Journal: Frontiers in Immunology

Article Title: Ligand-based targeting of c-kit using engineered γδ T cells as a strategy for treating acute myeloid leukemia

doi: 10.3389/fimmu.2023.1294555

Figure Lengend Snippet: Treatment of hSCF sBite-modified γδ T cells only moderately prolongs survival in vivo , despite aggressive treatment regimen. (A) Experimental design. Briefly, NSG mice were pre-conditioned with 20 mg/kg busulfan IP on day -1, then injected with 5 × 10 6 CMK cells via tail-vein injection in the morning on day 0. Beginning in the afternoon on day 0, and then once daily for the next 3 days for a total of 4 doses, 1 × 10 7 γδ T cells were injected via tail-vein injection. Mice were subjected to bioluminescence imaging for the following 3 weeks, then followed for survival until they met endpoint. n = 8 untreated, n = 6 mock T treated, n = 6 hSCF sBite treated, n = 3 mSCF CAR treated. (B) Bioluminescence images. (C) Peripheral blood leukocytes were collected 3 weeks after the start of treatment and assessed for presence of CD33 + CMK cells. Error bars represent SD. Statistical analysis represents Student’s t test (ns, p > 0.05). (D) MFI of c-kit on CMK cells within the periphery. Statistical analysis represents Student’s t test (ns, p > 0.05). (E) Kaplan-Meier survival analysis. Untreated and mock γδ T treated groups were combined as a control group. Statistical analysis represents log rank (Mantel-Cox) test (ns > 0.05). P-value is shown. (F) Representative flow plots of hCD33 + hCD45 + CMK cells in the bone marrow of an untreated mouse sacrificed near end-point.

Article Snippet: Bioluminescence was quantified using Living Image Software (PerkinElmer, Waltham, MA, USA) or Aura Software (Spectral Instruments Imaging, Tucson, AZ, USA).

Techniques: Modification, In Vivo, Injection, Imaging, Control

Journal: International Journal of Molecular Sciences

Article Title: The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells

doi: 10.3390/ijms24109008

Figure Lengend Snippet: DC-SIGN engagement induces autophagy and associates with LC3. ( A ) HEK293T cells were co-transfected with plasmids expressing DC-SIGN WT and GST-expressing constructs as indicated. Cells were engaged or not with DC-SIGN mAbs (5 μg/mL) for 30 min before pull-down. Lysates (input control) and GST pull-down were immunoblotted with anti-DC-SIGN ( upper panels), anti-GST ( middle panels), and anti-GAPDH for input control. This experiment is representative of 4. ( B ) Lysates from MoDC treated for indicated times with DC-SIGN specific mAbs were immunoblotted with anti-LC3, and loading was controlled with anti-GAPDH ( upper blots). Negative control experiments were performed using lysates of cells incubated with non-specific mouse serum (Isotype Ab) and immunoblotted as before ( lower blots). Pre-treatments of MoDC with chloroquine (50 μM) were performed to validate functional autophagy flux as evidenced by LC3-II increase. Densitometry values and LC3-II/GAPDH ratio obtained from lysates of primary MoDC treated with DC-SIGN mAbs (n = 6) or Isotype mouse Abs (n = 3) were obtained for each time point of each condition and graphically reported on the right. The same experiment as above was performed but with lysates from MoDC treated with ( C ) ManLAM (2 μg/mL) (n = 4) or ( D ) challenged with HIV-1-R5 (MOI of 2) (n = 3) for indicated times. LC3-II/GAPDH ratio from densitometry analyses was graphically represented as before. Statistical significance: * = p < 0.05; ** = p < 0.01.

Article Snippet: Chemiluminescence was acquired (Chemidoc, Bio-Rad Laboratories, Hercules, CA, USA), followed by densitometry analysis (Image Lab TM software; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Transfection, Expressing, Construct, Control, Negative Control, Incubation, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells

doi: 10.3390/ijms24109008

Figure Lengend Snippet: ATG9 is required for DC-SIGN-mediated autophagy flux activation. Primary MoDC were treated with irrelevant siRNA (siCtrl) or siRNA against ATG9 (siATG9), and ATG9 expression was controlled by RT-qPCR ( left graph). Lysates from MoDC transfected as above and pre-treated with bafilomycin A1 (50 nM) for 1 h before stimulation with ManLAM (2 μg/mL) for 2 h were immunoblotted with anti-LC3 ( upper blot). The loading control was performed with anti-actin ( lower blot). LC3-II/actin ratio from densitometry analyses obtained from 3 independent experiments (n = 3) was normalized to untreated controls of each siRNA condition and graphically represented ( right graph). Statistical significance: * = p < 0.05.

Article Snippet: Chemiluminescence was acquired (Chemidoc, Bio-Rad Laboratories, Hercules, CA, USA), followed by densitometry analysis (Image Lab TM software; Bio-Rad Laboratories, Hercules, CA, USA).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Control