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