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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

Mechanisms of B cell developmental defects in adolescent Sf mice. (A) Genetic lineage tracing of Foxp3 reporter activity in peripheral B cells. Putative populations of CD19+YFP+ cells were FACS-purified from spleen (top) and pooled mLNs and scLNs (bottom) of adult BAC-Foxp3Cre-GFP × R26-Y mice. Flow cytometric presort analysis (left) and post-sort analysis (right) is depicted. Note that for presort analysis numbers in dot plots indicate percentages of cells within the respective gates, whereas for post-sort analysis numbers indicate percentages of CD19+YFP+ cells among total cell populations prior to FACS purification. (B) Differentiation capacity of Pro/Pre-B-I cells from moribund Sf mice in vitro. Initially sIgM−, B220+c-kit+ Pro/Pre-B-I cells, either freshly isolated from BM of 3-week-old WT (blue circles) or Sf (red squares) mice (left), or isolated from IL-7-supplemented cultures after proliferative expansion of respective Pro/Pre-B-I cell populations (right), were cultured in the absence of added IL-7. The capacity to differentiate into sIgM+ cells was assessed at different time points after initiation of cultures, as indicated. Symbols represent mean values of replicate wells ± SEM. (C) B cell development in Sf mice that lack CD4+ and CD8+ T cells. Flow cytometry of indicated B cell developmental stages in the BM of 4-week-old WT (top) and Sf (bottom) mice that additionally lacked Tcra gene expression (TCRα−/−). Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of three independent experiments. (D) Flow cytometric analysis of immature B220lowsIgM+ and mature/recirculating B220highsIgM+ B cell compartments in the BM of 4-week-old Sf mice that were either proficient (IL-6+/−, top) or deficient (IL-6−/−, bottom) for IL-6. Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of two independent experiments. (E) Impact of inflammatory cytokines on IL-7-mediated survival of Pro/Pre-B-I cells in vitro. FACS-purified populations of BM-derived B220+c-kit+ Pro/Pre-B-I cells from WT mice were cultured with IL-7, either alone or in the presence of titrating amounts of IL-17A or IFN-γ, as indicated. At day 4 of cultures, numbers of B220+c-kit+ cells recovered from cultures with and without added inflammatory cytokines was calculated as fold difference. Shown are mean and range of duplicate wells. Data are representative of two independent experiments. (F,G) The expression of mRNAs encoding (F) indicated inflammatory cytokines and (G) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing whole tissues harvested from 4-week-old WT (blue) or Sf (red) mice, as indicated. Note that, prior to RNA extraction, prepared single cell suspensions of respective tissues were pooled from three mice per group. HPRT was used for normalization. Shown are mean and range of duplicate samples. Data are representative of at least three independent experiments.
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Figure 2: Mechanisms of B cell developmental defects in adolescent Sf mice. (A) Genetic lineage tracing of Foxp3 reporter activity in peripheral B cells. Putative populations of CD19+YFP+ cells were FACS-purified from spleen (top) and pooled mLNs and scLNs (bottom) of adult BAC-Foxp3Cre-GFP × R26-Y mice. Flow cytometric presort analysis (left) and post-sort analysis (right) is depicted. Note that for presort analysis numbers in dot plots indicate percentages of cells within the respective gates, whereas for post-sort analysis numbers indicate percentages of CD19+YFP+ cells among total cell populations prior to FACS purification. (B) Differentiation capacity of Pro/Pre-B-I cells from moribund Sf mice in vitro. Initially sIgM−, B220+c-kit+ Pro/Pre-B-I cells, either freshly isolated from BM of 3-week-old WT (blue circles) or Sf (red squares) mice (left), or isolated from IL-7-supplemented cultures after proliferative expansion of respective Pro/Pre-B-I cell populations (right), were cultured in the absence of added IL-7. The capacity to differentiate into sIgM+ cells was assessed at different time points after initiation of cultures, as indicated. Symbols represent mean values of replicate wells ± SEM. (C) B cell development in Sf mice that lack CD4+ and CD8+ T cells. Flow cytometry of indicated B cell developmental stages in the BM of 4-week-old WT (top) and Sf (bottom) mice that additionally lacked Tcra gene expression (TCRα−/−). Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of three independent experiments. (D) Flow cytometric analysis of immature B220lowsIgM+ and mature/recirculating B220highsIgM+ B cell compartments in the BM of 4-week-old Sf mice that were either proficient (IL-6+/−, top) or deficient (IL-6−/−, bottom) for IL-6. Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of two independent experiments. (E) Impact of inflammatory cytokines on IL-7-mediated survival of Pro/Pre-B-I cells in vitro. FACS-purified populations of BM-derived B220+c-kit+ Pro/Pre-B-I cells from WT mice were cultured with IL-7, either alone or in the presence of titrating amounts of IL-17A or IFN-γ, as indicated. At day 4 of cultures, numbers of B220+c-kit+ cells recovered from cultures with and without added inflammatory cytokines was calculated as fold difference. Shown are mean and range of duplicate wells. Data are representative of two independent experiments. (F,G) The expression of mRNAs encoding (F) indicated inflammatory cytokines and (G) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing whole tissues harvested from 4-week-old WT (blue) or Sf (red) mice, as indicated. Note that, prior to RNA extraction, prepared single cell suspensions of respective tissues were pooled from three mice per group. HPRT was used for normalization. Shown are mean and range of duplicate samples. Data are representative of at least three independent experiments.

Mentions: Previous studies employing mice with Foxp3-dependent fluorochrome reporter activity indicated that high levels of Foxp3 expression are limited to αβ T cells (Fontenot et al., 2005; Wan and Flavell, 2005), with undetectable fluorochrome expression in B220+ cells from BM (Leonardo et al., 2010) and secondary lymphoid organs such as LNs (Wan and Flavell, 2005) and spleen (Leonardo et al., 2010). Whether Foxp3 can be expressed in other hematopoietic (Bernard et al., 2009; Manrique et al., 2011) and non-hematopoietic (Chang et al., 2005; Hinz et al., 2007; Zuo et al., 2007; Chen et al., 2008; Karanikas et al., 2008; Chung et al., 2010) lineages has been actively debated (Liston et al., 2007; Mayer et al., 2012). To formally exclude the possibility that observed alterations in B cell development of Sf mice are due to a B cell-intrinsic function of Foxp3, we tracked B cells in peripheral lymphoid organs of BAC-Foxp3Cre-GFP × R26-Y mice that exhibit YFP expression in cells only after Foxp3-dependent Cre recombinase activity and resultant excision of a loxP-flanked STOP cassette (Zhou et al., 2008). Flow cytometric analysis revealed a minute population of CD19+ B cells (≤0.1%) in spleen and LNs of BAC-Foxp3Cre-GFP × R26-Y mice that appeared to co-express YFP (Figure 2A, left). However, post-sort analysis of FACS-purified cells established that this putative CD19+YFP+ population mainly consisted of doublets of CD19+YFP− and CD19−YFP+ cells, demonstrating that the potential population size of CD19+ cells that are truly YFP+ is negligible (spleen: ≤0.003%; LNs: ≤0.008%; Figure 2A, right).


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)

Mechanisms of B cell developmental defects in adolescent Sf mice. (A) Genetic lineage tracing of Foxp3 reporter activity in peripheral B cells. Putative populations of CD19+YFP+ cells were FACS-purified from spleen (top) and pooled mLNs and scLNs (bottom) of adult BAC-Foxp3Cre-GFP × R26-Y mice. Flow cytometric presort analysis (left) and post-sort analysis (right) is depicted. Note that for presort analysis numbers in dot plots indicate percentages of cells within the respective gates, whereas for post-sort analysis numbers indicate percentages of CD19+YFP+ cells among total cell populations prior to FACS purification. (B) Differentiation capacity of Pro/Pre-B-I cells from moribund Sf mice in vitro. Initially sIgM−, B220+c-kit+ Pro/Pre-B-I cells, either freshly isolated from BM of 3-week-old WT (blue circles) or Sf (red squares) mice (left), or isolated from IL-7-supplemented cultures after proliferative expansion of respective Pro/Pre-B-I cell populations (right), were cultured in the absence of added IL-7. The capacity to differentiate into sIgM+ cells was assessed at different time points after initiation of cultures, as indicated. Symbols represent mean values of replicate wells ± SEM. (C) B cell development in Sf mice that lack CD4+ and CD8+ T cells. Flow cytometry of indicated B cell developmental stages in the BM of 4-week-old WT (top) and Sf (bottom) mice that additionally lacked Tcra gene expression (TCRα−/−). Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of three independent experiments. (D) Flow cytometric analysis of immature B220lowsIgM+ and mature/recirculating B220highsIgM+ B cell compartments in the BM of 4-week-old Sf mice that were either proficient (IL-6+/−, top) or deficient (IL-6−/−, bottom) for IL-6. Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of two independent experiments. (E) Impact of inflammatory cytokines on IL-7-mediated survival of Pro/Pre-B-I cells in vitro. FACS-purified populations of BM-derived B220+c-kit+ Pro/Pre-B-I cells from WT mice were cultured with IL-7, either alone or in the presence of titrating amounts of IL-17A or IFN-γ, as indicated. At day 4 of cultures, numbers of B220+c-kit+ cells recovered from cultures with and without added inflammatory cytokines was calculated as fold difference. Shown are mean and range of duplicate wells. Data are representative of two independent experiments. (F,G) The expression of mRNAs encoding (F) indicated inflammatory cytokines and (G) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing whole tissues harvested from 4-week-old WT (blue) or Sf (red) mice, as indicated. Note that, prior to RNA extraction, prepared single cell suspensions of respective tissues were pooled from three mice per group. HPRT was used for normalization. Shown are mean and range of duplicate samples. Data are representative of at least three independent experiments.
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Figure 2: Mechanisms of B cell developmental defects in adolescent Sf mice. (A) Genetic lineage tracing of Foxp3 reporter activity in peripheral B cells. Putative populations of CD19+YFP+ cells were FACS-purified from spleen (top) and pooled mLNs and scLNs (bottom) of adult BAC-Foxp3Cre-GFP × R26-Y mice. Flow cytometric presort analysis (left) and post-sort analysis (right) is depicted. Note that for presort analysis numbers in dot plots indicate percentages of cells within the respective gates, whereas for post-sort analysis numbers indicate percentages of CD19+YFP+ cells among total cell populations prior to FACS purification. (B) Differentiation capacity of Pro/Pre-B-I cells from moribund Sf mice in vitro. Initially sIgM−, B220+c-kit+ Pro/Pre-B-I cells, either freshly isolated from BM of 3-week-old WT (blue circles) or Sf (red squares) mice (left), or isolated from IL-7-supplemented cultures after proliferative expansion of respective Pro/Pre-B-I cell populations (right), were cultured in the absence of added IL-7. The capacity to differentiate into sIgM+ cells was assessed at different time points after initiation of cultures, as indicated. Symbols represent mean values of replicate wells ± SEM. (C) B cell development in Sf mice that lack CD4+ and CD8+ T cells. Flow cytometry of indicated B cell developmental stages in the BM of 4-week-old WT (top) and Sf (bottom) mice that additionally lacked Tcra gene expression (TCRα−/−). Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of three independent experiments. (D) Flow cytometric analysis of immature B220lowsIgM+ and mature/recirculating B220highsIgM+ B cell compartments in the BM of 4-week-old Sf mice that were either proficient (IL-6+/−, top) or deficient (IL-6−/−, bottom) for IL-6. Numbers in dot plots indicate the percentage of cells in the respective gate. Data are representative of two independent experiments. (E) Impact of inflammatory cytokines on IL-7-mediated survival of Pro/Pre-B-I cells in vitro. FACS-purified populations of BM-derived B220+c-kit+ Pro/Pre-B-I cells from WT mice were cultured with IL-7, either alone or in the presence of titrating amounts of IL-17A or IFN-γ, as indicated. At day 4 of cultures, numbers of B220+c-kit+ cells recovered from cultures with and without added inflammatory cytokines was calculated as fold difference. Shown are mean and range of duplicate wells. Data are representative of two independent experiments. (F,G) The expression of mRNAs encoding (F) indicated inflammatory cytokines and (G) IL-7 and CXCL12 was assessed by quantitative RT-PCR employing whole tissues harvested from 4-week-old WT (blue) or Sf (red) mice, as indicated. Note that, prior to RNA extraction, prepared single cell suspensions of respective tissues were pooled from three mice per group. HPRT was used for normalization. Shown are mean and range of duplicate samples. Data are representative of at least three independent experiments.
Mentions: Previous studies employing mice with Foxp3-dependent fluorochrome reporter activity indicated that high levels of Foxp3 expression are limited to αβ T cells (Fontenot et al., 2005; Wan and Flavell, 2005), with undetectable fluorochrome expression in B220+ cells from BM (Leonardo et al., 2010) and secondary lymphoid organs such as LNs (Wan and Flavell, 2005) and spleen (Leonardo et al., 2010). Whether Foxp3 can be expressed in other hematopoietic (Bernard et al., 2009; Manrique et al., 2011) and non-hematopoietic (Chang et al., 2005; Hinz et al., 2007; Zuo et al., 2007; Chen et al., 2008; Karanikas et al., 2008; Chung et al., 2010) lineages has been actively debated (Liston et al., 2007; Mayer et al., 2012). To formally exclude the possibility that observed alterations in B cell development of Sf mice are due to a B cell-intrinsic function of Foxp3, we tracked B cells in peripheral lymphoid organs of BAC-Foxp3Cre-GFP × R26-Y mice that exhibit YFP expression in cells only after Foxp3-dependent Cre recombinase activity and resultant excision of a loxP-flanked STOP cassette (Zhou et al., 2008). Flow cytometric analysis revealed a minute population of CD19+ B cells (≤0.1%) in spleen and LNs of BAC-Foxp3Cre-GFP × R26-Y mice that appeared to co-express YFP (Figure 2A, left). However, post-sort analysis of FACS-purified cells established that this putative CD19+YFP+ population mainly consisted of doublets of CD19+YFP− and CD19−YFP+ cells, demonstrating that the potential population size of CD19+ cells that are truly YFP+ is negligible (spleen: ≤0.003%; LNs: ≤0.008%; Figure 2A, right).

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