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Targeting immune co-stimulatory effects of PD-L1 and PD-L2 might represent an effective therapeutic strategy in stroke.

Bodhankar S, Chen Y, Lapato A, Vandenbark AA, Murphy SJ, Offner H - Front Cell Neurosci (2014)

Bottom Line: We found that antibody neutralization of PD-1 and CTLA-4 signaling post-MCAO resulted in higher proliferation in WT CD8(+) and CD4(+) T-cells, confirming an inhibitory role of PD-1 and CTLA-4 on T-cell activation.PD-L2 was crucial in modulating CD4(+) T-cell responses, whereas PD-L1 regulated both CD8(+) and CD4(+) T-cells.PD-L2- but not PD-L1-deficient recipients of IL-10(+) B-cells had markedly reduced infarct volumes, indicating a regulatory role of PD-L2 on Bregs.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Research, Portland Veterans Affairs Medical Center Portland, OR, USA ; Department of Neurology, Oregon Health and Science University Portland, OR, USA.

ABSTRACT
Stroke outcome is worsened by the infiltration of inflammatory immune cells into ischemic brains. Our recent study demonstrated that PD-L1- and to a lesser extent PD-L2-deficient mice had smaller brain infarcts and fewer brain-infiltrating cells vs. wild-type (WT) mice, suggesting a pathogenic role for PD-ligands in experimental stroke. We sought to ascertain PD-L1 and PD-L2-expressing cell types that affect T-cell activation, post-stroke in the context of other known co-stimulatory molecules. Thus, cells from male WT and PD-L-deficient mice undergoing 60 min of middle cerebral artery occlusion (MCAO) followed by 96 h of reperfusion were treated with neutralizing antibodies to study co-stimulatory and co-inhibitory interactions between CD80, cytotoxic T-lymphocyte antigen-4 (CTLA-4), PD-1, and PD-Ls that regulate CD8(+) and CD4(+) T-cell activation. We found that antibody neutralization of PD-1 and CTLA-4 signaling post-MCAO resulted in higher proliferation in WT CD8(+) and CD4(+) T-cells, confirming an inhibitory role of PD-1 and CTLA-4 on T-cell activation. Also, CD80/CD28 interactions played a prominent regulatory role for the CD8(+) T-cells and the PD-1/PD-L2 interactions were dominant in controlling the CD4(+) T-cell responses in WT mice after stroke. A suppressive phenotype in PD-L1-deficient mice was attributed to CD80/CTLA-4 and PD-1/PD-L2 interactions. PD-L2 was crucial in modulating CD4(+) T-cell responses, whereas PD-L1 regulated both CD8(+) and CD4(+) T-cells. To establish the contribution of PD-L1 and PD-L2 on regulatory B-cells (Bregs), infarct volumes were evaluated in male PD-L1- and PD-L2-deficient mice receiving IL-10(+) B-cells 4h post-MCAO. PD-L2- but not PD-L1-deficient recipients of IL-10(+) B-cells had markedly reduced infarct volumes, indicating a regulatory role of PD-L2 on Bregs. These results imply that PD-L1 and PD-L2 differentially control induction of T- and Breg-cell responses after MCAO, thus suggesting that selective targeting of PD-L1 and PD-L2 might represent a valuable therapeutic strategy in stroke.

No MeSH data available.


Related in: MedlinePlus

Bregs decrease infarct volumes in male PD-L2-/- mice, but are dispensable when transferred to PD-L1-/- mice 4 h after MCAO. (A) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L1-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative 2,3,5-triphenyltetrazolium chloride (TTC) stained cerebral sections 96 h following 60 min of MCAO. (B) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L2-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative TTC stained cerebral sections 96 h following 60 min of MCAO. Significance values represent mean ± SEM. *p < 0.05, **p < 0.01. Splenocytes from sham-treated and MCAO-subjected WT, PD-L1-/- and PD-L2-/- mice were harvested 96 h after MCAO (60 min) and assessed for expression of: (C) CD1dhiCD5+CD19+ (Breg) cells and (D) IL-10 production by gated Breg cells. Data are representative of two independent experiments with spleens processed from four to five individual mice (mean ± SEM). Significant differences between sample means are indicated (*p ≤ 0.05 compared to the PD-L1-/- mice, post MCAO). Significant differences within the strains are indicated (#p ≤ 0.05 and ###p ≤ 0.001 as compared to its respective sham-treated counterparts).
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Figure 8: Bregs decrease infarct volumes in male PD-L2-/- mice, but are dispensable when transferred to PD-L1-/- mice 4 h after MCAO. (A) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L1-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative 2,3,5-triphenyltetrazolium chloride (TTC) stained cerebral sections 96 h following 60 min of MCAO. (B) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L2-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative TTC stained cerebral sections 96 h following 60 min of MCAO. Significance values represent mean ± SEM. *p < 0.05, **p < 0.01. Splenocytes from sham-treated and MCAO-subjected WT, PD-L1-/- and PD-L2-/- mice were harvested 96 h after MCAO (60 min) and assessed for expression of: (C) CD1dhiCD5+CD19+ (Breg) cells and (D) IL-10 production by gated Breg cells. Data are representative of two independent experiments with spleens processed from four to five individual mice (mean ± SEM). Significant differences between sample means are indicated (*p ≤ 0.05 compared to the PD-L1-/- mice, post MCAO). Significant differences within the strains are indicated (#p ≤ 0.05 and ###p ≤ 0.001 as compared to its respective sham-treated counterparts).

Mentions: B cells obtained from spleens of IL-10-GFP reporter mice were purified by negative selection and cultured in vitro for 48 h in presence of LPS. This in vitro culture enriched the B cells to produce high amounts of IL-10 cytokine. These IL-10-enriched Bregs were transferred to PD-L1-/- and PD-L2-/- recipients. As shown in Figure 8A, PD-L1-/- mice that received IL10+ B-cells (n = 8) 4 h after MCAO exhibited no differences in the infarct volumes in each of the cortex, striatum and hemisphere regions after 60 min MCAO followed by 96 h of reperfusion compared to no-cell transferred vehicle (RPMI) controls (n = 7). However, when IL-10+ B-cells were transferred to the PD-L2-/- mice (n = 10) 4 h after MCAO, there was as significant reduction in cortical (p ≤ 0.05) and total hemisphere (p ≤ 0.01) infarct volumes after 60 min MCAO followed by 96 h of reperfusion compared to no-cell transferred vehicle (RPMI) controls (n = 11). Representative cerebral sections from PD-L1-/- and PD-L2-/- mice treated with RPMI or IL10+ B-cells are shown in Figures 8A,B.


Targeting immune co-stimulatory effects of PD-L1 and PD-L2 might represent an effective therapeutic strategy in stroke.

Bodhankar S, Chen Y, Lapato A, Vandenbark AA, Murphy SJ, Offner H - Front Cell Neurosci (2014)

Bregs decrease infarct volumes in male PD-L2-/- mice, but are dispensable when transferred to PD-L1-/- mice 4 h after MCAO. (A) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L1-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative 2,3,5-triphenyltetrazolium chloride (TTC) stained cerebral sections 96 h following 60 min of MCAO. (B) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L2-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative TTC stained cerebral sections 96 h following 60 min of MCAO. Significance values represent mean ± SEM. *p < 0.05, **p < 0.01. Splenocytes from sham-treated and MCAO-subjected WT, PD-L1-/- and PD-L2-/- mice were harvested 96 h after MCAO (60 min) and assessed for expression of: (C) CD1dhiCD5+CD19+ (Breg) cells and (D) IL-10 production by gated Breg cells. Data are representative of two independent experiments with spleens processed from four to five individual mice (mean ± SEM). Significant differences between sample means are indicated (*p ≤ 0.05 compared to the PD-L1-/- mice, post MCAO). Significant differences within the strains are indicated (#p ≤ 0.05 and ###p ≤ 0.001 as compared to its respective sham-treated counterparts).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Bregs decrease infarct volumes in male PD-L2-/- mice, but are dispensable when transferred to PD-L1-/- mice 4 h after MCAO. (A) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L1-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative 2,3,5-triphenyltetrazolium chloride (TTC) stained cerebral sections 96 h following 60 min of MCAO. (B) Intravenous transfer of 5 million IL10+ B-cells 4 h after surgery to induce MCAO in PD-L2-/- mice 96 h following 60 min of MCAO compared to intravenous transfer of RPMI vehicle (no cells) and its representative TTC stained cerebral sections 96 h following 60 min of MCAO. Significance values represent mean ± SEM. *p < 0.05, **p < 0.01. Splenocytes from sham-treated and MCAO-subjected WT, PD-L1-/- and PD-L2-/- mice were harvested 96 h after MCAO (60 min) and assessed for expression of: (C) CD1dhiCD5+CD19+ (Breg) cells and (D) IL-10 production by gated Breg cells. Data are representative of two independent experiments with spleens processed from four to five individual mice (mean ± SEM). Significant differences between sample means are indicated (*p ≤ 0.05 compared to the PD-L1-/- mice, post MCAO). Significant differences within the strains are indicated (#p ≤ 0.05 and ###p ≤ 0.001 as compared to its respective sham-treated counterparts).
Mentions: B cells obtained from spleens of IL-10-GFP reporter mice were purified by negative selection and cultured in vitro for 48 h in presence of LPS. This in vitro culture enriched the B cells to produce high amounts of IL-10 cytokine. These IL-10-enriched Bregs were transferred to PD-L1-/- and PD-L2-/- recipients. As shown in Figure 8A, PD-L1-/- mice that received IL10+ B-cells (n = 8) 4 h after MCAO exhibited no differences in the infarct volumes in each of the cortex, striatum and hemisphere regions after 60 min MCAO followed by 96 h of reperfusion compared to no-cell transferred vehicle (RPMI) controls (n = 7). However, when IL-10+ B-cells were transferred to the PD-L2-/- mice (n = 10) 4 h after MCAO, there was as significant reduction in cortical (p ≤ 0.05) and total hemisphere (p ≤ 0.01) infarct volumes after 60 min MCAO followed by 96 h of reperfusion compared to no-cell transferred vehicle (RPMI) controls (n = 11). Representative cerebral sections from PD-L1-/- and PD-L2-/- mice treated with RPMI or IL10+ B-cells are shown in Figures 8A,B.

Bottom Line: We found that antibody neutralization of PD-1 and CTLA-4 signaling post-MCAO resulted in higher proliferation in WT CD8(+) and CD4(+) T-cells, confirming an inhibitory role of PD-1 and CTLA-4 on T-cell activation.PD-L2 was crucial in modulating CD4(+) T-cell responses, whereas PD-L1 regulated both CD8(+) and CD4(+) T-cells.PD-L2- but not PD-L1-deficient recipients of IL-10(+) B-cells had markedly reduced infarct volumes, indicating a regulatory role of PD-L2 on Bregs.

View Article: PubMed Central - PubMed

Affiliation: Neuroimmunology Research, Portland Veterans Affairs Medical Center Portland, OR, USA ; Department of Neurology, Oregon Health and Science University Portland, OR, USA.

ABSTRACT
Stroke outcome is worsened by the infiltration of inflammatory immune cells into ischemic brains. Our recent study demonstrated that PD-L1- and to a lesser extent PD-L2-deficient mice had smaller brain infarcts and fewer brain-infiltrating cells vs. wild-type (WT) mice, suggesting a pathogenic role for PD-ligands in experimental stroke. We sought to ascertain PD-L1 and PD-L2-expressing cell types that affect T-cell activation, post-stroke in the context of other known co-stimulatory molecules. Thus, cells from male WT and PD-L-deficient mice undergoing 60 min of middle cerebral artery occlusion (MCAO) followed by 96 h of reperfusion were treated with neutralizing antibodies to study co-stimulatory and co-inhibitory interactions between CD80, cytotoxic T-lymphocyte antigen-4 (CTLA-4), PD-1, and PD-Ls that regulate CD8(+) and CD4(+) T-cell activation. We found that antibody neutralization of PD-1 and CTLA-4 signaling post-MCAO resulted in higher proliferation in WT CD8(+) and CD4(+) T-cells, confirming an inhibitory role of PD-1 and CTLA-4 on T-cell activation. Also, CD80/CD28 interactions played a prominent regulatory role for the CD8(+) T-cells and the PD-1/PD-L2 interactions were dominant in controlling the CD4(+) T-cell responses in WT mice after stroke. A suppressive phenotype in PD-L1-deficient mice was attributed to CD80/CTLA-4 and PD-1/PD-L2 interactions. PD-L2 was crucial in modulating CD4(+) T-cell responses, whereas PD-L1 regulated both CD8(+) and CD4(+) T-cells. To establish the contribution of PD-L1 and PD-L2 on regulatory B-cells (Bregs), infarct volumes were evaluated in male PD-L1- and PD-L2-deficient mice receiving IL-10(+) B-cells 4h post-MCAO. PD-L2- but not PD-L1-deficient recipients of IL-10(+) B-cells had markedly reduced infarct volumes, indicating a regulatory role of PD-L2 on Bregs. These results imply that PD-L1 and PD-L2 differentially control induction of T- and Breg-cell responses after MCAO, thus suggesting that selective targeting of PD-L1 and PD-L2 might represent a valuable therapeutic strategy in stroke.

No MeSH data available.


Related in: MedlinePlus