Anti-CD154 and DST distinctly alter alloreactive CD8⁺ T cell programming into cytokine-producing effector cells.

<p>B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. A. Representative flow plots of TNF and IFN-γ producing CD8⁺ T cells after <i>ex vivo</i> restimulation with BALB/c splenocytes, isolated from spleens of mice at day 7 post-transplantation. B. Absolute count of total IFN-γ producing CD8⁺ T cells in the spleen over time following <i>ex vivo</i> restimulation. C. Pie charts represent total activated CD44^high CD62L^low CD8⁺ T cells. The black wedges represent the frequency of activated CD44^high CD62L^low CD8⁺ T cells that produce IFN-γ on day 7 post-transplantation (p<0.05). D. Pie charts represent all IFN-γ producing CD8⁺ T cells. The striped wedges represent the IFN-γ-only producing population and black segments represent the TNF/IFN-γ double producing population in untreated vs. DST treated mice on day 7 (p = 0.0028). Data are summary of two experiments with three mice per group. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Efficacy of selective CD28 blockade in inhibiting donor-reactive CD8⁺ T cell cytokine production is independent of its ability to inhibit ICOS expression.

<p>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.</p

Anti-CD154 and DST independently alter the expansion kinetics of activated CD44^high CD62L^low CD8⁺ T cells.

<p>B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. A. Representative flow plots of CD44^high and CD62L^low CD8⁺ T cells isolated from spleens of mice on day 7 post-transplantation. B. Expansion kinetics of activated CD8⁺ T cells after allo-transplantation. C. Accumulation of CD44^high CD62L^low CD8⁺ T cells on day 10, 14, and 50 post-transplantation. Data are summary of two experiments with three mice per group. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Anti-CD154 treatment increases the frequency of KLRG-1^high short-lived CD8⁺ effectors.

<p>B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. A. Flow plots of KLRG-1 expression on antigen experienced CD44^high CD62L^low CD8⁺ T cells at day 7 post-transplantation. B. Frequency of KLRG-1^high antigen experienced CD44^high CD62L^low CD8⁺ T cells on day 7. C. Absolute count of alloreactive CD4⁺ T cells producing IFN-γ on day 7. Data are summary of two experiments with three mice per group. Values are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

2B4 impacts ICOS expression in the setting of selective CD28 blockade in a cell-intrinsic manner.

<p>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.</p

Upregulation of 2B4 (CD244) following selective CD28 blockade is independent of degree of ICOS expression.

<p>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⁺ OT-II T cells and then challenged with an OVA-expressing skin graft. Groups of animals were left untreated or treated with anti-CD28 dAb. Animals were sacrificed at day 10 post transplant, and graft-draining LN T cells were analyzed for the expression of the magnitude of the CD8⁺ Thy1.1⁺ response. A, Representative flow cytometry plots depicting CD62L expression on control (left panel) or ICOSrg (right panel) Thy1.1⁺ CD8⁺ T cells in the treatment groups indicated. B, Summary data of CD62L expression (MFI) on control or ICOSrg Thy1.1⁺ CD8⁺ T cells (three independent experiments with a total of n = 6–9 animals/group). C, Representative flow cytometry plots depicting 2B4 expression on ICOSrg Thy1.1⁺ CD8⁺ T cells in the treatment groups indicated. B, Summary data of 4 expression (MFI) on ICOSrg Thy1.1⁺ CD8⁺ T cells (three independent experiments with a total of n = 6–9 animals/group). *p<0.05.</p

Anti-CD154 and DST interact to protect allogeneic grafts from rejection.

<p>A. B6-Ly5.2/Cr mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c splenocytes (DST) and/ or anti-CD154 monoclonal antibody (500 μg on D0, 2, 4, 6), where indicated. B. Allo-skin grafts in untreated mice had an MST of 13 days. Monotherapy with either CD40/CD154 pathway blockade or DST led to rapid rejection of the allograft with MSTs of 17.5d (p = 0.039) and 13d (p = n.s.), respectively. Anti-CD154 and DST combined treated significantly prolonged allograft survival to 50 days (p = 0.0002). Data are summary of two experiments of 4–5 mice per group. *p<0.05, ***p<0.001.</p

Generation of retrogenic graft-specific CD8⁺ T cells that constitutively express ICOS.

<p>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.</p

Efficacy of selective CD28 blockade in controlling donor-reactive CD8⁺ T cell expansion is independent of its ability to inhibit ICOS expression.

<p>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 then challenged with an OVA-expressing skin graft. Groups of animals were left untreated or treated with anti-CD28 dAb. Animals were sacrificed at day 10 post transplant, and graft-draining LN T cells were analyzed for the expression of the magnitude of the CD8⁺ Thy1.1⁺ response. A, Representative flow cytometry plots depicting frequencies of CD44^hi Thy1.1⁺ CD8⁺ T cells in the treatment groups indicated. B, Summary data of the frequencies of CD44^hi Thy1.1⁺ CD8⁺ T cells (three independent experiments with a total of n = 6–9 animals/group). C, Summary data of the absolute numbers of CD44^hi Thy1.1⁺ CD8⁺ T cells (three independent experiments with a total of n = 6–9 animals/group). D, Summary data of the frequencies of CD44^hi Thy1.1⁺ CD4⁺ T cells (three independent experiments with a total of n = 6–9 animals/group). C, Summary data of the absolute numbers of CD44^hi Thy1.1⁺ CD4⁺ T cells (three independent experiments with a total of n = 6–9 animals/group). *p<0.05, **p<0.01.</p

CD154 blockade decreases CXCL1, CCL3, and CCL5 expression in allografts.

<p>B6.SJL mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/ or anti-CD154 mAb, where indicated. On day 7, skin grafts were explanted and processed for RNA extraction. Real time PCRs for chemokines CXCL1/ KC, CCL3/ MIP-1α, and CCL5/ RANTES were performed from cDNA. Data are summary of two experiments with three mice per group. Values are mean ± SEM. **p<0.01, ***p<0.0001.</p