cd4  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Lymphedema pathogenesis involves antigen-driven expansion of CD4+ T cells in skin

doi: 10.3389/fimmu.2025.1620571

Figure Lengend Snippet: TCR repertoire analysis of CD4+ T cells in paired lymphedema and normal skin biopsies. (A) Schematic of collection and sequencing of CD4+ T cells in human skin samples (n=11), genomic(g) DNA isolated and high-throughput sequencing (HTS) performed using 2-step bias-controlled PCR. (B-D) Unique counts, percent unique counts, and clonality index of TCRs in normal and lymphedema (LE) samples. (E) CDR3 AA length distribution in normal skin. (F) CDR3 AA length distribution in LE skin. (G) Overlap of CDR3 length usage in normal and LE skin. (H) TCRβV gene usage in normal and LE skin. Student’s paired t-test; ***p<0.0001.

Article Snippet: The following anti-human antibodies were used for staining: CD4+ (1:1000; cat#AF379NA; R&D Systems), CD45RO (1:400; cat# MA511532 ; Thermo Fisher Scientific), and IR (1:1000; cat# AB137747 ; Abcam).

Techniques: Sequencing, Isolation, Next-Generation Sequencing

Journal: Frontiers in Immunology

Article Title: Lymphedema pathogenesis involves antigen-driven expansion of CD4+ T cells in skin

doi: 10.3389/fimmu.2025.1620571

Figure Lengend Snippet: CD4+ T cells in lymphedema exhibit an effector memory phenotype response to insulin peptide. (A) Immunofluorescence images showing effector memory CD4+ T cells (CD4+CD45RO+) and IR-activated effector memory CD4+ T cells (CD4+CD45RO+IR+) in normal and lymphedema (LE) skin biopsies. (B, C) Quantification (D) Gating strategy and frequency percent of antigen-activated (CD45RO+IR+) CD4+ T cell populations in LE liposuction fluid. A full gating strategy for panel (D) can be viewed in Supplementary File 3 . (E, F) Frequency (%) insulin responsive T cell populations between human LE fluid T cells (hLE) and human blood T cells (hBL) in two single human donors with LE and BMI of <25 (E) and >25 (F) . The full gating strategy for panels (E, F) may be viewed in Supplementary File 4 . Data analyzed by One-way ANOVA. *p<0.05, **p<0.005; ns, not significant.

Article Snippet: The following anti-human antibodies were used for staining: CD4+ (1:1000; cat#AF379NA; R&D Systems), CD45RO (1:400; cat# MA511532 ; Thermo Fisher Scientific), and IR (1:1000; cat# AB137747 ; Abcam).

Techniques: Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Lymphedema pathogenesis involves antigen-driven expansion of CD4+ T cells in skin

doi: 10.3389/fimmu.2025.1620571

Figure Lengend Snippet: Oligoclonality is demonstrated in a lymphedema mouse model. (A) Representative tail images of sham and tail-operated mice at 6 weeks. (B) Clonality index of CD4+ TCRs sequenced in tail skin of sham and surgery mice. A two-tailed unpaired t-test was performed. (C) CDR3 AA length distribution in sham (blue) and surgery (red) mice. (D) TCRβV gene usage in sham and surgery mice. Data analyzed by a two-tailed multiple unpaired t-test. (E) Experimental schematic for mouse studies: Effector T cells are sorted from PLND and sham controls (left panel). Irradiated APCs are plated with effector T cells at a 1:5 ratio, with or without insulin antigen, and treated for 48 hrs (middle). Samples are analyzed by flow cytometry for antigen-activated effector populations. (F) Gating strategy of populations of interest. The full gating strategy for panel (F) may be viewed in Supplementary File 5 . (G) Comparison of effector T cell populations expressing CD154. Data analyzed by one-way ANOVA. *p<0.05; ****p<0.0001.

Article Snippet: The following anti-human antibodies were used for staining: CD4+ (1:1000; cat#AF379NA; R&D Systems), CD45RO (1:400; cat# MA511532 ; Thermo Fisher Scientific), and IR (1:1000; cat# AB137747 ; Abcam).

Techniques: Two Tailed Test, Irradiation, Flow Cytometry, Comparison, Expressing