Journal: Immunity

Article Title: Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals

doi: 10.1016/j.immuni.2017.06.013

Figure Lengend Snippet: Human PC-Ms Accumulate in the Gut together with ME-M B Cells and Include a Circulating Counterpart Expressing Gut-Homing Receptors (A) Flow cytometry (FCM) of IgM and IgA on CD19 + CD38 hi CD10 − PCs from human ileum and colon. (B) Frequency of PC-Ms among total PCs, assessed by FCM. (C) FCM of selected surface molecules on naive (N) B cells, PC-Ms, and switched PCs (PC-SW) from human ileum. Numbers indicate mean fluorescence intensity (MFI). (D) Immunofluorescence analysis (IFA) of IgM (green), IgA (red), and DNA (blue) in human ileum and mouse small intestine (SI) lamina propria (LP). Original magnification, 20×. Scale bars, 50 μm. (E) Number of PC-Ms (top), PC-As (center) per mm 2 of LP, and PC-M/PC-A ratio (bottom) from human or mouse SI assessed following tissue IFA. Data summarize six different tissue samples where at least four high-power microscopic fields were analyzed. (F and G) Representative FCM (F) and frequency (G) of β7 + CCR9 + cells in human circulating PC-Ms, PC-As, and PC-G/Es. (H) Representative FCM showing IgM versus IgD staining on CD19 + CD38 − CD10 − B cells from different human tissues. (I) Frequency of ME-M B cells from tissues shown in (H). (J) FCM of IgD, CD24, CD27, and CD148 on naive, ME-M, and ME-SW B cells from ileum. Data show one representative result (A, F, H) of 12 (B), 8 (G), or 52 (I) experiments or are from one experiment of at least 3 with similar results (C, D, J). Results are presented as mean ± SEM; two-tailed unpaired Student’s t test (B and E) and one-way ANOVA with Tukey’s post hoc test (I). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. See also Figure S1 .

Article Snippet: Anti-human CD27 PerCP/Cy5.5 (clone: M-T271) , BD Biosciences , 560612, RRID: AB_1727457.

Techniques: Expressing, Flow Cytometry, Fluorescence, Immunofluorescence, Staining, Two Tailed Test

Journal: Immunity

Article Title: Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals

doi: 10.1016/j.immuni.2017.06.013

Figure Lengend Snippet: Human Gut ME-M B Cells Undergo Proliferation, PC Differentiation, IgM Secretion, and IgA Class Switching in Response to TD or TI Signals (A and B) FCM of CFSE dilution profiles (A) and GeoMean (% of max) of CFSE staining (B) in naive (N) and ME-M B cells from human ileum cultured for 5 days as indicated. Ctrl, medium alone. (C) FCM of CFSE and CD38 on naive (top) and ME-M (bottom) B cells from human ileum cultured as in (A) and (B). Numbers indicate percent of newly formed CD38 hi CFSE lo PCs. (D) FCM of IgM and IgA on CD38 hi CFSE lo plasmablasts emerging upon stimulation as in (A) and (B). (E) ELISA of IgM and IgA secreted by naive (N) and ME-M B cells from human ileum cultured for 5–7 days as in (A) and (B). (F) FCM of CD38, CD27, IgM, and IgA on sorted FcRL4 − (left) or FcRL4 + (right) ME-M B cells from human ileum cultured for 5 days with medium alone (ctrl) or CD40L, IL-21, and IL-10. (G) qRT-PCR analysis of mRNA encoding AID ( AICDA ) in naive, FcRL4 − ME-M, and FcRL4 + ME-M B cells from human ileum. Data represent one representative experiment of two with similar results (A–D, F) or summarize at least three different experiments (E, G). Results are presented as mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (two-tailed unpaired Student’s t test).

Article Snippet: Anti-human CD27 PerCP/Cy5.5 (clone: M-T271) , BD Biosciences , 560612, RRID: AB_1727457.

Techniques: Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Immunity

Article Title: Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals

doi: 10.1016/j.immuni.2017.06.013

Figure Lengend Snippet:

Article Snippet: Anti-human CD27 PerCP/Cy5.5 (clone: M-T271) , BD Biosciences , 560612, RRID: AB_1727457.

Techniques: Recombinant, Blocking Assay, Fluorsave, Giemsa Stain, Sample Prep, DNA Purification, SYBR Green Assay, Microarray, Transformation Assay, Software

Journal: Nature Cell Biology

Article Title: A single-cell atlas of non-haematopoietic cells in human lymph nodes and lymphoma reveals a landscape of stromal remodelling

doi: 10.1038/s41556-022-00866-3

Figure Lengend Snippet: a , Strategies used to identify malignant B-cell components in FL samples in silico . Shown are representative cases with light chain kappa (FL 4; top) or lambda (FL 8; bottom) restrictions confirmed by flow cytometry analysis (data not shown). After identifying B-cell components by detecting CD79A expression, we assessed expression of IGKC (for light chain kappa) and IGLC2 (for light chain lambda). Clusters with cells expressing IGKC and those expressing IGLC2 were considered non-malignant B cells, while clusters with cells expressing only one of these genes were considered malignant B cells. b , Scatter plot showing clear discrimination of malignant (filled circles) from non-malignant (empty circles) B-cell clusters in each FL sample, based on the ratio of cells expressing IGLC2 (expression level >1; y-axis) to those expressing IGKC (expression level >2; x-axis,). Red-shaded areas indicate regions in which the ratio was >2.0 or <0.25. c , Representative UMAP plots showing B cells from FL 4 according to B-cell types (beige; non-malignant, red; malignant) (left panel) or malignant B-cell signature score (right panel). d , Violin plots showing malignant B-cell signature score in extracted non-malignant and malignant B cells, according to different FL samples (FL 2–10). *** P = 1.1 × 10 −204 (FL 2), *** P = 0 (FL 3), *** P = 3.3 × 10 −176 (FL 4), *** P = 0 (FL 5), *** P = 4.2 × 10 −122 (FL 6), *** P = 0 (FL 7), *** P = 4.2 × 10 −161 (FL 8), *** P = 3.7 × 10 −256 (FL 9), *** P = 2.5 × 10 −81 (FL 10) (two-sided Wilcoxon Rank-Sum test with Bonferroni correction). e , Violin plots showing the expression of CD27 in non-malignant and malignant B cells. *** P = 0 (two-sided Wilcoxon Rank-Sum test with Bonferroni correction). f , Comparison of CD27 mean fluorescence intensity (MFI) between FL CD19 + CD10 − (non-malignant B-cell fraction) and CD19 + CD10 + (malignant B-cell fraction) cells. Circles represent biologically independent samples ( n = 8; FL 11–18). * P = 0.039 (two-sided Wilcoxon matched-pairs signed rank test). g , Flow cytometry analysis of CD27 expression on CD19 + CD10 − and CD19 + CD10 + cells of a representative FL sample (FL 14). The statistical source data are provided.

Article Snippet: To block CD70–CD27 binding, cells were incubated in the presence of anti-CD27 blocking antibody (R&D systems, MAB382) or isotype mouse IgG1 (R&D systems, MAB002) for 30 min at 4 °C before binding.

Techniques: In Silico, Flow Cytometry, Expressing, Comparison, Fluorescence

Journal: Nature Cell Biology

Article Title: A single-cell atlas of non-haematopoietic cells in human lymph nodes and lymphoma reveals a landscape of stromal remodelling

doi: 10.1038/s41556-022-00866-3

Figure Lengend Snippet: a , Enhanced interactions across FL NHC subclusters and malignant B cells (B malignant ). Circle size indicates the negative log 10 of adjusted P values . Circles are coloured when a stroma-derived factor is upregulated in relevant FL subclusters. b , IF staining for MECA-79 (cyan), DCN (red) and CD70 (green) in MFLN and FL samples. Scale bars, 200 μm. Representative images from one of three independent experiments are shown. c , Proportions of CD70 + area in medullary and adventitia regions of MFLN ( n = 3) and FL ( n = 3) samples. Circles represent biologically independent samples. Bars indicate the median. ** P = 0.0095 (two-sided unpaired t -test). d , Binding of FL CD19 + CD10 + cells to CD70-Fc protein with an anti-CD27 blocking antibody or isotype human IgG. The histograms represent three independent experiments (FL 13) with the count in arbitrary units. e , Blocking of FL CD19 + CD10 + cell binding to CD70-Fc protein after treating cells with an anti-CD27 blocking antibody ( n = 3) or isotype mouse IgG1 ( n = 3) in CD27 + FL samples (FL 11–FL 14). Proportions of cells bound to CD70-Fc protein were adjusted by subtracting nonspecific binding observed with human IgG. CD70-Fc protein binding to cells treated with isotype mouse IgG1 was set to 100% in each experiment. Circles represent independent experiments. Bars indicate the median. ** P = 0.0022, *** P = 7.3 × 10 −4 (FL 11), *** P = 2.2 × 10 −4 (FL 12), *** P = 7.6 × 10 −4 (FL 13) (two-sided paired t -test). f , Representative malignant B-enriched cell (FL 14) adhesion to medullary regions of FL in the presence of an isotype mouse IgG1 or anti-CD27 antibody. Orange dots indicate adherent cells. Yellow dashed lines indicate medullary regions. Scale bars, 200 μm. g , Blocking of malignant B-enriched cell (FL 11, FL 13 and FL 14) adhesion to FL medullary regions (per mm 2 ) after treating cells with an anti-CD27 blocking antibody ( n = 3) or isotype mouse IgG1 ( n = 3). Adhesion of cells treated with isotype mouse IgG1 was set to 100% in each experiment. Circles represent independent experiments. Bars indicate the median. * P = 0.041 (FL 11), * P = 0.027 (FL 14), ** P = 0.0050 (two-sided paired t -test). Statistical source data are provided.

Article Snippet: To block CD70–CD27 binding, cells were incubated in the presence of anti-CD27 blocking antibody (R&D systems, MAB382) or isotype mouse IgG1 (R&D systems, MAB002) for 30 min at 4 °C before binding.

Techniques: Derivative Assay, Staining, Binding Assay, Blocking Assay, Protein Binding

Journal: Scientific Reports

Article Title: A hexamerization-enhanced, Fc-silenced agonistic CD27 antibody amplifies T-cell effector functions as single agent and in combination with PD-1 blockade

doi: 10.1038/s41598-025-11990-z

Figure Lengend Snippet: A CD27 antibody with an Fc-silencing and a hexamerization-enhancing mutation drives CD27 agonist activity. ( a ) Proposed mechanism of action of HexaBody-CD27. Binding of HexaBody-CD27 to CD27 on T cells is hypothesized to result in the formation of hexameric antibody structures through Fc-Fc interactions between membrane-bound antibodies. Hexamer formation drives clustering and activation of CD27 receptors, resulting in enhanced T cell activation, differentiation and proliferation through CD27 agonism. ( b ) CD27 signaling in CD27-positive Jurkat reporter cells, after incubation with 10 µg/mL anti-CD27 antibody variants with a wild-type Fc (IgG1-CD27) or one or two Fc mutations: a P329R Fc-silencing mutation and/or an E430G or E345R hexamerization-enhancing mutation, in the absence or presence of 60 µg/mL purified C1q. Data shown are mean relative light units (RLU) +/− SD of three independent experiments, normalized by subtraction of non-binding control RLU values at 10 µg/mL (0%) and relative to 10 µg/mL of HexaBody-CD27 (P329R-E345R, 100%) in the presence of C1q.

Article Snippet: 96-well MULTI ARRAY standard plates (Meso Scale Discovery [MSD], L15XA-3) were coated with human CD27 TNFRSF7 protein (His Tag) (Sino Biological, 10039-H08B1) diluted in PBS for 16 to 24 h at 2 to 8 °C, washed and blocked for 60 min using 3% Blocker-A (Blocker-A 9%, MSD, R93AA 1) in PBS-T.

Techniques: Mutagenesis, Activity Assay, Binding Assay, Membrane, Activation Assay, Incubation, Purification, Control

Journal: Scientific Reports

Article Title: A hexamerization-enhanced, Fc-silenced agonistic CD27 antibody amplifies T-cell effector functions as single agent and in combination with PD-1 blockade

doi: 10.1038/s41598-025-11990-z

Figure Lengend Snippet: HexaBody-CD27 acts independently of Fcγ receptor-mediated crosslinking. ( a ) Macrophage-mediated phagocytosis. Human T cells isolated from PBMCs were cultured for four hours with autologous human monocyte-derived macrophages (hMDMs) in the presence or absence of 0.3 µg/mL CD27 antibodies. Shown is the mean percentage of phagocytosing hMDMs + SD of four donors tested in two independent experiments. Data of T-cell viability assessed over a concentration range of 0.000003–3 µg/mL anti-CD27 antibodies is shown in Supplementary Fig. .*, q < 0.05, nd, q ≥ 0.05, Friedmans test with FDR correction (Benjamini and Hochberg). ( b ) Setup scheme for the bioluminescence resonance energy transfer (BRET) assay (created with BioRender.com). ( c ) Molecular proximity determined by BRET analysis between HexaBody-CD27 molecules on the cell surface of K562_hCD27 cells. Cells were incubated with mixtures of NanoLuc-(NL, donor) and HaloTag-(Halo, acceptor) tagged antibodies (5 µg/mL each). Data shown are mean + SD of donor bleed-through corrected BRET in milliBRET units (mBU) from duplicate wells of one representative experiment out of three performed. ( d ) CD27 agonist activity in the presence or absence of FcγRIIb-expressing cells. NFκB-luc2/CD27 Jurkat reporter cells were cultured for five to six hours without or with FcγRIIb-expressing CHO-K1 cells at a reporter to FcγRIIb + cell ratio of 4:1 or 1:2 in the presence of CD27 antibodies or non-binding control antibody. Data shown are single measurements or the mean of duplicates ± SD of background-subtracted relative luminescence units from a representative experiment out of two performed.

Article Snippet: 96-well MULTI ARRAY standard plates (Meso Scale Discovery [MSD], L15XA-3) were coated with human CD27 TNFRSF7 protein (His Tag) (Sino Biological, 10039-H08B1) diluted in PBS for 16 to 24 h at 2 to 8 °C, washed and blocked for 60 min using 3% Blocker-A (Blocker-A 9%, MSD, R93AA 1) in PBS-T.

Techniques: Isolation, Cell Culture, Derivative Assay, Concentration Assay, Bioluminescence Resonance Energy Transfer, Incubation, Activity Assay, Expressing, Binding Assay, Control

Journal: Scientific Reports

Article Title: A hexamerization-enhanced, Fc-silenced agonistic CD27 antibody amplifies T-cell effector functions as single agent and in combination with PD-1 blockade

doi: 10.1038/s41598-025-11990-z

Figure Lengend Snippet: HexaBody-CD27 enhances T-cell proliferation, differentiation, cytokine secretion and cytotoxic activity in vitro. ( a ) Expansion of CTV-labeled PBMCs polyclonally activated with anti-CD3 antibody and cultured in the presence of 6.6 μg/mL of the indicated antibodies for four days. Dots represent the mean expansion index of single or duplicate wells derived from three individual donors (see Supplementary Fig. 4 for antibody concentration range). ( b ) Percentages of naïve and central memory T cells in CTV-labeled healthy donor T cells, at baseline and after incubation with anti-CD3 antibody and 10 μg/mL HexaBody-CD27 or non-binding control antibody. Bars represent mean + SD from three individual donors. ( c , d ) T-cell proliferation and cytokine secretion in an antigen-specific proliferation assay. CFSE-labeled CD8 + T cells, electroporated with RNA encoding a CLDN6-specific TCR, either alone (endogenous PD-1) or together with 5 µg RNA encoding PD-1 (PD-1 overexpression), were co-cultured with autologous CLDN6- or mock-electroporated dendritic cells in the presence or absence of 10 μg/mL of the indicated antibodies. CFSE dilution was analyzed by flow cytometry c and cytokine concentrations in the supernatants were determined by ECLIA ( d ). Data shown are expansion index values (different symbols indicate donors) + SD and fold changes in cytokine secretion relative to medium control of four individual donors, measured in triplicate. *, p < 0.05; **, p < 0.01. Friedman test with Dunn’s multiple comparisons test. If not indicated, differences were not significant after testing. ( e , f ) T-cell cytotoxicity in an antigen-specific tumor cell killing assay performed with three to six individual donors. CLDN6-TCR-electroporated CD8 + T cells were co-cultured with CLDN6-expressing MDA-MB-231 cells and 10 µg/mL of the indicated antibodies. ( e ) The frequency of CD8 + T cells showing positive staining for CD107a and granzyme B (GzmB) + SD was analyzed by flow cytometry after two days. ( f ) CD8 + T-cell mediated cytotoxic activity was evaluated by real-time cell analysis during five days. Left panel shows cell index + SD of duplicate wells from one exemplary donor normalized to the time point of co-culture start. Right panel shows AUC of cell index from three to six donors normalized to the non-binding control antibody of each donor. Dots represent individual donors. Additional exemplary donors are depicted in Supplementary Fig. .

Article Snippet: 96-well MULTI ARRAY standard plates (Meso Scale Discovery [MSD], L15XA-3) were coated with human CD27 TNFRSF7 protein (His Tag) (Sino Biological, 10039-H08B1) diluted in PBS for 16 to 24 h at 2 to 8 °C, washed and blocked for 60 min using 3% Blocker-A (Blocker-A 9%, MSD, R93AA 1) in PBS-T.

Techniques: Activity Assay, In Vitro, Labeling, Cell Culture, Derivative Assay, Concentration Assay, Incubation, Binding Assay, Control, Proliferation Assay, Over Expression, Flow Cytometry, Expressing, Staining, Cell Analysis, Co-Culture Assay

Journal: Scientific Reports

Article Title: A hexamerization-enhanced, Fc-silenced agonistic CD27 antibody amplifies T-cell effector functions as single agent and in combination with PD-1 blockade

doi: 10.1038/s41598-025-11990-z

Figure Lengend Snippet: HexaBody-CD27 promotes expansion of tumor-infiltrating lymphocytes ex vivo. Tumor fragments obtained from human NSCLC specimens were cultured in the presence of 60 U/mL IL-2 and with or without 1 µg/mL HexaBody-CD27. After 14 to 17 days, absolute counts of the indicated cell subsets were determined by flow cytometry. ( a ) Average cell counts of four replicate wells + SD are shown for each cell subset individually, from one exemplary NSCLC patient out of five tested. ( b ) Fold-change of average cell counts after treatment with HexaBody-CD27 compared to no antibody control across ex vivo cultures derived from 5 different NSCLC patients. Symbols represent individual donors, lines represent mean values, dotted line represents expansion of TILs cultured without antibody.

Article Snippet: 96-well MULTI ARRAY standard plates (Meso Scale Discovery [MSD], L15XA-3) were coated with human CD27 TNFRSF7 protein (His Tag) (Sino Biological, 10039-H08B1) diluted in PBS for 16 to 24 h at 2 to 8 °C, washed and blocked for 60 min using 3% Blocker-A (Blocker-A 9%, MSD, R93AA 1) in PBS-T.

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Control, Derivative Assay

Journal: Scientific Reports

Article Title: A hexamerization-enhanced, Fc-silenced agonistic CD27 antibody amplifies T-cell effector functions as single agent and in combination with PD-1 blockade

doi: 10.1038/s41598-025-11990-z

Figure Lengend Snippet: Combination of HexaBody-CD27 with pembrolizumab enhances T-cell effector functions compared to either single-agent treatment. ( a ) T-cell proliferation and cytokine secretion in an antigen-specific T-cell proliferation assay. The assay was performed with CD8 + T cells that were electroporated with RNA encoding a CLDN6-specific T-cell receptor and RNA encoding PD-1, in the presence of pembrolizumab, non-binding control antibody, or HexaBody-CD27 with or without pembrolizumab, for four days. CFSE dilution in T cells was analyzed by flow cytometry and cytokine concentrations in the supernatants were determined by ECLIA. Data shown are mean values + SD of seven donors tested. Symbols represent individual donors. ( b ) Mature monocyte-derived dendritic cells were co-cultured with CD8 + T cells from allogeneic donors in the presence of non-binding control mAb, HexaBody-CD27 and/or a pembrolizumab analog for five days. IFN-γ concentrations in supernatants of mixed lymphocyte reaction assays were determined by ECLIA. Data shown are the synergy scores of the combination of HexaBody-CD27 and 10 μg/mL pembrolizumab analog according to the Highest Single Agent (HSA) synergy model of five donor pairs tested. Scores of > 10 suggest synergy. Dose–response curves of all donor pairs are shown in Supplementary Fig. . ( c ) Percentage of CD8 + T cells expressing both GzmB and CD107a. A tumor cell killing assay was performed as described in Fig. E, with the exception that the CD8 + T cells were in addition electroporated with RNA encoding PD-1. Data from 13 donors tested in six individual experiments is shown. ( d ) CD8 + T-cell mediated cytotoxic activity was evaluated by real-time cell analysis over a 137 h period. Left: Normalized cell index values of one exemplary donor out of 14 donors tested were derived from impedance measurements. Right: Area under the curve (AUC) of normalized cell index values was calculated and normalized to the non-binding control mAb of the respective donor. Error bars indicate SD. ( a , c , d )*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Friedman test with Dunn’s multiple comparisons test.

Article Snippet: 96-well MULTI ARRAY standard plates (Meso Scale Discovery [MSD], L15XA-3) were coated with human CD27 TNFRSF7 protein (His Tag) (Sino Biological, 10039-H08B1) diluted in PBS for 16 to 24 h at 2 to 8 °C, washed and blocked for 60 min using 3% Blocker-A (Blocker-A 9%, MSD, R93AA 1) in PBS-T.

Techniques: Proliferation Assay, Binding Assay, Control, Flow Cytometry, Derivative Assay, Cell Culture, Expressing, Activity Assay, Cell Analysis

Journal:

Article Title: Identification of Homeodomain Proteins, PBX1 and PREP1, Involved in the Transcription of Murine Leukemia Virus

doi: 10.1128/MCB.23.3.831-841.2003

Figure Lengend Snippet: CDKI-regulated cellular genes—putative regulators of MLV

Article Snippet: Since our goal in this study was to identify cellular genes affecting viral transcription, we focused only on the candidate genes involved in regulation of transcription. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Gene product Avg difference value for condition a : DMSO Flavo Purv MeO-Ros PBX1 1,572 377 769 1,307 High-mobility group protein HMG I-C 1,959 1,452 485 427 CCAAT-box-binding transcription factor 431 103 374 110 Putative myelin regulatory factor 1 1,402 519 697 389 Forkhead box protein G1B 301 −36 350 50 DEAD-box protein 3 690 147 395 189 DNA-directed RNA polymerase II, largest subunit 415 24 380 −34 Histone H1 1,747 151 1,506 625 Histone H3 2,526 871 2,260 1,185 Serine/threonine protein phosphatase PP1-γ catalytic subunit 1,117 412 609 303 Serine/threonine-protein kinase PLK 431 67 232 101 Prohibitin 294 213 85 126 Protein translation factor SUI1 homolog 960 403 2,586 1,259 Casein kinase II, alpha chain 906 397 1,015 206 Myristoylated alanine-rich C-kinase substrate 403 193 336 121 Mus musculus , similar to RAS p21 protein activator 270 74 223 98 Quaking 748 204 635 251 Nucleolar protein GU2 273 121 262 94 Cell division cycle 20 homolog ( Saccharomyces cerevisiae ) 1,757 864 1,297 869 Secretory leukocyte protease inhibitor 280 703 699 735 Ubiquitin-conjugating enzyme E2, 23 kDa 133 310 266 268 Growth arrest and DNA damage-inducible protein GADD45 β 131 222 272 473 Cd27 binding protein 413 843 610 969 Phosphodiesterase 4B, cyclic AMP specific 11 233 255 162 Wild-type p53-induced gene 1 307 485 617 672 Open in a separate window a Affymetrix average difference values for means of duplicate chips.

Techniques: Protease Inhibitor, Binding Assay