antigen presentation system  (ATCC)

Journal: bioRxiv

Article Title: CD4⁺ T cells confer transplantable rejuvenation via Rivers of telomeres

doi: 10.1101/2025.11.14.688504

Figure Lengend Snippet: ( a, left ) Extracellular telomeres in vivo . Reactive lymph-nodes were harvested from 6-week-old mice, 7 days after recall responses to ovalbumin then analyzed by IF-FISH staining (telomere probe) plus CD3 and Cpt1a antibodies. Nuclei staining demonstrated by DAPI. Representative of 3 separate mice. Scale bar, 50 μM. ( a, middle ) Magnification of extracellular telomeres as in (a; scale bar, 2 μM) and quantification of extracellular telomeres in Cpt1a Bright and Cpt1a Low (DMFICpt1a> 23%) T cells. Note lack of extracellular telomeres in T cell with lower Cpt1a expression. ( a, right ) Presence of extracellular telomeres in lymph node vessels, with examples denoted by red arrows, throughout. Scale bar, 50 μM. Three unchallenged animals were used as control (no Ag, Supplementary information 1). ( b ) Discovery of telomere release in efferent vessels of lymph nodes of mice vaccinated twice with ovalbumin (OVA; 30 µg) then analyzed 7 days after the recall response. Representative of 3 separate mice. DAPI, blue; TelC, white. Quantification of River per LN section is shown. Representative FACS profiles and quantifications of circulating telomere rivers in mouse ( c ; n = 8 mice) and human ( d , n = 5 donors) sera and in plant xilema ( e , n = 4 orchids). PKH67, lipid dye and TelC probe were used for telomere River identification across kingdoms. For quantification, each dot is an individual donor. Note that centromere probes had no detection (see also Supplementary information 1). One-way Anova with Bonferroni post-correction for multiple comparisons in ( a ) and unpaired Student’s t-test ( c-e ). ****P < 0.0001. Error bars indicate s.e.m.

Article Snippet: CD3− antigen-presenting cells (APCs) were obtained by CD3 depletion (Miltenyi, 130-050-101).

Techniques: In Vivo, Staining, Expressing, Control

Journal: bioRxiv

Article Title: CD4⁺ T cells confer transplantable rejuvenation via Rivers of telomeres

doi: 10.1101/2025.11.14.688504

Figure Lengend Snippet: ( a ) Synapse-formation on planar bilayers of T sen transduced with CTRL or CPT1A-expressing vectors showing that CPT1A-driven FAO restores TCR centralization at the synapse. Note that functional synapses have centrally-accumulated TCRs with lower CD3 fluorescence. Representative data (left) and quantification (right) of n = 35 synapses per group ( n = 4 donors). Scale bars, 10 μM. ( b ) Ceramidase links FAO to the immune synapse. FAO activates ceramidase by blocking ceramide synthesis that in turn generates PE needed to centralize TCRs at the synapse. In contrast, short (20 min) treatment of T sen with the PE precursors sphingosine or sphingosine-1 phosphate (S1P) did not centralize TCRs. Data are from n = 35 synapses per group (4 donors). Scale bar, 10 μM ( c ) Quantification of telomere release 24h after antigen-pulsed APC activation on TCR-coated bilayers. Data pooled from 4 donors (T erl , T sen , T sen PE) or 2 donors (T sen CPT1A). T sen synapse competence was restored either by adding PE for 20 min or rescuing CPT1A expression by lenti-vectors for 10 days prior to synapse formation on bilayers. Antigen free APCs are shown, as control. ( d ) Telomere elongation by qPCR in metabolic competent T cells obtained by CPT1A expression or PE supplementation prior to conjugation with autologous antigen-pulsed APCs. T cells were purified from conjugates and analyzed 48 hours after synaptic activation ( n = 4 donors). In ( a ) Mann-Whitney test and one-way Anova with Bonferroni post-correction for multiple comparisons in ( b - d ). *P<0.05, ****P < 0.0001. Error bars indicate s.e.m.

Article Snippet: CD3− antigen-presenting cells (APCs) were obtained by CD3 depletion (Miltenyi, 130-050-101).

Techniques: Transduction, Expressing, Functional Assay, Fluorescence, Blocking Assay, Activation Assay, Control, Conjugation Assay, Purification, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD4⁺ T cells confer transplantable rejuvenation via Rivers of telomeres

doi: 10.1101/2025.11.14.688504

Figure Lengend Snippet: ( a ) Proteomic comparison by DIA. Telomere vesicles and circulating Rivers were analyzed by LC–MS/MS with the data-independent acquisition (DIA) approach. Human APCs from each donor were stimulated with ionomycin overnight to induce vesicle release, and matched serum was collected in parallel. Heatmaps show differential abundance of glycolytic versus stem-related proteins ( n = 3 donors per group). ( b ) Proteomics by DDA. Telomere-bound proteins were purified by TelC-biotin immunoprecipitation among and analyzed by LC–MS/MS with data-dependent acquisition (DDA). Rivers displayed enrichment of stemness factors and loss of GAPDH relative to APC telomere vesicles ( n = 8 donors). ( c ) AD-like CD4 + T cell behavior. Representative IF–FISH images (top) and quantification from pooled microscopy (bottom; 17 images, n = 3 donors) showing asymmetric CPT1A distribution in T erl cells after stimulation with anti-CD3/CD28 for 48 h, with or without telomere vesicles. Cytokinesis was blocked at 44 h; CPT1A asymmetry was assessed 4 hours later. Scale bar, 2 µm. Images were z-stacked and analyzed by confocal microscope. Scale bar, 2 μm ( d ) Flow cytometry of AD-like cells. Representative gating ( n = 9 donors) showing asymmetric CPT1A distribution in telomere recipient (Tel⁺) versus non-recipient (Tel⁻) CD4⁺ T cells 48 h after conjugation with TelC-labeled APCs. Cells were tracked with CTV dyes, followed by flow-FISH (TelC) and CPT1A staining. GAPDH staining confirmed differential segregation in daughter cells. ( e ) Spontaneous loss of transferred APC telomeres. CPT1A ^bright versus GAPDH ^bright stem-like CD4⁺ T cells were analyzed 48–72 h after APC conjugation, with or without GW4869 (10 µM). Endogenous T-cell telomeres were elongated and remained stable, whereas transferred APC telomeres were selectively lost, indicating selective disposal of supernumerary APC telomeres ( n = 5 donors). Telomere loss was calculated as ΔTelC fluorescence (48–72 h). ( f ) Release of Rivers by post-synaptic CD4⁺ T cells. After 24 h of APC–T cell co-culture, CD4⁺ T cells were freed by negative selection and cultured in isolation for 48 h. Supernatants were then analyzed for released telomeric particles. Rivers were defined as PKH67⁺ TelC⁺ GAPDH⁻, whereas APC vesicles were PKH67⁺ TelC⁺ GAPDH⁺. Rivers incorporated stemness factors (WNT5A, RUNX2; Extended data fig. 2 ). Pre-treatment with GW4869 or cytochalasin D blocked River release; by contrast cytochalasin D promoted River production when added 44 h after initial activation. Data are from ( n = 6 donors). One-way Anova with Bonferroni post-correction for multiple comparisons in ( c , d , f left ) and paired Student’s t-test in ( b, f right ) were used. *P<0.05, ***P<0.001, ****P < 0.0001. Error bars indicate s.e.m.

Article Snippet: CD3− antigen-presenting cells (APCs) were obtained by CD3 depletion (Miltenyi, 130-050-101).

Techniques: Comparison, Liquid Chromatography with Mass Spectroscopy, Data-independent acquisition, Purification, Immunoprecipitation, Data-dependent acquisition, Microscopy, Flow Cytometry, Conjugation Assay, Labeling, Staining, Fluorescence, Co-Culture Assay, Selection, Cell Culture, Isolation, Activation Assay