Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 1. Circulating CXCL10 in leprosy patients and healthy con- trols. Each point represents one sample from a different individual. The groups were healthy controls, borderline tuberculoid without re- action (BT), borderline lepromatous without reaction (BL), polar lep- romatous without reaction (LL), BT with reaction (BT plus RR), BL with reaction (BL plus RR), and LL patients with T2R (erythema nodosum leprosum). For patients with reaction (BT plus RR, BL plus RR, and T2R), the sample was taken at the time of reaction, prior to treatment. For LL, n 6; for all other groups, n 10. The horizontal lines indicate median values. The median level of CXCL10 in LL patients with type 2 reaction was not significantly elevated (P 0.9).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques:

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 2. Circulating CXCL10 in repeated samples from leprosy patients who developed T1R. Each graph depicts the measurements made from serum samples obtained at 1 month intervals in borderline tuberculoid (A, no. 1 to 10) and borderline lepromatous (B, no. 11 to 20) patients. All patients with clinical T1R received standard multidrug treatment starting at the first visit. The arrows indicate the visits at which T1R was diagnosed. The number above each time point indicates the dose of prednisone started on that day, tapered monthly as indicated; in some cases, corticosteroids were not used initially but were started 1 to 2 months after the initial clinical diagnosis of T1R. Each serum specimen was obtained before corticosteroid treatment was initiated. CXCL10 was significantly elevated during T1R in the 10 BT patients (A) (P 0.0006) and the 10 BL patients (B) (P 0.0001). In patients 12, 16, and 17 (B), T1R was diagnosed by histopathological criteria only; when they were excluded from the analysis, the association of T1R with CXCL10 remained significant (P 0.05).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Biomarker Discovery

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 3. Expression levels of CXCL10 and IFN- genes in skin biopsy specimens from leprosy patients. (A to H) Real-time quantitative RT-PCR was performed on cDNA obtained from sequential skin biopsy specimens from individual leprosy patients who were placed on MDT, none of whom had T1R at the time of the first biopsy. Five patients (A to E) had clinical symptoms of T1R at the time of the second biopsy; two of these (C and D) had a third biopsy after the reaction had resolved. Three patients (F to H) had no clinical symptoms of T1R at the time of the initial or second biopsy. Data were obtained using the standard-curve method and were normalized using 18S rRNA values, repeated three times for all determinations. The results are presented as the mean standard deviation.

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Cell death and differentiation

Article Title: RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8(+) T cells.

doi: 10.1038/cdd.2014.96

Figure Lengend Snippet: Figure 1 RLH activation induces secretion of proinflammatory cytokines and induction of apoptosis in murine pancreatic cancer cells. (a) Panc02 cells were stimulated with indicated amounts of ppp-RNA, poly(I:C) or left untreated. OH-RNA served as transfection control. IFN-b levels were analyzed with qRT-PCR relative to HPRT and secretion of CXCL10 or IL-6 was measured with ELISA; (b) Panc02 cells were stimulated with RNA (24 h for poly(I:C) and 48 h for ppp-RNA) and viability was assessed by FACS analysis using annexin V/PI staining; (c) Panc02 cells were incubated with siRNA specific for RIG-I or MDA5 for 24 h and subsequently stimulated with ppp-RNA or poly(I:C). Induction of apoptosis was measured by annexin V/PI staining. Silencing efficacy, as assessed by western blot, is shown; (d) activated caspase-9 (green) was visualized using green FLICA caspase-9 assay kit. Cell membranes were costained with cholera toxin B subunit (red) and nuclei with DAPI (blue); (e and f) Panc02 cells were treated as indicated for 48 h. Full length PARP-1 (116 kDa) and the cleaved large fragment of PARP-1 (89 kDa) (e) as well as the autophagy markers LC3B-I and LC3B-II (f) were analyzed by western blot. Results are representative of at least three independent experiments

Article Snippet: After fixation with paraformaldehyde and permeabilization with saponin (both Sigma), DCs were stained for IL-6 (PE, clone MP5-20F3; BioLegend) or goat anti-mouse CXCL10 (R&D Systems).

Techniques: Activation Assay, Transfection, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: C-X-C Motif Chemokine 10 Contributes to the Development of Neuropathic Pain by Increasing the Permeability of the Blood–Spinal Cord Barrier

doi: 10.3389/fimmu.2020.00477

Figure Lengend Snippet: CCI induces CXCL10/CXCR3 signaling activation. (A) Upregulated CXCL10 expression is observed in the ipsilateral spinal cord after CCI injury (** p < 0.01; independent t -test, n = 3). (B) Immunohistochemistry reveals the CCI-mediated induction of CXCR3 activation. (C) The spinal CXCR3 protein level increased during days 3–7 after CCI as shown by western blotting (** p < 0.01; *** p < 0.001; ANOVA, n = 3). The data are presented as means ± standard errors. GAPDH was used as a loading control. ANOVA, analysis of variance; CCI, chronic constriction injury; CXCL10, C-X-C motif chemokine 10; Contra, contralateral to CCI.

Article Snippet: A 20-μL volume of normal saline or a solution containing an anti-CXCL10 antibody (200 ng/rat, Proteintech) was injected into the subarachnoid space between the L5 and L6 vertebrae with a 30-gauge needle.

Techniques: Activation Assay, Expressing, Immunohistochemistry, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: C-X-C Motif Chemokine 10 Contributes to the Development of Neuropathic Pain by Increasing the Permeability of the Blood–Spinal Cord Barrier

doi: 10.3389/fimmu.2020.00477

Figure Lengend Snippet: Neutralizing CXCL10 alleviated hyperalgesia and reduced CCI-induced BSCB disruption. (A,B) Hindpaw PWTs in CCI + anti-CXCL10 antibodies group and CCI + saline group rats on various experimental days (* p < 0.05, ** p < 0.01, *** p < 0.001; ANOVA, n = 5–6). (C) Lumbar spinal cord NaFlu concentrations in the CCI + anti-CXCL10 antibodies group, CCI + saline group, and sham surgery group rats on experimental day 3 (* p < 0.01, CCI + NS group vs. sham surgery group; Mann–Whitney U- test, n = 4–6; ## p < 0.01, CCI + anti-CXCL10 antibodies group vs. CCI + NS group; independent t- test, n = 6). The data are shown as means ± standard errors. ANOVA, analysis of variance; BSCB, blood-spinal cord barrier; CCI, chronic constriction injury; CXCL10, C-X-C motif chemokine 10; NaFlu, sodium fluorescein; PWT, paw withdrawal threshold; NS, normal saline.

Article Snippet: A 20-μL volume of normal saline or a solution containing an anti-CXCL10 antibody (200 ng/rat, Proteintech) was injected into the subarachnoid space between the L5 and L6 vertebrae with a 30-gauge needle.

Techniques: Disruption, Saline, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: C-X-C Motif Chemokine 10 Contributes to the Development of Neuropathic Pain by Increasing the Permeability of the Blood–Spinal Cord Barrier

doi: 10.3389/fimmu.2020.00477

Figure Lengend Snippet: Neutralizing CXCL10 reduced CCI-induced T cell infiltration of the dorsal horn. (A,B) CD3-positive T cell levels as percentages of CCI-ipsilateral dorsal horn mononuclear cell populations in the sham surgery group and CCI + NS group rats (** p < 0.01; independent t- test, n = 3–4). (B,C) CD3-positive T cell levels as percentages of CCI-ipsilateral dorsal horn mononuclear cell populations in CCI + NS group and CCI + anti-CXCL10 antibodies group rats ( # p < 0.05; independent t- test, n = 3). (D) Bar chart indicating the statistical analysis. The data are shown as means ± standard errors. CCI, chronic constriction injury; CXCL10, C-X-C motif chemokine 10; NS, normal saline.

Article Snippet: A 20-μL volume of normal saline or a solution containing an anti-CXCL10 antibody (200 ng/rat, Proteintech) was injected into the subarachnoid space between the L5 and L6 vertebrae with a 30-gauge needle.

Techniques: Saline

Journal: Cellular and Molecular Immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: The synergistic interaction between LCN2 and TWEAK in keratinocytes. A Heatmap showing differential expressed genes after co-stimulating with TWEAK and LCN2 in keratinocytes. B Relative expression levels of IL6 , TNFa , IL17A, CCL5, CXCL5, CXCL10 and 24P3R were detected from HFKs using RT-qPCR. C , D Western blot was used to detect p-ERK1/2, p-JNK, p-p38, p-TRAF2, 24P3R and Fn14 protein level in keratinocytes after stimulation with LCN2, TWEAK, or both. One-way ANOVA was used for comparison between groups: ns ( P > 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: CXCL10 (1:1000) were bought from GeneTex (GeneTex, Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Comparison

Journal: Cellular and Molecular Immunology

Article Title: Synergistic effects of LCN2 and TWEAK on the progression of psoriasis

doi: 10.1038/s41423-025-01292-9

Figure Lengend Snippet: Lcn 2 –/– mice attenuate the pro-proliferative and inflammatory effects of IMQ on the epidermis. A After being treated with imiquimod and LCN2, the expression levels of Fn14, p-ERK1/2 and in the skin of wild-type mice were analyzed by Western blotting ( n = 3 per group). B Relative expression levels of Tnfsf12(Tweak) , Tnfrsf12a(Fn14) , Il6 , Tnfa , Il1b , Tgfb1 , Krt5 , Krt10 , and Krt17 from mice skin were detected using RT-qPCR. C , D Photographs of skin lesions in mice and the mouse psoriasis severity score difference between wild-type imiquimod mice and Lcn2 –/– imiquimod model mice. Body weight changes in mice modeled with imiquimod for six consecutive days. E Mouse serum levels of TWEAK were assayed by ELISA. F Western blotting was used to detect KRT1, KRT5, KRT6, KRT10, KRT14, KRT16, KRT17, Loricrin, Fn14, CXCL10, LCN2, p-ERK1/2, p-JNK, p-p38 in wild-type and Lcn2 –/– mice after treated with imiquimod ( n = 3 per group). G Relative mRNA expression levels of Lcn2 , Tnfsf12 , Tnfrsf12a , Il6 , Tnfa , Tgfb1 , Krt5 , Krt10 , Krt17 , 24p3r , Mc4r , Il1b were detected by RT-qPCR ( n = 3–5 per group). H Immunostaining of KRT5 and KRT14 were performed on paraffin sections. Data are shown as mean ± SEM ( n = 3–5 per group). ANOVA was used for comparison between groups: ns ( P > 0.05); * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: CXCL10 (1:1000) were bought from GeneTex (GeneTex, Shanghai, China).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunostaining, Comparison

Journal: Frontiers in Immunology

Article Title: Tumor-intrinsic interferon signaling drives pancreatic cancer resistance to tumor mucin1-targeted CAR T cell therapy

doi: 10.3389/fimmu.2025.1618415

Figure Lengend Snippet: Suppressing IFN signaling in PDA reduces ICAM-1 and CXCL10 levels. PDA cell lines were plated overnight for attachment. On the following day, PDA cells were treated with JAKi at 10μM and 25μM overnight. After JAKi treatment, it was removed, and tMUC1-CAR T cells or Mock T cells were added at E:T ratio of 5:1 in the absence of JAKi. The E:T ratio was calculated based on the initial number of PDA cells plated. After 24hr, co-culture supernatants were collected and assayed for cytokine levels of (A) ICAM-1 and (B) CXCL10 by ELISA. Data are presented as the mean ± SD of triplicate. Baseline levels of ICAM-1 and CXCL10 were low (<50pg/ml and <10pg/ml, respectively) in either CAR T cell-only or in PDA-only cultures, which were detected by ELISA. The statistical comparison was conducted between CAR T cell treatment in JAKi-pretreated PDA and CAR T cell treatment in PDA without JAKi pretreatment. *p<0.05, ****p<0.0001 (unpaired t test with Welch’s correction).

Article Snippet: On the following day, PDA cells were pre-incubated with PD-L1 blocking antibody (10μg/ml) or its isotype control antibody (10μg/ml; Cat# 400348; BioLegend), anti-ICAM-1 neutralizing antibody (5μg/ml; Cat# AF720; R&D Systems), anti-CXCL10 neutralizing antibody (5μg/ml; Cat# MAB266-100; R&D Systems), or fresh media alone for 2hr.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Tumor-intrinsic interferon signaling drives pancreatic cancer resistance to tumor mucin1-targeted CAR T cell therapy

doi: 10.3389/fimmu.2025.1618415

Figure Lengend Snippet: Suppressing IFN signaling in CAR T cells slightly decreases ICAM-1 level. PDA cell lines were plated overnight for attachment. On the same day, tMUC1-CAR T cells were treated with or without JAKi at 10μM and 25μM overnight. On the following day, JAKi-pretreated CAR T cells were washed with fresh media to remove JAKi before being added to PDA cells at E:T ratio of 5:1 for co-culture. After 24hr, co-culture supernatants were collected and assayed for cytokine levels of (A) ICAM-1 and (B) CXCL10 by ELISA. Data are presented as the mean ± SD of triplicate. The statistical comparison was conducted between JAKi-pretreated CAR T cell group and media-pretreated CAR T cell group in PDA. *p<0.05, **p<0.01 (unpaired t test with Welch’s correction).

Article Snippet: On the following day, PDA cells were pre-incubated with PD-L1 blocking antibody (10μg/ml) or its isotype control antibody (10μg/ml; Cat# 400348; BioLegend), anti-ICAM-1 neutralizing antibody (5μg/ml; Cat# AF720; R&D Systems), anti-CXCL10 neutralizing antibody (5μg/ml; Cat# MAB266-100; R&D Systems), or fresh media alone for 2hr.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Tumor-intrinsic interferon signaling drives pancreatic cancer resistance to tumor mucin1-targeted CAR T cell therapy

doi: 10.3389/fimmu.2025.1618415

Figure Lengend Snippet: Engagement of CAR T cell with PDA induces function loss of CAR T cells and up-regulation of immune checkpoints. (A) Loss of CAR T cell cytotoxicity. CAR T cells or Mock T cells were co-cultured with MiaPaCa-2 or HPAFII cells at E:T ratio of 5:1 or cultured with media alone for overnight. Then the live CAR T cells and Mock T cells were isolated from the first round co-culture, and they were added to the fresh MiaPaCa-2 or HPAFII plates at E:T ratio of 2:1 or 5:1 for 24hr as the second round of co-culture. At the end of culture, tumor cell lysis against MiaPaCa-2 (left panel) or against HPAFII (right panel) was determined using MTT assay. The percentage of lysis was calculated using the formula: [(OD of co-culture with Mock T cells– OD of co-culture with CAR T cells)/OD of co-culture with Mock T cells] ×100. The mock T cells and CAR T cells are from the same pair for calculation. Data are presented as the mean ± SD from quadruplicate. The statistical difference was conducted for cytolysis of CAR T cells retrieved from co-culture with PDA cells when compared with cytolysis of CAR T cells retrieved from culture with media alone. ****p<0.0001 (Multiple unpaired t tests with Welch’s correction). (B) Increase of CD25 expression on CAR T cells after engagement with PDA. CAR T cells were co-cultured with MiaPaCa-2 or HPAFII cells at E:T ratio of 5:1 or cultured with media alone for overnight. Then CAR T cells were stained and analyzed for CD25 expression on CAR-positive and CAR-negative live cells after gating on CD4 + T cells and CD8 + T cells. (C) Expression of ICs on CAR T cells. Cell culture was performed same as in (B) . IC expression on CAR-positive and CAR-negative live cells were displayed after gating on CD4 + T cells and CD8 + T cells. (D) The retaining of tMUC1 and up-regulation of PD-L1 on PDA. After rinsing off suspension cells in co-culture from (B) , adherent MiaPaCa-2 or HPAFII cells were stained and analyzed for tMUC1 and PD-L1. PDA cells cultured in media alone were included as baseline control. (E) Suppressing tumor IFN signaling blocks CAR T cells-induced PD-L1 increase. MiaPaCa-2 or HPAFII cells were pre-treated with JAKi at 25μM overnight. Then JAKi was removed, and CAR T cells were added in the absence of JAKi at E:T ratio of 5:1, calculated based on the initial number of PDA cells plated. After 24hr treatment with CAR T cells, live adherent PDA cells were stained and analyzed for PD-L1 expression. (F) Effectiveness of PD-L1 blocking antibody. CAR T cells-treated HPAFII cells were pre-incubated with PD-L1 blocking antibody at the indicated doses, followed by PD-L1-PE staining. (G) Blocking PD-L1 partially reversed HPAFII resistance to CAR T cell lysis. MiaPaCa-2 or HPAFII cells were treated with CAR T cells or Mock T cells in the presence of PD-L1 blocking antibody or its isotype control for 24hr. PDA cell lysis was determined using MTT assay. Data are presented as the mean ± SD from quadruplicate. The statistical difference was conducted for cytolysis of CAR T cells with PD-L1 blocking antibody when compared with cytolysis of CAR T cells with media alone. ****p<0.0001 (unpaired t tests with Welch’s correction). (H) Involvement of ICAM-1 and CXCL10 in CAR T cell cytotoxicity. MiaPaCa-2 or HPAFII cells were treated with CAR T cells or Mock T cells in the presence of anti-ICAM-1 or anti-CXCL10 antibodies for 24hr. PDA cell lysis was determined using MTT assay. Data are presented as the mean ± SD from quadruplicate. The statistical difference was conducted for cytolysis of CAR T cells with neutralizing antibody when compared with cytolysis of CAR T cells with media alone. **p<0.01 (unpaired t tests with Welch’s correction).

Article Snippet: On the following day, PDA cells were pre-incubated with PD-L1 blocking antibody (10μg/ml) or its isotype control antibody (10μg/ml; Cat# 400348; BioLegend), anti-ICAM-1 neutralizing antibody (5μg/ml; Cat# AF720; R&D Systems), anti-CXCL10 neutralizing antibody (5μg/ml; Cat# MAB266-100; R&D Systems), or fresh media alone for 2hr.

Techniques: Cell Culture, Isolation, Co-Culture Assay, Lysis, MTT Assay, Expressing, Staining, Suspension, Control, Blocking Assay, Incubation