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Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(pos)CD25(high) T cells for immunotherapy.

Peters JH, Hilbrands LB, Koenen HJ, Joosten I - PLoS ONE (2008)

Bottom Line: Efficient increases in cell numbers were obtained.Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent.This approach is of particular interest for recipients of HLA mismatched transplants.

View Article: PubMed Central - PubMed

Affiliation: Department of Bloodtransfusion and Transplantation Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

ABSTRACT

Background: Regulatory T cell (Treg) based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy.

Methodology/principal findings: Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4(pos)CD25(high) Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent.

Conclusions/significance: The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.

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Related in: MedlinePlus

Determination of optimal length of primary and secondary Treg expansion cycles.(A) Optimal length of primary Treg expansion cycles. Treg were stimulated with CFSE-labeled alloantigen (solid line, solid circle) or polyclonal (dotted line, open circle) stimulus in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. (B) Optimal length of secondary Treg expansion cycles. Treg were primed with alloantigen (solid lines) or polyclonal (dotted lines) stimulation as indicated in the presence of IL-2 and IL-15 and rested for 2 days prior to restimulation. Cells were restimulated with CFSE-labeled alloantigen (solid circles) or polyclonal stimulus (open circles) in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. Data are representative of three independent experiments.
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pone-0002233-g006: Determination of optimal length of primary and secondary Treg expansion cycles.(A) Optimal length of primary Treg expansion cycles. Treg were stimulated with CFSE-labeled alloantigen (solid line, solid circle) or polyclonal (dotted line, open circle) stimulus in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. (B) Optimal length of secondary Treg expansion cycles. Treg were primed with alloantigen (solid lines) or polyclonal (dotted lines) stimulation as indicated in the presence of IL-2 and IL-15 and rested for 2 days prior to restimulation. Cells were restimulated with CFSE-labeled alloantigen (solid circles) or polyclonal stimulus (open circles) in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. Data are representative of three independent experiments.

Mentions: To determine the optimal cycle length, cell numbers were counted at several time points during primary and secondary expansion cycles. Expansion values were calculated and are depicted in Figure 6. In primary Treg expansion cycles with either alloantigen or polyclonal stimulation, cultures reached maximal cell numbers after 10–12 days. In secondary expansion cycles with alloantigen stimulation, peak cell numbers were found at day 10. In secondary expansion cycles with polyclonal stimulation, peak cell numbers were found at day 10 if cells had been primed with alloantigen. However, when Treg had been primed with polyclonal stimulation, a plateau in cell numbers in a secondary polyclonal stimulation cycle was reached around day 7. Since our prime interest was to obtain alloantigen-specific Treg, the strategy comprising two subsequent cycles with polyclonal stimulation actually served as a control. Consequently, we standardized the expansion cycle length at 10 days for all strategies.


Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(pos)CD25(high) T cells for immunotherapy.

Peters JH, Hilbrands LB, Koenen HJ, Joosten I - PLoS ONE (2008)

Determination of optimal length of primary and secondary Treg expansion cycles.(A) Optimal length of primary Treg expansion cycles. Treg were stimulated with CFSE-labeled alloantigen (solid line, solid circle) or polyclonal (dotted line, open circle) stimulus in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. (B) Optimal length of secondary Treg expansion cycles. Treg were primed with alloantigen (solid lines) or polyclonal (dotted lines) stimulation as indicated in the presence of IL-2 and IL-15 and rested for 2 days prior to restimulation. Cells were restimulated with CFSE-labeled alloantigen (solid circles) or polyclonal stimulus (open circles) in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376059&req=5

pone-0002233-g006: Determination of optimal length of primary and secondary Treg expansion cycles.(A) Optimal length of primary Treg expansion cycles. Treg were stimulated with CFSE-labeled alloantigen (solid line, solid circle) or polyclonal (dotted line, open circle) stimulus in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. (B) Optimal length of secondary Treg expansion cycles. Treg were primed with alloantigen (solid lines) or polyclonal (dotted lines) stimulation as indicated in the presence of IL-2 and IL-15 and rested for 2 days prior to restimulation. Cells were restimulated with CFSE-labeled alloantigen (solid circles) or polyclonal stimulus (open circles) in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSEpos allogeneic stimulator cells) and related to Treg numbers at initial set up. Data are representative of three independent experiments.
Mentions: To determine the optimal cycle length, cell numbers were counted at several time points during primary and secondary expansion cycles. Expansion values were calculated and are depicted in Figure 6. In primary Treg expansion cycles with either alloantigen or polyclonal stimulation, cultures reached maximal cell numbers after 10–12 days. In secondary expansion cycles with alloantigen stimulation, peak cell numbers were found at day 10. In secondary expansion cycles with polyclonal stimulation, peak cell numbers were found at day 10 if cells had been primed with alloantigen. However, when Treg had been primed with polyclonal stimulation, a plateau in cell numbers in a secondary polyclonal stimulation cycle was reached around day 7. Since our prime interest was to obtain alloantigen-specific Treg, the strategy comprising two subsequent cycles with polyclonal stimulation actually served as a control. Consequently, we standardized the expansion cycle length at 10 days for all strategies.

Bottom Line: Efficient increases in cell numbers were obtained.Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent.This approach is of particular interest for recipients of HLA mismatched transplants.

View Article: PubMed Central - PubMed

Affiliation: Department of Bloodtransfusion and Transplantation Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

ABSTRACT

Background: Regulatory T cell (Treg) based immunotherapy is a potential treatment for several immune disorders. By now, this approach proved successful in preclinical animal transplantation and auto-immunity models. In these models the success of Treg based immunotherapy crucially depends on the antigen-specificity of the infused Treg population. For the human setting, information is lacking on how to generate Treg with direct antigen-specificity ex vivo to be used for immunotherapy.

Methodology/principal findings: Here, we demonstrate that in as little as two stimulation cycles with HLA mismatched allogeneic stimulator cells and T cell growth factors a very high degree of alloantigen-specificity was reached in magnetic bead isolated human CD4(pos)CD25(high) Treg. Efficient increases in cell numbers were obtained. Primary allogeneic stimulation appeared a prerequisite in the generation of alloantigen-specific Treg, while secondary allogeneic or polyclonal stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies enriched alloantigen-specificity and cell yield to a similar extent.

Conclusions/significance: The ex vivo expansion protocol that we describe will very likely increase the success of clinical Treg-based immunotherapy, and will help to induce tolerance to selected antigens, while minimizing general immune suppression. This approach is of particular interest for recipients of HLA mismatched transplants.

Show MeSH
Related in: MedlinePlus