Journal: Journal for Immunotherapy of Cancer

Article Title: Switch receptor T3/28 improves long-term persistence and antitumor efficacy of CAR-T cells

doi: 10.1136/jitc-2021-003176

Figure Lengend Snippet: T3/28 CAR-T cells expanded ex vivo and maintain a superior phenotype. Flow cytometry was performed to detect expression of activation markers CD69 (A) and CD127 (B) and low-differentiation associated costimulatory molecules CD27 (C) and CD28 (D) on T cells stimulated with tumor cells. T3/28 CAR-T cells, 19BBz CAR-T cells, and UNT cells were expanded until day 18, and activation and differentiation markers were evaluated at indicated time points. (E) The T CM compartment, with a CD45RO + CD62L + phenotype, was assayed using flow cytometry. (F) CD4:CD8 ratio of CAR-T cells stimulated with tumor cells at day 9 post transduction was evaluated using flow cytometry. (G) Quantitative PCR was used to analyze memory stem-like-associated transcription factors TCF7 and LEF1 in CAR-T and control cells. Data presented are the mean±SD of three separate experiments. ns means no significant difference, *p＜0.05, **p＜0.01, ***p＜0.001 compared with indicated group. CAR, chimeric antigen receptor; mRNA, messenger RNA; T CM , central memory T; T E , effector T; T EM , effector memory T; T N , naive T; UNT, untreated T cells.

Article Snippet: The phenotype of primary cells and cell lines was determined using the following anti-human antibodies: CD3-APC (clone HIT3a, BioLegend), CD3-Pcy5.5 (clone OKT3, BioLegend), CD4-PC7 (clone OKT4, BioLegend), CD8-BV421 (RPA-T8, BioLegend), CD19-FITC (clone HIB19, BD), CD19-APC (clone HIB19, BD), CD25-APC (clone 2A3, BD), CD107a-APC (clone H4A3, BioLegend), TIM3-APC (clone F38-2E2, BioLegend), LAG3-APC (clone 7H2C65, BioLegend), PD1-PE/dazzle (clone EH12.2H7, BioLegend), PD1-PC5.5 (clone EH12.1, BD), Foxp3-PE (clone 259D, BioLegend), Tigit-PC5.5 (clone A15153G, BioLegend), CD127-PE (clone HIL-7R-M21, BD), CD27-PC5.5 (clone M-T271, BD), CD28-PE (clone CD28.2, BD), CD45RO Alexa Fluor 700 (clone UCHL1, BD), CD62L-PE (clone DREG-56, BD), CD66 (Ceacam1)-BV421 (clone ASL-32, BioLegend), Gal-9-PE (clone ASL-32, BioLegend), Ki67-BV421 (clone Ki-67, BioLegend), and CD69-APCcy7 (clone PN50, BioLegend).

Techniques: Ex Vivo, Flow Cytometry, Expressing, Activation Assay, Transduction, Real-time Polymerase Chain Reaction

Journal: Journal of Immunology Research

Article Title: Tissue-Resident Memory-Like CD8 + T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion

doi: 10.1155/2021/6643808

Figure Lengend Snippet: Analysis of surface markers on distinct CD8 + T cell subsets. Coexpression of CD45RO and CD103 or CD45RO and CD69 in distinct samples, the representative dot plots were shown (a). The expression of (b, c) CD45RO and (d, e) other surface markers on distinct subsets distinguished based on CD103 and CD69 expression in resting PFMCs was analyzed; the representative dot plots and histogram graphs and statistical data were shown ( n = 8); ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.005.

Article Snippet: The following monoclonal antibodies were used for phenotypes and intracellular cytokine analyses: PE-CF594 anti-CD3 (UCHT1), APC-Cy7 anti-CD4 (RPA-T4), phycoerythrin (PE) anti-CD69 (FN50), PE anti-CXCR3 (CXCR3-173), PE anti-CD15 (HI98), PE-Cy7 anti-CD69 (FN50), PE-Cy7 anti-CCR4 (1G1), PE-Cy7 anti-CD103 (Ber-ACT8), PE-Cy7 anti-CCR7 (3D12), PE-Cy7 anti-CD19 (HIB19), PE-Cy7 anti-CD56 (B159), PE-Cy7 anti-CD14 (M5E2), Percp-Cy5.5 anti-CD3 (UCHT1), Percp-Cy5.5 anti-CD8 (RPA-T8), Percp-Cy5.5 anti-CD45RO (UCHL1), allophycocyanin (APC) anti-CD8 (RPA-T8), APC anti-CD27 (M-T271), APC anti-CCR6 (11A9), APC anti-CD62L (SK11), APC anti-IFN- γ (B27), APC CTLA-4 (BN13), APC anti- γδ TCR (B1), APC CD16 (3G8), APC-H7 anti-CD8 (RPA-T8), Alexa Fluor® 647 anti-Granzyme B (GB11), Alexa Fluor® 488 anti-CXCR5 (RF8B2), Alexa Fluor® 700 anti-CD45RO (UCHL1), fluorescein isothiocyanate (FITC) anti-CD103 (Ber-ACT8), FITC anti-CD127 (HIL-7R-M21), FITC anti-CD25 (M-A251), and BV510 anti-CD4 (OKT4) were purchased from BD Biosciences (Franklin Lakes, NJ, USA); APC anti-FoxP3 (236A/E7) and FITC anti-CXCR3 (CXCR3-173) were purchased from eBioscience (San Diego, CA, USA); PE anti-TCRv α 24 (6B11) was purchased from Beckman Coulter (Brea, CA, USA).

Techniques: Expressing

Journal: Journal of Immunology Research

Article Title: Tissue-Resident Memory-Like CD8 + T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion

doi: 10.1155/2021/6643808

Figure Lengend Snippet: Higher frequencies of CD103 + or CD69 + subsets in memory CD8 + T cells from PFMCs compared with PBMCs. Gated on CD45RO + CD8 + T cells, the frequencies of CD69 − CD103 − , CD69 + CD103 − , CD69 + CD103 + , and CD69 − CD103 + cells in PFMCs were detected; the (a) representative dot plots and (b) statistical data were shown; the frequencies of CD69 − CD103 − , CD69 + CD103 − , CD69 + CD103 + , and CD69 − CD103 + cells in different samples were compared; the (c) representative dot plots and (d) statistical data were shown ( n = 8); ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: The following monoclonal antibodies were used for phenotypes and intracellular cytokine analyses: PE-CF594 anti-CD3 (UCHT1), APC-Cy7 anti-CD4 (RPA-T4), phycoerythrin (PE) anti-CD69 (FN50), PE anti-CXCR3 (CXCR3-173), PE anti-CD15 (HI98), PE-Cy7 anti-CD69 (FN50), PE-Cy7 anti-CCR4 (1G1), PE-Cy7 anti-CD103 (Ber-ACT8), PE-Cy7 anti-CCR7 (3D12), PE-Cy7 anti-CD19 (HIB19), PE-Cy7 anti-CD56 (B159), PE-Cy7 anti-CD14 (M5E2), Percp-Cy5.5 anti-CD3 (UCHT1), Percp-Cy5.5 anti-CD8 (RPA-T8), Percp-Cy5.5 anti-CD45RO (UCHL1), allophycocyanin (APC) anti-CD8 (RPA-T8), APC anti-CD27 (M-T271), APC anti-CCR6 (11A9), APC anti-CD62L (SK11), APC anti-IFN- γ (B27), APC CTLA-4 (BN13), APC anti- γδ TCR (B1), APC CD16 (3G8), APC-H7 anti-CD8 (RPA-T8), Alexa Fluor® 647 anti-Granzyme B (GB11), Alexa Fluor® 488 anti-CXCR5 (RF8B2), Alexa Fluor® 700 anti-CD45RO (UCHL1), fluorescein isothiocyanate (FITC) anti-CD103 (Ber-ACT8), FITC anti-CD127 (HIL-7R-M21), FITC anti-CD25 (M-A251), and BV510 anti-CD4 (OKT4) were purchased from BD Biosciences (Franklin Lakes, NJ, USA); APC anti-FoxP3 (236A/E7) and FITC anti-CXCR3 (CXCR3-173) were purchased from eBioscience (San Diego, CA, USA); PE anti-TCRv α 24 (6B11) was purchased from Beckman Coulter (Brea, CA, USA).

Techniques:

Journal: Journal of Immunology Research

Article Title: Tissue-Resident Memory-Like CD8 + T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion

doi: 10.1155/2021/6643808

Figure Lengend Snippet: Heterogeneous Granzyme B expression by distinct CD8 + T RM -like cells from resting PFMCs. PFMCs were stimulated with BCG plus anti-CD28 for 12 hours in the presence of BFA, the coexpression of IFN- γ , CD69, and CD103 was detected, and the representative dot plots were shown (a) ( n = 6). After stimulation with BCG plus anti-CD28, the frequencies of CD69 − CD103 − , CD69 + CD103 − , CD69 + CD103 + , and CD69 − CD103 + cells in IFN- γ + CD8 + T cells were detected; the (b) representative dot plots and (c) statistical data were shown ( n = 9). The coexpression of Granzyme B and CD45RO, CD103, or CD69 was observed; the (d) representative dot plots were shown, and the correlations between Granzyme B and CD45RO, CD103, or CD69 in resting PFMCs were analyzed ( n = 27) (e). The expression of Granzyme B by different CD45RO + CD8 + T subsets distinguished based on CD103 and CD69 expression was analyzed; the (f) representative dot plots and (f) histogram graphs and (g) statistical data were shown ( n = 5). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.005.

Article Snippet: The following monoclonal antibodies were used for phenotypes and intracellular cytokine analyses: PE-CF594 anti-CD3 (UCHT1), APC-Cy7 anti-CD4 (RPA-T4), phycoerythrin (PE) anti-CD69 (FN50), PE anti-CXCR3 (CXCR3-173), PE anti-CD15 (HI98), PE-Cy7 anti-CD69 (FN50), PE-Cy7 anti-CCR4 (1G1), PE-Cy7 anti-CD103 (Ber-ACT8), PE-Cy7 anti-CCR7 (3D12), PE-Cy7 anti-CD19 (HIB19), PE-Cy7 anti-CD56 (B159), PE-Cy7 anti-CD14 (M5E2), Percp-Cy5.5 anti-CD3 (UCHT1), Percp-Cy5.5 anti-CD8 (RPA-T8), Percp-Cy5.5 anti-CD45RO (UCHL1), allophycocyanin (APC) anti-CD8 (RPA-T8), APC anti-CD27 (M-T271), APC anti-CCR6 (11A9), APC anti-CD62L (SK11), APC anti-IFN- γ (B27), APC CTLA-4 (BN13), APC anti- γδ TCR (B1), APC CD16 (3G8), APC-H7 anti-CD8 (RPA-T8), Alexa Fluor® 647 anti-Granzyme B (GB11), Alexa Fluor® 488 anti-CXCR5 (RF8B2), Alexa Fluor® 700 anti-CD45RO (UCHL1), fluorescein isothiocyanate (FITC) anti-CD103 (Ber-ACT8), FITC anti-CD127 (HIL-7R-M21), FITC anti-CD25 (M-A251), and BV510 anti-CD4 (OKT4) were purchased from BD Biosciences (Franklin Lakes, NJ, USA); APC anti-FoxP3 (236A/E7) and FITC anti-CXCR3 (CXCR3-173) were purchased from eBioscience (San Diego, CA, USA); PE anti-TCRv α 24 (6B11) was purchased from Beckman Coulter (Brea, CA, USA).

Techniques: Expressing

Journal: Journal of Immunology Research

Article Title: Tissue-Resident Memory-Like CD8 + T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion

doi: 10.1155/2021/6643808

Figure Lengend Snippet: Inhibitory characteristics of CD103 + CD8 + T cells. (a) The phenotype of CTLA-4 + CD8 + T cells from PFMCs, the expression of CTLA-4 on different CD45RO + CD8 + T subsets distinguished based on CD103 and CD69 was detected, and the (b) representative dot plots and (c) statistical data were shown. (d) The phenotype of FoxP3 + CD25 + CD8 + T cells from PFMCs, the coexpression of FoxP3 and CD25 in different CD45RO + CD8 + T subsets distinguished based on CD103 and CD69, and the (e) representative dot plots and (f) statistical data were shown ( n = 5); ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: The following monoclonal antibodies were used for phenotypes and intracellular cytokine analyses: PE-CF594 anti-CD3 (UCHT1), APC-Cy7 anti-CD4 (RPA-T4), phycoerythrin (PE) anti-CD69 (FN50), PE anti-CXCR3 (CXCR3-173), PE anti-CD15 (HI98), PE-Cy7 anti-CD69 (FN50), PE-Cy7 anti-CCR4 (1G1), PE-Cy7 anti-CD103 (Ber-ACT8), PE-Cy7 anti-CCR7 (3D12), PE-Cy7 anti-CD19 (HIB19), PE-Cy7 anti-CD56 (B159), PE-Cy7 anti-CD14 (M5E2), Percp-Cy5.5 anti-CD3 (UCHT1), Percp-Cy5.5 anti-CD8 (RPA-T8), Percp-Cy5.5 anti-CD45RO (UCHL1), allophycocyanin (APC) anti-CD8 (RPA-T8), APC anti-CD27 (M-T271), APC anti-CCR6 (11A9), APC anti-CD62L (SK11), APC anti-IFN- γ (B27), APC CTLA-4 (BN13), APC anti- γδ TCR (B1), APC CD16 (3G8), APC-H7 anti-CD8 (RPA-T8), Alexa Fluor® 647 anti-Granzyme B (GB11), Alexa Fluor® 488 anti-CXCR5 (RF8B2), Alexa Fluor® 700 anti-CD45RO (UCHL1), fluorescein isothiocyanate (FITC) anti-CD103 (Ber-ACT8), FITC anti-CD127 (HIL-7R-M21), FITC anti-CD25 (M-A251), and BV510 anti-CD4 (OKT4) were purchased from BD Biosciences (Franklin Lakes, NJ, USA); APC anti-FoxP3 (236A/E7) and FITC anti-CXCR3 (CXCR3-173) were purchased from eBioscience (San Diego, CA, USA); PE anti-TCRv α 24 (6B11) was purchased from Beckman Coulter (Brea, CA, USA).

Techniques: Expressing

Journal: Virology

Article Title: Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins

doi: 10.1016/j.virol.2017.12.036

Figure Lengend Snippet: HIV-1 infection of CD4 T cells in vitro demonstrates lower levels of HIV-1 DNA and gag mRNA in Tregs compared to Tconvs and Tmems. (A) FLOW contour plot shows the cell sorting strategy. The in vitro bulk infected ((B) and (C)) or freshly isolated ((D)) CD4 T cells were stained and sorted into Tregs (CD25high CD127low), Tconvs (CD25low CD127high), Tmems (CD25(−) CD45RO(+)), and Tnaives (CD25(−)CD45RA(+)) populations. (B) Semi-quantitative real-time PCR plots of HIV-1 viral DNA (top) and mRNA (bottom) levels, relative to control β-globin DNA and 18 S RNA, for Tmems (Tm), Tconvs (Tc), Tregs (Tr), and Tnaives (Tn) populations. (C) Bar graphs depict the mean results of viral DNA (top) and mRNA (bottom) levels from three independent experiments. Standard error bars are shown along with statistically significant comparisons between CD4 T cell populations. (D) Bar graph shows the viral DNA levels over two different time points as indicated post infection (n=3).

Article Snippet: The CD4 T subpopulations of Tregs (CD4+, CD25high, CD127low), conventional effectors or Tconvs (CD4+, CD25+, CD127high), memory or Tmems (CD4+, CD25−, CD45RO+), and Tnaives (CD4+, CD25−, CD45RA+) were purified from patient PBMC directly by staining for CD4 T cell surface markers CD3 (PerCP), CD4 (FITC), CD25 (APC), CD127 (PE), CD45RA (V450), and CD45RO (Alexa Fluor 700), and sorting the stained cells on an ARIA cell sorter (BD Biosciences, San Jose, CA, USA) as shown in .

Techniques: Infection, In Vitro, FACS, Isolation, Staining, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: CD45RB Status of CD8 + T Cell Memory Defines T Cell Receptor Affinity and Persistence

doi: 10.1016/j.celrep.2020.01.016

Figure Lengend Snippet: CD8+ T Cell Memory Contains CD45RBhi and CD45RBlo Populations with Distinct TCR Affinity and Repertoire Diversity. (A and B) FACS plots (A) and summary data (B) of CD45RB expression on CD44lo naive or CD44hi virus-specific populations following LCMV Armstrong infection., (C) Expression of CD45RA, CD45RB, and CD45RC on Db np396 at the indicated time points following infection with LCMV. See also Figure S1D. (D) Frequency of CD45RBhi cells within total Db np396 and Db gp276 populations at 6 weeks post-infection. (E) CD45RA and CD45RC expression among CD44lo naive or CD44hi CD8+ T cell populations at week 6 post-infection. (F) Frequency of the CD45RO transcript as a frequency of total Ptprc transcripts among naive and Db np396+ memory populations. (G) Representative frequency of IFN-g response following np396 peptide stimulation, normalized to maximum response, at week 6 post-infection. (H) EC50 from multiple mice analyzed as in (F). (I) Relative 2D micropipette adhesion assay values for Db np396 of FACS-isolated CD45RBhi and CD45RBlo memory CD8+ T cells at weeks 6–10 post-infection. (J) Clonal space homeostasis plots of CD45RBhi and CD45RBlo memory cells, depicting the proportion of T cell clones in three frequency ranges (1.0%–10%, 0.1%–0.01%, and 0.001%–0.0001%) within each memory population. Both the size of each clonal group and the radius reflect the proportion of the total. (K) Inverse Simpson’s diversity index for three populations of CD45RBhi and CD45RBlo memory cells. In (E), summary data depict 9 mice/group. For (J) and (K), each data point represents FACS-isolated populations of three pooled mice. Error bars represent mean ± SEM. Significance is defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Anti-human CD45RO Alexa Fluor 700 monoclonal antibody , BD PharMingen , Clone UCHL1.

Techniques: Expressing, Infection, Cell Adhesion Assay, Isolation, Clone Assay

Journal: Cell reports

Article Title: CD45RB Status of CD8 + T Cell Memory Defines T Cell Receptor Affinity and Persistence

doi: 10.1016/j.celrep.2020.01.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-human CD45RO Alexa Fluor 700 monoclonal antibody , BD PharMingen , Clone UCHL1.

Techniques: Recombinant, Flow Cytometry, Staining, Software