic potentials and are committed towards the lympho-myeloid lineage [8, 10]. The lympho-myeloid lineage can be subdivided into CD34+CD133+CD45RA+CD38low/-CD10, CD34+CD133+CD45RA+CD38+CD10 and CD34+CD133+ CD45RA+CD10+ cells (Fig. 1). Cells with the capability to create all lympho-myeloid cell fates are highly enriched in the CD34+CD133+CD45RA+CD38low/−CD10 cell fraction accordingly named lymphoid-myeloid primed progenitors (LMPPs). CD34+CD133+CD45RA+CD38+CD10 cells lack lymphoid potentials but are able to create neutrophils and macrophages and thus correspond to granulocyte-macrophage progenitors (GMPs). In contrast, CD34+CD133+CD45RA+CD38low/−CD10+ cells, the multi-lymphoid progenitors (MLPs), are able to create all lymphocytes and macrophages, but no neutrophils [8, 12].

Fig. 1
figure1

Lineage relationship and phenotypic characteristics of human hematopoietic progenitor cells according to our proposed revised model of human hematopoiesis (according to Görgens et al. [8]). Of note, all progenitors depicted express CD34 and are negative for common lineage antigens

Previously, we demonstrated that more than 80% of the MPPs divide asymmetrically, with each of them creating an LMPP and a CD34+CD133CD45RA daughter cell [10]. The latter largely correspond to the CMP which inherit erythroid, megakaryocytic as well as basophil and eosinophilic potentials, but against the previous assumptions have lost neutrophil-forming capabilities [8, 10]. Accordingly, the name CMP appears no longer justifiable. Consequently, we suggested renaming these cells to erythro-myeloid progenitor (EMP) cells [8]. Taken these data into account, we previously proposed a revised model of hematopoiesis, in which CMPs do not exist [8, 10, 13]. Meanwhile the proposed lineage relationships have been confirmed in mouse hematopoiesis [14]. Recently, our model has been substantiated by single cell transcriptomics of human bone marrow (BM)-derived steady-state HSPCs [15]. However, only few studies dissected the CD34+ progenitor pool of the BM following successful alloSCT. Consequently, we decided to analyze the presence of the different CD34+ progenitors in BM samples of successfully treated patients following alloSCT. In total, we included 21 PBSC-grafts and the 21 respective adult PBSC recipients in our study. BM was analyzed 4 weeks after alloSCT. For comparison, the progenitor pool was also analyzed in BM samples of five healthy donors (HD).

Material and methods

Specimen collection

The proportions of HSPC subpopulations were measured in 21 allogeneic PBSC-grafts. All donors were stimulated with G-CSF. At our institution, patients routinely undergo BM examination around day +28 after alloSCT. Accordingly, BM specimens of the 21 respective patients were investigated 4 weeks after alloSCT at the University Hospital of Essen (Germany). All patients had signed informed consent in accordance to the Declaration of Helsinki. The results of the measurements in the PBSC-grafts and Patient-BM were compared to measurements in BM of five healthy BM donors registered at the Westdeutsche SpenderZentrale (WSZE), Ratingen, Germany, who had signed informed consent in accordance to the Declaration of Helsinki, too.

Preparation of cells and flow cytometry analyses

Lysis of erythrocytes in the PBSC-grafts and in the BM specimens was carried out using VersaLyse® according to the manufacturer’s instructions (Beckman Coulter, Krefeld, Germany). PBSC-grafts, BM of the patients and of the HD were not treated with Ficoll density gradient centrifugation. Flow cytometry (FCM) analyses were performed in samples of 21 different PBSC-grafts and in BM of the patients having received these PBSC-grafts. Fifty microliters prepared BM or PBSC samples were stained with a cocktail of five antibodies (10 µl each) and incubated for 15 min light-shielded at room temperature. The following five antibodies were used according to the manufacturer’s recommendations: CD45RA-FITC (clone ALB 11, Beckman Coulter), CD133-PE (clone AC133, Miltenyi, Bergisch Gladbach, Germany), CD34-ECD (clone 581, Beckman Coulter), CD10-PC5 (clone ALB 1, Beckman Coulter) and CD38-PC7 (clone LA 198-4-3, Beckman Coulter). FCM analyses were carried out on an FC500 flow cytometer (Beckman Coulter). The gating strategy is depicted in Fig. 2. If possible, in each measurement 5000 CD34+ cells were recorded. Isotype controls were performed.

Fig. 2
figure2

Gating strategy in a PBSC grafts, b patient-BM 4 weeks after alloSCT and c healthy BM donors. BM bone marrow, alloSCT allogeneic hematopoietic stem cell transplantation

Statistical analysis

Statistical analysis was performed using GraphPad (San Diego, USA) Prism version 6. Mean percentage and SEM of percentage values were evaluated using the t test. P values < 0.05 were considered to be statistically significant. Results obtained from five BM samples of HD were used as controls.

Results

Patient characteristics

The median age of the patients was 54 years (range: 23–72 years). All detailed patient and alloSCT characteristics are listed in Table 1. Two patients received PBSC-grafts from related donors; the remaining 19 patients received PBSC-grafts from unrelated donors. All patients received myeloablative conditioning. The median CD34+ cell count transplanted was 6.58 × 106 per kg body weight of the patient. The majority of the patients (19) received in vivo T-cell depletion with anti-thymocyte globuline (ATG), two patients were not treated with ATG. Sixteen alloSCT were performed with HLA-identical PBSC-grafts, in five cases there was an HLA-mismatch. GvHD prophylaxis consisted of standard Cyclosporine A (CSA) and Methotrexate in 19 patients, two patients received CSA and mycophenolate mofetil (MMF), one patient tacrolimus and MMF. Hematological reconstitution occurred in all patients with a median recovery of neutrophils more than 500/µl after 16 days (range: 10–37 days) and of thrombocytes more than 50,000/µl after 14 days (range: 12–31 days).

Table 1 Patient and alloSCT characteristics

Comparison of the CD34+ cell content in PBSC-grafts and in reconstituted BM

At first, we compared the CD34+ cell content within the PBSC-grafts and the reconstituted BM (rBM). On average the PBSC-grafts had a content of 1.01 ± 0.08% CD34+ cells (n = 21; all results are given as mean ± SEM). Four weeks after alloSCT the mean proportion of CD34+ cells in Pat-BM was significantly lower (0.59 ± 0.17%, n = 21, p = 0.0456). In detail, the patient with the highest content of CD34+ cells had a proportion of 3.60% CD34+ cells in the BM and the patient with the lowest content a proportion of 0.05% CD34+ cells. In 18 out of the 21 patients, the content of CD34+ cells was higher in the PBSC-graft than in the BM; only three patients had a higher content of CD34+ cells in their BM than corresponding PBSC-grafts (Fig. 3).

Fig. 3
figure3

PBSC grafts contain significant higher frequencies of CD34+ cell than reconstituted BM. Individual comparison of the CD34+ cell content in PBSC-grafts (n = 21) and in rBM of respective patients (n = 21) and mean frequencies in healthy donors (HD-BM, n = 5). p values < 0.05 were considered to be statistically significant. BM bone marrow, rBM reconstituted BM

Previously, we reported a content of 4.2 ± 2.8% CD34+ cells (n = 5) within the mononuclear cell fraction of healthy donor BM that was prepared by Ficoll density gradient centrifugation followed by lysis of remaining red blood cells [11]. Here, however, related to the differences in the protocols we use for basic research and for the clinical routine, BM cells were prepared without Ficoll gradient. Consequently, we analyzed the CD34+ cell content of BM samples of healthy donors that were prepared with the same protocol as the rBM samples. On average, the BM samples of HDs had a content of 0.61 ± 0.05% CD34+ cells (n = 5), similar to the frequencies of CD34+ cells within the rBMs (0.59 ± 0.17%); significant differences in the CD34+ cell content between reconstituted and HD-BM were not observed, p = 0.9465) (Table 2, Fig. 4).

Table 2 HSPC subpopulations: immune phenotypic (IPT) characteristics (according to Görgens et al. [8]) and frequencies in PBSC grafts, in reconstituted Patient-BM (Pat-BM) and in BM of healthy donors (HD-BM). All values of the CD34+ subpopulations were calculated as percentage of the total CD34+ fraction
Fig. 4
figure4

Massive reduction of primitive HSPCs in patient-BM after alloSCT. Frequencies of HSC progenitors in bone marrow in patients after alloSCT (Pat-BM, n = 21), G-CSF-stimulated PBSC grafts (PBSC, n = 21) and bone marrow of healthy donors (HD-BM, n = 5). All values of the CD34+ subpopulations were calculated as percentage of the total CD34+ fraction. p values < 0.05 were considered to be statistically significant. HSPC hematopoietic stem and progenitor cell, alloSCT allogeneic hematopoietic stem cell transplantation

PBSC-grafts contain higher frequencies of primitive progenitors than BM of healthy donors

Next, the CD34+ compartment was dissected by means of the CD34+ cells’ expression of CD133, CD45RA, CD38 and CD10. We subdivided the CD34+ compartment according to our previous studies [8, 10] into MPPs (CD34+CD133+CD45RACD10CD38low/−), LMPPs (CD34+CD133+CD45RA+CD10CD38low/−), MLPs (CD34+CD133+CD45RA+CD10+CD38low/−), GMPs (CD34+CD133+CD45RA+CD10CD38+) and EMPs (CD34+CD133CD45RACD10CD38+). Furthermore, we analyzed frequencies of two different population consisting of late progenitors, which were identified as CD34+C133CD45RA+CD10CD38+ cells (“LP-1”) or as CD34+C133CD45RA+CD10+CD38+ cells (“LP-2”), respectively. The frequencies of the different progenitor types have been calculated as percentage of the total CD34+ fraction in the PBSC-grafts, in HD-BM and in the recipients (Table 2 and Fig. 4), thus, equalizing protocol-dependent parameters affecting the CD34+ frequencies in prepared samples.

Confirming our previous analyses [11], we demonstrate that the CD34+ compartment in the PBSC-grafts contain significant higher levels of more primitive HSPCs than HD-BM. We observed 43.12 ± 1.67% MPPs within the CD34+ cell compartment of PBSCs which was significantly higher (p < 0.0001) than in BM of HDs (14.57 ± 4.06%). The content of LMPPs was 6.02 ± 0.56% in PBSCs and 1.73 ± 0.46% in HD-BM (p = 0.0013). More mature progenitors were higher enriched in HD-BM compared to the PBSC-grafts with 18.60 ± 1.34% GMPs, 7.63 ± 1.83% LP-1 and 19.25 ± 5.86% LP-2 in HD-BM vs. 10.32 ± 0.82% GMPs (p = 0.0001), 2.37 ± 0.28% LP-1 (p < 0.0001) and 1.67 ± 0.24% LP-2 (p < 0.0001) in the PBSC-grafts. The content of EMPs was comparable in HD-BM (19.37 ± 1.34%) and in PBSCs-grafts (19.19 ± 0.93%; p = 0.9302). In addition to the overall CD34+ fraction, only the MLPs frequency was lower in the HD-BM (0.39 ± 0.09%) compared to the PBSC-grafts (1.42 ± 0.20%; p = 0.0219).

Following alloSCT the HSPC pool shifts towards more mature HSPC types

Upon comparing the frequencies of the different HSPC types, we learned that despite the high frequency of MPPs in the PBSC-grafts (43.12 ± 1.67%) MPPs were dramatically reduced in the BM after alloSCT (2.14 ± 0.47%; p < 0.0001). Similarly, the frequency of LMPPs in the PBSC-graft (6.02 ± 0.59%) was reduced in the rBMs (1.17 ± 0.22%, p < 0.0001). Consequently, more mature progenitor types including EMPs were observed in higher proportions in Pat-BM than in the PBSC-grafts (EMPs: 28.00 ± 3.14% in Pat-BM vs. 19.19 ± 0.93% in PBSC-grafts, p = 0.0178; LP-1: 18.88 ± 3.15% in Pat-BM vs. 2.37 ± 0.28% in the PBSC-grafts, p < 0.0001; LP-2: 18.97 ± 4.97% in Pat-BM vs. 1.67 ± 0.24% in PBSC-grafts, p = 0.0024; Table 2, Figs. 4 and 5). Thus, upon comparing the composition of the HSCP pool in PBSC grafts and rBM, we observe a massive shift from primitive HSCPs towards more mature HSPC types.

Fig. 5
figure5

Reduction of primitive HSPCs is compensated by a shift towards more mature HSPC types. Frequencies of HSC progenitors in G-CSF-stimulated PBSC grafts (PBSC, n = 21) and in bone marrow of patients after alloSCT (Pat-BM, n = 21). All values of the CD34+ subpopulations were calculated as percentage of the total CD34+ fraction. Sum does not add up to 100% since not all possible subpopulations were calculated, e.g. in the CD34+CD133+CD45RA fraction only the CD10/CD38neg/low subpopulation was determined. HSPC hematopoietic stem and progenitor cell, HSC hematopoietic stem cell, alloSCT allogeneic hematopoietic stem cell transplantation

The HSPC pool in reconstituted BM has a more mature composition than the BM of healthy donors

As the HSPC pool is differently composed in BM and in PBSC grafts, we next compared the HSPC pool of reconstituted and normal BM. As in the previous comparison, we observed significantly lower MPP frequencies in Pat-BM (2.14 ± 0.47%) than in HD-BM (14.57 ± 4.06%, p < 0.0001). In contrast to the PBSC-graft, LMPP frequencies were comparable (p = 0.2851) in Pat-BM (1.17 ± 0.22%) and HD-BM (1.73 ± 0.46%). Although not being significant, MLP, EMP and LP-1 frequencies were higher in rBM than in HD-BM (MLP: 0.94 ± 0.31% vs. 0.39 ± 0.09%, p = 0.4080; EMP: 28.00 ± 3.14% vs. 19.37 ± 1.34% vs., p = 0.2013; LP-1: 18.88 ± 3.15% vs. 7.63 ± 1.83%, p = 0.1013). In contrast, GMP and LP-2 frequencies were lower in reconstituted than in HD-BM (GMP: 14.71 ± 2.37% vs. 18.60 ± 1.34%, p = 0.4411; LP-2: 18.97 ± 4.97% vs. 19.25 ± 5.86%, p = 0.9794; Table 2 and Fig. 4).

The amount of transplanted MPPs does not affect the composition of the HSPC pool in Pat-BM

To learn whether MPP numbers critically affect the composition of rBM, we correlated the amount of transplanted MPPs with the assembly of the primitive HSPC pool following transplantation. In detail, the absolute cell numbers of MPPs and the other progenitor types were multiplied with their frequencies and that of CD34+ cells in PBSC-grafts and in rBM, respectively. Upon applying linear regression analysis, we could not observe any statistical correlation between the amount of MPPs in PBSC-grafts and the amount of MPPs, LMPPs, EMPs and GMPs in the rBM (Fig. 6).

Fig. 6
figure6

No correlation of the amount of transplanted MPPs in PBSC-grafts with the frequencies of MPPs, LMPPs, EMPs and GMPs in patient-BM. Linear regression analysis of phenotypically defined CD34+ subsets in PBSC grafts and the corresponding patient bone marrow. To estimate the absolute counts of MPPs and the other progenitor subtypes, we multiplied the frequencies of progenitor subtypes with the CD34+ cell frequencies in the PBSC graft (n = 21) and the Pat-BM (n = 21), respectively. MPP multipotent hematopoietic progenitor, LMPP lymphoid multipotent progenitor, EMP erythro-myeloid progenitor, GMP progenitor of neutrophils and macrophages

The HSPC-composition of Pat-BMs does not correlate with GvHD

Finally, we explored whether the HSPC-composition of rBM affects the incidence and severity of acute and chronic GvHD. In this context, we retrospectively analyzed and categorized the GvHD manifestations of the 21 patients according to accepted standards [16,17,18]. For acute GvHD the patients were grouped in two cohorts: one with no or only mild acute GvHD symptoms (grade I−II) and one cohort with severe acute GvHD (grade III−IV). With respect to chronic GvHD, also two cohorts were formed: no or only mild symptoms and moderate/severe symptoms of chronic GvHD.

Accordingly, 17 patients had no or only mild acute GvHD and four patients severe acute GvHD. Regarding chronic GvHD, 13 patients showed no or only mild symptoms and eight patients suffered from moderate or severe chronic GvHD (Suppl. Table 1). No associations between the HSPC subpopulations and the GvHD manifestations were observed (Suppl. Fig. 1a/1b). Due to the small number of severe GvHD patients in the investigated cohort, more detailed analyses regarding the HSCP-composition and patient-related data such as their steroid-responsiveness, i.e. steroid-responsive vs. -refractory, could not be performed.

Discussion

In the clinical setting, CD34+ cell frequencies are used as HSC surrogates without further dissecting the HSPC pool. Here, we systematically have dissected the HSPC pool of rBMs in patients after alloSCT in more detail and compared its composition with that of PBSC-grafts and BM of healthy donors, respectively. Furthermore, we correlated the composition of the HSCP pools of PBSC-grafts and rBM with the clinical incidence of GvHD. We confirmed previously reported differences in the HSPC composition of PBSC-grafts and BM of HDs [11], including a slightly higher expression of CD34 on HSPCs of PBSC than of BM [19]. Strikingly, we observed a massive decline of very primitive HSPCs in rBM, especially of cells of the CD34+CD133+CD45RACD10CD38low/− cell population (MPP), the progenitor population highly enriched for multipotent progenitor cells and HSCs [8, 10]. We did not find any correlation between the composition of the HSCP pool of the rBMs with the incidence and severity of acute or chronic GvHD. Obviously, there are other determinants that exert more influence on GvHD manifestations than the composition of the HSPCs. Yet, due to the small number of patients with severe GvHD in this cohort, further research is warranted to elucidate a possible association of HSPC composition with GvHD.

The results regarding the reduction of the MPP frequencies may appear surprising, as it is conventionally assumed that HSCs and MPPs can massively expand within the BM stem cell niche. Our findings being based on the direct comparison of donor and patient pairs are in accordance to the study of Dmytrus et al., who dissected the HSPC pool of rBM in children 1 year after alloSCT mainly in an unpaired manner [20]. Both studies observed a massive shift from more primitive to more mature progenitor types. Pointing towards the same effect, two early studies tested for the presence of HSPCs with long-term culture initiating cell (LTC-IC) activities up to 20 years following allogeneic BM transplantation. Despite normal peripheral blood cell counts, the LTC-IC rates were much lower in rBM and remained low over the years [21]. Upon comparing the composition of HSPCs of rBM with HD-BM eightfold lower LTC-IC frequencies were recorded in rBM than in HD-BM [22]. Since only MPPs and LMPPs contain LTC-IC potentials [8], these data point towards the same direction as the data reported here and by Dmytrus et al. [20]. Overall, these studies suggest that following transplantation the frequencies of primitive HSPCs are massively reduced.

A reduction of MPP frequencies in recipient BM could, in principle, be the result of reduced MPP homing capabilities. However, apparently MPPs have much higher homing and engraftment capabilities than more committed progenitors. Previously, we observed engraftment rates of approximately 20% of freshly isolated CB-derived CD34+ cells. Upon cultivation in the presence of early acting cytokines for 1 day, engraftment rates were increased up to 70% [13]. However, when cells were cultured under the same condition for 3 days, in which most of them performed at least one cell division [5, 10], engraftment rates dropped down to approximately 2% [8]. In 2014, we demonstrated that in vitro more than 80% of MPPs divide asymmetrically to create a pair of specified daughter cells [10]. Thus, the loss of MPPs correlates well with the loss of respective homing and engraftment potentials. Of course, the loss of MPPs after homing into the BM of mice or patients might be attributed to environmental conditions, e.g. increased oxidative stress or the presence of proinflammatory cytokines [23, 24]. However, maybe being more relevant, the mode of MPP division occurring after homing needs to be considered.

For now, under all conditions we have tested so far, including coculture with different stromal cells [25] (Radtke et al., in revision) or any of different published cytokine/small molecule conditions (Vitoriano et al., in preparation), failed to even maintain MPPs. Eventually, MPPs are intrinsically committed to divide asymmetrically to create a pair of committed daughter cells irrespective of the environmental conditions. Such lineage specifying asymmetric cell divisions occur in many developmental biological processes, e.g. the development of the peripheral sensory organs of Drosophila, a developmental process that had already been compared with early hematopoiesis some 20 years ago [26].

Although, we do not have the final proof, our data challenge the classical view of self-renewal of multipotent HSPCs. For now, our data would be compatible to a model in which the vast majority of multipotent HSPCs are committed to divide asymmetrically and create LMPP and EMP daughter cell pairs. As self-renewal apparently can occur in the LMPP compartment, long-term self-renewal activities should also occur within the EMP lineage. However, apart from the description of megakaryocyte biased HSCs [27, 28], we are not aware of the description of any progenitor type committed to any of the EMP lineage cell fates which reveals extensive self-renewal capabilities, either in vitro or in vivo. Detailed analyses of rBM offer the perspective to increase our overall understanding about human hematopoiesis. Furthermore, such studies may also help to understand better transplantation-related complications, such as graft failure.

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Acknowledgements

We are grateful to Iina Fischer from the Deutsches Rotes Kreuz for providing bone marrow samples of five Westdeutsche SpenderZentrale (WSZE) donors as healthy controls and to Martina Franke for performing the flow cytometry analyses. André Görgens is an International Society for Advancement of Cytometry (ISAC) Marylou Ingram Scholar (2019–2023).

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Correspondence to Lambros Kordelas or Bernd Giebel.

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Kordelas, L., Görgens, A., Radtke, S. et al. Allogeneic transplantation of peripheral blood stem cell grafts results in a massive decrease of primitive hematopoietic progenitor frequencies in reconstituted bone marrows. Bone Marrow Transplant 55, 100–109 (2020). https://doi.org/10.1038/s41409-019-0645-7

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