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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

Subtle changes in the proliferative capacities in the PD-L2-/- mice indicate a minor role of PD-L2 in ischemic stroke. T-cells were purified, by negative sort, from the spleens of MCAO-subjected PD-L2-/- mice, by labeling with specific microbeads and separating on the AutoMACSTM. The purified T-cells were CSFE-labeled and then cultured with non-T-cells from the same mice at a 1:1 ratio (T:APC) in the presence of anti-CD3 antibody (2.5 μg/mL; baseline) and other neutralizing Abs (10 μg/mL) to co-stimulatory molecules, in 96-well plates. After 72 h of culture, cells were washed and evaluated by FACS Calibur for (A) CD8+ and (B) CD4+ T-cell expression and CFSE dilution. Data represent the stimulation indices of the CD8 and CD4 T-cells in the presence of neutralizing Abs as compared to the control (T:APC+anti-CD3 Ab) condition). The data are represented such that the baseline value is 1 and all other values are adjusted relative to the baseline. Data presented are representative of splenocytes obtained from seven PD-L2-/- mice, with at least three separate experiments and each experiment comprising duplicates or triplicates of the given neutralizing Ab condition. Significant differences between sample means are indicated as *p ≤ 0.05, **p ≤ 0.01 as compared to the baseline condition.
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Figure 4: Subtle changes in the proliferative capacities in the PD-L2-/- mice indicate a minor role of PD-L2 in ischemic stroke. T-cells were purified, by negative sort, from the spleens of MCAO-subjected PD-L2-/- mice, by labeling with specific microbeads and separating on the AutoMACSTM. The purified T-cells were CSFE-labeled and then cultured with non-T-cells from the same mice at a 1:1 ratio (T:APC) in the presence of anti-CD3 antibody (2.5 μg/mL; baseline) and other neutralizing Abs (10 μg/mL) to co-stimulatory molecules, in 96-well plates. After 72 h of culture, cells were washed and evaluated by FACS Calibur for (A) CD8+ and (B) CD4+ T-cell expression and CFSE dilution. Data represent the stimulation indices of the CD8 and CD4 T-cells in the presence of neutralizing Abs as compared to the control (T:APC+anti-CD3 Ab) condition). The data are represented such that the baseline value is 1 and all other values are adjusted relative to the baseline. Data presented are representative of splenocytes obtained from seven PD-L2-/- mice, with at least three separate experiments and each experiment comprising duplicates or triplicates of the given neutralizing Ab condition. Significant differences between sample means are indicated as *p ≤ 0.05, **p ≤ 0.01 as compared to the baseline condition.

Mentions: When similar studies were conducted using splenocytes from MCAO-subjected PD-L2-/- mice, the responses, although more subtle, strongly support results obtained from WT and PD-L1-/- mice. In the absence of PD-L2, proliferation of CD8+ T-cells was diminished by treatment with neutralizing anti-CD28 Ab and with the combination of anti-CD28 + anti-CD80 Abs (p ≤ 0.01 for each, Figure 4A), again implicating CD28/CD80 as the major co-stimulatory pathway that is enabled by co-expression of PD-L1. Similarly, CD4+ T-cells demonstrated significantly decreased proliferative responses with anti-CD28 Ab alone (p ≤ 0.01) and with the combination of anti-CD28 + anti-CD80 Abs (p ≤ 0.05). Moreover, the proliferation response of CD4+ T-cells was inhibited by treatment with the combination of anti-CTLA-4 + anti-CD80 Abs (p ≤ 0.01, Figure 4B), thus implicating a new co-stimulatory pathway that emerged in the absence of PD-L2. Notably, in the absence of PD-L2, there was no significant neutralization of any tested co-inhibitory molecules, particularly PD-1, thus confirming the requirement for PD-L2 in co-inhibitory regulation of proliferation responses for both CD4+ and CD8+ T-cells.


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)

Subtle changes in the proliferative capacities in the PD-L2-/- mice indicate a minor role of PD-L2 in ischemic stroke. T-cells were purified, by negative sort, from the spleens of MCAO-subjected PD-L2-/- mice, by labeling with specific microbeads and separating on the AutoMACSTM. The purified T-cells were CSFE-labeled and then cultured with non-T-cells from the same mice at a 1:1 ratio (T:APC) in the presence of anti-CD3 antibody (2.5 μg/mL; baseline) and other neutralizing Abs (10 μg/mL) to co-stimulatory molecules, in 96-well plates. After 72 h of culture, cells were washed and evaluated by FACS Calibur for (A) CD8+ and (B) CD4+ T-cell expression and CFSE dilution. Data represent the stimulation indices of the CD8 and CD4 T-cells in the presence of neutralizing Abs as compared to the control (T:APC+anti-CD3 Ab) condition). The data are represented such that the baseline value is 1 and all other values are adjusted relative to the baseline. Data presented are representative of splenocytes obtained from seven PD-L2-/- mice, with at least three separate experiments and each experiment comprising duplicates or triplicates of the given neutralizing Ab condition. Significant differences between sample means are indicated as *p ≤ 0.05, **p ≤ 0.01 as compared to the baseline condition.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Subtle changes in the proliferative capacities in the PD-L2-/- mice indicate a minor role of PD-L2 in ischemic stroke. T-cells were purified, by negative sort, from the spleens of MCAO-subjected PD-L2-/- mice, by labeling with specific microbeads and separating on the AutoMACSTM. The purified T-cells were CSFE-labeled and then cultured with non-T-cells from the same mice at a 1:1 ratio (T:APC) in the presence of anti-CD3 antibody (2.5 μg/mL; baseline) and other neutralizing Abs (10 μg/mL) to co-stimulatory molecules, in 96-well plates. After 72 h of culture, cells were washed and evaluated by FACS Calibur for (A) CD8+ and (B) CD4+ T-cell expression and CFSE dilution. Data represent the stimulation indices of the CD8 and CD4 T-cells in the presence of neutralizing Abs as compared to the control (T:APC+anti-CD3 Ab) condition). The data are represented such that the baseline value is 1 and all other values are adjusted relative to the baseline. Data presented are representative of splenocytes obtained from seven PD-L2-/- mice, with at least three separate experiments and each experiment comprising duplicates or triplicates of the given neutralizing Ab condition. Significant differences between sample means are indicated as *p ≤ 0.05, **p ≤ 0.01 as compared to the baseline condition.
Mentions: When similar studies were conducted using splenocytes from MCAO-subjected PD-L2-/- mice, the responses, although more subtle, strongly support results obtained from WT and PD-L1-/- mice. In the absence of PD-L2, proliferation of CD8+ T-cells was diminished by treatment with neutralizing anti-CD28 Ab and with the combination of anti-CD28 + anti-CD80 Abs (p ≤ 0.01 for each, Figure 4A), again implicating CD28/CD80 as the major co-stimulatory pathway that is enabled by co-expression of PD-L1. Similarly, CD4+ T-cells demonstrated significantly decreased proliferative responses with anti-CD28 Ab alone (p ≤ 0.01) and with the combination of anti-CD28 + anti-CD80 Abs (p ≤ 0.05). Moreover, the proliferation response of CD4+ T-cells was inhibited by treatment with the combination of anti-CTLA-4 + anti-CD80 Abs (p ≤ 0.01, Figure 4B), thus implicating a new co-stimulatory pathway that emerged in the absence of PD-L2. Notably, in the absence of PD-L2, there was no significant neutralization of any tested co-inhibitory molecules, particularly PD-1, thus confirming the requirement for PD-L2 in co-inhibitory regulation of proliferation responses for both CD4+ and CD8+ T-cells.

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