Limits...
Ex Vivo Expanded Autologous Polyclonal Regulatory T Cells Suppress Inhibitor Formation in Hemophilia.

Sarkar D, Biswas M, Liao G, Seay HR, Perrin GQ, Markusic DM, Hoffman BE, Brusko TM, Terhorst C, Herzog RW - Mol Ther Methods Clin Dev (2014)

Bottom Line: Although transplanted Treg became undetectable within two weeks, suppression persisted for >2 months.Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg.The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT
Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft vs host disease in bone marrow transplantation. Here we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e. hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express GFP-marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 80-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within two weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

No MeSH data available.


Related in: MedlinePlus

Isolation and ex vivo expansion of murine CD4+CD25+FoxP3+ Treg. (a) Splenocytes are isolated from BALB/c mice with a GFP reporter gene knock-in at FoxP3 locus. GFP+ cells are sorted to 95–98% purity from magnetically purified CD4+ splenocytes. (b) Expansion of Treg (starting with 1 × 106 cells) after two rounds of stimulation with anti-CD3/-CD28 beads. Data are average ±SD from 5 independent experiments. (c) Percent GFP+ (i.e., FoxP3 expressing) cells as a function of days in culture.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4213815&req=5

fig1: Isolation and ex vivo expansion of murine CD4+CD25+FoxP3+ Treg. (a) Splenocytes are isolated from BALB/c mice with a GFP reporter gene knock-in at FoxP3 locus. GFP+ cells are sorted to 95–98% purity from magnetically purified CD4+ splenocytes. (b) Expansion of Treg (starting with 1 × 106 cells) after two rounds of stimulation with anti-CD3/-CD28 beads. Data are average ±SD from 5 independent experiments. (c) Percent GFP+ (i.e., FoxP3 expressing) cells as a function of days in culture.

Mentions: In contrast to the advanced protocols and reagents available for the in vitro expansion of human Treg, such approaches for the expansion of high purity murine Treg (to avoid contamination and outgrowth of effector T cells) are lacking. To develop a murine model of Treg therapy we used a BALB/c strain with a green fluorescent protein (GFP) reporter, expressed in conjunction with transcription factor FoxP3 (FoxP3-IRES-GFP+ BALB/c), to facilitate enrichment of a highly pure Treg population. Indeed, magnetic pre-enrichment for splenic CD4+ T cells from these FoxP3-IRES-GFP+ BALB/c mice followed by flow cytometry sorting of GFP+ (FoxP3+) cells resulted in Treg preparations with 95–99% purity upon post-sort analysis (Figure 1a). We were able to expand these Treg 7- to 10-fold after a first stimulation with anti-CD3/CD28 coated microbeads, with a second restimulation yielding an overall 50- to 100-fold expansion (Figure 1b). The second stimulation with anti-CD3/-CD28 beads was typically performed on day 7, but was varied by ±1 day depending on cell size and activation state of Treg. Proliferating cells typically assumed an irregular morphology and were larger in size. Cells were restimulated once they returned to their prestimulation size and when proliferation caused a substantial increase in the ratio of cells to beads.


Ex Vivo Expanded Autologous Polyclonal Regulatory T Cells Suppress Inhibitor Formation in Hemophilia.

Sarkar D, Biswas M, Liao G, Seay HR, Perrin GQ, Markusic DM, Hoffman BE, Brusko TM, Terhorst C, Herzog RW - Mol Ther Methods Clin Dev (2014)

Isolation and ex vivo expansion of murine CD4+CD25+FoxP3+ Treg. (a) Splenocytes are isolated from BALB/c mice with a GFP reporter gene knock-in at FoxP3 locus. GFP+ cells are sorted to 95–98% purity from magnetically purified CD4+ splenocytes. (b) Expansion of Treg (starting with 1 × 106 cells) after two rounds of stimulation with anti-CD3/-CD28 beads. Data are average ±SD from 5 independent experiments. (c) Percent GFP+ (i.e., FoxP3 expressing) cells as a function of days in culture.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4213815&req=5

fig1: Isolation and ex vivo expansion of murine CD4+CD25+FoxP3+ Treg. (a) Splenocytes are isolated from BALB/c mice with a GFP reporter gene knock-in at FoxP3 locus. GFP+ cells are sorted to 95–98% purity from magnetically purified CD4+ splenocytes. (b) Expansion of Treg (starting with 1 × 106 cells) after two rounds of stimulation with anti-CD3/-CD28 beads. Data are average ±SD from 5 independent experiments. (c) Percent GFP+ (i.e., FoxP3 expressing) cells as a function of days in culture.
Mentions: In contrast to the advanced protocols and reagents available for the in vitro expansion of human Treg, such approaches for the expansion of high purity murine Treg (to avoid contamination and outgrowth of effector T cells) are lacking. To develop a murine model of Treg therapy we used a BALB/c strain with a green fluorescent protein (GFP) reporter, expressed in conjunction with transcription factor FoxP3 (FoxP3-IRES-GFP+ BALB/c), to facilitate enrichment of a highly pure Treg population. Indeed, magnetic pre-enrichment for splenic CD4+ T cells from these FoxP3-IRES-GFP+ BALB/c mice followed by flow cytometry sorting of GFP+ (FoxP3+) cells resulted in Treg preparations with 95–99% purity upon post-sort analysis (Figure 1a). We were able to expand these Treg 7- to 10-fold after a first stimulation with anti-CD3/CD28 coated microbeads, with a second restimulation yielding an overall 50- to 100-fold expansion (Figure 1b). The second stimulation with anti-CD3/-CD28 beads was typically performed on day 7, but was varied by ±1 day depending on cell size and activation state of Treg. Proliferating cells typically assumed an irregular morphology and were larger in size. Cells were restimulated once they returned to their prestimulation size and when proliferation caused a substantial increase in the ratio of cells to beads.

Bottom Line: Although transplanted Treg became undetectable within two weeks, suppression persisted for >2 months.Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg.The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA.

ABSTRACT
Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft vs host disease in bone marrow transplantation. Here we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e. hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express GFP-marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 80-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within two weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

No MeSH data available.


Related in: MedlinePlus