Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 https://doi.org/10.1007/s00005-022-00666-5 ORIGINAL ARTICLE Impact of Betamethasone Pretreatment on Engrafment of Cord Blood‑Derived Hematopoietic Stem Cells David Perna‑Barrull1 · Laia Gomez‑Muñoz1 · Silvia Rodriguez‑Fernandez1 · Anna Gieras2 · Rosa M. Ampudia‑Carrasco1 · Lidia Almenara‑Fuentes3 · Ruth M. Risueño4 · Sergi Querol5 · Eva Tolosa2 · Marta Vives‑Pi1 Received: 28 July 2022 / Accepted: 28 October 2022 © The Author(s) 2022 Abstract Hematopoietic stem cell (HSC) transplantation is crucial to cure hematologic malignancies. Umbilical cord blood (UCB) is a source of stem cells, but 90% of UCB units are discarded due to low cellularity. Improving the engraftment capacities of ­CD34+ stem cells would allow the use of UCB that were so far rejected. Betamethasone induces long-term transcriptomic and epigenomic changes in immune cells through glucocorticoid receptor. We hypothesize that discarded UCB could be used owing to improvements induced by betamethasone. Isolated C ­ D34+ HSC from UCB were exposed to the synthetic glucocorticoids betamethasone and fluticasone for 20 h, and cell phenotype was determined before transplantation. NSG mice were sub-lethally irradiated (1 Gy or 2 Gy) 6 h before intravenously transferring 2–5 × ­105 ­CD34+ HSC. The peripheral blood engraftment levels and the leukocyte subsets were followed up for 20 weeks using flow cytometry. At end point, the engraftment and leukocyte subsets were determined in the spleen and bone marrow. We demonstrated that betamethasone has surprising effects in recovering immune system homeostasis. Betamethasone and fluticasone increase CXCR4 and decrease HLA class II and CD54 expression in ­CD34+ HSCs. Both glucocorticoids-exposed cells showed a similar engraftment in 2 Gy-irradiated NSG mice. Interestingly, betamethasone-exposed cells showed enhanced engraftment in 1 Gy-irradiated NSG mice, with a trend to increase regulatory T cell percentage when compared to control. Betamethasone induces altera- tions in ­CD34+ HSCs and improve the engraftment, leading to a faster immune system recovery, which will contribute to engrafted cells survival. Keywords Betamethasone · Cord blood · Hematopoietic stem cell · CD34+ · Transplantation · Immune system Introduction Synthetic glucocorticoids (e.g., prednisolone, fluticasone, or betamethasone) are drugs widely used for their immu- nomodulatory effects. Their mechanism of action is through * Marta Vives‑Pi the interaction with transcription factors related to inflamma- mvives@igtp.cat tion, such as NF-κB and AP-1 (Cain and Cidlowski 2017), inhibiting their function. These molecules also induce anti- 1 Immunology Department, Germans Trias I Pujol Research inflammatory pathways such as interleukin (IL)-10 produc- Institute, Autonomous University of Barcelona, Badalona, Spain tion (Mozo et al. 2004). Short-term exposure to synthetic 2 glucocorticoids induces long-term changes in gene expres- Department of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany sion of immune system cells (Franco et al. 2019; Hong et al. 3 2020; Wang et al. 2019). Moreover, glucocorticoids can Ahead Therapeutics SL, Barcelona, Spain exert a rapid, non-genomic effect on cells through kinases 4 Josep Carreras Leukaemia Research Institute, Campus and the release of accessory proteins (Ramamoorthy and IGTP-ICO, Badalona, Spain Cidlowski 2016). Betamethasone—usually administered to 5 Cell Therapy Services and Cord Blood Bank, Catalan Blood pregnant women at risk of pre-term delivery—induces fetal and Tissue Bank, Barcelona, Spain 13 Vol.:(0123456789) 1 Page 2 of 14 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 lung maturation, thus reducing mortality and morbidity in of Germans Trias i Pujol Research Institute. NSG mice pre-term newborns (Liggins and Howie 1972). Betametha- were kept under specific pathogen-free conditions, in a 12 h sone targets the ubiquitous glucocorticoid receptor, present dark/12 h light cycle with food and water ad libitum. Mice in almost all cell types, including stem cells and leukocytes, were anesthetized using Isoflurane (Forane, Abbvie pharma- and may therefore affect the development of the immune ceutics, Madrid, Spain) and euthanized by cervical disloca- system (Solano et al. 2016). tion. All animal studies were approved by the institutional We previously demonstrated that betamethasone increases animal ethics committee. self-tolerance and arrests autoimmunity in experimental type 1 diabetes (T1D) by inducing changes in leukocytes and islet Cell Isolation β cells (Gieras et al. 2017; Perna-Barrull et al. 2019). In this sense, betamethasone alters gene expression and causes UCB samples were obtained from the Blood and Tissue long-term changes through histone acetylation and DNA Research Biobank (Barcelona, Spain). All samples were methylation (Kim et al. 2022; Seckl 2004). These altera- obtained between 20 and 72 h after delivery. To isolate tions modify the accessibility to enhancers that control the ­CD34+ cells, UCB were incubated with RosetteSep (STEM- expression of relevant molecules. Other synthetic glucocorti- CELL Technologies, Vancouver, Canada) for 20 min prior coids, such as fluticasone propionate—usually administered to density gradient purification using Ficoll-Paque (GE to patients with asthma—induce similar effects. Healthcare, Uppsala, Sweden) and centrifugation at 700g Hematopoietic stem cell transplantation (HSCT) refers to for 30 min. ­CD34+ cells were selected from the mononuclear the transplantation of stem cells for the treatment of hemato- cells fraction using the EasySep human cord blood ­CD34+ logic, autoimmune, and genetic diseases. Malignant hemato- selection kit II (STEMCELL Technologies, Vancouver, logic diseases have an incidence of 40/100,000 inhabitants Canada). Briefly, mononuclear cells were incubated with per year in Europe and are particularly dramatic in children, anti-human CD34 antibodies and FcR-blocking antibodies who show a survival rate of 45–50% five years after HSCT for 10 min. Then, dextran microbeads were added to the (Copelan 2009; Styczyński et al. 2020). The main source of cell suspension and incubated for 1 min at room tempera- stem cells for allogeneic transplantation is bone marrow or ture. The immunomagnetic selection was performed using mobilized peripheral blood from adult donors, but the proce- EasySep Magnets (STEMCELL Technologies) according to dure is limited since it is restricted by the human leukocyte the manufacturer’s instructions. Purity, viability, and cell antigen (HLA) matching. Umbilical cord blood (UCB) is an number were then assessed by flow cytometry using anti- alternative source of stem cells for allogeneic transplantation human CD34-PE (BD Biosciences, San Jose, CA, USA), with clear benefits, including a lower HLA match require- anti-human CD45-FITC (BD Biosciences), 7-amino-actin- ment and the ease of retrieval in a non-invasive manner. omycin-D (7-AAD, BD Biosciences) and Perfect Count Engraftment success of UCB correlates with the number of Microbeads (Cytognos, Salamanca, Spain). Only samples stem cells (­ CD34+) transplanted, being the reason why only with cell purity higher than 75% and viability higher than 10% of the cord blood donations are used for HSCT (Rebulla 90% were used. et al. 2022; Wagner et al. 2002). Here, we provide a strategy to improve the engraftment Cell Culture capacities of UCB stem cells using betamethasone as a pre- conditioning treatment. We show that cord blood C ­ D34+ CD34+ stem cells were cultured at a density of 1 × ­105 hematopoietic stem cells (HSC) exposed to betamethasone ­CD34+/mL with RPMI-1640 (Biowest, Nuaille, France) increase the expression of C-X-C chemokine receptor type complete medium (CM) containing 10% fetal bovine serum 4 (CXCR4) and decrease the expression of HLA class II and (Gibco, Invitrogen, Carlsbad, CA, USA), penicillin (100 U/ CD54 without compromising their viability and function. ml, Normon SA, Madrid, Spain), streptomycin (100 ug/mL, These changes correlate with an improvement of C ­ D34+ cell Reig Jofre, Sant Joan Despí, Spain), glutamine (2 mmol/ engraftment in suboptimal (low-irradiation) conditions. mL, Sigma-Aldrich, St. Louis, MO, USA), sodium pyruvate (1 mmol/L, Gibco) and beta-mercaptoethanol (1 mmol/L, Sigma-Aldrich). Moreover, CM was supplemented with the Materials and Methods following human growth factors: Stem cell factor (100 ng/ mL), FMS-related tyrosine kinase 3 ligand (100 ng/mL), Mice IL-6 (50 ng/mL) and thrombopoietin (40 ng/mL; Prepro- tech, London UK). Depending on the group condition, cells Immunodeficient NOD.SCID-IL2Rɣ −/− (NSG) mice were cultured with betamethasone (100 nM, Sigma-Aldrich), were obtained from Charles River Laboratories (Barce- fluticasone propionate (100 nM, Fluticasone, Selleckchem, lona, Spain) and maintained in the Animal Care Facility Houston, TX, USA), or phosphate-buffered saline (PBS). 13 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Page 3 of 14 1 Glucocorticoid concentration was chosen based on our pre- the tail vein. For each experimental group, 3–5 mice were vious results (Perna-Barrull et al. 2019). Cells were incu- transplanted. bated for 20 h at 37 ºC in a humidified atmosphere contain- ing 5% ­CO2. Before transplantation, viability and ­CD34+ Flow Cytometry purity were assessed by flow cytometry using anti-human CD34-PE (BD Biosciences), anti-human CD45-FITC (BD To assess changes induced by betamethasone in leukocyte Biosciences), and 7-AAD (BD Biosciences). In addition, subsets of transplanted mice, peripheral blood was obtained cell number was determined using Perfect Count Microbeads every 1–2 weeks for 20 weeks. Moreover, bone marrow and (Cytognos, Salamanca, Spain). spleens were harvested at the endpoint of the study, and single-cell suspensions were obtained by mechanical dis- ruption. Cells were stained with anti-human CD3 BV650, Pre‑Engraftment Phenotype of ­CD34+ Cells CD4 AF488, CD8a AF700, CD1c PE, CD14 BV711, CD16 BV605, CD20 AF700, CD25 BV421, CD45 BV510, CD56 The phenotype of ­CD34+ HSC exposed to betamethasone APC, CD123 PE-Cy7, CD127 PerCP Cy5.5, HLA-DR or fluticasone after 20 h of culture was determined by flow BV785 (all from Biolegend, San Diego, CA, USA), CD11c cytometry. To that end, 20.000 C ­ D34+ cells of each group PE-efluor610 (eBioscience, San Diego, CA, USA), TCR gd (control, betamethasone, and fluticasone) were stained with PE-Cy7 (BD Biosciences), anti-mouse CD45 AF700 (eBi- anti-human CD34 PE (BD Biosciences), HLA-ABC FITC oscience) and Alexa Fluor 750 (Thermo Fisher Scientific, (Immunotools), CD45 PercP (BD Biosciences), CD40 APC Waltham, MA, EUA) for viability. Peripheral blood cells (Immunotools), CD54 PE-Cy7, CXCR4 APC-Cy7 and were directly stained, and RBC lysis/Fixation solution (Bio- HLA-DR V500 (BD Biosciences), and median fluorescence legend) was used before acquisition. The acquisition was intensity values were obtained using FACS Canto II cytom- performed using LSR Fortessa cytometer (BD Biosciences). eter (BD Biosciences). Data were analyzed using FlowJo Data were analyzed using FlowJo software (Tree Star Inc). software (Tree Star Inc., Ashland, OR, USA). Chimerism percentages were calculated as human leukocyte counts ­(HuCD45+) with respect to the total leukocyte counts CD34+ Cord Blood Cell Transplantation ­(HuCD45+ and ­muCD45+). Human leukocyte subsets were defined as specified in Table 1. Mice were transplanted at 8–10 weeks of age. Before trans- plantation, mice were sub-lethally whole-body irradiated at Statistical Analysis 1 or 2 Gy using a cesium source (Scherin, IBL 437 C). The standard irradiation in NSG is from 2 to 4 Gy (McDermott Statistical analysis was performed using Prism 9.3 soft- et al. 2010). We considered 2 Gy as optimal irradiation and ware (GraphPad Software Inc., San Diego, CA, USA). 1 Gy as suboptimal irradiation. After irradiation, mice were For comparison between groups, a Friedman’s test with randomized into treatment groups. Six hours after irradia- a posthoc Dunn’s test or Mixed-effect analysis with Geis- tion, mice transplantation was performed by transferring ser–Greenhouse correction and a post hoc Tukey’s multi- 2–5 × ­105 ­CD34+ cells obtained after 20 h of cell culture ple comparison test were used. For comparisons between exposed to betamethasone (100 nM, Sigma-Aldrich) or treatments in the follow-up experiments, a Wilcoxon test fluticasone (100 nM, Selleckchem). As a control, the same for the area under the curve (AUC) was used, and differ- dose of non-glucocorticoid exposed C ­ D34+ cells from the ences between weeks in the follow-up experiment for each same UCB was used. Cells were injected intravenously into group were assessed with a Tukey’s (three treatments) or Table 1  List of markers of Leukocyte subset Phenotype different leukocyte subsets analyzed Monocytes CD45+ ­CD20− ­CD3− HLA-DR+ ­CD14+ Conventional dendritic cells ­CD1c+ CD45+ ­CD20− ­CD3− HLA-DR+ ­CD123− ­CD14− ­CD11c+ ­CD1c+ (cDC2) Plasmacytoid dendritic cells CD45+ ­CD20− ­CD3− HLA-DR+ ­CD123hi B cells CD45+ ­CD20+ ­CD3− T cells αβ CD45+ ­CD20− ­CD3+ ­CD56− TCRγδ− CD4+ T cells CD45+ ­CD20− ­CD3+ ­CD56− TCRγδ− ­CD4+ ­CD8– CD8+ T cells CD45+ ­CD20− ­CD3+ ­CD56− TCRγδ− ­CD4– ­CD8+ Regulatory T cells CD45+ ­CD20− ­CD3+ ­CD56− TCRγδ− ­CD4+ ­CD8−CD127− ­CD25+ 13 1 Page 4 of 14 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Wilcoxon (two treatments) multiple comparisons test. For Betamethasone‑Treated ­CD34+ Cells Improve paired data, a non-parametric Wilcoxon test was used. Engraftment in Suboptimal Conditions Because myeloablative irradiation has hematopoietic, gas- trointestinal, and neurological side effects, we evaluated the Results ability of betamethasone-treated C ­ D34+ cells to engraft in suboptimal conditions of irradiation (sublethal low irradia- Betamethasone Alters the Phenotype of ­CD34+ Cord tion, 1 Gy). Using 1 Gy the niche space in the bone mar- Blood Cells row will be limited, and HSC will compete with other cells. Before transplantation, both betamethasone-exposed and The direct effect of betamethasone on C ­ D34+ cord blood unexposed ­HuCD34+ cells showed viability higher than cells was tested in vitro. The viability of C ­ D34+ cells was 90%. Figure 4a shows peripheral blood chimerism over a not impaired after 20 h in culture with betamethasone at period of 20 weeks post transplantation. We considered suc- ­1 0 2, ­1 0 4 or ­1 0 5 nM (Fig. 1a). By contrast, fluticasone, cessful engraftment when more than 2% of ­HuCD45+ of another synthetic glucocorticoid with described effect total leukocytes were detected 10 weeks after transplanta- on ­C D34 + cord blood cells, significantly decreased the tion. In the control group, only one mouse showed positive viability of ­CD34+ cells at 1­ 05 nM (p < 0.05). Then, phe- engraftment, whereas 4/5 mice transplanted with betameth- notypic changes induced by 20 h exposure to betametha- asone-exposed ­CD34+ cells displayed positive engraftment. sone and fluticasone were assessed. Figure 1b shows a Thus, a clear trend to improve the chimerism was observed significant increase in CXCR4, and a significant reduc- at weeks 7, 12, 14, and 20 when cells were treated with bet- tion in CD54 and HLA-DR by betamethasone (p < 0.0001) amethasone before transplantation (p = 0.0625). The AUC and fluticasone (p < 0.01, 0.05 and 0.001, respectively) on (Fig. 4b) confirmed this biological effect, although the dif- ­CD34+ cells in comparison to the control group. No dif- ference was not significant (p = 0.0625). At the end of the ferences were found in HLA class I and CD40 expression. study (20 weeks post transplantation), chimerism was also In summary, betamethasone alters C ­ D34+ cells toward a assessed in the spleen and bone marrow. The percentage and less immunogenic phenotype and a potentially improved number of H ­ uCD45+ cells with respect to total leukocytes repopulation. tended to be higher in the spleen (Fig. 4c, p = 0.0625) and bone marrow (Fig. 4d) of mice transplanted with ­CD34+ exposed to betamethasone. Betamethasone Allows Experimental ­CD34+ Cell Engraftment Follow‑up of the Development of Human Immune Cell Subsets after Transplant with Human ­CD34+ To assess the effect of betamethasone on ­C D34 + HSC Exposed to Betamethasone engraftment in optimal conditions, mice were irradiated at 2 Gy, and cord blood ­CD34+ HSC were cultured with We next aimed at assessing the quality of immune cell betamethasone or fluticasone at 100 nM for 20 h before reconstitution by determining the percentages of different transplantation. Fluticasone-treated cells were used as a human immune cell subsets in peripheral blood of low- positive control because it has been described that this irradiated transplanted mice during the study follow-up. drug improves cell engraftment (Guo et al. 2017). Before Table 1 shows the phenotype of the different leukocyte transplantation, the purity of the ­H uCD34 + fraction subsets analyzed. Figure 5 shows the evolution of differ- was 93.02% ± 2.80% (mean ± SD) and viability was ent cell subsets in NSG mice transplanted with ­C D34 + always > 90% in the three conditions (PBS, Bmet, and cord blood cells exposed to betamethasone in compari- Fase). In the present study, the gating strategy (Fig. 2) son to unexposed cells. All subsets showed improved was used to determine the engraftment and leukocyte sub- biological expansion after betamethasone exposure. The sets, as defined in Table 1. No significant differences were percentage of monocytes, conventional dendritic cells observed in the engraftment between the control (PBS), ­CD1c+ (cDC2s), and plasmacytoid dendritic cells (pDCs) betamethasone, and fluticasone groups (Fig. 3a). The per- showed a trend to increase 2–4 weeks after transplant, centage of human chimerism at 2, 4, 6, and 8 weeks after decreasing then from week 6 in both groups. By contrast, transplantation was similar in all conditions. These results B cells were detected 4 weeks after transplant reaching demonstrate that betamethasone-treated cells do not impair a plateau at weeks 7–9. At that point, the median (inter- ­CD34+ HSC engraftment in this experimental model. The ­ uCD45+ cells) was quartile range) B cell percentage (of H analysis of the AUC confirmed these results (Fig. 3b). 75.26 (34.36–92.86) in the betamethasone group and 1.95 (1.43–47.93) in the control group. As expected, T cells 13 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Page 5 of 14 1 Fig. 1  Effect of betamethasone on ­CD34+ phenotype. (a) Per- centage of viable ­CD34+ cells (annexin-V ­PE–, ­7aad–) after 20 h cultured with betametha- sone (left panel) and flutica- sone (right panel) at ­102, ­104 and ­105 nM. Dots show three independent experiments and lines connect paired experi- ments (*p ≤ 0.05, Dunn’s test, Friedman test). (b) Relative median fluorescence intensity (MFI) of CXCR4, CD54, MHC class II (HLA-DR), MHC class I, and CD40 expression on the membrane of C ­ D34+ cells. PBS represents control conditions. Squares and triangles represent ­CD34+ cells cultured with 100 nM betamethasone (bmet) or 100 nM fluticasone (fase) respectively. Lines show the mean of n = 6–18 independ- ent experiments. (*p ≤ 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Mixed-effect analysis with the Geisser– Greenhouse correction, Tukey’s multiple comparison test) ­ D4+ and ­CD8+, arise 8 weeks after transplantation, both C always to a greater extent in the betamethasone group. increasing in percentage until the end of the follow-up and ­CD4+ regulatory T cells (Treg) were detected only in the betamethasone group from 12 to 14 weeks after transplant. 13 1 Page 6 of 14 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Fig. 2  Representative gating strategy for the analysis of the engraft- spleen and bone marrow to determine the engraftment and leukocyte ment and leukocyte subsets. The gating strategy was used during subsets as defined in Table 1 the follow-up in peripheral blood, and at the end of the study in the Spleen and Bone Marrow Repopulation Increases Although non-significant, these results reflect a biological with Betamethasone‑Exposed ­CD34+ Cord Blood effect. In the betamethasone group, B cells were the most Cells abundant (6.900.000, median of five mice), followed by T lymphocytes (308.000), pDCs (69.000), cDC2s (21.000), To characterize the potential of C­ D34+ betamethasone- and monocytes (13.000), which correlates well to the human exposed cells in spleen and bone marrow repopulation, immune cell subsets found in peripheral blood during the immune cell subsets were analyzed in these two lymphoid follow-up. Figure 7 shows the total cell numbers of human organs 20 weeks after transplantation. Figure 6 shows total cell subsets in the bone marrow. Similar to the spleen, all cell numbers of splenic monocytes, cDC2s, pDCs, B lym- subsets displayed higher counts in the betamethasone group phocytes, T lymphocytes (both ­CD4+ and C­ D8+), and C ­ D4+ than in the PBS group. In the betamethasone group, B cells Treg cells. All subsets showed a trend to increase in the were the most abundant (850.000, median of five mice), betamethasone group in comparison to the control group. followed by pDCs (200.000), monocytes (80.000), cDC2s (70.000), and T lymphocytes (15.000). In summary, a clear 13 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Page 7 of 14 1 Fig. 3  Repopulation kinetics in mice irradiated with 2 Gy (opti- casone (Fase, green triangle and line show the median, IQR) after mal conditions). Human chimerism was determined at 2, 4, 6, and 8 weeks. At the end of the follow-up, the median (IQR) percentage 8 weeks post transplantation in peripheral blood (three animals per of chimerism reached 30.91 (11.65–31.71), 14.10 (8.52–34.03) and group). (a) The percentage of chimerism in peripheral blood of mice 53.16 (23.41–62.35), respectively (Tukey’s multiple comparison test). ­ uCD34+ cultured for 20 h with PBS (orange dot transplanted with H (b) Area under the curve (AUC) at week 8 post transplantation, bars and line show the median and interquartile range, IQR), betametha- show the median (IQR) of three mice (Dunn’s test, Friedman test) sone (Bmet, blue square and line show the median, IQR) or fluti- shift in the repopulation density was observed both in lym- insulin-producing β cells by T lymphocytes, we found a pre- phoid and myeloid populations due to the effect of beta- ventive effect of this drug. The mechanism of action was methasone at the end of the study. mainly through long-lasting alterations on the T cell recep- tor repertoire, thus protecting against β cell autoimmunity (Gieras et al. 2017; Perna-Barrull et al. 2019). These results Discussion point to a direct effect of betamethasone on HSC that will be maintained later in life. In humans, a preliminary study In this work, we demonstrate for the first time that betameth- in a Spanish cohort shows that the developing immune sys- asone modifies human cord blood C ­ D34+ HSC phenotype tem plasticity can be influenced by prenatal betamethasone, enhancing engraftment ability. Phenotypic changes induced which could contribute to preventing autoimmune diabetes by the short exposure of HSC to betamethasone would pro- in subjects at risk (Perna-Barrull et al. 2022). Based on these mote the migration to bone marrow, thus improving engraft- data, we hypothesized that betamethasone could also exert ment in a competitive environment (suboptimal irradiation). beneficial effects on HSC to improve engraftment. Moreover, these C ­ D34+ cells treated with betamethasone To assess that, we first determined the effect of beta- can differentiate in all the immune system cell lineages. methasone in cord blood HSC. We found that betamethasone Betamethasone was selected as pre-treatment for C ­ D34+ alters ­CD34+ cell phenotype, mainly by increasing CXCR4 experimental transplantation for two reasons: (1) it is a com- and reducing CD54 and HLA class II membrane expres- mercially available and safe synthetic glucocorticoid used sion. The increase of CXCR4 expression in C ­ D34+ cells in the late phase of pregnancy to prevent respiratory dis- is a biomarker of enhanced migration potential to the bone tress syndrome in pre-term newborns (Sweet et al. 2019), marrow and spleen. It has been previously demonstrated that and (2) glucocorticoids are involved in the ontogeny of the different glucocorticoids increase CXCR4 expression (Guo fetal immune system and other organs. Therefore, the fine- et al. 2017; Kahn et al. 2004; Kollet et al. 2001), but it has tuning of glucocorticoids is crucial since alterations in their not been reported using betamethasone. Also, impairing the concentration, for example, due to exogenous administration recognition of transplanted cells by the immune system of or aberrant endogenous production, can hugely impact fetal the host by reducing HLA class II molecules and CD54 will development (Solano et al. 2016). Moreover, these effects contribute to avoiding alloreactivity, reducing graft rejec- are maintained after birth due to epigenetic modifications tion, and increasing graft survival (Dustin 2014; Koga et al. (Bose et al. 2015; Zannas and Chrousos 2017). 2020). These changes in the phenotype should contribute to We have previously described prenatal betametha- the improved engraftment of betamethasone-exposed cord sone’s effect on autoimmune diseases. In experimental blood ­CD34+ cells in suboptimal conditions. T1D, an autoimmune disease caused by the destruction of 13 1 Page 8 of 14 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Fig. 4  Repopulation kinetics in mice irradiated with 1 Gy (subopti- parisons test). (b) Area under the curve (AUC) at week 20 post trans- mal conditions). Human chimerism was determined at 1–20 weeks plantation chimerism in peripheral blood of betamethasone-exposed post transplantation in peripheral blood. At week 20, chimerism was (Bmet) or unexposed (PBS) H ­ uCD34+ cells, bars show the median determined in the spleen and bone marrow (five animals per group). (IQR) of five mice (Wilcoxon matched-paired test). (c) Percentage (a) The repopulation of 20 h unexposed (PBS) or betamethasone- (left panel) and total cell number (right panel) of betamethasone- exposed (Bmet) ­CD34+ cells at week 20 post transplantation reached exposed (blue squares) or unexposed (orange dots) ­HuCD34+ cells in a median (interquartile range, IQR) percentage of chimerism of 1.12 the spleen of mice 20 weeks after transplantation or (d) in the bone (0.71–24.05) and 42.29 (21.21–54.40), respectively. Lines show the marrow. Bars show the median (IQR) percentage of leukocyte subset median percentage of chimerism in 5 mice (Wilcoxon multiple com- chimerism of five mice (Wilcoxon matched-paired test) 13 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Page 9 of 14 1 Then, we determined the effect of betamethasone on HSC blood, as reported (Beyer and Muench 2017). This fact engraftment. By irradiating mice with 2 Gy, we observed could be due, at least in part, to immunological differences similar engraftment in mice transplanted with betameth- in terms of chemokines that will hamper the mobilization asone-exposed or unexposed C ­ D34+ cells, comparable to of immune cells. reported studies (Casamayor-Genescà et al. 2017). At the Interestingly, our results show a trend to increase Treg end of the follow-up, fluticasone and betamethasone show cells in the betamethasone-exposed ­CD34+ HSC, fitting analogous engraftment, fitting well with similar changes in well with previous results (Chen et al. 2006). It is well the phenotype observed in vitro, and despite a described known that Treg cells are crucial for the immunosuppres- higher affinity of fluticasone for the glucocorticoid recep- sive effects of glucocorticoids (Kim et al. 2020). At the same tor when compared to betamethasone (Daley-Yates 2015). time, glucocorticoids promote the expression of FOXP3 in It would be interesting to determine the differences induced T cells, which induces Treg cell differentiation and function in the gene expression profile by these two glucocorticoids (Karagiannidis et al. 2004; Ugor et al. 2018). Thus, a Treg and to correlate those results with their engraftment ability. increase could be crucial to avoid rejection and graft versus However, in a more graft-restrictive environment due host disease. Since betamethasone is prenatally administered to low irradiation (1 Gy), betamethasone-exposed ­CD34+ and that fact correlates with protection against autoimmunity cells tended to improve engraftment results when com- (Gieras et al. 2017; Perna-Barrull et al. 2022), it is reason- pared to non-treated cells. The phenotype of betametha- able to speculate that Tregs will play a role in homeostasis. sone-exposed HSC points to this functional improvement, This fact suggests that betamethasone-induced changes in mediated, at least in part, by CXCR4 overexpression. HSC will be long-term maintained. Although glucocorti- This could contribute to a quick repopulation in the bone coid-driven epigenetic changes have been described, the marrow niche before the host cells recover from the low specific effects of betamethasone on HSC have not been irradiation, a condition that could be optimal for the host fully characterized. Morante-Palacios et al. (2022) reported in a clinical setting. This fact can explain the differences the epigenetic mechanism of glucocorticoids that induce the observed in the total number of graft cells expanded differentiation from monocytes to tolerogenic dendritic cells. between groups. Understanding the role of this molecule in the development The percentages of the different subsets from 8 weeks of immune system cells could help in the evolution of future after transplantation and until the end of the follow-up therapeutic strategies. were similar to those previously reported (Audigé et al. We are well aware of the limitations of the study. First, 2017; Haworth et al. 2017). However, the use of adult NSG this is a preliminary study, and the number of transplanted mice allowed us to perform an exhaustive follow-up of the mice is scarce. Also, functional studies of the cell subsets immune system cells differentiated just after transplanta- differentiated in vivo would help to understand the descrip- tion. Indeed, this is the first report on the evolution of the tive results of longstanding betamethasone effects. Another engraftment of C­ D34+ HSC from cord blood during the first limitation was that the UCB units used in this study were 4 weeks after transplantation. Myeloid cells (dendritic cells the ones discarded for clinical use and this is not the optimal and monocytes) compose the first wave of human cells in substrate for the study and a clinical scale-up. Further vali- mouse peripheral blood. In human HSCT, a similar recon- dation in this setting will be required for clinical translation. stitution has been described, being donor dendritic cells and Despite these limitations, these novel results show a clear monocytes detected 2–4 weeks after transplantation (Auffer- benefit of using betamethasone in HSC transplantation. An mann-Gretzinger et al. 2002; Turcotte et al. 2016). It would important clinical implication would be that discarded UCB be interesting to analyze the ability of these myeloid cells units or those banked with low ­CD34+ cell numbers could be to colonize the different secondary lymphoid organs and, at recovered with a short-term treatment using betamethasone, the same time, to determine their role in the establishment of as a pre-conditioning strategy. the germinal centers. Because fetal cDC2s are immunosup- In conclusion, betamethasone induces alterations in pressive, but in adults, these cells induce T cell responses ­CD34+ HSC that improve engraftment in competitive con- (McGovern et al. 2017), betamethasone could maintain this ditions, leading to a faster recovery of the immune system fetal tolerogenic action. We also observed higher engraft- and promoting the Treg lymphocyte subset that in turn will ment in the spleen and bone marrow than in peripheral contribute to the survival of the engrafted cells. 13 1 Page 10 of 14 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Fig. 5  Engraftment of human immune cell subsets in peripheral ­ D4+ Treg cells from mice transplanted with betameth- T cells, and C blood samples. Percentage of human subsets assessed by flow cytom- asone-exposed ­HuCD34+ cells (Bmet, blue squares, the blue line etry at 1–20 weeks after transplantation in mice (five animals per shows the median percentage of subsets in five mice) or unexposed group). The analyzed subsets (as defined in Table 1) were monocytes, ­HuCD34+ cells (PBS, orange dots, the orange line shows the median ­ D1c+ (cDC2s), plasmacytoid dendritic conventional dendritic cells C percentage of subsets in five mice) (Wilcoxon multiple comparisons cells (pDCs), B lymphocytes, T lymphocytes, ­CD4+ T cells, ­CD8+ test) 13 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Page 11 of 14 1 Fig. 6  Total cell number of human immune cell subsets in the spleen. cytes, conventional dendritic cells ­ CD1c+ (cDC2s), plasmacytoid The total cell number of human subsets was assessed by flow cytom- dendritic cells (pDCs), B lymphocytes (B cells), T lymphocytes (T etry in the spleen of mice at week 20 after transplantation. Bars show cells), ­CD4+ T cells, C ­ D8+ T cells, and C ­ D4+ Treg cells (Wilcoxon the median (interquartile range) number of subset cells in five animals matched-paired test) per group. The analyzed subsets (as defined in Table 1) were mono- 13 1 Page 12 of 14 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Fig. 7  Total cell number of human immune cell subsets in the bone marrow. The total cell number of human subsets was assessed by flow cytometry in the bone marrow of mice at week 20 after transplantation (bars show the median (interquartile range) of five animals per group). The analyzed subsets (as defined in Table 1) were monocytes, conventional dendritic cells ­CD1c+ (cDC2s), plasmacytoid dendritic cells (pDCs), B lymphocytes (B cells), T lymphocytes (T cells), ­CD4+ T cells, ­CD8+ T cells, and C ­ D4+ Treg cells (Wilcoxon matched-paired test) 13 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:1 Page 13 of 14 1 Acknowledgements We thank Mr. Marco Fernandez (Flow Cytometry References Plattform, IGTP) and Dr Adrian Villalba (Immunology Section, IGTP) for technical assistance. Special thanks to Dr Joaquim Vives, from Audigé A, Rochat MA, Li D et al (2017) Long-term leukocyte recon- Banc de Sang i Teixits, Barcelona, for fruitful discussions. stitution in NSG mice transplanted with human cord blood hemat- opoietic stem and progenitor cells. BMC Immunol 18:28. https://​ Author contributions DPB, AG, ET, SQ, RMR and MVP designed doi.​org/​10.​1186/​s12865-​017-​0209-9 the study and interpreted the data; DPB, and MVP wrote the manu- Auffermann-Gretzinger S, Lossos IS, Vayntrub TA et al (2002) Rapid script; SQ provided the cord blood units; DPB, LGM, SRF, and LAF establishment of dendritic cell chimerism in allogeneic hemat- performed the experiments; RMAC and RMR were the responsible of opoietic cell transplant recipients. Blood 99:1442–1448. https://​ mice; and DPB, LGM, and MVP analyzed the data. All authors read doi.​org/​10.​1182/​blood.​v99.4.​1442 and approved the final manuscript. Beyer AI, Muench MO (2017) Comparison of human hematopoietic reconstitution in different strains of immunodeficient mice. Stem Funding Open Access Funding provided by Universitat Autonoma de Cells Dev 26:102–112. https://​doi.​org/​10.​1089/​scd.​2016.​0083 Barcelona. This work was supported by a grant from the Deutsche Bose R, Spulber S, Kilian P et al (2015) Tet3 mediates stable gluco- Forschungsgemeinschaft (KFO296) and by a donation from Naval- corticoid-induced alterations in DNA methylation and Dnmt3a/ potro-Cardús family (Amics de Can Ruti crowdfunding campaign). Dkk1 expression in neural progenitors. Cell Death Dis 6:e1793. LGM is supported by the PERIS PIF-Salut Program, Departament de https://​doi.​org/​10.​1038/​cddis.​2015.​159 Salut, Generalitat de Catalunya (Grant # SLT017/20/000049). LAF is Cain DW, Cidlowski JA (2017) Immune regulation by glucocorticoids. supported by AGAUR, Generalitat de Catalunya (Grant DI2021-69). Nat Rev Immunol 17:233–247. https://d​ oi.o​ rg/1​ 0.1​ 038/n​ ri.2​ 017.1 Casamayor-Genescà A, Pla A, Oliver-Vila I et al (2017) Clinical-scale Data availability The datasets generated and/or analyzed during the expansion of CD34+ cord blood cells amplifies committed pro- current study are available from the corresponding author upon rea- genitors and rapid scid repopulation cells. N Biotechnol 35:19–29. sonable request. https://​doi.​org/​10.​1016/j.​nbt.​2016.​10.​011 Chen X, Oppenheim JJ, Winkler-Pickett RT et al (2006) Glucocorti- Declarations coid amplifies IL-2-dependent expansion of functional FoxP3(+) CD4(+)CD25(+) T regulatory cells in vivo and enhances their Conflict of interest MVP is co-founder and SEO of Ahead Therapeu- capacity to suppress EAE. Eur J Immunol 36:2139–2149. https://​ tics SL. LAF and SRF are employees of this company. 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