Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: CyTOF antibody panel

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques:

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Selected genes involved in Tfh cell biology

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Activation Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Distinct CD4 + T cell subsets contribute to the generation of Tfh with heterogeneous functional profiles (A) Mean fluorescence intensity of the CXCR5 marker expressed by CXCR5 + PD-1 + cells derived from (1) naive, (2) MemPD-1 neg , (3) MemPD-1 neg and Tfh. (B) Frequency of IL-21- and/or IFNγ-positive cells among CXCR5 + PD-1 + cells at day 3. (C – E) Representative flow plots showing CXCR3, ICOS, and CD40L expression by CXCR5 + PD-1 + cells (left panel) and frequency of CXCR3-, ICOS-, and CD40L-positive cells among CXCR5 + PD-1 + cells at day 3 (right panel). (F) Ex vivo cells or their respective Tfh D3 counterparts obtained after 3 days of splenocyte culture were co-cultured with autologous B cells for 7 days. (G) Box plots represent the frequency of CD27 + CD38 + cells among CD19 + cells, the concentration of total immunoglobulins and the absolute number of live B cells after co-culture. (H) Quantification of IgG1, IgG4, and IgA in the co-culture supernatants. Each symbol (A–H) represents an individual donor. (A–H) A Wilcoxon matched pairs test was performed, ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.001.

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Functional Assay, Fluorescence, Marker, Derivative Assay, Expressing, Ex Vivo, Cell Culture, Concentration Assay, Co-Culture Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: Vio Bright FITC anti-human CXCR3 , Miltenyi Biotec , Cat# 130-118-673; RRID:AB_2734057.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software

Journal: Cell reports. Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects.

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet: Figure 2. CD4+ anti-TGF-b CAR T cells exhibit memory-like T cell phenotypes in xenografts (A) 2D projection of the sample distribution (left) and subclusters (right) of purified splenic CAR+(GFP+) CD4+ T cells from the T28zT2 group (blue) and G28zT2 (red) group using t-SNE. T28zT2 clusters contain clusters 8–15, defined as IL7R+PD-1LAG3 T cell subsets; G28zT2 clusters contain clusters 1–6, defined as IL7RPD-1+LAG3+ T cell subsets or IL7RPD-1+LAG3 T cell subsets. (B) PhenoGraph cluster distribution comparing CAR+CD4+ T cells between the T28zT2 group (blue) and G28zT2 (green) group. (C) Differences in gene expression between the T28zT2 and G28zT2 groups of individual purified splenic CAR+(GFP+) CD4+ T cells in the t-SNE projection, including CD4, IL7R (CD127), TCF-1, CXCR3, PD-1, and LAG3. (D) Volcano plot of DEGs showing upregulated (red) and downregulated (blue) DEGs and non-DEGs (gray) identified by RNA-seq in T28zT2 CAR+CD4+ T cells compared to G28zT2 cells. Adjustment for the false discovery rate (FDR) results in an adjusted p value called the q value. The y axis shows the significance value after log10 transformation of the FDR (Log10(FDR)). The x axis shows the fold difference threshold between the T28zT2 and G28zT2 groups (Log2(T28zT2/ G28zT2)).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human CD25 Antibody R&D systems Cat# AF-223-NA RRID: AB_354408 Purified anti-human CXCR3 (Maxpar Ready) Antibody Fluidigm Cat# 3163004B Purified anti-human CCR6 (Maxpar Ready) Antibody Fluidigm Cat# 3141003A Anti-Human CD45 (HI30)-89Y Fluidigm Cat# 3089003B TIGIT Monoclonal Antibody (MBSA43), Functional Grade, eBioscienceTM Thermo Cat# 16-9500-82 RRID: AB_10718831 FOXP3 Monoclonal Antibody (PCH101), Functional Grade, eBioscienceTM Thermo Cat# 14-4776-82 RRID: AB_467553 APC/Cyanine7 anti-human CD279 (PD-1) Antibody Biolegend Cat# 367416 RRID: AB_2616744 APC anti-human CD279 (PD-1) Antibody Biolegend Cat# 367406 RRID: AB_2566067 PE/Cyanine7 anti-human CD223 (LAG-3) Antibody Biolegend Cat# 369208 RRID: AB_2629835 PerCP/Cyanine5.5 anti-human CD223 (LAG-3) Antibody Biolegend Cat# 369216 RRID: AB_2910413 APC anti-human CD3 Antibody Biolegend Cat# 300439 RRID: AB_2562045 PE/Cyanine7 anti-human CD3 Antibody Biolegend Cat# 300420 RRID: AB_439781 APC/Cyanine7 anti-human CD4 Antibody Biolegend Cat# 317418 RRID: AB_571947 PE anti-human CD8 Antibody Biolegend Cat# 303804 RRID: AB_2860786 APC anti-human CD25 Antibody Biolegend Cat# 356110 RRID: AB_2561977 PE/Cyanine7 anti-human CD69 Antibody Biolegend Cat# 310912 RRID: AB_314847 APC anti-human CD69 Antibody Biolegend Cat# 310910 RRID: AB_314845 PE anti-human CD69 Antibody Biolegend Cat# 310906 RRID: AB_314841 PE anti-human NKG2D Antibody Biolegend Cat# 320806 RRID: AB_492960 APC anti-human MICA/B Antibody Biolegend Cat# 320908 RRID: AB_493196 Human ULBP-2/5/6 APC-conjugated Antibody R&D systems Cat# FAB1298A RRID: AB_2257142 Human Mesothelin APC-conjugated Antibody R&D systems Cat# FAB32652A RRID: AB_2298058 APC Mouse IgG1, k Isotype Ctrl Antibody Biolegend Cat# 400120 RRID: AB_2888687 APC anti-mouse IgG2a Antibody Biolegend Cat# 407110 RRID: AB_2561754 SMAD2/3(D7G7) XP Rabbit mAb Cell Signaling Technology Cat# 8685 RRID: 10891619 Phospho-SMAD2(Ser465/467)/SMAD3 (Ser423/425) (D27F4) Rabbit mAb Cell Signaling Technology Cat# 8828 RRID: 2631089 DRP1 (D6C7) Rabbit mAb Cell Signaling Technology Cat# 8570 RRID: AB_10950498 (Continued on next page) e2 Cell Reports Medicine 6, 102020, March 18, 2025

Techniques: Gene Expression, RNA Sequencing, Transformation Assay

Journal: The Journal of Neuroscience

Article Title: Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice

doi: 10.1523/jneurosci.2262-16.2017

Figure Lengend Snippet: Figure1. CXCR3expressionisincreasedmainlyinspinalneuronsafterSNL.A,TimecourseofCxcr3mRNAexpressionintheipsilateraldorsalhorninnaive,sham-operated,andSNLmice.Cxcr3 expression at 3, 10, and 21 d increased more in SNL mice than in sham-operated mice. *p 0.05, Student’s t test. n 4–5 mice/group. B, Western blotting shows the increase of CXCR3 protein inthespinalcord10dafterSNL.*p 0.05,Student’sttest,n 4mice/group.C–E,RepresentativeimagesofCXCR3immunofluorescenceinthespinalcordfromnaiveandSNLmice.CXCR3was constitutivelyexpressedinnaivemice(C),remarkablyincreasedintheipsilateraldorsalhornofSNLmice(D),butnotinthecontralateralside(E).F–H,InsituhybridizationofCxcr3mRNAshowsthat nosignalswerefoundinspinalsectionsincubatedwithCxcr3senseprobe(F),butpositivesignalswereapparentinsectionsincubatedwithantisenseprobe(G,H).H,High-magnificationimageof G. I–K, In situ hybridization of Cxcr3 mRNA and immunofluorescence staining of NeuN (I), IBA-1 (J), and GFAP (K) shows that Cxcr3 mRNA was predominantly colocalized with neuronal marker NeuN, rarely with microglia marker IBA-1, and not at all with astrocyte marker GFAP. L, Double staining of NK1R and CXCR3 in the spinal dorsal horn. M, N, Immunofluorescence staining of CXCR3 on the spinal cord from SST-Tomato (M) and GAD2-Tomato (N) mice 10 d after SNL.

Article Snippet: Cellular localization of Cxcr3 was performed using a mouse Cxcr3 mRNA in situ hybridization assay kit (Boster Biological Technology) as described previously (Zhang et al., 2013).

Techniques: Expressing, Western Blot, In Situ Hybridization, Immunofluorescence, Staining, Marker, Double Staining

Journal: The Journal of Neuroscience

Article Title: Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice

doi: 10.1523/jneurosci.2262-16.2017

Figure Lengend Snippet: Figure 2. Demethylation of Cxcr3 gene promoter region after SNL. A, Schematic of a CpG island showing the locations of the nine CpG sites (in red) in the Cxcr3 gene promoter area. B, Representative PCR results of the Cxcr3 promoter region using primers for methylation-specific or unmethylation-specific amplifications of genomic DNA from the spinal cord of sham-operated or SNL-operatedmice.SNLreducedtheratioofmethylated(M)tounmethylated(U)productsofCpGsites.*p 0.05,SNLversussham,Student’sttest,n 3mice/group.C,Bisulfitesequencingdata ofCxcr3promoterofspinaldorsalhornfromSNL(n 4)andsham-operated(n 4)mice.Tencloneswererandomlycollectedfromeachmouse.Filledcircles,MethylatedCpGsites;unfilledcircles, unmethylated CpG sites. D, Overall methylation of Cxcr3 promoter was decreased in SNL mice. *p 0.05, SNL versus sham, two-way ANOVA, n 4 mice/group. E, Luciferases assay using coelenterazineshowsthattheluciferaseactivitywasincreasedwhenHEK293cellsweretransfectedwithunmethylatedpCpG-free-Cxcr3-promoter-Luciavectorcomparedwiththattransfectedwith methylated pCpG-free-Cxcr3-promoter-Lucia vector. *p 0.05, unmethylated versus methylated, n 3/group. F, Dnmt1 mRNA and Dnmt3a mRNA were not changed 10 d after SNL. However, Dnmt3b mRNA level decreased at days 1, 3, and 10 after SNL. *p 0.05, one-way ANOVA, n 5–6 mice/group. G, DNMT3b protein was decreased in the spinal cord 10 d after SNL. *p 0.05, Student’s t test, n 3 mice/group. H, I, Intraspinal injection of LV-Dnmt3b, 7 d before SNL, attenuated SNL-induced mechanical allodynia (H) and heat hyperalgesia (I). *p 0.05, LV-Dnmt3b versus LV-Con, two-way repeated-measures ANOVA followed by Bonferroni’s tests, n 7 mice/group. J, Pretreatment with LV-Dnmt3b increased the methylation of Cxcr3 promoter in the spinal cord 10 d after SNL. *p 0.05, LV-Dnmt3b vs LV-Con, Student’s t test, n 4–6 mice/group.

Article Snippet: Cellular localization of Cxcr3 was performed using a mouse Cxcr3 mRNA in situ hybridization assay kit (Boster Biological Technology) as described previously (Zhang et al., 2013).

Techniques: Methylation, Plasmid Preparation, Injection

Journal: The Journal of Neuroscience

Article Title: Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice

doi: 10.1523/jneurosci.2262-16.2017

Figure Lengend Snippet: Figure3. C/EBPincreasesCXCR3expressioninthespinalcordafterSNL.A,SchematicofCxcr3genepromotershowinglocationsofthethreebindingsitesforC/EBPwithCxcr3withintheCpG islands (left). P1 and P2 were ChIP-PCR primer pairs. Right, The logos of the standard C/EBP motif and three predicted C/EBP binding sites in Cxcr3 gene promoter. B, Cotransfection of C/EBP-expressingvectorwithunmethylatedpCpG-free-Cxcr3-promoter-Luciavectordramaticallyincreasedtheluciferaseactivity.*p 0.05,two-wayANOVA.C,ChIPassayshowstheincreased bindingofC/EBPwiththebindingsites1and2atCxcr3promoterafterSNL.*p 0.05,Student’sttest,n 5–6mice/group.D,ThebindingofDMNT3bwithCxcr3promoteronthebindingsite 2wasdecreasedafterSNL.*p 0.05,Student’sttest,n 4mice/group.E,CebpamRNAexpressionwasincreasedatdays3,10,and21afterSNL.p 0.05,one-wayANOVA,n 3mice/group. F,C/EBPproteinwasincreased3dinthespinalcordafterSNL.p 0.05,Student’sttest.G,ImmunofluorescencestainingofC/EBPandinsituhybridizationofCxcr3mRNAshowsthatC/EBP washighlycolocalizedwithCxcr3inthespinalcorddorsalhorn10dafterSNL.H,I,IntrathecalinjectionofC/EBPsiRNA1dbeforeSNLattenuatedSNL-inducedmechanicalallodynia(H)andheat hyperalgesia(I)for2d.*p 0.05,two-wayrepeated-measuresANOVAfollowedbyBonferroni’stests.J,PretreatmentwithCebpasiRNAreducedtheexpressionofCebpamRNAandCxcr3mRNA inthespinalcord2dafterSNL.*p 0.05,Student’sttest,n 5mice/group.K–M,IntrathecalinjectionofC/EBPsiRNA10dafterSNLattenuatedmechanicalallodynia(K)andheathyperalgesia (L), and also reduced mRNA expression of Cebpa and Cxcr3 in the spinal cord (M). *p 0.05, n 5–6 mice/group.

Article Snippet: Cellular localization of Cxcr3 was performed using a mouse Cxcr3 mRNA in situ hybridization assay kit (Boster Biological Technology) as described previously (Zhang et al., 2013).

Techniques: Binding Assay, Cotransfection, Expressing

Journal: The Journal of Neuroscience

Article Title: Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice

doi: 10.1523/jneurosci.2262-16.2017

Figure Lengend Snippet: Figure4. Cxcr3KOmicearenormalinbasalpainandintheexpressionofcellularmarkersandneurochemicalmarkers.A–D,Cxcr3KOmiceshownormalacutepainthresholdandmotorfunction. Acute pain thresholds tested by tail immersion (A), Hargreaves test (B), and von Frey test (C). D, Motor function assessed by recording the falling latency on a rotarod. E–P, The distribution of the cellular marker NeuN (E, H), GFAP (F, I), and IBA-1(G, J), and of neurochemical markers PKC (K, N), IB4 (L, O), and CGRP (M, P) in the spinal dorsal horn of WT and Cxcr3 KO mice.

Article Snippet: Cellular localization of Cxcr3 was performed using a mouse Cxcr3 mRNA in situ hybridization assay kit (Boster Biological Technology) as described previously (Zhang et al., 2013).

Techniques: Marker

Journal: The Journal of Neuroscience

Article Title: Promoted Interaction of C/EBPα with Demethylated Cxcr3 Gene Promoter Contributes to Neuropathic Pain in Mice

doi: 10.1523/jneurosci.2262-16.2017

Figure Lengend Snippet: Figure 9. Schematic shows the regulation of CXCR3 expression and the involvement of CXCL10/CXCR3 in neuropathic pain. A, SNL decreases the expression of DNMT3b and increased the expressionofC/EBPintheneuronsofthespinaldorsalhorn.ThedecreasedDNMT3bcausesDNAdemethylationofCxcr3promoter,whichincreasesthebindingofC/EBPtoCxcr3promoterand furtherincreasesthetranscriptionofCxcr3mRNAandtheexpressionofCXCR3proteinoncytoplasmandmembrane.B,SNLincreasesCXCL10expressioninspinalastrocytesandneurons.Thereleased CXCL10actsonneuronalCXCR3,whichactivatesERK.ActivationofERKmayphosphorylateNR2BtoenhanceNMDAreceptoractivity(Huetal.,2015)andfurtherenhanceAMPAreceptoractivity(Lu et al., 2001), thus increasing excitatory synaptic transmission and contributing to the pathogenesis of neuropathic pain.

Article Snippet: Cellular localization of Cxcr3 was performed using a mouse Cxcr3 mRNA in situ hybridization assay kit (Boster Biological Technology) as described previously (Zhang et al., 2013).

Techniques: Expressing, Transmission Assay