Journal: bioRxiv

Article Title: Tim-3 co-stimulation promotes short-term effector T cells, restricts memory precursors and is dispensable for T cell exhaustion

doi: 10.1101/179002

Figure Lengend Snippet: A , CD25-depleted T cells from Nur77 GFP mice were stimulated for three days with anti-CD3/CD28 mAbs, followed by a seven day rest with IL-2. After three rounds of stimulation, cells were stained for PD-1 and Tim-3 and analyzed by flow cytometry. Representative of 5 technical replicates per experiment, repeated with n=5 mice. B , WT C57BL/6 mice were infected with LCMV Arm. After 30d, spleens were harvested for flow cytometry (n=5 mice, mean ± SD). representative of three independent experiments C , Tim-3 + vs. Tim-3 − CD8 + cells were further analyzed for expression of activation and differentiation markers shown in the histograms. D , splenocytes from the same experiments as panels b-c were stained with LCMV tetramers plus α Tim-3. (n=5 mice, mean ± SD) representative of three independent experiments. **p<0.01 by two-tailed paired Student’s t test. E , C57Bl/6 mice previously infected with LCMV-Arm (>30d.p.i.) were challenged with LM-GP33 and Tet − vs. Tet + CD8 + splenocytes were analyzed for KLRG1 and Tim-3 four days post-challenge. Representative of three independent experiments (n=5).

Article Snippet: CD25 depletion was done using a CD25 microbead kit (Miltenyi).

Techniques: Staining, Flow Cytometry, Infection, Expressing, Activation Assay, Two Tailed Test

Journal: Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation

Article Title: Functional divergence of antigen-specific T-lymphocyte responses in syngeneic graft-versus-host disease.

doi: 10.1016/j.bbmt.2004.05.003

Figure Lengend Snippet: Figure 2. Flow cytometric selection of CLIP antigen-specific T-cell subsets. Splenic T lymphocytes harvested on day 14 after cessation of cyclosporine treatment were subjected to 3-color immunofluorescence. The cells were stained with the biotinylated soluble MHC class II immunoglobulin construct loaded with the N- or C-terminal CLIP variants (counterstained with CyChrome/streptavidin) and with fluorescein isothiocyanate (FITC)–and phycoerythrin (PE)–conjugated antibodies to CD8 and CD25, respectively. The percentage of cells staining with the construct is shown in the histogram; the dot plot displays CD8 and CD25 expression of the cells reacting with the peptide-loaded construct.

Article Snippet: The shaded area, however, repr etween N- and C-terminal flanking domain dependence. eric molecule loaded with unrelated peptides was [ B & M T neffective in isolating CLIP-specific T cells [23]. low cytometric multicolor analysis was used to isoate the antigen-specific T cells and to confirm expresion of the CD4, CD8, and CD25 cell-surface markrs (phycoerythrin- or fluorescein isothiocyanate– onjugated mouse anti-rat CD8, anti-CD4, and CD25 onoclonal antibodies; Serotec, Harlan Bioproducts or Science, Indianapolis, IN).

Techniques: Selection, Staining, Construct, Expressing

Journal: Arthritis Research & Therapy

Article Title: Therapeutic effect of a novel histone deacetylase 6 inhibitor, CKD-L, on collagen-induced arthritis in vivo and regulatory T cells in rheumatoid arthritis in vitro

doi: 10.1186/s13075-017-1357-2

Figure Lengend Snippet: Increased cytotoxic T-lymphocyte antigen-4 ( CTLA-4 ) expression by CKD-L in Treg cells from C57BL/6 mice ( n = 3). CD4 + CD25 – T cells were isolated from splenocytes of C57BL/6 mice. CD4 + CD25 – T cells were incubated with vehicle or HDAC6 inhibitors (1 to 10 μM) in the presence of antiCD3/CD28 beads and recombinant TGF-β2 in a 48-well plate for 6 days. CTLA-4 expression in CD4 + CD25 + Foxp3 + T cells was analyzed by mean fluorescence intensity ( MFI ; mean ± SEM) using flow cytometry. * p < 0.05, ** p < 0.001, vs vehicle

Article Snippet: Isolated CD4 + cells were incubated with microbead conjugated to a monoclonal antihuman CD25 antibody (MACS®; Miltenyi Biotec) for 15 min at 4 °C.

Techniques: Expressing, Isolation, Incubation, Recombinant, Fluorescence, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: PD-1 or CTLA-4 blockade promotes CD86-driven Treg responses upon radiotherapy of lymphocyte-depleted cancer in mice

doi: 10.1172/JCI171154

Figure Lengend Snippet: ( A – D ) TC-1 tumor–bearing mice were treated with 20 Gy RT ( n = 10) or control (0 Gy, n = 4–6) when tumors reached approximately 20 mm 2 in size (day 0). FTY720 or vehicle (NaCl) was administered orally on days –1, 3, and 5. On day 8, the CD8 + T cell response was analyzed by flow cytometry in the TdLN ( A and B ) and tumor ( C and D ). ( A and C ) Representative concatenated flow cytometric plots showing IFN-γ + cells among CD8 + T cells in the TdLN ( A ) and tumor ( C ). ( B and D ) Frequency of CD44 + TCF-1 – , GZB + , and IFN-γ + cells among CD8 + T cells in the TdLN ( B ) and tumor ( D ). IFN-γ was measured after in vitro PMA/Ionomycin stimulation. ( E and F ) Monitoring of the (FOXP3 + CD25 + ) Treg response to 20 Gy RT ( n = 6–8) or control (0 Gy, n = 6) in TC-1 tumor–bearing mice on day 8 after treatment. ( E ) Treg frequency among CD4 + T cells in the non-TdLN and TdLN, or among CD45 + cells within the tumor. ( F ) Percentage of Tregs among live cells in blood at the indicated time points ( n = 6/group). ( G ) Frequency of Tregs in the indicated tissues on day 8 following 20 Gy RT ( n = 10) or control (0 Gy, n = 4–6) with or without FTY720 treatment. ( H ) CD8 + T cell/Treg ratio in the TdLN and tumor after RT. ( I ) OS of TC-1 tumor–bearing mice treated with 0 Gy ( n = 5) or 20 Gy ( n = 11–14/group) RT in combination with a CD25-depleting mAb or vehicle (PBS) administered i.p. on day –1 and on day 5 after RT. * P < 0.05 (Mantel-Cox analysis). Data are from 1 experiment and are representative of at least 2 experiments. Error bars indicate the SD. * P < 0.05 and ** P < 0.01, by 2-way ANOVA with Bonferroni’s post hoc test ( B , D , F and G ) and Mann-Whitney U test ( E and H ).

Article Snippet: For Treg depletion experiments, mice were injected i.p. with 250 μg depleting mouse IgG2a isotype CD25 mAbs ( ) (modified clone of PC61, Evitria) in 100 μL PBS on day –1 prior to RT and on day 5.

Techniques: Flow Cytometry, In Vitro, MANN-WHITNEY