mouse cd8 t cell enrichment columns  (R&D Systems)

Journal: Journal of Nanobiotechnology

Article Title: Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy

doi: 10.1186/s12951-024-02314-w

Figure Lengend Snippet: Immunogenic cell death (ICD) of Aposomes in cell culture system. a Fluorescence images CRT-stained CT26 cells after treated with free DOX, DOXIL or Aposomes (2 µM based on DOX contents) for 48 h. b Relative amount of HMGB1 and ATP released from CT26 cells treated with free DOX, DOXIL or Aposomes (2 µM based on DOX contents) for 48 h. c Fluorescence images of CellTracker Green-labeled BMDMs co-cultured with free DOX-, DOXIL- or Aposome-treated CT26 cells (pHrodo labeled) for 2 h. d Percentage of mature DCs (CD11c + CD40 + CD86 + ) and e cytotoxic T cells (CD45 + CD3 + CD8 + ) within the lymphocytes after co-culture with CT26 cells treated with free free DOX, DOXIL or Aposomes for 48 h. f Relative amount of TNF-α, IFN-γ and IL-17 in the cell culture medium after co-culture of lymphocytes with CT26 cells treated with free free DOX, DOXIL or Aposomes for 48 h. Significance was determined by Tukey − Kramer post-hoc test

Article Snippet: M1 macrophages were prepared by incubating M0 macrophages with LPS (1 µg/ml) for 5 h. CD8 + T cells were collected from BALB/c mice using a CD8 + T cell enrichment column, followed by activation for 5 days via a T cell activation/expansion kit (cat# 130-093-627, Miltenyi Biotechnology (Bergisch Gladbach, Germany).

Techniques: Cell Culture, Fluorescence, Staining, Labeling, Co-Culture Assay

Journal: Journal of Nanobiotechnology

Article Title: Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy

doi: 10.1186/s12951-024-02314-w

Figure Lengend Snippet: Antitumor efficacy and immune response of Aposomes in the colon tumor models. a Tumor growth curves of CT26 colon tumor-bearing mice during treatment with an equivalent 3 mg/kg DOX dose of free DOX, DOXIL or Aposomes once every three days. b Tumor tissues stained with TUNEL after 13 days of treatment. c Expression levels of IAP in cancer cells after 13 days of treatment with free DOX, DOXIL or Aposomes. d Survival of mice during treatment with an equivalent 3 mg/kg DOX dose of free DOX, DOXIL or Aposomes once every three days. e The percentage of PD-L1 + tumor cells (CD45 − PD-L1 + ) in the tumor tissues on day 13 after treatment. (f) The amount of HMGB1 and ATP in tumor supernatants on day 13 after treatment. g–j The percentage of g mature DCs (CD11c + CD40 + CD86 + ), h cytotoxic T cells (CD45 + CD3 + CD8 + ) and i , j regulatory T cells (CD45 + CD3 + CD4 + CD25 +) in the tumor tissues on day 13 after treatment. k Relative amount of TNF-α, IFN-γ and IL-17 in tumor supernatants on day 13 after treatment. Significance was determined by Tukey − Kramer post-hoc and log-rank tests

Article Snippet: M1 macrophages were prepared by incubating M0 macrophages with LPS (1 µg/ml) for 5 h. CD8 + T cells were collected from BALB/c mice using a CD8 + T cell enrichment column, followed by activation for 5 days via a T cell activation/expansion kit (cat# 130-093-627, Miltenyi Biotechnology (Bergisch Gladbach, Germany).

Techniques: Staining, TUNEL Assay, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy

doi: 10.1186/s12951-024-02314-w

Figure Lengend Snippet: Combination of Aposomes and ICB therapy in the colon tumor models. a Tumor growth curves of CT26 colon tumor-bearing mice during treatment with DOXIL plus αPD-L1 Ab, Aposomes plus αPD-L1 Ab or αPD-L1 Ab only. Aposomes and DOXIL were injected with an equivalent 3 mg/kg DOX dose once every three days, and αPD-L1 Ab (10 mg/kg) was simultaneously administered via tail vein. b Tumor tissues stained with TUNEL of H&E after 13 days of treatment. c The percentage of cytotoxic T cells (CD45 + CD3 + CD8 + ) in the tumor tissues on day 13 after treatment. d Relative amount of TNF-α, IFN-γ and IL-17 in tumor supernatants on day 13 after treatment. e The percentage of splenic effector/memory CD8 + T cells (CD8 + CD44 + CD62L low ) in CR and naive mice. f Tumor growth curves in naive and CR mice rechallenged with CT26 cells. g Cytokine levels in plasma on day 20 after CR mice were rechallenged with secondary tumors compared to naive mice. Significance was determined by Tukey − Kramer post-hoc test

Article Snippet: M1 macrophages were prepared by incubating M0 macrophages with LPS (1 µg/ml) for 5 h. CD8 + T cells were collected from BALB/c mice using a CD8 + T cell enrichment column, followed by activation for 5 days via a T cell activation/expansion kit (cat# 130-093-627, Miltenyi Biotechnology (Bergisch Gladbach, Germany).

Techniques: Injection, Staining, TUNEL Assay

Journal: Science Advances

Article Title: Xenogenization of tumor cells by fusogenic exosomes in tumor microenvironment ignites and propagates antitumor immunity

doi: 10.1126/sciadv.aaz2083

Figure Lengend Snippet: ( A , B , D , and E ) On day 6 after tumor inoculation, EL4-Ova tumor models were treated (intratumorally) twice at a 1-day interval with 100 μg of mVSVG-Exo, wtVSVG-Exo, Con-Exo, or PBS. Tumor tissues or tumor-draining LN cells were extracted at day 18. (A) The percentage of H2kb-OVA + in CD11c + cells was analyzed by flow cytometric analysis ( n = 4 or 5). (B) The average levels of CD40 or CD86 on DCs (CD11c + cells) were analyzed by flow cytometric analysis. Data are presented as the relative MFI toward the control ( n = 4 or 5). ( C ) IFN-β production of BMDCs treated with 10 μg of mVSVG-Exo, wtVSVG-Exo, Con-Exo, or PBS was assessed by enzyme-linked immunosorbent assay (ELISA) ( n = 2). (D) Macrophages (F4/80 + cells) or DCs (CD11c + cells) of tumor tissues were cocultured for 72 hours with OT-I cells, and the amount of IFN-γ was assessed by ELISA ( n = 3 to 5). (E) The percentage of CD8 + T cells (CD45.2 + CD3 + CD8 + ) and CD4 + T cells (CD45.2 + CD3 + CD4 + ) was evaluated by flow cytometric analysis in tumor-draining LN (DLN) ( n = 6). P values were determined by one-way ANOVA with Tukey's post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The CD8 + T cell enrichment columns and IFN-γ and IFN-β enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Xenogenization of tumor cells by fusogenic exosomes in tumor microenvironment ignites and propagates antitumor immunity

doi: 10.1126/sciadv.aaz2083

Figure Lengend Snippet: ( A ) EL4-Ova tumor-bearing nude mice, ( C ) Batf3 KO mice, or ( D ) CD8 + -depleted mice were injected (intratumorally) twice with 100 μg of mVSVG-Exo, Con-Exo, or PBS. Tumor size (mm 3 ) profiles ( n = 5 to 6). ( B ) EL4-Ova tumor-bearing nude mice were treated (intratumorally) four times with 100 μg of mVSVG-Exo, Con-Exo, or PBS. Tumor size (mm 3 ) profiles ( n = 6 to 8). Arrows indicate treatment time points. ( E and F ) On day 6 after tumor inoculation, EL4-Ova tumor-bearing mice were treated (intratumorally) twice at a 1-day interval with 100 μg of mVSVG-Exo, wtVSVG-Exo, Con-Exo, or PBS. Tumor tissues of EL4-Ova tumor models were extracted at day 18. (E) Representative immunofluorescence images (left) of CD8 + T cell infiltration in the tumor tissues from treated mouse. The number of CD8 + T cells per mm 2 was analyzed using ImageJ software (right) ( n = 10 to 12). Scale bars, 50 μm. (F) The average levels of IFN-γ, TNF-α, or IL-2 on CD8 + T cells in tumor tissues were evaluated by flow cytometry. Data are presented as means of relative MFI toward the control ( n = 3). P values were determined by one-way ANOVA with Tukey's post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The CD8 + T cell enrichment columns and IFN-γ and IFN-β enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems.

Techniques: Injection, Immunofluorescence, Software, Flow Cytometry

Journal: Science Advances

Article Title: Xenogenization of tumor cells by fusogenic exosomes in tumor microenvironment ignites and propagates antitumor immunity

doi: 10.1126/sciadv.aaz2083

Figure Lengend Snippet: ( A ) mVSVG-Exo fuses with the tumor cell membrane in an LDLR- and pH-dependent manner. ( B ) mVSVG-edited tumor cells are more easily engulfed by phagocytes. ( C ) mVSVG-Exo can activate DC functions by stimulating TLR4 signaling. This strategy enhances cross-prime ability, which can further increase CD8 + T cell immunity against cancer.

Article Snippet: The CD8 + T cell enrichment columns and IFN-γ and IFN-β enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems.

Techniques: Membrane

Journal: Cancer Management and Research

Article Title: LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression

doi: 10.2147/CMAR.S210545

Figure Lengend Snippet: LOX-1+ PMN is a population of PMN-MDSC in GBM patient PB. ( A, B ) Suppressive function of LOX-1+ and LOX-1− PMN sorted from PB of GBM patients in allogeneic MLR. The data indicate the mean±sem of three independent experiments. CD4+T cell ( A ) and CD8+ T cell ( B ) proliferation was determined in triplicates using 3H-thymidine uptake. ( C ) Production of ROS in LOX-1+ and LOX-1− PMN from six patients with GBM. Production of ROS was detected by staining with DCFDA. ( D, E ) ARG1 ( D ) and iNOS ( E ) expression in LOX-1- and LOX-1+ PMN from six patients with GBM detected by qPCR. ( F, G ) Effect of 1 μM of N-acetyl L-cysteine (NAC), 1,000 U/mL of catalase and 20 μM nor-NOHA on immune suppressive function of LOX-1+ PMN. The data indicate the mean±sem of three independent experiments. Allogeneic MLR and 1:1 PMN: T cell ratio was used in all experiments. CD4+T cell ( F ) and CD8+ T cell ( G ) proliferation were determined in triplicates by 3H-thymidine incorporation. ** P <0.01, *** P <0.001. Abbreviations: LOX-1, lectin-type oxidized LDL receptor 1; PMN, polymorphonuclear neutrophil; GBM, glioblastoma multiforme; PB, peripheral blood; CPM, counts per minute.

Article Snippet: The isolation of CD4+ or CD8+ T cells from the PBMC of the same patients as LOX-1+ PMN was performed using human CD4+ or CD8+ T Cell Enrichment Column Kit (Miltenyi).

Techniques: Staining, Expressing

Journal: Cancer Management and Research

Article Title: LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression

doi: 10.2147/CMAR.S210545

Figure Lengend Snippet: LOX-1+ PMN is negatively correlation with effector immune cells in GBM patient PB. ( A ) Typical example of the analysis of the proportion of LOX-1+ PMN, IFN-γ+CD4+ T cells and IFN-γ+CD8+ T cells in two patients with GBM. ( B, C ) Quantification showing the associations between LOX-1+ PMN and IFN-γ+CD4+ T cells ( B ) and IFN-γ+CD8+ T cells in 10 patients with GBM ( C ). Abbreviations: LOX-1, lectin-type oxidized LDL receptor 1; PMN, polymorphonuclear neutrophil; GBM, glioblastoma multiforme; PB, peripheral blood.

Article Snippet: The isolation of CD4+ or CD8+ T cells from the PBMC of the same patients as LOX-1+ PMN was performed using human CD4+ or CD8+ T Cell Enrichment Column Kit (Miltenyi).

Techniques:

Journal: Cancer Management and Research

Article Title: LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression

doi: 10.2147/CMAR.S210545

Figure Lengend Snippet: LOX-1 defines a subset of PMN-MDSC in GBM patient tissues. ( A) Typical example of the analysis of PMN in GBM patient tissue. ( B, C ) Suppressive function of LOX-1+ and LOX-1− PMN sorted from tumor tissue of GBM patients in allogeneic MLR. CD4+T cell ( B ) and CD8+ T cell ( C ) proliferation was determined in triplicates using 3H-thymidine uptake. ( D ) Production of ROS in LOX-1+ and LOX-1− PMN from five patients with GBM. Production of ROS was detected by staining with DCFDA. ( E, F ) ARG1 ( E ) and iNOS ( F ) expression in LOX-1- and LOX-1+ PMN from five patients with GBM detected by qPCR. * P <0.05, ** P <0.01, *** P <0.001. Abbreviations: LOX-1, lectin-type oxidized LDL receptor 1; PMN, polymorphonuclear neutrophil; GBM, glioblastoma multiforme; MDSC, myeloid-derived suppressor cell; DCFDA, dichlorodihydrofluorescein diacetate; MLR, mixed lymphocyte reaction; n.s, not significant.

Article Snippet: The isolation of CD4+ or CD8+ T cells from the PBMC of the same patients as LOX-1+ PMN was performed using human CD4+ or CD8+ T Cell Enrichment Column Kit (Miltenyi).

Techniques: Staining, Expressing, Derivative Assay

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: Depletion of CD8+ T cells results in more severe thrombocytopenia and impairs responsiveness to steroid therapy in vivo. For the active model, WT mice were transplanted with immunized β3−/− splenocytes with or without depletion of CD8+ T cells. DEX treatment began at day 6. (A) Thrombocytopenia was more severe in mice given CD8+ T cell–depleted splenocytes compared with those transplanted with nondepleted splenocytes as indicated by &&&P < .001. Mice transplanted with CD8+ T cell–depleted immunized β3−/− splenocytes were less responsive to oral DEX compared with mice transplanted with nondepleted splenocytes as indicated by ###P < .001. ***P < .001, nondepleted splenocytes vs nondepleted splenocytes + DEX. N = 8. In our passive mouse model, CD8+ T cells were depleted from WT mice by injection of anti-CD8 mAb (400 μg intravenously) before inducing passive ITP with anti-β3 mAb (9D2, 1 μg intraperitoneally). Platelet counts were not significantly affected by anti-CD8 mAb depletion of CD8+ T cells. DEX was administered (10 mg/kg/day, intraperitoneally, daily) beginning at 4 hours after anti-β3 mAb injection. (B) Thrombocytopenia was more severe in CD8+ T cell–depleted mice compared with those given the anti-β3 integrin mAb alone. N = 6. (C) CD8+ T cell–depleted thrombocytopenic mice were less responsive to DEX compared with mice with normal levels of CD8+ T cells. N = 6. ***P < .001. Mean ± SD.

Article Snippet: CD8 + T-cell purification and transfusion For in vitro suppression assays and adoptive transfer experiments, CD8 + T cells were enriched (>90%) using a CD8 + T-cell enrichment column (StemCell Technologies).

Techniques: In Vivo, Injection

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ T-cell transfusion therapeutically attenuated platelet clearance and enhanced response to DEX in passive murine model of ITP. In the passive model of ITP, mice were injected with anti-β3 integrin mAb (9D2, 1 μg, intraperitoneally) at day 0 and transfused with 106 CD8+ T cells from either WT, naïve β3−/−, or immunized β3−/− mice. Control cells are remaining splenocytes following CD8+ purification. (A) CD8+ T cells significantly increased platelet count in the absence of any DEX treatment. N = 6. *P < .05, **P < .01, ***P < .001 vs PBS. (B) DEX (10 mg/kg) was administered at 4 hours after mAb injection and CD8+ T-cell transfusion. N = 6. *P < .05, **P < .01, ***P < .001 vs PBS or indicated groups. Mean ± SD.

Article Snippet: CD8 + T-cell purification and transfusion For in vitro suppression assays and adoptive transfer experiments, CD8 + T cells were enriched (>90%) using a CD8 + T-cell enrichment column (StemCell Technologies).

Techniques: Injection, Control, Purification

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: Steroid treatment selectively increased CD8+ Tregs in both the passive and active models of ITP. (A) In the passive model of ITP, mice were injected with anti-β3 mAb (9D2, 1 μg, intraperitoneally) at day 0. In some mice, 10 mg/kg DEX was administered via intraperitoneal injection 4 hours after antibody injection. On indicated days, mice were bled, and different CD8+ subsets were detected via flow cytometry. N = 8 to 10. (B) In the active model, mice were transfused with immunized β3−/− splenocytes on day 0. DEX treatment (oral, 10 mg/kg) or control water was initiated on day 6. Mice were bled on the indicated days, and CD8+ subsets were detected as above. N = 6, *P < .05, **P < .01, ***P < .001. Mean ± SD.

Article Snippet: CD8 + T-cell purification and transfusion For in vitro suppression assays and adoptive transfer experiments, CD8 + T cells were enriched (>90%) using a CD8 + T-cell enrichment column (StemCell Technologies).

Techniques: Injection, Flow Cytometry, Control

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ T cells suppressed platelet apoptosis and clearance in vitro. (A-B) CD8+ T cells from immunized β3−/− mice were added to CD8+-depleted splenocytes and then cocultured with WT platelets. Control has no CD8+ T cells added. (A) After 72 hours, platelets remaining in culture were identified by forward and side scatter characteristics and gated on 7-AAD− and assessed for apoptosis with caspase 3/7 and Annexin V. N = 4. (B) CD8+ T cells from immunized β3−/− mice were cocultured with platelets for 72 hours, and then the remaining platelets in solution were counted, with flow cytometry as events per second. More platelets remained in the coculture system after incubation with CD8+ T cells from immunized β3−/− mice compared with control or naïve β3−/− CD8+ T cells. N = 4. (C) Platelets were labeled with 9D2 primary antibody and FITC anti-mouse IgG. Platelets were then cocultured with macrophages with or without CD8+ T cells. After 24 hours, FITC-positive macrophages were detected by flow cytometry. CD8+, CD8+CD25+, CD8+CD122+, and CD8+CD103+ T cells from immunized β3−/− and WT mice significantly suppressed phagocytosis of platelets by macrophages. Furthermore, the CD8+ T cells from immunized β3−/− mice suppressed phagocytosis more effectively (P < .05). This inhibitory function was also dose dependent (supplemental Figure 4) *P < .05, **P < .01. Mean ± SEM.

Article Snippet: CD8 + T-cell purification and transfusion For in vitro suppression assays and adoptive transfer experiments, CD8 + T cells were enriched (>90%) using a CD8 + T-cell enrichment column (StemCell Technologies).

Techniques: In Vitro, Control, Flow Cytometry, Incubation, Labeling

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ Tregs from immunized β3−/− mice inhibit the adaptive antiplatelet immune response in vitro. For proliferation assays, purified splenic CD8+ T cells or CD8+ Tregs from WT, naïve β3−/−, or WT platelet immunized β3−/− mice were added to CD8+ T cell–depleted splenocytes in the presence of 1 × 107 WT platelets per well to induce T- or B-cell proliferation. Control cells were CD8+-depleted splenocytes with PBS or platelets (PLT) as indicated. Both immunized β3−/− CD8+ T cells and CD8+ Tregs inhibited (A,E) CD4+ T-cell proliferation, (B,F) CD19+ B-cell proliferation, and (C,G) platelet-associated IgG production. (D) Immunized β3−/− CD8+ T cells also inhibited IL-10 cytokine production dose dependently. N = 8. Imm, immunized. *P < .05, **P < .01. Mean ± SEM.

Article Snippet: CD8 + T-cell purification and transfusion For in vitro suppression assays and adoptive transfer experiments, CD8 + T cells were enriched (>90%) using a CD8 + T-cell enrichment column (StemCell Technologies).

Techniques: In Vitro, Purification, Control

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ Tregs from immunized β3−/− mice inhibit CD8+ cytotoxic activity against platelets. Platelets (5 × 106) were cultured with purified 1 × 105 cytotoxic CD8+ T cells (CD8+CD178+) with or without 5 × 105 CD8+ Treg cells (according to the original ratio) for 24 hours. Platelet cytotoxicity was measured with (A) remaining platelet number in supernatant, determined as number of platelets per second counted by flow cytometry69 and (B) caspase 3/7 expression. N = 4. PLT, platelet; CTL, cytotoxic CD8+ T cells. *P < .05, **P < .01. Mean ± SEM.

Article Snippet: CD8 + T-cell purification and transfusion For in vitro suppression assays and adoptive transfer experiments, CD8 + T cells were enriched (>90%) using a CD8 + T-cell enrichment column (StemCell Technologies).

Techniques: Activity Assay, Cell Culture, Purification, Expressing