Journal: Open biology

Article Title: Internal regulation between constitutively expressed T cell co-inhibitory receptors BTLA and CD5 and tolerance in recent thymic emigrants.

doi: 10.1098/rsob.240178

Figure Lengend Snippet: Figure 1. BTLA expression in the thymus and spleen is inversely related to CD5 expression. (a) Relative fluorescent intensities (RFIs) of BTLA (upper row) or CD5 (lower row) in CD4 SP T cells (left column) and CD8 SP T cells (right column) in the thymus and spleen (BTLA, n = 39; CD5, n = 24). To calculate the RFI of BTLA or CD5, mean fluorescence intensity (MFI) data were normalized to the average MFI of BTLA or CD5 of the thymic SP T cells in each individual experiment. (b) Representative histograms showing the proportion of BTLA+ CD4 SP T cells (upper row) in the thymus and spleen. Lower row shows the proportion of BTLA+ CD4 SP T cells (lower left) and BTLA+ CD8 SP T cells (lower right) in the thymus and spleen (n = 39). (c) Representative flow cytometry dot plot (left) and histogram (right) of the indicated markers in splenic TCRβ+ cells from 7 to 10 week old B6.Rag2pGFP mice. (d) RFIs of BTLA (top panels) and CD5 (lower panels) on mature (GFP−) or newly generated (GFP+) SP T cells in the spleen (n = 11). Data are normalized to the average BTLA MFI or CD5 MFI of the GFP negative splenic SP T cells in each individual experiment. (e) CD5 expression on CD4 SP T cells and CD8 SP T cells from thymus (left) or spleen (right) and their corresponding representative histograms (lower rows) from WT (n = 24; B6.Foxp3GFP and B6.Nur77GFP), Btla−/− (n = 22; B6.Foxp3GFP Btla−/− and B6.Nur77GFP Btla−/−) and Pdcd1−/− (n = 6; B6.Foxp3EGFP Pdcd1−/−) mice. (f) Nur77-GFP expression. Dots indicate individual mice from six to nine independent experiments (a,b,e) or two independent experiments (c,d,f). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Resources resource source identifier antibodies Alexa Fluor 488 Goat anti-rat IgG Life Technologies A11006 APC-eFluor 780 Anti-TCRβ clone H57−597 ThermoFisher 47-5961-82 Alexa Fluor 700 Anti-CD4 clone RM4-5 ThermoFisher 56-0042-82 PerCP-Cyanine5.5 Anti-CD5 clone 53-7.3 ThermoFisher 45-0051-82 APC Anti-FoxP3 clone FJK-16s ThermoFisher 17-5773-82 PerCP-Cyanine5.5 Anti-CD62L clone MEL-4 ThermoFisher 45-0621-82 PE-Cyanine7 Anti-Mouse CD44 clone IM7 ThermoFisher 25-0441-82 PE Anti-BTLA clone 6F7 ThermoFisher 12-5950-82 APC Anti-PD−1 clone J43 ThermoFisher 17-9985-82 PE Anti-TCR-Vß5 clone MR9-4 BD Pharmingen 5 53 190 eFluor 450 Anti-TCR-Vα2 clone B20.1 eBioscience 48-5812-82 Super Bright 600 Anti-Mouse CD8a clone 53-6.7 ThermoFisher 63-0081-82 Brilliant Violet 421 Anti-CD19 clone 6D5 BioLegend 115537 PE Anti-HVEM clone HMHV-1B18 BioLegend 136304 Anti-CD16/32 antibody clone 2.4G2 Bioxcell BE0307 Rat anti-Mouse CD8α clone 53-6.7 Biolegend 100701 Rat anti-Mouse CD4 Bio-Rad MCA2691 Purified anti-BTLA clone 6A6 Bioxcell BE0132 (Continued.)

Techniques: Expressing, Fluorescence, Flow Cytometry, Generated

Journal: Open biology

Article Title: Internal regulation between constitutively expressed T cell co-inhibitory receptors BTLA and CD5 and tolerance in recent thymic emigrants.

doi: 10.1098/rsob.240178

Figure Lengend Snippet: Figure 2. Loss of BTLA in adult mice leads to increased T cell CD5 and PD1 expression. (a) Adult B6Cre/ERT2+/− or B6Cre/ERT2+/− Btlafl/fl mice received five doses of tamoxifen on days 0, 1, 3, 5 and 6 (highlighted in red). (b) Representative histograms (top) of BTLA expression and the MFI of CD5 (bottom) of BTLA+ and BTLA− cells in the CD4 gated (left) and CD8 gated (right) T cells in the peripheral blood at one week post-tamoxifen. (c) Representative dot plots of BTLA expression in the splenic CD4 SP T cells (top) and CD8 T cells (bottom) in the B6Cre/ERT2+/− mice (left) and B6Cre/ERT2+/− Btlafl/fl mice (right) at two weeks post-tamoxifen. (d) RFI of CD5 and PD-1 in the thymic (top row) CD4 and CD8 or splenic (bottom row) CD4 and CD8 T cells of B6Cre/ERT2+/− (WT) and B6Cre/ERT2+/− Btlafl/fl (fl/fl) mice at four weeks post-tamoxifen. Dots indicate individual mice from two independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Resources resource source identifier antibodies Alexa Fluor 488 Goat anti-rat IgG Life Technologies A11006 APC-eFluor 780 Anti-TCRβ clone H57−597 ThermoFisher 47-5961-82 Alexa Fluor 700 Anti-CD4 clone RM4-5 ThermoFisher 56-0042-82 PerCP-Cyanine5.5 Anti-CD5 clone 53-7.3 ThermoFisher 45-0051-82 APC Anti-FoxP3 clone FJK-16s ThermoFisher 17-5773-82 PerCP-Cyanine5.5 Anti-CD62L clone MEL-4 ThermoFisher 45-0621-82 PE-Cyanine7 Anti-Mouse CD44 clone IM7 ThermoFisher 25-0441-82 PE Anti-BTLA clone 6F7 ThermoFisher 12-5950-82 APC Anti-PD−1 clone J43 ThermoFisher 17-9985-82 PE Anti-TCR-Vß5 clone MR9-4 BD Pharmingen 5 53 190 eFluor 450 Anti-TCR-Vα2 clone B20.1 eBioscience 48-5812-82 Super Bright 600 Anti-Mouse CD8a clone 53-6.7 ThermoFisher 63-0081-82 Brilliant Violet 421 Anti-CD19 clone 6D5 BioLegend 115537 PE Anti-HVEM clone HMHV-1B18 BioLegend 136304 Anti-CD16/32 antibody clone 2.4G2 Bioxcell BE0307 Rat anti-Mouse CD8α clone 53-6.7 Biolegend 100701 Rat anti-Mouse CD4 Bio-Rad MCA2691 Purified anti-BTLA clone 6A6 Bioxcell BE0132 (Continued.)

Techniques: Expressing

Journal: Open biology

Article Title: Internal regulation between constitutively expressed T cell co-inhibitory receptors BTLA and CD5 and tolerance in recent thymic emigrants.

doi: 10.1098/rsob.240178

Figure Lengend Snippet: Figure 5. Loss of BTLA early in T cell ontogeny generates autoimmune disease. (a) We adoptively transferred 20 × 106 FLC pooled from 8 to 10 embryonic day 14–16 B6Cre/ERT2+/− (WT) or B6Cre/ERT2+/− Btlafl/fl (fl/fl) foetuses to 7 week old Rag–/– mice on day 0 (n = 3 recipients per group), followed by tamoxifen injection on days 0, 1, 3, 5 and 6. Recipient mice were monitored for signs of disease for eight weeks post-FLC transfer. (b) MFI of CD5 in CD4 T cells (top) and CD8 T cells (bottom) with respective representative histograms of peripheral T cells in the recipients of FLC from B6Cre/ERT2+/− and B6Cre/ERT2+/− Btlafl/fl at eight weeks post-tamoxifen. Dots indicate data from individual mice; **p < 0.01. (c) Left panel: disease incidence in recipients of B6Cre/ERT2+/− (blue line) B6Cre/ERT2+/− Btlafl/fl (black dashed line) FLC. Survival curves were significantly different, p = 0.02. The grey rectangle indicates the range, in days, at which the first T cells were detected in the peripheral blood after FLC transfer. Right panel: weight changes in recipients of B6Cre/ERT2+/− Btlafl/fl FLC or B6Cre/ERT2+/− FLC. The red box on the X-axes indicates the tamoxifen treatment period. The presence (filled) or absence (empty) of disease signs is depicted on the far-right panel. (d) Flow cytometry gating (top). A representative histogram of BTLA expression in the T and B cells populating the periphery of B6Cre/ERT2+/− Btlafl/fl FLC recipient mice (middle) or B6Cre/ERT2+/− Btlafl/fl FLC recipient mice (bottom) at four weeks post-FLC transfer is shown.

Article Snippet: Resources resource source identifier antibodies Alexa Fluor 488 Goat anti-rat IgG Life Technologies A11006 APC-eFluor 780 Anti-TCRβ clone H57−597 ThermoFisher 47-5961-82 Alexa Fluor 700 Anti-CD4 clone RM4-5 ThermoFisher 56-0042-82 PerCP-Cyanine5.5 Anti-CD5 clone 53-7.3 ThermoFisher 45-0051-82 APC Anti-FoxP3 clone FJK-16s ThermoFisher 17-5773-82 PerCP-Cyanine5.5 Anti-CD62L clone MEL-4 ThermoFisher 45-0621-82 PE-Cyanine7 Anti-Mouse CD44 clone IM7 ThermoFisher 25-0441-82 PE Anti-BTLA clone 6F7 ThermoFisher 12-5950-82 APC Anti-PD−1 clone J43 ThermoFisher 17-9985-82 PE Anti-TCR-Vß5 clone MR9-4 BD Pharmingen 5 53 190 eFluor 450 Anti-TCR-Vα2 clone B20.1 eBioscience 48-5812-82 Super Bright 600 Anti-Mouse CD8a clone 53-6.7 ThermoFisher 63-0081-82 Brilliant Violet 421 Anti-CD19 clone 6D5 BioLegend 115537 PE Anti-HVEM clone HMHV-1B18 BioLegend 136304 Anti-CD16/32 antibody clone 2.4G2 Bioxcell BE0307 Rat anti-Mouse CD8α clone 53-6.7 Biolegend 100701 Rat anti-Mouse CD4 Bio-Rad MCA2691 Purified anti-BTLA clone 6A6 Bioxcell BE0132 (Continued.)

Techniques: Injection, Flow Cytometry, Expressing

Journal: Open biology

Article Title: Internal regulation between constitutively expressed T cell co-inhibitory receptors BTLA and CD5 and tolerance in recent thymic emigrants.

doi: 10.1098/rsob.240178

Figure Lengend Snippet: Figure 6. Autoimmune disease in Btla–/– thymocyte recipients requires CD4+ T cells and MHC II. (a) We adoptively transferred 3 × 106 MACS-sorted CD4 or CD8 SP thymocytes pooled from seven 8–10 week old B6.Foxp3EGFP × Btla−/− (left column) or B6.Foxp3EGFP (right column) mice i.v. to 8–10 week old Rag−/− mice (Btla–/–

Article Snippet: Resources resource source identifier antibodies Alexa Fluor 488 Goat anti-rat IgG Life Technologies A11006 APC-eFluor 780 Anti-TCRβ clone H57−597 ThermoFisher 47-5961-82 Alexa Fluor 700 Anti-CD4 clone RM4-5 ThermoFisher 56-0042-82 PerCP-Cyanine5.5 Anti-CD5 clone 53-7.3 ThermoFisher 45-0051-82 APC Anti-FoxP3 clone FJK-16s ThermoFisher 17-5773-82 PerCP-Cyanine5.5 Anti-CD62L clone MEL-4 ThermoFisher 45-0621-82 PE-Cyanine7 Anti-Mouse CD44 clone IM7 ThermoFisher 25-0441-82 PE Anti-BTLA clone 6F7 ThermoFisher 12-5950-82 APC Anti-PD−1 clone J43 ThermoFisher 17-9985-82 PE Anti-TCR-Vß5 clone MR9-4 BD Pharmingen 5 53 190 eFluor 450 Anti-TCR-Vα2 clone B20.1 eBioscience 48-5812-82 Super Bright 600 Anti-Mouse CD8a clone 53-6.7 ThermoFisher 63-0081-82 Brilliant Violet 421 Anti-CD19 clone 6D5 BioLegend 115537 PE Anti-HVEM clone HMHV-1B18 BioLegend 136304 Anti-CD16/32 antibody clone 2.4G2 Bioxcell BE0307 Rat anti-Mouse CD8α clone 53-6.7 Biolegend 100701 Rat anti-Mouse CD4 Bio-Rad MCA2691 Purified anti-BTLA clone 6A6 Bioxcell BE0132 (Continued.)

Techniques: