Journal: Journal for immunotherapy of cancer

Article Title: Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

doi: 10.1136/jitc-2023-006783

Figure Lengend Snippet: Figure 2 Q-2361 dose-dependently rescues mouse CD8 T-cell function in the presence of tacrolimus. Mouse T cells were labeled with CellTrace Violet and cocultured with CD3/CD28 Dynabeads. For the first hour, they were incubated with 0.6 ng/ mL of tacrolimus (left panels) or vehicle control (central and right panels) alone. Subsequently, the indicated concentrations of (A) VX-710 or (B) Q-2361 were added. After 3 days, CD8 T-cell proliferation was determined by flow cytometry through the measurement of CellTrace Violet dilution (left and central panels), and cell viability was determined using the 7AAD live/dead discrimination dye (right panels). Results in (A) and (B) show representative data from two experiments each with similar results. One-way analysis of variance followed by Tukey’s multiple comparison test, **p<0.01; ***p<0.001; ****p<0.0001. n=3/group. TAC, tacrolimus.

Article Snippet: In experiments involving CD8 T- cell depletion, mice were injected with 250 μg of anti- CD8β (clone 53–5.8, Bio X Cell) antibody or isotype control antibody (clone HRPN, Bio X Cell) intraperitoneal on days 8 and 15 post tumor challenge, and 100 μg of antibody on day 22 post tumor challenge.

Techniques: Cell Function Assay, Labeling, Incubation, Control, Flow Cytometry, Comparison

Journal: Journal for immunotherapy of cancer

Article Title: Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

doi: 10.1136/jitc-2023-006783

Figure Lengend Snippet: Figure 4 Regression of tacrolimus-dependent squamous cell carcinoma (SCC) growth. (A–D) Description of model and experimental set up. (A) Creation of a transplantable SCC cell line. (B) Growth characteristics of SCC in immune-competent mice receiving normal or tacrolimus-containing diet. (C) Research concept for local Q-2361 treatment in tacrolimus-suppressed mice, and (D) experimental outline. (E) Left: Tumor volume following intratumoral (IT) injection of Q-2361 or vehicle solution. Groups were further subdivided by receipt of anti-CD8β antibody (mAb) to deplete CD8 T cells, or receipt of an isotype control mAb. Right: Confirmation of CD8 T-cell depletion in the blood on day 10 (IT Vehicle+Isotype mAb, IT Vehicle+anti-CD8β, IT Q-2361+anti-CD8β; CD45+TCR-β+CD8α+ T cells as a percentage of all CD45+TCR-β+ T cells). Mice with poorly established tumors<0.07 cm3 on day 14 and/or fold growth was <1.15 from day 9 to 14 were excluded from the study; excluded data not included in analysis. Dotted line in (E) represents start of two times per day injections on day 11. Error bars represent SEM, n=8–12; (F) Body weight on day 25. One-way analysis of variance on day 32 (E); Left, or day 10 (E); Right, p<0.0001 (****), or day 25 (F) followed by Tukey’s multiple comparison test.

Article Snippet: In experiments involving CD8 T- cell depletion, mice were injected with 250 μg of anti- CD8β (clone 53–5.8, Bio X Cell) antibody or isotype control antibody (clone HRPN, Bio X Cell) intraperitoneal on days 8 and 15 post tumor challenge, and 100 μg of antibody on day 22 post tumor challenge.

Techniques: Injection, Control, Comparison

Journal: Journal for immunotherapy of cancer

Article Title: Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

doi: 10.1136/jitc-2023-006783

Figure Lengend Snippet: Figure 5 Q-2361 treatment increases CD8 T-cell activation and effector function within squamous cell carcinoma (SCC) tumors. (A) Impact of systemic tacrolimus withdrawal on SCC tumor growth. (B) Impact of local Q-2361 treatment on SCC tumor growth in immunosuppressed mice. In (A), mice with poorly established tumors<0.06 cm3 on day 12 and/or fold growth was <1 from day 12 to 19 were excluded from the study, and in (B) mice with poorly established tumors<0.07 cm3 on day 13 and/or fold growth was <1.15 from day 9 to 13 were excluded from the study; excluded data not included in analysis. Dotted line in (A) represents TAC diet removal on day 14 and in (B) represents start of two times per day injections on day 8. (C,D) Analysis of tumor weight and flow cytometry analysis of intratumoral T-cell abundance, activation, and effector molecule production in tumors harvested from (A) and (B), respectively. Data for T cells, CD8 T cells, and CD69+CD8 T cells represent number of cells per gram of tumor. Error bars represent SEM, (A/C) n=6, (B/D) n=10. (A/B) One-way analysis of variance on day 26 (A) or day 19 (B) followed by Tukey’s multiple comparison test. (C/D) Unpaired student’s t-test, p<0.05 (*), p<0.01 (**), p<0.001 (***), and p<0.0001 (****). IT, intratumoral; ns, not significant; TAC, tacrolimus.

Article Snippet: In experiments involving CD8 T- cell depletion, mice were injected with 250 μg of anti- CD8β (clone 53–5.8, Bio X Cell) antibody or isotype control antibody (clone HRPN, Bio X Cell) intraperitoneal on days 8 and 15 post tumor challenge, and 100 μg of antibody on day 22 post tumor challenge.

Techniques: Activation Assay, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Representative dot plots and quantification of CD44 and CD62L expression in CD8 + T cells from the indicated non lymphoid organs in conditional SUV39H1 KO (Cre+) or WT (Cre-) littermates. B. t-SNE plots of merged flow cytometry data gated on total CD8 + T cells from the liver of 5 conditional SUV39H1 KO (Cre+) and 5 WT littermates (Cre-) at steady state. See Supp Table 1. C. Representative dot plots and quantification of CD69 + CD103 - and CD49d + Ly6c - populations in CD44 + CD8 + T cells from liver of conditional SUV39H1 KO mice (Cre+) or WT littermates (Cre-) at steady state. D. Representative dot plots and quantification of intravascular staining negative (iv-) cells among lung CD8 + T cells and CD69 + CD103 - or CD49d + Ly6c - populations in iv-CD44 + CD8 + T cells from conditional SUV39H1 KO mice (Cre+) or WT littermates (Cre-) at steady state. Each dot represents a mouse, different shapes represent different experiments. * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by multiple unpaired t test with Holm-Šídák correction ( A and B ) or Mann Whitney test ( C and D ).

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Expressing, Flow Cytometry, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots of WT (CD45.1-) and SUV39H1 KO (CD45.1+) OTI cells (Vα2+) in the indicated organs 33 days after adoptive transfer. C. Quantification of the ratio between SUV39H1 KO and WT OTI cells within each organ. D. Representative dot plot of intravascular (iv) staining ( left ) and expression of CD49d and Ly6c ( right ) in each population at day 34 post transfer in CD8 + T cells from lungs from Exp. 2 in C . E. Quantification of the percentage and absolute numbers of CD49d + Ly6c - cells among SUV39H1KO or WT OTI cells in the indicated organs from Exp 1 in C . Numbers represent the fold increase between KO and WT condition for each organ. F. Histogram of CD69 expression in iv-CD49d + Ly6c - CD8 + T cells and proportions of CD69+ cells among iv+ and iv-CD49d + Ly6c - CD8 + T cells in lungs from Exp. 4 in C . Numbers represent p values. Each dot represents a mouse, different shapes represent different experiments. * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; **** p<0.0001 ; ns: not significant by one sample t test compared to 1 ( C ), multiple ratio paired t test with Holm-Šídák correction ( E ) and multiple paired t test ( F ).

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Adoptive Transfer Assay, Staining, Expressing

Journal: bioRxiv

Article Title: Epigenetic control of CD8 + T cell tissue homing and tissue resident memory T cell precursors by the histone methyltransferase SUV39H1

doi: 10.1101/2024.05.13.593994

Figure Lengend Snippet: A. Scheme of the experiment. B. Representative dot plots and quantification of the normalized numbers of iv-CD44 + CD8 + T cells and the normalized percentages of CD49d + Ly6c - cells among iv-CD44 + CD8 + T cells in lungs 28 days post X31 infection ( Memory ). Values were normalized to the mean of X31-infected WT group for each experiment and then pooled. C. Representative dot plots and quantification of the number of iv-CD44 + CD8 + T cells in lungs 3 days post PR8 infection ( Recall ). D. Representative dot plots and quantification of the number of CD49d + Ly6c - and CD69 + CD103 - iv-CD44 + CD8 + T cells in lungs 3 days post PR8 infection ( Recall ). * p<0.05 ; ** p<0.01 ; *** p< 0.001 ; ns: not significant by ANOVA test with Šídák’s multiple comparisons test.

Article Snippet: Cell suspensions were prepared in PBS and stained with Near IR (Fisher Scientific L34975) or Acqua LIVE/DEAD™ Fixable Cell Stain Kit (Fisher Scientific L34957) according to manufacturer’s instructions to exclude dead cells and then labelled in FACS buffer with the following antibodies: V500 Rat anti-Mouse CD4 (RM4-5, BD), eF450 CD5 Monoclonal Antibody (53-7.3, eBiosciences), FITC anti-CD8α (53-6.7, BD), PerCP-Cy5.5 CD8 α (53-6.7, BD), PE CD8 α Monoclonal Antibody (53-6.7, Ficher Scientific), BUV395 Rat Anti-Mouse CD8 β (H35-17.2, BD), APC anti-mouse CD25 Antibody (PC61, Biolegend), PerCP-Cy5.5 Rat Anti-Mouse CD44 (IM7, BD), APC Rat Anti-Mouse CD44 (IM7, BD), APC-R700 Rat Anti-Mouse CD44 (IM7, BD), eFluor 450 CD45.1 Monoclonal Antibody (A20, Fisher Scientific), PE Mouse Anti-Mouse CD45.1 (A20, BD), APC-Cy7 CD45.1 (A20, BD), PE Mouse Anti-Mouse CD45.2 (104, BD), APC Mouse anti-Mouse CD45.2 (104, BD), BV605 Hamster Anti-Rat/Mouse CD49a (Ha31/8, BD), BV786 Rat Anti-Mouse CD49d (R1-2, BD), PE/Cyanine7 anti-mouse CD49d Antibody (R1-2, Biolegend), PE-Cy7 Rat Anti-Mouse CD62L (MEL-14, BD), APC Rat Anti-Mouse CD62L (MEL-14, BD), APC/Cyanine7 anti-mouse CD62L Antibody (MEL-14, Biolegend), PerCP-Cy5.5 Hamster Anti-Mouse CD69 (H1.2F3, BD), PE-Cy7 Hamster Anti-Mouse CD69 (H1.2F3, BD), Brilliant Violet 605™ anti-mouse CD73 Antibody (TY/11.8, Biolegend), eFluor 450 CD103 Monoclonal Antibody (2E7, Fisher Scientific), eF660 CD127 (A7R34, eBiosciences), Brilliant Violet 711 anti-mouse CX3CR1 (SA011F11, Biolegend), BV711 CXCR3 (CXCR3-173, BD), Brilliant Violet 650™ anti-mouse Ly-6C Antibody (HK1.4, Biolegend), FITC anti-mouse Ly-6C (HK1.4, Biolegend), APC Ly-6C Monoclonal Antibody (HK1.4, eBiosciences), PE-CF594 Hamster Anti-Mouse KLRG1 (2F1, BD), BUV737 Hamster Anti-Mouse TCR β Chain (H57-597, BD), Alexa-Fluor®488 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-Cy™7 Hamster Anti-Mouse TCR β Chain (H57-597, BD), PE-CF594 TCR β Chain (H57-597, BD), eFluor 450 TRCR V alpha 2 (B20.1, Invitrogen), PerCP/Cy5.5 anti-mouse TCR Vα2 (B20.1, Biolegend).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: CD3ε + Cells in Pigs With Severe Combined Immunodeficiency Due to Defects in ARTEMIS

doi: 10.3389/fimmu.2020.00510

Figure Lengend Snippet: SCID pig CD3ε + cells are primarily cytotoxic T and memory T cell in circulation. (A) Four SCID pigs (p4 [ ART16/16 ], p5 [ ART16/16 ], p6 [ ART12/16 ], and p7 [ ART12/12 ]) were monitored over a three to four-month period for circulating CD3ε + cells. P6 and p7 had variable levels of CD3ε + cells throughout this period. (B) CD3ε + cells from p6 and p7 were assed for expression of SWC6, CD4α, CD8α, and CD8β. CD3ε + cells within the SCID pigs were primarily CD8α + CD8β + CD4α − and CD8α + CD8β − CD4α + .

Article Snippet: PBMCs and MNCs were stained with the following antibodies: anti-pig CD16 (G7, Bio-Rad) with a goat anti-mouse IgG1 secondary antibody, anti-pig CD3ε (BB23-8E6-8C8), anti-human CD79α (HM7), anti-pig CD8β (PPT23), anti-pig CD8α (76-12-4), anti-pig CD4α (74-12-4), and anti-pig SWC6 (MAC320).

Techniques: Expressing