cd45 pe antibody  (Miltenyi Biotec)

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Intratumoral delivery of 4–1BBL boosts IL-12-triggered anti-glioma immunity

doi: 10.1016/j.ymthe.2025.08.028

Figure Lengend Snippet: (A) IL12Rβ1 , IL12Rβ2 , and STAT4 expression in immune cells of GB patients. Distinct cell types were clustered, annotated, and visualized with a high-resolution color-coded UMAP projection. To visualize IL12Rβ1 , IL12Rβ2 , and STAT4 expression, dot plots were used. No expression was observed in the tumor compartment, but it was detected in the immune cell compartment. (B) Representative flow cytometry plots of STAT4p levels in CT-2A-FLuc-cultured cells. CT-2A-FLuc cells were exposed to sham or rIL-12 for 24 h, STAT4p expression was measured by flow cytometry and no differences were observed (dataset from Miller et al. ). (C) Positive IL12RB1 receptor staining in brain tissues implanted with CT-2A cells. Immunohistochemistry of IL12RB1-positive cells (brown) in the TME of the CT-2A tumor (TU) cells (blue) (magnification 20×). Scale bar, 100 μm. (D) Decoupling non-immune cells and Mo/Mϕ/MG cells from other immune cells in GB-bearing mouse brains to explore Il-12R expression. A schematic display shows the sequential method used to isolate CD11b-enriched (EN) tumor (TU) cells, CD45 EN TU cells, and CD45-deprived (DEPR) TU cells derived from the tumor hemisphere (TH) of mouse brains post-rIL-12 treatment (left). I.t. Il12rb2 expression was analyzed 8 days after rIL-12 treatement. Il12rb2 was expressed significantly higher in CD45 EN TU compared with CD11b EN TU ( p = 0.0043) and CD45 DEPR TU cells ( p = 0.0001). CD45 EN TU Il12rb2 levels were significantly higher ( p = 0.0136) than CD45 DEPR TU cells. Data represent CT values normalized to β-actin. Data represent three independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 (right). (E) Expression of Il12rb1 , Il12rb2 , and STAT4p in immune cell populations of TME of mouse GB models. scRNA-seq datasets of CD45 POS -sorted tumor cells derived from mouse GB tumor (TU) (CT-2A, n = 3) were analyzed (dataset from Tomaszewski et al. ). Distinct cell types were clustered, annotated, and visualized with a high-resolution color-coded UMAP projection. To visualize Il12rb1 , Il12rb2 , and STAT4p expression in different datasets, feature plots were used to display the expression in NK/T cluster (marked in dotted lines) (datasets from Pombo Antunes et al. Tomaszewski et al. and Chen et al. ). (F) STAT4p levels in CD8 T cell, CD4 T cell, and NK cell populations post-rIL-12 treatment. CD8 T cells, CD4 T cells, and NK1.1 cells were isolated from CT-2A-FLuc tumor (TU)-bearing, rIL-12-treated mice on day 18 post-tumor implantation. Representative flow plots showed STAT4p POS and STAT4p NEG levels as the percentage of max. All three cell types express STAT4p. (G) Schematic illustration of the T cell depletion strategy in rIL-12-treated GB mouse. On day 0, 100,000 GB cells (CT-2A-FLuc) were implanted i.c. into the left striatum. Anti-CD8 or IgG control was injected i.v. on day 9 (50 μg, retro-orbitally). On day 10, mice were injected with 50 ng rIL-12 or sham control i.t. at the tumor site and anti-CD8 or IgG control was injected i.v. (100 μg, retro-orbitally) to deplete endogenous CD8 POS T cells systemically. (H) Importance of CD8 POS T cell recruitment for survival benefit in anti-GB therapy with rIL-12. Kaplan-Meier curves showing survival outcome of tumor-bearing mice injected with IgG and rIL-12 (gray), with CD8 depletion (anti-CD8) and rIL-12 (red), with CD4 depletion (anti-CD4) and rIL-12 (turquoise) and with NK cells depletion (anti-NK1.1) and rIL-12 (orange) ( n = 6–8 mice per group). IgG control treated with rIL-12 had a median survival (MS) of 25 days, whereas anti-CD8 had a median survival of 21 days, anti-CD4 34 days, and anti-NK 40.5 days. IgG control did not differ compared with anti-CD4 but had a significantly improved survival compared with anti-CD8 ( p = 0.0372), and anti-NK ( p = 0.0479). Anti-CD8 had significant improved median survival compared with anti-NK ( p = 0.0122) and anti-CD4 ( p = 0.0055). Data represent two independent experiments and were analyzed using log rank (Mantel-Cox) test, * p < 0.05; ** p < 0.001. Median survival in days (MS).

Article Snippet: Samples were then loaded onto a series of LS columns containing microbeads conjugated to anti-mouse CD11b and anti-mouse CD45 (Miltenyi Biotec), respectively, and separated into CD11b POS , CD11b NEG CD45 POS , and CD11b NEG CD45 NEG (non-immune) cell populations using the MACS multi-stand (Milteny Biotec).

Techniques: Expressing, Flow Cytometry, Cell Culture, Staining, Immunohistochemistry, Derivative Assay, Isolation, Tumor Implantation, Control, Injection

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Intratumoral delivery of 4–1BBL boosts IL-12-triggered anti-glioma immunity

doi: 10.1016/j.ymthe.2025.08.028

Figure Lengend Snippet: (A) IFN-γ production by primary CT-2A-associated CD8 POS T cells following rIL-12 exposure and tumor Ag presentation via MHC class I. Representative images of the IFN-γ Elispot assay demonstrate an increased number of spots when primary CD8 POS T cells, isolated from GL261-bearing mice brain, were co-cultured with naive splenocyte-derived DCs that MHCI-present mImp3, a GL261-specific neopeptide, for 24 h. This increase was observed only in the presence of rIL-12 and not with the sham control (sham = Fc control). Each spot corresponds to an IFN-γ-releasing T cell. The accompanying bar graph quantifies the number of spots (mean number of spots: 22 for sham; 153 for rIL-12). Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using an unpaired t test, **** p < 0.0001. (B) MHCI-expressing DCs at the tumor site express regulatory factors, including Il12b and Tnfsf9, capable of modulating CD8 POS T cell activity. Heatmap showing co-expression of genes that are expressed by CT-2A-associated DCs involved in tumor Ag cross-presentation (dataset from Tomaszewski et al. ). The listed genes are selected based on their ability to modulate CD8 POS T cell activity through stimulatory factors and/or inhibitory factors. H2-D1 encodes for H-2Db that is an MHC-I class molecule responsible for mImp3 presentation. MHC-I-expressing DCs co-express high levels of CD8 POS T cell activity modulating factors, including Il12b (encoded by IL-12p40), inhibitory factors, including Cd274 (encoding for PD-L1), and co-stimulatory factors, including Tnfsf9 (encoding for 4–1BBL). CT-2A (CD45 POS ) tumor (TU). (C) MHCI-expressing DCs at the tumor site co-express Il12 receptor and Tnfrsf9 . Heatmap illustrating that Il12b POS DCs co-express Il12rb1 (encoding the IL12 receptor subunit) and Tnfrsf9 (encoding 4–1BB) at the CT-2A tumor site. Based on Ccr7 expression, these Il12b POS DCs can be classified as CCR7 POS DCs, which display a distinct transcriptional profile compared with (less/non-activated) conventional DCs (cDC1 and cDC2) and plasmacytoid DCs (pDCs). CT-2A (CD45 POS ) tumor (TU) (dataset from Tomaszewski et al. , ). (D) Il12b expression is restricted to a subcluster of DCs. UMAP clustering shows expression of Il12b in distinct population of the DC cluster in CT-2A (CD45 POS ) tumor (TU) (dataset from Tomaszewski et al. , ). (E) Il12b POS DCs have migratory signatures. The cells positive in (D, marked with a dotted line) match with the migratory factors Fscn1 , Ccr7 , and Ccl22 . DC cluster in CT-2A (CD45 POS ) tumor (TU) (dataset from Tomaszewski et al. , ). (F) IL-12- expressing cells are recruited to the TME of rIL-12 -treated GB tumors. High numbers of eYFP-expressing cells (in green) were observed in both the CT-2A tumor (TU) border (white dotted line) as well as the tumor itself in IL-12b-eYFP reporter mice treated on day 10 post-tumor implantation with sham control (50 ng of Fc) or 50 ng rIL-12. Mice were sacrificed on day 18 post-tumor implantation (4× magnification). Scale bar, 50 μm. (G) IL-12b-eYFP- expressing cells recruited to the GB TME post- rIL-12 treatment are DCs. CT-2A-Fluc tumor (TU)-bearing mice were treated with rIL-12 and sacrificed on day 18 post-tumor implantation. Samples generated in (F) were stained with anti-CD11c, confirming that eYFP-expressing cells are DCs. Scale bars, 10 μm (10× magnification) and 50 μm (40× magnification). (H) Tumor-engaging and Ag-presenting DCs at the GB tumor site express PD-L1. Flow cytometry contour plots of MHC-II versus PD-L1 expression in DCs on day 18 post-tumor (TU) implantation i.t. treated with sham (gray) or rIL-12 (blue). Distinct DC subsets (CD11c POS ) were identified: PD-L1 NEG MHC-II NEG (box 1), PD-L1 NEG MHC-II POS (box 2), and PD-L1 POS MHC-II POS (box 3), the latter representing activated DCs. (I) Ag-presenting PD-L1 POS DCs respond to rIL-12 by increasing 4–1BB expression. DCs were isolated from CT-2A tumor (TU) mice on day 18 post-tumor implantation i.t. treated with sham or rIL-12. DCs responsive to rIL-12 were identified based on STAT4p expression and were mainly present in PD-L1 POS MHC-II POS cells (box 3 of H, represented by the red “3”) and not in the PD-L1 NEG MHC-II NEG and PD-L1 NEG MHC-II POS (boxes 1 and 2 of H, represented by the red “1” and “2”) after rIL-12 treatment. 4–1BB was increased in the PD-L1 POS MHC-II POS pSTAT POS cells (top). Quantification of STAT4p POS -expressing cells in 4–1BB POS DCs post-treatment with rIL-12 showed an increased expression of 54.71% ± 15.41% (mean ± SEM) in cells pre-gated for PD-L1 POS MHC-II POS (box 3 of H) were found in rIL-12-treated cells compared with sham ( n = 6–7 mice per group) (bottom). The 4–1BB POS DCs are a subset of the STAT4p POS DCs. Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using multiple comparison two-way ANOVA, **** p < 0.0001, n.s.

Article Snippet: Samples were then loaded onto a series of LS columns containing microbeads conjugated to anti-mouse CD11b and anti-mouse CD45 (Miltenyi Biotec), respectively, and separated into CD11b POS , CD11b NEG CD45 POS , and CD11b NEG CD45 NEG (non-immune) cell populations using the MACS multi-stand (Milteny Biotec).

Techniques: Enzyme-linked Immunospot, Isolation, Cell Culture, Derivative Assay, Control, Expressing, Activity Assay, Tumor Implantation, Generated, Staining, Flow Cytometry, Comparison

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Intratumoral delivery of 4–1BBL boosts IL-12-triggered anti-glioma immunity

doi: 10.1016/j.ymthe.2025.08.028

Figure Lengend Snippet: (A) Higher number of CD8 POS T cells around rIL-12-treated CT-2A-FLuc tumor. Immunofluorescent imaging of CD8b at the tumor border (white dotted line) after 18 days of CT-2A-FLuc implantation comparing sham and rIL-12-treated mice (magnification 20×). Scale bar, 100 μm. (B) Increase of CD8 POS T cells at tumor site with intratumoral rIL-12. Representative flow cytometry plots of CD45 EN TU CT-2A tumor cells show the gating for live cells based on uptake of the viability dye ZombieBlue staining. Isolation of Thy1.2 POS and CD8 POS T cells, pre-gated for CD45 POS CD11b NEG in brains of tumor-bearing mice on day 18 post-tumor implantation, comparing rIL-12-treated and sham control mice (left). The brain tissue was enzymatically digested and enriched for CD45 EN tumor cells. The bar graph represents the quantification of CD8 POS T cell numbers in ipsilateral and contralateral hemispheres ( n = 6 mice per group) as a percentage of CD45 POS cells in rIL-12- or sham-treated mice (right, bar graph). Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using unpaired t test, * p < 0.05. (C) Increased CD8 POS T cell differentiation upon rIL-12 treatment of GB. Overlaid flow cytometry plots of CD8 POS T cells expressing TCF-1 against TIM-3 while comparing rIL-12 (blue) and sham (gray) treatment (left). Tumor tissue was harvested on day 18 post-tumor (CT-2A CD45 EN TU) implantation. Quantification of the percentage of CD8 POS T cells comparing TCF-1 POS (box 1, effector-like), TCF-1 NEG TIM-3 NEG (box 2), and TIM-3 POS (box 3, stemness-like) showed a 32.2% increase of TIM-3 POS after rIL-12 (65.5%) treatment compared with sham (33.2%) ( n = 6–7 mice per condition, right bar graph). Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using two-way ANOVA ** p < 0.01, n.s. (D) The activation-induced marker PD-1 is highly expressed in effector-like CD8 POS T cells. Percent of maximum PD-1 expression within TCF-1 POS (box 1), TCF-1 NEG TIM-3 NEG (box 2), and TIM-3 POS (box 3) populations on day 18 post-tumor implantation (CT-2A CD45 EN TU), of CD8 POS T cells when treated with rIL-12 (left). The black dotted vertical line represents the FMO signal. Quantification of PD-1 levels per CD8 POS T cell by flow cytometry after rIL-12 treatment within TCF-1 POS (box 1 in C, MFI 2679), TCF-1 NEG TIM-3 NEG (box 2 in C, MFI 1280), and TIM-3 POS (box 3 in C, MFI 652) populations. TIM-3 POS cells express significantly more PD-1 compared with TCF-1 POS and TIM-3 NEG TCF-1 NEG cells. In contrast, TIM-3 NEG TCF-1 NEG CD8 POS T cells express significantly more PD-1 per cell, compared with TCF-1 POS ones (right). Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using two-way ANOVA, ** p < 0.01, *** p < 0.001. (E) Cytotoxic GZM-B is highly expressed in effector-like CD8 POS T cells. Percent of maximum GZM-B expression within TCF-1 POS (box 1 in C), TCF-1 NEG TIM-3 NEG (box 2 in C), and TIM-3 POS (box 3 in C) populations on day 18 post-tumor (CT-2A CD45 EN TU) implantation, of CD8 POS T cells when treated with rIL-12 (left). The black dotted vertical line represents the FMO signal. Quantification of GZM-B levels per CD8 POS T cell by flow cytometry after rIL-12 treatment within TCF-1 POS (box 1 in C, MFI 1229), TCF-1 NEG TIM-3 NEG (box 2 in C, MFI 277.3) and TIM-3 POS (box 3 in C, MFI 93.5) populations ( n = 5–6 mice per group) (right). Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using two-way ANOVA, **** p < 0.0001.

Article Snippet: Samples were then loaded onto a series of LS columns containing microbeads conjugated to anti-mouse CD11b and anti-mouse CD45 (Miltenyi Biotec), respectively, and separated into CD11b POS , CD11b NEG CD45 POS , and CD11b NEG CD45 NEG (non-immune) cell populations using the MACS multi-stand (Milteny Biotec).

Techniques: Imaging, Flow Cytometry, Staining, Isolation, Tumor Implantation, Control, Cell Differentiation, Expressing, Activation Assay, Marker

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Intratumoral delivery of 4–1BBL boosts IL-12-triggered anti-glioma immunity

doi: 10.1016/j.ymthe.2025.08.028

Figure Lengend Snippet: (A) Survival probability of de novo TNFRSF9. Kaplan-Meier survival curves showing the survival outcomes over a period of 5 years of 63 GB patients (IDH-WT) per group with high (red) or low (blue) levels of TNFRSF9 , each group had a median of ~2 years (based on Miller et al. ). No differences were observed between groups. Log rank (Mantel-Cox) test, p = 0.6, n.s. (B) Immunohistochemistry shows 4–1BB expression at the tumor border. The CT-2A-bearing brain treated with rIL-12 was isolated at day 18 post-implantation and stained for 4–1BB. Brown color represents HRP signal (magnification 20×). Scale bar 100 μm. (C) CD8 POS T cells expressing 4–1BB are recruited at the tumor upon rIL-12 treatment. Immunofluorescence shows that 4–1BB (cyan) was expressed in CD8 POS T cells (pink) post-rIL-12 treatment at day 18 post-implantation at the CT-2A-Fluc-GFP (green) tumor (TU) border (white dotted line) (20× magnification). Scale bar, 50 μm. (D) 4–1BB expression in CD8 POS T cells. scRNA-seq analysis displaying TNFRSF9 expression in CD3 POS tumor (TU) cells of de novo human glioma and Tnfrsf9 expression in the NK/T cluster of CD45 POS tumor (TU) cells in CT-2A murine GB. The red dotted line represents the CD8a population. (E) 4–1BB is expressed by effector-like CD8 POS T cells during rIL-12 treatment. Percent of maximum 4–1BB expression (left histogram plot) within TCF-1 POS (box 1 in ), TCF-1 NEG TIM-3 NEG (box 2 in ), and TIM-3 POS (box 3 in ) populations on day 18 post-tumor (CT-2A CD45 EN TU) implantation of CD8 POS T cells when treated with rIL-12. The black dotted vertical line represents the FMO signal. Quantification by 4–1BB levels in CD8 POS T cells by flow cytometry (right bar graph) after rIL-12 treatment within TCF-1 POS (box 1 in , MFI: 251.9), TCF-1 NEG TIM-3 NEG (box 2 in , MFI: 48.4) and TIM-3 POS (box 3 in , MFI: 46.3) populations ( n = 4–7 mice per group). Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using two-way ANOVA, **** p < 0.0001 (datasets from Tomaszewski et al. and Miller et al. , ). (F) Effector-like CD8 POS T cells can be identified by PD-1 and GZM-B during rIL-12 treatment. Counter plots of GZM-B expression against PD-1 within PD-1 NEG (box 1), PD-1 POS GZM-B NEG (box 2), and PD-1 POS GZM-B POS , representing effector-like cells (box 3) populations on day 18 post-tumor (CT-2A CD45 EN TU) implantation of CD8 POS T cells when treated with sham (top) or rIL-12 (bottom). (G) 4–1BB POS cells are more present in the CD8 POS T cell effector-like subset that is STAT4 phosphorylated during rIL-12 treatment. Cells were pre-gated for 4–1BB versus STAT4p comparing sham and rIL-12-treated conditions (top). Quantification by flow cytometry as shown in bar graphs of percentage of 4–1BB POS cells post-rIL-12 treatment showing STAT4 phosphorylation of CD8 POS T cells within PD-1 NEG (box 1 in F), PD-1 POS GZM-B NEG (box 2 in F), and PD-1 POS GZM-B POS (box 3 in F) populations (bottom) ( n = 3–7 mice per group). Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using two-way ANOVA, * p < 0.05.

Article Snippet: Samples were then loaded onto a series of LS columns containing microbeads conjugated to anti-mouse CD11b and anti-mouse CD45 (Miltenyi Biotec), respectively, and separated into CD11b POS , CD11b NEG CD45 POS , and CD11b NEG CD45 NEG (non-immune) cell populations using the MACS multi-stand (Milteny Biotec).

Techniques: Immunohistochemistry, Expressing, Isolation, Staining, Immunofluorescence, Flow Cytometry, Phospho-proteomics

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Intratumoral delivery of 4–1BBL boosts IL-12-triggered anti-glioma immunity

doi: 10.1016/j.ymthe.2025.08.028

Figure Lengend Snippet: (A) GB in mice express high levels of PD-L1 in non-immune compartment after rIL-12 treatment. CD11b EN , CD45 EN , and CD45 DEPR TU populations were isolated from CT-2A-FLuc tumor-bearing hemisphere of brains using CD11b beads and CD45 beads (see ). Cd274 (encoding the PD-L1 gene) was expressed at significantly higher levels in CD45 DEPR tumor (TU) cells. No differences were observed between sham (gray) and rIL-12 treatment (blue). Gene expression levels were normalized to β-actin ( n = 4 mice per group). Data represent four independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using one-way ANOVA, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Lentivirus vector constructs expressing Tnfsf9 or null. Schematic display of Tnfsf9 lentivirus vectors (LVV); LVV null, a GFAP promotor followed mCherry and WPRE (left); LVV Tnfsf9 containing mCherry labeled Tnfsf9 and 3xFLAG-tag driven by a GFAP promotor (right). (C) Tnfsf9 expression in GB mouse cells. CT-2A-FLuc and 005-FLuc cells transduced with the LVV Tnfsf9 showed significant enhanced gene expression levels of Tnfsf9 (encoding for 4–1BBL) compared with cells transduced with LVV null and non-transduced cells, normalized to β-actin. Data represent three independent in vitro experiments and are presented as the mean ± SEM (error bars). Data were analyzed using one-way ANOVA, **** p < 0.0001. (D) 4–1BBL protein expression in CT-2A cells. 3xFLAG-tag protein levels (37.5 kDa) were only present in CT-2A-FLuc cells transduced with the LVV Tnfsf9 compared with in vitro CT-2A cells transduced with LVV null, normalized to β-actin. 3xFLAG-tag detection enabled detection of transgene 4–1BBL and not endogenous 4–1BBL. (E) Homogenous 4–1BBL expression in transduced GB mouse cell line for brain implantation experiments. Immunofluorescent images of 4–1BBL overexpressing CT-2A cells post-LVV Tnfsf9 transduction in culture stained for DAPI (blue), 3xFLAG-tag (green), mCherry (red), and 4–1BBL (pink) with a merged image. Scale bar, 50 μm. Tumor-bearing mouse brains confirmed transgene expression (mCherry-positive cells) co-localized with 3xFLAG-tag and 4–1BBL (40× magnification). Scale bar, 50 μm. (F) Experimental outline to test therapeutic effect of local expression of Tnfsf9 and rIL-12 treatment. The in vivo approach is schematically displayed: CT-2A-FLuc-null, CT-2A-FLuc- Tnfsf9 , 005-FLuc-null, and 005-FLuc- Tnfsf9 cells (100,000 cells) were implanted i.c. and mice were treated i.t. with rIL-12 or sham (PBS or Fc control) 10 days after tumor implantation. (G) Survival benefit of local Tnfsf9 expression in CT-2-FLuc-bearing mice post-rIL-12 treatment. Kaplan-Meier curves showing survival outcomes following treatment of CT-2A-FLuc-control with rIL-12 (solid gray) or CT-2A-FLuc- Tnfsf9 treated with rIL-12 (solid pink) ( n = 4–5 mice per group). Mice injected with CT-2A-FLuc- Tnfsf9 tumor cells treated with rIL-12 (50 ng) had a median survival of >50 days ( p = 0.0037) compared with mice implanted with tumor cells lacking 4–1BBL, median survival of 17.5 days. Data represent at least two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using log rank (Mantel-Cox) test, ** p < 0.01. Median survival in days (MS). (H) Survival benefit of local Tnfsf9 expression in 005-FLuc-bearing mice post-rIL-12 treatment. Kaplan-Meier curves showing survival outcomes following treatment of 005-FLuc-control with rIL-12 (solid gray) or 005-FLuc- Tnfsf9 treated with rIL-12 (solid pink) ( n = 4–5 mice per group). Mice injected with 005-FLuc- Tnfsf9 tumor cells treated with rIL-12 (50 ng) had a 100% survival ( p = 0.0045) compared with mice implanted with tumor cells lacking Tnfsf9 , median survival of 38 days. Data represent at least two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using log rank (Mantel-Cox) test, ** p < 0.01. Median survival in days (MS). (I) Experimental outline to test CD8 T cell dependency of Tnfsf9 and rIL-12 combination treatment. Schematic display shows i.v. injection with or without CD8 T cell depletion (αCD8 or IgG control, respectively) on days 9 and 10 (50 and 100 μg on days 9 and 10, respectively) post-tumor (CT-2A-FLuc- Tnfsf9 , 005-Fluc- Tnfsf9 , 100,000 cells) implantation. Mice were injected i.t. with rIL-12 50 ng on day 10. (J) GB mouse survival benefit from Tnfsf9 and rIL-12 combination treatment is CD8 T cell dependent. Kaplan-Meier curves of Il12 +/+ mice showing survival outcomes of CT-2A-FLuc- Tnfsf9 tumor-bearing mice all i.t. treated with rIL-12, after treatment with αCD8 (red) or IgG control (blue). Mice ( n = 5–6 mice per group) treated with IgG control had a median survival of 23.5 days ( p = 0.0283), compared with 20 days for mice treated with αCD8. Data represent two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using log rank (Mantel-Cox) test, * p < 0.05. Median survival in days (MS). (K) GB mouse survival benefit from CD8 T cell recruitment induced by the Tnfsf9 and rIL-12 combination treatment is not dependent on endogenous IL-12. Kaplan-Meier curves of Il12 −/− mice showing survival outcomes of CT-2A-FLuc- Tnfsf9 tumor-bearing mice all treated with i.t. rIL-12, after treatment with αCD8 (red) or IgG control (blue). Mice ( n = 5–6 mice per group) treated with IgG control had a median survival of 35 days ( p = 0.0285), compared with 21 days for mice treated with αCD8. Data represent at least two independent experiments and are presented as the mean ± SEM (error bars). Data were analyzed using log rank (Mantel-Cox) test, * p < 0.05. Median survival in days (MS).

Article Snippet: Samples were then loaded onto a series of LS columns containing microbeads conjugated to anti-mouse CD11b and anti-mouse CD45 (Miltenyi Biotec), respectively, and separated into CD11b POS , CD11b NEG CD45 POS , and CD11b NEG CD45 NEG (non-immune) cell populations using the MACS multi-stand (Milteny Biotec).

Techniques: Isolation, Gene Expression, Plasmid Preparation, Construct, Expressing, Labeling, Transduction, In Vitro, Staining, In Vivo, Control, Tumor Implantation, Injection