Journal: The Scientific World Journal

Article Title: Immunomodulatory Effects of Hemagglutinin- (HA-) Modified A20 B-Cell Lymphoma Expanded as a Brain Tumor on Adoptively Transferred HA-Specific CD4 + T Cells

doi: 10.1155/2014/165265

Figure Lengend Snippet: Adoptively transferred HA-specific CD4 + Thy1.1 + T cells demonstrate early activation and following depletion in cervical lymph nodes of A20HA brain tumor-bearing mice. ((a), (b), (c)) Flow cytometry of CD4 + Thy1.1 + CD44 + T cells from cervical lymph nodes on days 2, 9, and 16 after adoptive transfer. HA-specific CD4 + Thy1.1 + T cells were injected i.v. into A20HA brain tumor-bearing mice 5 days after i.c. tumor inoculation (A20HA + T cells; left column) and tumor-free control mice (T cells; middle column). Naive mice represented a negative control (Neither; right column). Each group included three mice. Cervical lymph nodes were analyzed individually from each mouse by three-color FACS assay. A representative experiment among three equivalents is shown. (a) Dot plots show gated HA-specific CD4 + Thy1.1 + T cells (upper right quadrants; numbers in the quadrants indicate percent cells in each). Bars show (b) percent of HA-specific CD4 + Thy-1.1 + T cells and (c) intensity of CD44 expression in the gated population of CD4 + Thy-1.1 + T cells. Data are represented as the mean ± SD, n = 3. * P < 0.05.

Article Snippet: Cy-Chrome-conjugated rat anti-mouse CD4 (RM4-5), FITC-conjugated rat anti-mouse CD44 (IM7), and PE- or perCp-conjugated mouse anti-rat/mouse Thy1.1 (OX-7) antibodies were purchased from PharMingen (San Diego, CA).

Techniques: Activation Assay, Flow Cytometry, Adoptive Transfer Assay, Injection, Negative Control, Expressing

Journal: The Scientific World Journal

Article Title: Immunomodulatory Effects of Hemagglutinin- (HA-) Modified A20 B-Cell Lymphoma Expanded as a Brain Tumor on Adoptively Transferred HA-Specific CD4 + T Cells

doi: 10.1155/2014/165265

Figure Lengend Snippet: HA-specific CD4 + Thy1.1 + T cells from cervical lymph nodes of A20HA brain tumor-bearing mice show increased response to restimulation in vitro with MHC class II synthetic HA peptide. ((a), (b), (c)) Flow cytometry of CD4 + Thy1.1 + CD44 + T cells from cervical lymph nodes on day 2 after adoptive transfer. HA-specific CD4 + Thy1.1 + T cells were injected i.v. into A20HA brain tumor-bearing mice 5 days after i.c. tumor inoculation (A20HA + T cells; dot plots in the upper row) and a tumor-free control group (T cells; dot plots in the middle row). A20HA brain tumor-bearing mice in the absence of HA-specific CD4 + Thy1.1 + T cells represented a negative control (A20HA; dot plots in the lower row). Cervical lymph nodes were pooled and analyzed for CD4 + Thy1.1 + CD44 + T cells (a) before incubation ( in vivo column; right upper quadrants) and ((b), (c)) after 72 h incubation in vitro in triplicates (b) without ( in vitro column; right upper quadrants) and (c) with ( in vitro column; right upper quadrants) HA peptide. Histograms under the dot plots show the level of CD44 expression in the gated populations of CD4 + Thy-1.1 + T cells (shown in the right upper quadrants). Numbers above histograms indicate percent of gated CD4 + Thy-1.1 + T cells in the designed dot plots (left numbers) and intensity of CD44 expression by these cells (right numbers). One representative experiment of equivalent two is shown.

Article Snippet: Cy-Chrome-conjugated rat anti-mouse CD4 (RM4-5), FITC-conjugated rat anti-mouse CD44 (IM7), and PE- or perCp-conjugated mouse anti-rat/mouse Thy1.1 (OX-7) antibodies were purchased from PharMingen (San Diego, CA).

Techniques: In Vitro, Flow Cytometry, Adoptive Transfer Assay, Injection, Negative Control, Incubation, In Vivo, Expressing

Journal: The Scientific World Journal

Article Title: Immunomodulatory Effects of Hemagglutinin- (HA-) Modified A20 B-Cell Lymphoma Expanded as a Brain Tumor on Adoptively Transferred HA-Specific CD4 + T Cells

doi: 10.1155/2014/165265

Figure Lengend Snippet: Response of HA-specific CD4 + Thy-1.1 + T cells from cervical lymph nodes of A20HA brain tumor-bearing mice to HA peptide in vitro is reduced in process of the tumor progression and controversial to response of CD4 + T cells from spleen. ((a), (b)) Percent of CD4 + Thy-1.1 + T cells (upper) and intensity of CD44 expression (lower) by cells isolated from (a) cervical lymph nodes and (b) spleens on days 2, 9, and 16 after the adoptive transfer. HA-specific CD4 + Thy1.1 + T cells were injected i.v. into A20HA brain tumor-bearing mice 5 days after i.c. tumor inoculation (A20HA + T cells) and tumor-free control mice (T cells). A20HA brain tumor-bearing mice in the absence of HA-specific CD4 + Thy1.1 + T cells represented a negative control (background staining of a specified area was lesser than 0.01%; data not shown). Cervical lymph nodes and spleens were pooled and analyzed by FACS for CD4 + Thy1.1 + CD44 + T cells before incubation and after 72 h incubation without (w/o) and with HA peptide. FACS data are presented as a percent of double positive cells, CD4 versus Thy1.1, and the level of CD44 expression in the CD4 + Thy1.1 + T-cell population. One representative experiment of equivalent two is shown. * P < 0.05.

Article Snippet: Cy-Chrome-conjugated rat anti-mouse CD4 (RM4-5), FITC-conjugated rat anti-mouse CD44 (IM7), and PE- or perCp-conjugated mouse anti-rat/mouse Thy1.1 (OX-7) antibodies were purchased from PharMingen (San Diego, CA).

Techniques: In Vitro, Expressing, Isolation, Adoptive Transfer Assay, Injection, Negative Control, Staining, Incubation

Journal: The Scientific World Journal

Article Title: Immunomodulatory Effects of Hemagglutinin- (HA-) Modified A20 B-Cell Lymphoma Expanded as a Brain Tumor on Adoptively Transferred HA-Specific CD4 + T Cells

doi: 10.1155/2014/165265

Figure Lengend Snippet: Virus HA vaccination at late stages of A20HA brain tumor progression stimulates activation of residual HA-specific CD4 + T cells in vivo . ((a), (b)) Percent of CD4 + Thy-1.1 + T cells (upper) and intensity of CD44 expression (lower) by cells isolated from (a) cervical lymph nodes and (b) spleens from separated asymptomatic (As) and symptomatic (Sy) mice on day 18 after the adoptive transfer. HA-specific CD4 + Thy1.1 + T cells were injected i.v. to A20HA brain tumor-bearing mice on day 5 after i.c. tumor inoculation. Virus HA-Vac was delivered i.p. on day 15 after the adoptive transfer. Individual cervical lymph nodes and spleens of asymptomatic and symptomatic mice (three mice per group) were analyzed by FACS for CD4 + CD44 + Thy1.1 + T cells. A20HA + T cells + HA-Vac, mice that received A20HA tumor cells, HA-specific CD4 + Thy1.1 + T cells and virus HA-Vac; A20HA + T cells, mice that received A20HA tumor cells and HA-specific CD4 + Thy1.1 + T cells; T cells + HA-Vac, mice that received HA-specific CD4 + Thy1.1 + T cells and virus HA-Vac; T cells, mice that received only HA-specific CD4 + Thy1.1 + T cells. Data are represented as the mean ± SD, n = 3. * P < 0.05.

Article Snippet: Cy-Chrome-conjugated rat anti-mouse CD4 (RM4-5), FITC-conjugated rat anti-mouse CD44 (IM7), and PE- or perCp-conjugated mouse anti-rat/mouse Thy1.1 (OX-7) antibodies were purchased from PharMingen (San Diego, CA).

Techniques: Activation Assay, In Vivo, Expressing, Isolation, Adoptive Transfer Assay, Injection

Journal: The Scientific World Journal

Article Title: Immunomodulatory Effects of Hemagglutinin- (HA-) Modified A20 B-Cell Lymphoma Expanded as a Brain Tumor on Adoptively Transferred HA-Specific CD4 + T Cells

doi: 10.1155/2014/165265

Figure Lengend Snippet: HA-specific activation stimulates migration of CD4 + T cells into the brain of A20HA brain tumor-bearing mice. (a) Percent (left bars) and CD44 intensity (right bars) of transgenic CD4 + Thy1.1 + T cells and nontransgenic CD4 + Thy1.1 − T cells from brains of A20HA brain tumor-bearing mice and A20HA tumor-free mice measured by FACS on day 18 after the adoptive transfer and HA vaccination. HA-specific CD4 + Thy1.1 + T cells were injected i.v. on day 5 after A20HA i.c. inoculation, and virus HA-Vac was delivered i.p. at the same day. Brain pools of three mice in each group were analyzed by FACS 18 days later. A20HA + T cells + HA-Vac, mice that received A20HA tumor cells, HA-specific CD4 + Thy1.1 + T cells, and HA-Vac; A20HA + T cells, mice that received A20HA tumor cells and HA-specific CD4 + Thy1.1 + T cells; T cells + HA-Vac, mice that received HA-specific CD4 + Thy1.1 + T cells and HA-Vac; T cells, mice that received only HA-specific CD4 + Thy1.1 + T cells. (b) Photomicrographs of eosin and hematoxylin stained coronal sections of the brains on days 2, 9, and 18 after the adoptive transfer showing growth dynamics of A20HA B-cell lymphoma in BALB/c mice that received HA-specific CD4 + T cells and HA-Vac on day 5 after A20HA i.c. inoculation. Magnifications ×1 (upper row; arrows show the tumor areas) and ×40 (lower row, arrows show single lymphocytes in the tumor areas). * P < 0.05.

Article Snippet: Cy-Chrome-conjugated rat anti-mouse CD4 (RM4-5), FITC-conjugated rat anti-mouse CD44 (IM7), and PE- or perCp-conjugated mouse anti-rat/mouse Thy1.1 (OX-7) antibodies were purchased from PharMingen (San Diego, CA).

Techniques: Activation Assay, Migration, Transgenic Assay, Adoptive Transfer Assay, Injection, Staining

Journal: Stem Cell Research & Therapy

Article Title: TNFAIP6 defines the MSC subpopulation with enhanced immune suppression activities

doi: 10.1186/s13287-022-03176-5

Figure Lengend Snippet: TNFAIP6 is a potential MSC marker-expression validation. A The mRNA level of TNFAIP6 was plotted on single-cell RNA sequencing data of bone marrow cells. B The mRNA level of TNFAIP6 was plotted on single-cell RNA sequencing data of placenta cells. C The mRNA level of TNFAIP6 was plotted on single-cell RNA sequencing data of fat tissue cells. D The mRNA level of CD44 was plotted on single-cell RNA sequencing data of bone marrow cells. E The mRNA level of CD44 was plotted on single-cell RNA sequencing data of placenta cells. F The mRNA level of CD44 was plotted on single-cell RNA sequencing data of fat tissue cells. G The protein levels of TNFAIP6 and CD44 were determined by flow cytometry after cell fixation and permeabilization. H The cell surface expression levels of TNFAIP6 and CD44 were determined by flow cytometry. TNFAIP6 tumor necrosis factor alpha-induced protein 6; BM bone marrow

Article Snippet: Briefly, for cell surface protein staining, the freshly isolated cells were incubated with 5% FBS in PBS (Thermo Fisher Scientific) for 30 min, and then, the cells were stained with anti-TNFAIP6 (Cat. No. PA5-75,332, Thermo Fisher Scientific) for 60 min on ice, followed by 3 times washes, and then FITC-conjugated anti-rabbit IgG secondary (Cat. No. F-2765, Thermo Fisher Scientific) antibody for 30 min. For CD45 staining, the cells were stained with PE-conjugated rat anti-mouse CD45 (Cat. No. 567111, BD Biosciences) or PE Rat IgG2b, κ Isotype Control (Cat. No. 555848, BD Biosciences) for 30 min. For CD44 staining, the cells were stained with FITC-conjugated rat anti-mouse CD44 (Cat. No. 561859, BD Biosciences) or FITC Rat IgG2b, κ Isotype Control (Cat. No. 553988, BD Biosciences) for 30 min. For whole cell protein staining, the cells were fixed with 4% PFA (Paraformaldehyde, Sigma), permeablized with 0.2% Triton-X 100 in PBS with 10% FBS for 30 min, and then subjected to antibody staining.

Techniques: Marker, Expressing, RNA Sequencing Assay, Flow Cytometry

Journal: Stem Cell Research & Therapy

Article Title: TNFAIP6 defines the MSC subpopulation with enhanced immune suppression activities

doi: 10.1186/s13287-022-03176-5

Figure Lengend Snippet: TNFAIP6 defines a subpopulation of mouse MSCs with enhanced immune suppression activities. A Cell morphology of CD45 − TNFAIP6 − and CD45 − TNFAIP6 + MSCs. B Left panel: mRNA level of TNFAIP6 was assessed by qPCR (n = 3); Right panel: protein level of TNFAIP6 was assessed by western blot. C Protein level of TNFAIP6 secreted from the CD45 − TNFAIP6 − or CD45 − TNFAIP6 + MSCs (n = 3). D Splenocytes proliferation assay after co-culture with CD45 − TNFAIP6 − or CD45 − TNFAIP6 + MSCs (n = 3). Both high cell number (5 × 10 4 ) and low cell number (2 × 10 4 ) were assessed. E Splenocytes proliferation assay after co-culture with CD45 − TNFAIP6 − or CD45 − TNFAIP6 + MSCs was assessed with CFSE approach. High cell number (5 × 10 4 ) was used. F CD44 expression of CD45 − TNFAIP6 − or CD45 − TNFAIP6 + MSCs, determined by flow cytometry. G Cell proliferation of CD45 − TNFAIP6 − or CD45 − TNFAIP6 + MSCs was determined by doubling time (n = 3). H The adipocytes differentiation efficiency was assessed by Oil Red O staining. I The adipocytes differentiation efficiency was quantified by Oil Red O staining and qPCR analysis of gene Lpl and Pparγ (n = 3). J The osteocytes differentiation efficiency was assessed by Alizarin Red staining. K The osteocytes differentiation efficiency was quantified by Alizarin Red staining and qPCR analysis of gene Osterix and Runx2 (n = 3). L The chondrocytes differentiation efficiency was assessed by Alcian Blue staining. M The chondrocytes differentiation efficiency was quantified by Alcian Blue staining and qPCR analysis of gene Sox9 and Bmp2 (n = 3). * indicates P < 0.05. TNFAIP6 tumor necrosis factor alpha-induced protein 6; MSCs mesenchymal stromal/stem cells; Lpl lipoprotein lipase; Pparγ peroxisome proliferator-activated receptor gamma; Osterix Sp7 transcription factor; Runx2 RUNX family transcription factor 2; Sox9 SRY-box transcription factor 9; and Bmp2 bone morphogenetic protein 2

Article Snippet: Briefly, for cell surface protein staining, the freshly isolated cells were incubated with 5% FBS in PBS (Thermo Fisher Scientific) for 30 min, and then, the cells were stained with anti-TNFAIP6 (Cat. No. PA5-75,332, Thermo Fisher Scientific) for 60 min on ice, followed by 3 times washes, and then FITC-conjugated anti-rabbit IgG secondary (Cat. No. F-2765, Thermo Fisher Scientific) antibody for 30 min. For CD45 staining, the cells were stained with PE-conjugated rat anti-mouse CD45 (Cat. No. 567111, BD Biosciences) or PE Rat IgG2b, κ Isotype Control (Cat. No. 555848, BD Biosciences) for 30 min. For CD44 staining, the cells were stained with FITC-conjugated rat anti-mouse CD44 (Cat. No. 561859, BD Biosciences) or FITC Rat IgG2b, κ Isotype Control (Cat. No. 553988, BD Biosciences) for 30 min. For whole cell protein staining, the cells were fixed with 4% PFA (Paraformaldehyde, Sigma), permeablized with 0.2% Triton-X 100 in PBS with 10% FBS for 30 min, and then subjected to antibody staining.

Techniques: Western Blot, Proliferation Assay, Co-Culture Assay, Expressing, Flow Cytometry, Staining