Journal: Journal for Immunotherapy of Cancer

Article Title: Combined innate and adaptive immunotherapy overcomes resistance of immunologically cold syngeneic murine neuroblastoma to checkpoint inhibition

doi: 10.1186/s40425-019-0823-6

Figure Lengend Snippet: Treated 9464D-GD2 tumors have significantly fewer T regulatory cells, with a higher CD8+ T cell to Treg ratio, and more CD4+ T cells and monocytes/macrophages compared to untreated tumors. Untreated 9464D-GD2 tumors and tumors treated with 12 Gy and combined ½ dose IT-IC, anti-CTLA-4 (CTLA), CpG, and anti-CD40 (CD40) were harvested on treatment day 13, and tumor microenvironment was analyzed by flow cytometry ( a ). Representative dot plots of Treg populations (defined as CD25 + FoxP3+ of CD45 + CD4+ live cells) are shown for three representative untreated ( b ) and treated ( c ) tumors (numerical values shown are the % of CD45 + CD4+ live cells that are Tregs)

Article Snippet: For cell surface staining, cells were incubated with anti-GD2-APC (clone 14G2a; BioLegend), anti-CD45-eF450 (clone 30-F11; eBioscience), anti-CD3-Alexa700 (clone 17A2; BioLegend), anti-CD4-PE-Dazzle594 (clone GK1.5; BioLegend), anti-CD8a-APC-eFluor780 (clone 53–6.7; eBioscience), anti-CD11b-BB700 (clone M1/70; BD Horizon), anti-Ly6G-BV711 (clone 1A8; BioLegend), anti-CD25-BB515 (clone PC61; BD Horizon), anti-FoxP3-PE-Cy7 (clone FJK-16 s; eBioscience), and Ghost Dye Violet 510 (Tonbo Biosciences).

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Nanoparticles Containing an Insulin-ChgA Hybrid Peptide Protect from Transfer of Autoimmune Diabetes by Shifting the Balance between Effector T cells and Tregs

doi: 10.4049/jimmunol.1900127

Figure Lengend Snippet: A–C: Single-cell suspensions were prepared from the pancreas and spleen of control and 2.5HIP-PLG treated mice euthanized at 2 or 8 weeks post adoptive transfer, followed by staining with I-Ag7/2.5HIP tetramer and antibodies. Gates were set on live, CD45+, lineage-negative (lin−), CD4+, 2.5HIP tet+ cells. A: Representative example of Foxp3 and CD25/Foxp3 staining from the pancreas of a 2.5HIP-PLG treated mouse euthanized at 8 weeks. B: Summary of 2–3 independent experiments for control 2 wks (n = 6), 2.5HIP-PLG 2 wks (n = 6), and 2.5HIP-PLG 8 wks (n = 4). Each symbol represents an individual mouse. *P < 0.05, **P < 0.01. C: Data are representative of 3 independent experiments (n = 6) from mice euthanized at 2 weeks. **P < 0.01, ***P < 0.001.

Article Snippet: Other antibodies used included: anti-CD45:BUV395 (30-F11, BD), anti-CD4:BV711 (GK1.5, Biolegend), anti-Foxp3:PE and eFluor450 (FJK-16s, eBioscience), anti-CD25:BB515 (PC61, BD), anti-CTLA-4:PE-Cy7 (4C10–4B9, Biolegend), anti-GITR:BV510 (DTA-1, BD), anti-ICOS:BV605 (7E.17G9, BD), anti-CD127:PE-Cy7 (SB/199, eBioscience), anti-CD103:eFluor450 (2E7, eBioscience), anti-CD44:BV510 (IM7, BD), anti-CD73:BV605 (TY/11.8, Biolegend), anti-FR4:PE-Cy7 (12A5, eBioscience), anti-IFN-γ:APC (XMG1.2, BD), anti-TNF-α: FITC (MP6-XT22, eBioscience), and anti-T-bet: BV605 (4B10, Biolegend).

Techniques: Adoptive Transfer Assay, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Nanoparticles Containing an Insulin-ChgA Hybrid Peptide Protect from Transfer of Autoimmune Diabetes by Shifting the Balance between Effector T cells and Tregs

doi: 10.4049/jimmunol.1900127

Figure Lengend Snippet: A–C: Single-cell suspensions were prepared from the pancreas and spleen of control and 2.5HIP-PLG-treated mice euthanized at 2 or 8 weeks post adoptive transfer, followed by staining with I-Ag7/2.5HIP tetramer and antibodies. Gates were set on live, CD45+, lineage-negative (lin−), CD4+, 2.5HIP tet+ cells, CD25+ Foxp3+ cells. A. Representative example of CTLA-4, GITR, and ICOS staining on bulk 2.5HIP tet+ or tet+ Treg cells from the pancreas of a 2.5HIP-PLG-treated mouse euthanized at 8 weeks. B: Summary of 2 independent experiments for control 2 wks (n = 4), 2.5HIP-PLG 2 wks (n = 3–4), and 2.5HIP-PLG 8 wks (n = 3–4). Each symbol represents an individual mouse. C: Geometric mean fluorescence intensity (gMFI) data from mice euthanized at 2 weeks from 1 experiment is shown (n = 2).

Article Snippet: Other antibodies used included: anti-CD45:BUV395 (30-F11, BD), anti-CD4:BV711 (GK1.5, Biolegend), anti-Foxp3:PE and eFluor450 (FJK-16s, eBioscience), anti-CD25:BB515 (PC61, BD), anti-CTLA-4:PE-Cy7 (4C10–4B9, Biolegend), anti-GITR:BV510 (DTA-1, BD), anti-ICOS:BV605 (7E.17G9, BD), anti-CD127:PE-Cy7 (SB/199, eBioscience), anti-CD103:eFluor450 (2E7, eBioscience), anti-CD44:BV510 (IM7, BD), anti-CD73:BV605 (TY/11.8, Biolegend), anti-FR4:PE-Cy7 (12A5, eBioscience), anti-IFN-γ:APC (XMG1.2, BD), anti-TNF-α: FITC (MP6-XT22, eBioscience), and anti-T-bet: BV605 (4B10, Biolegend).

Techniques: Adoptive Transfer Assay, Staining, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Nanoparticles Containing an Insulin-ChgA Hybrid Peptide Protect from Transfer of Autoimmune Diabetes by Shifting the Balance between Effector T cells and Tregs

doi: 10.4049/jimmunol.1900127

Figure Lengend Snippet: A–D: Single-cell suspensions were prepared from the pancreas and spleen of control and 2.5HIP-PLG-treated mice euthanized at 2 or 8 weeks post adoptive transfer, followed by staining with I-Ag7/2.5HIP tetramer and antibodies. Gates were set on live, CD45+, lineage-negative (lin−), CD4+, 2.5HIP tet+ cells, CD25+ Foxp3+ cells. A: Representative example of CD127 staining on tet+ Treg cells from the pancreas of a control (CTR) or 2.5HIP-PLG-treated (TOL) mouse euthanized at 2 weeks. C: Representative example of CD103 staining on bulk 2.5HIP tet+ or tet+ Treg cells from the pancreas of a 2.5HIP-PLG-treated mouse euthanized at 8 weeks. B&D: Summary of 2–3 independent experiments for control 2 wks (n = 4–6), 2.5HIP-PLG 2 wks (n = 4–6), and 2.5HIP-PLG 8 wks (n = 3–4). Each symbol represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Other antibodies used included: anti-CD45:BUV395 (30-F11, BD), anti-CD4:BV711 (GK1.5, Biolegend), anti-Foxp3:PE and eFluor450 (FJK-16s, eBioscience), anti-CD25:BB515 (PC61, BD), anti-CTLA-4:PE-Cy7 (4C10–4B9, Biolegend), anti-GITR:BV510 (DTA-1, BD), anti-ICOS:BV605 (7E.17G9, BD), anti-CD127:PE-Cy7 (SB/199, eBioscience), anti-CD103:eFluor450 (2E7, eBioscience), anti-CD44:BV510 (IM7, BD), anti-CD73:BV605 (TY/11.8, Biolegend), anti-FR4:PE-Cy7 (12A5, eBioscience), anti-IFN-γ:APC (XMG1.2, BD), anti-TNF-α: FITC (MP6-XT22, eBioscience), and anti-T-bet: BV605 (4B10, Biolegend).

Techniques: Adoptive Transfer Assay, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Central nervous system demyelinating disease protection by the human commensal Bacteroides fragilis depends on polysaccharide A expression.

doi: 10.4049/jimmunol.1001443

Figure Lengend Snippet: FIGURE 4. Bacteroidesreconstitution maintains the enhanced frequenciesofTreg cells found inmicetreatedorally with antibiotics. Flow cytometry wasusedto compare the frequencies of Foxp3+CD25+ among total CD4+ T cells (A, B) gated on cervical LNs in PBS-treated mice, mice treated with antibiotics, and mice reconstituted with either WTor DPSA B. fragilis, 0, 3, and 7 d after treatments, and CD25+ T cells or CD25highCD4+ T cells (C, D) 7 d after bacterial reconstitution. Depicted are results for a representative experiment (A, C) and the means for a representative experiment from two separate experiments (n = 8 per group; B, D).

Article Snippet: To inactivate CD25+CD4+ T cells, mice were given 0.3 mg anti-CD25 mAb (ATCC No. TIB-222, clone PC 61.5.3) on days 4 and 2 before EAE challenge (24).

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Central nervous system demyelinating disease protection by the human commensal Bacteroides fragilis depends on polysaccharide A expression.

doi: 10.4049/jimmunol.1001443

Figure Lengend Snippet: FIGURE 5. Bacteroides reconstitution depends on Treg cells to induce protection. In vivo depletion of CD25+ cells restores the susceptibility to EAE in animals reconstituted with WT B. fragilis. To inactivate CD25+T cells, mice were given 0.3 mg anti-CD25 mAb (clone PC 61.5.3) on days 4 and 2 before EAE induction. As a control group, treated and naive mice received 0.3 mg purified rat IgG Ab. When EAE was induced, protection observed in mice treated with antibiotics and in mice reconstituted with WT B. fragilis was lost. Depicted are the means for a representative experiment from two separate experiments (n = 4 per group; the combination of all data [n = 8] from the experimentalgroupsare representedinTable II).pp,0.01for PBS/IgGversus WTB.fragilis-reconstituted–IgG,PBS/IgG-versusantibiotic-treated/IgG,and PBS/IgG- versus PBS/aCD25. dp , 0.01, for WT B. fragilis reconstituted/IgG versus WT B. fragilis reconstituted/aCD25, and DPSA B. fragilis recon- stituted/IgG versus DPSA B. fragilis reconstituted/aCD25.

Article Snippet: To inactivate CD25+CD4+ T cells, mice were given 0.3 mg anti-CD25 mAb (ATCC No. TIB-222, clone PC 61.5.3) on days 4 and 2 before EAE challenge (24).

Techniques: In Vivo, Control

Journal: EBioMedicine

Article Title: Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

doi: 10.1016/j.ebiom.2020.102913

Figure Lengend Snippet: Butyrate increased T FR cells in CoPs (a, b) Follicular T cells in the DLNs and CoPs from CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Representative flow cytometry contour plots of CXCR5 and intracellular Bcl-6 staining (a), and the frequency (b) of Bcl-6 + CXCR5 + follicular T cells within CD4 + TCRβ + gate ( n = 11, 10). (c–g) T FR and T FH cells in the DLNs and CoPs from CIA mice fed the control HAMS or HAMSB diet two weeks after the initial immunization. Representative flow cytometry contour plots of CD25 and intracellular Foxp3 staining (c), and the frequency of CD25 − Foxp3 + T FR (d) and Foxp3 − T FH (e) cells within Bcl-6 + CXCR5 + follicular T cell gate and the total number of CD25 − Foxp3 + T FR (f) and Foxp3 − T FH (g) cells ( n = 11, 10). Gating strategy is depicted in Figure S4. (h) T FR /T FH ratio calculated using the total number of CD25 − Foxp3 + T FR and Foxp3 − T FH cells in F and G. (i, j) B cell class switching to IgG 1 by T FH cells. IgG − CD19 + B cells sort-purified from the DLNs of CIA mice fed the control HAMS or HAMSB diet (described as HAMS-B or HAMSB-B respectively) two weeks after the initial immunization were cultured alone with 100 µg/ml type II collagen (CII),and IgG − CD19 + B cells sort-purified from the DLNs of CIA mice fed the control HAMS diet were co-cultured with CXCR5 + ICOS + CD4 + T FH cells sort-purified from DLNs of CIA mice fed the control HAMS or HAMSB diet (described as HAMS-T FH or HAMSB-T FH respectively) in the presence or absence of CII. Representative flow cytometry contour plots of GL7 and intracellular IgG 1 staining six days after cultivation (i, n = 6), and the frequency of GL7 + IgG1 + B cells within CD19 + gate (j, n = 6). (k–n) Differentiation of T FR and T FH cells in CoPs of Tcrb −/− Tcrd −/− mice transferred with Foxp3-hCD2 + iT REG cells. Sort-purified CD45.1 + Foxp3-hCD2 + T cells cultured for five days under iT REG conditions were injected intravenously with CD45.2 + CD4 + T cells into Tcrb −/− Tcrd −/− mice fed the control HAMS or HAMSB diet. Representative flow cytometry contour plots of hCD2 (Foxp3) and CD25 staining (k), and the frequency of Foxp3 − , CD25 + Foxp3 + and CD25 − Foxp3 + cells (L) within CD45.1 + CD4 + TCRβ + gate ( n = 7). Representative flow cytometry contour plots of CXCR5 and PD-1 staining among Foxp3 − , CD25 + Foxp3 + and CD25 − Foxp3 + gate (m), and the frequency of CXCR5 + PD-1 + cells (N) among Foxp3 − (T FH cells), CD25 + Foxp3 + (CD25 + T FR cells) and CD25 − Foxp3 + (CD25 − T FR cells) gates (n, n = 7). Results show one representative experiment of at least two experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (b, d–h, j, l, n, Welch's t -test or unpaired two-tailed Student's t -test).

Article Snippet: Follicular T cell staining was performed with mAbs including BUV737-conjugated anti-TCRβ-chain (H57–597; BD Biosciences, Franklin Lakes, NJ, USA), BUV395-conjugated anti-CD25 (PC61; BD Biosciences), Brilliant Violet 510 (BV510)-conjugated anti-CD45 (30-F11; BioLegend), PE-eFluor 610-conjugated anti-Nrp-1 (3DS304M; Thermo Fisher Scientific, Waltham, MA, USA), PE-conjugated anti-CXCR5 (L138D7; BioLegend), and redFluor710-conjugated anti-CD4 (RM4–5; Tonbo Biosciences, San Diego, CA, USA), followed by dead cell staining with Fixable Viability Stain 780 (FVS780; BD Biosciences).

Techniques: Flow Cytometry, Staining, Purification, Cell Culture, Injection, Two Tailed Test

Journal: EBioMedicine

Article Title: Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

doi: 10.1016/j.ebiom.2020.102913

Figure Lengend Snippet: In vitro T FR (iT FR ) cell differentiation (a) Schematic of iT FR and iT REG cell differentiation culture. (b) Relative mRNA expression of Pdcd1, Cxcr5, Bcl6 , and Tcf7 in sort-purified TCRβ + CD4 + Foxp3-hCD2 + cells fro, iT FR or iT REG cell culture conditions ( n = 5). Sort-purified naïve CD4 + T cells from Foxp3 hCD2 reporter mice were used for the culture. (c, d) Expression of CD25 and Foxp3 by cells from iT FR or iT REG cultures. Representative flow cytometry contour plots of CD25 and Foxp3-hCD2 staining (c), and the frequency of CD25 + Foxp3 + and CD25 − Foxp3 + cells (d) within CD4 + TCRβ + gate ( n = 5). (e, f) Expression of T FR cell phenotypic markers by cells from iT FR or iT REG cultures. Representative flow cytometry histograms of FSC, PD-1, CXCR5, Bcl-6, and TCF-1 expression (e), and the gMFI of PD-1, CXCR5, Bcl-6, and TCF-1 (f) within CD25 + Foxp3 + and CD25 − Foxp3 + gate ( n = 5). (g, h) Expression of Bcl-6-tdTomato reporter by cells from iT FR or iT REG cultures. Sort-purified naïve CD4 + T cells from Bcl-6-tdTomato Foxp3 hCD2 double reporter mice or Foxp3 hCD2 reporter mice (described as control) were used for the culture. Representative flow cytometry histograms of Bcl-6-tdTomato reporter and control expression (g), and Bcl-6-tdTomato gMFI (h) within CD25 + Foxp3 + and CD25 − Foxp3 + gate ( n = 5). (i–l) B cell class switching to IgG 1 and IgG 2a in suppression assays. IgG − CD43 − CD19 + resting B cells, Foxp3-hCD2 − CXCR5 + Bcl-6-Eyfp + T FH cells, and CD25 − Foxp3-hCD2 + CXCR5 + Bcl-6-Eyfp + iT FR cells sort-purified from Bcl-6-Eyfp Foxp3 hCD2 double reporter mice were used.The resting B cells from were co-cultured with T FH cells alone, T FH and T FR cells, or T FH and sort-purified CD25 − Foxp3-hCD2 + CXCR5 + Bcl-6-Eyfp + iT FR cells under the exsitance of 5 μg ml −1 anti-IgM and 2 μg ml − 1 anti-CD3ε Abs. Representative flow cytometry histograms of Fixable Viability Stain 780 (FVS780) staining (i), and the frequency of FVS780 − live cells (j) within GL7 + CD19 + B cells ( n = 3–5). Representative flow cytometry histograms of intracellular IgG 1 and IgG 2a staining (k), and the frequency of IgG 1 + and IgG 2a + cells (l) within GL7 + CD19 + B gate ( n = 3–5). Results show one representative experiment of at least two experiments.*** P < 0.001 (b, d, Welch's t -test or unpaired two-tailed Student's t -test; j, l, one-way ANOVA followed by Dunnett's post-hoc test; g, h, two-way ANOVA followed by Sidak's post-hoc test).

Article Snippet: Follicular T cell staining was performed with mAbs including BUV737-conjugated anti-TCRβ-chain (H57–597; BD Biosciences, Franklin Lakes, NJ, USA), BUV395-conjugated anti-CD25 (PC61; BD Biosciences), Brilliant Violet 510 (BV510)-conjugated anti-CD45 (30-F11; BioLegend), PE-eFluor 610-conjugated anti-Nrp-1 (3DS304M; Thermo Fisher Scientific, Waltham, MA, USA), PE-conjugated anti-CXCR5 (L138D7; BioLegend), and redFluor710-conjugated anti-CD4 (RM4–5; Tonbo Biosciences, San Diego, CA, USA), followed by dead cell staining with Fixable Viability Stain 780 (FVS780; BD Biosciences).

Techniques: In Vitro, Cell Differentiation, Expressing, Purification, Cell Culture, Flow Cytometry, Staining, Two Tailed Test

Journal: EBioMedicine

Article Title: Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

doi: 10.1016/j.ebiom.2020.102913

Figure Lengend Snippet: Butyrate induces iT FR cell differentiation (a-d) iT FR cell differentiation with SCFAs treatment. Sort-purified naïve CD4 + T cells from Bcl-6-tdTomato Foxp3 hCD2 double reporter mice were cultivated under iT FR -cell culture conditions in the presence of sodium acetate (SA), sodium propionate (SP), or sodium butyrate (SB) at 10 μM or 100 μM. Representative flow cytometry contour plots of hCD2 (Foxp3) and CD25 staining (a), and the frequency of CD25 + Foxp3 + (upper panel) and CD25 − Foxp3 + (lower panel) cells within CD45 + CD4 + TCRβ + gate (b, n = 5). Representative flow cytometry contour plots of Bcl-6-tdTomato reporter signal and CXCR5 staining among CD25 + Foxp3 + and CD25 − Foxp3 + gate (c), and the frequency of Bcl-6-tdTomato + CXCR5 + cells among CD25 + Foxp3 + (upper panel) and CD25 − Foxp3 + (lower panel) gates in (b) (d, n = 5). (e) gMFI of Bcl-6-tdTomato reporter in CXCR5 + and CXCR5 − populations among CD25 + Foxp3 + (upper panel) and CD25 − Foxp3 + (lower panel) gates of iT FR culture in (c). CXCR5 + and CXCR5 − populations are shown as areas equal to or higher than, or less than the dotted lines in (c), respectively ( n = 5). (f, g) Expression of TCF-1 among CD25 + Foxp3 + (upper panel) and CD25 − Foxp3 + (lower panel) gates of iT FR culture in (c). iT FR culture was treated with 100 μM SA, SP or SB. Representative flow cytometry histograms of TCF-1 expression (f), and the gMFI of TCF-1 (g, n = 5). (h, i) iT FR cell differentiation with pan-HDAC inhibitor treatment. Sort-purified naïve CD4 + T cells from Bcl-6-tdTomato Foxp3 hCD2 double reporter mice were cultivated under iT FR -cell culture conditions with the treatment of suberoylanilide hydroxamic acid (SAHA, 50 or 200 nM), trichostatin A (TSA, 2.5 or 10 nM), or vehicle control 0.01% dimethyl sulfoxide (DMSO). The frequency of Bcl-6-tdTomato + CXCR5 + cells (h) and the gMFI of TCF-1 (i) among CD25 + Foxp3 + CD4 + TCRβ + (upper panel) and CD25 − Foxp3 + CD4 + TCRβ + (lower panel) gates ( n = 5). (j, k) Differentiation of T FR cells in the inguinal lymph node (InLN) of mice treated with SAHA. C57BL/6 J mice were subcutaneously immunized with human insulin and intravenously injected with SAHA (20 mg/kg body weight) or vehicle control DMSO every day after immunization. The frequency of Bcl-6 + CXCR5 + follicular T cells within CD4 + TCRβ + gate (j, n = 6), and CD25 + Foxp3 + T FR cells within Bcl-6 + CXCR5 + gate (k, n = 6) from InLN 10 days after immunization. (l) Accumulation of H3K27 acetylation (ac) at the promoter region of T FR cell-related genes in Foxp3 + cells from iT FR or iT REG cultures. ChIP quantitative RT-PCR (qPCR) analysis of H3K27ac levels in the promoters of Bcl6, Cxcr5, and Tcf7 in sort purified Foxp3 + T cells cultured for the total three days under iT FR or iT REG -cell polarizing conditions (see Fig. 5 a) with or without the treatment of 100 µM SB. (m) iT FR cell differentiation using G-protein coupled receptor-deficient mice. Sort-purified naïve CD4 + T cells from Ffar3 −/− , Ffar2 −/− , or Hcar2 −/− mice were cultivated under iT FR -cell culture conditions with or without the treatment of 100 µM S. The frequency of Bcl-6 + CXCR5 + cells within CD25 − Foxp3 + CD4 + TCRβ + gate ( n = 5). Results show one representative experiment of at least two experiments.* P < 0.05, ** P < 0.01, *** P < 0.001 (b, c, d, f-h, one-way ANOVA followed by Dunnett's post-hoc test; l -m Welch's t -test or unpaired two-tailed Student's t -test).

Article Snippet: Follicular T cell staining was performed with mAbs including BUV737-conjugated anti-TCRβ-chain (H57–597; BD Biosciences, Franklin Lakes, NJ, USA), BUV395-conjugated anti-CD25 (PC61; BD Biosciences), Brilliant Violet 510 (BV510)-conjugated anti-CD45 (30-F11; BioLegend), PE-eFluor 610-conjugated anti-Nrp-1 (3DS304M; Thermo Fisher Scientific, Waltham, MA, USA), PE-conjugated anti-CXCR5 (L138D7; BioLegend), and redFluor710-conjugated anti-CD4 (RM4–5; Tonbo Biosciences, San Diego, CA, USA), followed by dead cell staining with Fixable Viability Stain 780 (FVS780; BD Biosciences).

Techniques: Cell Differentiation, Purification, Cell Culture, Flow Cytometry, Staining, Expressing, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: On CD28/CD40 Ligand Costimulation, Common γ -Chain Signals, and the Alloimmune Response 1

doi:

Figure Lengend Snippet: Acquisition of primed phenotype by dividing CD4+ and CD8+ T cells in vivo. A, CFSE-labeled B6AF1 cells were recovered from irradiated DBA/2 hosts 3 days after adoptive transfer. Cells were stained with CyChrome anti-mouse CD4 and anti-CD8 as well as PE-conjugated mAbs against CD25, CD44, and CD62L. Expression of the activation markers was analyzed by gating onto CFSE+CD4+ or CFSE+CD8+ cells. Cells stained with isotype control Abs were used as a control to set up the quadrants for analysis. B, Expression of CD25, CD44, and CD62L in each cell division was analyzed, and the mean fluorescence intensity (MFI) was calculated and plotted against the number of cell divisions. Representative data of five experiments shown.

Article Snippet: Hybridoma cell lines secreting a hamster anti-mouse CD40L mAb (MR1, IgG) and a rat anti-mouse CD25 mAb (TIB 222, IgG1) were obtained from American Type Culture Collection (Manassas, VA).

Techniques: In Vivo, Labeling, Irradiation, Adoptive Transfer Assay, Staining, Expressing, Activation Assay, Control, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: On CD28/CD40 Ligand Costimulation, Common γ -Chain Signals, and the Alloimmune Response 1

doi:

Figure Lengend Snippet: Effect of anti-CD25 and mutant IL-15/Fc on expansion of CD8+ T cells in vivo. Irradiated DBA/2 mice were injected with CFSE-labeled B6AF1 cells and treated with anti-CD25 or anti-CD25 plus mutant IL-15/Fc. Cells were recovered 3 days later from the host spleen, and cell division in vivo was analyzed by gating onto the CFSE+CD8+ population. Representative data of three experiments shown.

Article Snippet: Hybridoma cell lines secreting a hamster anti-mouse CD40L mAb (MR1, IgG) and a rat anti-mouse CD25 mAb (TIB 222, IgG1) were obtained from American Type Culture Collection (Manassas, VA).

Techniques: Mutagenesis, In Vivo, Irradiation, Injection, Labeling