Journal: Advanced Healthcare Materials

Article Title: Endogenous/Exogenous Nanovaccines Synergistically Enhance Dendritic Cell‐Mediated Tumor Immunotherapy

doi: 10.1002/adhm.202203028

Figure Lengend Snippet: DC maturation triggered by MXP‐mediated PTT in vitro. A) Schematic illustration of the coculture system of B16‐OVA tumor cells and mutuDCs. B16‐OVA cells were cultured in the upper wells, and DCs were cultured in the bottom wells. B16‐OVA cells were treated with MXP and NIR irradiation in the MXP+NIR group. FCM analysis and quantification of B,C) CD40 + , D,E) CD80 + , and F,G) CD86 + DCs. H) FCM analysis and I) quantification of SIINFEKL‐H‐2Kb + DCs. J) Schematic illustration of MXP‐mediated PTT through increasing the maturation process of DCs.

Article Snippet: Briefly, SIINFEKL peptide‐restimulated splenocytes were stained with anti‐CD3 and anti‐CD8 antibodies for surface staining, fixed and permeabilized using a BD Cytofix/Cytoperm kit, and intracellularly stained with an anti‐IFN‐ γ antibody in BD Perm/Wash buffer.

Techniques: In Vitro, Cell Culture, Irradiation

Journal: Advanced Healthcare Materials

Article Title: Endogenous/Exogenous Nanovaccines Synergistically Enhance Dendritic Cell‐Mediated Tumor Immunotherapy

doi: 10.1002/adhm.202203028

Figure Lengend Snippet: The MXP@OC vaccine enhances lymphatic drainage and DC‐mediated immune responses. A) FITC + cells in pLNs and iLNs after the administration of PBS, OC, or MXP@OC. Blue, nuclei; green, OVA‐FITC. B) Schematic illustration of the experimental schedule used to evaluate the immune responses to the nanovaccines. C) OVA‐specific immunoglobulin (IgG) production in the harvested serum. D) Ratios of CD11c + SIINFEKL‐H‐2Kb + cells in LNs as measured by FCM. E) Statistical analysis of CD11c + SIINFEKL‐H‐2Kb + cells. F,G) Representative FCM plots and statistical analysis of CD11c + CD80 + cells. H,I) Representative FCM plots and statistical analysis of CD11c + CD86 + cells. J) Representative FCM images of CD3 + CD8 + IFN‐ γ + T cells in the splenocytes of immunized mice after restimulation with the SIINFEKL peptide. The data are presented as the means ± SD (n = 5/group). For statistical significance, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Briefly, SIINFEKL peptide‐restimulated splenocytes were stained with anti‐CD3 and anti‐CD8 antibodies for surface staining, fixed and permeabilized using a BD Cytofix/Cytoperm kit, and intracellularly stained with an anti‐IFN‐ γ antibody in BD Perm/Wash buffer.

Techniques:

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

Article Title: Switch Region Identity Plays an Important Role in Ig Class Switch Recombination

doi: 10.4049/jimmunol.1000507

Figure Lengend Snippet: Expression of γ3 GLT from intact and Sγ1 replacement alleles. GLT γ3, γ1, and AID expression were analyzed by semiquantitative RT-PCR using cDNAs derived from purified B cells or splenocytes that were either unstimulated (U) or activated with LPS (L) alone or LPS plus IL-4lo (1 ng/ml) or LPS plus IL-4hi (10 ng/ml) for 48 h. RT-PCR analyses of the Gapd or Hprt transcripts were used as loading controls. A, Purified B cells were unstimulated or stimulated and analyzed as indicated. B, WT F1 and chimeric (Chi 28) splenocyte cultures, activated as indicated, and then analyzed for γ3 or γ1 GLT expression. C, LPS-activated WT F1 and chimeric (Chi D, E, F) splenocyte cultures are analyzed for γ3 GLTs. Allele-specific products were distinguished by PstI digestion, which generates 180- and 125-bp restriction fragments derived from the C57/B6 (b) and 129 (a) alleles, respectively. Samples in lanes 3–7 were digested with PstI. Samples are m.w. marker, M (lane 1), intact γ3 GLT (lane 2), and γ3 GLT from the I29 a allele (lane 3), the C57BL/6 b allele (lane 4), the 129 × C57BL/6 (F1) a and b alleles (lane 5), and Chi mice (lanes 6–8). D, WT F1 and chimeric (Chi 1) splenocyte cultures activated as indicated were assessed for allele-specific γ3 GLT by RT-PCR followed by Southern blot analysis.

Article Snippet: C and D , DC-PCR analysis of DNA from splenocytes from WT F1 and three independent chimeras (Chi D, E, F) that were stimulated with LPS alone or in the presence of LPS and IL-4 lo or IL-4 hi for 5 d. All DC-PCR products were harvested at 26, 29, and 32 cycles.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Purification, Marker, Southern Blot

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

Article Title: Switch Region Identity Plays an Important Role in Ig Class Switch Recombination

doi: 10.4049/jimmunol.1000507

Figure Lengend Snippet: Active μ→γ3 CSR in Sγ1 replacement splenocytes following LPS plus IL-4 induction. Purified B cells (A) or splenocytes from C57BL/6 × 129 (WT-F1) or Sγ1 replacement chimeric mice (B) were cultured for 5 d with LPS (L) in the presence or absence of IL-4lo or IL-4hi, as indicated. PST Iμ-Cγ3 and Iμ-Cγ1 were analyzed by semiquantitative RT-PCR. RT-PCR analyses of the Gapd or Hprt transcripts were used as loading controls. A, Purified B cells were unstimulated (U) or stimulated and analyzed as indicated. B, WT-F1 and chimeric (Chi D, E, F) splenocyte cultures, activated as indicated, and then analyzed for γ3 or γ1 PST expression.

Article Snippet: C and D , DC-PCR analysis of DNA from splenocytes from WT F1 and three independent chimeras (Chi D, E, F) that were stimulated with LPS alone or in the presence of LPS and IL-4 lo or IL-4 hi for 5 d. All DC-PCR products were harvested at 26, 29, and 32 cycles.

Techniques: Purification, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing

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

Article Title: Switch Region Identity Plays an Important Role in Ig Class Switch Recombination

doi: 10.4049/jimmunol.1000507

Figure Lengend Snippet: DC-PCR analysis demonstrates that μ→γ3 CSR occurs on the Sγ1 replacement allele in response to LPS plus IL-4 activation. A, The DC-PCR strategy for detection of CSR at the endogenous γ3 locus is schematically illustrated. EcoRI sites (RI) flank the 5′ and 3′ ends of the S regions. Genomic DNA is digested with EcoRI and ligated under low concentration conditions that favor fragment circularization. The positions and orientations of the nested primer sets are shown postligation. The nAChR gene is used as a control. B, Allele-specific μ→γ3 DC-PCR is demonstrated using allele-specific primers and genomic DNA from the 129 a allele (μ→γ3a) or C57BL/6 b allele (μ→γ3b) mice. C and D, DC-PCR analysis of DNA from splenocytes from WT F1 and three independent chimeras (Chi D, E, F) that were stimulated with LPS alone or in the presence of LPS and IL-4lo or IL-4hi for 5 d. All DC-PCR products were harvested at 26, 29, and 32 cycles. C, Allele-specific μ→γ3a (WT) or μ→γ3a(KI) (Sγ1 replacement allele) and μ→γ3b (intact b allele) and μ→γ1 (WT a and b alleles) DC-PCR products. Templates were concentrated 5-fold and reanalyzed for the intact b allele as indicated (μ→γ3bp). D, Allele-specific DC-PCR products μ→γ3a(KI), μ→γ3b, or μ→γ1 for Chi 1.

Article Snippet: C and D , DC-PCR analysis of DNA from splenocytes from WT F1 and three independent chimeras (Chi D, E, F) that were stimulated with LPS alone or in the presence of LPS and IL-4 lo or IL-4 hi for 5 d. All DC-PCR products were harvested at 26, 29, and 32 cycles.

Techniques: Activation Assay, Concentration Assay, Control

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

Article Title: Switch Region Identity Plays an Important Role in Ig Class Switch Recombination

doi: 10.4049/jimmunol.1000507

Figure Lengend Snippet: S/S junction frequency analysis indicates IL-4–induced targeting of the γ3a(KI) allele for μ→γ3 CSR. Splenocytes were stimulated with LPS in the presence or absence of IL-4lo for 5 d. S-S junctions from WT F1 (A) and chimeric (B) mice were amplified from genomic DNA using primers located 5′ of Sμ and in the Sγ3-Cγ3 intron as indicated by the arrows under each locus schematic. The locations of the recombination breakpoints were determined by DNA sequence analysis of the cloned PCR products and are indicated by the symbols beneath the locus diagrams. S-S junction sequences are shown in Supplemental Figs. 2 and 3 and are analyzed in Table I. In the Sγ1 replacement allele, indicated as γ3a(KI), the remaining loxP site is shown by the black triangle upstream of the Sγ1 replacement region.

Article Snippet: C and D , DC-PCR analysis of DNA from splenocytes from WT F1 and three independent chimeras (Chi D, E, F) that were stimulated with LPS alone or in the presence of LPS and IL-4 lo or IL-4 hi for 5 d. All DC-PCR products were harvested at 26, 29, and 32 cycles.

Techniques: Amplification, Sequencing, Clone Assay