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BDC12-4.1 T-cell receptor transgenic insulin-specific CD4 T cells are resistant to in vitro differentiation into functional Foxp3+ T regulatory cells.

Sarikonda G, Fousteri G, Sachithanantham S, Miller JF, Dave A, Juntti T, Coppieters KT, von Herrath M - PLoS ONE (2014)

Bottom Line: We found that in vitro polarization toward Foxp3(+) iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3.Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4(+) T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice.These results highlight the disconnect between an in vitro acquired Foxp3(+) cell phenotype and its associated in vivo regulatory potential.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

ABSTRACT
The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4(+) T cells of the BDC12-4.1 clone to convert into Foxp3(+) iTreg cells. We found that in vitro polarization toward Foxp3(+) iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3(+) BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4(+) T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3(+) cell phenotype and its associated in vivo regulatory potential.

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BDC12-4.1 TCR transgenic T cells exhibit robust insB:9-23-specific responses.CD4+ T cells purified from splenocytes of BDC12-4.1/RAGKO or 2H6 mice were plated in triplicate, in round-bottom 96-well plates. 5–10×104 T cells per well were incubated with an equal number of APCs for 48 hours. Native InsB:9-23 peptide (open bars) or R22E mimotope ([14]) of the InsB:9-23 peptide (filled bars) were used at the indicated concentration. T cells incubated alone or with only APCs (without added antigen) served to measure spontaneous proliferation. Mean and SEM values for average of the triplicate wells derived from pooled mice (n = 3) is shown.
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pone-0112242-g001: BDC12-4.1 TCR transgenic T cells exhibit robust insB:9-23-specific responses.CD4+ T cells purified from splenocytes of BDC12-4.1/RAGKO or 2H6 mice were plated in triplicate, in round-bottom 96-well plates. 5–10×104 T cells per well were incubated with an equal number of APCs for 48 hours. Native InsB:9-23 peptide (open bars) or R22E mimotope ([14]) of the InsB:9-23 peptide (filled bars) were used at the indicated concentration. T cells incubated alone or with only APCs (without added antigen) served to measure spontaneous proliferation. Mean and SEM values for average of the triplicate wells derived from pooled mice (n = 3) is shown.

Mentions: To confirm the insulin recognition properties of the two InsB:9-23 specific T cells, we assessed their proliferative capacity in response to peptide stimulation. BDC12-4.1 T-cells exhibited robust proliferation in a dose-dependent manner (Fig. 1A). Exposure to R22E mimotope [14] of the InsB:9-23 peptide resulted in an enhanced dose-dependent proliferation. These results confirm the recognition of InsB:9-23 peptide as the cognate antigen for BDC12-4.1 T cells. On the other hand, we could not obtain convincing evidence of insulin-specific responses from 2H6 T cells. Addition of antigen presenting cells (APCs) to 2H6 TCR Tg cells in the absence of antigen resulted in non-specific proliferation. Such proliferation did not increase in magnitude upon the addition of the native InsB:9-23 peptide or the R22E mimotope (Fig. 1B). Further, although 2H6 T cells exhibited Ca+2 mobilization in response to InsB:9-23 stimulation, in some but not all of our experiments, (Fig. S1), they did not bind to I-Ag7 tetramer (data not shown) loaded with the modified InsB:9-23 peptide [15]. Taken together, these results show that only T cells from the BDC12-4.1 clone respond to cognate antigen-specific stimulation and therefore we focused on generating iTregs only from BDC12-4.1 TCR Tg mice.


BDC12-4.1 T-cell receptor transgenic insulin-specific CD4 T cells are resistant to in vitro differentiation into functional Foxp3+ T regulatory cells.

Sarikonda G, Fousteri G, Sachithanantham S, Miller JF, Dave A, Juntti T, Coppieters KT, von Herrath M - PLoS ONE (2014)

BDC12-4.1 TCR transgenic T cells exhibit robust insB:9-23-specific responses.CD4+ T cells purified from splenocytes of BDC12-4.1/RAGKO or 2H6 mice were plated in triplicate, in round-bottom 96-well plates. 5–10×104 T cells per well were incubated with an equal number of APCs for 48 hours. Native InsB:9-23 peptide (open bars) or R22E mimotope ([14]) of the InsB:9-23 peptide (filled bars) were used at the indicated concentration. T cells incubated alone or with only APCs (without added antigen) served to measure spontaneous proliferation. Mean and SEM values for average of the triplicate wells derived from pooled mice (n = 3) is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231041&req=5

pone-0112242-g001: BDC12-4.1 TCR transgenic T cells exhibit robust insB:9-23-specific responses.CD4+ T cells purified from splenocytes of BDC12-4.1/RAGKO or 2H6 mice were plated in triplicate, in round-bottom 96-well plates. 5–10×104 T cells per well were incubated with an equal number of APCs for 48 hours. Native InsB:9-23 peptide (open bars) or R22E mimotope ([14]) of the InsB:9-23 peptide (filled bars) were used at the indicated concentration. T cells incubated alone or with only APCs (without added antigen) served to measure spontaneous proliferation. Mean and SEM values for average of the triplicate wells derived from pooled mice (n = 3) is shown.
Mentions: To confirm the insulin recognition properties of the two InsB:9-23 specific T cells, we assessed their proliferative capacity in response to peptide stimulation. BDC12-4.1 T-cells exhibited robust proliferation in a dose-dependent manner (Fig. 1A). Exposure to R22E mimotope [14] of the InsB:9-23 peptide resulted in an enhanced dose-dependent proliferation. These results confirm the recognition of InsB:9-23 peptide as the cognate antigen for BDC12-4.1 T cells. On the other hand, we could not obtain convincing evidence of insulin-specific responses from 2H6 T cells. Addition of antigen presenting cells (APCs) to 2H6 TCR Tg cells in the absence of antigen resulted in non-specific proliferation. Such proliferation did not increase in magnitude upon the addition of the native InsB:9-23 peptide or the R22E mimotope (Fig. 1B). Further, although 2H6 T cells exhibited Ca+2 mobilization in response to InsB:9-23 stimulation, in some but not all of our experiments, (Fig. S1), they did not bind to I-Ag7 tetramer (data not shown) loaded with the modified InsB:9-23 peptide [15]. Taken together, these results show that only T cells from the BDC12-4.1 clone respond to cognate antigen-specific stimulation and therefore we focused on generating iTregs only from BDC12-4.1 TCR Tg mice.

Bottom Line: We found that in vitro polarization toward Foxp3(+) iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3.Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4(+) T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice.These results highlight the disconnect between an in vitro acquired Foxp3(+) cell phenotype and its associated in vivo regulatory potential.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

ABSTRACT
The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4(+) T cells of the BDC12-4.1 clone to convert into Foxp3(+) iTreg cells. We found that in vitro polarization toward Foxp3(+) iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3(+) BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4(+) T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3(+) cell phenotype and its associated in vivo regulatory potential.

Show MeSH
Related in: MedlinePlus