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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

Phenotypical characterization of expanded Treg.Treg and Tconv were expanded according the schedule in Figure 1 and rested for two days. Cell surface expression of CD25, CD127, CD27, CD70 and CD62L, and intracellular expression of FoxP3 was analyzed on Treg (black filled histograms) or Tconv (grey line histogram). Data are representative of four to seven independent experiments.
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pone-0002233-g008: Phenotypical characterization of expanded Treg.Treg and Tconv were expanded according the schedule in Figure 1 and rested for two days. Cell surface expression of CD25, CD127, CD27, CD70 and CD62L, and intracellular expression of FoxP3 was analyzed on Treg (black filled histograms) or Tconv (grey line histogram). Data are representative of four to seven independent experiments.

Mentions: A potential risk of Treg expansion is the outgrowth of contaminating cell types such as CD8pos T cells or NK-cells. In our experiments, we did not find major contaminations, since typically >90% of the expanded cells were CD4pos T cells. The majority of Treg expanded with either strategy was CD25high, and CD127neg (Figure 8). The majority of Treg retained expression of FoxP3pos after two cycles of expansion. Tconv cells were largely FoxP3neg. Previously, we and others have shown that Treg constitutively expressing CD27 are stronger suppressors [17], [27]. Typically, after expansion, a portion of Treg retained a CD27pos phenotype, while the remaining Treg shifted towards a CD27neg phenotype. Treg expanded with two cycles of polyclonal stimulation repeatedly showed the highest percentage of CD27pos cells (50 to 80%), while cells expanded with two subsequent alloantigen stimulation cycles had the lowest percentage of CD27pos cells (10 to 40%). The two strategies with alternated alloantigen and polyclonal stimulation cycles showed intermediate percentages of CD27pos cells. The expression of CD70, the ligand for CD27, was reversely correlated with CD27 expression. CD62L is also described as a marker for Treg with high suppressive potency [24]. Whereas expression of CD62L was lost on expanded Tconv cells, a substantial portion of Treg retained expression of this marker.


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)

Phenotypical characterization of expanded Treg.Treg and Tconv were expanded according the schedule in Figure 1 and rested for two days. Cell surface expression of CD25, CD127, CD27, CD70 and CD62L, and intracellular expression of FoxP3 was analyzed on Treg (black filled histograms) or Tconv (grey line histogram). Data are representative of four to seven independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002233-g008: Phenotypical characterization of expanded Treg.Treg and Tconv were expanded according the schedule in Figure 1 and rested for two days. Cell surface expression of CD25, CD127, CD27, CD70 and CD62L, and intracellular expression of FoxP3 was analyzed on Treg (black filled histograms) or Tconv (grey line histogram). Data are representative of four to seven independent experiments.
Mentions: A potential risk of Treg expansion is the outgrowth of contaminating cell types such as CD8pos T cells or NK-cells. In our experiments, we did not find major contaminations, since typically >90% of the expanded cells were CD4pos T cells. The majority of Treg expanded with either strategy was CD25high, and CD127neg (Figure 8). The majority of Treg retained expression of FoxP3pos after two cycles of expansion. Tconv cells were largely FoxP3neg. Previously, we and others have shown that Treg constitutively expressing CD27 are stronger suppressors [17], [27]. Typically, after expansion, a portion of Treg retained a CD27pos phenotype, while the remaining Treg shifted towards a CD27neg phenotype. Treg expanded with two cycles of polyclonal stimulation repeatedly showed the highest percentage of CD27pos cells (50 to 80%), while cells expanded with two subsequent alloantigen stimulation cycles had the lowest percentage of CD27pos cells (10 to 40%). The two strategies with alternated alloantigen and polyclonal stimulation cycles showed intermediate percentages of CD27pos cells. The expression of CD70, the ligand for CD27, was reversely correlated with CD27 expression. CD62L is also described as a marker for Treg with high suppressive potency [24]. Whereas expression of CD62L was lost on expanded Tconv cells, a substantial portion of Treg retained expression of this marker.

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