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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

2B4 impacts ICOS expression in the setting of selective CD28 blockade in a cell-intrinsic manner.106 CD45.2+ Thy1.2+ OT-I (black) or 106 CD45.2+ Thy1.1+ 2B4-/- OT-I (red) were adoptively transferred along with 106 CD45.2+ Thy1.1+ OT-II T cells into naïve CD45.1+ B6 recipients, which were then challenged with an OVA expressing skin graft in the presence of either control dAb or anti-CD28 dAb (A). Graft-draining LN were harvested on day 10 post-transplant and analyzed by flow cytometry. B, ICOS expression on WT (black, Thy1.1-) and 2B4-/- (red, Thy1.1+) donor-reactive CD8+ Thy1.1+ T cells in the lymph nodes and spleens of animals treated with anti-CD28 dAb. C-D, Summary data of ICOS MFI on WT Thy1.2+ and 2B4-/- Thy1.1+ T cells in the spleens on day 10 of untreated and anti-CD28 dAb treated grafted animals (C) and frequencies of WT Thy1.2+ ICOS+ and 2B4-/- Thy1.1+ ICOS+ cells (D) in the spleens on day 10 of naïve (no skin graft), untreated grafted, anti-CD28 dAb treated grafted animals (two independent experiments with a total of n = 8 animals/group). E, Model of the cell intrinsic role for 2B4 in modulating ICOS expression in the setting of selective CD28 blockade. During an unmodified immune response, CD28 costimulatory signals predominate and result in ICOS expression following T cell activation (left panel). However, in the setting of CD28 dAb, 2B4 is upregulated and contribute to decreased ICOS expression in a cell-intrinsic manner. *p<0.05, ns = not significant.
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pone.0130490.g006: 2B4 impacts ICOS expression in the setting of selective CD28 blockade in a cell-intrinsic manner.106 CD45.2+ Thy1.2+ OT-I (black) or 106 CD45.2+ Thy1.1+ 2B4-/- OT-I (red) were adoptively transferred along with 106 CD45.2+ Thy1.1+ OT-II T cells into naïve CD45.1+ B6 recipients, which were then challenged with an OVA expressing skin graft in the presence of either control dAb or anti-CD28 dAb (A). Graft-draining LN were harvested on day 10 post-transplant and analyzed by flow cytometry. B, ICOS expression on WT (black, Thy1.1-) and 2B4-/- (red, Thy1.1+) donor-reactive CD8+ Thy1.1+ T cells in the lymph nodes and spleens of animals treated with anti-CD28 dAb. C-D, Summary data of ICOS MFI on WT Thy1.2+ and 2B4-/- Thy1.1+ T cells in the spleens on day 10 of untreated and anti-CD28 dAb treated grafted animals (C) and frequencies of WT Thy1.2+ ICOS+ and 2B4-/- Thy1.1+ ICOS+ cells (D) in the spleens on day 10 of naïve (no skin graft), untreated grafted, anti-CD28 dAb treated grafted animals (two independent experiments with a total of n = 8 animals/group). E, Model of the cell intrinsic role for 2B4 in modulating ICOS expression in the setting of selective CD28 blockade. During an unmodified immune response, CD28 costimulatory signals predominate and result in ICOS expression following T cell activation (left panel). However, in the setting of CD28 dAb, 2B4 is upregulated and contribute to decreased ICOS expression in a cell-intrinsic manner. *p<0.05, ns = not significant.

Mentions: Given the above findings suggesting that the lack of ICOS-mediated signals on graft-specific CD8+ T cells isolated from anti-CD28 dAb treated mice was not responsible for the observed increased 2B4 expression on these cells, we next interrogated the reciprocal possibility: whether 2B4 coinhibitory signals were instead functionally important for the observed reduction in ICOS signals on anti-CD28 dAb treated CD8+ T cell populations. In order to accomplish this, Thy1.1+ OT-I animals were bred onto a 2B4-/- background (a kind gift of Dr. Cox Terhorst). We have previously published that 2B4 coinhibitory signals are functionally important for controlling the expansion, accumulation, and effector function of graft-specific CD8+ T cells during transplantation [15], and here we use a co-adoptive transfer approach to determine whether 2B4 coinhibitory signals function in a cell-intrinsic manner to limit ICOS expression in the setting of selective CD28 blockade. Briefly, Thy1.1+ 2B4-/- T cells were co-adoptively transferred into naïve CD45.1+ B6 recipients along with an identical number of Thy1.2+ WT OT-I T cells, and animals were grafted with OVA-expressing skin grafts and treated with anti-CD28 dAb (Fig 6A). Graft-specific CD8+ T cell responses could be identified on the basis of their expression of CD45.2, and ICOS expression was interrogated on WT Thy1.2+ OT-I cells as compared to 2B4-/- Thy1.1+ OT-I cells. In examining the WT Thy1.2 population, we observed that as expected ICOS expression was downregulated on a subset of Thy1.2+ WT CD8+ T cells isolated from both the spleens and lymph nodes of animals treated with anti-CD28 dAb (Fig 6B–6D). In contrast, ICOS was highly expressed on virtually all Thy1.1+ 2B4-/- T cells in untreated recipients even in the presence of anti-CD28dAb (Fig 6B–6D). These results suggest that 2B4 mediated coinhibitory T cells on antigen-specific CD8+ T cells function in a cell-intrinsic manner to result in reduced ICOS expression observed in the setting of selective CD28 blockade.


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)

2B4 impacts ICOS expression in the setting of selective CD28 blockade in a cell-intrinsic manner.106 CD45.2+ Thy1.2+ OT-I (black) or 106 CD45.2+ Thy1.1+ 2B4-/- OT-I (red) were adoptively transferred along with 106 CD45.2+ Thy1.1+ OT-II T cells into naïve CD45.1+ B6 recipients, which were then challenged with an OVA expressing skin graft in the presence of either control dAb or anti-CD28 dAb (A). Graft-draining LN were harvested on day 10 post-transplant and analyzed by flow cytometry. B, ICOS expression on WT (black, Thy1.1-) and 2B4-/- (red, Thy1.1+) donor-reactive CD8+ Thy1.1+ T cells in the lymph nodes and spleens of animals treated with anti-CD28 dAb. C-D, Summary data of ICOS MFI on WT Thy1.2+ and 2B4-/- Thy1.1+ T cells in the spleens on day 10 of untreated and anti-CD28 dAb treated grafted animals (C) and frequencies of WT Thy1.2+ ICOS+ and 2B4-/- Thy1.1+ ICOS+ cells (D) in the spleens on day 10 of naïve (no skin graft), untreated grafted, anti-CD28 dAb treated grafted animals (two independent experiments with a total of n = 8 animals/group). E, Model of the cell intrinsic role for 2B4 in modulating ICOS expression in the setting of selective CD28 blockade. During an unmodified immune response, CD28 costimulatory signals predominate and result in ICOS expression following T cell activation (left panel). However, in the setting of CD28 dAb, 2B4 is upregulated and contribute to decreased ICOS expression in a cell-intrinsic manner. *p<0.05, ns = not significant.
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Related In: Results  -  Collection

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pone.0130490.g006: 2B4 impacts ICOS expression in the setting of selective CD28 blockade in a cell-intrinsic manner.106 CD45.2+ Thy1.2+ OT-I (black) or 106 CD45.2+ Thy1.1+ 2B4-/- OT-I (red) were adoptively transferred along with 106 CD45.2+ Thy1.1+ OT-II T cells into naïve CD45.1+ B6 recipients, which were then challenged with an OVA expressing skin graft in the presence of either control dAb or anti-CD28 dAb (A). Graft-draining LN were harvested on day 10 post-transplant and analyzed by flow cytometry. B, ICOS expression on WT (black, Thy1.1-) and 2B4-/- (red, Thy1.1+) donor-reactive CD8+ Thy1.1+ T cells in the lymph nodes and spleens of animals treated with anti-CD28 dAb. C-D, Summary data of ICOS MFI on WT Thy1.2+ and 2B4-/- Thy1.1+ T cells in the spleens on day 10 of untreated and anti-CD28 dAb treated grafted animals (C) and frequencies of WT Thy1.2+ ICOS+ and 2B4-/- Thy1.1+ ICOS+ cells (D) in the spleens on day 10 of naïve (no skin graft), untreated grafted, anti-CD28 dAb treated grafted animals (two independent experiments with a total of n = 8 animals/group). E, Model of the cell intrinsic role for 2B4 in modulating ICOS expression in the setting of selective CD28 blockade. During an unmodified immune response, CD28 costimulatory signals predominate and result in ICOS expression following T cell activation (left panel). However, in the setting of CD28 dAb, 2B4 is upregulated and contribute to decreased ICOS expression in a cell-intrinsic manner. *p<0.05, ns = not significant.
Mentions: Given the above findings suggesting that the lack of ICOS-mediated signals on graft-specific CD8+ T cells isolated from anti-CD28 dAb treated mice was not responsible for the observed increased 2B4 expression on these cells, we next interrogated the reciprocal possibility: whether 2B4 coinhibitory signals were instead functionally important for the observed reduction in ICOS signals on anti-CD28 dAb treated CD8+ T cell populations. In order to accomplish this, Thy1.1+ OT-I animals were bred onto a 2B4-/- background (a kind gift of Dr. Cox Terhorst). We have previously published that 2B4 coinhibitory signals are functionally important for controlling the expansion, accumulation, and effector function of graft-specific CD8+ T cells during transplantation [15], and here we use a co-adoptive transfer approach to determine whether 2B4 coinhibitory signals function in a cell-intrinsic manner to limit ICOS expression in the setting of selective CD28 blockade. Briefly, Thy1.1+ 2B4-/- T cells were co-adoptively transferred into naïve CD45.1+ B6 recipients along with an identical number of Thy1.2+ WT OT-I T cells, and animals were grafted with OVA-expressing skin grafts and treated with anti-CD28 dAb (Fig 6A). Graft-specific CD8+ T cell responses could be identified on the basis of their expression of CD45.2, and ICOS expression was interrogated on WT Thy1.2+ OT-I cells as compared to 2B4-/- Thy1.1+ OT-I cells. In examining the WT Thy1.2 population, we observed that as expected ICOS expression was downregulated on a subset of Thy1.2+ WT CD8+ T cells isolated from both the spleens and lymph nodes of animals treated with anti-CD28 dAb (Fig 6B–6D). In contrast, ICOS was highly expressed on virtually all Thy1.1+ 2B4-/- T cells in untreated recipients even in the presence of anti-CD28dAb (Fig 6B–6D). These results suggest that 2B4 mediated coinhibitory T cells on antigen-specific CD8+ T cells function in a cell-intrinsic manner to result in reduced ICOS expression observed in the setting of selective CD28 blockade.

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