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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 strength of alloantigen Treg stimulation.(A) Treg were stimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 5. Data are representative of three independent experiments. (B) Treg primed with alloantigen were restimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 3. Data are representative of three independent experiments. (C) Efficiency of primary alloantigen stimulated expansion is related to the number of HLA-DRB1 mismatches. Freshly isolated Treg were expanded in one cycle in the presence of IL-2 and IL-15 and stimulation by gamma irradiated allogeneic PBMC with one (N = 10) or two (N = 6) mismatches on HLA class II DRB1 genes. Expansion values were calculated by relating the number of cells set up in the initial culture to the number of cells after expansion (after two days rest). Expansion was higher with alloantigen mismatched on two HLA-DRB1 genes as compared to alloantigen with one mismatch. This difference was found to be statistically significant in a Mann Whitney test (P = 0.025).
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pone-0002233-g003: Determination of optimal strength of alloantigen Treg stimulation.(A) Treg were stimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 5. Data are representative of three independent experiments. (B) Treg primed with alloantigen were restimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 3. Data are representative of three independent experiments. (C) Efficiency of primary alloantigen stimulated expansion is related to the number of HLA-DRB1 mismatches. Freshly isolated Treg were expanded in one cycle in the presence of IL-2 and IL-15 and stimulation by gamma irradiated allogeneic PBMC with one (N = 10) or two (N = 6) mismatches on HLA class II DRB1 genes. Expansion values were calculated by relating the number of cells set up in the initial culture to the number of cells after expansion (after two days rest). Expansion was higher with alloantigen mismatched on two HLA-DRB1 genes as compared to alloantigen with one mismatch. This difference was found to be statistically significant in a Mann Whitney test (P = 0.025).

Mentions: For alloantigen driven expansion, the optimal strength of alloantigen stimulation was determined by titrating irradiated HLA mismatched allogeneic PBMC into Treg cultures, in the presence of exogenous IL-2 and IL-15. In a primary MLR, proliferation was maximal with stimulator∶responder ratios of 4∶1 (Figure 3A). Secondary stimulation with alloantigen showed similar results (Figure 3B). The data shown in Figure 3B were obtained with Treg primed with alloantigen, but similar results were observed when polyclonally primed Treg were used (data not shown). Consequently, for expansion experiments, a stimulator∶responder ratio of 4∶1 was used. Interestingly, a higher number of HLA-DRB1 mismatches between stimulator and responder resulted in higher expansion rates (P<0.05) (Figure 3C). Mismatches at other HLA genes did not significantly correlate with expansion efficiency (data not shown).


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 strength of alloantigen Treg stimulation.(A) Treg were stimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 5. Data are representative of three independent experiments. (B) Treg primed with alloantigen were restimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 3. Data are representative of three independent experiments. (C) Efficiency of primary alloantigen stimulated expansion is related to the number of HLA-DRB1 mismatches. Freshly isolated Treg were expanded in one cycle in the presence of IL-2 and IL-15 and stimulation by gamma irradiated allogeneic PBMC with one (N = 10) or two (N = 6) mismatches on HLA class II DRB1 genes. Expansion values were calculated by relating the number of cells set up in the initial culture to the number of cells after expansion (after two days rest). Expansion was higher with alloantigen mismatched on two HLA-DRB1 genes as compared to alloantigen with one mismatch. This difference was found to be statistically significant in a Mann Whitney test (P = 0.025).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2376059&req=5

pone-0002233-g003: Determination of optimal strength of alloantigen Treg stimulation.(A) Treg were stimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 5. Data are representative of three independent experiments. (B) Treg primed with alloantigen were restimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [3H]Thymidine incorporation at day 3. Data are representative of three independent experiments. (C) Efficiency of primary alloantigen stimulated expansion is related to the number of HLA-DRB1 mismatches. Freshly isolated Treg were expanded in one cycle in the presence of IL-2 and IL-15 and stimulation by gamma irradiated allogeneic PBMC with one (N = 10) or two (N = 6) mismatches on HLA class II DRB1 genes. Expansion values were calculated by relating the number of cells set up in the initial culture to the number of cells after expansion (after two days rest). Expansion was higher with alloantigen mismatched on two HLA-DRB1 genes as compared to alloantigen with one mismatch. This difference was found to be statistically significant in a Mann Whitney test (P = 0.025).
Mentions: For alloantigen driven expansion, the optimal strength of alloantigen stimulation was determined by titrating irradiated HLA mismatched allogeneic PBMC into Treg cultures, in the presence of exogenous IL-2 and IL-15. In a primary MLR, proliferation was maximal with stimulator∶responder ratios of 4∶1 (Figure 3A). Secondary stimulation with alloantigen showed similar results (Figure 3B). The data shown in Figure 3B were obtained with Treg primed with alloantigen, but similar results were observed when polyclonally primed Treg were used (data not shown). Consequently, for expansion experiments, a stimulator∶responder ratio of 4∶1 was used. Interestingly, a higher number of HLA-DRB1 mismatches between stimulator and responder resulted in higher expansion rates (P<0.05) (Figure 3C). Mismatches at other HLA genes did not significantly correlate with expansion efficiency (data not shown).

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