Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: The sequences of primers and siRNAs

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques:

Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: CD69 is indispensable to maintain iTregs functio n. ( A ) CD69 fl/fl or Foxp3 YFP-Cre CD69 fl/fl mice were administrated with 2% DSS in drinking water for 9 days (n = 7 for each group). The average body weight is shown as a percentage relative to the initial value. ( B ) The disease activity index is analyzed. The length of the colon ( C ) and histological appearance ( D ) after 9 days of colitis induction. ( E and F ) Naïve CD4 + T cells isolated from CD69 fl/fl or CD4 Cr e CD69 fl/fl mice were cultured for 3 days under Treg-polarization conditions and then analyzed by flow cytometry. Foxp3 YFP-Cre CD69 fl/fl mice after 2 days of DSS administration were intravenously injected with iTregs from CD69 fl/fl and CD4 Cr e CD69 fl/fl mice (n = 7 for each group). The mice's body weight ( G )and the DAI ( H ) were recorded every day. The average body weight is shown as a percentage relative to the initial value. ( I )The average length of the colons. ( J ) Hematoxylin and eosin staining of the colon sections. Scale bar:500 μm (whole colon section) and 100 μm (enlarged insets). ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the control or the indicated group.

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques: Activity Assay, Isolation, Cell Culture, Flow Cytometry, Injection, Staining

Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: HSF1 is important for CD69 transcription in CD4 + T cells. ( A ) Schematic of the nucleotide sequence of CD69 promoter, indicating the locations of the putative HSE sites. The potential HSF1 binding sites are shown in boxes. ( B ) HEK293 cells were infected for 48 h with luciferase reporter plasmids as shown on the left as well as a construct that encodes HSF1 or the empty vector, and then, cells were harvested to detect luciferase activity. ( C ) ChIP analysis of HSF1 binding to the CD69 promoter in CD4 + T cells with or without anti-CD3/CD28 mAb stimulation. ( D ) Overall 1×10 6 /mL CD4 + T cells were added to 8 μg polybrene and transfected with control lentivirus or lentivirus-HSF1 at an MOI of 100 for 48 h. CD69 protein levels were determined using flow cytometry. ( E ) CD4 + T cells isolated from HSF1 +/+ and HSF1 +/- mice were stimulated with different doses of anti-CD3/CD28 antibodies, and the mRNA levels of CD69 were detected by qRT-PCR. ( F ) 1 × 10 6 /mL CD4 + T cells were stimulated with or without anti-CD3/CD28 antibodies and treated with HSF1 siRNA for the indicated time. The relative levels of CD69 expression in different groups of CD4 + T cells were determined by qRT-PCR. ( G ) CD4 + T cells isolated from HSF1 +/+ and HSF1 +/- mice were stimulated with or without anti-CD3/CD28 antibodies following the treatment with KRIBB11 for 24 h, and then the expression of CD69 + CD4 + T cell was analyzed by FACS. Representative images of the data expressed as mean ± SD of three independent experiments (n = 7 per group). ns, not significant. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as analyzed by ANOVA or Student's t -test.

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques: Sequencing, Binding Assay, Infection, Luciferase, Construct, Plasmid Preparation, Activity Assay, Transfection, Flow Cytometry, Isolation, Quantitative RT-PCR, Expressing

Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: HSF1 is necessary for CD69 + Treg differentiation. Naive CD4 + T cells were cultured in Treg polarization conditions with or without KRIBB11. The frequency of iTreg ( A ) and CD69 + iTreg ( B ) was analyzed by FACS. Induced Tregs were sorted and injected i.v into IBD mice treated with KRIBB11 on day 2. The body weights ( C ) were measured and DAI ( D ) was analyzed daily. ( E ) H&E-stained images of colon sections. Scale bar: 100 μm. ( F-G ) The mice were treated with KRIBB11 and then administration with 2% DSS, body weight and DAI were analyzed daily. ( H ) H&E-stained images of colon sections. Scale bar: 100 μm. Representative images of the data expressed as mean ± SD of three independent experiments (n = 7 per group). ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as analyzed by ANOVA and Student's t -test.

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques: Cell Culture, Injection, Staining

Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: MG132 prompts the activation of HSF1 and the expression of CD69. 1 × 10 7 CD4 + T cells were isolated and incubated with 10 µM of MG132 at 37°C for 2 h and then cultured in T lymphocyte culture medium for 24 h. ( A ) All the cells were harvested and protein levels of HSF1 and CD69 were analyzed by western blot. ( B ) The relative levels of CD69 were analyzed by qRT-PCR. ( C ) CD4 + T cells were incubated with MG132 for 2 h and then washed with 1640 RPMI, followed by culture under Treg polarization for 24 h. Cells were then subjected to a ChIP assay using the indicated antibodies. The precipitated DNA was analyzed by quantitative PCR using primer pairs corresponding to the indicated genomic regions. ( D ) CD4 + naive T cells were sorted from HSF1 +/+ o r HSF1 +/- mice and pretreated with MG132 and induced iTregs while HSF1 inhibitor was added at the same time to block HSF1 expression. The flow cytometric analysis of iTregs and CD69 + iTregs expression. Representative images of the data expressed as mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant, as analyzed by ANOVA or Student's t -test.

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques: Activation Assay, Expressing, Isolation, Incubation, Cell Culture, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Blocking Assay

Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: Proteasome inhibitors showed a milder therapeutic effect on IBD. ( A ) Density plots showing CD69 expression in gated CD4 + Foxp3 + cells from freshly isolated spleen, MLN and colonic LPL in mice treated with MG132 and bortezomib. Acute colitis was induced in animals by administering 2% DSS in their drinking water for 9 days. Mice were treated with or without MG132 and bortezomib for 2 days at indicated doses. Changes in body weight ( B ), disease activity index ( C ), and colon length ( D ) and histological sections of inflamed colons ( E ) during the course of DSS treatment in each group of mice. Scale bar:100 μm. ( F ) Survival rates of each group of mice after the initiation of DSS-induced acute colitis were recorded daily (n=10 per group). Representative images of the data expressed as mean ± SD of three independent experiments. * p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0. 0001.ns, not significant, as analyzed by ANOVA or Student's t -test.

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques: Expressing, Isolation, Activity Assay

Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: Proteasome inhibitors prompt CD69 expression in vitro . Overall 1 × 10 6 /ml freshly isolated naive CD4 + T cells incubated with or without MG132 for 2 h at 37°C and washed three times with 1640 RPMI. The cells were then stimulated with 2 µg/ml anti-CD3/CD28 antibody, 10ng/ml TGF-β1, and 50 IU/ml of IL-2 for 72 h. ( A ) The levels of IL-10 and TGF-β1 in the supernatants of cultured cells were measured by ELISA. ( B ) Total RNA was extracted from isolated PSI-iTregs and Control-iTregs while the gene expression profile was analyzed using the microarray analysis. The heat map of the gene expression is also shown. ( C and F ) The expression levels of CTLA-4 and ICOS in PSI-iTregs/CD69 + iTregs and Control-iTregs/CD69 + iTregs were analyzed using flow cytometry with indicated antibodies. ( D and G ) 1 × 10 6 /ml of CD4 + CD25 - T cells were labeled with CFSE and co-cultured with PSI-iTregs/CD69 + iTregs and Control-iTregs/CD69 + iTregs at a ratio of 1:1, 1: 2, or 1: 4 in the presence of 1 µl anti-CD3/CD28 coated beads for 3 days. The proliferation of CD4 + T cells was analyzed by flow cytometry. The cells were first gated on living lymphocytes and then on CFSE + T cells (n = 3). ( E ) Naive CD4 + T cells incubated with or without MG132 or Bortezomib then cultured under Treg porlarization condition, the relative frequency of iTregs and CD69 + iTregs was analyzed using FACS (n = 3). Representative images of the data were expressed as mean ± SD of three independent experiments while Student's t -test was used for statistical analysis. * p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques: Expressing, In Vitro, Isolation, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Microarray, Flow Cytometry, Labeling

Journal: Theranostics

Article Title: HSF1 promotes CD69 + Treg differentiation to inhibit colitis progression

doi: 10.7150/thno.78078

Figure Lengend Snippet: Adoptive transfer of PSI-iTregs attenuated the severity of DSS-induced IBD in mice. IBD was induced by administering DSS in drinking water for 10 days. For the treatment of IBD, 1 × 10 6 PSI-iTregs or Control-iTregs were sorted and intravenously injected into mice on day 2. ( A ) The loss in body weight was recorded daily. Each point represents the average weight data pooled from eight mice ± SD. Control group: the mice fed with normal water; PBS group, the mice were drinking water containing 2% DSS and intravenously treated with PBS on day 2. ( B ) The DAI was evaluated daily. Each point represents the average DAI data pooled from eight mice. ( C ) Appearance and statistical analysis of colon length on day 9. ( D ) Representative H&E staining of the colon mice of different groups. ( E-F ) Weight loss and DAI in mice transferred with PSI-CD69 + iTregs and Control-CD69 + iTregs. ( G-H ) The length of colon and H&E staining of the colon from mice from the different groups. Representative images of the data expressed as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, ns, not significant. ANOVA or Student's t -test was used to determine the significance.

Article Snippet: Antibodies including APC-anti-CD4 (Invitrogen, Clone: RM4-5, Catalog: 17-0043-82) or PE-Cy7-anti-CD4 (Invitrogen, Clone: GK1.5, Catalog: 25-0041-82) and PE-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 12-0691-82) or PE-Cy5-anti-CD69 (Invitrogen, Clone: H1.2F3, Catalog: 15-0691-82), PE-anti-Foxp3 (Invitrogen, Clone: FJK-16s, Catalog: 12-5773-82), APC-anti-CD25 (Invitrogen, Clone: PC61.5, Catalog: 17-0251-82), PE-anti-CTLA-4 (Invitrogen, Clone: UC10-4B9, Catalog: 12-1522-82), PE-anti-ICOS (Invitrogen, Clone: C398.41, Catalog: 12-9949-81) or isotype controls(Invitrogen, USA) were used to analyze the phenotype of various Tregs.

Techniques: Adoptive Transfer Assay, Injection, Staining

Journal: Frontiers in Immunology

Article Title: Ex vivo modulation of intact tumor fragments with anti-PD-1 and anti-CTLA-4 influences the expansion and specificity of tumor-infiltrating lymphocytes

doi: 10.3389/fimmu.2023.1180997

Figure Lengend Snippet: Tumor-specific recognition and phenotype is largely unchanged between expansion conditions. Epitope-specific recognition of tumor-shared antigens numerated with fluorescent combinatorial encoding of pMHC tetramers. All epitopes were restricted to HLA-A*02:01. Protein expression of CD69 and CD39 in bulk CD8 T cells overlaid by data from each specific population as shown in (A) . Enumeration of tumor-specific ie tetramer double positive CD8 T cells (B) . All populations for each donor are plotted individually. (C) Enumeration of total tumor-specific CD8 T cells i.e. the sum of all individual tetramer double positive CD8 T cells per donor and expansion condition. (D) Stem-like and activated phenotypes for bulk CD8 T cells for each condition of interested described previously . (E) Terminally differentiated phenotypes among tumor-specific CD8 T cells. (F) Terminally differentiated phenotypes among total tumor-specific CD8 T cells. (G) Individual enumeration of each tumor-specific population of interest for each condition with each dot representing individual donors and their responses. ELA, ELAGIGILTV-MART-1. LLF, LLFGLALIEV-MAGEC2. YLE, YLEPGPVTA-gp100. AML, AMLGTHTMEV-gp100. SVY, SVYDFFVWL-TRP-2. ITD, ITDQVPFSV-gp100. KAS, KASEKIFYV-SSX-2. MLA, MLAVISCAV-HERV-K. P values were evaluated using unpaired kruskal-wallis test.

Article Snippet: The fluorochrome-labeled monoclonal antibodies (from BD Biosciences, unless indicated otherwise) BV786 CD3 (563800), BV711 and PE-AF700 CD4 (563033 and MHCD0424, Invitrogen), PerCP-Cy5.5 and QDot605 CD8 (565310 and Q10009, Invitrogen), APC-R700 CD27 (565116), PE-Cy7 CD28 (560684), BV421 CD39 (563679), SB702 CD56 (67-0566-42, Life Technologies), PE-CF594 CD57 (562488), PE-Cy5.5 CD69 (MHCD6918, Invitrogen), BV421 B- and T-lymphocyte attenuator (BTLA, 564802), BV510 CCR7 (353232, Biolegend), BV570 CD45RO (304226, Biolegend), FITC lymphocyte-activation gene 3 (LAG-3, LS-B2237, LS Bioscience), PE-Cy7 PD-1 (561272), BV650 T-cell immunoglobulin and mucin-domain containing-3 (TIM-3, 565564) were used for surface staining.

Techniques: Expressing

Journal: ImmunoHorizons

Article Title: Enhancing Mucosal-Associated Invariant T Cell Function and Expansion with Human Selective Serum

doi: 10.4049/immunohorizons.2200082

Figure Lengend Snippet: ( A ) Frequency of MAIT cells after 20 h with or without 10 MOIs of E. coli stimulation in Phx- or FBS-supplemented RPMI. ( B – F ) Comparison of frequency of CD69 + , CD25 + , LAG-3 + , PD-1 + , and TIM-3 + MAIT cells after 20 h with or without 10 MOIs of E. coli stimulation in Phx- or FBS-supplemented RPMI. ( G – I ) Gating strategy and frequency of TNF-α + and IFN-α + MAIT cells following 20 h with or without 10 MOIs of E. coli stimulation in Phx- or FBS-supplemented RPMI. ( J ) Frequency of granzyme B + MAIT cells after 20 h with or without 10 MOIs of E. coli stimulation in Phx- or FBS-supplemented RPMI. ( K ) Comparison of mean fluorescence intensity (MFI) of T-bet expression in MAIT cells after 20 h with or without 10 MOIs of E. coli stimulation in Phx- or FBS-supplemented RPMI. These data are reflective of two independent experiments with n = 6 each. * p < 0.05, ** p < 0.01.

Article Snippet: We stimulated PBMCs with 10 multiplicities of infection (MOIs) of strain 1100–2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. We performed extracellular staining with fixable viability dye eFluor 780 (eBioscience), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BV510 (BioLegend), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD25-BV650 (BioLegend), anti-PD-1-PerCPCy-5.5 (BioLegend), anti-CD161-PE/Dazzle-594 (BioLegend), anti-CD69-PE-Cy5 (Invitrogen), anti-CD161-allophycocyanin (BioLegend), and anti-human PE-MR1–5-OP-RU tetramer (NIH Tetramer Core Facility).

Techniques: Fluorescence, Expressing