Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Nutrients

Article Title: Long-Term Consumption of a Sugar-Sweetened Soft Drink in Combination with a Western-Type Diet Is Associated with Morphological and Molecular Changes of Taste Markers Independent of Body Weight Development in Mice

doi: 10.3390/nu14030594

Figure Lengend Snippet: Transcriptome analysis of 59 selected genes associated with oral chemosensation, displayed as a heatmap showing mean fold changes in gene expression of the SSB-fed diet groups in relation to the respective water-fed group (=1) in the form of a color code. The gene expression was analyzed using one customized cDNA microarray per group from pooled RNA samples of the CV from mice that received either a standard diet (chow, n = 10–11) or Western-type diet (WD, n = 7–8) with water (Water) or a sugar-sweetened beverage (SSB) as a drink for 24 weeks.

Article Snippet: The isolated RNA samples per mouse were reverse transcribed to cDNA using the LunaScript RT Supermix Kit (New England Biolabs GmbH, Frankfurt am Main, Germany).

Techniques: Expressing, Microarray, Western Blot

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , Scheme showing the localization of epidermal melanocytes (in red) in the human skin. B, C , Immunostaining for MITF (green, left panel) and SOX9 (red, right panel) in the human skin demonstrating the lack of SOX9 expression in the epidermal melanocytes. Inserts show higher magnification images of MITF and SOX9 immunostainings. Scale bars, 25 μm. D , Scheme showing the localization of melanocytes (in red) within the hair follicle. E , Immunostaining for MITF (green) and SOX9 (red) in the human skin reveals the expression of SOX9 in the cells of outer root sheath but not in the MITF-positive melanoblasts/melanocytes. Scale bar 100 μm. F, G , High magnification images of immunostaining for MITF and SOX9 in the upper part of human hair follicle ( F ) and the follicular bulb ( G ). H , Analysis of SOX9 (red, left panel) and SOX10 (red, right panel) expression in the patients with human giant congenital naevi demonstrates the lack of SOX9 expression in the SOX10-positive giant congenital naevi cells. Inserts show higher magnification. I , Representative examples of immunostaining for SOX9 (green) and SOX10 (red) in a tissue microarray of primary melanoma samples are shown. J-K , Distribution of SOX10 vs. SOX9 expression in human melanoma (based on TCGA database). 334 melanoma patients were divided in two groups, namely SOX10 High/ SOX9 Low and SOX10 Low / SOX9 high based on SOX10 and SOX9 expression levels. DP, dermal papilla; HF, hair follicle; M, melanocytes; ORS, outer root sheath. Scale bars, 25 μm.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Immunostaining, Expressing, Microarray

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , A schematic representation of the experimental strategy used to analyze the expression of Sox9, Sox10, Mitf and Tyr genes in the melanocytic lineage in vivo . B , Results of RNA-seq analysis demonstrating high Sox10 and low Sox9 expression in melanocytic cells at various stages of development. C , A schematic representation of the anatomical location of the melanocyte stem cells, melanoblasts and differentiated melanocytes within the hair follicle in the mouse skin. D , X-Gal staining (blue) combined with Sox9 immunostaining (red) in skin sections of Dct::LacZ mice demonstrating the lack of the Sox9 expression in the melanocyte stem cells located in the bulge region of the hair follicle (upper panels) and in the differentiated melanocytes located in the hair follicular bulb (lower panels). E , Skin sections of Dct::LacZ mice stained for Sox10 (red) in combination with X-Gal staining (blue) reveal the expression of Sox10 in the melanocyte stem cells (upper panels) as well as in the differentiated melanocytes (lower panels). Dashed lines demarcate HFs. Insets show high magnification views. F-I , Experimental strategy used to analyze the effect of the lack of Sox9 ( F ) and Sox10 ( H ) expression in the mouse melanocytic lineage. Pictures of two representative mice at 6 months of age lacking Sox9 gene ( G ) and Sox10 gene ( I ) demonstrating the effects on hair graying. Bg, bulge; HF, hair follicle; MSCs, melanocyte stem cells; Mo, months; E 15.5, embryonic day 15.5; P0, postnatal day 0; SG, sebaceous gland. Scale bars, 25 μm.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Expressing, In Vivo, RNA Sequencing Assay, Staining, Immunostaining

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A-D , Immunostaining for Sox9 ( A, C ) and Sox10 ( B, D ) in the skin sections of Tyr::Nras Q61K and Tyr::Nras Q61K INK4a −/− mice. E-H , Experimental strategy used to abrogate the expression of Sox9 (E) and Sox10 (G) in the mouse melanocytic lineage. Pictures of two representative mice 1 year after tamoxifen injections reveal no reduction in the skin hyperpigmentation in Tyr::Nras Q61K Sox9 fl/+ Tyr-CreERT2 mice as compared to their Tyr::Nras Q61K littermates (F) in contrast to a pronounced skin whitening observed upon Sox10 loss (H). BF, bright field; HF, hair follicle; mo, months; P0, postnatal day 0. Scale bars, 25 μm.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Immunostaining, Expressing

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , Experimental design used to investigate the level of SOX9 and SOX10 expression in vitro . Cultured human keratinocytes, melanocytes, cells derived from biopsies of patients with giant congential naevi and melanoma cells (M010817 cell line) were subjected to RNA isolation and subsequent Q-RT-PCR analysis. Keratinocytes were used as a control. B, C , Quantitative real-time PCR analysis showing the decline of SOX9 expression ( C ) and increase of SOX10 expression ( B ) that correlate with the acquisition of malignant state by human NRAS Q61K -mutated cells. Data are presented as the mean fold change and are normalized over levels found in melanocytes. D, E , SOX10 and SOX9 expression in a large set of proliferative and invasive cell lines analysed by gene expression using microarrays ( D ) and Western blot ( E ) techniques. F , Experimental design used to deregulate SOX10 expression in human cells derived from the biopsy of a patient with NRAS Q61K -mutated giant congenital naevus. G, H , Quantitative real-time PCR analysis of SOX10 ( G ) and SOX9 ( H ) expression after the knockdown of SOX10. I , Experimental design used to analyze the expression of Sox9 in the melanocytic lineage from Tyr::Nras Q61K and Tyr::Nras Q61K Sox10 LacZ/+ mice. K, L , Cells were isolated from the trunk skin of Tyr::Nras Q61K and Tyr::Nras Q61K Sox10 LacZ/+ mice and stained for Melan-a and c-Kit antibodies. FACS-sorted cells were subsequently used for the RNA isolation and quantitative real-time PCR with primers specific for the coding regions of Sox9 gene. Data are presented as the mean fold change and are normalized to the control. Kerat, keratinocytes; M, melanocytes; Nev, naevus cells; Mel, melanoma cells; KD, knock down.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Expressing, In Vitro, Cell Culture, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Staining

Journal: PLoS Genetics

Article Title: Antagonistic Cross-Regulation between Sox9 and Sox10 Controls an Anti-tumorigenic Program in Melanoma

doi: 10.1371/journal.pgen.1004877

Figure Lengend Snippet: A , SOX9 overexpression in human melanoma cells closely resembles the gene expression signature of SOX10 knockdown as revealed by unsupervised hierarchical clustering of control M010817 melanoma cells, SOX9 overexpressing M010817 cells and SOX10 knock down M010817 cells. Microarray gene expression accession number: GSE37059. B , Western blot analysis showing that SOX10 expression is downregulated upon overexpression of SOX9 in two independent human melanoma cell lines (A375 and M010817). C , Chromatin immunoprecipitation assay demonstrating the binding of SOX9 to the promoter of SOX10 in human melanoma M010817 cells. D, E , Quantitative real-time PCR analysis of SOX10 ( E ) and SOX9 ( F ) expression after the knockdown of SOX10 and after the double knockdown of SOX10 and SOX9 in M010817 cell line. F , Quantification of number of Annexin V-positive cells based on the FACS analysis in the melanoma M010817 cells upon SOX9 KD, SOX10 KD or double SOX9/SOX10 KD. OE, overexpression; KD, knock down; ChIP, chromatin immunoprecipitation; prom, promoter.

Article Snippet: The following primary antibodies were used: anti-Sox10 (goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox10 (mouse, 1:200, R&D), anti-Sox9 (rabbit, 1:100, sc-20095, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Sox9 (rabbit, 1:100, ab36748, Abcam), anti-Sox9 (M00006662, Abnova), anti-Sox9 (AB5535, Millipore), anti-Sox9 (GTX 109661, GenTex), anti-MITF (mouse, clone 6D3, 1:500) was a kind gift from Heinz Arnheiter (NIH, USA).

Techniques: Over Expression, Expressing, Microarray, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–E) ATs from naïve C57BL/6 mice were analyzed by flow cytometry. (A) Representative plots of transcription factor expression or cytokine production by CD8+ or CD4+ T cells from the mAT after PMA/ionomycin restimulation with BFA. (B) Numbers of CD8+ T cells and non-Treg CD4+ T cells from the mAT producing the indicated cytokine. (C) Frequencies of IFN-γ- and TNF-α-producing CD8+ and CD4+ T cells after PMA/ionomycin restimulation with BFA isolated from gAT, scAT, and mAT. (D) CD8+ (top) and CD4+ (bottom) T cells isolated from the mAT. Left: representative plots of CD44 and CD62L expression, Right: representative histograms of CD69 expression on CD44+CD62L− (red) or CD44−CD62L+ (blue) T cells. (E) Numbers of central memory (Tcm: CD44+CD62L+), effector memory (Tem: CD44+CD62L−CD69−) and resident memory (Trm: CD44+CD62L−CD69+) CD8+ and CD4+ T cells per gram of gAT, scAT, and mAT. (F) Conjoined pairs of naïve CD45.1 and CD45.2 congenic C57BL/6 mice were analyzed 6 weeks after parabiosis surgery to quantify the origin of CD8+ (left) and CD4+ (right) memory T cell subsets (described in (E)) in the spleen (Spl), mLN, siLP, and mAT. The percentage of cells originating from host (black bars) or donor (white bars) animals is shown. nd not detected. (G–K) mAT isolated from healthy rhesus macaques was analyzed by flow cytometry. (G) Representative plot (gated on CD3+CD8+ T cells) indicating the gating strategy for naïve (CD95−CD28lo), central memory or stem cell memory (Tcm/scm) (CD95+CD28+), and Tem/rm (CD95+CD28−) T cells. (H) Representative plots showing CD8+ (left) and CD4+ (right) naïve, Tcm/scm, and Tem/rm T cells. (I) Representative histograms of CD69 expression on CD8+ (left) and CD4+ (right) naïve (shaded) and Tem/rm T cells (blue). (J) Frequencies of CD8+ (left) and CD4+ (right) memory T cell subsets from (HI). (K) Representative plots (gated on CD3+ T cells) from concatenation of all samples, showing cytokine production by T cell subsets. Numbers in representative plots indicate mean±SD. In all bar graphs, error bars represent standard deviation. Data are representative of at least 2 experiments with at least 4 mice, 3 pairs of parabiotic animals, or a total of 3 rhesus macaques. See Figure S1.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Flow Cytometry, Expressing, Isolation, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–D) C57BL/6 mice were orally infected with T. gondii. 6 weeks post-infection, T cell populations were evaluated by flow cytometry. (A) Number of IFN-γ+ CD8+ (left) and IFN-γ+ CD4+ (right) T cells from the mAT post PMA/ionomycin activation with BFA. (Student’s t test) (B) Representative plots show T. gondii-specific CD8+ (left) and CD4+ (right) T cells. (C) Representative plots of IFN-γ production by CD44+tgd057:Kb+ CD8+ T cells (left) and TGME49:I-Ab+ CD4+ T cells (right) after PMA/ionomycin stimulation with BFA in the indicated organ. (D) The mean fluorescence intensity (MFI) of IFN-γ+ tgd057:Kb+ CD8+ T cells (left) and IFN-γ+ TGME49:I-Ab+ CD4+ T cells (right) from (C). (E–G) C57Bl/6 mice were orally infected with Yptb WT. At the indicated time points post-infection, T cells were analyzed by flow cytometry. (E) Representative plots of YopE:Kb+ CD8+ T cells in the indicated organs at 15 days post-infection. (F) Time course of the numbers of CD44+YopE:Kb+CD8+ T cells in the mLN, siLP, and mAT. (G) Frequencies of Tcm, Tem, and Trm cells within YopE:Kb+CD8+ T cells in the mAT 31 days post-infection. (H–J) C57BL/6 mice were orally infected with Yptb WT or Yptb ΔyopM. At the indicated time points post-infection, T cell populations were analyzed by flow cytometry. (H) Representative plots of YopE:Kb+CD8+ T cells from the mLN or mAT 31 days post-infection. (I) Numbers of CD44+YopE:Kb+CD8+ T cells from the mAT 31 days post-infection. (J) Representative plots of YopE:Kb+ CD8+ T cells from the mAT and gAT >4 weeks post-infection with Yptb ΔyopM. (K) Representative plots of tgd057:Kb+ CD8+ T cells (left) and TGME49:I-Ab+ CD4+ T cells (right) from the gAT and mAT of mice >6 weeks post oral T. gondii infection. (L) Pairs consisting of one naïve and one previously infected (>4 weeks post Yptb ΔyopM infection) congenic C57BL/6 mouse were conjoined and analyzed 6 weeks after parabiosis surgery. The number of YopE:Kb+ CD8+ T cells isolated from the indicated organs of the naive (white bars) or previously infected (black bars) mouse in the pair was calculated. (One way Anova adjusted for multiple comparisons). Numbers in representative plots indicate mean±SD. Error bars in all bar graphs represent standard deviation. Data are representative of at least 2 experiments with at least 3 mice per group or 3 pairs of parabiotic animals. ns not significant, *p<0.05, ****p<0.0001. See Figure S2.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Flow Cytometry, Activation Assay, Fluorescence, Isolation, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–B) Mice were orally infected with Yptb ΔyopM. >4 weeks post-infection, Tem and Trm memory CD8+ T cells were sorted for gene expression analysis by RNA-Seq. (A) Numbers of genes with a ≥2 fold increase (red) or decrease (blue) in expression levels between the indicated populations. (B) Pathway analysis was performed and gene pathways were organized into clusters, represented here as clustergrams showing gene pathways differentially regulated between Trm cells in the siLP and mAT. (C–G) Mice infected with Yptb ΔyopM were analyzed at >4 weeks post-infection. (C) Ki67 expression by Tem and Trm YopE:Kb+CD8+ T cells was determined by flow cytometric analysis. Left hand plots show YopE:Kb+ expression on CD8+ T cells. Center plots show CD69 expression on YopE:Kb+CD8+ cells. Right hand plots show Ki67 expression on Tem and Trm cells. Bar graphs show frequencies of Ki67+YopE:Kb+CD8+ Tem (left) and Trm (right) cells. (D) Representative Vybrant DyeCycle Violet staining of YopE:Kb+CD44+CD8+ T cells from the siLP (blue), spleen (green), and mAT (red). Bar graphs show the percentage of YopE:Kb+CD44+CD8+ Tem and Trm cells in the indicated cell cycle stages in the siLP (left), spleen (center), and mAT (right). (E) >4 weeks post-infection with the Yptb ΔyopM, mice received BrdU for 12 days. The percentage of BrdU+YopE:Kb+CD44+ CD8+ T cells found in the intraepithelial lymphocytes (IEL), lungs, salivary gland (SG), siLP, liver, mAT, and gAT is quantified in the bar graph. (F) >4 weeks post-infection, cells from the siLP, spleen, and mAT were incubated with Bodipy FL C16. Representative histogram indicates Bodipy uptake in YopE:Kb+CD8+ Trm cells (red) or YopE:Kb+CD8+ Tem cells (blue) from the siLP (open) and mAT (shaded). Bar graphs show the quantification of the bodipy MFI. (Student’s t test). (G) >4 weeks post-infection, cells from the siLP, spleen, and mAT were incubated with Mitotracker Deep Red. Representative histograms indicate Mitotracker Deep Red staining in YopE:Kb+CD8+ Trm cells (red) or YopE:Kb+CD8+ Tem cells (blue) from the siLP (open histogram) and mAT (shaded histograms). Bar graphs show the quantification of the Mitotracker Deep Red MFI. Numbers in representative plots indicate mean±SD. Error bars in all graphs represent mean±SD. Data are representative of at least 2 experiments with ≥3 mice per group. One way Anova adjusted for multiple comparisons. **p<0.01, ****p<0.0001 See Figure S3.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Gene Expression, RNA Sequencing, Expressing, Staining, Incubation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–D) Mice were either naïve, 6 days post-infection with Yptb (Yptb WT 6 days), >4 weeks post-infection with Yptb ΔyopM (Yptb ΔyopM), or >4 weeks post-infection with Yptb ΔyopM followed by challenge with Yptb for 6 days (Yptb ΔyopM + Yptb WT) before imaging or isolation of cells for flow cytometry. (A) Representative images of the entire mAT from actin-DsRed reporter mice are shown. White arrows indicate FALCs. (B) Number of FALCs per mAT represented as fold change over the number of FALCs per naïve mAT. (C) mATs from CD11c–YFP reporter mice were stained for CD8 and LYVE-1 and imaged by confocal microscopy. CD8, CD11c, and LYVE-1 staining (top) or CD8+ T cells alone (bottom) in areas of the mAT with (left) or without (right) FALCs are shown. (D) Numbers of CD44+YopE:Kb+CD8+ T cells from mAT. (E) Pooled mAT, scAT and gAT isolated from either naïve donors or donors >4 weeks post-infection with Yptb ΔyopM were subcutaneously transplanted into Rag1−/− mice. Rag1−/− mice receiving ATs from previously infected mice were either left untreated or injected with anti-CD4 and anti-CD8 depleting antibodies. 2 weeks post surgery, animals were challenged i.v. with 200 CFU of Yptb WT. Data are representative of at least 2 experiments with ≥5 mice per group. Error bars in all bar graphs represent standard deviation. Statistical comparisons in (B) and (D) were performed using one way Anova adjusted for multiple comparisons. Statistical comparisons in (E) were performed using Log-rank (Mantel Cox) test. ns not significant, *p<0.05, **p<0.01, ****p<0.0001. See Figure S4.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Imaging, Isolation, Flow Cytometry, Staining, Confocal Microscopy, Injection, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–B) >4 weeks post-infection with Yptb ΔyopM, mice were injected i.v. with YopE69–77 peptide or vehicle control (ctrl.) and evaluated by flow cytometry 1 hour post-injection. (A) Representative histogram shows expression of CD69 on YopE:Kb+CD8+ T cells after vehicle control (dotted line) or peptide (gray shading) injection (left) and MFI of CD69 in the indicated organs after the indicated treatment is quantified (right). (B) Representative plots show IFN-γ and TNF-α production by YopE:Kb+CD8+ T cells 1 hour after vehicle control (left) or peptide (right) injection in the indicated organs. Frequencies of IFN-γ+ TNF-α+ YopE:Kb+ CD8+ T cells in the indicated organs are shown in the bar graph. (C) Mice were infected orally with a fluorescent reporter strain of T. gondii. 6 weeks post-infection, mice were injected i.v. with MHC-I and MHC-II peptides or vehicle control and analyzed by flow cytometry 1 hour post injection. Representative plots show IFN-γ and TNF-α in CD44+CD8+ T cells in the indicated organ. Bar graphs show the frequencies of IFN-γ+TNF-α+ cells within the CD44+ CD8+ T cells. (D–E) >4 weeks post-infection with Yptb ΔyopM, mice were injected i.v. with YopE69-77 peptide or an equal volume of vehicle control and evaluated by flow cytometry at 1 and 4 hours post-injection. (D) Representative plots show neutrophils in the indicated organs (gated on live CD45+TCRβ−Siglec F−NK1.1−B220− cells) 4 hours after injection. Bar graph shows frequency of neutrophils at 1 and 4 hours post-injection in the siLP and mAT. (E) Representative plots show monocytes (gated on live CD45+TCR-β−Siglec F−NK1.1−B220−CD11b+Ly-6G−CD64+CCR2+ cells) 4 hours after injection in the indicated organs. Bar graph shows the frequency of monocytes at 1 and 4 hours post-injection in the siLP and mAT. Numbers in representative plots indicate mean±SD. Error bars in all bar graphs represent standard deviation and statistics are calculated using one way Anova adjusted for multiple comparisons. Data are representative of at least 2 experiments with 2–6 mice per group. ns not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Figure S5.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Injection, Control, Flow Cytometry, Expressing, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: DATA AND SOFTWARE AVAILABILITY

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Software, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, SYBR Green Assay, Staining, Microarray