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: 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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: KLHL22 is a major PD-1–associated protein. (A) PD-1–associated proteins in Jurkat cells were identified by MS. Jurkat cells stably expressing PD-1–FLAG were purified with FLAG-M2 beads and analyzed by MS. Jurkat cells was stimulated by PMA (50 ng/mL) and ionomycin (1 μM) for 12 h. The table lists the selected proteins identified by MS. The full protein list is provided in Dataset S1. (B) PD-1–associated proteins in HEK293T cells were identified by TAP/MS. HEK293T cells stably expressing PD-1–SFB were purified by TAP/MS. The table lists the selected proteins identified by MS. The full protein list is provided in Dataset S2. (C and D) Venn diagram showing the overlap of two MS results. Proteins appearing in the MS results of both the Jurkat PD-1–FLAG cells and HEK293T PD-1–SFB cells. The results from the overlap shown in C are presented in a 2D graph in D. The full protein list is provided in Dataset S3. (E) 6-Kelch repeats in KLHL22 are required for the interaction between KLHL22 and PD-1. HEK293T cells were cotransfected with untagged PD-1 (PD-1 without an artificial tag) and PD-L1-SFB, SFB-KLHL22, or SFB-KLHL22Δ6K. The cell lysates were subjected to pull-down assays with S protein Sepharose and immunoblotted with the indicated antibodies. (F) HEK293T cells were cotransfected with PD-1–SFB and Myc-KLHL22, Myc-KLHL22Δ6K, Myc-KLHL9, or Myc-KLHL13. The cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies. (G) Lysates of Jurkat cells stably expressing PD-1–FLAG were immunoprecipitated with FLAG-M2 beads or protein G beads with IgG and subjected to immunoblotting with the indicated antibodies. Jurkat cells were stimulated by PMA (50 ng/mL) and ionomycin (1 μM) for 12 h. (H) The endogenous interaction of PD-1 and KLHL22 in healthy human PBMCs using KLHL22 antibody pull-down. Human healthy PBMCs lysates were immunoprecipitated with an anti-KLHL22 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) for 12 h, 24 h, or 36 h. (I) Endogenous PD-1 associates with endogenous KLHL22 in healthy human PBMCs. Human healthy PBMCs lysates were immunoprecipitated with an anti–PD-1 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (1 μg/mL) or anti-CD3 (1 μg/mL)/anti-CD28 (2 μg/mL) for 24 h. The third group was also treated with PD-1 antibody (2 μg/mL) for 24 h. (J) Endogenous KLHL22 associates with endogenous PD-1 in healthy human PBMCs. CD28 and CTLA4 were tested simultaneously and showed negative results. Human healthy PBMCs lysates were immunoprecipitated with an anti-KLHL22 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (0.5 μg/mL or 1 μg/mL) and anti-CD28 (2 μg/mL) for 24 h.

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Stable Transfection, Expressing, Purification, Immunoprecipitation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: Loss of KLHL22 leads to up-regulation of PD-1 at the protein level. (A) Depletion of KLHL22 increases PD-1 expression on the surface of Jurkat cells. Jurkat cells infected with lentivirus containing control or KLHL22-specific shRNA were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) for 24 h and subjected to flow cytometry to measure PD-1 expression on the cell surface. n = 3 biological independent samples, **P < 0.01, ***P < 0.001, unpaired Student’s t test. (B) Knockdown of KLHL22 increases PD-1 expression in HEK293T cells stably expressing PD-1–SFB. HEK293T cells stably expressing PD-1–SFB were infected with lentivirus containing control or KLHL22-specific shRNA and subjected to immunoblotting to detect PD-1 expression. (C and D) Cell-surface expression of PD-1 is higher in activated CD8+ (C) and CD4+ (D) T cells from Klhl22 KO mice than in those from WT mice. Naïve T cells from WT and Klhl22 KO mice were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) for 24 h. n = 4 mice per group. **P < 0.01, unpaired Student’s t test. (E) Transcription levels of Pdcd1 in CD3+ T cells from WT and Klhl22 KO mice showed no differences. CD3+ T cells were isolated from lymph nodes, and qRT-PCR analysis was used to measure the mRNA level of Pdcd1. n = 3 mice per group; ns, not significant, unpaired Student’s t test. (F) Cell-surface expression levels of LAG-3 in activated CD8+ T cells from WT and Klhl22 KO mice showed no differences. Naïve T cells from WT and Klhl22 KO mice were stimulated with anti-CD3 and anti-CD28 antibodies for 24 h. n = 3 mice per group; ns, not significant, unpaired Student’s t test.

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Expressing, Infection, Control, shRNA, Flow Cytometry, Knockdown, Stable Transfection, Western Blot, Isolation, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: KLHL22 mediates the degradation of PD-1 before it is transported to the cell surface. (A) Cell-surface levels of PD-1 of CD8+ T cell in activated healthy human PBMCs with or without MG132 treatment were detected by flow cytometry. PBMCs were stimulated with anti-CD3 (1 μg/mL), anti-CD28 (2 μg/mL) and treated with MG132 (1 μM) for indicated hours. n = 3 repeats, *P < 0.05, ***P < 0.001, unpaired Student’s t test. (B) KLHL22 does not localize to the cell membrane in healthy human PBMCs with or without stimulation. PBMCs were incubated in the presence or absence of anti-CD3 (1 μg/mL), anti-CD28 (2 μg/mL), and subjected to immunostaining with KLHL22 antibodies. (Scale bar, 2 μm.) On the right, no significant difference in cell membrane localization of KLHL22 before and after activation. Immunofluorescence staining for KLHL22 was performed in PBMCs. MATLAB was used to identify KLHL22 near membrane location in multiple pictures and quantify the percentage of KLHL22 localization near the cell membrane. ns, not significant, unpaired Student’s t test. (C) Simultaneous treatment with MLN4924 and BFA increased the levels of incompletely glycosylated PD-1. HEK293T cells stably expressing untagged PD-1 were treated with BFA (1 µM), MLN4924 (1 µM), and MG132 (1 µM) as indicated for 12 h. The three arrows on the right side of the figure indicate fully glycosylated PD-1 (top), incompletely glycosylated PD-1 (middle), and newly synthesized PD-1 (bottom). (D) KLHL22 down-regulation leads to more accumulation of incompletely glycosylated PD-1 than fully glycosylated PD-1. The expression level of PD-1 in HEK293T cells subjected to BFA treatment and/or KLHL22 shRNA lentivirus infection was detected. HEK293T cells stably expressing PD-1–SFB were infected with lentivirus containing control or KLHL22-specific shRNA and incubated in the presence or absence of BFA (1 µM, 12 h). Cells were then subjected to immunoblotting with the indicated antibodies. (E) The amount of cytoplasmic PD-1 is higher in CD3+ T cells from Klhl22 KO mice cells than in those from WT mice. The Golgi apparatus was extracted from CD3+ T cells from WT and Klhl22 KO mice and subjected to immunoblotting to detect the protein level of PD-1 in the Golgi apparatus. Na+/K+ ATPase served as a cell membrane marker, whereas GM130 served as a Golgi apparatus marker. (F) KLHL22 has a higher affinity for incompletely glycosylated PD-1 than for fully glycosylated PD-1. HEK293T cells stably expressing untagged PD-1 were transfected with the indicated plasmids in the presence or absence of BFA (1 µM, 12 h), and cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies. (G) PD-1 colocalizes with KLHL22 in the cytoplasm in Jurkat cells. Colocalization of PD-1 and endogenous KLHL22 in Jurkat cells stably expressing PD-1–FLAG was confirmed by immunostaining with anti-KLHL22 and anti-FLAG antibodies. Jurkat cells were stimulated with PMA (50 ng/mL 12 h)/ionomycin (1 µM 12 h) and treated with BFA (1 µM, 6 h). Pearson’s r = 0.76. (Scale bar, 2 μm.) (H) PLA was used to detect the colocalization of PD-1 and endogenous KLHL22 in Jurkat PD-1–FLAG stable cell lines in the presence or absence of BFA (1 µM, 6 h). Using of only one antibody (anti-KLHL22 or anti-FLAG) served as the negative control groups. (Scale bar, 2 μm.)

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Flow Cytometry, Membrane, Incubation, Immunostaining, Activation Assay, Immunofluorescence, Staining, Stable Transfection, Expressing, Synthesized, shRNA, Infection, Control, Western Blot, Marker, Transfection, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: KLHL22 mediates polyubiquitination-directed degradation of incompletely glycosylated PD-1. (A) PD-1 ubiquitination is inhibited by KLHL22 depletion. Control or KLHL22-specific siRNA was transfected into HEK293T cells stably expressing PD-1–SFB in the presence of MG132 (1 µM 24 h). Cell lysates were subjected to pull-down assays by S-protein Sepharose and immunoblotted with the indicated antibodies. (B) PD-1 ubiquitination is inhibited upon MLN4924 treatment. HEK293T cells stably expressing PD-1–SFB were treated with BFA (1 µM), MLN4924 (1 µM), and MG132 (1 µM) as indicated for 12 h, and cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with anti-FLAG and antiubiquitin antibodies. (C) Incompletely glycosylated PD-1 is unstable in vivo. PD-1–SFB stable cells were incubated in medium containing 10 μg/mL cycloheximide (CHX) in the presence or absence of BFA (1 µM) for the indicated time. Western blotting was carried out using the indicated antibodies. (D) The KLHL22/CUL3/RBX1 complex ubiquitinates PD-1 in vivo. CUL3, RBX1, and either KLHL22 or KLHL22Δ6K were overexpressed in HEK293T cells stably expressing PD-1–SFB, and cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies. (E) Only 48K ubiquitin can be conjugated to PD-1. For ubiquitination mutants transfected into HEK293T cells stably expressing PD-1–SFB, all lysine’s were mutated to arginine except Lys48 (48K) or Lys63 (63K). Cells were treated with MG132 (1 µM, 24 h). Ubiquitination of PD-1 was detected by immunoblotting with antiubiquitin antibody. Cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with antiubiquitin and anti-FLAG antibodies. (F) The ubiquitination of PD-1 on K210R and K233R is significantly reduced. PD-1–SFB (WT), PD-1–SFB (K210R), or PD-1–SFB (K233R) was transfected into HEK293T cells treated with BFA (1 µM), MLN4924 (1 µM) and MG132 (1 µM) as indicated for 12 h. The resulting cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies.

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Ubiquitin Proteomics, Control, Transfection, Stable Transfection, Expressing, In Vivo, Incubation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: KLHL22 regulates T cell antitumor immunity. (A–C) Klhl22 KO mice showed faster tumor progression. WT and Klhl22 KO mice were subcutaneously injected with B16F10 melanoma cells. Tumor growth was monitored over a period of 17 d. The tumors were isolated from mice killed on day 17 and measured. n = 7 mice per group, ***P < 0.001, (B) two-way ANOVA, (C) unpaired Student’s t test. (D) Klhl22 KO mice inoculated with tumors have a shorter survival time. WT mice and Klhl22 KO mice were subcutaneously injected with B16F10 melanoma cells, and the survival was assessed in WT and Klhl22 KO mice. n = 7 mice per group, *P < 0.05, log-rank (Mantel–Cox) test. (E and F) The CD8+/CD4+ ratio (E) and regulatory T cell ratio (F) of tumor-infiltrating T cells were not significantly different in tumor tissues from WT mice and those from Klhl22 KO mice. Tumor-infiltrating T cells from WT and Klhl22 KO mice were isolated on day 17 and subjected to flow cytometry to measure the CD4/CD8 ratio and T cell ratio. n = 7 mice per group; ns, not significant, unpaired Student’s t test. (G) PD-1 expression in tumor-infiltrating T cells from Klhl22 KO mice is significantly higher than that in cells from WT mice. Tumor-infiltrating T cells from WT and Klhl22 KO mice were isolated on day 17 and subjected to flow cytometry. n = 7 mice per group, *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t test. (H–J) Cytokine production by tumor-infiltrating CD8+ and CD4+ T cells from Klhl22 KO mice is inhibited. Tumor-infiltrating T cells from WT and Klhl22 KO mice were isolated on day 17 and subjected to flow cytometry. n = 7 or 6 mice per group, *P < 0.05, **P < 0.01, unpaired Student’s t test. (K) The proliferation of tumor-infiltrating CD8+ T cells was decreased in Klhl22 KO mice. Tumor-infiltrating T cells from WT and Klhl22 KO mice were isolated on day 17 and subjected to flow cytometry. n = 6 mice per group, *P < 0.05, **P < 0.01, unpaired Student’s t test. (L and M) PD-1 antibody treatment dramatically diminished the difference in tumor growth between WT and KO mice where there was no statistically significant difference. WT and Klhl22 mice were subcutaneously injected with B16F10 melanoma cells. Tumor growth was monitored over a period of 18 d. Mice were intraperitoneally injected with PBS or anti–PD-1 (RMP1-14, 200 μg per mouse, dissolved in PBS) every 3 d (three times in total) 8 d after B16F10 inoculation and tumor sizes were recorded every 2 d afterward (n = 6). ns, not significant, ***P < 0.001, two-way ANOVA.

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Injection, Isolation, Flow Cytometry, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: T cell activation and the tumor microenvironment regulate KLHL22 expression. (A) The transcription levels of Klhl22 in mouse CD3+ T cells increase dramatically upon T cell activation. CD3+ T cells were isolated from the lymph nodes of C57BL6 WT mice and stimulated with anti-CD3 (2 µg/mL) and/or anti-CD28 (4 µg/mL); the cells were then subjected to total RNA extraction and qRT-PCR analysis. n = 3 independent biological samples per group, **P < 0.01, unpaired Student’s t test. (B) The transcription levels of KLHL22 increase over time in activated Jurkat cells. Jurkat cells were stimulated with PMA (50 µg/mL) and ionomycin (1 µM) for the indicated time and then subjected to total RNA extraction and qRT-PCR analysis. n = 3 independent biological samples per group; ns, not significant, *P < 0.05, unpaired Student’s t test. (C) The level of KLHL22 protein was markedly decreased in tumor-infiltrating T cells of CRC patients. Immunohistochemical staining of KLHL22 and CD3e was performed using a colorectal tissue microarray of CRC patients. CD3e staining was used to mark CD3+ T cells, and KLHL22 staining was used to measure KLHL22 expression in CD3+ T cells. Selected samples from the tissue microarrays show KLHL22 expression in CD3+ T cells. Normal colon tissue (Left), atypical hyperplasia (Center), and colon cancer (Right). The number of atypical hyperplasia samples was too low to be included in the subsequent analyses. The 2× magnified image is displayed in the lower right corner. (Scale bar, 20 μm.) (D) Semiquantitative analysis of KLHL22 levels in CRC patients’ CD3+ T cells. Correlation analyses of KLHL22 expression between tumor-infiltrating CD3+ T cells and CD3+ T cells that infiltrated normal adjacent tissues. (E) Bubble chart showing the individual quantities of samples at different levels. Samples with low KLHL22 expression in tumor-infiltrating T cells are in the upper left quadrant of the graph, represented by red bubbles. Samples with high KLHL22 expression in tumor-infiltrating T cells are in the lower right quadrant of the graph, represented by blue bubbles. Larger bubbles indicate a larger number of samples; smaller bubbles are paler in color. Wilcoxon matched-pairs signed rank test: P < 0.0001.

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Activation Assay, Expressing, Isolation, RNA Extraction, Quantitative RT-PCR, Immunohistochemical staining, Staining, Microarray

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: 5-FU increases the expression of PD-1 by decreasing KLHL22 mRNA levels. (A) Treatment with 5-FU rather than other chemotherapeutic drugs increases PD-1 protein levels. Jurkat cells stably expressing PD-1–FLAG were stimulated with PMA (50 ng/mL 24 h) and ionomycin (1 µM 24 h) in the presence of 5-FU (100 µM 24 h), Etoposide (10 µM, 6 h), MMC (100 nM, 24 h), or CPT-11(10 µM, 24 h), as indicated. Cell-surface levels of PD-1 were measured by flow cytometry. ns, not significant, *P < 0.05, unpaired Student’s t test. (B) 5-FU treatment represses KLHL22 transcription. Jurkat cells were stimulated with PMA (50 ng/mL 24 h) and ionomycin (1 µM 24 h) in the presence of 5-FU (100 µM, 24 h), Etoposide (10 µM, 6 h), Olaparib (10 µM, 24 h), or irradiation (1 Gr) as indicated, and the KLHL22 transcription level was measured by qRT-PCR. ns, not significant, ***P < 0.001, unpaired Student’s t test. (C) 5-FU treatment represses only Klhl22 mRNA levels. CD3+ T cells were freshly isolated from the lymph nodes of WT mice and stimulated with anti-CD3 (1 μg/mL, 24 h) and anti-CD28 (2 μg/mL, 24 h) in the presence or absence of 5-FU (100 µM, 24 h). Klhl22 and Pdcd1 transcription levels were measured by qRT-PCR. ns, not significant, *P < 0.05, unpaired Student’s t test. (D) Deletion of KLHL22 abolishes the effect of 5-FU on PD-1 expression. CD3+ T cells were freshly isolated from the lymph nodes of WT or Klhl22 KO mice and stimulated with anti-CD3 (1 μg/mL, 24 h) and anti-CD28 (2 μg/mL, 24 h) in the presence or absence of 5-FU (100 µM, 24 h). Flow cytometry was used to measure PD-1 levels on the surface of CD8+ or CD4+ T cells. ns, not significant, **P < 0.01, unpaired Student’s t test. (E) 5-FU represses KLHL22 expression in a dose-dependent manner. Jurkat cells were stimulated with PMA (50 ng/mL 24 h) and ionomycin (1 µM 24 h) and treated with a concentration gradient of 5-FU. KLHL22 protein levels were measured by Western blotting. (F) 5-FU increases PD-1 expression in a dose-dependent manner. Jurkat cells were stimulated with PMA (50 ng/mL 24 h) and ionomycin (1 µM 24 h) and treated with a concentration gradient of 5-FU. Cell-surface expression of PD-1 was measured by flow cytometry, *P < 0.05, ***P < 0.001, unpaired Student’s t test. (G) PD-1 is expressed at high levels and KLHL22 is expressed at low levels in patients who received chemotherapy containing 5-FU. Patients were divided into two groups based on whether they had received 5-FU (chemotherapy and no chemotherapy). Tumor-infiltrating lymphocytes were isolated from fresh tumor samples and subjected to flow cytometry analysis to measure cell-surface expression of PD-1 and intracellular expression of KLHL22 in CD3+ T cells. Each point in the figure represents the KLHL22 and PD-1 expression levels of a single patient. Data points corresponding to the chemotherapy group are concentrated in the left region of the figure (red circle), indicating the grouping of patients with low KLHL22 expression and high PD-1 expression, while the no chemotherapy group is concentrated in the lower region of the figure (blue circle), indicating the grouping of patients with high KLHL22 expression and relatively low PD-1 expression. n = 5 patients received chemotherapy and n = 7 patients has not received chemotherapy. Hotelling’s T2 test: P < 0.05. (H) After 5-FU treatment, CD3+ T cells showed reduced KLHL22 expression and increased PD-1 expression compared to that of untreated cells from the same individual patient. PBMCs were isolated from the CRC patients’ blood and divided into two equal volumes. Both were treated with PMA (50 ng/mL 12 h) and ionomycin (1 µM 12 h) in the presence or absence of 5-FU (5-FU and Ctrl, respectively). Flow cytometry analysis was used to measure cell-surface expression on PD-1 and intracellular expression of KLHL22. n = 7 patients, *P < 0.05, paired Student’s t test.

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Expressing, Stable Transfection, Flow Cytometry, Irradiation, Quantitative RT-PCR, Isolation, Concentration Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

doi: 10.1073/pnas.2004570117

Figure Lengend Snippet: Working model. The biogenesis of PD-1 contains multiple steps of transportation and modification along the ER–Golgi–plasma membrane trafficking axis. KLHL22 is a major interacting protein of PD-1 and recognizes incompletely glycosylated PD-1, subsequently ubiquitinating and degrading PD-1 before it is transported to the cell surface. T cell activation upon TCR stimulation simultaneously promotes PD-1 and KLHL22 expression. KLHL22 degrades incompletely glycosylated PD-1 and maintains PD-1 homeostasis, preventing excessive suppression of T cells. KLHL22 deficiency, as well as deregulation of KLHL22 in response to the tumor microenvironment or 5-FU treatment, leads to excessive accumulation of PD-1 on the T cell surface and the repression of the antitumor immunity activity of T cells.

Article Snippet: The Klhl22 KO mouse was created using a CRISPR/Cas9-mediated genome-editing system from Cyagen Biosciences; exon 4 was selected as the target site.

Techniques: Modification, Clinical Proteomics, Membrane, Activation Assay, Expressing, Activity Assay