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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 T cells expand and display memory and Treg cell phenotypes in the periphery.(A and B) Age-associated increases in total CD4+ T cell numbers in the spleen and PLN of BDC12-4.1.RAGKO mice. Splenocytes and PLN lymphocytes from BDC12-4.1.RAGKO mice were analyzed at different ages by flow cytometry for the frequency of CD4+Vβ2+ T cells. The percentage of CD4+Vβ2+ cells was multiplied with the total number of cells isolated from the spleen or PLN to calculate the total number of CD4+ T cells. Trypan blue was used for the exclusion of dead cells prior to counting. (C and D) Representative FACS plots and data for activated T cells, CD44hiCD62Llow, and Treg cells, CD25+Foxp3+, from the spleens of donor mice that were used to generate Foxp3+ Treg cells in vitro are shown.
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pone-0112242-g002: BDC12-4.1 T cells expand and display memory and Treg cell phenotypes in the periphery.(A and B) Age-associated increases in total CD4+ T cell numbers in the spleen and PLN of BDC12-4.1.RAGKO mice. Splenocytes and PLN lymphocytes from BDC12-4.1.RAGKO mice were analyzed at different ages by flow cytometry for the frequency of CD4+Vβ2+ T cells. The percentage of CD4+Vβ2+ cells was multiplied with the total number of cells isolated from the spleen or PLN to calculate the total number of CD4+ T cells. Trypan blue was used for the exclusion of dead cells prior to counting. (C and D) Representative FACS plots and data for activated T cells, CD44hiCD62Llow, and Treg cells, CD25+Foxp3+, from the spleens of donor mice that were used to generate Foxp3+ Treg cells in vitro are shown.

Mentions: Few BDC12-4.1 T cells escape thymic deletion [2] and undergo positive selection. Surprisingly, we found that CD4+CD8+ double positive thymocytes from BDC12-4.1 mice also express elevated levels of CD69 suggesting that some antigen-specific recognition is ongoing during positive selection (data not included). We previously showed that the majority of BDC12-4.1 TCR Tg cells acquire memory and Foxp3+ Treg phenotype in the spleen and PLN. Here, we also examined the number of total CD4+ T cells and found that in both spleen (Fig. 2A) and PLN (Fig. 2B) CD4+ T cells show a trend to expand with age. Prior to in vitro polarization into Foxp3 Treg, we evaluated the presence of CD44hiCD62Llow effector memory and Foxp3+ Treg cells in the spleens of the donor mice. As depicted in Fig. 2C and D, on average, 60% of CD4+ splenocytes displayed activated-memory phenotype and 5% expressed Foxp3. Whereas this data confirm our previous observations, it also shows that the starting CD4+ T cell population in the in vitro T cell cultures contains a great amount of already differentiated CD4+ T cells. Subsequently, we tested whether these cells can be polarized into functional Foxp3+ Treg cells.


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 T cells expand and display memory and Treg cell phenotypes in the periphery.(A and B) Age-associated increases in total CD4+ T cell numbers in the spleen and PLN of BDC12-4.1.RAGKO mice. Splenocytes and PLN lymphocytes from BDC12-4.1.RAGKO mice were analyzed at different ages by flow cytometry for the frequency of CD4+Vβ2+ T cells. The percentage of CD4+Vβ2+ cells was multiplied with the total number of cells isolated from the spleen or PLN to calculate the total number of CD4+ T cells. Trypan blue was used for the exclusion of dead cells prior to counting. (C and D) Representative FACS plots and data for activated T cells, CD44hiCD62Llow, and Treg cells, CD25+Foxp3+, from the spleens of donor mice that were used to generate Foxp3+ Treg cells in vitro are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112242-g002: BDC12-4.1 T cells expand and display memory and Treg cell phenotypes in the periphery.(A and B) Age-associated increases in total CD4+ T cell numbers in the spleen and PLN of BDC12-4.1.RAGKO mice. Splenocytes and PLN lymphocytes from BDC12-4.1.RAGKO mice were analyzed at different ages by flow cytometry for the frequency of CD4+Vβ2+ T cells. The percentage of CD4+Vβ2+ cells was multiplied with the total number of cells isolated from the spleen or PLN to calculate the total number of CD4+ T cells. Trypan blue was used for the exclusion of dead cells prior to counting. (C and D) Representative FACS plots and data for activated T cells, CD44hiCD62Llow, and Treg cells, CD25+Foxp3+, from the spleens of donor mice that were used to generate Foxp3+ Treg cells in vitro are shown.
Mentions: Few BDC12-4.1 T cells escape thymic deletion [2] and undergo positive selection. Surprisingly, we found that CD4+CD8+ double positive thymocytes from BDC12-4.1 mice also express elevated levels of CD69 suggesting that some antigen-specific recognition is ongoing during positive selection (data not included). We previously showed that the majority of BDC12-4.1 TCR Tg cells acquire memory and Foxp3+ Treg phenotype in the spleen and PLN. Here, we also examined the number of total CD4+ T cells and found that in both spleen (Fig. 2A) and PLN (Fig. 2B) CD4+ T cells show a trend to expand with age. Prior to in vitro polarization into Foxp3 Treg, we evaluated the presence of CD44hiCD62Llow effector memory and Foxp3+ Treg cells in the spleens of the donor mice. As depicted in Fig. 2C and D, on average, 60% of CD4+ splenocytes displayed activated-memory phenotype and 5% expressed Foxp3. Whereas this data confirm our previous observations, it also shows that the starting CD4+ T cell population in the in vitro T cell cultures contains a great amount of already differentiated CD4+ T cells. Subsequently, we tested whether these cells can be polarized into functional Foxp3+ Treg cells.

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