Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: Galectin-9 inhibits mycobacterial growth directly . A. Profile of Mtb H37Rv (Rv) grown at 37°C in Middlebrook 7H9 liquid medium with different concentration of Galectin-9 (Gal9, 0, 0.01, 0.1, 1, 10 μg/mL). Growth curve was measured using a Bioscreen Growth Curve Instrument. Optical density was measured at absorbance at 600 nm every 2 h. B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with10μg/mL galectin-9 (Gal9) or inactivated galectin-9 (Gal9 HK, heat killed at 95 [for 5 min). C. CFU of Mtb H37Rv (Rv) on Middlebrook 7H10 solid medium with or without 10μg/mL Galectin-9 (Gal9). Cultures were grown at 37°C for 4-8 weeks. D. Growth profile of Mycobacterium smegmatis (MS) in Middlebrook 7H9 liquid medium with different concentrations of Galectin-9 (Gal9, 0, 0.01, 0.1, 1 μg/mL). E. Concentrations of galectin-9 in sera of healthy donors (n = 40) and active TB patients (n = 40). F. Confocal microscopy of M. bovis BCG-DsRed (BCG-DsRed, red) and Galectin[9 (Anti-Gal9, green) in THP-1 cells. Nuclei was stained with DAPI (blue). G. Percent of cells with galectin-9 positive (gal9 + ) BCG in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. H. Confocal microscopy of Mtb H37Rv-GFP (Rv-GFP, green) and Galectin[9 (Anti-Gal9, red) in THP-1 cells. Nuclei were stained with DAPI (blue). I. Percent of cells with galectin9 positive (gal9 + ) Mtb H37Rv in total infected THP-1 cells. Symbols indicate colocalization ratio of at least 12 fields in each experiment. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Data are representative of three independent experiments with similar results (F and H). Two-tailed unpaired Student’s t test (A-D, G, and I) or Mann-Whitney U test (E). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Concentration Assay, Confocal Microscopy, Staining, Infection, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and lactose (1 μg/mL). B. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and D-glucose (10 μg/mL). C. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with or without galectin-9 (Gal9, 10 μg/mL) and AG (1 μg/mL). D. Growth profile of Mtb H37Rv (Rv) in Middlebrook 7H9 liquid medium with 1μg/mL CRD1 or CRD2 of galectin-9. Data are shown as mean ± SD, n = 3 biologically independent experiments performed in triplicate (A-D). Two-tailed unpaired Student’s t test (A-D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Cell-autonomous targeting of arabinogalactan by host immune factors inhibits mycobacterial growth

doi: 10.1101/2023.10.05.561003

Figure Lengend Snippet: A. Morphologic characteristics for Mtb H37Rv strain grown in liquid culture with or without anti-AG mAbs (1 μg/mL) observed by ×[2 magnifier. B. Bacterial shape of Mtb H37Rv strain treated as in (A) observed by acid fast staining under a Leica DM2500 microscope using the 100× oil microscopy. EMB, Ethambutol. Scale bar, 20μm. C. Ultrastructural morphology of Mtb H37Rv treated as in (A) analyzed by transmission electron microscopy (TEM). The cell wall was labeled with red arrows. D. Cell wall thickness of bacteria in (C). E. Schematic presentation of Mtb growth arrest by Galectin-9 or anti-AG antibodies. Data are representative of three independent experiments with similar results (A, B and C). Data are means ± SD of 11 bacteria, representatives of three independent experiments (D). Two-tailed unpaired Student’s t test (D). P < 0.05 was considered statistically significant.

Article Snippet: Cells were stained with the anti-galectin-9 antibody (Cell Signaling Technology, Cat#54330, RRID:AB_2799456), antibodies at a dilution of 1:200 in 5% BSA in PBS overnight at 4[°C and then incubated with Alexa Fluor 488 or 555 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A-11008; RRID: AB_143165; Cat# A32732, RRID:AB_2633281) at a dilution of 1:1000 for 2[h at R.T..

Techniques: Staining, Microscopy, Transmission Assay, Electron Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: PLoS Pathogens

Article Title: TIM-3 Does Not Act as a Receptor for Galectin-9

doi: 10.1371/journal.ppat.1003253

Figure Lengend Snippet: A) Characterization of T cell stimulator cells. Upper and middle panel: Expression of membrane-bound anti-CD3 antibody fragments (mb-aCD3) on T cell stimulator cells was detected with Dylight-649-labelled goat-anti-mouse IgG (H+L-chain specific) antibodies (grey histograms). Open histogram: reactivity with control Bw cells. Lower panel: T cell stimulator cells engineered to express human galectin-9 were probed with galectin 9 antibodies (grey histogram) or isotype control antibodies (open histogram). B) Human T cells derived from healthy donors were co-cultured with T cell stimulator cells expressing human galectin-9 or no human costimulatory molecule (control). 3 [H]-methyl-thymidine uptake was assessed following 3 days of co-culture (cpm, counts per minute). The results summarize 16 experiments with different donors. C) Culture supernatant derived from T cells activated with galectin-9 expressing stimulator cells or control stimulator cells was harvested after 48 h and subjected to multiplex cytokine measurement. Data show +/− SD of triplicates from one experiment representative for 16 independently performed.

Article Snippet: Mouse and human TIM-3 immunoglobulin (Ig) fusion proteins, human ILT5 Ig, human TROP2 Ig, human TREML2 Ig, mouse CTLA4 Ig fusion proteins - all expressed in NSO-cells – and recombinant human (rh) and recombinant mouse (rm) galectin-9 were purchased from R&D systems.

Techniques: Expressing, Membrane, Derivative Assay, Cell Culture, Co-Culture Assay, Multiplex Assay

Journal: PLoS Pathogens

Article Title: TIM-3 Does Not Act as a Receptor for Galectin-9

doi: 10.1371/journal.ppat.1003253

Figure Lengend Snippet: A, D) Bw control cells (open histograms) or Bw cells transduced to express human (A) or murine (D) galectin-9 (grey histograms) were probed with galectin-9 antibodies or with immunoglobulin fusion proteins representing human TIM-3 (TIM3-Ig; A) or murine TIM-3 (m-TIM-3-Ig; D) or a control fusion protein (B7-H3-Ig). Bound antibodies were detected with PE-labelled donkey-anti-goat antibodies. Immunoglobulin fusion proteins were detected with PE-labelled goat antibodies to the Fc-part of human IgG. B, E) Bw control cells (open histogram) or Bw cells transduced to express human (B) or murine (E) TIM-3 (grey histograms) were probed with appropriate anti-TIM-3 mAbs or with biotin-labelled recombinant human (B) or murine (E) galectin-9. Bound anti-human TIM-3 mAb and anti-murine TIM-3 mAb were detected with PE-labelled goat-anti-mouse IgG and APC-labelled goat-anti-rat IgG, respectively. SA-PE was used as secondary reagent for biotin-labelled recombinant human and murine galectin-9. C, F) Recombinant human (C) or murine (F) galectin-9 was immobilized on ELISA plates and probed with immunoglobulin fusion proteins (Ig) representing the extra-cellular domain of human TIM-3 (TIM-3-Ig; C) or murine TIM-3 (m-TIM-3-Ig; F) at the indicated concentrations. HRP-conjugated goat-anti-human IgG-Fc-specific antibodies were used for detection. Binding buffer only (PBS) or the indicated immunoglobulin fusion proteins were used as controls. All binding experiments were repeated three times with similar outcome.

Article Snippet: Mouse and human TIM-3 immunoglobulin (Ig) fusion proteins, human ILT5 Ig, human TROP2 Ig, human TREML2 Ig, mouse CTLA4 Ig fusion proteins - all expressed in NSO-cells – and recombinant human (rh) and recombinant mouse (rm) galectin-9 were purchased from R&D systems.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay