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Severe Developmental B Lymphopoietic Defects in Foxp3-Deficient Mice are Refractory to Adoptive Regulatory T Cell Therapy.

Riewaldt J, Düber S, Boernert M, Krey M, Dembinski M, Weiss S, Garbe AI, Kretschmer K - Front Immunol (2012)

Bottom Line: However, marginal zone B and B-1a cells were absent throughout ontogeny.Developmental B lymphopoietic defects largely correlated with defective thymopoiesis.Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12.

View Article: PubMed Central - PubMed

Affiliation: Center for Regenerative Therapies Dresden, Technical University Dresden Dresden, Germany.

ABSTRACT
The role of Foxp3-expressing regulatory T (T(reg)) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of T(reg) cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of T(reg) cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3(+) T(reg) cells.

No MeSH data available.


Related in: MedlinePlus

Adoptive Tregcell transfer in Sf mice. (A) Kaplan-Meier survival curves of CD45.2 Sf mice that had either been left untreated (red squares; n = 14) or injected with total populations of CD4+ T cells from Foxp3-proficient CD45.1+ Foxp3GFP mice (green triangles; n = 11), as indicated by the arrow. 6–8 weeks after cell transfer, recipient Sf mice were subjected to analysis by flow cytometry and RT-PCR. Data were collected from 2 (B,C) or 3 (F) independent experiments. (B,C) Congenically marked T cells were tracked in primary and secondary lymphoid organs of recipient mice. (B) Absolute numbers and (C) flow cytometry of CD4+CD45.1+ cells (left) for Foxp3GFP expression among gated CD4+CD45.1+ cells (right) in indicated organs. Lines with arrowheads illustrate the gating scheme. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively. Numbers in dot plots without parentheses indicate the percentage of gated cells within the respective gates. Parenthesized numbers indicate mean percentages ± SD of CD45.1+ cells among gated CD4+ cells (left) or mean percentages ± SD of Foxp3+ cells among gated CD4+CD45.1+ cells (right). (D,E) Impact of adoptive Treg cell transfer on in situ production of cytokines. The expression of mRNAs encoding (D) indicated inflammatory cytokines and (E) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing total RNA extracted from indicated whole tissues of Sf mice that had previously been injected with Treg cells. Each dot represents an individual Sf recipient mouse, and horizontal bars indicate mean values. Bars depict mean values from pooled single cell suspensions of three individual untreated 4-week-old Sf mice. Blue hatched lines indicate mRNA expression levels in WT animals for comparison (see also Figures 2F,G). HPRT was used for normalization. Data are representative of at least three independent experiments. (F) Impact of adoptive Treg cell transfer on T cell development in the thymus of Sf mice. Representative flow cytometry of CD4 and CD8 expression on thymocytes from WT mice (6-week-old; top) and Sf mice that were either left untreated (4-week-old; middle) or treated with adoptive Treg cell transfer (6–8-week-old; bottom). Numbers in dot plots indicate the percentages of gated cells within the respective gates. Graphs depict total thymic cellularity (top), as well as percentages (middle) and numbers (bottom) of DP thymocytes of WT mice (blue circles), as well as Sf mice that were either left untreated (red squares) or treated with adoptive Treg cell transfer (green triangles). Symbols and horizontal lines in graphs indicate individual mice and mean values, respectively. (One-way ANOVA: ***p ≤ 0.01; **p ≤ 0.01; *p ≤ 0.05; ns, not significant).
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Figure 5: Adoptive Tregcell transfer in Sf mice. (A) Kaplan-Meier survival curves of CD45.2 Sf mice that had either been left untreated (red squares; n = 14) or injected with total populations of CD4+ T cells from Foxp3-proficient CD45.1+ Foxp3GFP mice (green triangles; n = 11), as indicated by the arrow. 6–8 weeks after cell transfer, recipient Sf mice were subjected to analysis by flow cytometry and RT-PCR. Data were collected from 2 (B,C) or 3 (F) independent experiments. (B,C) Congenically marked T cells were tracked in primary and secondary lymphoid organs of recipient mice. (B) Absolute numbers and (C) flow cytometry of CD4+CD45.1+ cells (left) for Foxp3GFP expression among gated CD4+CD45.1+ cells (right) in indicated organs. Lines with arrowheads illustrate the gating scheme. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively. Numbers in dot plots without parentheses indicate the percentage of gated cells within the respective gates. Parenthesized numbers indicate mean percentages ± SD of CD45.1+ cells among gated CD4+ cells (left) or mean percentages ± SD of Foxp3+ cells among gated CD4+CD45.1+ cells (right). (D,E) Impact of adoptive Treg cell transfer on in situ production of cytokines. The expression of mRNAs encoding (D) indicated inflammatory cytokines and (E) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing total RNA extracted from indicated whole tissues of Sf mice that had previously been injected with Treg cells. Each dot represents an individual Sf recipient mouse, and horizontal bars indicate mean values. Bars depict mean values from pooled single cell suspensions of three individual untreated 4-week-old Sf mice. Blue hatched lines indicate mRNA expression levels in WT animals for comparison (see also Figures 2F,G). HPRT was used for normalization. Data are representative of at least three independent experiments. (F) Impact of adoptive Treg cell transfer on T cell development in the thymus of Sf mice. Representative flow cytometry of CD4 and CD8 expression on thymocytes from WT mice (6-week-old; top) and Sf mice that were either left untreated (4-week-old; middle) or treated with adoptive Treg cell transfer (6–8-week-old; bottom). Numbers in dot plots indicate the percentages of gated cells within the respective gates. Graphs depict total thymic cellularity (top), as well as percentages (middle) and numbers (bottom) of DP thymocytes of WT mice (blue circles), as well as Sf mice that were either left untreated (red squares) or treated with adoptive Treg cell transfer (green triangles). Symbols and horizontal lines in graphs indicate individual mice and mean values, respectively. (One-way ANOVA: ***p ≤ 0.01; **p ≤ 0.01; *p ≤ 0.05; ns, not significant).

Mentions: We initially focused on the assessment of B and T lymphopoietic activity in adolescent Sf mice. In this context, it is important to note that all Sf mice in our colony housed under specific pathogen-free conditions develop external signs of severe autoimmune disease (general failure to thrive, exfoliative dermatitis, etc.) by 4 weeks of age (data not shown), with a median survival of 27 days (see also Figure 5A). Flow cytometric analysis of moribund 4-week-old Sf mice revealed an up to 10-fold reduction in both percentages and numbers of early B220+c-kit+ Pro/Pre-B-I cells (nomenclature according to Rolink et al., 1999), as compared to age-matched wild-type (WT) control animals (Figure 1A, left). In WT mice, developmental progression of Pre-B-I cells gives rise to a pronounced compartment of B220+CD25+ Pre-B-II cells, which ultimately generate substantial populations of immature B220lowsIgM+ B cells (Figure 1A, top). In contrast, in Sf mice, regardless of the existence of a clearly discernable, albeit dramatically reduced population of Pro/Pre-B-I cells, subsequent Pre-B-II and immature sIgM+ B cell stages were consistently found to be below the level of detection (Figure 1A, middle and right). Similar results were obtained with adolescent Foxp3ΔEGFP mice (data not shown) that lack functional Treg cells due to a targeted insertion of GFP-encoding sequence into the Foxp3 locus, resulting in the expression of a non-functional Foxp3 protein fused to GFP (Lin et al., 2007). Overall, these data are largely consistent with previous reports (Leonardo et al., 2010; Chang et al., 2012) in that B cell development in the BM of adolescent Foxp3-deficient mice is severely abrogated.


Severe Developmental B Lymphopoietic Defects in Foxp3-Deficient Mice are Refractory to Adoptive Regulatory T Cell Therapy.

Riewaldt J, Düber S, Boernert M, Krey M, Dembinski M, Weiss S, Garbe AI, Kretschmer K - Front Immunol (2012)

Adoptive Tregcell transfer in Sf mice. (A) Kaplan-Meier survival curves of CD45.2 Sf mice that had either been left untreated (red squares; n = 14) or injected with total populations of CD4+ T cells from Foxp3-proficient CD45.1+ Foxp3GFP mice (green triangles; n = 11), as indicated by the arrow. 6–8 weeks after cell transfer, recipient Sf mice were subjected to analysis by flow cytometry and RT-PCR. Data were collected from 2 (B,C) or 3 (F) independent experiments. (B,C) Congenically marked T cells were tracked in primary and secondary lymphoid organs of recipient mice. (B) Absolute numbers and (C) flow cytometry of CD4+CD45.1+ cells (left) for Foxp3GFP expression among gated CD4+CD45.1+ cells (right) in indicated organs. Lines with arrowheads illustrate the gating scheme. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively. Numbers in dot plots without parentheses indicate the percentage of gated cells within the respective gates. Parenthesized numbers indicate mean percentages ± SD of CD45.1+ cells among gated CD4+ cells (left) or mean percentages ± SD of Foxp3+ cells among gated CD4+CD45.1+ cells (right). (D,E) Impact of adoptive Treg cell transfer on in situ production of cytokines. The expression of mRNAs encoding (D) indicated inflammatory cytokines and (E) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing total RNA extracted from indicated whole tissues of Sf mice that had previously been injected with Treg cells. Each dot represents an individual Sf recipient mouse, and horizontal bars indicate mean values. Bars depict mean values from pooled single cell suspensions of three individual untreated 4-week-old Sf mice. Blue hatched lines indicate mRNA expression levels in WT animals for comparison (see also Figures 2F,G). HPRT was used for normalization. Data are representative of at least three independent experiments. (F) Impact of adoptive Treg cell transfer on T cell development in the thymus of Sf mice. Representative flow cytometry of CD4 and CD8 expression on thymocytes from WT mice (6-week-old; top) and Sf mice that were either left untreated (4-week-old; middle) or treated with adoptive Treg cell transfer (6–8-week-old; bottom). Numbers in dot plots indicate the percentages of gated cells within the respective gates. Graphs depict total thymic cellularity (top), as well as percentages (middle) and numbers (bottom) of DP thymocytes of WT mice (blue circles), as well as Sf mice that were either left untreated (red squares) or treated with adoptive Treg cell transfer (green triangles). Symbols and horizontal lines in graphs indicate individual mice and mean values, respectively. (One-way ANOVA: ***p ≤ 0.01; **p ≤ 0.01; *p ≤ 0.05; ns, not significant).
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Related In: Results  -  Collection

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Figure 5: Adoptive Tregcell transfer in Sf mice. (A) Kaplan-Meier survival curves of CD45.2 Sf mice that had either been left untreated (red squares; n = 14) or injected with total populations of CD4+ T cells from Foxp3-proficient CD45.1+ Foxp3GFP mice (green triangles; n = 11), as indicated by the arrow. 6–8 weeks after cell transfer, recipient Sf mice were subjected to analysis by flow cytometry and RT-PCR. Data were collected from 2 (B,C) or 3 (F) independent experiments. (B,C) Congenically marked T cells were tracked in primary and secondary lymphoid organs of recipient mice. (B) Absolute numbers and (C) flow cytometry of CD4+CD45.1+ cells (left) for Foxp3GFP expression among gated CD4+CD45.1+ cells (right) in indicated organs. Lines with arrowheads illustrate the gating scheme. Dots and horizontal lines in graphs indicate individual mice and mean values, respectively. Numbers in dot plots without parentheses indicate the percentage of gated cells within the respective gates. Parenthesized numbers indicate mean percentages ± SD of CD45.1+ cells among gated CD4+ cells (left) or mean percentages ± SD of Foxp3+ cells among gated CD4+CD45.1+ cells (right). (D,E) Impact of adoptive Treg cell transfer on in situ production of cytokines. The expression of mRNAs encoding (D) indicated inflammatory cytokines and (E) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing total RNA extracted from indicated whole tissues of Sf mice that had previously been injected with Treg cells. Each dot represents an individual Sf recipient mouse, and horizontal bars indicate mean values. Bars depict mean values from pooled single cell suspensions of three individual untreated 4-week-old Sf mice. Blue hatched lines indicate mRNA expression levels in WT animals for comparison (see also Figures 2F,G). HPRT was used for normalization. Data are representative of at least three independent experiments. (F) Impact of adoptive Treg cell transfer on T cell development in the thymus of Sf mice. Representative flow cytometry of CD4 and CD8 expression on thymocytes from WT mice (6-week-old; top) and Sf mice that were either left untreated (4-week-old; middle) or treated with adoptive Treg cell transfer (6–8-week-old; bottom). Numbers in dot plots indicate the percentages of gated cells within the respective gates. Graphs depict total thymic cellularity (top), as well as percentages (middle) and numbers (bottom) of DP thymocytes of WT mice (blue circles), as well as Sf mice that were either left untreated (red squares) or treated with adoptive Treg cell transfer (green triangles). Symbols and horizontal lines in graphs indicate individual mice and mean values, respectively. (One-way ANOVA: ***p ≤ 0.01; **p ≤ 0.01; *p ≤ 0.05; ns, not significant).
Mentions: We initially focused on the assessment of B and T lymphopoietic activity in adolescent Sf mice. In this context, it is important to note that all Sf mice in our colony housed under specific pathogen-free conditions develop external signs of severe autoimmune disease (general failure to thrive, exfoliative dermatitis, etc.) by 4 weeks of age (data not shown), with a median survival of 27 days (see also Figure 5A). Flow cytometric analysis of moribund 4-week-old Sf mice revealed an up to 10-fold reduction in both percentages and numbers of early B220+c-kit+ Pro/Pre-B-I cells (nomenclature according to Rolink et al., 1999), as compared to age-matched wild-type (WT) control animals (Figure 1A, left). In WT mice, developmental progression of Pre-B-I cells gives rise to a pronounced compartment of B220+CD25+ Pre-B-II cells, which ultimately generate substantial populations of immature B220lowsIgM+ B cells (Figure 1A, top). In contrast, in Sf mice, regardless of the existence of a clearly discernable, albeit dramatically reduced population of Pro/Pre-B-I cells, subsequent Pre-B-II and immature sIgM+ B cell stages were consistently found to be below the level of detection (Figure 1A, middle and right). Similar results were obtained with adolescent Foxp3ΔEGFP mice (data not shown) that lack functional Treg cells due to a targeted insertion of GFP-encoding sequence into the Foxp3 locus, resulting in the expression of a non-functional Foxp3 protein fused to GFP (Lin et al., 2007). Overall, these data are largely consistent with previous reports (Leonardo et al., 2010; Chang et al., 2012) in that B cell development in the BM of adolescent Foxp3-deficient mice is severely abrogated.

Bottom Line: However, marginal zone B and B-1a cells were absent throughout ontogeny.Developmental B lymphopoietic defects largely correlated with defective thymopoiesis.Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12.

View Article: PubMed Central - PubMed

Affiliation: Center for Regenerative Therapies Dresden, Technical University Dresden Dresden, Germany.

ABSTRACT
The role of Foxp3-expressing regulatory T (T(reg)) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of T(reg) cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of T(reg) cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive T(reg) cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3(+) T(reg) cells.

No MeSH data available.


Related in: MedlinePlus