Journal: Frontiers in Immunology

Article Title: Post-Transplantation Cyclophosphamide Uniquely Restrains Alloreactive CD4 + T-Cell Proliferation and Differentiation After Murine MHC-Haploidentical Hematopoietic Cell Transplantation

doi: 10.3389/fimmu.2022.796349

Figure Lengend Snippet: CY uniquely restrains T-cell differentiation at both early and later timepoints. Mice were transplanted, treated with PBS or a chemotherapeutic on days +3 and +4, and euthanized for flow cytometric assessment at day +7 or +21 as in Figure 3 . (A) At day +7, CY decreased percentages of CD4 + Foxp3 - Vβ6 + T cells that were phenotypically effector/effector memory (CD62L - ). Consequently, percentages of naïve (CD44 - CD62L + ) and central memory (CD44 + CD62L + ) CD4 + Foxp3 - Vβ6 + T cells were increased by CY at day +7. This same effect was achieved to a lesser extent after both MTX and ARA-C. (B) This restrained differentiation was persistent after CY at day +21 but was completely lost after MTX, wherein differentiation seemed to be overall accelerated. Combined results from two independent experiments are shown with n = 4/group/experiment except for VCR (n = 6 total) in (B) due to excess early deaths. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 on one-way ANOVA followed by the Holm-Sidak post hoc test using the vehicle-treated group as the control. Only significant results are shown; all other comparisons between treatment groups and the vehicle group are non-significant.

Article Snippet: Fluorochrome-conjugated monoclonal antibodies used for flow cytometry included BUV395 anti-CD3 (clone 145-2C11), BV786 anti-CD8a (clone 53-6.7), PE-CF594 anti-CD25 (clone PC61), AF700 anti-CD44 (clone IM7), BUV737 anti-CD62L (clone MEL-14), PE anti-H2k k (clone 36-7-5), and BV711 anti-H2k k (clone AF3-12.1) from BD Biosciences; PE-Cy5 anti-CD8 (clone 53-6.7), PE-Cy7 anti-H2k d (clone SF1-1.1), and BV605 anti-Ki67 (clone 16A8) from BioLegend; and APC-eFluor780 anti-CD4 (clone GK1.5), PerCP-eFluor710 anti-Vβ6 (clone RR4-7), eFluor450 anti-Foxp3 (clone FJK-16s), and PE anti-phospho-STAT5 (Tyr694) (clone SRBCZX) from Invitrogen.

Techniques: Cell Differentiation

Journal: Scientific Reports

Article Title: Virus infection drives IL-2 antibody complexes into pro-inflammatory agonists in mice

doi: 10.1038/srep37603

Figure Lengend Snippet: IL-2 Ab Cx or PBS was administered to CHIKV-infected mice on 3, 4 and 5 dpi. The popliteal lymph node (pLN) was isolated from these mice on 6 dpi. N.I mice were added as a control. Prophylactic treatment of IL-2 Ab Cx was included. (a) Representative scatterplots show Foxp3 and CD25 gated on CD4 + T cells. Foxp3 + Tregs and Foxp3 − Teffs are represented in red and blue respectively in the scatterplots. Numbers in scatterplots indicate percentage of Foxp3 + Tregs (in red) and Foxp3 − Teffs (in blue) in total CD4 + T cells. Histogram profiles of activation markers CD44 (left), CTLA-4 (middle) and CD25 (right) on (b) Foxp3 − Teffs and (c) Foxp3 + Tregs were also determined. The experiment was performed 3 times independently.

Article Snippet: Staining was performed using rat anti-CD45 (30-F11, BD Pharmingen), rat anti-CD4 (RM4–5, BD Pharmingen), rat anti-CD3 (17A2, Biolegend), rat anti-CD25 (PC61.5, eBioscience), hamster anti-CTLA-4 (UC10-4B9, eBioscience) and rat anti-CD44 (IM7, eBioscience) antibodies for 15 min. Stained cells were then washed with PBS and fixed using IC Fixation Buffer (eBioscience).

Techniques: Infection, Isolation, Activation Assay

Journal: Scientific Reports

Article Title: Virus infection drives IL-2 antibody complexes into pro-inflammatory agonists in mice

doi: 10.1038/srep37603

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated from 7 healthy donors and stimulated with increasing concentrations of human IL-2 (1 to 1000 ng/ml) stimulation. pSTAT5 signaling in CD4 + T cells were measured. (a) Representative histograms of pSTAT5 signaling in Teffs (top) and Tregs (bottom) in response to increasing concentration of IL-2. Line graph shows average percentage of pSTAT5 + Tregs and Teffs with respect to increasing concentration of IL-2 stimulation. (b) Representative histograms of pSTAT5 signaling in naive (CD45RA + ) (top) and memory (CD45RA − ) (bottom) CD4 + T cells in response to increasing concentration of IL-2. Line graph shows average percentage of pSTAT5 + naive and memory CD4 + T cells with respect to increasing concentration of IL-2 stimulation (n = 4). Data was presented as mean ± SD. (c) Blood was collected from 261 healthy donors and assessed for the percentage of memory population (CD45RA − ) in peripheral CD4 + T cells, CD4 + Teffs and CD4 + Tregs using flow cytometry. The percentage of peripheral memory CD4 + T cells population (CD44 + ) in mice was also assessed in comparison. (d) CD25 expression on naïve (CD45RA + ) and memory (CD45RA − ) population from (c) was also measured.

Article Snippet: Staining was performed using rat anti-CD45 (30-F11, BD Pharmingen), rat anti-CD4 (RM4–5, BD Pharmingen), rat anti-CD3 (17A2, Biolegend), rat anti-CD25 (PC61.5, eBioscience), hamster anti-CTLA-4 (UC10-4B9, eBioscience) and rat anti-CD44 (IM7, eBioscience) antibodies for 15 min. Stained cells were then washed with PBS and fixed using IC Fixation Buffer (eBioscience).

Techniques: Isolation, Concentration Assay, Flow Cytometry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Continuous Activation of Autoreactive CD4 + CD25 + Regulatory T Cells in the Steady State

doi: 10.1084/jem.20030686

Figure Lengend Snippet: Analysis of turnover of T reg by BrdU incorporation. BALB/c mice were treated with BrdU administered continuously for 7 d using osmotic pumps. Then, peripheral LN cells and splenocytes were analyzed for cell surface expression of CD4, CD25, and CD44, and BrdU incorporated into DNA of divided cells. Levels of BrdU incorporations were quantified on gated CD4 + CD25 + CD44 high cells and CD4 + CD25 + CD44 low cells in LNs and the spleen as defined in A. Background BrdU staining levels were obtained from untreated mice (B). Representative results are shown and values indicate the mean ± SD of BrdU + cells from data from two independent experiments (seven mice). The percentage of CD25 + CD4 + T cells was not statistically different in mice receiving osmotic pump with BrdU, mice receiving osmotic pump with PBS, and unmanipulated mice. In A, CD25 + CD4 + T cells represented 8.2% of CD4 + cells.

Article Snippet: Then, LN cells and splenocytes were stained with anti-CD4 CyChrome (GK1.5; BD Biosciences), anti–CD25-PE (PC61; BD Biosciences), anti–CD44-biotin (IM7.8.1; Caltag), and streptavidin-allophycocyanin (BD Biosciences).

Techniques: BrdU Incorporation Assay, Expressing, BrdU Staining

Journal: The Journal of Experimental Medicine

Article Title: Continuous Activation of Autoreactive CD4 + CD25 + Regulatory T Cells in the Steady State

doi: 10.1084/jem.20030686

Figure Lengend Snippet: Phenotypic Characterization of the Two T reg Subsets and CD4 + CD25 − Cells

Article Snippet: Then, LN cells and splenocytes were stained with anti-CD4 CyChrome (GK1.5; BD Biosciences), anti–CD25-PE (PC61; BD Biosciences), anti–CD44-biotin (IM7.8.1; Caltag), and streptavidin-allophycocyanin (BD Biosciences).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Continuous Activation of Autoreactive CD4 + CD25 + Regulatory T Cells in the Steady State

doi: 10.1084/jem.20030686

Figure Lengend Snippet: The activated T reg population contains autoreactive cells. CFSE-labeled CD62L high T reg purified from Thy-1.1 TCR-HA transgenic mice were intravenously injected into Thy-1.2 ins-HA transgenic mice. (A) The proportion of CD4 + Thy-1.1 + donor cells was determined by flow cytometry in pancreatic (•) and peripheral (○) LNs 3, 5, 7, and 11 d after transfer. The graph, which represents the percentage of donor T reg (CD4 + Thy-1.1 + ) per organ, shows the mean of two to three mice per time point and is representative of three independent experiments. (B and C) Representative phenotypic analysis of the donor cells on day 7 after transfer in the indicated LNs. Gated on CD4 + Thy-1.1 + cells, cells were analyzed for CFSE staining and expression of the anti-HA–specific TCR using the 6.5 mAb or of CD44 and CD62L. Values ± SD indicate the percentages of 6.5 + divided cells.

Article Snippet: Then, LN cells and splenocytes were stained with anti-CD4 CyChrome (GK1.5; BD Biosciences), anti–CD25-PE (PC61; BD Biosciences), anti–CD44-biotin (IM7.8.1; Caltag), and streptavidin-allophycocyanin (BD Biosciences).

Techniques: Labeling, Purification, Transgenic Assay, Injection, Flow Cytometry, Staining, Expressing

Journal: International Journal of Nanomedicine

Article Title: Ether lipid vesicle-based antigens impart protection against experimental listeriosis

doi: 10.2147/IJN.S25875

Figure Lengend Snippet: Augmentation of CD8 + /CD4 + T cell effector memory response upon immunization with archaeosome-entrapped SAgs. The CD4 + and CD8 + T cells were harvested and their purity was depicted as described in the ‘Materials and methods’ section. The ( A ) CD4 + CD44 high CD62L low/high and ( B ) CD8 + CD44 high CD62L low/high phenotypes were analyzed using flow cytometry at 2 weeks post-challenge, representing various immunized groups: ( i ) Sham archaeosomes as control; ( ii ) free culture supernatant SAgs; ( iii ) Sham + SAgs as aphysical mixture; ( iv ) archaeosome-entrapped culture supernatant SAgs; and ( v ) isotype control. The bar graph depicts the population of ( C ) CD4 + CD44 high CD62L high/low and ( D ) CD8 + CD44 high CD62L high/low and is representative of three independent experiments and presented as mean ± SD. Notes: Archaeosome-entrapped SAgs vs free SAgs *** P < 0.001 (CD4 + CD44 high CD62L high ; CD4 + CD44 high CD62L low ), P < 0.001 (CD8 + CD44 high CD62L high ; CD8 + CD44 high CD62L low ); physical mixture vs free SAgs * P < 0.05 CD4 + CD44 high CD62L high ; P = NS CD4 + CD44 high CD62L low , P < 0.05 CD8 + CD44 high CD62L high ; P = NS CD8 + CD44 high CD62L low . Abbreviations: SD, standard deviation; SAgs, secretory protein antigens; NS, not significant.

Article Snippet: The following: fluorochrome-labeled anti-mouse antibodies; fluorescein isothiocyanate-conjugated CD4 (GK 1.5) and CD8 (53.67); PerCP-conjugated CD62L (MEL-14); phycoerythrin-conjugated CD44 (IM7), CD80 (B7-1), and CD86 (GL1); and IgG2a (R35-95) isotype control were procured from eBiosciences (San Diego, CA).

Techniques: Flow Cytometry, Standard Deviation