Raw data for the figures in Franka Klatte-Schulz et al. Increased CD4+ to CD8+ T Cell Ratio as a Driver of Impaired Achilles Tendon Healing in Human Patients
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This dataset contains raw data underlying the main and supplementary figures of the manuscript titled “Increased CD4+ to CD8+ T cell ratio as a driver of impaired Achilles tendon healing in human patients.” The data were generated through a combination of clinical follow-up, flow cytometry, cell culture, and molecular biology techniques.
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Clinical study and sample collection: Peripheral blood and hematoma samples were obtained from patients undergoing minimally invasive Achilles tendon repair surgery (Charité – Universitätsmedizin Berlin). Clinical outcome parameters such as ATRS, pain/function VAS, Hannover score, muscle volume, and tendon elongation (Matles test) were assessed preoperatively and at 6 weeks, 6 months, and 12 months post-surgery. MRI imaging was used to quantify tendon length, muscle volume, and fatty degeneration. Flow cytometry: T cell subpopulations (CD3, CD4, CD8, CD11a, CD28, CD57) were analyzed by multicolor flow cytometry (BD FACSCanto II, BD Biosciences) after erythrocyte lysis. Data were acquired and analyzed using standard gating strategies with FMO controls. Frequencies of subsets were calculated as percentages of parent populations (e.g., CD4+ of CD3+ T cells). In vitro co-culture experiments: Primary tenocytes were isolated from surgical Achilles tendon specimens via enzymatic digestion (0.3% collagenase) and cultured in DMEM/Ham’s F12 medium. Autologous CD4+ and CD8+ T cells were sorted from PBMCs after polarization toward IL-17 or IFNγ phenotypes using established cytokine cocktails (IL-6, IL-23, IL-1β, TGFβ for IL-17; IL-2, IL-12, anti-IL4 for IFNγ). T cell polarization was confirmed by ELISA (IFNγ, IL-17). Co-cultures were conducted in both 2D (scratch assay) and 3D (collagen gel contraction assay) formats. After 45–65 hours, wound closure and gel area were quantified using ImageJ. Cell lysates and supernatants were harvested for gene expression (qRT-PCR) and protein quantification (ELISA for MMP1/2/3, TIMP1). Gene expression and ELISA: RNA from tenocytes was isolated (NucleoSpin kit) and reverse-transcribed (qScript cDNA). Quantitative PCR was performed with SYBR Green chemistry using gene-specific primers (Col1, Col3, IL17RA/C, IL1β, MMPs). Protein secretion into supernatants was measured via DuoSet ELISA kits according to manufacturer protocols. Data processing: Statistical analyses were performed using GraphPad Prism 7.0. Data are presented as individual values per patient or replicate, with appropriate groupings (e.g., successful vs. poor healers) or correlations (e.g., CD4+ T cells vs. ATRS).
Institutions
- Berliner Institut fur Gesundheitsforschung