anti lag3 antibody  (Bio X Cell)

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Design of a SHEDTAC library spanning five anti-ADAM10 VHH and three anti-LAG3 VHH, all targeting distinct epitopes. Diverse configurations are achieved through N→C or C→N terminal VHH fusions, affording a library of thirty unique LAG3/ADAM10 bispecific combinations. ( b ) Reducing SDS-PAGE analysis of purified SHEDTACs used to treat cells in ( c,d ). ( c ) T cell surface LAG3 shedding by the protease ADAM10, which is accelerated by SHEDTACs ( d ) Western blot analysis of T cell pellets indicating levels of intact LAG3 on cells following 24h treatment, quantified in ( e ). Intensity is expressed as percent of vehicle (V). “+” indicates the addition of ionomycin (10µg/ml) to induce ADAM10 activity.

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: SDS Page, Purification, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Reducing SDS-PAGE indicating SHEDTAC#8, selected for its high activity shown in . ( b-e ) Flow cytometry contour plots showing LAG3 abundance on CD3+ADAM10+ PBMCs following 1h treatment with ( b ) vehicle, ( c ) 500nM SHEDTAC, ( d,e ) equimolar TEV-proteolyzed SHEDTAC, serving as monospecific controls. ( f ) Flow cytometry contour plots showing LAG3 abundance on CD3+ADAM10+ PBMCs treated with SHEDTAC (left) or equimolar TEV-digested SHEDTAC (right). Prior to treatment, cells were incubated for 2h at 37°C with vehicle (left plots), proteasome inhibitor (MG132, 10µM, middle plots), or lysosome inhibitor (Dynasore, 50µM, right plots). ( g ) Western blot analysis of cell pellets and conditioned cell supernatants treated with SHEDTAC, sampled every 10 minutes for 60 minutes, indicating time-dependent decreases in full-length (∼70kDa) LAG3 and concomitant increases in soluble LAG3 (sLAG3, ∼60kDa) ectodomain released into the growth medium by ADAM10. ( h ) quantification of data from ( g ) normalized to GAPDH and expressed as percent control of cell pellet at t=0. ( i ) Cells from ( g ) following 24h SHEDTAC treatment. ( j ) Ratio of LAG3:ADAM10 as determined by flow cytometry over a range of SHEDTAC concentrations.

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: SDS Page, Activity Assay, Flow Cytometry, Incubation, Western Blot, Control

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) LAG3 suppresses T cell signaling through homodimer formation, and interactions with the TCR on T cells and MHCII on antigen presenting cells (APCs) (left). LAG3-SHEDTACs catalyze LAG3 proteolysis by endogenous protease ADAM10 to restore TCR signaling and induce a luciferase reporter (right). ( b ) Flow cytometry contour plots indicating LAG3 abundance on ADAM10(+) luciferase reporter Jurkat cells treated with isotype control or LAG3-SHEDTAC. ( c ) Dose-dependent luminescence increases following treatment with SHEDTAC at the indicated concentration, illustrating enhanced TCR signaling that is afforded through LAG3 shedding. RLU = relative luminescence units

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Luciferase, Flow Cytometry, Control, Concentration Assay

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Gating strategy for activated CD3+ADAM10+LAG3+ PBMCs

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques:

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Flow cytometry TEV-normalization scheme. SHEDTACs were normalized to their equimolar TEV-proteolyzed controls, and significant shedding was indicated wherever this ratio ‘x’ was x<1. In contrast, TEV-normalized shedding where x≥1 indicates low SHEDTAC activity, or VHH competition with LAG3 detection reagents, confirmed by western blot

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Flow Cytometry, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Comparison of western blot versus flow cytometry analyses to assess LAG3 shedding by ADAM10 following treatment with SHEDTACs

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Comparison, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Soluble LAG3 (sLAG3) generation through receptor shedding. ( b ) primary amino acid sequence analysis showing transmembrane and intracellular regions totaling ∼8.8kDa. ( c ) Dose-dependent loss of LAG3 abundance on T cells following treatment with SHEDTACs. Contour plots correspond to data plotted in . ( d ) Concomitant soluble LAG3 production in conditioned supernatants from cells treated in ( c ).

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Immune cells adapt to distinct stem cell niches to govern tissue homeostasis

doi: 10.64898/2026.01.28.701831

Figure Lengend Snippet: a , UMAP plots displaying distribution of Ltb, Areg and Lag3 transcripts across different cell types in mouse skin. Each dot is one cell, color-coded based on transcript expression levels. b, Schematic of the Ltb-CreER x R26-tdT mouse line. c, Left: Representative whole mount immunofluorescence showing that double positive (tdT + and TCRγδ + ) LTB-expressing DETCs reside primarily within the uHF compartment as do other LTB-expressing lymphocytes (tdT + and TCRγδ neg ). Epithelial SCs are marked by K14. Scale bar 30μm. Right: quantifications of (left) the probability of finding LTB + DETCs (tdT + and TCRγδ + ) in uHF vs IFE and (right) probability of finding any LTB + cell (tdT + ) in the uHF region vs IFE. Each circle represents data from one mouse (3-8 fields of view). d-e , Representative FACS plots and quantifications showing that most DETCs expressing AREG (d) and LAG-3 (e) are uLIPSTIC neg (IFE-DETC) and not uLIPSTIC + (uHF-DETC). MFI, mean fluorescence intensity. f, Percentage of LTB-tdT + , AREG + or LAG-3 + DETC out of total DETC as quantified by FACS of the skins of mice at the indicated postnatal time points. Each point represents the mean with SEM of 2-7 mice. g, Percentage of AREG + DETC in the embryonic thymus of E17-18 pups and from the back skin of adult second telogen mice as quantified by FACS. h, Quantifications of total skin intraepithelial Ltb transcripts determined by qPCR, and AREG + or LAG-3 + DETC determined by FACS when mice are kept in specific pathogen free (SPF) or germ-free (GF) facility. i, Schematic summarizing the compartmentalization of the two DETC niche programs in steady state. Data are representative of two-four (panel h), two (panel e), three (panel c, d, g) or three-seven (panel f) independent experiments and, unless indicated, each circle represents one mouse. Data in c, d, g and h are analysed by unpaired two-tailed Student’s t- test. p- values are indicated in each figure and data are represented as mean with SEM. Further details on statistics and reproducibility in Methods. See for additional supporting experiments.

Article Snippet: monoclonal antibodies against mouse LAG-3 (clone C9B7W, BioXcell) were injected intraperitoneally every other day (for a total of three injections) in wild-type C57BL/6 animals.

Techniques: Expressing, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Immune cells adapt to distinct stem cell niches to govern tissue homeostasis

doi: 10.64898/2026.01.28.701831

Figure Lengend Snippet: a , Percentage of LAG-3 + cells gated on DETC, ILC or TCRαβ + T cells in wild type back skin as determined by FACS. b, Left: strategy to block LAG-3 in vivo . Right: quantifications reveal that DETCs but not TCRαβ + T cells increase in numbers in the skin when anti-LAG-3 antibodies are delivered intraperitoneally to mice. c, Left: representative max projections of whole mount immunofluorescence of IFE and uHF compartments showing that the increase in DETC (in yellow) resulting from anti-LAG-3 blocking antibodies occurs largely in the IFE-SC and not the uHF-SC niche. Scale bar 50μm. * denote autofluorescence. d, Normalized concentration of AREG determined by ELISA shows elevated AREG in the intraepithelial tissue, consistent with the elevation in DETCs caused by LAG-3 inhibition. e, Normalized percentage of EdU + SCs determined by FACS after a 3-hour pulse in mice injected with either PBS or anti-LAG-3 blocking antibodies. f, Top: strategy to deliver recombinant AREG (rAREG) systemically in vivo by osmotic pump implantation; bottom: concentration of AREG determined by ELISA in the intraepithelial tissue of mice implanted with pumps containing either rAREG or PBS as a control. g, Percentage of EdU + SCs determined by FACS after a 4-hour pulse in mice implanted with pumps containing either rAREG or PBS as a control. Data are representative of two (panel f), three (panel c, d, g), five (panel a, e) or seven (panel b) independent experiments and each circle represents one mouse. Data in b, c, d, e, f and g are analyzed by unpaired two-tailed Student’s t- test. Data in panel a are analyzed by ordinary one-way ANOVA with Tukey’s multiple comparison post-test. p- values are indicated in each figure and data are represented as mean with SEM. Further details on statistics and reproducibility in Methods. See and for additional supporting experiments.

Article Snippet: monoclonal antibodies against mouse LAG-3 (clone C9B7W, BioXcell) were injected intraperitoneally every other day (for a total of three injections) in wild-type C57BL/6 animals.

Techniques: Blocking Assay, In Vivo, Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Injection, Recombinant, Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Immune cells adapt to distinct stem cell niches to govern tissue homeostasis

doi: 10.64898/2026.01.28.701831

Figure Lengend Snippet: a , UMAP plots showing expression pattern of H2-ab1 (MHC-II) and Lgals3 (Galectin-3) across different cell type from mouse back skin. b , Quantification of the percentage of MHC-II + and mean fluorescence intensity (MFI) of Galectin-3 in Bulge-SC, uHF-SC and IFE-SC from wild type mice determined by FACS. Each dot represents one mouse. c , Percentage of LAG-3 + cells gated on CD8 + CD44 + splenocytes from wild type mice injected with PBS or with anti-LAG3 blocking antibodies. Each dot represents one mouse from two independent experiments. d , qPCR analysis of mKi67 expression in the intraepithelial compartment of wild type mice injected with PBS or with anti-LAG3 blocking antibodies. Each dot represents one mouse from three independent experiments. Data in c and d are analysed by unpaired two-tailed Student’s t- test. Data in b are analysed by ordinary one-way ANOVA with Tukey’s multiple comparison post-test. p -values are indicated in each figure and data are represented as mean with SEM.

Article Snippet: monoclonal antibodies against mouse LAG-3 (clone C9B7W, BioXcell) were injected intraperitoneally every other day (for a total of three injections) in wild-type C57BL/6 animals.

Techniques: Expressing, Fluorescence, Injection, Blocking Assay, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Immune cells adapt to distinct stem cell niches to govern tissue homeostasis

doi: 10.64898/2026.01.28.701831

Figure Lengend Snippet: Representative whole mount max projections of the IFE compartment confirms that immigrant T cells (CD3+) occupy the IFE in FVB-TAC (mutant for DETC-cognate ligand, Skint1 ). FVB-JAX mice are shown as WT controls. Scale bar 100μm. b, Bar plot showing relative proportions of Vg5+, Vg5neg, TCRab+ T cells and ILCs in the intraepithelial compartment of FVB JAX or TAC mice. c, Left: percentage of AREG+ Vg5neg TCRgd T cells detected by FACS in the intraepithelial compartment of FVB JAX or TAC mice. Middle: ELISA showing comparable AREG levels in the intraepithelial fractions from FVB JAX and FVB TAC mice. Right: Representative FACS plots and quantifications of percentage of LAG-3+ Vg5neg TCRgd T cells from the intraepithelial fraction of FVB JAX or TAC mice. d, Representative whole mount max projections of the IFE compartment from wild type and Rag2-/-mice showing intraepithelial CD45+ immune cells (red) and CD3+ T cells (green). Scale bar 100μm. * denote autofluorescence. e, Quantifications reveal that ILCs (CD45+CD90+ TCRgdneg TCRbneg) compensate for the loss of DETCs (CD45+CD90+ TCRgdhigh) when all T cells are ablated. f, Left: FACS analyses of the intraepithelial compartment reveal that ILCs become AREG+ when they occupy the IFE-SC niche vacated by DETC loss. Middle: ELISA reveals that the level of AREG in the intraepithelial fraction is largely independent of the immune resident in the IFE niche. Right: Representative FACS plots and quantifications of percentage of LAG-3+ ILCs from the intraepithelial fraction of wild type (WT) and Rag2-/- mice. g, Schematic showing immune adaptation within distinct SC niches and homeostatic mechanisms to keep niche size and activity in check. Data are representative of two (panel b, c, e) or three (f) independent experiments, and each circle represents one mouse. Data in c and f are analysed by unpaired two-tailed Student’s t- test. p- values are indicated in each figure and data are represented as mean with SEM. Further details on statistics and reproducibility in Methods. See for additional supporting experiments.

Article Snippet: monoclonal antibodies against mouse LAG-3 (clone C9B7W, BioXcell) were injected intraperitoneally every other day (for a total of three injections) in wild-type C57BL/6 animals.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test