Journal: bioRxiv

Article Title: CD3e-immunotoxin spares CD62L lo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3e hi T cells

doi: 10.1101/2022.07.25.501205

Figure Lengend Snippet: (A) The diagram shows an S-CD3e-IT treatment and intravascular staining strategy to analyze the effect of S-CD3e-IT efficacy on different Tissue-resident T cells The flow cytometry gating strategy shows circulating (IVS+) and tissue-resident cells (IVS-) separation in peripheral blood, mesenteric LNs, and spleen. (B) Absolute number of CD3+ cells (y-axis) is shown for the peripheral blood (IVS+) and tissue-resident (IVS-) portions of spleen, mesenteric LNs, Peyer’s patches, lung, thymus, and bone marrow. Absolute cell numbers were calculated based on CountBright Absolute counting beads and total cell counts per organ. (C-D) Flow cytometry for CD4+ and CD8+ T cells (C) and the ratios of CD4+ to CD8+ for all tested organs (D) are shown. (E-F) Flow cytometry for CD4+Foxp3- and CD4+Foxp3+ T cells (E) and % of these cells in CD4+ cells (F) are shown for all tested organs. Nonimmunized mice treated with PBS ( n = 4∼5, depending on organs), or S-CD3e-IT ( n = 4∼6), OVA-immunized mice treated with PBS ( n = 3∼4), or S-CD3e-IT ( n = 7∼8), were compared. (* p < 0.05 and ** p < 0.01).

Article Snippet: Anti-murine version of S-CD3e-IT was prepared by conjugating biotinylated-S-CD3e-mAb with streptavidin-ZAP (Advanced Targeting System, San Diego, Calif) in a 1:1 molar ratio as previously described( ).

Techniques: Staining, Flow Cytometry

Journal: bioRxiv

Article Title: CD3e-immunotoxin spares CD62L lo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3e hi T cells

doi: 10.1101/2022.07.25.501205

Figure Lengend Snippet: (A) Symbols of mesenteric (white), inguinal (light blue), mandibular (blue), mediastinal (red), and lumber (green) LNs are shown. (B) Fold reductions of CD4+ or CD8+ T cells (y-axis; S-CD3e-IT/PBS) and CD3e MFI of these cells in each LNs (x-axis) are shown. There was no positive correlation between CD3e MFI and CD4+ and CD8+ cell depletion rates. (C) Fold reductions of CD4+Foxp3-or CD4+Foxp3+ T cells (y-axis; S-CD3e-IT/PBS) and CD3e MFI of these cells in each LNs (x-axis) are shown. There was no positive correlation between CD3e MFI and CD4+Foxp3-cells. (D) CD62L lo and CD62L hi (% in CD4+Foxp3+ cells) are shown for these LNs. The surviving cells were enriched in CD4+Foxp3+ cell pools with the CD62L lo phenotype. (E) The Tfr-to-Tfh ratios (% in CD4+) are shown for these LNs. S-CD3e-IT-treated mice show higher Tfr-to-Tfh ratios in most of these LNs. Nonimmunized mice were treated with PBS ( n = 2∼4, depending on LN types) or S-CD3e-IT ( n = 2∼6). OVA-immunized mice were treated with PBS ( n = 3∼4) or S-CD3e-IT) ( n = 4∼8) * p < 0.05 and ** p < 0.01.

Article Snippet: Anti-murine version of S-CD3e-IT was prepared by conjugating biotinylated-S-CD3e-mAb with streptavidin-ZAP (Advanced Targeting System, San Diego, Calif) in a 1:1 molar ratio as previously described( ).

Techniques:

Journal: bioRxiv

Article Title: CD3e-immunotoxin spares CD62L lo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3e hi T cells

doi: 10.1101/2022.07.25.501205

Figure Lengend Snippet: (A) CD3e expression (x-axis) on the CD8+, CD4+, CD4+Foxp3-, and CD4+Foxp3+ cells (y-axis, normalized to mode) are shown for the peripheral blood and tissue-resident portions (IVS-) of spleen, mesenteric LNs, and lungs. (B) Heatmaps show CD3e mean fluorescent intensities (MFI) for CD4+, CD8+, CD4+Foxp3- and CD4+Foxp3+ cells cells of all tested organs. Statistical comparison of CD3e MFI on two groups, CD4 (PBS) vs CD8 (PBS) and PBS-treated vs S-CD3e-IT-treated mice, is denoted with an asterisk (*) on the heat map. (* p < 0.05 and ** p < 0.01). (C) The diagram shows the symbol of all tested organs in (D) and (E). (D) A positive correlation between the fold reductions of CD4+ or CD8+ cell numbers in different organs after CD3e-IT treatment (y-axis) and CD3e MFI of these cells in PBS-treated animals (x-axis). CD4+ and CD8+ lymphocytes for the peripheral blood (red), and tissue-resident (IVS-) portions of spleen (green), mesenteric LNs (white), Peyer’s patches (purple), lung (black), thymus (cross), and bone marrow (asterisk) are compared. (E) A positive correlation between the fold reductions of CD4+Foxp3-cell numbers in different organs after CD3e-IT treatment (y-axis) and CD3e MFI of these cells in PBS-treated animals (x-axis). Note that this correlation was not evident for CD3+Foxp3+ T regs. Nonimmunized mice (PBS n = 4∼5, and S-CD3e-IT n = 4∼6, depending on organs); OVA-immunized mice (PBS n = 3∼4, and S-CD3e-IT n = 7∼8, depending on organs)

Article Snippet: Anti-murine version of S-CD3e-IT was prepared by conjugating biotinylated-S-CD3e-mAb with streptavidin-ZAP (Advanced Targeting System, San Diego, Calif) in a 1:1 molar ratio as previously described( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: CD3e-immunotoxin spares CD62L lo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3e hi T cells

doi: 10.1101/2022.07.25.501205

Figure Lengend Snippet: (A) The flow cytometry gating strategy for CD4+Foxp3+CD62L lo and CD4+Foxp3+CD62L hi . (B) CD62L lo and CD62L hi (% in CD4+Foxp3+ cells) are shown for the peripheral blood (IVS+) and tissue-resident (IVS-) portions of spleen, mesenteric LNs, peyer’s patches, lungs, thymus, and bone marrow. (C) The heatmap of CD3e MFI (yellow to red) on CD4+Foxp3+CD62L lo and CD4+Foxp3+CD62L hi cells in tested organs. CD3e MFI on CD62L lo was higher than CD62L hi in all tested organs. A statistical comparison of CD3e MFI of two groups, CD62L lo (PBS) vs CD62L hi (PBS) and PBS-treated vs S-CD3e-IT-treated mice, is denoted with an asterisk (*) on the heat map. The heatmap of the CD62L lo -to-CD62L hi ratios (white to blue). The ratio increased after S-CD3e-IT treatment except in peripheral blood and bone marrow. Nonimmunized mice were treated with PBS ( n = 4∼5, depending on organs) or S-CD3e-IT ( n = 4∼6). OVA-immunized mice were treated with PBS ( n = 3∼4) or S-CD3e-IT ( n = 7∼8). (* p < 0.05 and ** p < 0.01)

Article Snippet: Anti-murine version of S-CD3e-IT was prepared by conjugating biotinylated-S-CD3e-mAb with streptavidin-ZAP (Advanced Targeting System, San Diego, Calif) in a 1:1 molar ratio as previously described( ).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: CD3e-immunotoxin spares CD62L lo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3e hi T cells

doi: 10.1101/2022.07.25.501205

Figure Lengend Snippet: (A) S-CD3e-IT treatment enriched CD25+ Tregs cells. CD25+ population (% in CD4+ cells) is shown for the peripheral blood and tissue-resident (IVS-) portions of spleen, mesenteric LNs, and thymus in PBS and S-CD3e-IT treated mice. (B) The flow gating strategy is shown to define CD44+, CXCR5+, Ki67+, and PD-1+ cells in CD4+Foxp3- and CD4+Foxp3+ cells. The heat map shows four functional phenotypic changes (light red to dark red) of CD4+Foxp3- and CD4+Foxp3+ T cells following S-CD3e-IT treatment. Statistical comparison between PBS-treated vs S-CD3e-IT-treated mice is denoted with an asterisk (*) on the Heat map. (C) The flow cytometry gating strategy is shown to define CXCR5+PD-1+ Tfr or Tfh. The CXCR5+PD-1+ Tfr-to-CXCR5+PD-1+ Tfh ratios are shown forperipheral blood and tissue-resident (IVS-) portions of the spleen, mesenteric LN, and thymus. Nonimmunized mice were treated with PBS ( n = 4∼5, depending on organs) or S-CD3e-IT ( n = 4∼6). OVA-immunized mice were treated with PBS ( n = 3∼4) or S-CD3e-IT ( n = 7∼8). (* p < 0.05 and ** p < 0.01).

Article Snippet: Anti-murine version of S-CD3e-IT was prepared by conjugating biotinylated-S-CD3e-mAb with streptavidin-ZAP (Advanced Targeting System, San Diego, Calif) in a 1:1 molar ratio as previously described( ).

Techniques: Functional Assay, Flow Cytometry