Design on What Was Actually Done

 Quick Analysis Plan Copied

 Long Analysis Plan

Experimental Design: Validating Inflammaging-Driven CD8 T Cell Remodeling

Phase 1 — Cohort and Single-Cell Profiling. Enroll 30 young (20–35 y) and 30 aged (≥65 y) donors. Isolate PBMC and perform paired scRNA-seq + TCR-seq. Cluster CD8 T cells into naive, effector-memory, exhausted, and senescent states. Quantify CD226 expression to discriminate functional from dysfunctional CD8 subsets, as loss of CD226 marks dysfunctional cells unable to recognize target cells CD226 distinguishes effector CD8 states. Also quantify TCR clonal expansion (Gini index, Shannon entropy) and infer pseudo-time trajectories with scVelo.

Phase 2 — Inflammaging Cytokine Milieu. Measure plasma IL-6, TNF-α, IL-4, IL-13, and CRP by multiplex immunoassay. The rationale for IL-4 and IL-13 is that both cytokines down-regulate CD8 expression levels, dampening T cell avidity and polyfunctionality IL-4/IL-13 suppress CD8 avidity. Correlate circulating IL-4/IL-13 with CD8 surface density (MFI) and TCR avidity (pMHC tetramer dissociation k_off).

Phase 3 — Functional and Tissue-Level Validation. Perform ex vivo co-culture of aged vs young CD8 T cells with peptide-pulsed APCs; read out IFN-γ, TNF-α, GzmB by intracellular staining and ELISA. In parallel, quantify tissue-infiltrating CD8 T cells and FOXP3⁺ Tregs by IHC in paired tissue biopsies to compute the CD8/Treg ratio, a metric that collapses from ~5.2 (healthy tissue) to ~0.3 (end-stage diseased tissue) in progressive immune remodeling CD8/Treg ratio collapses with tissue progression. This ratio serves as a quantitative remodeling readout.

Phase 4 — Computational Pipeline. Use Scanpy for scRNA-seq QC/clustering, gseapy for GSEA of inflammaging hallmark pathways, Milo for differential abundance, and statsmodels for multi-predictor regression (age, IL-6, IL-4, CD226 MFI → avidity). The pipeline is organized below:

Expected effect sizes: Based on the sourced data, CD8/Treg ratio is expected to decline by ~94% between healthy and end-stage remodeling tissue CD8/Treg collapse quantified. CD8 expression MFI is expected to decrease with rising IL-4/IL-13, paralleling loss of avidity IL-4/IL-13 reduce CD8 MFI.

Blind spots and limitations: No supplied evidence directly measures inflammaging in aged human cohorts; the sourced data are from mouse viral infection, cancer tissue, and in vitro cytokine perturbation. Cross-species extrapolation is a major uncertainty. Sample sizes in the sourced studies ranged from 10 cases (B-CLL) CD8 in B-CLL: small cohort to 123 patients (CRC) CRC cohort: 123 patients. Age-confounded comorbidities, tissue source heterogeneity, and batch effects in scRNA-seq remain uncontrolled risks.