Journal: Frontiers in Immunology

Article Title: Allergic T H 2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4 + T Cells

doi: 10.3389/fimmu.2018.00750

Figure Lengend Snippet: Role of Bcl6-mediated MPT cell functions in NAT H 2 differentiation in an allergic murine model. [ (A) top] Mixture of purified KJ1-26 − MPT cells ( Bcl6 -WT or Bcl6 -KO) and KJ1-26 + WT naïve CD4 + T cells were transferred into BALB/c nu/nu mice intravenously (day 0). These mice were immunized with alum-conjugated OVA and then intratracheally challenged with OVA. [ (A) bottom] Absolute cell numbers of Neu, Eos, AM, and Lym in BALF, (B) hematoxylin and eosin-stained, formalin-fixed lung sections (magnification: 200×), and (C) T H 2 cytokine levels in the BALF of recipient mice 48 h after the last OVA challenge. (D) Relative Il4, Il5 , and Il13 expression mRNA in splenic KJ1-26 + T cells restimulated with anti-CD3 monoclonal antibodies 5 days after the last challenge. (E) OVA-specific IgE antibody titers in sera from each recipient of Bcl6 -WT NAT H 2 cells, plus MPT H 2 cells transferred from Bcl6 -TG, Bcl6 -WT, or Bcl6 -KO mice 2 days after the last challenge. All results are representative of four independent experiments with similar outcomes. Data are presented as the mean ± SEM ( n = 5–7). * P < 0.05, ** P < 0.01, comparison between two groups is indicated. AM, alveolar macrophages; BALF, bronchoalveolar lavage fluid; Bcl6, B-cell lymphoma 6; Eos, eosinophils; KJ + , KJ1-26-positive; KO, knockout; Lym, lymphocytes; MPT cell, memory phenotype CD4 + T cell; MPT H 2 cell, MPT cell-derived T H 2 cell; NAT H 2 cell; naïve CD4 + T cell-derived T H 2 cell; Neu, neutrophils; NS, not significant; OVA, ovalbumin; TG, transgenic; WT, wild-type.

Article Snippet: IgE anti-OVA Abs were detected using a mouse anti-OVA IgE Antibody Assay Kit (Chondrex, Redmond, WA, USA).

Techniques: Purification, Staining, Expressing, Knock-Out, Derivative Assay, Transgenic Assay

Journal: ACS Central Science

Article Title: Injectable Hydrogels for Sustained Codelivery of Subunit Vaccines Enhance Humoral Immunity

doi: 10.1021/acscentsci.0c00732

Figure Lengend Snippet: Antibody concentration and affinity following immunization. (a) Timeline of the experimental setup shows subcutaneous (SC) injection of a model vaccine containing OVA and Poly(I:C) in a gel or bolus formulation at day 0, antibody analysis over time following a single administration, boost with a bolus vaccine formulation at day 90, and analysis of the immune response 15 days after the boost. (b) Serum anti-OVA IgG1 concentrations from day 0 to day 90 after a single injection of vaccines ( n = 5 to 19; one to four independent experiments; mean ± s.e.m.). *** p < 0.001 and **** p < 0.0001 compared to bolus, # p < 0.05, ## p < 0.005 compared to 1:5, determined by mixed-effects analysis with Tukey’s post hoc test. Serum anti-OVA (c) IgG end point titer, (d) IgG1 concentration, (e) IgG2b concentration, and (f) IgG2c concentration 15 days after bolus boost on day 90 for animals receiving either bolus, 1:5 gel, or 2:10 gel vaccines ( n = 4 to 5; mean ± s.d.). Reported p values determined by one-way ANOVA with Tukey’s post hoc test. (g) Model comparing competitive binding data with K D ranging from 1 to 10 4 nM. (h) Representative competitive binding curves for bolus, 1:5 gel, and 2:10 gel vaccine groups after the day 90 boost compared to an mAb reference competing with the same mAb. (i) Calculated K D values from fitted binding curves for bolus, 1:5 gel, and 2:10 gel vaccine groups ( n = 4; mean ± s.d.). p values determined by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Anti-OVA IgG2b and IgG2c antibody concentrations were measured using mouse anti-OVA antibody assay kits for IgG2b (Chondrex, 3016) and IgG2c (Chondrex, 3029).

Techniques: Concentration Assay, Injection, Formulation, Vaccines, Binding Assay

Journal: ACS Central Science

Article Title: Injectable Hydrogels for Sustained Codelivery of Subunit Vaccines Enhance Humoral Immunity

doi: 10.1021/acscentsci.0c00732

Figure Lengend Snippet: Germinal center response to single vaccine administration. (a) Immunohistochemistry (IHC) of explanted inguinal lymph node 15 days after OVA + Poly(I:C) vaccine administration in 2:10 and bolus groups to visualize germinal centers (GC) (red) and naïve B cells (green). (b,c) The frequency of germinal center B cells (GCBCs) within total B cells at day 15 (b) and day 30 (c) after prime and (d,e) frequency of IgG1 + GCBCs within total GCBCs at day 15 (d) and day 30 (e) after prime in the inguinal lymph nodes were measured by flow cytometry ( n = 5 to 10). (f) Schematic of the GC response. (g) The percent of T follicular helper cells (Tfh) out of the CD4 + cell population and (h) and the ratio of lights zone (LZ) to dark zone (DZ) GCBCs in the inguinal lymph nodes at day 15 after vaccinatation ( n = 5 to 10). For (b,d,h), data come from two independent experiments, and all other graphs represent one independent experiment. All error bars are mean ± s.d.; p values are determined by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Anti-OVA IgG2b and IgG2c antibody concentrations were measured using mouse anti-OVA antibody assay kits for IgG2b (Chondrex, 3016) and IgG2c (Chondrex, 3029).

Techniques: Immunohistochemistry, Flow Cytometry

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

Article Title: Th17 Cells Induce Th1 Polarizing Monocyte-Derived Dendritic Cells

doi: 10.4049/jimmunol.1203201

Figure Lengend Snippet: A) Bone-marrow monocytes were cultured at a ratio of 10:1 with activated bulk CD4+ T-cells (ThBulk) for 5 days in the presence of 10ng/ml IL-2 (DCThBulk), or as controls, monocytes were cultured alone, with GM-CSF and IL-4, or with M-CSF. The cells were then imaged with DIC microscopy. The scale bar represents 50 microns. B) Cell surface phenotype of DAPI−, Thy1.2−, CD11b+ cells after 5 days of culture with GM-CSF and IL-4 (solid gray), with ThBulk cells and IL-2 (solid black) or fresh after isolation (gray line). Median fluorescence intensity (MFI) for each marker is shown in the table above. Data are representative of more than three independent experiments. C and D) DCTh or fresh monocytes were purified, pulsed with MHC II-restricted OVA peptide and 1μg/ml LPS for 90 minutes, washed and then cultured in triplicate with T-cells for 3 days and pulsed with H3-thymidine for the final 18 hours. The dotted line shows the mean proliferation of the T-cells in the absence of APC. The data shown are representative of more than 3 independent experiments. C) DCThBulk, fresh bone marrow monocytes and GM IL-4 DC were pulsed with 2.5μg/ml OVA peptide and cultured at a ratio of 1 APC: 15 naïve T-cells from OT-II RAG KO mice. D) Cells were pulsed with 2.5μg/ml MOG peptide and cultured at various ratios with naïve T-cells from 2D2 mice. Error bars represent SEM.

Article Snippet: Purified DC Th were pulsed with 2.5μg/ml MHC II restricted ISQ peptide (OVA 323–339) (New England Peptides) or 2.5μg/ml MHC II restricted MOG peptide ( 33 – 55 ) in complete media for 90 minutes at 37°C.

Techniques: Cell Culture, Microscopy, Isolation, Fluorescence, Marker, Purification

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

Article Title: Th17 Cells Induce Th1 Polarizing Monocyte-Derived Dendritic Cells

doi: 10.4049/jimmunol.1203201

Figure Lengend Snippet: A) CD8 depleted splenocytes were activated with 1μg/ml αCD3 in the presence of various polarizing cytokines and blocking antibodies to skew the T-cells toward a Th1, Th2 or Th17 phenotype. After 96 hours, the cells were activated and stained intracellularly for IFNγ, IL-4 and IL-17A. Cells shown are gated on Live/Dead Blue−, Thy1.2+ B) Bone-marrow monocytes as used in Figure 1 were cultured 10:1 with Th1, Th2, or Th17 polarized T-cells for 5 days in the presence of 10ng/ml IL-2. The cultures were then imaged with DIC microscopy. C) Cell surface phenotype of DAPI−, Thy1.2−, CD11b+ cells after 5 days of culture with Th1 (blue), Th2 (green), Th17 polarized T-cells (orange), or fresh after isolation. MFI for each marker is shown in the table above. This experiment was performed at the same time and under identical conditions as Figure 1C. Data shown are representative of more than 3 independent experiments. D) DCTh1, DCTh2, DCTh17, M-CSF macrophages or fresh monocytes were purified, pulsed with MHC II-restricted OVA peptide and 1μg/ml LPS for 90 minutes, washed and then cultured at a ratio of 1 APC:15 naïve T-cells from OT-II RAG KO mice in triplicate for 3 days and pulsed with H3 thymidine for the last 18 hours. The dotted line shows the mean proliferation of the T-cells in the absence of APC. Results are representative of 2 independent experiments. Error bars represent SEM.

Article Snippet: Purified DC Th were pulsed with 2.5μg/ml MHC II restricted ISQ peptide (OVA 323–339) (New England Peptides) or 2.5μg/ml MHC II restricted MOG peptide ( 33 – 55 ) in complete media for 90 minutes at 37°C.

Techniques: Blocking Assay, Staining, Cell Culture, Microscopy, Isolation, Marker, Purification

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

Article Title: Th17 Cells Induce Th1 Polarizing Monocyte-Derived Dendritic Cells

doi: 10.4049/jimmunol.1203201

Figure Lengend Snippet: A) DCTh subsets or control APCs were purified with magnetic microbeads and then pulsed with 2.5μg/ml MHC II-restricted OVA peptide and 1μg/ml LPS for 90 minutes. The cells were washed twice and then cultured at a ratio of 1 APC:15 naïve T-cells from OT-II RAG KO mice. On day 4 of culture, the cells were restimulated with PMA/ionomycin and BFA for 4 hours prior to intracellular staining. Cells shown are gated on live/dead blue−, Thy1.2+ cells. Data are representative of 2 independent experiments. B) Th17 cells were rested in media for 2 days and then cultured 1 DC:5 T-cells with DCTh1 or DCTh17 that had been pulsed with PBS, LPS alone or LPS and MHC II-restricted OVA peptide for 90 minutes. After 3 days of coculture, the T-cells were activated with PMA/ionomycin and BFA for 4 hours and then intracellularly stained for IL-17A and IFNγ. Cells were gated on live/dead blue−, Thy1.2+ cells. One replicate for each condition is shown. Data are representative of 2 independent experiments. C) IL-17A secreting OT-II T-cells were sorted from in vitro Th17 cultures, rested in the absence of IL-2 for 3 days and then cocultured with ISQ and LPS pulsed DCTh1 or DCTh17. After 4 days the ex-IL-17A+ cells were evaluated for IL-17A and IFNγ expression by intracellular cytokine staining. Done in duplicate. D) The percentage of IFNγ and IL-17A single positive T-cells in each condition are shown. Error bars represent SEM.

Article Snippet: Purified DC Th were pulsed with 2.5μg/ml MHC II restricted ISQ peptide (OVA 323–339) (New England Peptides) or 2.5μg/ml MHC II restricted MOG peptide ( 33 – 55 ) in complete media for 90 minutes at 37°C.

Techniques: Control, Purification, Cell Culture, Staining, In Vitro, Expressing

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

Article Title: Th17 Cells Induce Th1 Polarizing Monocyte-Derived Dendritic Cells

doi: 10.4049/jimmunol.1203201

Figure Lengend Snippet: A) CD4+ T-cells from EAE mice spinal cords or a healthy wild-type spleen cells were cultured 1:10 with monocytes alone or monocytes in the presence of 10ng/ml IL-2 for 4 days and then imaged with bright field microscopy. The scale bar represents 50 microns. The results are representative of 4 independent experiments. B) Cells from A) were gated on CD4−, CD11b+, DAPI− and analyzed by flow cytometry. MFI for each marker is shown in the table above. Results are representative of 3 independent experiments. C) Cells from A) were harvested, pulsed with MHC II-restricted OVA peptide and then cultured in triplicate at a ratio of 1 APC:33 naïve T-cells from OT-II RAG KO mice for 3 days and then pulsed with H3 thymidine for 20 hours. The dotted horizontal line represents the mean proliferation of OT-II T-cells in the absence of APC. The solid horizontal line represents the mean proliferation of the DCThEAE without OT-II T-cells. Cultures were performed in triplicate. D) Cells prepared as in A) were stimulated overnight with 1μg/ml of LPS. The cell-free supernatant was assayed in duplicate by ELISA for IL-12p70. Data shown are representative of 2 independent experiments. ND = Not Detected. E) CD11b+ DCThEAE selected via MACS were pulsed with or without 1μg/ml LPS and 2.5μg/ml OVA peptide for 90 minutes, washed and then cultured at a ratio of 1 APC: 5 naïve T-cells from OT-II RAG KO mice for 5 days. Supernatant was collected and assayed via ELISA for IFNγ. Done in triplicate. F) BM chimeric mice were generated using wild-type or IL-12p35 KO BM. 10 weeks later, the mice were injected with Th17 skewed encephalitogenic cells to induce EAE, and mice were monitored for signs of disease on a daily basis. N = 4–5 mice per group. Data are presented as means ± SEM, with significance determined by a two-tailed Mann-Whitney test. Day 17 p = 0.27, Day 18 p = 0.36, Day 19 p = 0.37, Day 20 p = 0.45

Article Snippet: Purified DC Th were pulsed with 2.5μg/ml MHC II restricted ISQ peptide (OVA 323–339) (New England Peptides) or 2.5μg/ml MHC II restricted MOG peptide ( 33 – 55 ) in complete media for 90 minutes at 37°C.

Techniques: Cell Culture, Microscopy, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, Generated, Injection, Two Tailed Test, MANN-WHITNEY

Journal: ACS Omega

Article Title: Dendritic Cell Membrane-Derived Nanovesicles for Targeted T Cell Activation

doi: 10.1021/acsomega.2c04420

Figure Lengend Snippet: Phenotypic characterization of unstimulated and IFN-γ-stimulated DC2.4 cells. DC2.4 cells were untreated or treated with 20 ng/mL IFN-γ overnight. Expression of CD11c, CD40, CD54, CD80, CD86, MHC class I, MHC class II, and MHC class I-bound SIINFEKL peptide (red) by untreated and IFN-γ treated DC2.4 was analyzed by flow cytometry. Isotype-matched antibodies (black) were used as controls. Following IFN-γ stimulation, DC2.4 expression of the lineage marker CD11c remained constant, and that of maturation markers CD40, CD54, CD80, CD86, and MHC class I increased, while that of the maturation marker MHC class II decreased. Presentation of SIINFEKL peptide by MHC class I also increased following IFN-γ stimulation.

Article Snippet: When indicated, DC2.4 cells were cultured with 20 ng/mL recombinant mouse IFN-γ (Invitrogen) with or without 5 μg/mL SIINFEKL peptide (AnaSpec) overnight.

Techniques: Expressing, Flow Cytometry, Marker

Journal: ACS Omega

Article Title: Dendritic Cell Membrane-Derived Nanovesicles for Targeted T Cell Activation

doi: 10.1021/acsomega.2c04420

Figure Lengend Snippet: Characterization of CDNV surface proteins by western blot. Western blot of precursor DC2.4 cell and DC2.4-derived CDNVs incubated with 5 μg/mL SIINFEKL peptide and with or without 20 ng/mL IFN-γ overnight. Molecules required for T cell activation, namely, CD80, CD86, and SIINFEKL-presenting MHC class I, were observed on CDNVs from both immature and mature parental DC2.4 cells.

Article Snippet: When indicated, DC2.4 cells were cultured with 20 ng/mL recombinant mouse IFN-γ (Invitrogen) with or without 5 μg/mL SIINFEKL peptide (AnaSpec) overnight.

Techniques: Western Blot, Derivative Assay, Incubation, Activation Assay

Journal: ACS Omega

Article Title: Dendritic Cell Membrane-Derived Nanovesicles for Targeted T Cell Activation

doi: 10.1021/acsomega.2c04420

Figure Lengend Snippet: SIINFEKL-presenting CDNVs promote direct activation of T cells. (A) Schematic demonstrating the proposed mechanism of CDNV production and direct activation of T cells. In this approach, immature DC2.4 cells are pulsed with 20 ng/mL IFN-γ to stimulate maturation and 5 μg/mL SIINFEKL peptide for cross-presentation. CDNVs are then generated from mature, peptide-presenting DC2.4 cells by nitrogen cavitation and isolated via differential ultracentrifugation. Direct CD8 + T cell activation is mediated by interaction of pMHC complexes and costimulatory molecules on CDNV SIIN + /IFN - γ + with TCR and costimulatory receptors on recipient CD8 + T cells. (B) Phenotypic analysis of the T cell early activation marker CD69 following CD8 + T cell treatment with CDNVs generated from immature (IFN-γ – ) or mature (IFN-γ + ) DCs incubated with (SIIN + ) or without (SIIN – ) SIINFEKL peptide. ( C ) Expression of CD69, measured as the geometric mean fluorescence intensity (Geo. MFI) following incubation of CDNVs generated from immature (IFN-γ – ) or mature (IFN-γ + ) DCs incubated with (SIIN + ) or without (SIIN – ) SIINFEKL peptide. Statistical analysis was performed using one-way ANOVA with Tukey’s HSD test comparing immature to mature CDNVs (* p > 0.05, ** p > 0.01, and *** p > 0.001) or CDNV mixture ( # p > 0.05, ## p > 0.01, and ### p > 0.001) (mean ± SEM, n = 3).

Article Snippet: When indicated, DC2.4 cells were cultured with 20 ng/mL recombinant mouse IFN-γ (Invitrogen) with or without 5 μg/mL SIINFEKL peptide (AnaSpec) overnight.

Techniques: Activation Assay, Generated, Isolation, Marker, Incubation, Expressing, Fluorescence

Journal: ACS Omega

Article Title: Dendritic Cell Membrane-Derived Nanovesicles for Targeted T Cell Activation

doi: 10.1021/acsomega.2c04420

Figure Lengend Snippet: SIINFEKL-presenting CDNVs promote indirect activation of T cells through delivery of antigen to recipient APCs. (A) Schematic demonstrating the mechanism of CDNV production and delivery of antigenic peptide to bystander APCs, conferring the ability to activate T cells. As before, immature DC2.4 cells were pulsed with 20 ng/mL IFN-γ and 5 μg/mL SIINFEKL peptide, which then underwent nitrogen cavitation and differential centrifugation to generate and isolate peptide-presenting CDNVs. Upon DC2.4 treatment with peptide-presenting CDNVs, recipient DC2.4 cells may take up and present antigenic peptides in MHC I complexes on their surface through cross-presentation or cross-dressing. Antigenic peptide-presenting DC2.4 cells then interact with naïve CD8 + T cells, stimulating T cell activation. (B) Phenotypic analysis of T cell activation by T cell early activation marker CD69 expression following CD8 + T cell treatment with DC2.4 cells pulsed with CDNV SIIN + /IFN - γ – , CDNV SIIN + /IFN - γ + , or 1:1 mixture of CDNV SIIN + /IFN - γ – + CDNV SIIN + /IFN - γ + (CDNV Mix ). ( C ) Expression of the early T cell activation marker CD69, measured as Geo. MFI, following incubation with DC2.4 cells pulsed with CDNV SIIN + /IFN - γ – , CDNV SIIN + /IFN - γ + , or CDNV Mix . Statistical analysis was performed using one-way ANOVA with Tukey’s HSD test comparing immature to mature CDNVs (* p > 0.05, ** p > 0.01, and *** p > 0.001) or CDNV mixture ( # p > 0.05, ## p > 0.01, and ### p > 0.001) (mean ± SEM, n = 3).

Article Snippet: When indicated, DC2.4 cells were cultured with 20 ng/mL recombinant mouse IFN-γ (Invitrogen) with or without 5 μg/mL SIINFEKL peptide (AnaSpec) overnight.

Techniques: Activation Assay, Centrifugation, Marker, Expressing, Incubation