Journal: Clinical and Translational Medicine

Article Title: Granulocyte colony‐stimulating factor induced T‐cell hyporesponsiveness via modulation of CD177 + S100A hi neutrophils in unexplained recurrent pregnancy loss

doi: 10.1002/ctm2.70508

Figure Lengend Snippet: Immunosuppressive capacity of circulating CD177 + S100A hi neutrophils. (A) Schematic representation of T cells and neutrophils co‐culture system. CD3/CD28‐primed pan‐T cells from healthy donors were co‐cultured in the absence or presence of CD177 + S100A hi neutrophils from G‐PB of early pregnancy. (B and C) Representative images of proliferative alterations (B, left), proportion of proliferative CD4 + T cells (B, right top) and CD8 + T cells (B, right bottom), secretion of TNF‐α (C, top) and IFN‐γ (C, bottom) in co‐cultures of pan‐T cells with or without CD177 + S100A hi ‐LDNs and CD177 + S100A hi ‐NDNs (ratio of 1:5) at Day 3.5 ( n = 7). Data are represented as mean ± SD. The symbol ‘–’ indicates T cells without CD3/CD28 stimulation, whereas ‘+’ designates T cells primed with CD3/CD28. (D) Boxplot showing proportion of proliferative CD4 + T cells (left) and CD8 + T cells (right) in co‐cultures of pan‐T cells with CD177 + S100A hi ‐LDNs and CD177 + S100A hi ‐NDNs (ratio of 1:5) under a Transwell device at Day 3.5 ( n = 3). Data are represented as mean ± SD. (E) Quantification of apoptosis of CD4 + T cells and CD8 + T cells at a co‐culture ratio of 1:5 ( n = 4). Data are represented as mean ± SEM. (F) Enriched GO terms for the downregulated proteins in T cells from co‐cultures of pan‐T cells with CD177 + S100A hi ‐LDNs. The thickness of the string is proportional to LogFC. (G) Violin plot of average expression of co‐inhibitory receptor molecules TIGIT, TIM‐3, LAG‐3 and PSGL‐1 in T cells exposed to CD177 + S100A hi ‐LDNs compared with cultured alone. The y ‐axis represents the value of the log2 protein abundance. (H) L‐Arg concentration in supernatant from co‐cultures of pan‐T cells with or without CD177 + S100A hi ‐LDNs and CD177 + S100A hi ‐NDNs (ratio of 1:5) at Day 3.5 ( n = 7). Data are represented as mean ± SD. (I and J) The proportion of proliferative CD4 + T cells (left) and CD8 + T cells (right) in co‐cultures of pan‐T cells with or without CD177 + S100A hi ‐LDNs and CD177 + S100A hi ‐NDNs (ratio of 1:5), with or without: 200 µg/mL L‐Arg (I); 200 U/mL CAT (J, top); 2 mM NAC (J, bottom). Data are represented as mean ± SD. p ‐values are calculated using the one‐way ANOVA with Tukey's post hoc test and Student's t ‐test. G‐PB, G‐CSF‐mobilised peripheral blood; LDNs, low density neutrophils; NDNs, normal density neutrophils; TNF‐α, tumour necrosis factor‐α; IFN‐γ, interferon‐γ; ARG1, arginase 1; ROS, reactive oxygen species; L‐Arg, L‐arginine; CAT, catalase.

Article Snippet: CD2 + CD3 + CD5 + CD7 + ‐pan‐T cells were collected from the PBMCs of healthy donors using the ‘Pan‐T‐cell Isolation kit’ (Miltenyi Biotec) and Lymphoprep (Stemcell).

Techniques: Co-Culture Assay, Cell Culture, Expressing, Quantitative Proteomics, Concentration Assay

Journal: Clinical and Translational Medicine

Article Title: Granulocyte colony‐stimulating factor induced T‐cell hyporesponsiveness via modulation of CD177 + S100A hi neutrophils in unexplained recurrent pregnancy loss

doi: 10.1002/ctm2.70508

Figure Lengend Snippet: Diminished CD177 + S100A hi neutrophils in PB of URPL. (A) Representative flow cytometry plots showing distribution of neutrophils in PBMCs and NDNs layers of HCs, patients with URPL and URPL receiving G‐CSF treatment. NDNs and LDNs are gated based on CD45 and side‐scatter properties in PBMCs and NDNs fractions, respectively. Reliable neutrophils are identified as CD15 + cells among NDNs/LDNs. (B) Representative flow cytometry plots and quantification showing percentage of CD177 + S100A hi ‐LDNs in HCs and URPL patients receiving G‐CSF treatment ( n = 8/each group). Data are represented as mean ± SD. (C) Representative flow cytometry plots and quantification showing percentage of CD177 + S100A hi ‐NDNs in HCs ( n = 8), patients with URPL ( n = 6) and URPL receiving G‐CSF treatment ( n = 8). Data are represented as mean ± SD. (D) Flow cytometry of ARG1 expression in HC, patients with URPL and URPL receiving G‐CSF treatment ( n = 5/each group). Data are represented as mean ± SD. (E) Serum L‐Arg concentrations in HCs ( n = 13), patients with URPL ( n = 18) and URPL receiving G‐CSF treatment ( n = 18). Data are represented as mean ± SD. (F) The percentage of circulating neutrophils detected in PB before and after G‐CSF administration ( n = 16/each group). (G) Relative mRNA expression of CD177, S100A8, S100A9 and S100A12 between G‐CSF‐mobilised and non‐mobilised neutrophils ( n = 16/each group). (H) The percentage of circulating CD3 + T cells detected in PBMCs before and after G‐CSF administration ( n = 8/each group). (I) Expression of inflammation‐related factors TNF‐α, IFN‐γ, IL‐6 and IL‐1β in pregnant women with URPL history before and after G‐CSF administration ( n = 13/each group). p ‐values are calculated using the one‐way ANOVA with Tukey's post hoc test and Student's t ‐test. PBMCs, peripheral blood mononuclear cells; HCs, healthy pregnant controls; URPL, unexplained recurrent pregnancy loss; LDNs, low density neutrophils; NDNs, normal density neutrophils; ARG1, arginase 1; MFI, mean fluorescence intensity; L‐Arg, L‐arginine; TNF‐α, tumour necrosis factor‐α; IFN‐γ, interferon‐γ; IL‐6, interleukin‐6.

Article Snippet: CD2 + CD3 + CD5 + CD7 + ‐pan‐T cells were collected from the PBMCs of healthy donors using the ‘Pan‐T‐cell Isolation kit’ (Miltenyi Biotec) and Lymphoprep (Stemcell).

Techniques: Flow Cytometry, Expressing, Fluorescence

Journal: HemaSphere

Article Title: Inhibiting the alarmin‐driven hematopoiesis‐stromal cell crosstalk in primary myelofibrosis ameliorates bone marrow fibrosis

doi: 10.1002/hem3.70179

Figure Lengend Snippet: S100A9‐inhibition using tasquinimod reveals novel transcriptome reprogramming and reversal of TGFβ activation. (A) Experimental scheme of RNAseq sorting strategy for JAK2 WT or JAK2 V617F CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells, either treated with vehicle or tasquinimod ( n = 3 mice/group/cell population). (B) Principal component analysis of JAK2 WT or JAK2 V617F CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells, either treated with vehicle or tasquinimod. (C) Heatmap representation of PROGENy analysis in CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells. (D) Heatmap representation of extracellular matrix (ECM)‐related Hallmark pathways in CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells, comparing JAK2 V617F vehicle versus JAK2 WT vehicle (labeled as “V617F veh”) and JAK2 V617F tasquinimod versus JAK2 V617F vehicle (labeled as “V617F Tasq”). (E) Overall cell‐to‐cell interactions between CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells. (F) Top 30 deregulated interactions mediated by TGFB1 between CD41 + , CD11b + Gr1 − , and Lin − Sca1 + cells. Interactions ordered based on the difference in mean LR expression between JAK2 V617F tasquinimod and JAK2 V617F vehicle.

Article Snippet: The cells were lineage‐depleted using biotinylated antibodies directed against lineages (CD5, CD45R, CD11b, Gr1, 7‐4, and Ter119) (Miltenyi Biotec) and additionally added CD45‐ and CD71‐biotin antibodies (BioLegend).

Techniques: Inhibition, Activation Assay, Labeling, Expressing

Journal: HemaSphere

Article Title: Inhibiting the alarmin‐driven hematopoiesis‐stromal cell crosstalk in primary myelofibrosis ameliorates bone marrow fibrosis

doi: 10.1002/hem3.70179

Figure Lengend Snippet: Tasquinimod treatment abrogates Myc activation and induces JAK2 V617F ‐specific apoptosis. (A) Hallmark gene set enrichment analysis (GSEA) of Gr1 − CD11b + monocytes and (B) Lin − Sca1 + stromal cells, comparing JAK2V617F vehicle versus JAK2WT vehicle (labeled as “V617F vehicle”) and JAK2V617F tasquinimod versus JAK2V617F vehicle (labeled as “V617F tasquinimod”). (C) Frequency of GFP + cells in peripheral blood over time. (D) Flow analysis of early‐stage cell apoptosis (Annexin V + ), and late‐stage apoptosis (Annexin V + 7‐AAD + ) in JAK2 WT ‐HoxB8‐Flt3 or JAK2 V617F ‐HoxB8‐Flt3 cells treated with 50 µM tasquinimod or DMSO control for 24 h. (E) Heatmaps of differential analysis focused on damage‐associated molecular pattern (DAMP)‐associated genes. All comparisons are JAK2 V617F vehicle versus JAK2 WT vehicle (labeled as “V617F vehicle”) and JAK2 V617F tasquinimod versus JAK2 V617F vehicle (labeled as “V617F tasquinimod”). (F) Enzyme‐linked immunosorbent assay (ELISA) analysis of the S100A8/S100A9 heterodimer on murine plasma samples from JAK2 WT or JAK2 V617F animals, either treated with vehicle or tasquinimod drinking water ( n = 7–10/group). (G) Sankey plot showing S100a8‐Tlr4 ligand–receptor interactions in Gr1 − CD11b + monocytes. (H) CARNIVAL‐based pathway inference of the effect of S100a8/S100a9 binding to Tlr4.

Article Snippet: The cells were lineage‐depleted using biotinylated antibodies directed against lineages (CD5, CD45R, CD11b, Gr1, 7‐4, and Ter119) (Miltenyi Biotec) and additionally added CD45‐ and CD71‐biotin antibodies (BioLegend).

Techniques: Activation Assay, Labeling, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Binding Assay