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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

Isolation and characterization of lentiviral transduced primary human CD4+ T cells expressing the tyrosinase-reactive TCR.(A) Human CD4+ T cells were isolated by negative selection from fresh peripheral blood and activated for 48 h in the presence of anti-CD3 and anti-CD28 coated microbeads and human IL-2 (300 IU/ml). Cells were spin infected 48 h post-transduction and assessed for TCR Vβ12 and GFP expression by FACS 72 hr post-transduction (A, upper-left hand plot). Individual cell populations (column A plots) were FACS sorted based on expression TCR Vβ12 and GFP (Vβ12+GFP+, upper-right quadrant; Vβ12−GFP+, lower-right; Vβ12−GFP−, lower-left). Each sorted population was expanded in culture for 12 d and then assessed for stability of (B) TCR Vβ12 and GFP expression by FACS and (C) proliferation assessed by 3H-thymidine incorporation in response to peptide presented in the context of HLA-A*0201 expressing aAPCs. T cell populations (2×104 cells/well) were stimulated by culturing 5×105 irradiated (10,000 rads) aAPCs with soluble peptide as indicated (C and D). Proliferation is graphed as the mean±SD from triplicate wells. Data are representative of two independent experiments.
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pone-0011726-g002: Isolation and characterization of lentiviral transduced primary human CD4+ T cells expressing the tyrosinase-reactive TCR.(A) Human CD4+ T cells were isolated by negative selection from fresh peripheral blood and activated for 48 h in the presence of anti-CD3 and anti-CD28 coated microbeads and human IL-2 (300 IU/ml). Cells were spin infected 48 h post-transduction and assessed for TCR Vβ12 and GFP expression by FACS 72 hr post-transduction (A, upper-left hand plot). Individual cell populations (column A plots) were FACS sorted based on expression TCR Vβ12 and GFP (Vβ12+GFP+, upper-right quadrant; Vβ12−GFP+, lower-right; Vβ12−GFP−, lower-left). Each sorted population was expanded in culture for 12 d and then assessed for stability of (B) TCR Vβ12 and GFP expression by FACS and (C) proliferation assessed by 3H-thymidine incorporation in response to peptide presented in the context of HLA-A*0201 expressing aAPCs. T cell populations (2×104 cells/well) were stimulated by culturing 5×105 irradiated (10,000 rads) aAPCs with soluble peptide as indicated (C and D). Proliferation is graphed as the mean±SD from triplicate wells. Data are representative of two independent experiments.

Mentions: Given our ability to express a functional TCR in Jurkat T cells, we next sought to validate expression in primary human CD4+ T cells. Freshly isolated human CD4+ T cells were activated for 48 hr with anti-CD3 and anti-CD28-coated beads prior to transduction with lentivirus expressing the TyrTCR. GFP and TCR Vβ12 co-expressing cells were isolated by fluorescence-based cell sorting (Figure 2A). Expression of surface TCR was stable following in vitro expansion (Figure 2B) and the TyrTCR-transduced cells responded specifically to tyrosinase peptide in a dose-responsive fashion. No proliferation was observed from the native GFP−Vβ12+ sorted population stimulated with tyrosinase peptide or from the GFP+Vβ12+ T cell population incubated with an irrelevant HLA-A*201-restricted control peptide (MART-1) (Figure 2C,D). Cytokine production (IFN-γ, IL-10) by TyrTCR-transduced cells was also detected following co-culture with a melanoma tumor cell line expressing tyrosinase naturally processed and presented in the context of HLA-A*0201 (data not shown). The successful de novo expression of a functional TCR in primary human CD4+ T cells facilitated the subsequent targeting of this receptor to Tregs.


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)

Isolation and characterization of lentiviral transduced primary human CD4+ T cells expressing the tyrosinase-reactive TCR.(A) Human CD4+ T cells were isolated by negative selection from fresh peripheral blood and activated for 48 h in the presence of anti-CD3 and anti-CD28 coated microbeads and human IL-2 (300 IU/ml). Cells were spin infected 48 h post-transduction and assessed for TCR Vβ12 and GFP expression by FACS 72 hr post-transduction (A, upper-left hand plot). Individual cell populations (column A plots) were FACS sorted based on expression TCR Vβ12 and GFP (Vβ12+GFP+, upper-right quadrant; Vβ12−GFP+, lower-right; Vβ12−GFP−, lower-left). Each sorted population was expanded in culture for 12 d and then assessed for stability of (B) TCR Vβ12 and GFP expression by FACS and (C) proliferation assessed by 3H-thymidine incorporation in response to peptide presented in the context of HLA-A*0201 expressing aAPCs. T cell populations (2×104 cells/well) were stimulated by culturing 5×105 irradiated (10,000 rads) aAPCs with soluble peptide as indicated (C and D). Proliferation is graphed as the mean±SD from triplicate wells. Data are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908680&req=5

pone-0011726-g002: Isolation and characterization of lentiviral transduced primary human CD4+ T cells expressing the tyrosinase-reactive TCR.(A) Human CD4+ T cells were isolated by negative selection from fresh peripheral blood and activated for 48 h in the presence of anti-CD3 and anti-CD28 coated microbeads and human IL-2 (300 IU/ml). Cells were spin infected 48 h post-transduction and assessed for TCR Vβ12 and GFP expression by FACS 72 hr post-transduction (A, upper-left hand plot). Individual cell populations (column A plots) were FACS sorted based on expression TCR Vβ12 and GFP (Vβ12+GFP+, upper-right quadrant; Vβ12−GFP+, lower-right; Vβ12−GFP−, lower-left). Each sorted population was expanded in culture for 12 d and then assessed for stability of (B) TCR Vβ12 and GFP expression by FACS and (C) proliferation assessed by 3H-thymidine incorporation in response to peptide presented in the context of HLA-A*0201 expressing aAPCs. T cell populations (2×104 cells/well) were stimulated by culturing 5×105 irradiated (10,000 rads) aAPCs with soluble peptide as indicated (C and D). Proliferation is graphed as the mean±SD from triplicate wells. Data are representative of two independent experiments.
Mentions: Given our ability to express a functional TCR in Jurkat T cells, we next sought to validate expression in primary human CD4+ T cells. Freshly isolated human CD4+ T cells were activated for 48 hr with anti-CD3 and anti-CD28-coated beads prior to transduction with lentivirus expressing the TyrTCR. GFP and TCR Vβ12 co-expressing cells were isolated by fluorescence-based cell sorting (Figure 2A). Expression of surface TCR was stable following in vitro expansion (Figure 2B) and the TyrTCR-transduced cells responded specifically to tyrosinase peptide in a dose-responsive fashion. No proliferation was observed from the native GFP−Vβ12+ sorted population stimulated with tyrosinase peptide or from the GFP+Vβ12+ T cell population incubated with an irrelevant HLA-A*201-restricted control peptide (MART-1) (Figure 2C,D). Cytokine production (IFN-γ, IL-10) by TyrTCR-transduced cells was also detected following co-culture with a melanoma tumor cell line expressing tyrosinase naturally processed and presented in the context of HLA-A*0201 (data not shown). The successful de novo expression of a functional TCR in primary human CD4+ T cells facilitated the subsequent targeting of this receptor to Tregs.

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