anti cxcl10 neutralizing antibody  (R&D Systems)

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

Journal: Microorganisms

Article Title: The Absence of CXCL10 Activity Does Not Affect the Capability of CD8 + T Cells to Migrate and Eliminate the Tissue Cysts of Toxoplasma gondii from the Brains of Chronically Infected Mice

doi: 10.3390/microorganisms12112172

Figure Lengend Snippet: Neutralization of CXCL10 does not inhibit the effector function of CD8 + immune T cells to eliminate T. gondii cysts from the brains of chronically infected SCID mice following the adoptive transfer of T cells. SCID mice were infected with 10 cysts and treated with sulfadiazine beginning at 7 days after infection to control tachyzoite proliferation and maintain chronic infection in their brains. CD8 + T cells purified from the spleens of chronically infected WT mice were injected (2 × 10 6 cells/mouse) intravenously into two groups of chronically infected SCID mice at 3 weeks after infection. One of those two groups of infected SCID mice was injected intraperitoneally with anti-CXCL10 mAb (100 μg) every other day beginning at one day before the CD8 + T-cell transfer. Another group was injected with isotype control Ab in the same manner. As a negative control, one group of the infected SCID mice did not receive any T cells or any Ab treatment. Their brains were obtained at 7 days after the T-cell transfer. Cerebral mRNA levels for ( A ) bradyzoite (cyst)-specific BAG1, ( B ) cyst-specific CST-1, and ( C ) bradyzoite-specific SAG2C were measured by RT-PCR. There were 4 mice in each experimental group. The date from two independent experiments were combined. *** p < 0.001, and **** p < 0.0001 by Student’s t test or Mann–Whitney U test. The latter was used when the variations in data within the experimental group significantly differed between the two groups compared. N.S.: Not significant.

Article Snippet: Infected and sulfadiazine-treated SCID mice were injected intraperitoneally with 100 μg of anti-CXCL10 neutralizing mAb (clone 1F11, BioXcell, Lebanon, NH, USA) every other day beginning at one day before the transfer of CD8 + immune T cells.

Techniques: Neutralization, Infection, Adoptive Transfer Assay, Control, Purification, Injection, Negative Control, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Microorganisms

Article Title: The Absence of CXCL10 Activity Does Not Affect the Capability of CD8 + T Cells to Migrate and Eliminate the Tissue Cysts of Toxoplasma gondii from the Brains of Chronically Infected Mice

doi: 10.3390/microorganisms12112172

Figure Lengend Snippet: Neutralization of CXCL10 does not inhibit recruitment of CD8 + immune T cells into the brain and their cytotoxic activity in this organ following the adoptive transfer of the T cells into T. gondii -infected SCID mice. SCID mice were infected with 10 cysts and treated with sulfadiazine beginning at 7 days after infection to control tachyzoite proliferation and maintain chronic infection in their brains. CD8 + T cells purified from the spleens of chronically infected WT mice were injected (2 × 10 6 cells/mouse) intravenously into two groups of chronically infected SCID mice at 3 weeks after infection. One of those two groups of infected SCID mice was injected intraperitoneally with anti-CXCL10 mAb (100 μg) every other day beginning at one day before the CD8 + T-cell transfer. Another group was injected with isotype control Ab in the same manner. As a negative control, one group of the infected SCID mice did not receive any T cells or any Ab treatment. Their brains were obtained at 7 days after the T-cell transfer. Cerebral mRNA levels for ( A ) CD8β, ( B ) perforin, and ( C ) GzmB were measured by RT-PCR. Relative ratios of ( D ) perforin mRNA levels to CD8β mRNA levels and ( E ) GzmB mRNA levels to CD8β mRNA levels were also calculated. There were 4 mice in each experimental group. The date from two independent experiments were combined. * p < 0.05, ** p < 0.01, and **** p < 0.0001 by Student’s t test or Mann–Whitney U test. The latter was used when the variations in data within the experimental group significantly differed between the two groups compared. N.S.: Not significant.

Article Snippet: Infected and sulfadiazine-treated SCID mice were injected intraperitoneally with 100 μg of anti-CXCL10 neutralizing mAb (clone 1F11, BioXcell, Lebanon, NH, USA) every other day beginning at one day before the transfer of CD8 + immune T cells.

Techniques: Neutralization, Activity Assay, Adoptive Transfer Assay, Infection, Control, Purification, Injection, Negative Control, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Microorganisms

Article Title: The Absence of CXCL10 Activity Does Not Affect the Capability of CD8 + T Cells to Migrate and Eliminate the Tissue Cysts of Toxoplasma gondii from the Brains of Chronically Infected Mice

doi: 10.3390/microorganisms12112172

Figure Lengend Snippet: Blocking of CXCL10 activity by a neutralizing mAb does not increase the mRNA levels for this chemokine to compensate for the neutralization of the activity of this chemokine in the brains of T. gondii -infected SCID mice following adoptive transfer of CD8 + immune T cells. SCID mice were infected with 10 cysts and treated with sulfadiazine beginning at 7 days after infection to control tachyzoite proliferation and maintain chronic infection in their brains. CD8 + T cells purified from the spleens of chronically infected WT mice were injected (2 × 10 6 cells/mouse) intravenously into two groups of chronically infected SCID mice at 3 weeks after infection. One of these two groups of infected SCID mice was injected intraperitoneally with anti-CXCL10 mAb (100 μg) every other day beginning at one day before the CD8 + T-cell transfer. Another group of infected SCID mice was injected with isotype control Ab in the same manner. As a negative control, one group of infected SCID mice did not receive any T cells or any Ab treatment. Their brains were obtained at 7 days after the T-cell transfer. The cerebral mRNA levels for CXCL10 were measured by RT-PCR. There were 4 mice in each experimental group. The date from two independent experiments were combined. ** p < 0.01, and **** p < 0.0001 by Student’s t test or Mann–Whitney U test. The latter was used when the variations in data within the experimental group significantly differed between the two groups compared. N.S.: Not significant.

Article Snippet: Infected and sulfadiazine-treated SCID mice were injected intraperitoneally with 100 μg of anti-CXCL10 neutralizing mAb (clone 1F11, BioXcell, Lebanon, NH, USA) every other day beginning at one day before the transfer of CD8 + immune T cells.

Techniques: Blocking Assay, Activity Assay, Neutralization, Infection, Adoptive Transfer Assay, Control, Purification, Injection, Negative Control, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Microorganisms

Article Title: The Absence of CXCL10 Activity Does Not Affect the Capability of CD8 + T Cells to Migrate and Eliminate the Tissue Cysts of Toxoplasma gondii from the Brains of Chronically Infected Mice

doi: 10.3390/microorganisms12112172

Figure Lengend Snippet: The absence of the CXCL10 gene in T cell-deficient RAG1 −/− mice infected with T. gondii rather enhances the elimination of cerebral tissue cysts of the parasite after receiving an adoptive transfer of CD8 + immune T cells from chronically infected WT mice. RAG1 −/− and RAG1 −/− CXCL10 −/− mice were infected with 10 cysts and treated with sulfadiazine beginning at 7 days after infection to control tachyzoite proliferation and maintain chronic infection in their brains. CD8 + T cells purified from the spleens of chronically infected WT mice were injected (2 × 10 6 cells/mouse) intravenously into one group of each of the chronically infected RAG1 −/− and RAG1 −/− CXCL10 −/− mice at 3 weeks after infection. Another group of each of these two strains of infected mice did not receive the T cells as a control. Their brains were obtained at 7 days after the T-cell transfer. ( A ) Cerebral mRNA levels for the cyst (bradyzoite)-specific BAG1 were measured by RT-PCR. ( B ) Decreases in BAG1 mRNA levels by the transfer of CD8 + immune T cells were measured by calculating differences in the mean value of BAG1 mRNA levels in the control mice with no T-cell transfer and the BAG1 mRNA level of the mice that had received CD8 + T cells in each of RAG1 −/− and RAG1 −/− CXCL10 −/− groups. There were 4 mice in each experimental group. * p < 0.05, *** p < 0.001, and **** p < 0.0001 by Student’s t test or Mann–Whitney U test. The latter was used when the variations in data within the experimental group significantly differed between the two groups compared. N.S.: Not significant.

Article Snippet: Infected and sulfadiazine-treated SCID mice were injected intraperitoneally with 100 μg of anti-CXCL10 neutralizing mAb (clone 1F11, BioXcell, Lebanon, NH, USA) every other day beginning at one day before the transfer of CD8 + immune T cells.

Techniques: Infection, Adoptive Transfer Assay, Control, Purification, Injection, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Microorganisms

Article Title: The Absence of CXCL10 Activity Does Not Affect the Capability of CD8 + T Cells to Migrate and Eliminate the Tissue Cysts of Toxoplasma gondii from the Brains of Chronically Infected Mice

doi: 10.3390/microorganisms12112172

Figure Lengend Snippet: The absence of the CXCL10 gene in T cell-deficient RAG1 −/− mice infected with T. gondii does not affect the recruitment of adoptively transferred CD8 + immune T cells into the brain but enhances the GzmB mRNA expression in their brains. RAG1 −/− and RAG1 −/− CXCL10 −/− mice were infected with 10 cysts and treated with sulfadiazine beginning at 7 days after infection to control tachyzoite proliferation and maintain chronic infection in their brains. CD8 + T cells purified from the spleens of chronically infected WT mice were injected (2 × 10 6 cells/mouse) intravenously into one group of each of the chronically infected RAG1 −/− and RAG1 −/− CXCL10 −/− mice at 3 weeks after infection. Another group of each of these two strains of infected mice did not receive the T cells as a control. Their brains were obtained at 7 days after the T-cell transfer. Cerebral mRNA levels for ( A ) CD8β, ( B ) perforin, and ( C ) GzmB were measured by RT-PCR. Relative ratios of ( D ) perforin mRNA levels to CD8β mRNA levels and ( E ) GzmB mRNA levels to CD8β mRNA levels were also calculated. There were 4 mice in each experimental group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by Student’s t test or Mann–Whitney U test. The latter was used when the variations in data within the experimental group significantly differed between the two groups compared. N.S.: Not significant.

Article Snippet: Infected and sulfadiazine-treated SCID mice were injected intraperitoneally with 100 μg of anti-CXCL10 neutralizing mAb (clone 1F11, BioXcell, Lebanon, NH, USA) every other day beginning at one day before the transfer of CD8 + immune T cells.

Techniques: Infection, Expressing, Control, Purification, Injection, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cellular and Molecular Immunology

Article Title: The BCL-2 inhibitor APG-2575 resets tumor-associated macrophages toward the M1 phenotype, promoting a favorable response to anti-PD-1 therapy via NLRP3 activation

doi: 10.1038/s41423-023-01112-y

Figure Lengend Snippet: APG-2575 remodels the transcriptomic landscape of macrophages and CD8+ T cells and promotes CD8+ T-cell infiltration via the chemokines CCL5 and CXCL10. A UMAP plot from merged data of tumor-infiltrating Mo/MF populations. B UMAP plots with annotated clusters of Mo/MF cells from the control and APG-2575 groups. C A heatmap showing the differentially expressed genes (rows) among Mo/MF subpopulations (columns). Representative genes from each cluster are highlighted (right). D Ratios of the proportions of Mo/MF clusters across different regimens. E GSEA using genes differentially expressed between IL-4-activated RAW264.7 cells with control or APG-2575 treatment. F ‒ I Scatter plot showing the results of Pearson correlation analysis of CCL5 and CXCL10 expression and the infiltration of CD8+ T cells and M1 macrophages in TCGA cohorts. J Growth of LLC tumors in C57BL/6 mice treated with the indicated regimens. ( K – M ) Flow cytometric analysis of CD8+, CD8 + GZMB+ and CD8+ TNF-α+ T cells in C57BL/6 mouse xenograft tumors treated with the indicated regimens. N ‒ Q Immunohistochemical staining of CD8 and GZMB in C57BL/6 mouse xenograft tumors

Article Snippet: Murine or human T-cell migration was assessed with medium, APG-2575-treated supernatant alone, supernatant plus 10 μg/mL anti-CCL5 neutralizing antibodies, and/or 10 μg/mL anti-CXCL10 neutralizing antibody (R&D Systems).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cellular and Molecular Immunology

Article Title: The BCL-2 inhibitor APG-2575 resets tumor-associated macrophages toward the M1 phenotype, promoting a favorable response to anti-PD-1 therapy via NLRP3 activation

doi: 10.1038/s41423-023-01112-y

Figure Lengend Snippet: Graphical summary of the results. APG-2575 can synergize with ICIs through a mechanism involving the repolarization of TAMs from the M2 to the M1 phenotype, further enhancing CD8+ T-cell recruitment into the TME via the augmentation of CCL5 and CXCL10 secretion and thereby improving tumor immunosuppression

Article Snippet: Murine or human T-cell migration was assessed with medium, APG-2575-treated supernatant alone, supernatant plus 10 μg/mL anti-CCL5 neutralizing antibodies, and/or 10 μg/mL anti-CXCL10 neutralizing antibody (R&D Systems).

Techniques:

Journal: Frontiers in Immunology

Article Title: Latent Cytomegalovirus-Driven Recruitment of Activated CD4+ T Cells Promotes Virus Reactivation

doi: 10.3389/fimmu.2021.657945

Figure Lengend Snippet: CXCL10, CCL8 and cIL-10 are upregulated in the secretome from latently infected CD14+ monocytes. A Volcano plot summarizing the protein array results from 3 independent day 7 secretomes, significant (p<0.05) highly upregulated (values > 4-fold) proteins are indicated by the red circle and label. Data was analyzed as described in the methods. (A) Confirmatory ELISA results for the production of CXCL10 (B) and CCL8 (C) show that both cytokines are significantly upregulated in the HCMV latent infected secretome at day 7. Seven and six independent secretomes were analyzed for CXCL10 and CCL8 production respectively. Friedman 1-way ANOVA results are shown in black on the graph; CXCL10 p=0.0012** and CCL8 p=0.0031**. Post hoc Dunn’s test results are shown in blue CXCL10 p=0.004** and CCL8 p=0.0133*.

Article Snippet: CXCL10 neutralization assays were performed using supernatants or supernatants treated with anti-CXCL10 neutralizing antibodies or IgG2a isotype control (R & D Systems) for 1 hour using the recommended neutralization procedure and dose of the manufacturer, prior to being used in the migration assays.

Techniques: Infection, Protein Array, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Latent Cytomegalovirus-Driven Recruitment of Activated CD4+ T Cells Promotes Virus Reactivation

doi: 10.3389/fimmu.2021.657945

Figure Lengend Snippet: Production of CCL8 and CXCL10 by latently infected CD14+ monocytes is independent of interferon signaling. Neutralization of IFNα, IFNβ and IFNγ does not prevent the production of either CCL8 (A) or CXCL10 (B) from latent infected CD14+ cells at day 7 post infection. CXCL10 and CCL8 was measured by ELISA, with background chemokine production in response to mock infection subtracted and fold-change results above UV irradiated infection from 2 CMV sero-negative donors CMV401 and CMV405 shown. There was no significant (ns) change in the production of CCL8 or CXCL10 in the presence of neutralizing antibodies (Kruskall-Wallis test). (C) Freshly isolated monocytes were infected with TB40/E virus as described, cell supernatant was removed and replaced (indicated on x-axis) over a 20-day period, error bars represent SEM. This shows that CXCL10 is only reproduced by the latent infected monocytes over this time period. (D) Treatment of monocytes by recombinant IFNγ protein shows CXCL10 is detectable at day 3 but lost at later time points. Freshly isolated monocytes were stimulated with decreasing amount of IFNγ from 100IU/ml for 4 hours. Supernatants were collected on days 3 (white), 6 (grey) and 10 (black). Levels of CXCL10 were assayed using ELISA. Positive control consisted of continuous 100IU/ml IFNγ and negative control was media alone. This is the representative results of three repeats, error bars represent SEM.

Article Snippet: CXCL10 neutralization assays were performed using supernatants or supernatants treated with anti-CXCL10 neutralizing antibodies or IgG2a isotype control (R & D Systems) for 1 hour using the recommended neutralization procedure and dose of the manufacturer, prior to being used in the migration assays.

Techniques: Infection, Neutralization, Enzyme-linked Immunosorbent Assay, Irradiation, Isolation, Virus, Recombinant, Positive Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Latent Cytomegalovirus-Driven Recruitment of Activated CD4+ T Cells Promotes Virus Reactivation

doi: 10.3389/fimmu.2021.657945

Figure Lengend Snippet: CXCR3 is upregulated on activated immune cell subsets and neutralization of CXCL10 abrogates migration of these cells. Histograms from flow cytometry analysis of CXCR3 expression on resting (left-hand histogram) and activated (right-hand histogram) NK cells (A) , CD8+ T cells (B) and CD4+ T cells (C) showing increased expression of CXCR3 on activated cell subsets (value shown on graph normalized geo-mean of CXCR3 expression for all cells analyzed). Representative results from 6 individual donors shown. Transwell migration assays were performed on the latent infected monocyte secretomes in the presence of CXCL10 neutralizing antibody or isotype controls showing that neutralization of CXCL10 significantly abrogates migration of activated NK cells (D) , CD8+ T cells (E) and CD4+ T cells (F) (1-way ANOVA with Sidak’s multiple comparison results shown in blue on the graphs; NK p<0.0001****, CD8 p=0.0488* and CD4 p=0.0032**, non-significant (ns) comparisons are also shown.).

Article Snippet: CXCL10 neutralization assays were performed using supernatants or supernatants treated with anti-CXCL10 neutralizing antibodies or IgG2a isotype control (R & D Systems) for 1 hour using the recommended neutralization procedure and dose of the manufacturer, prior to being used in the migration assays.

Techniques: Neutralization, Migration, Flow Cytometry, Expressing, Infection, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Hedgehog signaling promotes tumor-associated macrophage polarization to suppress intratumoral CD8 + T cell recruitment

doi: 10.1172/JCI128644

Figure Lengend Snippet: (A and B) Quantification of tumor-infiltrating CD8+ T cells by immunofluorescent staining in Hepa1-6 cells implanted s.c. into Smofl/fl and SmoΔM mice (A) and in the autochthonous HCC model (B). (C) Chemotaxis of CD8+ T cells toward macrophages treated with IFN-γ (control) and IFN-γ plus SHH. (D) Ccl3, Ccl4, Ccl5, Cxcl9, and Cxcl10 mRNA levels in macrophages treated with IFN-γ or with IFN-γ plus SHH were measured by qRT-PCR. Expression was normalized to Actb and compared with untreated. (E) Chemotaxis of CD8+ T cells toward macrophages treated with IFN-γ alone, IFN-γ plus CXCL9 and/or CXCL10-neutralizing antibodies, and IFN-γ plus SHH. (F and G) Expression of Cxcl9 and Cxcl10 in Smofl/fl or SmoΔM TAMs was measured by qRT-PCR (F) and ELISA (G). Expression of mRNAs was normalized to Actb and compared with that of Smofl/fl TAMs (F). (H) Tumor growth of Hepa1-6 in Smofl/fl and SmoΔM mice injected with CXCR3-blocking antibody or isotype control. (I) Percentage of tumor CD8+ T cell infiltration quantified by FACS. (J) Frozen tissue sections were stained for CD8+ T cells and quantified under high-power field (hpf). Values are the mean ± SEM of a minimum of 3 independent experiments. **P < 0.005; ***P < 0.0005. n = 8 biological replicates per group (A and B); n = 5 technical replicates per group (C and E); n = 5 biological replicates per group (D, F–J). Two-tailed Student’s t test (A–D, F, and G); 1-way ANOVA (E); Kruskal-Wallis test (H); 2-way ANOVA (I and J). α, anti.

Article Snippet: Murine T cell migration was assessed with medium, 50 U/mL IFN-γ–treated supernatant alone, supernatant plus 10 μg/mL anti-CXCL9 neutralizing antibodies, and/or 10 μg/mL anti-CXCL10 neutralizing antibodies (R&D Systems).

Techniques: Staining, Chemotaxis Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Blocking Assay, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: Hedgehog signaling promotes tumor-associated macrophage polarization to suppress intratumoral CD8 + T cell recruitment

doi: 10.1172/JCI128644

Figure Lengend Snippet: (A) Klf2, Klf4, Stat6, Pparg, and Cebpb mRNA levels in control and SHH-treated macrophages were measured by qRT-PCR. Expression was normalized to Actb and compared with control. (B) Klf4 mRNA levels in Smofl/fl and SmoΔM TAMs were measured by qRT-PCR. Expression was normalized to reference gene Actb and compared with that of Smofl/fl. (C) Gli1 transcription factor binds to the Klf4 promoter region as demonstrated by ChIP. Gli1 activity was inhibited using 5 μM GANT61 or constitutively activated using SmoCM macrophages. (D) Tumor volumes of Hepa1-6 hepatoma cells inoculated s.c. in Klf4fl/fl and Klf4ΔM mice on day 18 at sacrifice. (E) Expression of Arg1, Mrc1, Il10, and Tnf mRNAs in Klf4fl/fl and Klf4ΔM TAMs was quantified by qRT-PCR. Expression was normalized to reference gene Actb and compared with that of Klf4fl/fl TAMs. (F) Percentages of tumor CD8+ T cell infiltration into tumors from Klf4fl/fl and Klf4ΔM mice. (G) CXCL9 and CXCL10 production by SmoCM and SmoCMKlf4ΔM TAMs was measured by ELISA. (H) Tumor volumes of Hepa1-6 hepatoma cells inoculated s.c. in SmoCM and SmoCMKlf4ΔM mice on day 18 at sacrifice. (I) Expression of Arg1, Mrc1, Il10, and Tnf mRNAs in SmoCM and SmoCMKlf4ΔM TAMs was quantified by qRT-PCR. Expression was normalized to reference gene Actb and compared with that of SmoCM TAMs. (J) Percentages of tumor CD8+ T cell infiltration in tumors from SmoCM and SmoCMKlf4ΔM mice. Values are the mean ± SEM of a minimum of 3 independent experiments. *P < 0.05; **P < 0.005; ***P < 0.0005. n = 5 biological replicates per group (A and B); n = 3 technical replicates per group (C); n = 6 biological replicates per group (D–J). Two-tailed Student’s t test (A, B, D, and J).

Article Snippet: Murine T cell migration was assessed with medium, 50 U/mL IFN-γ–treated supernatant alone, supernatant plus 10 μg/mL anti-CXCL9 neutralizing antibodies, and/or 10 μg/mL anti-CXCL10 neutralizing antibodies (R&D Systems).

Techniques: Quantitative RT-PCR, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test