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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

Efficacy of selective CD28 blockade in inhibiting donor-reactive CD8+ T cell cytokine production is independent of its ability to inhibit ICOS expression.Naïve B6 animals were adoptively transferred with 106 congenically labeled ICOSrg Thy1.1+ OT-I T cells (or pMY Thy1.1+ OT-I controls) along with 106 Thy1.1+ CD4+ WT OT-II T cells and were then grafted with an OVA-expressing skin graft in the presence or absence of selective CD28 blockade. Ten days post-transplant, splenocytes were restimulated for 4h in vitro with cognate SIINFEKL peptide and assessed for their ability to secrete IFN-γ, TNF, and IL-2 via intracellular cytokine staining. A, Representative flow cytometry plots depicting IFN-γ and TNF staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ TNF+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). C, Representative flow cytometry plots depicting IFN-γ and IL-2 staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ IL-2+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). *p<0.05.
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pone.0130490.g004: Efficacy of selective CD28 blockade in inhibiting donor-reactive CD8+ T cell cytokine production is independent of its ability to inhibit ICOS expression.Naïve B6 animals were adoptively transferred with 106 congenically labeled ICOSrg Thy1.1+ OT-I T cells (or pMY Thy1.1+ OT-I controls) along with 106 Thy1.1+ CD4+ WT OT-II T cells and were then grafted with an OVA-expressing skin graft in the presence or absence of selective CD28 blockade. Ten days post-transplant, splenocytes were restimulated for 4h in vitro with cognate SIINFEKL peptide and assessed for their ability to secrete IFN-γ, TNF, and IL-2 via intracellular cytokine staining. A, Representative flow cytometry plots depicting IFN-γ and TNF staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ TNF+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). C, Representative flow cytometry plots depicting IFN-γ and IL-2 staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ IL-2+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). *p<0.05.

Mentions: Given the above results, we next questioned whether reduced ICOS expression was required for the ability of anti-CD28 dAb to effectively control the functionality of donor-reactive CD8+ T cell responses during transplantation. To test this, a similar experiment was conducted in which ICOSrg Thy1.1+ OT-I T cells (or pMY Thy1.1+ OT-I controls) were adoptively transferred into naïve B6 animals, which were then grafted with an OVA-expressing skin graft in the presence or absence of selective CD28 blockade. Ten days post-transplant, spleens and draining LN were harvested and restimulated in vitro with cognate peptide and assessed for their ability to secrete IFN-γ, TNF, and IL-2 via intracellular cytokine staining. Similar to what we observed in terms of donor-reactive CD8+ T cell expansion and accumulation, we observed that the constitutive overexpression of ICOS on donor-reactive T cells did not result in increased functionality in terms of their ability to secrete IFN-γ, TNF (Fig 4A and 4B), or IL-2 (Fig 4C and 4D). As expected, anti-CD28 dAb potently inhibited the frequencies of IFN-γ+ TNF+ double producers and IFN-γ+ IL-2+ double producers in recipients of pMY graft-specific OT-I T cells. Importantly, the efficacy of anti-CD28 dAb in inhibiting the acquisition of cytokine-producing effector function by donor-reactive OT-I T cells was not altered in the ICOSrg OT-I T cells (Fig 4A–4D).


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)

Efficacy of selective CD28 blockade in inhibiting donor-reactive CD8+ T cell cytokine production is independent of its ability to inhibit ICOS expression.Naïve B6 animals were adoptively transferred with 106 congenically labeled ICOSrg Thy1.1+ OT-I T cells (or pMY Thy1.1+ OT-I controls) along with 106 Thy1.1+ CD4+ WT OT-II T cells and were then grafted with an OVA-expressing skin graft in the presence or absence of selective CD28 blockade. Ten days post-transplant, splenocytes were restimulated for 4h in vitro with cognate SIINFEKL peptide and assessed for their ability to secrete IFN-γ, TNF, and IL-2 via intracellular cytokine staining. A, Representative flow cytometry plots depicting IFN-γ and TNF staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ TNF+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). C, Representative flow cytometry plots depicting IFN-γ and IL-2 staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ IL-2+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476729&req=5

pone.0130490.g004: Efficacy of selective CD28 blockade in inhibiting donor-reactive CD8+ T cell cytokine production is independent of its ability to inhibit ICOS expression.Naïve B6 animals were adoptively transferred with 106 congenically labeled ICOSrg Thy1.1+ OT-I T cells (or pMY Thy1.1+ OT-I controls) along with 106 Thy1.1+ CD4+ WT OT-II T cells and were then grafted with an OVA-expressing skin graft in the presence or absence of selective CD28 blockade. Ten days post-transplant, splenocytes were restimulated for 4h in vitro with cognate SIINFEKL peptide and assessed for their ability to secrete IFN-γ, TNF, and IL-2 via intracellular cytokine staining. A, Representative flow cytometry plots depicting IFN-γ and TNF staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ TNF+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). C, Representative flow cytometry plots depicting IFN-γ and IL-2 staining in the indicated treatment groups. B, Summary data of frequencies of IFN-γ+ IL-2+ double producers in the indicated treatment groups (three independent experiments with a total of n = 6–9 animals/group). *p<0.05.
Mentions: Given the above results, we next questioned whether reduced ICOS expression was required for the ability of anti-CD28 dAb to effectively control the functionality of donor-reactive CD8+ T cell responses during transplantation. To test this, a similar experiment was conducted in which ICOSrg Thy1.1+ OT-I T cells (or pMY Thy1.1+ OT-I controls) were adoptively transferred into naïve B6 animals, which were then grafted with an OVA-expressing skin graft in the presence or absence of selective CD28 blockade. Ten days post-transplant, spleens and draining LN were harvested and restimulated in vitro with cognate peptide and assessed for their ability to secrete IFN-γ, TNF, and IL-2 via intracellular cytokine staining. Similar to what we observed in terms of donor-reactive CD8+ T cell expansion and accumulation, we observed that the constitutive overexpression of ICOS on donor-reactive T cells did not result in increased functionality in terms of their ability to secrete IFN-γ, TNF (Fig 4A and 4B), or IL-2 (Fig 4C and 4D). As expected, anti-CD28 dAb potently inhibited the frequencies of IFN-γ+ TNF+ double producers and IFN-γ+ IL-2+ double producers in recipients of pMY graft-specific OT-I T cells. Importantly, the efficacy of anti-CD28 dAb in inhibiting the acquisition of cytokine-producing effector function by donor-reactive OT-I T cells was not altered in the ICOSrg OT-I T cells (Fig 4A–4D).

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