Journal: Nature Communications

Article Title: PD-L1 + plasma cells suppress T lymphocyte responses in patients with sepsis and mouse sepsis models

doi: 10.1038/s41467-025-57706-9

Figure Lengend Snippet: a Representative examples of flow cytometry gating of plasma cells (PCs) in one Sham and one CLP animal. b Morphology of sorted CD138+ B220low splenic cells from a CLP mouse. c , d Proportions and absolute counts of PCs among splenocytes in 19 Sham and 24 CLP mice. e mRNA expressions of transcription factors bcl6 , pax5 , irf4 , and prdm1 (coding for BLIMP1) measured in B cells and PCs purified from 6 CLP mice. f BLIMP1 expression as median fluorescence intensity (MFI) on splenic B cells and PCs in 7 CLP mice. g Percentages of cell surface IgM-expressing PCs (among the entire PC population) in 8 Sham and 9 CLP mice. Representative examples of IgM intracellular expression profile in B220+ splenocytes from 2 CLP mice h Expressions on PCs in Sham and CLP mice of CD138 ( n = 10 and 13), B220 ( n = 10 and 13), CD86 ( n = 6 and 10) and MHC II ( n = 6 and 10) as median fluorescence intensities (MFI). i Heatmap of mRNA expressions of selected transcripts related to cell differentiation, BCR signaling, or migration measured in PCs purified from 6 Sham and 6 CLP mice. The red and blue colors indicate over- and under-expressed markers, respectively, and the color intensity specifies the expression level. j Splenic T cells from Sham mice ( n = 8) were co-cultured with Sham ( n = 8) or CLP ( n = 17) splenic PCs at 1:1 ratio for 72 h, with or without anti-CD3/CD28 antibodies-coated beads. T-cell proliferation was measured among viable cells by flow cytometry. k IFNγ was measured in culture supernatants of Sham T cells cultured either alone ( n = 5) or with PCs from Sham ( n = 6) or CLP ( n = 10) mice. Red squares represent results in CLP mice (either B cells or PCs), and white circles in Sham animals. Geometric means with standard deviations (SD) and individual values are shown. Two-sided Mann–Whitney tests were performed, and only significant p values < 0.05 are shown.

Article Snippet: PCs were purified from splenocytes by positive selection with magnetic beads (EasySepTM Mouse CD138 Positive Selection Kit; STEMCELL), while B and T cells were purified by negative selection (EasySepTM Mouse B and T Cell Isolation Kits; STEMCELL).

Techniques: Flow Cytometry, Clinical Proteomics, Purification, Expressing, Fluorescence, Cell Differentiation, Migration, Cell Culture, MANN-WHITNEY

Journal: Nature Communications

Article Title: PD-L1 + plasma cells suppress T lymphocyte responses in patients with sepsis and mouse sepsis models

doi: 10.1038/s41467-025-57706-9

Figure Lengend Snippet: a Percentages of SSC Int CD19 low plasma cells (PCs) in septic patients ( n = 107) at D1 ( n = 92), D3 ( n = 87), D5 ( n = 92), D14 ( n = 61), D28 ( n = 46), D60 ( n = 40) and healthy volunteers (HV, n = 175). b Percentages of PCs between D14 survivors and non-survivors at D1 ( n = 79 vs 13), D3 ( n = 78 vs 9), D5 ( n = 87 vs 5). c Percentages of CD38 + CD138 + PCs in septic ( n = 67) and COVID-19 patients ( n = 33) at D1 ( n = 41 and 31), D3 ( n = 61 and 20), D5 ( n = 40 and 11) and HVs ( n = 33). d Median fluorescence intensity (MFI) and percentage of PD-L1 + B cells and PCs in 15 HVs and 17 septic patients at D3. e MFI and percentage of PD-L1 + B cells and PCs from 2 HVs and 21 COVID-19 patients. f Representative histograms of CD138/IgM CD19 + SSC low lymphocytes in 2 septic patients. Intracellular IgM staining was detected in >95% PCs (red dots). g BLIMP1 MFI in B cells and PCs from 8 septic patients. h Stimulated T cells proliferation ( n = 8 HVs; white squares) with or without purified PCs ( n = 8; squares) or B cells ( n = 6, dots) from septic ( n = 7) or COVID-19 patient ( n = 1). i IFNγ in supernatants of HVs T cells cultured alone ( n = 11) or with B cells ( n = 5) or PCs ( n = 11) from septic patients. Results are shown as box plots (box between first and third quartiles with horizontal line at median value, horizontal lines representing upper and lower adjacent values, values higher than the upper and lower adjacent values shown as dots). b – e , g – i individual values are also represented. Two-sided Mann–Whitney tests were performed to compare results obtained in patients versus HVs, and two-sided Wilcoxon paired tests to compare results of T-cell proliferation and IFNγ concentration between culture conditions. Only significant p values < 0.05 are shown.

Article Snippet: PCs were purified from splenocytes by positive selection with magnetic beads (EasySepTM Mouse CD138 Positive Selection Kit; STEMCELL), while B and T cells were purified by negative selection (EasySepTM Mouse B and T Cell Isolation Kits; STEMCELL).

Techniques: Clinical Proteomics, Fluorescence, Staining, Purification, Cell Culture, MANN-WHITNEY, Concentration Assay

Journal: Haematologica

Article Title: Multi-parametric single cell evaluation defines distinct drug responses in healthy hematologic cells that are retained in corresponding malignant cell types

doi: 10.3324/haematol.2019.217414

Figure Lengend Snippet: Overview of the study. Schematic diagram summarizing the study design, datasets and analytical framework of the study. Bone marrow (BM) and peripheral blood (PB) samples from both healthy individuals and cancer patients were subjected to drug sensitivity assessment. Single cell drug sensitivity assay using the iQue® Screener PLUS flow cytometer was performed in 96-and 384-well plates to monitor drug effects on ten and six hematopoietic cell subtypes, respectively. Immunophenotypic details and cellular proportions of the analyzed cell types are provided in Online Supplementary Figure S1A-D, Figure S2 and Online Supplementary Table S3 , respectively. 71 drugs in 384-well plates and six drugs in 96-well plates were tested. Proteomic analysis was performed on three cell subsets (monocytes, T and B cells) from two healthy individuals and four myeloma patients. Basal phosphorylation of nine signaling proteins involved in MAPK, JAK-STAT, PI3K-AKT-mTOR and NF-κB signaling was monitored in 14 samples. Healthy BM samples from four healthy individuals were subjected to CD34, CD3, CD14, CD19 and CD138 cell enrichment and tested against 71 small molecules with cell viability as the end point readout using the CellTiter-Glo ® assay. A comparison of ex vivo drug response in healthy and corresponding malignant cell types was performed for six drugs in 281 primary patient samples representing different hematologic malignancies. Samples included both published and unpublished datasets from chronic myeloid leukemia (CML, n=13), , , chronic myelomonocytic leukemia (CMML, n=11), myelodysplastic syndromes (MDS, n=4), acute myeloid leukemia (AML, n=145), , B-cell acute lymphoblastic leukemia (BALL, n=14), chronic lymphocytic leukemia (CLL, n=4), T-cell prolymphocytic leukemia (T-PLL, n=40), multiple myeloma (MM, n=50), and other hematologic malignancies (n=6). PLL: prolymphocytic leukemia.

Article Snippet: Four healthy BM aspirates subjected to magnetic bead-based enrichment using EasySepTM human CD138, CD3, CD19, CD14 and CD34 positive selection kits (StemCell Technologies), served as healthy cell-of-origin samples for comparison against the malignant cell counterparts.

Techniques: Sensitive Assay, Flow Cytometry, Phospho-proteomics, Glo Assay, Comparison, Ex Vivo

Journal: Haematologica

Article Title: Multi-parametric single cell evaluation defines distinct drug responses in healthy hematologic cells that are retained in corresponding malignant cell types

doi: 10.3324/haematol.2019.217414

Figure Lengend Snippet: Distinct drug responses observed in immune subsets are tied to cell lineages. (A) A drug sensitivity score (DSS), which is a modified form of the area under the curve (AUC) calculation, was used to quantitate drug responses among the different detected cell populations. Higher DSS values (DSS>10) indicate higher sensitivity to the individual drug. The heatmap displays a summary view of hierarchical clustering analysis with DSS scores for six cell subsets in three peripheral blood (PB) samples from healthy controls (marked as HC-1, 2 and 3). Immunophenotyping of hematopoietic cell types was performed on the basis of their known surface antigen expression profiles (see also Online Supplementary Figure S1A ). Monocytes and T-cell subsets formed two separate clusters. B and natural killer (NK) cells had similar drug response patterns. Small molecules that were tested along with their functional classes are displayed in Online Supplementary Figure S3 . DSS scores and IC50 values for all 71 drugs are provided in Online Supplementary Table S2 . (B) Differential effect of bortezomib, dexamethasone, clofarabine, venetoclax, navitoclax and omipalisib on hematopoietic cell subsets presented as mean values from 16 samples derived from three healthy, three acute myeloid leukemia (AML) and ten multiple myeloma (MM) patients (Cohort I). Highest variation was observed for BCL2 inhibitors (venetoclax and navitoclax) and dexamethasone between myeloid and lymphoid lineages. Monocytes were resistant to both of these drug classes. A concentration-dependent increase in numbers of CD3 + cells was observed for omipalisib at 10 and 100 nM. The proportion of cells detected in these analyzed samples are presented in Online Supplementary Table S3 .

Article Snippet: Four healthy BM aspirates subjected to magnetic bead-based enrichment using EasySepTM human CD138, CD3, CD19, CD14 and CD34 positive selection kits (StemCell Technologies), served as healthy cell-of-origin samples for comparison against the malignant cell counterparts.

Techniques: Modification, Expressing, Functional Assay, Derivative Assay, Concentration Assay

Journal: Haematologica

Article Title: Multi-parametric single cell evaluation defines distinct drug responses in healthy hematologic cells that are retained in corresponding malignant cell types

doi: 10.3324/haematol.2019.217414

Figure Lengend Snippet: Variable dose-dependent activity of venetoclax on leukocytes. (A) Scatter diagram displaying dose-dependent cytotoxicity of venetoclax (1-10,000 nM) in CD45 + (upper panel) and CD45 + CD19 + (lower panel) cells for a single patient. (B) Averaged dose response graphs generated for different immune cell subtypes derived from healthy (n=3), acute myeloid leukemia (AML) (n=3), and multiple myeloma (MM) (n=10) samples showed venetoclax sensitivity in CD19 + /B cells with an IC 50 <1 nM. CD3 + CD4 − cytotoxic T cells were more sensitive compared to CD3 + CD4 + T-helper cells. Data are presented as mean±standard error of mean responses for the tested samples in each disease group. Mean IC 50 values for the analyzed samples are listed in Online Supplementary Table S4 .

Article Snippet: Four healthy BM aspirates subjected to magnetic bead-based enrichment using EasySepTM human CD138, CD3, CD19, CD14 and CD34 positive selection kits (StemCell Technologies), served as healthy cell-of-origin samples for comparison against the malignant cell counterparts.

Techniques: Activity Assay, Generated, Derivative Assay

Journal: Haematologica

Article Title: Multi-parametric single cell evaluation defines distinct drug responses in healthy hematologic cells that are retained in corresponding malignant cell types

doi: 10.3324/haematol.2019.217414

Figure Lengend Snippet: Systematic comparison of drug responses in healthy cell-of-origin and corresponding cell types from hematologic malignancies. (A-F) Ex vivo drug responses presented as drug sensitivity scores (DSS) of healthy cell types (CD3, n=4; CD14, n=4; CD19, n=2; CD34, n=2; and CD138, n=3) were compared to malignant counterparts in a cohort of 281 primary samples for bortezomib, clofarabine, dexamethasone, omipalisib, venetoclax and navitoclax. Samples included both published and unpublished datasets generated at our facility for chronic myeloid leukemia (CML) (n=13), , chronic myelomonocytic leukemia (CMML) (n=11), myelodysplastic syndromes (MDS) (n=4), acute myeloid leukemia (AML) (n=145), , B-cell acute lymphoblastic leukemia (B-ALL) (n=14), chronic lymphocytic leukemia (CLL) (n=4), T-cell prolymphocytic leukemia (T-PLL) (n=40), multiple myeloma (MM) (n=50) and other hematologic malignancies (n=6). AML and MM samples were subdivided depending on whether they were derived from newly diagnosed (D) and relapsed (R) samples. T-PLL and MM samples were tested with enriched CD8 + and CD138 + cells. Results provide evidence that response in healthy cell subsets is predictive of responses observed in the malignant cell counterparts. A comparison between drug effects on CD14 + and CD34 + cells derived from healthy individuals and AML samples are displayed in Online Supplementary Figure S13 and S14 . B-PLL: B-cell prolymphocytic leukemia.

Article Snippet: Four healthy BM aspirates subjected to magnetic bead-based enrichment using EasySepTM human CD138, CD3, CD19, CD14 and CD34 positive selection kits (StemCell Technologies), served as healthy cell-of-origin samples for comparison against the malignant cell counterparts.

Techniques: Comparison, Ex Vivo, Generated, Derivative Assay