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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

Expression of tyrosinase TCR constructs by in vitro expanded human Treg and Tconv cells.Plots indicate expression of surface TCR, GFP, and FOXP3 following 14 day in vitro expansion period for mock or TyrTCR transduced Treg and Tconv cells. Fresh human PBMCs were sorted by FACS to yield CD4+CD127−/loCD25+CD45RA+ Tregs and CD4+CD127+CD25−CD45RA+ Tconv cell populations and stimulated with anti-CD3 and anti-CD28 microbeads for 48 hours prior to lentiviral spinoculation. Following eight days of in vitro expansion, mock transduced cells were restimulated with microbeads and TyrTCR transduced T cells were enriched by restimulation with aAPCs and soluble tyrosinase peptide (0.1 µg/ml). Plots indicate expression of eGFP (y-axis) and TCR Vβ12 or HLA-A2 tyrosinase(368–376) tetramer staining (x-axis) with (A, upper plots) representing mock transduced cells and (B, lower plots) showing TyrTCR transduced populations. Histograms overlays indicate isotype control staining (red histograms) or FOXP3 staining (green histograms) of mock or TyrTCR transduced populations. Data shown represent one subject of ten independent experiments following lentiviral TyrTCR transduction of Tregs.
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pone-0011726-g003: Expression of tyrosinase TCR constructs by in vitro expanded human Treg and Tconv cells.Plots indicate expression of surface TCR, GFP, and FOXP3 following 14 day in vitro expansion period for mock or TyrTCR transduced Treg and Tconv cells. Fresh human PBMCs were sorted by FACS to yield CD4+CD127−/loCD25+CD45RA+ Tregs and CD4+CD127+CD25−CD45RA+ Tconv cell populations and stimulated with anti-CD3 and anti-CD28 microbeads for 48 hours prior to lentiviral spinoculation. Following eight days of in vitro expansion, mock transduced cells were restimulated with microbeads and TyrTCR transduced T cells were enriched by restimulation with aAPCs and soluble tyrosinase peptide (0.1 µg/ml). Plots indicate expression of eGFP (y-axis) and TCR Vβ12 or HLA-A2 tyrosinase(368–376) tetramer staining (x-axis) with (A, upper plots) representing mock transduced cells and (B, lower plots) showing TyrTCR transduced populations. Histograms overlays indicate isotype control staining (red histograms) or FOXP3 staining (green histograms) of mock or TyrTCR transduced populations. Data shown represent one subject of ten independent experiments following lentiviral TyrTCR transduction of Tregs.

Mentions: We observed that increasing lentiviral titer led to only modest increases in the number of cells expressing functional receptors (data not shown). Therefore, we sought to enrich antigen-specific T cells while limiting non-specific growth, as previously described [42]. At day nine, cells expressing the TyrTCR were restimulated with aAPCs and tyrosinase peptide. This antigen-specific activation led to enrichment of the transduced cells with 66.1% of Treg and 50.3% of Tconv cells expressing both GFP and the TyrTCR (Figure 3A,B). Mock transduced cells were restimulated by anti-CD3 and anti-CD28 coated beads. The expansion capacity of TyrTCR Tregs expanded on aAPC and peptide was dependent upon the initial transduction efficiency (N = 3; median 480-fold, range 240 to 1088-fold). Of note, we were able to obtain >5200-fold expansion of TyrTCR+ Tregs yielding 1.18×109 cells (89.3% eGFP+, 91.7% FOXP3+), from an initial blood volume of 80 ml by extending the culture to day 20 (data not shown).


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)

Expression of tyrosinase TCR constructs by in vitro expanded human Treg and Tconv cells.Plots indicate expression of surface TCR, GFP, and FOXP3 following 14 day in vitro expansion period for mock or TyrTCR transduced Treg and Tconv cells. Fresh human PBMCs were sorted by FACS to yield CD4+CD127−/loCD25+CD45RA+ Tregs and CD4+CD127+CD25−CD45RA+ Tconv cell populations and stimulated with anti-CD3 and anti-CD28 microbeads for 48 hours prior to lentiviral spinoculation. Following eight days of in vitro expansion, mock transduced cells were restimulated with microbeads and TyrTCR transduced T cells were enriched by restimulation with aAPCs and soluble tyrosinase peptide (0.1 µg/ml). Plots indicate expression of eGFP (y-axis) and TCR Vβ12 or HLA-A2 tyrosinase(368–376) tetramer staining (x-axis) with (A, upper plots) representing mock transduced cells and (B, lower plots) showing TyrTCR transduced populations. Histograms overlays indicate isotype control staining (red histograms) or FOXP3 staining (green histograms) of mock or TyrTCR transduced populations. Data shown represent one subject of ten independent experiments following lentiviral TyrTCR transduction of Tregs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908680&req=5

pone-0011726-g003: Expression of tyrosinase TCR constructs by in vitro expanded human Treg and Tconv cells.Plots indicate expression of surface TCR, GFP, and FOXP3 following 14 day in vitro expansion period for mock or TyrTCR transduced Treg and Tconv cells. Fresh human PBMCs were sorted by FACS to yield CD4+CD127−/loCD25+CD45RA+ Tregs and CD4+CD127+CD25−CD45RA+ Tconv cell populations and stimulated with anti-CD3 and anti-CD28 microbeads for 48 hours prior to lentiviral spinoculation. Following eight days of in vitro expansion, mock transduced cells were restimulated with microbeads and TyrTCR transduced T cells were enriched by restimulation with aAPCs and soluble tyrosinase peptide (0.1 µg/ml). Plots indicate expression of eGFP (y-axis) and TCR Vβ12 or HLA-A2 tyrosinase(368–376) tetramer staining (x-axis) with (A, upper plots) representing mock transduced cells and (B, lower plots) showing TyrTCR transduced populations. Histograms overlays indicate isotype control staining (red histograms) or FOXP3 staining (green histograms) of mock or TyrTCR transduced populations. Data shown represent one subject of ten independent experiments following lentiviral TyrTCR transduction of Tregs.
Mentions: We observed that increasing lentiviral titer led to only modest increases in the number of cells expressing functional receptors (data not shown). Therefore, we sought to enrich antigen-specific T cells while limiting non-specific growth, as previously described [42]. At day nine, cells expressing the TyrTCR were restimulated with aAPCs and tyrosinase peptide. This antigen-specific activation led to enrichment of the transduced cells with 66.1% of Treg and 50.3% of Tconv cells expressing both GFP and the TyrTCR (Figure 3A,B). Mock transduced cells were restimulated by anti-CD3 and anti-CD28 coated beads. The expansion capacity of TyrTCR Tregs expanded on aAPC and peptide was dependent upon the initial transduction efficiency (N = 3; median 480-fold, range 240 to 1088-fold). Of note, we were able to obtain >5200-fold expansion of TyrTCR+ Tregs yielding 1.18×109 cells (89.3% eGFP+, 91.7% FOXP3+), from an initial blood volume of 80 ml by extending the culture to day 20 (data not shown).

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