Fig 2: Schematic illustration of the workflow for panel design and verification. (A) Theoretical panel design of the T cell immunophenotyping panel. Readout of the Cytek Full Spectrum Viewer (Cytek Biosciences) displaying the spectral signatures of the 22 fluorophores in the 3L configuration of the Cytek Aurora (left). Optical layout of the used markers and fluorophores showing the approximate peak emission wavelengths (nm) (right). (B) Overview of the verification strategy for the T cell immunophenotyping panel. Representative example of the antibody titration depicting CD28 conjugated to BV650. Left: Pseudo-color plot of the concatenated files of the 1:2 serial dilutions of CD28. On the x-axis, the respective concentration is shown as µl/test. The red box indicates the final titration result. Right top: The Stain Index (SI) is calculated as the difference between the median fluorescence intensity (MFI) of the positive and negative populations, divided by two times the robust standard deviation (rSD) of the negative population. MFI and rSD were extracted from FlowJo in.csv file format. Right bottom: Diagram depicting the calculated SI of CD28-BV650 over 1:2 serial dilution steps. The x-axis displays the respective concentration as µl/test. The red circle indicates the final titration result. (C) Dot plots showing an exemplary illustration of “unmixing” accuracy of CXCR5-BV750 and CCR6-BV711 utilizing single stained compensation beads compared to single stained whole PBMCs. (D) NxN plots depicting the quality control of “unmixing” accuracy in the multi-color stained sample, gated on lymphocytes. CCR7-BV421 is shown as a representative example of correct “unmixing”. In every NxN plot, CCR7 is represented on the y-axis and all other fluorophores of the panel on the x-axis. (E) Staining resolution of the single stained (SS) tube compared to the fully stained/multi-color (MC) tube on the example of CXCR5-BV750. (F) Loss of staining resolution resulted in fine-tuning of the staining protocol, such as separate staining steps and changes in the antibody concentrations. (G) Immunophenotyping results were manually gated and compared to published studies to evaluate the quality of the data.

Fig 3: Telomere length dynamics across T‐cell differentiation stages. (A) Relative telomere length (RTL) in CD4 (left) and CD8 (right) T cells across differentiation from naïve to terminally differentiated (TD) stages, showing a progressive telomere shortening with increasing differentiation. (B) Telomere length comparison in progressively senescent CD4 (left) and CD8 (right) T‐cell subsets stratified by CD57 and KLRG1 co‐expression. (C) Telomere length in naïve CD4 T cells (CD3⁺CD4⁺CD45RA⁺CD27⁺CCR7⁺) comparing recent thymic emigrants (RTEs, CD31⁺) and non‐RTE naïve cells (CD31−). (D) Telomere length in CD8 T cells with a replicative senescence (RS) phenotype (RS⁺; CD57⁺CD28−) compared with non‐senescent cells (RS−). A paired t‐test was used for analyses in (A), whereas the Wilcoxon test was applied in (B‐D).

Fig 4: Flow‐FISH gating strategy and relative telomere length calculation. (A) Gating strategy for T‐cell immunophenotyping and telomere length measurement by spectral flow‐FISH. Lymphocytes were identified by forward‐ and side‐scatter properties, and dead cells and cycling cells (G2/S) were excluded based on DNA content. CD3+ T cells were subdivided into CD4 and CD8 populations, followed by phenotypic characterization into naïve, central memory (CM), transitional memory (TM), effector memory (EM), TEMRA (CD45RA+ EM), and terminally differentiated (TD) subsets. Additional markers were used to define activation (HLA‐DR, CD38), exhaustion (PD‐1), recent thymic emigrants (RTEs, CD31), and senescence‐associated phenotypes (CD57, KRLG1, CD28). (B) Equation used to calculate relative telomere length (RTL) based on the mean fluorescence intensity (MFI) of the telomere probe, normalized to a control cell line and corrected for probe‐negative background.