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Inhibition of Donor-Reactive CD8+ T Cell Responses by Selective CD28 Blockade Is Independent of Reduced ICOS Expression.

Liu D, Suchard SJ, Nadler SG, Ford ML - PLoS ONE (2015)

Bottom Line: We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model.Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known.In this study, we created retrogenic donor-reactive CD8+ T cells that overexpress ICOS in order to determine whether reduced ICOS expression mechanistically underlies the increased efficacy of selective CD28 blockade in controlling graft-specific T cell responses as compared to conventional costimulation blockade with CTLA-4 Ig.

View Article: PubMed Central - PubMed

Affiliation: Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA 30322, United States of America.

ABSTRACT
Programmed T cell differentiation is critically influenced by the complement of costimulatory and coinhibitory signals transmitted during initial antigen encounter. We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model. Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known. In this study, we created retrogenic donor-reactive CD8+ T cells that overexpress ICOS in order to determine whether reduced ICOS expression mechanistically underlies the increased efficacy of selective CD28 blockade in controlling graft-specific T cell responses as compared to conventional costimulation blockade with CTLA-4 Ig. Results indicated that the ability of selective CD28 blockade to blunt donor-reactive CD8+ T cell expansion following transplantation was independent of its ability to inhibit ICOS expression. Furthermore, we have previously published that 2B4 coinhibitory signals are functionally important for controlling graft-specific CD8+ T cell responses in mice treated with CD28 blockade. Here we used a co-adoptive transfer approach to determine that 2B4 coinhibitory signals on antigen-specific CD8+ T cells function in a cell-intrinsic manner to limit ICOS expression in the setting of selective CD28 blockade.

No MeSH data available.


Related in: MedlinePlus

Generation of retrogenic graft-specific CD8+ T cells that constitutively express ICOS.To generate donor-reactive T cells that constitutively over-express ICOS, CD45.2+ Thy1.1+ OT-I bone marrow was transduced with a construct that expresses ICOS under a constitutively active viral promoter (A). The construct also contained an IRES-GFP to facilitate tracking the cells. At day 2 post transduction, ~5–10% of Thy1.1+ OT-I BM cells expressed either GFP alone (pMY control vector-transduced cells) or both GFP and ICOS (for ICOS vector-transduced cells). BM cells were then adoptively transferred into irradiated CD45.1+ Thy1.2+ animals. At 8–10 weeks post-in vivo transfer, Thy1.1+ OT-I T cells were detectable. B) GFP+ CD3+ Thy1.1+ (for pMY) or GFP+ ICOS+ CD3+ Thy1.1+ (for ICOSrg) OT-I T cells from spleen and LN of pMY or ICOSrg chimeric animals were FACS sorted and adoptively transferred (106 /recipient) into naïve B6 hosts. Animals also received 106 WT CD4+ Thy1.1+ OT-II T cells and were grafted with an OVA-expressing skin graft. C) PBL analyzed 10 days post skin grafts contained Thy1.1+ pMY and ICOSrg cells in the respective recipients. D) Assessment of ICOS expression on pMY vs. ICOSrg cells at day 10 post skin graft. ***p<0.0001.
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pone.0130490.g002: Generation of retrogenic graft-specific CD8+ T cells that constitutively express ICOS.To generate donor-reactive T cells that constitutively over-express ICOS, CD45.2+ Thy1.1+ OT-I bone marrow was transduced with a construct that expresses ICOS under a constitutively active viral promoter (A). The construct also contained an IRES-GFP to facilitate tracking the cells. At day 2 post transduction, ~5–10% of Thy1.1+ OT-I BM cells expressed either GFP alone (pMY control vector-transduced cells) or both GFP and ICOS (for ICOS vector-transduced cells). BM cells were then adoptively transferred into irradiated CD45.1+ Thy1.2+ animals. At 8–10 weeks post-in vivo transfer, Thy1.1+ OT-I T cells were detectable. B) GFP+ CD3+ Thy1.1+ (for pMY) or GFP+ ICOS+ CD3+ Thy1.1+ (for ICOSrg) OT-I T cells from spleen and LN of pMY or ICOSrg chimeric animals were FACS sorted and adoptively transferred (106 /recipient) into naïve B6 hosts. Animals also received 106 WT CD4+ Thy1.1+ OT-II T cells and were grafted with an OVA-expressing skin graft. C) PBL analyzed 10 days post skin grafts contained Thy1.1+ pMY and ICOSrg cells in the respective recipients. D) Assessment of ICOS expression on pMY vs. ICOSrg cells at day 10 post skin graft. ***p<0.0001.

Mentions: In light of the observation that selective CD28 blockade and preservation of CTLA-4 mediated coinhibitory signals were associated with profoundly decreased ICOS expression, we next determined whether this reduced ICOS expression was causally related to the efficacy of selective CD28 blockade. In order to test this, we utilized a retrogenic approach to generate donor-reactive T cells that constitutively over-express ICOS, and then interrogated their expansion and function following selective CD28 blockade. Briefly, CD45.2+ Thy1.1+ OT-I bone marrow was transduced with a construct that expresses ICOS under a constitutively active promoter (Fig 2A). The construct also contained an IRES-GFP to facilitate tracking the cells. As shown in Fig 2A, ~5–10% of Thy1.1 OT-I BM cells expressed either GFP alone (pMY control vector-transduced cells) or both GFP and ICOS (for ICOS vector-transduced cells) on day 3 following transduction. BM cells were then adoptively transferred into irradiated CD45.1+ animals. Using this approach, in 8–10 weeks we achieved reconstitution of the T cell compartment, and were able to detect Thy1.1+ OT-I T cells in recipients of both pMY and ICOSrg BM (Fig 2A). We then sorted GFP+ CD3+ Thy1.1+ OT-I T cells from spleen and LN of pMY or ICOSrg chimeric animals (Fig 2B), and of these, all are GFP+ (for pMY mice) or GFP+ ICOS+ (for ICOSrg mice), indicating the ICOS and GFP transgenes are stably expressed. pMY or ICOSrg Thy1.1+ OT-I T cells were then adoptively transferred (106 /recipient) into naïve B6 hosts. Animals also received 106 WT CD4+ Thy1.1+ OT-II T cells and were grafted with an OVA-expressing skin graft. Analysis of peripheral blood of these recipients 10 days after transfer confirmed that both pMY and ICOSrg cells were viable (Fig 2C), and that ICOSrg cells continued to express high levels of the ICOS receptor on the cell surface (Fig 2D). The ability of our ICOS protein to signal was confirmed in separate studies which showed that expression of this construct functioned to increase CD8+ T cell expansion and alter memory differentiation in a model of Listeria infection (data not shown).


Inhibition of Donor-Reactive CD8+ T Cell Responses by Selective CD28 Blockade Is Independent of Reduced ICOS Expression.

Liu D, Suchard SJ, Nadler SG, Ford ML - PLoS ONE (2015)

Generation of retrogenic graft-specific CD8+ T cells that constitutively express ICOS.To generate donor-reactive T cells that constitutively over-express ICOS, CD45.2+ Thy1.1+ OT-I bone marrow was transduced with a construct that expresses ICOS under a constitutively active viral promoter (A). The construct also contained an IRES-GFP to facilitate tracking the cells. At day 2 post transduction, ~5–10% of Thy1.1+ OT-I BM cells expressed either GFP alone (pMY control vector-transduced cells) or both GFP and ICOS (for ICOS vector-transduced cells). BM cells were then adoptively transferred into irradiated CD45.1+ Thy1.2+ animals. At 8–10 weeks post-in vivo transfer, Thy1.1+ OT-I T cells were detectable. B) GFP+ CD3+ Thy1.1+ (for pMY) or GFP+ ICOS+ CD3+ Thy1.1+ (for ICOSrg) OT-I T cells from spleen and LN of pMY or ICOSrg chimeric animals were FACS sorted and adoptively transferred (106 /recipient) into naïve B6 hosts. Animals also received 106 WT CD4+ Thy1.1+ OT-II T cells and were grafted with an OVA-expressing skin graft. C) PBL analyzed 10 days post skin grafts contained Thy1.1+ pMY and ICOSrg cells in the respective recipients. D) Assessment of ICOS expression on pMY vs. ICOSrg cells at day 10 post skin graft. ***p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476729&req=5

pone.0130490.g002: Generation of retrogenic graft-specific CD8+ T cells that constitutively express ICOS.To generate donor-reactive T cells that constitutively over-express ICOS, CD45.2+ Thy1.1+ OT-I bone marrow was transduced with a construct that expresses ICOS under a constitutively active viral promoter (A). The construct also contained an IRES-GFP to facilitate tracking the cells. At day 2 post transduction, ~5–10% of Thy1.1+ OT-I BM cells expressed either GFP alone (pMY control vector-transduced cells) or both GFP and ICOS (for ICOS vector-transduced cells). BM cells were then adoptively transferred into irradiated CD45.1+ Thy1.2+ animals. At 8–10 weeks post-in vivo transfer, Thy1.1+ OT-I T cells were detectable. B) GFP+ CD3+ Thy1.1+ (for pMY) or GFP+ ICOS+ CD3+ Thy1.1+ (for ICOSrg) OT-I T cells from spleen and LN of pMY or ICOSrg chimeric animals were FACS sorted and adoptively transferred (106 /recipient) into naïve B6 hosts. Animals also received 106 WT CD4+ Thy1.1+ OT-II T cells and were grafted with an OVA-expressing skin graft. C) PBL analyzed 10 days post skin grafts contained Thy1.1+ pMY and ICOSrg cells in the respective recipients. D) Assessment of ICOS expression on pMY vs. ICOSrg cells at day 10 post skin graft. ***p<0.0001.
Mentions: In light of the observation that selective CD28 blockade and preservation of CTLA-4 mediated coinhibitory signals were associated with profoundly decreased ICOS expression, we next determined whether this reduced ICOS expression was causally related to the efficacy of selective CD28 blockade. In order to test this, we utilized a retrogenic approach to generate donor-reactive T cells that constitutively over-express ICOS, and then interrogated their expansion and function following selective CD28 blockade. Briefly, CD45.2+ Thy1.1+ OT-I bone marrow was transduced with a construct that expresses ICOS under a constitutively active promoter (Fig 2A). The construct also contained an IRES-GFP to facilitate tracking the cells. As shown in Fig 2A, ~5–10% of Thy1.1 OT-I BM cells expressed either GFP alone (pMY control vector-transduced cells) or both GFP and ICOS (for ICOS vector-transduced cells) on day 3 following transduction. BM cells were then adoptively transferred into irradiated CD45.1+ animals. Using this approach, in 8–10 weeks we achieved reconstitution of the T cell compartment, and were able to detect Thy1.1+ OT-I T cells in recipients of both pMY and ICOSrg BM (Fig 2A). We then sorted GFP+ CD3+ Thy1.1+ OT-I T cells from spleen and LN of pMY or ICOSrg chimeric animals (Fig 2B), and of these, all are GFP+ (for pMY mice) or GFP+ ICOS+ (for ICOSrg mice), indicating the ICOS and GFP transgenes are stably expressed. pMY or ICOSrg Thy1.1+ OT-I T cells were then adoptively transferred (106 /recipient) into naïve B6 hosts. Animals also received 106 WT CD4+ Thy1.1+ OT-II T cells and were grafted with an OVA-expressing skin graft. Analysis of peripheral blood of these recipients 10 days after transfer confirmed that both pMY and ICOSrg cells were viable (Fig 2C), and that ICOSrg cells continued to express high levels of the ICOS receptor on the cell surface (Fig 2D). The ability of our ICOS protein to signal was confirmed in separate studies which showed that expression of this construct functioned to increase CD8+ T cell expansion and alter memory differentiation in a model of Listeria infection (data not shown).

Bottom Line: We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model.Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known.In this study, we created retrogenic donor-reactive CD8+ T cells that overexpress ICOS in order to determine whether reduced ICOS expression mechanistically underlies the increased efficacy of selective CD28 blockade in controlling graft-specific T cell responses as compared to conventional costimulation blockade with CTLA-4 Ig.

View Article: PubMed Central - PubMed

Affiliation: Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA 30322, United States of America.

ABSTRACT
Programmed T cell differentiation is critically influenced by the complement of costimulatory and coinhibitory signals transmitted during initial antigen encounter. We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model. Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known. In this study, we created retrogenic donor-reactive CD8+ T cells that overexpress ICOS in order to determine whether reduced ICOS expression mechanistically underlies the increased efficacy of selective CD28 blockade in controlling graft-specific T cell responses as compared to conventional costimulation blockade with CTLA-4 Ig. Results indicated that the ability of selective CD28 blockade to blunt donor-reactive CD8+ T cell expansion following transplantation was independent of its ability to inhibit ICOS expression. Furthermore, we have previously published that 2B4 coinhibitory signals are functionally important for controlling graft-specific CD8+ T cell responses in mice treated with CD28 blockade. Here we used a co-adoptive transfer approach to determine that 2B4 coinhibitory signals on antigen-specific CD8+ T cells function in a cell-intrinsic manner to limit ICOS expression in the setting of selective CD28 blockade.

No MeSH data available.


Related in: MedlinePlus