Journal: Cell reports

Article Title: STING is an intrinsic checkpoint inhibitor that restrains the T H 17 cell pathogenic program

doi: 10.1016/j.celrep.2022.110838

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat Anti-Mouse CD4-PerCP-Cy5.5 (clone RM4-5) , BD Biosciences , Cat# 550954; RRID:AB_393977.

Techniques: Recombinant, Modification, Staining, Cell Culture, SYBR Green Assay, Isolation, Chromatin Immunoprecipitation, Software

Journal: Journal of Neuroinflammation

Article Title: Sirt6 inhibition delays the onset of experimental autoimmune encephalomyelitis by reducing dendritic cell migration

doi: 10.1186/s12974-020-01906-1

Figure Lengend Snippet: Compound 1 administration in vivo inhibits SIRT6 enzymatic activity and reduces cell number in spleen and lymph node and lymphocyte infiltration in spinal cord. Mice were treated (or not) with 1 (administered twice/day), following the preventive protocol (see the “Materials and methods” section). a At 7 dpi, splenocytes were collected and homogenized, and Western blot analyses were performed to evaluate the level of acetylated H3K9 (H3K9-Ac). A representative Western blot analysis is shown, together with the normalized quantification of the band intensity ( n = 6). b , c At the indicated time points, the total cell number in spleen ( b ) and in lymph nodes ( c ) was evaluated. Data are expressed as mean ± SD from 6 animals. d At 15–17 dpi (i.e., 4 days post onset in vehicle-treated mice), spinal cords were collected from 1 -treated and vehicle-treated animals. Immunofluorescence analyses were performed, upon staining of the infiltrating lymphocytes with an anti-CD4 antibody. Representative images are shown, together with the quantification of at least 10 different images, from 3 animals for each conditions. * p < 0.05, ** p < 0.01 compared to the relative control. Data were analyzed by t test

Article Snippet: The antibody against CD4 (rat monoclonal anti-mouse CD4 RM4-5 clone (1:100, 550280 BD Pharmingen) was incubated over-night at 4 °C in PBS with 10% goat serum (Sigma-Aldrich) and 0.1% BSA (Sigma-Aldrich).

Techniques: In Vivo, Activity Assay, Western Blot, Immunofluorescence, Staining

Journal:

Article Title: Increased inducible apoptosis in CD4 + T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin

doi: 10.1046/j.1365-2567.1999.00765.x

Figure Lengend Snippet: (a)–(h) illustrates the results of typical three-colour flow cytometric analysis of a cell suspension obtained from spleen 24 hr following Sham-CLP (a,c,e,g) or CLP (b,d,f,h) and stimulated in vitro for 24 hr with 2·5 μg Con A/ml. The contour plot (a, b) of the total cell sample illustrates the typical cell cycle progression and the primary gate established on this population (R1). (c) and (d) are the cell cycle histograms of cell number versus DNA content generated from the R1 gated population in (a) and (b) respectively, which show the typical regions defined for these studies. With respect to the Sham, it can be seen that spleen cells extracted from a septic mouse at 24 hr exhibited a population of cells trailing off with lower DNA content, Ao+ (M1), from the G0/G1 peak (M2) as opposed to S/G2/M phase cells (M3)(a, c versus b, d). However, no marked shift in the phenotypic make-up of the stimulated splenocytes is evident in septic animal cells at 24 hr (e versus f). Histograms of DNA content produced from each of these gated phenotypic populations illustrate that the majority of the increase in apoptotic cells is present in the single-positive CD4+ stained cell population (g versus h).

Article Snippet: Cell staining and flow cytometric analysis In an attempt to correlate the changes in the percentage of A o + cells with their phenotypic expression, samples were stained with the combination of antibodies conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) and the DNA dye 4′,6-diamino-2-phenylindole dihydrochloride (DAPI, Molecular Bioprobes Inc., Eugene, OR) for cell cycle analysis according to the methods of Telford et al. 19 In a typical staining protocol 2×10 6 cells were incubated with 10 μg non-specific mouse immunoglobulin G (IgG)/ml phosphate-buffered saline (PBS) containing 1·0% bovine serum albumin and 0·1% sodium azide (PBS–BSA–Az buffer) for 15 min at 4°, washed by centrifugation, and incubated for 45 min at 4° with 25 μl of PBS–BSA–Az buffer containing 2 μg of FITC-conjugated monoclonal antibody against the murine T helper cell marker, CD4 (clone RM4-5, rat IgG2a) obtained from Pharmingen Inc., San Diego, CA.

Techniques: In Vitro, Generated, Produced, Staining

Journal:

Article Title: Increased inducible apoptosis in CD4 + T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin

doi: 10.1046/j.1365-2567.1999.00765.x

Figure Lengend Snippet: Neither (a) ex vivo (innate or unstimulated) nor (b) in vitro Con A-stimulated (24 hr) splenocytes harvested from mice 24 hr following CLP or Sham-CLP exhibit marked changes in their phenotypic make-up. (c) Neither the mixed (inset) nor those cells differentiated by their phenotypic (CD4 and/or CD8) expression from the ex vivo splenocytes harvested from mice 24 hr following CLP or Sham-CLP exhibited a marked increase in the percentage of Ao+ based on number of cells residing in the M1 region of the cell cycle figures depicted in Fig. 2(c) or (d). Phenotype and associated cell cycle analysis is gated as depicted in Fig. 2(e, g),(f, h). (d) Alternatively (inset) the mixed splenocytes harvested from septic mice show a significant increase (*P < 0·05 versus Sham group) in the percentage of cells which are Ao+ following 24 hr stimulation with Con A. Furthermore, when the CD4 and/or CD8 phenotypic expression was correlated with the extent of Ao+ expression, a marked increase (*P < 0·05 versus Sham group) in the percentage of single-positive CD4+, T helper cells, which were also apoptotic (Ao+) was observed. Mean ± SEM; n is six mice sampled/group.

Article Snippet: Cell staining and flow cytometric analysis In an attempt to correlate the changes in the percentage of A o + cells with their phenotypic expression, samples were stained with the combination of antibodies conjugated to either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) and the DNA dye 4′,6-diamino-2-phenylindole dihydrochloride (DAPI, Molecular Bioprobes Inc., Eugene, OR) for cell cycle analysis according to the methods of Telford et al. 19 In a typical staining protocol 2×10 6 cells were incubated with 10 μg non-specific mouse immunoglobulin G (IgG)/ml phosphate-buffered saline (PBS) containing 1·0% bovine serum albumin and 0·1% sodium azide (PBS–BSA–Az buffer) for 15 min at 4°, washed by centrifugation, and incubated for 45 min at 4° with 25 μl of PBS–BSA–Az buffer containing 2 μg of FITC-conjugated monoclonal antibody against the murine T helper cell marker, CD4 (clone RM4-5, rat IgG2a) obtained from Pharmingen Inc., San Diego, CA.

Techniques: Ex Vivo, In Vitro, Expressing, Cell Cycle Assay

Journal: STAR Protocols

Article Title: Flow cytometry and immunohistochemistry of the mouse dural meninges for immunological and virological assessments

doi: 10.1016/j.xpro.2023.102119

Figure Lengend Snippet:

Article Snippet: BV786 rat monoclonal anti-mouse CD4 (Clone RM4-5) (used at 1:200 dilution) , BD Biosciences , Cat# 563727, RRID: AB_2728707.

Techniques: Recombinant, Staining, Software

Journal: STAR Protocols

Article Title: Flow cytometry and immunohistochemistry of the mouse dural meninges for immunological and virological assessments

doi: 10.1016/j.xpro.2023.102119

Figure Lengend Snippet: Antibody mix for flow cytometry (2×)

Article Snippet: BV786 rat monoclonal anti-mouse CD4 (Clone RM4-5) (used at 1:200 dilution) , BD Biosciences , Cat# 563727, RRID: AB_2728707.

Techniques: Flow Cytometry, Concentration Assay

Journal: Nature Communications

Article Title: Lymphatic-preserving treatment sequencing with immune checkpoint inhibition unleashes cDC1-dependent antitumor immunity in HNSCC

doi: 10.1038/s41467-022-31941-w

Figure Lengend Snippet: a Representative immunohistochemical images of 4MOSC1-tongue tumors from animals subjected to neck dissection or sham surgery followed by αCTLA-4 therapy, harvested at day 10. Shown are whole tumor sections, representative high-power H&E, and anti-pan-CK stained sections (representative of n = 5 tumors/treatment group). b Top: Representative tSNE plots shown from time-of-flight mass cytometry (CyTOF), comparing 4MOSC1-tongue tumors from animals subjected to neck dissection or sham surgery followed by αCTLA-4, harvested at day 10; bottom: quantification of selected populations identified in the TIME of the aforementioned groups ( n = 3 samples/group). c Representative high-power IHC images probing for CD8+ or CD4+ cells in 4MOSC1-tongue tumors from animals subjected to neck dissection or sham surgery followed by αCTLA-4 therapy, harvested at day 10 (representative of n = 5 tumors/treatment group). d Flow plot and quantification comparing the CD4+ and CD8+ T-cell populations of MOC1 tongue tumors from animals subjected to neck dissection or sham surgery followed by αCTLA-4 therapy (red = sham surgery cohort, blue = neck dissection cohort, n = 5/group). e Heatmap comparing the expression of select chemokines and cytokines from the TIME of either control or αCTLA-4 treated 4MOSC1-tongue tumor-bearing animals at day 8 ( n = 4/group). f Experimental schema—( g ) 4MOSC1-LucOS or ( h ) MOC1-OVA tumors. Animals were randomized to receive sham surgery or neck dissection followed by treatment with αCTLA-4, after which tumors were harvested for flow cytometry to detect tumor-specific antigen tumor-infiltrating T cells. g Left: Representative flow cytometry plots and; right: quantification identifying TCRβ + OVA-H-2kb Tetramer+ CD8+ T cells from 4MOSC1-LucOS tumor-bearing animals harvested at day 10 after sham surgery or neck dissection and αCTLA-4 ( n = 5/group). h Left: Representative flow cytometry plots; and right: quantification identifying TCRβ + MuLVp15 Tetramer+ or OVA-H-2kb Tetramer+ CD8+ T cells from MOC1-OVA tongue tumor-bearing animals harvested at day 10 after sham surgery or neck dissection and αCTLA-4 ( n = 5/group). The differences between experimental groups were analyzed using independent, two-sided Student t tests ( b , d , e , g , h ). All data represent averages ± SEM, except where indicated. **** P < 0.0001. ns not statistically significant. Source data are provided as a Source Data file.

Article Snippet: Cells were stained with the following antibodies from Fluidigm: B220 (RA3-6B2), CD117(2B8), CD11c (N418), CD25 (3C7), CD4 (RM4-5), CD45 (30-F11), CD8a (53-6.7), MHCII (M5/114.15.2), NKP46 (29A1.4), CD169 (3D6.112), CD206 (C068C2), and TCRb (H57-597); or from BioLegend: CD103 (2E7), CD115 (AFS98), CD11b (M1/70), CD19 (6D5), CD3 (145-2C11), CD64 (X54-5/7.1), F4/80 (BM8), FR4 (TH6), Ly6C (HK1.4), Ly6G (1A8), and NK1.1 (PK136); or from eBioscience (ThermoFisher Scientific): Siglec-F (1RNM44N).

Techniques: Immunohistochemical staining, Dissection, Staining, Mass Cytometry, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Lymphatic-preserving treatment sequencing with immune checkpoint inhibition unleashes cDC1-dependent antitumor immunity in HNSCC

doi: 10.1038/s41467-022-31941-w

Figure Lengend Snippet: a Heatmap comparing the expression of select chemokines and cytokines from tumor-draining lymph nodes of either control or αCTLA-4 treated 4MOSC1 tumor-bearing animals, day 8 ( n = 4/group). b Representative multiplex immunofluorescence images identifying putative conventional type I dendritic cells (cDC1s) within the control or αCTLA-4 treated tdLNs from 4MOSC1 tumor-bearing animals, day 10 (representative of n = 10 tdLN/treatment group). c Representative flow cytometry contour plots identifying cDC1s (Ly6c-CD64-CD19-NK-CD11c + MHCIIhi CD11b-XCR1 +) from the tdLNs of control or αCTLA-4 treated 4MOSC1 tumor-bearing animals, day 10. d Quantification of cDC1s in tdLN after αCTLA-4 ( n = 3). e Quantification of cDC1 in tdLN after αPD-1 ( n = 4). f Quantification of activated CXCR3 + CD8+ T cells in the tdLNs of control or αCTLA-4-treated 4MOSC1 tumor-bearing animals, day 10 ( n = 3). g Quantification of CD4+ T cells in the tdLNs of control or αCTLA-4-treated 4MOSC1 tumor-bearing animals, day 10 ( n = 3). h IFNβ ELISA from tumor and tdLN from control or αCTLA-4 treated 4MOSC1 tumor-bearing animals, normalized to control, day 10 ( n = 3). i Left: Quantification of cDC1s in the tdLNs of 4MOSC1 tumor-bearing WT animals treated with αCTLA-4 (green), MAR1-5A3 blocking antibody (red lines) or combination (blue lines) ( n = 3 animals/group), day 10; (Right) and, in the tdLNs of WT (black), batf3 –/– (red), or ifnar –/– (purple) animals treated with αCTLA-4 ( n = 4 animals/group), day 10. j Tumor growth kinetics from 4MOSC1 tumor-bearing animals treated with αCTLA-4 (green lines, n = 7), MAR1-5A3 blocking antibody (red lines, n = 6), combination therapy (blue lines, n = 6) or control (black lines, n = 6). k Tumor growth kinetics from 4MOSC1 tumor-bearing WT control (black lines, n = 4) and αCTLA-4 treated animals (green lines, n = 5) versus αCTLA-4 treated batf3 –/– (red lines, n = 5) or ifnar −/− (purple lines, n = 4); bottom right: tumor volume normalized to control at day 13. l Left: Experimental schema: 5 μg of MAR1-5A3 blocking antibody or vehicle was injected into the tdLN every 2 days, beginning day 1, for a total of 4 doses. Following the development of conspicuous tumors, animals were randomized to receive αCTLA-4. Right: Tumor growth kinetics from 4MOSC1 buccal tumor-bearing animals treated with αCTLA-4 ICI and tdLN-injected local IFNAR blockade (red lines) or vehicle (green lines) ( n = 8 animals/treatment group). m Left: Experimental schema: 1 μg of diphtheria toxin or vehicle was injected into the tdLN every 3 days, beginning on day 3, for a total of three doses. Following the development of conspicuous tumors, XCR1 DTRVenus+/– animals were randomized to receive αCTLA-4. Right: Tumor growth kinetics from 4MOSC1 buccal tumor-bearing animals treated with αCTLA-4 ICI and tdLN-injected local diphtheria toxin (red lines, n = 7) or vehicle (green lines, n = 7) versus control (black lines, n = 8). n Left: Representative flow cytometry plots and; right: quantification identifying TCRβ + OVA-H-2kb Tetramer+ CD8+ T cells from 4MOSC1-LucOS tongue tumor-bearing WT or batf3 −/− animals, day 10 ( n = 5). The differences between experimental groups were analyzed using independent, two-sided Student t tests ( a , d – h , n , right), one-way ANOVA ( i , k , bottom right) or simple linear regression analysis ( j – m ). All data represent averages ± SEM, except where indicated. **** P < 0.0001. ns not statistically significant. Source data are provided as a Source Data file.

Article Snippet: Cells were stained with the following antibodies from Fluidigm: B220 (RA3-6B2), CD117(2B8), CD11c (N418), CD25 (3C7), CD4 (RM4-5), CD45 (30-F11), CD8a (53-6.7), MHCII (M5/114.15.2), NKP46 (29A1.4), CD169 (3D6.112), CD206 (C068C2), and TCRb (H57-597); or from BioLegend: CD103 (2E7), CD115 (AFS98), CD11b (M1/70), CD19 (6D5), CD3 (145-2C11), CD64 (X54-5/7.1), F4/80 (BM8), FR4 (TH6), Ly6C (HK1.4), Ly6G (1A8), and NK1.1 (PK136); or from eBioscience (ThermoFisher Scientific): Siglec-F (1RNM44N).

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Blocking Assay, Injection