cd183 pe  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Stable characteristics of intrapopulation heterogeneity in virus-specific Th1 cells during chronic viral challenge infection

doi: 10.1101/2025.09.15.676263

Figure Lengend Snippet: Naive Smarta CD4 + T cells from T-bet ZsGreen donors (Thy1.1 + ) were transferred into T-bet ZsGreen recipients (Thy1.2 + ). Recipient mice were infected with LCMV Arm (200 PFU). On day 10 p.i. the cells were harvested from the spleen and lymph nodes. T-bet ZsGreen + Smarta cells were electronically gated (egated) according to their T-bet reporter expression levels into T-bet high or T-bet low fractions, and all T-bet reporter positive cells (T-bet mock ) served as controls. A) Representative histogram of T-bet ZsGreen expression (grey = naïve endog. CD4 + T cells). Pooled and normalized T-bet ZsGreen MFI of each egated fraction. B) Representative histogram of T-bet protein expression (grey = naïve endog. CD4 + T cells). Pooled and normalized T-bet protein MFI of each egated fraction. C) Representative gating of CXCR3 and Ly6C of endogenous effector CD4 + T cells (grey) or the egated T-bet High , T-bet Low or T-bet Mock fractions of Smarta cells. Pooled frequencies of different subsets. D) Representative gating of PD-1 and CXCR5 of endogenous effector CD4 + T cells (grey) or the egated fractions of Smarta cells. Pooled frequencies of different subsets. E) Representative histogram of BCL6 expression (grey = naïve endog. CD4 + T cells, dark grey = endogenous effector GC Tfh (PD-1 + CXCR5 + ) cells). Pooled and normalized BCL6 MFI of each egated fraction. F) Representative histogram of TCF1 expression (grey = naïve endog. CD4 + T cells, dark grey = endogenous effector GC Tfh (PD-1 + CXCR5 + ) cells). Pooled and normalized TCF1 MFI of each egated fraction. Data are presented as mean ± SD. Each dot represents isolated Smarta T cells or endogenous CD4 + T cells (grey) from one individual recipient. 5 independent experiments were pooled (n=4-5 mice/experiment). For MFI comparison, MFI of T-bet High , T-bet Low , endogenous GC Tfh or naïve CD4 + T cells were normalized to the corresponding T-bet Mock sample. Statistical significance was determined by paired T-test or Wilcoxon test comparing T-bet low or mock to high Smarta cell fraction, statistical comparison to endogenous cells was not performed. p* <0.05, p**< 0.01, p****< 0.0001, ns = not significant.

Article Snippet: Naive CD4 + T cells from T-bet ZsGreen Smarta Thy1.1 + mice were purified by magnetic cell sorting in a negative enrichment approach with biotin-labeled antibodies against CD8 (53-6.7), NK1.1 (PK136), CD11b (M1/70), CD11c (HL3), CD25 (7D4), Gr-1 (RB6-8C5), CD19 (1D3), and CXCR3 (CXCR3-173) in combination with anti-biotin microbeads according to the manufacturer instructions (Miltenyi Biotec).

Techniques: Infection, Expressing, Isolation, Comparison

Journal: bioRxiv

Article Title: Stable characteristics of intrapopulation heterogeneity in virus-specific Th1 cells during chronic viral challenge infection

doi: 10.1101/2025.09.15.676263

Figure Lengend Snippet: Ten days p.i. with LCMV Arm, T-bet High , T-bet Low and T-bet Mock sorted Smarta cells (Thy1.1 + ) were transferred into individual naïve T-bet ZsGreen recipients (Thy1.2 + ). Two weeks post transfer, the recipients were infected with high dose LCMV Clone 13 (≥2×10 6 PFU) and 7 days post infection, the transferred cells were isolated from spleen and their phenotype was analyzed with flow cytometry. A) Experimental Layout. B) Representative histogram of T-bet ZsGreen expression (grey = naïve endog. CD4 + T cells). Normalized and pooled T-bet ZsGreen MFI of transferred cells day 7 p.i. with LCMV Cl13. C) Representative histogram of IFN-γ expression after GP64 restimulation (grey = unstimulated T-bet Mock cells). Normalized and pooled IFN-γ MFI of cytokine-positive transferred cells. D) Representative plots of CXCR3 and Ly6C staining of transferred cells and endogenous T-bet + primary effector CD4 + T cells, as indicated. Pooled frequencies of CXCR3 + Ly6C - and CXCR3 + Ly6 + Smarta cells. Data are presented as mean ± SD. Each dot represents isolated Smarta CD4 + T cells from one individual recipient. 3 independent experiments were pooled (n=4-5 mice/fraction/experiment). For MFI comparison, MFI of T-bet High or T-bet Low cells were normalized to the average of T-bet Mock samples in each experiment. Statistical significance was determined using unpaired T-test or Mann-Whitney test comparing T-bet low or mock to high fraction. p* <0.05, p***< 0.001, p****< 0.0001, ns = not significant.

Article Snippet: Naive CD4 + T cells from T-bet ZsGreen Smarta Thy1.1 + mice were purified by magnetic cell sorting in a negative enrichment approach with biotin-labeled antibodies against CD8 (53-6.7), NK1.1 (PK136), CD11b (M1/70), CD11c (HL3), CD25 (7D4), Gr-1 (RB6-8C5), CD19 (1D3), and CXCR3 (CXCR3-173) in combination with anti-biotin microbeads according to the manufacturer instructions (Miltenyi Biotec).

Techniques: Infection, Isolation, Flow Cytometry, Expressing, Staining, Comparison, MANN-WHITNEY