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

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.

Fig. 1

Co-transplantation of sT_reg cells and Lin⁻ BMCs facilitates therapeutic correction in HA mice. a Scheme of the experiment. Lin⁻ BMCs (0.25 × 10⁶ cells) from female FVB-GFP mice (H2K^q) were transplanted into HA mice (H2K^b) through the tail vein within 30 h of acute liver injury, and the recipient mice were grouped as HAT-A. The T_reg cells from HA mice were primed against donor MHC antigens by co-culturing with splenic DCs of FVB mice for 48 h to generate allo-antigen sensitized T_reg cells (sT_reg cells). To establish transplant tolerance, allogeneic Lin⁻ BMCs and sT_reg cells were co-transplanted into HA mice, and the recipient mice were grouped as HAT-AT. b Plasma FVIII activity was measured by in vitro coagulation assay performed by using a Coatest assay kit. FVIII activity as percentage of normal plasma in wild-type mice is shown. There was no significant increase in FVIII activity in HAT-A mice but the activity was much higher in HAT-AT mice (P < 0.0001, HAT-A versus HAT-AT mice) after 3 and 6 months of transplantation. c Capillary blood clotting time. WT, HA, HAT-A, and HAT-AT mice were subjected to the assay after 3 months of transplantation. d Tail-clip challenge test. Survival of mice after 24 h of tail clip was determined, and HAT-AT group showed no mortality. BM bone marrow, BMC bone marrow cell, DC dendritic cell, FVIII K/O factor VIII knockout, HA hemophilia A, 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, sT _reg sensitized regulatory T, WT wild-type

Fig. 2

In vitro functional characterization of sT_reg cells. a T-cell suppression (CFSE dilution) assay. DCs (1 × 10⁵ cells) of FVB/J mice and 1 × 10⁵ CFSE-labeled CD4⁺CD25⁻ T cells (HA mice) were added to set up T-cell suppression assay. The nT_reg or sT_reg cells were added in each well at different ratios of T cells and cultured for 5 days. The proliferation of T cells in mixed lymphocyte reaction was measured in terms of dilution of CFSE intensity as shown in the representative histograms. b A significant (*P = 0.0142) difference in suppression of T-cell proliferation between two groups of T_reg cells was noticed at 0.1:1, 0.25:1, 0.5:1, and 1:1 ratios of the regulatory to effector T cells. c IL-10 secretion in the culture supernatants (T_reg cells: T cells = 1:1) was measured by enzyme-linked immunosorbent assay. A significant increase in IL-10 levels was observed in cultures with sT_reg cells (P = 0.04). CFSE carboxyfluorescein succinimidyl ester, DC dendritic cell, HA hemophilia A, IL-10 interleukin-10, n number of experiments, nT _reg naïve regulatory T, sT _reg sensitized regulatory T, T _reg regulatory T (***) P < 0.0001, (*) P < 0.05

Fig. 3

sT_reg cells modulate systemic immune response with allo-specificity in vivo. a Time course analysis of T_reg 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 sT_reg 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, nT _reg naïve regulatory T, sT _reg sensitized regulatory T, T _reg regulatory T

Fig. 4

Sensitized regulatory T cells induce suppression of inflammatory responses in the liver. a Splenomagaly in HAT-A (1) mice in comparison with HAT-AT (2) and HA (3) mice. b Gross macroscopic abnormalities in mice. Mice with anomalies in various organs were counted, and percentage of mice with each anomaly was calculated. Most of the HAT-A mice showed abnormalities with respect to two to three indicators, whereas more than 90 % of the HAT-AT mice were normal. c Inflammation in the liver by histopathological analysis of HAT-A and HAT-AT mice after 120 days of transplantation. Representative images are shown for scoring of various inflammatory parameters: (1) portal inflammation, (2) periportal necro-inflammation, (3) endotheliasis, (4) necrosis, (5) sinusoidal lymphocyte infiltration, and (6) lobular necro-inflammation. Arrow indicated different immunoreactions with respect to the said parameters (scale of 100 μm; 200× magnification). d Comparative inflammatory scoring on the basis of histopathological analysis of the liver at different time intervals after transplantation in HAT-A and HAT-AT groups (*P < 0.05, **P < 0.01). 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 time point

Fig. 5

Long-term retention of transplant tolerance. a Migration of regulatory T cells to the liver. Representative photomicrographs show Foxp3-expressing cells (left panel, arrows) after 3 months of transplantation (scale of 100 μm; 400× magnification). Statistical analysis is shown in the right panel. b After 1 year of primary transplantation, a second dose of allogeneic 0.25 × 10⁶ Lin⁻ BMCs from FVB-GFP cells was given to HAT-A and HAT-AT mice after liver injury was inflicted. Representative photomicrographs show extensive immune reactions in HAT-A mice (scale of 100 μm; 200× magnification). Statistical analysis of inflammatory scores determined on the basis of histo-pathological analysis of liver is shown in the right panel. BMC bone marrow cell, 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 (***) P < 0.0001, (*) P < 0.05

Fig. 6

FVIII synthesis by donor-derived endothelial cells and Kupffer cells in HAT-AT liver. a Donor-derived endothelial cells indicated by white arrow (anti-GFP/donkey anti-mouse Alexa fluor 488 and anti-CD31/donkey anti-rat Alexa fluor 594) in HAT-AT liver (scale of 10 μm; 630 × 2.8 zoomed magnification). b Donor-derived Kupffer cells indicated by white arrow (anti-GFP/donkey anti-mouse Alexa fluor 488 and anti-F4/80/donkey anti-rat Alexa fluor 594) in HAT-AT liver (scale of 10 μm; 630 × 2.8 zoomed magnification). c Flow cytometric analysis of donor cells in non-parenchymal fraction of HAT-AT mouse liver after 1 month of transplantation. Representative dot-plots are given in the left panel, and quantitative analysis is shown in the right panel (n = 3). d FVIII(lc) gene expression in livers of HAT-AT, HA, and WT mice. e Immunocytochemical analysis of FVIII in endothelial and Kupffer cells from HA, WT, and HAT-AT mice (scale of 100 μm; 600× magnification). Relative cell fluorescence intensities are shown in the bar diagram on right. f Highly magnified confocal images (scale of 10 μm; 630× 2.8 zoomed magnification) show that donor-derived endothelial cells express FVIII indicated by white arrow (anti-GFP/donkey anti-mouse Alexafluor 488, anti-CD31/donkey anti-rat Alexafluor 594, anti-FVIII(lc)/donkey anti-rabbit Alexafluor 647). BMD bone marrow derived, DAPI 4′,6-diamidino-2-phenylindole, EC endothelial cell, GFP green fluorescent protein, FVIII factor VIII, FVIII(lc) factor VIII light chain, HA hemophilia A, HAT-AT hemophilia A mice transplanted with allogeneic and regulatory T cells, KC Kupffer cell, n number of mice in each experiment, NPC non-parenchymal cell, WT wild-type

Fig. 7

Engraftment and regeneration of liver by donor-derived hepatocytes. a Representative photomicrographs show immunohistochemistry analysis of liver sections of HAT-A and HAT-AT mice after 4 months of transplantation. Sections were stained with anti-GFP/donkey anti-mouse Alexafluor 488 (scale of 100 μm; 200× magnification). Statistical analysis of GFP⁺ cells (indicated by white arrow) is shown in the right panel. b Immunohistochemical analysis of HAT-AT mouse liver sections after 4 months of transplantation. Representative images (scale of 20 μm; 630× magnification) show donor derived hepatocytes (anti-GFP/donkey anti-mouse Alexa fluor 488 and anti-albumin/donkey anti-goat Alexa fluor 594). c Quantitative analysis of GFP⁺ and GFP⁺albumin⁺ cells in HAT-AT liver (200× magnification) after 45 and 120 days of transplantation (*P = 0.03). d Representative images for Ki67⁺ donor cells in HAT-AT liver after 45 and 120 days of transplantation (scale of 10, 20 μm; 630 × 2.8 magnification). e Analysis of serum AST levels in HAT-AT and HAT-A mice after 45 and 90 days of transplantation. f Analysis of serum ALT levels in HAT-AT and HAT-A mice after 45 and 90 days of transplantation. ALB albumin, ALT alanine aminotransferase, AST aspartate aminotransferase, GFP green fluorescent protein, HAT-A hemophilia A mice transplanted with allogeneic cells, HAT-AT hemophilia A mice transplanted with allogeneic and regulatory T cells, IU international units, n number of mice in each experiment (***) P < 0.0001, (*) P < 0.05