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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

Tyrosinase TCR transduced Tregs block antigen-specific effector T cell activity in vivo.Eight-weeks old HLA-A2/Kb transgenic mice were injected subcutaneously (s.c.) with EL4-A2/Kb tyrosinase protein expressing tumors (5 mm in diameter) in the inguinal fat pad. Murine T cells were transduced with MSCV-based retroviral vectors encoding the Tyrosinase TCR with (A) Firefly Luciferase (fLuc) reporter for Teff cells or HSVtk/GFP reporter for Tregs. Mice were randomized into three groups receiving in vitro expanded Tregs transduced with TyrTCR/TK/GFP vector (TyrTCR Tregs), polyclonal mock Tregs (Untrd, Treg), or mock T effector cells (Untrd, Teff). (B) Mock or HLA-A*0201(368–376) tetramer staining of TyrTCR transduced Tregs prior to injection. Following 24 hours, all mice received Teff cells transduced with Tyr-TCR/fLuciferase reporter. (C) Luciferase-based in vivo bioluminescence imaging (BLI) was performed for imaging of fLuc-transduced Teff cells. Fluorescence images of treatment groups are shown at day 8. (D) The mean number of photons per square centimeter per second per steradian in the region of interest (ROI) was determined on tumor-implanted sites for mice receiving TyrTCR Tregs (blue square), Untransduced Tregs (red circle), and Untrd Teff (green triangle). (E) Tumor site volume (mm3) assessed by caliper measurement for adoptive cell transfer treatment groups. Data shown is representative of two independent experiments with *P<0.05, **P<0.01, and ***P<0.0001 assessed by Two-way ANOVA with Bonferroni correction.
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pone-0011726-g005: Tyrosinase TCR transduced Tregs block antigen-specific effector T cell activity in vivo.Eight-weeks old HLA-A2/Kb transgenic mice were injected subcutaneously (s.c.) with EL4-A2/Kb tyrosinase protein expressing tumors (5 mm in diameter) in the inguinal fat pad. Murine T cells were transduced with MSCV-based retroviral vectors encoding the Tyrosinase TCR with (A) Firefly Luciferase (fLuc) reporter for Teff cells or HSVtk/GFP reporter for Tregs. Mice were randomized into three groups receiving in vitro expanded Tregs transduced with TyrTCR/TK/GFP vector (TyrTCR Tregs), polyclonal mock Tregs (Untrd, Treg), or mock T effector cells (Untrd, Teff). (B) Mock or HLA-A*0201(368–376) tetramer staining of TyrTCR transduced Tregs prior to injection. Following 24 hours, all mice received Teff cells transduced with Tyr-TCR/fLuciferase reporter. (C) Luciferase-based in vivo bioluminescence imaging (BLI) was performed for imaging of fLuc-transduced Teff cells. Fluorescence images of treatment groups are shown at day 8. (D) The mean number of photons per square centimeter per second per steradian in the region of interest (ROI) was determined on tumor-implanted sites for mice receiving TyrTCR Tregs (blue square), Untransduced Tregs (red circle), and Untrd Teff (green triangle). (E) Tumor site volume (mm3) assessed by caliper measurement for adoptive cell transfer treatment groups. Data shown is representative of two independent experiments with *P<0.05, **P<0.01, and ***P<0.0001 assessed by Two-way ANOVA with Bonferroni correction.

Mentions: We employed a tumor model system to assess the in vivo function of TyrTCR-redirected Tregs. EL-4-HLA-A2/Kb tumors expressing tyrosinase were transferred into HLA-A2/Kb transgenic mice. Mice were then separated into groups receiving in vitro expanded murine TyrTCR Tregs, mock vector-transduced Tregs, or control vector-transduced Teff cells via intravenous injection. All mice received antigen-specific Teff cells expressing the TyrTCR. These TyrTCR Teff cells could be tracked in vivo by a firefly-Luciferase (fLuc) reporter element encoded within the expression construct (Figure 5A). The murine TyrTCR expression construct was modified for optimized surface expression and improved tetramer reactivity (R. Koya, unpublished observations). Specifically, the TyrTCR construct was modified to express murine TCR constant regions and human TCR variable regions [34]. Additional modifications consisted of introducing cysteine residues to the TCR α and β chains to induce disulfide linkages, 2A peptide linker sequences, and leucine zipper motifs between TCR chains as described in Materials and Methods. The modified TyrTCR could be expressed on murine CD4+ T cells with expression by expanded Tregs verified by HLA-A*201-Tyrosinase(368–376) tetramer staining following in vitro expansion with anti-CD3 and anti-CD28-coated microbeads and IL-2 (Figure 5B).


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)

Tyrosinase TCR transduced Tregs block antigen-specific effector T cell activity in vivo.Eight-weeks old HLA-A2/Kb transgenic mice were injected subcutaneously (s.c.) with EL4-A2/Kb tyrosinase protein expressing tumors (5 mm in diameter) in the inguinal fat pad. Murine T cells were transduced with MSCV-based retroviral vectors encoding the Tyrosinase TCR with (A) Firefly Luciferase (fLuc) reporter for Teff cells or HSVtk/GFP reporter for Tregs. Mice were randomized into three groups receiving in vitro expanded Tregs transduced with TyrTCR/TK/GFP vector (TyrTCR Tregs), polyclonal mock Tregs (Untrd, Treg), or mock T effector cells (Untrd, Teff). (B) Mock or HLA-A*0201(368–376) tetramer staining of TyrTCR transduced Tregs prior to injection. Following 24 hours, all mice received Teff cells transduced with Tyr-TCR/fLuciferase reporter. (C) Luciferase-based in vivo bioluminescence imaging (BLI) was performed for imaging of fLuc-transduced Teff cells. Fluorescence images of treatment groups are shown at day 8. (D) The mean number of photons per square centimeter per second per steradian in the region of interest (ROI) was determined on tumor-implanted sites for mice receiving TyrTCR Tregs (blue square), Untransduced Tregs (red circle), and Untrd Teff (green triangle). (E) Tumor site volume (mm3) assessed by caliper measurement for adoptive cell transfer treatment groups. Data shown is representative of two independent experiments with *P<0.05, **P<0.01, and ***P<0.0001 assessed by Two-way ANOVA with Bonferroni correction.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908680&req=5

pone-0011726-g005: Tyrosinase TCR transduced Tregs block antigen-specific effector T cell activity in vivo.Eight-weeks old HLA-A2/Kb transgenic mice were injected subcutaneously (s.c.) with EL4-A2/Kb tyrosinase protein expressing tumors (5 mm in diameter) in the inguinal fat pad. Murine T cells were transduced with MSCV-based retroviral vectors encoding the Tyrosinase TCR with (A) Firefly Luciferase (fLuc) reporter for Teff cells or HSVtk/GFP reporter for Tregs. Mice were randomized into three groups receiving in vitro expanded Tregs transduced with TyrTCR/TK/GFP vector (TyrTCR Tregs), polyclonal mock Tregs (Untrd, Treg), or mock T effector cells (Untrd, Teff). (B) Mock or HLA-A*0201(368–376) tetramer staining of TyrTCR transduced Tregs prior to injection. Following 24 hours, all mice received Teff cells transduced with Tyr-TCR/fLuciferase reporter. (C) Luciferase-based in vivo bioluminescence imaging (BLI) was performed for imaging of fLuc-transduced Teff cells. Fluorescence images of treatment groups are shown at day 8. (D) The mean number of photons per square centimeter per second per steradian in the region of interest (ROI) was determined on tumor-implanted sites for mice receiving TyrTCR Tregs (blue square), Untransduced Tregs (red circle), and Untrd Teff (green triangle). (E) Tumor site volume (mm3) assessed by caliper measurement for adoptive cell transfer treatment groups. Data shown is representative of two independent experiments with *P<0.05, **P<0.01, and ***P<0.0001 assessed by Two-way ANOVA with Bonferroni correction.
Mentions: We employed a tumor model system to assess the in vivo function of TyrTCR-redirected Tregs. EL-4-HLA-A2/Kb tumors expressing tyrosinase were transferred into HLA-A2/Kb transgenic mice. Mice were then separated into groups receiving in vitro expanded murine TyrTCR Tregs, mock vector-transduced Tregs, or control vector-transduced Teff cells via intravenous injection. All mice received antigen-specific Teff cells expressing the TyrTCR. These TyrTCR Teff cells could be tracked in vivo by a firefly-Luciferase (fLuc) reporter element encoded within the expression construct (Figure 5A). The murine TyrTCR expression construct was modified for optimized surface expression and improved tetramer reactivity (R. Koya, unpublished observations). Specifically, the TyrTCR construct was modified to express murine TCR constant regions and human TCR variable regions [34]. Additional modifications consisted of introducing cysteine residues to the TCR α and β chains to induce disulfide linkages, 2A peptide linker sequences, and leucine zipper motifs between TCR chains as described in Materials and Methods. The modified TyrTCR could be expressed on murine CD4+ T cells with expression by expanded Tregs verified by HLA-A*201-Tyrosinase(368–376) tetramer staining following in vitro expansion with anti-CD3 and anti-CD28-coated microbeads and IL-2 (Figure 5B).

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