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: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

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: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

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: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

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: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

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: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

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: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: Activity Assay, Cell Culture, Purification, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Heterosubtypic influenza infection antagonizes elicitation of immunological reactivity to HA

doi: 10.4049/jimmunol.1203520

Figure Lengend Snippet: B10 CD8 T cells were depleted in vivo throughout the duration of secondary infection (days −2, 0, 2, 4, and 6). Eight days post infection, CD4 T cell reactivity to various epitopes was examined by IFNγ Elispot assay. Panel A depicts data obtained from MACS purified CD4 T cells isolated from the spleen. Panel B shows IFNγ Elispots obtained from unpurified MLN cells. Data are presented as spots per 106 CD4 T cells, with MLN data normalized for the percent of CD4 T cells present. Flow cytometric analysis of both spleen and MLN cells indicated complete depletion of CD8 T cells.

Article Snippet: CD8 antibody (Clone 2.43, BioXCell, West Lebanon, NH) was used for in vivo depletion of CD8 T cells.

Techniques: In Vivo, Infection, Enzyme-linked Immunospot, Purification, Isolation

Journal: Cell Reports Medicine

Article Title: Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity

doi: 10.1016/j.xcrm.2024.101531

Figure Lengend Snippet: LH05 significantly reduces systemic toxicity compared with LH01 (A and B) Female BALB/c mice were intraperitoneally injected with PBS, LH01 (5 mg/kg), or LH05 (10 mg/kg) every 3 days, with body weight changes (A) and survival (B) monitored ( n = 8). (C) Spleens of mice were extracted and weighed after euthanasia on day 5 ( n = 4). (D) The percentages of splenic CD8 + T cells are shown for populations of CD3 + lymphocytes ( n = 4). (E) The CD8 + T cells in peripheral blood were counted ( n = 4). (F) The percentages of splenic NK cells are shown for populations of CD45 + lymphocytes ( n = 4). (G) The NK cells in peripheral blood were counted ( n = 4). (H and I) Blood samples ( n = 4) were collected after euthanasia on day 5, and plasma cytokine levels were measured using ELISAs (H); ALT and AST plasma levels were also quantified (I). All graphs show the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant by an unpaired t test or one-way ANOVA. The log rank (Mantel-Cox) test was used to assess survival. See also Figure S3 .

Article Snippet: InVivoMAb anti-mouse CD8a (clone 2.43) , BioXCell , Cat#: BE0061; RRID: AB_1125541.

Techniques: Injection, Clinical Proteomics

Journal: Cell Reports Medicine

Article Title: Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity

doi: 10.1016/j.xcrm.2024.101531

Figure Lengend Snippet: LH05 induces both adaptive and innate immune cells activation (A) The spleens of RM-1 tumor-bearing mice were extracted and weighed after euthanasia ( n = 5). (B and C) The frequency of splenic CD4 + T cells (B) and CD8 + T cells (C) for CD3 + lymphocytes, respectively. (D) The ratio of CD4 + to CD8 + T cells was calculated. (E) The percentage of splenic NK cells for CD45 + lymphocytes was determined. (F) The expression of the memory cell markers CD62L and CD44 on splenic or intratumoral CD8 + T cells was assessed. (G–I) The percentage of intratumoral CD8 + T cells (G) within the population of CD3 + lymphocytes and the frequency of IFNγ + (H) or granzyme B + (I) CD8 + T cells were assessed. (J–L) The percentage of intratumoral NK cells (J) within the population of CD45 + lymphocytes and the frequency of IFNγ + (K) or granzyme B + (L) NK cells were determined. All graphs show the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant by an unpaired t test. See also Figure S5 .

Article Snippet: InVivoMAb anti-mouse CD8a (clone 2.43) , BioXCell , Cat#: BE0061; RRID: AB_1125541.

Techniques: Activation Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity

doi: 10.1016/j.xcrm.2024.101531

Figure Lengend Snippet: The recruited CD8 + T cells and NK cells contribute to LH05-mediated antitumor efficacy (A and B) Growth curves of RM-1 tumors of mice treated with IgG, LH05, and CD8 + T or NK cell depletion (A) or FTY720 (B) in the presence or absence of LH05 ( n = 6). (C and D) Tumor tissues were extracted for RNA-seq analysis ( n = 3). The heatmaps depict gene expression alterations of chemokines and receptors (C) and leukocyte-mediated cytotoxic effectors (D) in response to three LH05 treatments, as indicated by the log2 (fold change) values. (E and F) The expression levels of Pdl1 , Cxcl9 , and Cxcl10 (E) and Ifng , Tbet , and Tnf (F) in the TME were measured using quantitative real-time PCR ( n = 4). All graphs show the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant by an unpaired t test or one-way ANOVA. See also Figure S6 .

Article Snippet: InVivoMAb anti-mouse CD8a (clone 2.43) , BioXCell , Cat#: BE0061; RRID: AB_1125541.

Techniques: RNA Sequencing, Gene Expression, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity

doi: 10.1016/j.xcrm.2024.101531

Figure Lengend Snippet: LH05 exerts enhanced antitumor efficacy in U251 cold tumors and synergizes with an oncolytic virus or checkpoint blockade to control advanced and metastatic cold tumors (A) NOD CRISPR Prkdc Il2r Gamma mice were inoculated subcutaneously with 2 × 10 6 U251 cells and received 4.0 × 10 6 fresh human peripheral blood mononuclear cells intravenously on day 4. Mice were treated with PBS, LH03 (10 mg/kg), LH01 (3 mg/kg), or LH05 (10 mg/kg) intraperitoneally on days 5, 8, and 11 ( n = 5). Tumor volumes were measured. (B) On days 15, mice were euthanized, and tumors were removed and weighed. (C) Immunohistochemical staining for Ki67 was performed on the tumor tissues. (D and E) Treatment was initiated when tumors reached 200 mm 3 . On days 9, 12, and 15 ( n = 8), B16-F10 tumor-bearing mice were intravenously injected with IgG (10 mg/kg) or LH06 (10 mg/kg). An oncolytic virus (5 × 10 5 plaque-forming units) was injected intratumorally on days 9 and 12. Shown are tumor volumes (D) and immunofluorescence staining for CD8 (green) and Foxp3 (red) of tumor tissues (E) (scale bar, 50 μm; n = 2). (F and G) B16-F10 tumor cells (5 × 10 5 ) were intravenously inoculated into C57BL/6 mice. On days 5, 8, and 11 ( n = 8), mice were intravenously injected with IgG control (10 mg/kg), anti-PD-1 (10 mg/kg), LH06 (10 mg/kg), and LH06 (10 mg/kg) + anti-PD-1 (10 mg/kg). The mice were sacrificed on day 15. The colonies on the lungs were counted. (H) LH05 was incubated with human serum at 37°C for 24 or 72 h before the cleavage was measured by ELISA ( n = 4). (I) LH05 was incubated with human cancer homogenate or adjacent normal tissues homogenate at 37°C for 24 h, and the cleavage efficiency was detected by ELISA ( n = 3 for glioma, 8 for lung cancer, and 5 for colon cancer). Data are shown as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns, not significant by an unpaired t test or one-way ANOVA. See also Figure S7 .

Article Snippet: InVivoMAb anti-mouse CD8a (clone 2.43) , BioXCell , Cat#: BE0061; RRID: AB_1125541.

Techniques: Virus, Control, CRISPR, Immunohistochemical staining, Staining, Injection, Immunofluorescence, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity

doi: 10.1016/j.xcrm.2024.101531

Figure Lengend Snippet:

Article Snippet: InVivoMAb anti-mouse CD8a (clone 2.43) , BioXCell , Cat#: BE0061; RRID: AB_1125541.

Techniques: Blocking Assay, Virus, Recombinant, Fluorescence, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Quantitative Proteomics, Expressing, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator.

doi: 10.4049/jimmunol.177.8.5186

Figure Lengend Snippet: FIGURE 4. A, IL-4 (A) and IFN- (B) production by naive CD4 T cells after activation with anti-CD3 and anti-CD28 mAbs as determined by ELISA. C, Proliferation of naive CD4 T cells after activation with anti- CD3 and anti-CD28 mAbs as determined by [3H]thymidine incorporation. In vitro data presented are the means SEM of one representative exper- iment of two with cultures performed in triplicate. Statistical significance was determined by two-way ANOVA. , p 0.0001 compared with values in WT AHA-exposed mice.

Article Snippet: CD4 T cells were isolated from the spleen and lymph nodes (inguinal and mesenteric) by negative selection as previously described (30), using mAbs to CD8 (clone 53-6.72 and clone 2.43) (31), class II MHC I-Ad (clone 212.A1) (31), anti-NK1.1 (PK136; American Type Culture Collection) and anti-Mac-1 (BD Pharmingen) mAbs and anti-Ig-coated magnetic beads (Qiagen).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator.

doi: 10.4049/jimmunol.177.8.5186

Figure Lengend Snippet: FIGURE 3. A, Total lung real-time RT-PCR for IL-4 and IFN- after intranasal AHA exposure. IL-4 and IFN- expression is referenced to non- exposed WT mice. B, Cytokine production by MLN cells isolated from mice after intranasal AHA exposure were in vitro activated with anti-CD3 and anti-CD28 mAbs. C, Cytokine production by ex vivo AHA-activated CD4 T cells after in vivo intranasal AHA exposure. Data presented are the means SEM of one experiment of two with three mice per group. Sta- tistical significance was determined by the Student t test. , p 0.05 compared with values in WT AHA-exposed mice.

Article Snippet: CD4 T cells were isolated from the spleen and lymph nodes (inguinal and mesenteric) by negative selection as previously described (30), using mAbs to CD8 (clone 53-6.72 and clone 2.43) (31), class II MHC I-Ad (clone 212.A1) (31), anti-NK1.1 (PK136; American Type Culture Collection) and anti-Mac-1 (BD Pharmingen) mAbs and anti-Ig-coated magnetic beads (Qiagen).

Techniques: Quantitative RT-PCR, Expressing, Isolation, In Vitro, Ex Vivo, In Vivo

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator.

doi: 10.4049/jimmunol.177.8.5186

Figure Lengend Snippet: FIGURE 6. IL-4 (A) and IFN- (B) production by naive CD4 T cells after activation with anti-CD3 and anti-CD28 mAbs as determined by ELISA. C, Proliferation of naive CD4 T cells after activation with anti- CD3 and anti-CD28 mAbs as determined by [3H]thymidine incorporation. In vitro data presented are the means SEM of one representative exper- iment of three with cultures performed in triplicate. Statistical significance was determined by two-way ANOVA. , p 0.001 compared with values in WT AHA-exposed mice.

Article Snippet: CD4 T cells were isolated from the spleen and lymph nodes (inguinal and mesenteric) by negative selection as previously described (30), using mAbs to CD8 (clone 53-6.72 and clone 2.43) (31), class II MHC I-Ad (clone 212.A1) (31), anti-NK1.1 (PK136; American Type Culture Collection) and anti-Mac-1 (BD Pharmingen) mAbs and anti-Ig-coated magnetic beads (Qiagen).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator.

doi: 10.4049/jimmunol.177.8.5186

Figure Lengend Snippet: FIGURE 8. A, CD4 T cells from wild-type (WT) and F508 (F) mice were activated for 24 h in the presence of anti-CD3 and anti-CD28 mAbs. Nuclear extracts were prepared and examined for NFAT DNA- binding activity by EMSA. B, CD4 T cells from WT, CFTR KO (KO), and F508 (F) mice were activated as in A and nuclear extracts were prepared and examined for NFAT DNA-binding activity in the presence or absence of an anti-NFATc2 Ab by EMSA. CREB DNA binding was also examined as a control for protein loading. C, The densitometric ratio of NFAT complexes to CREB complexes.

Article Snippet: CD4 T cells were isolated from the spleen and lymph nodes (inguinal and mesenteric) by negative selection as previously described (30), using mAbs to CD8 (clone 53-6.72 and clone 2.43) (31), class II MHC I-Ad (clone 212.A1) (31), anti-NK1.1 (PK136; American Type Culture Collection) and anti-Mac-1 (BD Pharmingen) mAbs and anti-Ig-coated magnetic beads (Qiagen).

Techniques: Binding Assay, Activity Assay, Control

Journal: Frontiers in Immunology

Article Title: FLT3L combined with GM-CSF induced dendritic cells drive broad tumor-specific CD8 + T cell responses and remodel the tumor microenvironment to enhance anti-tumor efficacy

doi: 10.3389/fimmu.2025.1649891

Figure Lengend Snippet: FL/GM-DCs enhance the anti-tumor immune response. (a) BM cells, FL/GM-DCs, and GM-DCs were cultured in equal numbers, and OVA-FITC was added for 4 h at 37 °C. OVA uptake was quantified using flow cytometry by first gating on cells based on MHCII and CD11c expression, and the results are displayed as a histogram. Each group contained at least n = 3 biological replicates. (b, c) FL/GM-DCs (b) and GM/IL4-DCs (c) were cultured in equal numbers and co-cultured with OVA at concentrations of 1μg/ml, 10μg/ml, 100μg/ml, 500μg/ml, and 1000μg/ml at 37 °C for 24 hours. The expression levels of H - 2Kb OVA257 – 264 on DCs were measured by flow cytometry. Each group contained at least n = 3 biological replicates. (d) Flow cytometric analysis of expression levels of the chemokine receptor CCR7, gated within MHCII+CD11c+ cells. The analyzed cells were stimulated with polyI:C and R484 prior to analysis. Each group contained at least n = 3 biological replicates. (e) Flow cytometric analysis of expression levels of the Integrin CD103, gated within MHCII + CD11c + cells. Each group contained at least n = 3 biological replicates. (f) Flow cytometric analysis of expression levels of the costimulatory molecule CD86, gated within MHCII + CD11c + cells. Each group contained at least n = 3 biological replicates. (g) Cytokine and chemokine levels, including IL - 12p70, IL - 12p40, IL - 10, IL - 6, TNF-α, IFN-γ, CCL2, CCL4, CCL5, and CXCL1, were measured in the supernatants of FL/GM-DC and GM/IL4-DC cultures after antigen stimulation and maturation for 72 h (n=3). (h) Schematic of T cell proliferation assays using CFSE-labeled OT-I and OT-II T cells co-cultured with B16-OVA tumor lysate-pulsed DCs. (i) Flow cytometric analysis of CD8 + T cell proliferation and activation after 96 h of co-culture. (j) Flow cytometric analysis of CD4 + T cell proliferation and activation after 96 h of co-culture.

Article Snippet: Mice were intraperitoneally injected with 100μL of the following antibodies: anti-mouse F4/80 (Bio X Cell Cat# BE0206, CI:A3-1, RRID: AB_10949019), anti-mouse CD8a (Selleck Cat# A2102, 2.43, RRID: AB_3099521), anti-mouse CD4 (Selleck Cat# A2101, GK1.5, RRID: AB_3677296), or rat IgG2b isotype control (Selleck Cat# A2116, LTF - 2, RRID: AB_3662740).

Techniques: Cell Culture, Flow Cytometry, Expressing, Labeling, Activation Assay, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: FLT3L combined with GM-CSF induced dendritic cells drive broad tumor-specific CD8 + T cell responses and remodel the tumor microenvironment to enhance anti-tumor efficacy

doi: 10.3389/fimmu.2025.1649891

Figure Lengend Snippet: FL/GM-DCs exhibit migration and long-term survival in vivo . (a) Experimental protocol for tracking the fate of adoptively transferred FL/GM-DCs and GM/IL4-DCs in tumor-free mice (above) and MC38 tumor-bearing mice (below). In this experiment, each group consisted of 3 mice. (b) Flow cytometric analysis of CD45.1 + CD8 + T cell infiltration in the lymph nodes of tumor-free mice at 7 or 14 days after adoptive transfer of DCs and T cells. (c) Flow cytometric analysis of the proportion of CD45.1 + cells in the lymph nodes of MC38 tumor-bearing mice on days 7, 14, and 30 post-transfer. (d) Flow cytometric analysis of the proportion of CD45.1 + cells in MC38 tumor tissue on days 7, 14, and 30 post-transfer. (e) Flow cytometric analysis of infiltrating CD45.1 + DCs (MHCII + CD86 + ) in MC38 tumor tissue after adoptive transfer of CD45.1 + DCs. (f) Flow cytometry histogram showing XCR1 expression on MHCII + CD86 + DCs.

Article Snippet: Mice were intraperitoneally injected with 100μL of the following antibodies: anti-mouse F4/80 (Bio X Cell Cat# BE0206, CI:A3-1, RRID: AB_10949019), anti-mouse CD8a (Selleck Cat# A2102, 2.43, RRID: AB_3099521), anti-mouse CD4 (Selleck Cat# A2101, GK1.5, RRID: AB_3677296), or rat IgG2b isotype control (Selleck Cat# A2116, LTF - 2, RRID: AB_3662740).

Techniques: Migration, In Vivo, Adoptive Transfer Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: FLT3L combined with GM-CSF induced dendritic cells drive broad tumor-specific CD8 + T cell responses and remodel the tumor microenvironment to enhance anti-tumor efficacy

doi: 10.3389/fimmu.2025.1649891

Figure Lengend Snippet: The anti-tumor effect of FL/GM-DC in tumor models. (a) Experimental protocol for studying the anti-tumor effects of FL/GM-DCs and GM/IL4-DCs in the tumor models. Mice with tumors were vaccinated on days 3, 6, 9, 12, 15, and 21 with FL/GM-DCs or GM/IL4-DCs (Each mouse received 2.5 x 10 6 cells per administration). Measured the size of the tumor every 3 days. (b) Tumor size in MC38 tumor-bearing mice measured over time (mean ± SEM; NC, n=8; FL/GM-DC, n=8; GM/IL4-DC, n=8). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. (c) Flow cytometric analysis of the proportion of infiltrating DCs (MHCII + CD11c + ) in MC38 tumor tissue. (d) Representative fluorescence images of MC38 tumors from treated mice, showing CD11c (green), CD8a (orange), CD4 (pink), Foxp3 (red), and DAPI nuclear staining (blue). Scale bar, 100 μm. The bar chart represented the positive rate of CD8a + , CD4 + , and Foxp3 + cells in the tumors of each group of MC38 mice. (e) Experimental research protocol on the anti-tumor effects of FL/GM-DCs and GM/IL4-DCs in an antibody-blockaded MC38 tumor model. Mice with tumors were vaccinated on days 3, 6, 9, 12 with FL/GM-DCs or GM/IL4-DCs (Each mouse received 2.5 x 106 cells per administration) and received antibody blocking therapy (Each mouse received 200ug antibodies per administration) on the next day. Measured the size of the tumor every 3 days. (f) Tumor size in MC38 tumor-bearing mice measured over time (mean ± SEM; NC, n=6; FL/GM-DC+IgG2b, n=6; FL/GM-DC+antiCD8a, n=6; FL/GM-DC+antiCD4, n=6; FL/GM-DC+antiF4/80, n=6). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. (g) Each subplot displayed the tumor volume over time for individual mice in different treatment groups (NC, FL/GM-DC+IgG2b, FL/GM-DC+antiCD8a, FL/GM-DC+antiCD4, and FL/GM-DC+antiF4/80).

Article Snippet: Mice were intraperitoneally injected with 100μL of the following antibodies: anti-mouse F4/80 (Bio X Cell Cat# BE0206, CI:A3-1, RRID: AB_10949019), anti-mouse CD8a (Selleck Cat# A2102, 2.43, RRID: AB_3099521), anti-mouse CD4 (Selleck Cat# A2101, GK1.5, RRID: AB_3677296), or rat IgG2b isotype control (Selleck Cat# A2116, LTF - 2, RRID: AB_3662740).

Techniques: Fluorescence, Staining, Blocking Assay