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Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells.

Kochat V, Kanjirakkuzhiyil S, Baligar P, Nagarajan P, Mukhopadhyay A - Stem Cell Res Ther (2015)

Bottom Line: Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury.A highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin(-) BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells.This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India. veenak@nii.ac.in.

ABSTRACT

Introduction: Cell replacement therapy may be considered as an alternate approach to provide therapeutic dose of plasma factor VIII (FVIII) in patients with hemophilia A (HA). However, immune rejection limits the use of allogeneic cells in this mode of therapy. Here, we have examined the role of donor major histocompatibility complex (MHC)-stimulated host CD4(+)CD25(+) regulatory T (Treg) cells in suppressing immune responses against allogeneic uncommitted (Lin(-)) bone marrow cells (BMCs) for correction of bleeding disorder in HA mice.

Methods: Allogeneic donor Lin(-) BMCs were co-transplanted with allo-antigen sensitized Treg cells in HA mice having acetaminophen-induced acute liver injury. Plasma FVIII activity was determined by in vitro functional assay, and correction of bleeding phenotype was assessed on the basis of capillary blood clotting time and tail-clip challenge. The immunosuppression potential of the sensitized Treg cells on CD4(+) T cells was studied both in vitro and in vivo. Suppression of inflammatory reactions in the liver against the homed donor cells by sensitized Treg cells was analysed by histopathological scoring. Allo-specificity of sensitized Treg cells and long-term retention of immunosuppression were examined against a third-party donor and by secondary challenge of allogeneic donor cells, respectively. The engraftment and phenotype change of donor BMCs in the liver and their role in synthesis of FVIII and liver regeneration were also determined.

Results: Co-transplantation of allogeneic Lin(-) BMCs with sensitized Treg cells led to systemic immune modulation and suppression of inflammatory reactions in the liver, allowing better engraftment of allogeneic cells in the liver. Allo-antigen priming led to allo-specific immune suppression even after 1 year of transplantation. Donor-derived endothelial cells expressed FVIII in HA mice, leading to the correction of bleeding phenotype. Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury.

Conclusions: A highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin(-) BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells. This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.

No MeSH data available.


Related in: MedlinePlus

sTreg cells modulate systemic immune response with allo-specificity in vivo.a Time course analysis of Treg cells by flow cytometry in both HAT-AT and HA-SC mice. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. b CD4+ T cells in spleen of HAT-AT and HAT-A mice were analysed by flow cytometry. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. c Allo-specific activation of sTreg cells. T-cell activation in response to allogeneic Lin− BMCs of FVB-GFP and Balb/c mice were compared. Representative dot-plots show the levels of CD4+CD44+ T cells after 48 h of transplantation (left panel), and statistical analysis is shown in the right panel (n = 3). *P < 0.05, **P < 0.01. BMC bone marrow cell, HA-SC sham control mice in which liver was damaged but neither Lin− bone marrow cells nor sensitized regulatory T cells were transplanted, HAT-A hemophilia A mice transplanted with allogeneic cells, HAT-AT hemophilia A mice transplanted with allogeneic and regulatory T cells, n number of mice in each experiment, nTreg naïve regulatory T, sTreg sensitized regulatory T, Treg regulatory T
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Fig3: sTreg cells modulate systemic immune response with allo-specificity in vivo.a Time course analysis of Treg cells by flow cytometry in both HAT-AT and HA-SC mice. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. b CD4+ T cells in spleen of HAT-AT and HAT-A mice were analysed by flow cytometry. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. c Allo-specific activation of sTreg cells. T-cell activation in response to allogeneic Lin− BMCs of FVB-GFP and Balb/c mice were compared. Representative dot-plots show the levels of CD4+CD44+ T cells after 48 h of transplantation (left panel), and statistical analysis is shown in the right panel (n = 3). *P < 0.05, **P < 0.01. BMC bone marrow cell, HA-SC sham control mice in which liver was damaged but neither Lin− bone marrow cells nor sensitized regulatory T cells were transplanted, HAT-A hemophilia A mice transplanted with allogeneic cells, HAT-AT hemophilia A mice transplanted with allogeneic and regulatory T cells, n number of mice in each experiment, nTreg naïve regulatory T, sTreg sensitized regulatory T, Treg regulatory T

Mentions: Time course analysis of CD4+CD25+Foxp3+ Treg cells in the spleen of HAT-AT and HA-SC (sham control mice in which liver was damaged but neither Lin− BMCs nor sTreg cells were transplanted) mice showed a significant increase in the percentage of Treg cells in the HAT-AT group in comparison to the HAT-SC group of mice within the first 2 months of transplantation, and the percentages later became comparable (Fig. 3a). Furthermore, the percentage of splenic CD4+ T cells in HAT-A mice was 31.9 ± 3.62 %, which significantly (P < 0.01) decreased to 17.13 ± 1.15 % in HAT-AT within 10 days of co-transplantation with sTreg cells. Interestingly, this suppressive effect was continued throughout the course of the study (Fig. 3b). The above results suggest that effective immune suppression and development of tolerance are attributed to sTreg cells.Fig. 3


Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells.

Kochat V, Kanjirakkuzhiyil S, Baligar P, Nagarajan P, Mukhopadhyay A - Stem Cell Res Ther (2015)

sTreg cells modulate systemic immune response with allo-specificity in vivo.a Time course analysis of Treg cells by flow cytometry in both HAT-AT and HA-SC mice. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. b CD4+ T cells in spleen of HAT-AT and HAT-A mice were analysed by flow cytometry. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. c Allo-specific activation of sTreg cells. T-cell activation in response to allogeneic Lin− BMCs of FVB-GFP and Balb/c mice were compared. Representative dot-plots show the levels of CD4+CD44+ T cells after 48 h of transplantation (left panel), and statistical analysis is shown in the right panel (n = 3). *P < 0.05, **P < 0.01. BMC bone marrow cell, HA-SC sham control mice in which liver was damaged but neither Lin− bone marrow cells nor sensitized regulatory T cells were transplanted, HAT-A hemophilia A mice transplanted with allogeneic cells, HAT-AT hemophilia A mice transplanted with allogeneic and regulatory T cells, n number of mice in each experiment, nTreg naïve regulatory T, sTreg sensitized regulatory T, Treg regulatory T
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4513683&req=5

Fig3: sTreg cells modulate systemic immune response with allo-specificity in vivo.a Time course analysis of Treg cells by flow cytometry in both HAT-AT and HA-SC mice. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. b CD4+ T cells in spleen of HAT-AT and HAT-A mice were analysed by flow cytometry. Representative dot-plots are shown in the left panel, and statistical analysis in the right panel. c Allo-specific activation of sTreg cells. T-cell activation in response to allogeneic Lin− BMCs of FVB-GFP and Balb/c mice were compared. Representative dot-plots show the levels of CD4+CD44+ T cells after 48 h of transplantation (left panel), and statistical analysis is shown in the right panel (n = 3). *P < 0.05, **P < 0.01. BMC bone marrow cell, HA-SC sham control mice in which liver was damaged but neither Lin− bone marrow cells nor sensitized regulatory T cells were transplanted, HAT-A hemophilia A mice transplanted with allogeneic cells, HAT-AT hemophilia A mice transplanted with allogeneic and regulatory T cells, n number of mice in each experiment, nTreg naïve regulatory T, sTreg sensitized regulatory T, Treg regulatory T
Mentions: Time course analysis of CD4+CD25+Foxp3+ Treg cells in the spleen of HAT-AT and HA-SC (sham control mice in which liver was damaged but neither Lin− BMCs nor sTreg cells were transplanted) mice showed a significant increase in the percentage of Treg cells in the HAT-AT group in comparison to the HAT-SC group of mice within the first 2 months of transplantation, and the percentages later became comparable (Fig. 3a). Furthermore, the percentage of splenic CD4+ T cells in HAT-A mice was 31.9 ± 3.62 %, which significantly (P < 0.01) decreased to 17.13 ± 1.15 % in HAT-AT within 10 days of co-transplantation with sTreg cells. Interestingly, this suppressive effect was continued throughout the course of the study (Fig. 3b). The above results suggest that effective immune suppression and development of tolerance are attributed to sTreg cells.Fig. 3

Bottom Line: Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury.A highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin(-) BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells.This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India. veenak@nii.ac.in.

ABSTRACT

Introduction: Cell replacement therapy may be considered as an alternate approach to provide therapeutic dose of plasma factor VIII (FVIII) in patients with hemophilia A (HA). However, immune rejection limits the use of allogeneic cells in this mode of therapy. Here, we have examined the role of donor major histocompatibility complex (MHC)-stimulated host CD4(+)CD25(+) regulatory T (Treg) cells in suppressing immune responses against allogeneic uncommitted (Lin(-)) bone marrow cells (BMCs) for correction of bleeding disorder in HA mice.

Methods: Allogeneic donor Lin(-) BMCs were co-transplanted with allo-antigen sensitized Treg cells in HA mice having acetaminophen-induced acute liver injury. Plasma FVIII activity was determined by in vitro functional assay, and correction of bleeding phenotype was assessed on the basis of capillary blood clotting time and tail-clip challenge. The immunosuppression potential of the sensitized Treg cells on CD4(+) T cells was studied both in vitro and in vivo. Suppression of inflammatory reactions in the liver against the homed donor cells by sensitized Treg cells was analysed by histopathological scoring. Allo-specificity of sensitized Treg cells and long-term retention of immunosuppression were examined against a third-party donor and by secondary challenge of allogeneic donor cells, respectively. The engraftment and phenotype change of donor BMCs in the liver and their role in synthesis of FVIII and liver regeneration were also determined.

Results: Co-transplantation of allogeneic Lin(-) BMCs with sensitized Treg cells led to systemic immune modulation and suppression of inflammatory reactions in the liver, allowing better engraftment of allogeneic cells in the liver. Allo-antigen priming led to allo-specific immune suppression even after 1 year of transplantation. Donor-derived endothelial cells expressed FVIII in HA mice, leading to the correction of bleeding phenotype. Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury.

Conclusions: A highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin(-) BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells. This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.

No MeSH data available.


Related in: MedlinePlus