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Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.

Brusko TM, Koya RC, Zhu S, Lee MR, Putnam AL, McClymont SA, Nishimura MI, Han S, Chang LJ, Atkinson MA, Ribas A, Bluestone JA - PLoS ONE (2010)

Bottom Line: Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells.These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses.These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT

Background: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition.

Methodology/principal findings: To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging.

Conclusions/significance: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.

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Related in: MedlinePlus

In vitro suppressive capacity of lentiviral transduced Tregs expressing the tyrosinase TCR.TyrTCR transduced Treg and Tconv cells were sorted to enrich the GFP+Vβ12+ cell population (as indicated in Fig. 2A). The sorted Tregs were then tested for their capacity to suppress proliferation of autologous expanded Tconv cells during in vitro suppression assays following both polyclonal and antigen-specific T cell activation. (A) FACS sorted GFP+Vβ12+ Treg cells suppress proliferation of anti-CD3 and anti-CD28 stimulated GFP+Vβ12+ Tconv cells in a dose-responsive manner. (B) In vitro expanded Tregs suppress TyrTCR GFP+Vβ12+ Tconv cells stimulated by aAPCs and tyrosinase peptide. (C) A representative suppression assay demonstrating GFP+Vβ12+ Tregs are more potent at suppressing autologous hemagglutinin (HA)-reactive Teff cells when stimulated by their cognate antigen compared to an irrelevant HLA-A2 restricted peptide. HA-reactive Teff cells were stimulated by HLA-DR*0401 expressing APCs and HA peptide with Tregs stimulated with either tyrosinase peptide (C, closed bars) or an irrelevant HLA-A*0201 restricted peptide melan-A (MART-1) (C, open bars). (D) Percent suppression of HA-reactive Teff cells stimulated in the presence of Tyr-Tregs activated by tyrosinase peptide (black bars) or MART-1 (open bars) (N = 3 independent experiments, *P<0.05). Shown are mean±SEM.
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pone-0011726-g004: In vitro suppressive capacity of lentiviral transduced Tregs expressing the tyrosinase TCR.TyrTCR transduced Treg and Tconv cells were sorted to enrich the GFP+Vβ12+ cell population (as indicated in Fig. 2A). The sorted Tregs were then tested for their capacity to suppress proliferation of autologous expanded Tconv cells during in vitro suppression assays following both polyclonal and antigen-specific T cell activation. (A) FACS sorted GFP+Vβ12+ Treg cells suppress proliferation of anti-CD3 and anti-CD28 stimulated GFP+Vβ12+ Tconv cells in a dose-responsive manner. (B) In vitro expanded Tregs suppress TyrTCR GFP+Vβ12+ Tconv cells stimulated by aAPCs and tyrosinase peptide. (C) A representative suppression assay demonstrating GFP+Vβ12+ Tregs are more potent at suppressing autologous hemagglutinin (HA)-reactive Teff cells when stimulated by their cognate antigen compared to an irrelevant HLA-A2 restricted peptide. HA-reactive Teff cells were stimulated by HLA-DR*0401 expressing APCs and HA peptide with Tregs stimulated with either tyrosinase peptide (C, closed bars) or an irrelevant HLA-A*0201 restricted peptide melan-A (MART-1) (C, open bars). (D) Percent suppression of HA-reactive Teff cells stimulated in the presence of Tyr-Tregs activated by tyrosinase peptide (black bars) or MART-1 (open bars) (N = 3 independent experiments, *P<0.05). Shown are mean±SEM.

Mentions: The capacity of expanded TCR-transduced Tregs to suppress proliferation of freshly isolated conventional CD4+ T cells was examined following stimulation with anti-CD3 and anti-CD28. GFP+TyrTCR+ Tregs were FACS sorted following 14 days of in vitro expansion and mixed at indicated ratios of Tregs to Tconv cells. TyrTCR Tregs suppressed polyclonal T cell proliferation potently and in a dose-responsive fashion (Figure 4A). We next tested the capacity of TyrTCR Tregs to suppress antigen-specific CD4+CD127+CD25− Tconv cell responses. Following expansion, Treg and Tconv GFP+TyrTCR+ cells were sorted from expanded cultures and restimulated with tyrosinase peptide in the presence of aAPCs (Figure 4B). TyrTCR-expressing Tregs were capable of suppressing Teff cell responses in an antigen-specific fashion.


Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.

Brusko TM, Koya RC, Zhu S, Lee MR, Putnam AL, McClymont SA, Nishimura MI, Han S, Chang LJ, Atkinson MA, Ribas A, Bluestone JA - PLoS ONE (2010)

In vitro suppressive capacity of lentiviral transduced Tregs expressing the tyrosinase TCR.TyrTCR transduced Treg and Tconv cells were sorted to enrich the GFP+Vβ12+ cell population (as indicated in Fig. 2A). The sorted Tregs were then tested for their capacity to suppress proliferation of autologous expanded Tconv cells during in vitro suppression assays following both polyclonal and antigen-specific T cell activation. (A) FACS sorted GFP+Vβ12+ Treg cells suppress proliferation of anti-CD3 and anti-CD28 stimulated GFP+Vβ12+ Tconv cells in a dose-responsive manner. (B) In vitro expanded Tregs suppress TyrTCR GFP+Vβ12+ Tconv cells stimulated by aAPCs and tyrosinase peptide. (C) A representative suppression assay demonstrating GFP+Vβ12+ Tregs are more potent at suppressing autologous hemagglutinin (HA)-reactive Teff cells when stimulated by their cognate antigen compared to an irrelevant HLA-A2 restricted peptide. HA-reactive Teff cells were stimulated by HLA-DR*0401 expressing APCs and HA peptide with Tregs stimulated with either tyrosinase peptide (C, closed bars) or an irrelevant HLA-A*0201 restricted peptide melan-A (MART-1) (C, open bars). (D) Percent suppression of HA-reactive Teff cells stimulated in the presence of Tyr-Tregs activated by tyrosinase peptide (black bars) or MART-1 (open bars) (N = 3 independent experiments, *P<0.05). Shown are mean±SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908680&req=5

pone-0011726-g004: In vitro suppressive capacity of lentiviral transduced Tregs expressing the tyrosinase TCR.TyrTCR transduced Treg and Tconv cells were sorted to enrich the GFP+Vβ12+ cell population (as indicated in Fig. 2A). The sorted Tregs were then tested for their capacity to suppress proliferation of autologous expanded Tconv cells during in vitro suppression assays following both polyclonal and antigen-specific T cell activation. (A) FACS sorted GFP+Vβ12+ Treg cells suppress proliferation of anti-CD3 and anti-CD28 stimulated GFP+Vβ12+ Tconv cells in a dose-responsive manner. (B) In vitro expanded Tregs suppress TyrTCR GFP+Vβ12+ Tconv cells stimulated by aAPCs and tyrosinase peptide. (C) A representative suppression assay demonstrating GFP+Vβ12+ Tregs are more potent at suppressing autologous hemagglutinin (HA)-reactive Teff cells when stimulated by their cognate antigen compared to an irrelevant HLA-A2 restricted peptide. HA-reactive Teff cells were stimulated by HLA-DR*0401 expressing APCs and HA peptide with Tregs stimulated with either tyrosinase peptide (C, closed bars) or an irrelevant HLA-A*0201 restricted peptide melan-A (MART-1) (C, open bars). (D) Percent suppression of HA-reactive Teff cells stimulated in the presence of Tyr-Tregs activated by tyrosinase peptide (black bars) or MART-1 (open bars) (N = 3 independent experiments, *P<0.05). Shown are mean±SEM.
Mentions: The capacity of expanded TCR-transduced Tregs to suppress proliferation of freshly isolated conventional CD4+ T cells was examined following stimulation with anti-CD3 and anti-CD28. GFP+TyrTCR+ Tregs were FACS sorted following 14 days of in vitro expansion and mixed at indicated ratios of Tregs to Tconv cells. TyrTCR Tregs suppressed polyclonal T cell proliferation potently and in a dose-responsive fashion (Figure 4A). We next tested the capacity of TyrTCR Tregs to suppress antigen-specific CD4+CD127+CD25− Tconv cell responses. Following expansion, Treg and Tconv GFP+TyrTCR+ cells were sorted from expanded cultures and restimulated with tyrosinase peptide in the presence of aAPCs (Figure 4B). TyrTCR-expressing Tregs were capable of suppressing Teff cell responses in an antigen-specific fashion.

Bottom Line: Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells.These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses.These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT

Background: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition.

Methodology/principal findings: To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging.

Conclusions/significance: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.

Show MeSH
Related in: MedlinePlus