mem 181  (Bio-Rad)

Journal: bioRxiv

Article Title: Treg cell epitopes from α -tubulin: discovery and immunomodulatory features

doi: 10.1101/2025.01.08.631899

Figure Lengend Snippet: PBMCs from 15 subjects were stimulated with individual potential Treg cell epitopes for 7 days (NA 226 , RA 373 , RR 229 , LV 391 , IL 238 , PS 184 and GL 143 ) in RPMI complete medium in the presence of IL-2. Subsequently, cells were stained to analyze CD25, FoxP3, and IL-10 and in CD4 + T cells by flow cytometry. As negative controls, cells were cultured without peptides (Untreated), or with a peptide pool consisting of five peptides from C3 complement (CP pool). (A) Representative dot plot showing the percentage of CD4 + CD25 high FoxP3 + cells in response to individual predicted Treg cell epitopes (B) Percentage of CD4 + CD25 high FoxP3 + cells relative to untreated cells determined for each donor. (C) Representative dot plot showing the percentage of CD4 + FoxP3 + IL-10 + cells in response to individual predicted Treg cell epitopes. (D) Percentage of CD4 + FoxP3 + IL-10 + cells relative to untreated cells for each donor. In panels B and D, the dotted horizontal line marks the threshold that was used for considering positive responses (>3-fold increase). Peptides with arrows underneath met this criterion in at least 3 donors (n=15). These peptides were then selected to examine CD4 + FoxP3 + TGF-β + cells (E) Representative dot plot showing the percentage of CD4 + FoxP3 + TGF-β + cells in response to a pool of peptides consisting of validated Treg cell epitopes (αTBL pool) and its individual peptides. (F) Percentage of CD4 + FoxP3 + TGF-β + cells relative to untreated cells for each donor (n=6). In panels, B, D, and F, each symbol represents a different donor, and bars represent mean values.

Article Snippet: Finally, cells were washed with PBS by centrifugation at 300 g for 5 minutes and surface stained with antibodies anti-CD3 (HIT3a, PE/Cyanine5, Biolegend), anti-CD4 (MEM-241, FITC, Immunotools) and anti-CD25 (MEM-181, APC, Immunotools).

Techniques: Staining, Flow Cytometry, Cell Culture

Journal: bioRxiv

Article Title: Treg cell epitopes from α -tubulin: discovery and immunomodulatory features

doi: 10.1101/2025.01.08.631899

Figure Lengend Snippet: (A) Percentage of IL-10-producing CD4 + FoxP3 − cells in response to predicted Treg cell epitopes relative to the untreated cells of each donor (n=15). PBMCs were stimulated with individual potential Treg cell epitopes for 7 days (NA 226 , RA 373 , RR 229 , IL 238 , and LV 391 ) in RPMI complete medium in the presence of IL-2. Subsequently, cells were stained to analyze CD25, FoxP3, and IL-10 and in CD4 + cells by flow cytometry. Dotted line marks a 3-fold increase (B) Representative FACS plots showing the gating strategy to identify CD4 + LAG-3 + CD49b + FoxP3 − IL-10 + cells. (C) Representative dot plot showing CD4 + LAG- 3 + CD49b + FoxP3 − IL-10 + cells. PBMCs were stimulated with the αTBL pool or its peptides (NA 226 , RA 373 , RR 229 , IL 238 , and LV 391 ) and the CD4 + LAG-3 + CD49b + FoxP3 − IL-10 + Tr1 cell population analyzed by flow cytometry (details described in Materials and Methods). As negative controls, cells were cultured without peptides (Untreated), or cultured with a peptide pool consisting of five peptides from C3 complement protein (CP pool). (D) Percentage of CD4 + LAG-3 + CD49b + FoxP3 − IL-10 + cells expanded under different conditions relative to untreated cells (n=5). Dotted line marks a 3-fold increase. Each symbol represents a different donor, and the bars represent mean values.

Article Snippet: Finally, cells were washed with PBS by centrifugation at 300 g for 5 minutes and surface stained with antibodies anti-CD3 (HIT3a, PE/Cyanine5, Biolegend), anti-CD4 (MEM-241, FITC, Immunotools) and anti-CD25 (MEM-181, APC, Immunotools).

Techniques: Staining, Flow Cytometry, Cell Culture

Journal: bioRxiv

Article Title: Treg cell epitopes from α -tubulin: discovery and immunomodulatory features

doi: 10.1101/2025.01.08.631899

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from 18 donors were expanded and stimulated with the αTBL pool for 7 days. As controls cells were expanded with the CP pool or without peptides (Untreated). Finally, CD4 + CD25 high FoxP3 + cells and CD4 + FoxP3 + IL-10 + cells were evaluated by flow cytometry. (A) Representative dot plot showing CD4 + CD25 high FoxP3 + cells in different conditions. Gating was performed on CD3 + CD4 + T cells (B) Percentage of CD4 + CD25 high FoxP3 + cells stimulated by different peptide pools relative to untreated cells. (C) Representative dot plot showing IL-10-producing CD4 + FoxP3 + cells in response to different conditions. Gating was performed on CD4 + T cells ( D) Percentage of CD4 + FoxP3 + IL-10 + cells stimulated by different peptide pools relative to untreated cells. In panels B and D, the dotted horizontal line marks the threshold that was used for positive responses (>3-fold increase), each symbol represents a different donor, and the bars represent mean values. Statistically significant differences between conditions were obtained by applying Kruskal-Wallis tests followed by post-hoc Dunn’s tests and p -values are shown as follows: p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****).

Article Snippet: Finally, cells were washed with PBS by centrifugation at 300 g for 5 minutes and surface stained with antibodies anti-CD3 (HIT3a, PE/Cyanine5, Biolegend), anti-CD4 (MEM-241, FITC, Immunotools) and anti-CD25 (MEM-181, APC, Immunotools).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Treg cell epitopes from α -tubulin: discovery and immunomodulatory features

doi: 10.1101/2025.01.08.631899

Figure Lengend Snippet: (A) Experimental procedure to differentiate αTBL-specific FoxP3 + Treg cells. moDCs were differentiated from monocytes isolated from PBMCs by culturing them with IL-4 and GM-CSF for 6 days. moDCs were cultured with autologous naive CD4 + T cells and cultured with αTBL pool for 6 days in the presence of IL-2. αTBL pool and IL-2 were renewed every 2 days). Subsequently, Treg cells in the different co-cultures were evaluated by flow cytometry (panels B - D ), and Treg cells were sorted and used for bystander suppression assays (panel F and G ). (B) Representative dot plot showing CD4 + CD25 high FoxP3 + cells differentiated in co-cultures of moDCs and naive CD4 + T cells with αTBL and CP peptide pools and without peptides (Untreated). (C) Plot depicting the percentage of CD4 + CD25 high FoxP3 + cells differentiated under different conditions (n=5) (D) Representative dot plot showing CD4 + FoxP3 + IL-10 + cells differentiated with αTBL and CP peptides pools and without peptides (Untreated). (E) Plot depicting the percentage of CD4 + FoxP3 + IL-10 + cells differentiated under different conditions (n=5). (F) Bystander inhibition of cell proliferation by αTBL-specific Treg cells. Purified αTBL-specific FoxP3 + Treg cells were cultured with allogeneic CFSE-labelled PBMCs and stimulated with anti-CD3/CD28 Dynabeads. CFSE-dilution assay was used to measure T cell proliferation. Histograms show CFSE-staining in CD3-gated cells in non-stimulated PBMCs (Unstimulated) and CD3/CD28 stimulated PBMCS alone (PBMCs) or in culture with Treg cells (Treg:PBMC) and non-Treg cells (non-Treg:PBMCs). ( G ) Plot showing the percentage of T cells that proliferated after CD3/CD28 stimulation in different donors (n=3). Each symbol represents a different donor, and the bars represent mean values. Significant differences were obtained by applying One-way ANOVA tests followed by post hoc Tukey tests in panels C and E. Student t-tests was used in panel G shown as follows: p ≤ 0.05 (*), p ≤ 0.01 (**).

Article Snippet: Finally, cells were washed with PBS by centrifugation at 300 g for 5 minutes and surface stained with antibodies anti-CD3 (HIT3a, PE/Cyanine5, Biolegend), anti-CD4 (MEM-241, FITC, Immunotools) and anti-CD25 (MEM-181, APC, Immunotools).

Techniques: Isolation, Cell Culture, Flow Cytometry, Inhibition, Purification, Dilution Assay, Staining