1. by Silke Huber, Reinhard Hoffmann, Femke Muskens, and David Voehringer
Alternatively activated macrophages inhibit T-cell proliferation by Stat6-dependent expression of PD-L2
by Silke Huber, Reinhard Hoffmann, Femke Muskens, and David Voehringer
Blood
Volume 116(17):
October 28, 2010
©2010 by American Society of Hematology

2. IL-4–exposed macrophages suppress T-cell proliferation in a cell-cell contact–dependent manner.
IL-4–exposed macrophages suppress T-cell proliferation in a cell-cell contact–dependent manner. (A) CFSE-labeled wild-type (WT) splenocytes were left untreated (black line) or were stimulated with plate-bound anti-TCR/anti-CD28 (filled) and were either cultured alone (T cells only) or were cocultured with control or IL-4–treated (10 ng/mL) BMDM from WT or Stat6−/− mice at ratio of 1:3 (BMDM:splenocytes). Cells were collected after 4 days, stained for CD4 and CD8 and analyzed by flow cytometry. Histograms show the CFSE profile of gated CD4 and CD8 T cells. (B) CFSE-labeled WT splenocytes were cultured with untreated BMDM (black bars) or IL-4–treated BMDM (gray bars) from WT or Stat6−/− mice at ratios of 1:3, 1:6, and 1:10 (BMDM:splenocytes). Bars show the mean ± SD from 3 separate cultures per condition (**P < .001; *P < .05). (C) T-cell suppression is dependent on cell-cell contact. Supernatants of untreated or IL-4–treated WT and Stat6−/− BMDM were collected after 24 hours of stimulation and added to CFSE-labeled WT splenocytes that were left untreated (black line) or were stimulated (filled) as described in panel A. “T cells only” indicates cultures without addition of supernatant. The data are representative of 3 independent experiments.
Silke Huber et al. Blood 2010;116:
©2010 by American Society of Hematology

3. Stat6-regulated gene expression profile of macrophages.
Stat6-regulated gene expression profile of macrophages. (A) RNA was isolated from F4/80+-sorted (purity 99%), untreated (control), or IL-4–treated (10 ng/mL) wild-type (WT) and Stat6−/− BMDM. Gene expression profile was performed on high-density oligonucleotide arrays (Affymetrix). (B) Quantitative RT-PCR analysis of selected genes that appeared differently expressed in untreated (black bars) or IL-4–treated (gray bars) WT and Stat6−/− BMDM based on the microarray experiment. Samples were normalized to β-actin expression. The microarray data have been deposited to the Gene Expression Omnibus database under the accession number GSE20030.
Silke Huber et al. Blood 2010;116:
©2010 by American Society of Hematology

4. PD-L2 is the only B7 family member that is induced by IL-4 on BMDM in a Stat6-dependent manner.
PD-L2 is the only B7 family member that is induced by IL-4 on BMDM in a Stat6-dependent manner. (A) BMDM from wild-type mice (black line) and Stat6−/− mice (gray line) were cultured in medium alone (control) or were activated for 24 hours with IL-4 (10 ng/mL) or LPS (1 μg/mL). The cells were double-stained for F4/80 and PD-L1, PD-L2, CD80, CD86, or MHC-II. Histograms are gated on F4/80+ macrophages. Filled histograms show isotype control staining. Data shown are representative of 3 independent experiments. (B) BMDM from WT mice were cultured for 6 hours with IL-4 and sorted into PD-L2+ and PD-L2− populations. (C) PD-L2 expression correlates with higher expression levels of the AAM markers Retnla, Chi3l3, and Arg1. RNA was isolated from PD-L2+ (solid bars) or PD-L2− (open bars) BMDM and analyzed by quantitative RT-PCR. The graphs show means of 2 independent experiments, normalized to β-actin expression.
Silke Huber et al. Blood 2010;116:
©2010 by American Society of Hematology

5. PD-L2 on AAM is required and sufficient to mediate inhibition.
PD-L2 on AAM is required and sufficient to mediate inhibition. (A) Untreated (black line) or anti-TCR/anti-CD28 stimulated (filled) CFSE-labeled wild-type splenocytes were cultured with IL-4–treated (10 ng/mL) BMDM from wild-type mice in the absence (control) or presence of anti-PD-L2 (5 μg/mL) or isotype control (5 μg/mL) and analyzed after 4 days. Histograms are gated on CD4+ T cells. The experiment has been repeated with similar results. (B) BMDM from Stat6−/− mice were transduced with a bicistronic retroviral PD-L2/GFP expression vector (PD-L2 Mϕ) or empty GFP control vector (control Mϕ). Cells were stained for F4/80 and PD-L2. Dot plots are gated on F4/80+ cells. F4/80+GFP+ macrophages were sorted with > 94% purity. (C) CFSE-labeled wild-type splenocytes were left untreated (black line) or were stimulated with plate-bound anti-TCR/anti-CD28 (filled), cultured in the presence of GFP+-sorted macrophages and analyzed after 4 days. Histograms are gated on CD4+ or CD8+ cells as indicated. The data are representative of 3 independent experiments.
Silke Huber et al. Blood 2010;116:
©2010 by American Society of Hematology

6. Analysis of PD-L2 expression on macrophages in vivo during N brasiliensis infection.
Analysis of PD-L2 expression on macrophages in vivo during N brasiliensis infection. Lung (A) and BAL (B) from wild-type (WT) and Stat6−/− mice were analyzed before or at indicated time points after N brasiliensis infection for PD-L2 expression by flow cytometry. Dot plots show staining of F4/80 and PD-L2 or isotype control on CD11c+ autofluorescencehi-gated cells at day 13 after N brasiliensis infection. Graphs show the frequency and absolute number of PD-L2+ macrophages in WT (filled circles) and Stat6−/− (open circles) mice as mean ± SE with 3 individual mice from 2 independent experiments. (C) Detection of Ym1 (blue) and PD-L2 (red) expression in alveolar macrophages (arrows) in the lung of WT but not Stat6−/− mice that had been infected with N brasiliensis 13 days before. Original magnification of the objective was ×40. (D) T-cell–derived IL-4/IL-13 is required for PD-L2 expression on AAM in vivo. Analysis of PD-L2 expression on macrophages in BAL and lung (F4/80+ CD11c+ autofluorescencehi) in complete IL-4/IL-13–deficient mice (IL-4/IL-13−/−), mice deficient of IL-4/IL-13 only in T cells (CD4Cre/IL-4/IL-13F/F), and control mice (IL-4/IL-13F/F) 13 days after N brasiliensis infection. Bars show the frequency of PD-L2+ macrophages as means of 2 mice (dots).
Silke Huber et al. Blood 2010;116:
©2010 by American Society of Hematology

7. Analysis of Stat6 requirement for accumulation of macrophages during N brasiliensis infection.
Analysis of Stat6 requirement for accumulation of macrophages during N brasiliensis infection. (A) Congenic mixed BM chimeras were generated with BM cells from wild-type (Ly5.1+) and Stat6−/− (Ly5.1−) donor mice to analyze whether Stat6 expression in macrophages was required for survival and recruitment to the effector sites. The histogram demonstrates equal engraftment of both donor marrows at 8 weeks after reconstitution in the peripheral blood. (B) Peritoneal cells of naive mice (left dot plot) or of day 9 N brasiliensis-infected mice (right dot plot) that had been given BrdU for the past 36 hours were stained for F4/80, Ly5.1, and BrdU. Dot plots show F4/80-gated macrophages. (C-D) Macrophages in peritoneum (PEC), spleen (SP), BAL, and lung (LU) of wild-type (filled bars) and Stat6−/− (hatched bars) mice that had been infected with N brasiliensis 9 days before (infected) or were left untreated (naive) were analyzed 36 hours after BrdU administration. Panel C shows the frequency of BrdU+ cells among total macrophages, and panel D shows the frequency of macrophages among total cells. Macrophages were identified by staining for F4/80 and CD11c (lung and BAL), F4/80 and CD11b (spleen), or F4/80 (PEC). The bars show mean ± SE of 7 individual mice from 4 independent experiments (**P ≤ .001; *P ≤ .05).
Silke Huber et al. Blood 2010;116:
©2010 by American Society of Hematology

8. PD-1 expression on Th2 cells during N brasiliensis infection.
PD-1 expression on Th2 cells during N brasiliensis infection. Lung (A) and mesenteric lymph nodes (B) of 4get (WT) and 4get/Stat6−/− (Stat6−/−) mice were analyzed before or at indicated days after N brasiliensis infection. Dot plots are gated on CD4+ cells and show staining for PD-1 or isotype control versus expression of IL-4/eGFP at day 13 after N brasiliensis infection. The graphs show the frequency and absolute number of PD-1+ cells among Th2 cells (CD4+ IL-4/eGFP+ T cells) from WT (●) or Stat6−/− (○) mice as mean ± SE of 3 individual mice from 2 independent experiments (***P ≤ .001; **P ≤ .01; *P ≤ .05). (C) Effect of anti-PD-L2 mAb treatment during N brasiliensis infection on the Th2 response and PD-1 expression on T cells. Anti-PD-L2–treated or rat IgG-treated mice were analyzed at day 9 after infection. Dot plots are gated on CD4+ cells and show staining for PD-1 versus expression of IL-4/eGFP. Bar graphs show the frequency of PD-1+ cells among CD4+IL-4/eGFP+ cells (left) and the frequency of IL-4/eGFP+ cells among all CD4+ cells (right) as mean ± SE of 4 individual mice from 2 independent experiments (*P ≤ .05).
Silke Huber et al. Blood 2010;116:
©2010 by American Society of Hematology