Bone Marrow Transplantation (2016) 51, 596–597 © 2016 Macmillan Publishers Limited All rights reserved 0268-3369/16
OPEN
www.nature.com/bmt
LETTER TO THE EDITOR
The gender gap: discrepant human T-cell reconstitution after cord blood stem cell transplantation in humanized female and male mice Bone Marrow Transplantation (2016) 51, 596–597; doi:10.1038/ bmt.2015.290; published online 23 November 2015
models to evaluate the effects of new human vaccines and immune therapies on adaptive immune responses,1–3 but possible gender aspects were not considered. Notta et al., reported that female non-obese diabetic/severe combined immunodeficient (NOD/SCID)/IL2Rγc− / − (NSG) mice transplanted with limiting numbers of human CD34+CD38− cord blood hematopoietic stem
Several laboratories are exploring the use of humanized mice transplanted with human hematopoietic stem cells as in vivo
CD45+ cells in peripheral blood Experiment 1 (n=23) 60 50 40 30 20 10
60 50 40 30 20 10 10
20
15
10
Time in weeks
15
20
16
12
20
Time in weeks
Female Male
16
20
Time in weeks
T cells in peripheral blood (Experiment 1) Week 10
Week 15
Week 20
100
100
100
80
80
80
60
60
60
40 0.036
40 0.915 0.604 0.227
20
Female, n= 13 Male, n= 10 0.247
0
CD3/CD8
CD3/CD8
CD3/CD4
0
CD3/CD4
0
c
0.050
20
0.045
CD45
0.251
CD3/CD4
20
CD3/CD8
0.001
CD45
40
CD45
Mean percentage
12
Time in weeks
b
T cells and subtypes in peripheral blood (Experiment 2)
20
0.917 0.647 0.379
40
0.565 0.569 0.887 0.334 0.346 0.860
0.709
60
Week 20
Female, n= 12 Male, n= 14
0 CD45 CD3/CD4 CD3/CD8 CD4N CD4CM CD4EM CD8N CD8CM CD8EM
CD45 CD3/CD4 CD3/CD8 CD4N CD4CM CD4EM CD8N CD8CM CD8EM
0.141 0.020
20 0
0
80
CD45 CD3/CD4 CD3/CD8 CD4N CD4CM CD4EM CD8N CD8CM CD8EM
0.191
40
100 0.851 0.725 0.029
0.638 0.755
20
0.945
40
80 60
0.365
0.066
60
0.332 0.151
80
Week 16
100
0.010
Week 12
100
0.215
Mean percentage
Experiment 2 (n=26) 60 50 40 30 20 10
60 50 40 30 20 10
0.580 0.017
Mean percentage
a
Figure 1. Differential engraftment and kinetics of human lymphocytes in humanized female and male mice. 5-week-old NRG mice were irradiated with 450 cGy and i.v. injected with 2 × 105 (Experiment 1: 13 females and 10 males) or 1.5 × 105 (Experiment 2: 12 females and 14 males) human cord blood CD34+ cells. Mice were killed 20 weeks after transplantation. Immune reconstitution was assessed by flow cytometry analyses at different time points. (a) Error bars representing frequencies of human CD45+ cells in peripheral blood for each gender in experiments 1 and 2. Results represent least square means as percentages obtained from repeated measures analysis of variance (ANOVA) with between-subjects factor gender. Error bars represent 95% confidence interval. Engraftment in female mice is represented in red, and in male mice in blue. (b) Bar plots illustrating frequencies of lymphocyte types per gender in peripheral blood at different time points of experiment 1. P-value results are from two-sided t-tests for independent group comparisons. Results represent least square means as percentages obtained from repeated measures ANOVA with between-subjects factor gender. Tukey–Cramer adjustment was used for multiple comparisons. Female mice are represented in red, and male in blue. P-values o0.05 are indicated in italic. (c) Bar plots illustrating T-cell subtypes (naïve, central memory and effector memory) per gender in peripheral blood for different time points of experiment 2 accompanied by P-value results from twosided t-tests for independent group comparisons. Results represent least square means as percentages obtained from repeated measures ANOVA with between-subjects factor the gender. Tukey–Cramer adjustment was used for multiple comparisons. Female mice are represented in red, whereas males are represented in blue. P-values o0.05 are indicated in italic.
Letter to the Editor
cells (CB-HSCs) showed better engraftment of human CD45+ hematopoietic cells than males.4 A subsequent work reported faster growth of human tumor lines in NSG male mice, further supporting differences of xenograft cell engraftment and growth in female and male hosts.5 Here, we evaluated whether T-cell development after CD34+ CB-HSC transplantation into 5-week-old NOD-Rag1− / −-IL2Rγc− / − (NRG) mice was associated with gender. All experiments were performed in accordance with the guidelines and regulations regarding patient anonymity, informed consent and animal welfare. The protocols were approved by the Hannover Medical School (Ethics Committee) and State of Lower Saxony (Nds. Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Dezernat 33/Tierschutz). Different cohorts of immune deficient mice were transplanted with distinct cord blood donors and analyzed at several time points (for example, weeks 10/12, weeks 15/16 and week 20). Information was collected for various cells in blood, spleen and lymph nodes. We considered 10– 12 weeks after CB-HSCT in NRG mice as the time point to determine acceptable engraftment (for example, 45% huCD45+ cells in peripheral blood) for inclusion in the study. We examined 23 mice (13 female and 10 male) in the first experiment, and 26 mice (12 female and 14 male) in the second experiment. In agreement with Notta et al., we also observed that female mice transplanted with HSC from several cord blood units generally exhibited a higher frequency of huCD45+ cells than males at the 10–12 week time point (Figures 1a and b). However, after 15–16 weeks post CB-HSCT, the frequency of huCD45+ decreased in females but increased in males (Figure 1a). Remarkably, from weeks 10 to 16, females showed overall higher frequencies of T cells than males. Closer investigation of T-cell subsets from weeks 12–16 showed higher frequencies of naïve T cells in females, but higher frequencies of effector memory T cells in males (Figure 1c). Spleen and lymph nodes obtained from females at week 20 also contained higher frequencies of naive T cells, whereas memory T cells were more frequent in males (Supplementary Table 4). Taken together, these data from independent experiments indicated that higher thymic naïve T-cell output was supported in females post CB-HSCT, whereas peripheral activation/expansion of memory T cells was more dominant in males. Sex steroids were demonstrated to cause thymic atrophy with age in immune competent mice, and blockade of these steroids resulted in full reversal of thymic atrophy and enhanced regeneration of T cells.6 In patients, temporarily blocking sex steroids before stem cell transplantation increased thymus function and enhanced the rate of T-cell regeneration.7 In conclusion, CB-HSCT in humanized mice could potentially mirror the effect of murine sex hormones on human immune reconstitution, which should be taken as a note of caution for pre-clinical testing of vaccines and immune therapies. Thus, this emphasizes the need to separately analyze mice of both genders in experiments with humanized mice, as the quality of immune responses is likely to be different. In addition, the
597 influence of gender in immune reconstitution should be evaluated after CB-HSCT in patients. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by grants of the German Research Council DFG/SFB738project A6 and DFG/ REBIRTH—unit 6.4 (to RS).
V Volk1, A Schneider1, LM Spineli2, A Grosshennig2 and R Stripecke1 1 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany and 2 Institute of Biostatistics, Hannover Medical School, Hannover, Germany E-mail: [email protected] REFERENCES 1 Daenthanasanmak A, Salguero G, Sundarasetty BS, Waskow C, Cosgun KN, Guzman CA et al. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV. Mol Ther Methods Clin Dev 2015; 1: 14060. 2 Salguero G, Daenthanasanmak A, Munz C, Raykova A, Guzman CA, Riese P et al. Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation. J Immunol 2014; 192: 4636–4647. 3 Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol 2012; 12: 786–798. 4 Notta F, Doulatov S, Dick JE. Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgc-null recipients. Blood 2010; 115: 3704–3707. 5 Martin-Padura I, Agliano A, Marighetti P, Porretti L, Bertolini F. Sex-related efficiency in NSG mouse engraftment. Blood 2010; 116: 2616–2617. 6 Sutherland JS, Goldberg GL, Hammett MV, Uldrich AP, Berzins SP, Heng TS et al. Activation of thymic regeneration in mice and humans following androgen blockade. J Immunol 2005; 175: 2741–2753. 7 Sutherland JS, Spyroglou L, Muirhead JL, Heng TS, Prieto-Hinojosa A, Prince HM et al. Enhanced immune system regeneration in humans following allogeneic or autologous hemopoietic stem cell transplantation by temporary sex steroid blockade. Clin Cancer Res 2008; 14: 1138–1149.
This work is licensed under a Creative Commons AttributionNonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/4.0/
Supplementary Information accompanies this paper on Bone Marrow Transplantation website (http://www.nature.com/bmt)
© 2016 Macmillan Publishers Limited
Bone Marrow Transplantation (2016) 596 – 597
OPEN
www.nature.com/bmt
LETTER TO THE EDITOR
The gender gap: discrepant human T-cell reconstitution after cord blood stem cell transplantation in humanized female and male mice Bone Marrow Transplantation (2016) 51, 596–597; doi:10.1038/ bmt.2015.290; published online 23 November 2015
models to evaluate the effects of new human vaccines and immune therapies on adaptive immune responses,1–3 but possible gender aspects were not considered. Notta et al., reported that female non-obese diabetic/severe combined immunodeficient (NOD/SCID)/IL2Rγc− / − (NSG) mice transplanted with limiting numbers of human CD34+CD38− cord blood hematopoietic stem
Several laboratories are exploring the use of humanized mice transplanted with human hematopoietic stem cells as in vivo
CD45+ cells in peripheral blood Experiment 1 (n=23) 60 50 40 30 20 10
60 50 40 30 20 10 10
20
15
10
Time in weeks
15
20
16
12
20
Time in weeks
Female Male
16
20
Time in weeks
T cells in peripheral blood (Experiment 1) Week 10
Week 15
Week 20
100
100
100
80
80
80
60
60
60
40 0.036
40 0.915 0.604 0.227
20
Female, n= 13 Male, n= 10 0.247
0
CD3/CD8
CD3/CD8
CD3/CD4
0
CD3/CD4
0
c
0.050
20
0.045
CD45
0.251
CD3/CD4
20
CD3/CD8
0.001
CD45
40
CD45
Mean percentage
12
Time in weeks
b
T cells and subtypes in peripheral blood (Experiment 2)
20
0.917 0.647 0.379
40
0.565 0.569 0.887 0.334 0.346 0.860
0.709
60
Week 20
Female, n= 12 Male, n= 14
0 CD45 CD3/CD4 CD3/CD8 CD4N CD4CM CD4EM CD8N CD8CM CD8EM
CD45 CD3/CD4 CD3/CD8 CD4N CD4CM CD4EM CD8N CD8CM CD8EM
0.141 0.020
20 0
0
80
CD45 CD3/CD4 CD3/CD8 CD4N CD4CM CD4EM CD8N CD8CM CD8EM
0.191
40
100 0.851 0.725 0.029
0.638 0.755
20
0.945
40
80 60
0.365
0.066
60
0.332 0.151
80
Week 16
100
0.010
Week 12
100
0.215
Mean percentage
Experiment 2 (n=26) 60 50 40 30 20 10
60 50 40 30 20 10
0.580 0.017
Mean percentage
a
Figure 1. Differential engraftment and kinetics of human lymphocytes in humanized female and male mice. 5-week-old NRG mice were irradiated with 450 cGy and i.v. injected with 2 × 105 (Experiment 1: 13 females and 10 males) or 1.5 × 105 (Experiment 2: 12 females and 14 males) human cord blood CD34+ cells. Mice were killed 20 weeks after transplantation. Immune reconstitution was assessed by flow cytometry analyses at different time points. (a) Error bars representing frequencies of human CD45+ cells in peripheral blood for each gender in experiments 1 and 2. Results represent least square means as percentages obtained from repeated measures analysis of variance (ANOVA) with between-subjects factor gender. Error bars represent 95% confidence interval. Engraftment in female mice is represented in red, and in male mice in blue. (b) Bar plots illustrating frequencies of lymphocyte types per gender in peripheral blood at different time points of experiment 1. P-value results are from two-sided t-tests for independent group comparisons. Results represent least square means as percentages obtained from repeated measures ANOVA with between-subjects factor gender. Tukey–Cramer adjustment was used for multiple comparisons. Female mice are represented in red, and male in blue. P-values o0.05 are indicated in italic. (c) Bar plots illustrating T-cell subtypes (naïve, central memory and effector memory) per gender in peripheral blood for different time points of experiment 2 accompanied by P-value results from twosided t-tests for independent group comparisons. Results represent least square means as percentages obtained from repeated measures ANOVA with between-subjects factor the gender. Tukey–Cramer adjustment was used for multiple comparisons. Female mice are represented in red, whereas males are represented in blue. P-values o0.05 are indicated in italic.
Letter to the Editor
cells (CB-HSCs) showed better engraftment of human CD45+ hematopoietic cells than males.4 A subsequent work reported faster growth of human tumor lines in NSG male mice, further supporting differences of xenograft cell engraftment and growth in female and male hosts.5 Here, we evaluated whether T-cell development after CD34+ CB-HSC transplantation into 5-week-old NOD-Rag1− / −-IL2Rγc− / − (NRG) mice was associated with gender. All experiments were performed in accordance with the guidelines and regulations regarding patient anonymity, informed consent and animal welfare. The protocols were approved by the Hannover Medical School (Ethics Committee) and State of Lower Saxony (Nds. Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Dezernat 33/Tierschutz). Different cohorts of immune deficient mice were transplanted with distinct cord blood donors and analyzed at several time points (for example, weeks 10/12, weeks 15/16 and week 20). Information was collected for various cells in blood, spleen and lymph nodes. We considered 10– 12 weeks after CB-HSCT in NRG mice as the time point to determine acceptable engraftment (for example, 45% huCD45+ cells in peripheral blood) for inclusion in the study. We examined 23 mice (13 female and 10 male) in the first experiment, and 26 mice (12 female and 14 male) in the second experiment. In agreement with Notta et al., we also observed that female mice transplanted with HSC from several cord blood units generally exhibited a higher frequency of huCD45+ cells than males at the 10–12 week time point (Figures 1a and b). However, after 15–16 weeks post CB-HSCT, the frequency of huCD45+ decreased in females but increased in males (Figure 1a). Remarkably, from weeks 10 to 16, females showed overall higher frequencies of T cells than males. Closer investigation of T-cell subsets from weeks 12–16 showed higher frequencies of naïve T cells in females, but higher frequencies of effector memory T cells in males (Figure 1c). Spleen and lymph nodes obtained from females at week 20 also contained higher frequencies of naive T cells, whereas memory T cells were more frequent in males (Supplementary Table 4). Taken together, these data from independent experiments indicated that higher thymic naïve T-cell output was supported in females post CB-HSCT, whereas peripheral activation/expansion of memory T cells was more dominant in males. Sex steroids were demonstrated to cause thymic atrophy with age in immune competent mice, and blockade of these steroids resulted in full reversal of thymic atrophy and enhanced regeneration of T cells.6 In patients, temporarily blocking sex steroids before stem cell transplantation increased thymus function and enhanced the rate of T-cell regeneration.7 In conclusion, CB-HSCT in humanized mice could potentially mirror the effect of murine sex hormones on human immune reconstitution, which should be taken as a note of caution for pre-clinical testing of vaccines and immune therapies. Thus, this emphasizes the need to separately analyze mice of both genders in experiments with humanized mice, as the quality of immune responses is likely to be different. In addition, the
597 influence of gender in immune reconstitution should be evaluated after CB-HSCT in patients. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by grants of the German Research Council DFG/SFB738project A6 and DFG/ REBIRTH—unit 6.4 (to RS).
V Volk1, A Schneider1, LM Spineli2, A Grosshennig2 and R Stripecke1 1 Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany and 2 Institute of Biostatistics, Hannover Medical School, Hannover, Germany E-mail: [email protected] REFERENCES 1 Daenthanasanmak A, Salguero G, Sundarasetty BS, Waskow C, Cosgun KN, Guzman CA et al. Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV. Mol Ther Methods Clin Dev 2015; 1: 14060. 2 Salguero G, Daenthanasanmak A, Munz C, Raykova A, Guzman CA, Riese P et al. Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation. J Immunol 2014; 192: 4636–4647. 3 Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol 2012; 12: 786–798. 4 Notta F, Doulatov S, Dick JE. Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgc-null recipients. Blood 2010; 115: 3704–3707. 5 Martin-Padura I, Agliano A, Marighetti P, Porretti L, Bertolini F. Sex-related efficiency in NSG mouse engraftment. Blood 2010; 116: 2616–2617. 6 Sutherland JS, Goldberg GL, Hammett MV, Uldrich AP, Berzins SP, Heng TS et al. Activation of thymic regeneration in mice and humans following androgen blockade. J Immunol 2005; 175: 2741–2753. 7 Sutherland JS, Spyroglou L, Muirhead JL, Heng TS, Prieto-Hinojosa A, Prince HM et al. Enhanced immune system regeneration in humans following allogeneic or autologous hemopoietic stem cell transplantation by temporary sex steroid blockade. Clin Cancer Res 2008; 14: 1138–1149.
This work is licensed under a Creative Commons AttributionNonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/4.0/
Supplementary Information accompanies this paper on Bone Marrow Transplantation website (http://www.nature.com/bmt)
© 2016 Macmillan Publishers Limited
Bone Marrow Transplantation (2016) 596 – 597
