Journal: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 1 Downregulation of IRF4 expression in CAR T cells. (A) Schematic of CAR constructs. (B) CAR expression in T cells detected by staining with a phycoerythrin (PE)-labeled goat anti-human IgG antibody before (upper panels) and after (lower panels) magnetic cell separation (MACS). One representative donor out of four is shown. (C) Intracellular staining of IRF4 after stimulation with CEA+ BxPC-3 cells at the indicated time points in CD8+ (left panel) and CD4+ CAR T cells (right panel). CAR T cells were generated as described in the materials and methods section by activation of PBMCs followed by retroviral transduction. Untransduced cells were generated by activation of PBMCs and subsequent expansion with IL-2, but without retroviral transduction. Data represent means ± SEM of five donors, p values were calculated by Student´s t test, *indicates p ≤0.05, ***indicates p ≤0.001.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Expressing, Construct, Staining, Labeling, Magnetic Cell Separation, Generated, Activation Assay, Retroviral, Transduction

Journal: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 3 Downregulation of IRF4 enhances CAR T cell functionality. (A) CAR T cells (starting with 1 × 105 CAR T cells) underwent four rounds (R1-R4) of stimulation with GFP-labeled CEA+ BxPC-3 cells (1 × 105 tumor cells at the beginning of each round). At the end of each round, CAR T cells (live CD3+ CAR+) (left panel) and BxPC-3 cells (right panel) were quantified by flow cytometry. Data represent means ± SEM of six donors, p values were calculated by Student´s t test, ns indicates not significant, and * indicates p ≤0.05. (B-G) Phenotypic analysis of CD8+ CAR T cells during repetitive antigen stimulation. CAR T cells underwent three rounds (R1-R3) of antigen-stimulation with unlabeled BxPC-3 cells. At the end of each round, the CD8/CD4 T cell ratio was determined (B). CAR T cells were stained for CD8 and further characterized regarding TIM-3 (C), PD-1 (D), TIGIT (E) expression and effector-memory cell differentiation: SCM = T stem-cell-memory (CD45RO+ CD62L+), EM = effector-memory (CD45RO+ CD62L-), CM = central-memory (CD45RO+ CD62L+), E = effector (CD45RO- CD62L-) (F), and CD27 expression (G). Data represent geometric means of ± SEM of at least four donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤0.05, **indicates p ≤0.01.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Labeling, Cytometry, Staining, Expressing, Cell Differentiation

Journal: Scientific Reports

Article Title: Cysteine redox state regulates human β2-adrenergic receptor binding and function

doi: 10.1038/s41598-020-59983-4

Figure Lengend Snippet: Agonism of β2AR on human lung airway epithelial cells stimulates ROS generation. ( A ) CALU3 cells were preloaded with the intracellular ROS-sensitive fluorescent probe CM-H 2 DCFDA (10 µM) and stimulated with 10 μM ISO for 5 min or as otherwise shown, prior to fixation and confocal imaging. ISO stimulation elicited robust fluorescence, indicative of ROS generation, at the cell membrane, and this effect was blocked by pretreatment with alprenolol (10 µM). The overall degree of fluorescence was equivalent to that seen upon exogenous treatment with 1 mM H 2 O 2 . The upper panel is 40X image and the lower panel depicts further zoom on a single cell from that image. ( B ) Fluorescent microscopy of cells that were pretreated with the NADPH oxidase inhibitors apocynin (APO; 100 µM) and diphenyleneiodonium chloride (DPI; 10 µM) reveals marked decreases in the fluorescent intensity ( B ) and total number of fluorescent cells ( C ) in APO and DPI treated states. Images in ( B ) represent a 20X objective image capture with the top image representing FITC fluorescence, the middle image representing the respective transilluminated image, and the lower image representing the overlay. ( D , E ) Agonism of CALU3 cells with 1 μM ISO for 5 or 15 minutes oxidized cysteine residues on the β2AR enabling selective dimedone alkylation of these sulfenic acid residues with 5 mM dimedone (15 minutes) as can be seen by either β2AR immunoprecipitation followed by anti-Cys-dimedone immunoblotting ( D ) or Cys-dimedone immunoprecipitation followed by β2AR immunoblotting ( E ). Full length immunoblots are shown in Supplementary Fig. .

Article Snippet: FITC conjugated anti-mouse antibody was from Rockland Immunochemicals (Limerick, PA).

Techniques: Imaging, Fluorescence, Membrane, Microscopy, Immunoprecipitation, Western Blot

Journal: Vaccines

Article Title: Ethnic Comparisons of Spike-Specific CD4+ T Cells, Serological Responses, and Neutralizing Antibody Titers Against SARS-CoV-2 Variants

doi: 10.3390/vaccines13060607

Figure Lengend Snippet: Figure 2. Frequency of Spike-specific AIM+ (CD154+ and CD69+) CD4+ T cells and AIM+ (CD137+ and CD69+) CD8+ T cells across Wuhan, Delta, and Omicron subvariants among Black, Caucasian, and Asian participants. (a) Mann–Whitney unpaired t-test showed no difference in Spike-specific response among Wuhan, Delta, and Omicron/B1.1.529-specific CD154+ CD69+ CD4+ T cells. Yet significantly lower frequencies were detected in Asian participants as compared to the Caucasians against Omicron/BA2.86 (p < 0.001), Omicron/BA4.5 (p < 0.01), and Omicron/XBB.1 (p < 0.05). Additionally, the Asian group had significantly lower frequencies as compared to Black participants (p < 0.05). (b) Mann–Whitney unpaired t-test showed no difference among the groups (p > 0.05) when evaluated against Wuhan, Delta/B1.617.2, Omicron/B1.1.529, Omicron/BA2.86, Omicron/BA4.5, and Omicron/XBB.1-specific CD137+ CD69+ CD8+ T cell responses. Data are represented as Median ± 95 CI for the Black (N = 12–13), Caucasian (N = 17), and Asian (N = 14) participants. Significance is noted as p < 0.05 *, p < 0.01 **, p < 0.1 ***.

Article Snippet: Dilution Viability dye - Zombie NIR Biolegend surface 423105 1:1,250 CD8 SK1 SB550 Biolegend surface 344760 1:125 PD1 EH12.1 BB515 BD Bioscience surface 564494 1:10 CD45RA HI100 PerCP Biolegend surface 304156 1:25 CD154 (CD40L) 24-31 APC Biolegend surface 310810 1:50 CCR7 2-L1-A APCR700 Biolegend surface 566766 1:25 CD19 HIB19 APCEF780 Invitrogen surface 50-161-52 1:25 CD14 61D3 APCEF780 Invitrogen surface 47-0149-42 1:10 CD3 UCHT1 BV570 Biolegend surface 300436 1:10 CXCR5 J252D4 PE-DAZZLE Biolegend surface 356928 1:50 CD4 SK3 cFluor YG 584 Cytek surface R7-20041 1:100 CD134 (OX40) Ber-ACT35 PECY7 Biolegend surface 350012 1:250 IL2 MQ1-17H12 PERCP-EF 710 Invitrogen intracellular 50-161-00 1:50 IFNG 4SB3 BV711 Biolegend intracellular 502540 1:25 TNF MAB11 BV785 Biolegend intracellular 502948 1:25 GZB QA16A02 PE-CY5 Biolegend intracellular 372226 1:100 CD137 4B4-1 BV750 Biolegend intracellular 309844 1:50 CD69 FN50 BV510 Biolegend intracellular 310936 1:25 Figure A2.

Techniques: MANN-WHITNEY

Journal: Vaccines

Article Title: Ethnic Comparisons of Spike-Specific CD4+ T Cells, Serological Responses, and Neutralizing Antibody Titers Against SARS-CoV-2 Variants

doi: 10.3390/vaccines13060607

Figure Lengend Snippet: Figure 3. Frequency of T follicular Helper cells and AIM+, CD4+, and CD8+ Memory T cell subtypes gated on AIM+ and CD4+ double-activated T cells across variants from Wuhan, Delta, and Omicron subvariants among Black, Caucasian, and Asian participants. (a) Mann–Whitney unpaired t-test showed no difference among the groups (p > 0.05) for Wuhan, Delta/B1.617.2, Omicron/B1.1.529, Omicron/BA2.86, Omicron/BA4.5, and Omicron/XBB.1–specific CXCR5+, PD1+, CD154+, CD69+, and CD4+ T cell responses. Data are represented as Median ± 95 CI for the Black (N = 12–13), Caucasian (N = 17), and Asian (N = 14) participants. (b,c) AIM+ and CD4+ memory cells were gated on CD154+, CD69+, and CD4+ T cells and showed greater predominance for the expression of central and effector memory cell subtypes across all variants within all three groups, with a similar distribution pattern among the groups. AIM+ and CD8+ memory cells were gated on CD137+, CD69+, and CD8+ T cells and showed great diversity in the expression of memory cell subtypes, including central, effector, naïve, and terminal memory cells. Black participants show a diverse memory subtypes repertoire, including all four subtypes across all variants tested, whereas Caucasian participants had a profile of central and terminal effector memory predominantly expressed across all variants. The Asian participants show a higher expression of effector and terminal effector memory cell subtypes across all variants. AIM+, CD4+, or CD8+ memory T cells were defined by the combination of CD45RA and CCR7 markers, where central memory is CD45RA−and CCR7+, naïve memory is CD45RA+ and CCR7+, terminal effector memory is CD45RA+ and CCR7−, and effector memory is CD45RA−and CCR7−, while each population is expressed as % of the total cells.

Article Snippet: Dilution Viability dye - Zombie NIR Biolegend surface 423105 1:1,250 CD8 SK1 SB550 Biolegend surface 344760 1:125 PD1 EH12.1 BB515 BD Bioscience surface 564494 1:10 CD45RA HI100 PerCP Biolegend surface 304156 1:25 CD154 (CD40L) 24-31 APC Biolegend surface 310810 1:50 CCR7 2-L1-A APCR700 Biolegend surface 566766 1:25 CD19 HIB19 APCEF780 Invitrogen surface 50-161-52 1:25 CD14 61D3 APCEF780 Invitrogen surface 47-0149-42 1:10 CD3 UCHT1 BV570 Biolegend surface 300436 1:10 CXCR5 J252D4 PE-DAZZLE Biolegend surface 356928 1:50 CD4 SK3 cFluor YG 584 Cytek surface R7-20041 1:100 CD134 (OX40) Ber-ACT35 PECY7 Biolegend surface 350012 1:250 IL2 MQ1-17H12 PERCP-EF 710 Invitrogen intracellular 50-161-00 1:50 IFNG 4SB3 BV711 Biolegend intracellular 502540 1:25 TNF MAB11 BV785 Biolegend intracellular 502948 1:25 GZB QA16A02 PE-CY5 Biolegend intracellular 372226 1:100 CD137 4B4-1 BV750 Biolegend intracellular 309844 1:50 CD69 FN50 BV510 Biolegend intracellular 310936 1:25 Figure A2.

Techniques: MANN-WHITNEY, Expressing

Journal: Vaccines

Article Title: Ethnic Comparisons of Spike-Specific CD4+ T Cells, Serological Responses, and Neutralizing Antibody Titers Against SARS-CoV-2 Variants

doi: 10.3390/vaccines13060607

Figure Lengend Snippet: Figure 4. Frequency of TNFα+, IFNγ+, IL-2, and GZB+ gated on AIM+ (CD154+ and CD69+) CD4+ T cells across variants from Wuhan, Delta, and Omicron subvariants—specific among Black, Caucasian, and Asian participants. (a) Mann–Whitney unpaired t-test showed no difference among the groups for Wuhan and Delta/B1.617.2-specific TNFα+, CD154+, CD69+, and CD4+ T cells. The frequency of Omicron/B1.1.529-specific TNFα+, CD154+, CD69+, and CD4+ T cells was significantly increased in the Asian group as compared to the Caucasian participants (p < 0.05). Omicron/BA2.86- specific TNFα+ CD154+ CD69+ CD4+ T cell responses showed no difference among the groups (p > 0.05).

Article Snippet: Dilution Viability dye - Zombie NIR Biolegend surface 423105 1:1,250 CD8 SK1 SB550 Biolegend surface 344760 1:125 PD1 EH12.1 BB515 BD Bioscience surface 564494 1:10 CD45RA HI100 PerCP Biolegend surface 304156 1:25 CD154 (CD40L) 24-31 APC Biolegend surface 310810 1:50 CCR7 2-L1-A APCR700 Biolegend surface 566766 1:25 CD19 HIB19 APCEF780 Invitrogen surface 50-161-52 1:25 CD14 61D3 APCEF780 Invitrogen surface 47-0149-42 1:10 CD3 UCHT1 BV570 Biolegend surface 300436 1:10 CXCR5 J252D4 PE-DAZZLE Biolegend surface 356928 1:50 CD4 SK3 cFluor YG 584 Cytek surface R7-20041 1:100 CD134 (OX40) Ber-ACT35 PECY7 Biolegend surface 350012 1:250 IL2 MQ1-17H12 PERCP-EF 710 Invitrogen intracellular 50-161-00 1:50 IFNG 4SB3 BV711 Biolegend intracellular 502540 1:25 TNF MAB11 BV785 Biolegend intracellular 502948 1:25 GZB QA16A02 PE-CY5 Biolegend intracellular 372226 1:100 CD137 4B4-1 BV750 Biolegend intracellular 309844 1:50 CD69 FN50 BV510 Biolegend intracellular 310936 1:25 Figure A2.

Techniques: MANN-WHITNEY