H 2kb h 2db

Manufactured by BioLegend

H-2Kb/H-2Db is a lab equipment product that serves as a major histocompatibility complex (MHC) class I reagent. It is used for the detection and analysis of CD8+ T cell responses.

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APC anti-mouse H-2Kb/H-2Db PE anti-mouse H-2Kb/H-2Db H-2Db H-2Kb

6 protocols using h 2kb h 2db

Flow Cytometry Analysis of Immune Cell Markers

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Protein cell surface expression was confirmed by flow cytometry. For flow cytometry analysis, T cells were labeled with fluorescence-conjugated CD3 (# 100209, BioLegend – dilution 1/200), CD4 (# 100549, Biolegend – dilution 1/200), CD8a (# 301035, BioLegend – dilution 1/200), CD25 (# 101915, BioLegend – dilution 1/200), PD-1 (# 135205, BioLegend – dilution 1/200), FOXP₃ (# 15–5773-80, Invitrogen – dilution 3/200), CXCR4 (# 146505, BioLegend – dilution 1/200) antibodies, and cancer cells were labeled with fluorescence-conjugated PD-L1 (# 124315 – dilution 1.5/200), H-2Kb/H-2 Db (# 114607 – dilution 1/200) and CXCR4 (# 146505 – dilution 3/200) antibodies (BioLegend) and analyzed by FACS (BD LSRFortessa Cell Analyzer, # 647177).

Lin Y.N., Schmidt M.O., Sharif G.M., Vietsch E.E., Kiliti A.J., Barefoot M.E., Riegel A.T, & Wellstein A. (2022). Impaired CXCL12 signaling contributes to resistance of pancreatic cancer subpopulations to T cell-mediated cytotoxicity. Oncoimmunology, 11(1), 2027136.

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Multicolor Flow Cytometry Analysis

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Cells were stained for extracellular molecular expression patterns using mAbs against mouse CD3 (APC–Cy5 conjugated), CD4 (Pacific Blue conjugated), CD8 (V450 conjugated), CD45R/B220 (PE conjugated), CD11b (PE–Cy5 conjugated), F4/80 (PE–Cy7 conjugated; V450 conjugated), Ly6G (APC conjugated), CD25 (APC conjugated), FOXP3 (PE conjugated), H-2D^b (Alexa Fluor-647 conjugated; BioLegend), and H-2K^b/H-2D^b (PE conjugated; BioLegend). All antibodies were purchased from BD. The frequency of positive cells was analyzed using a gate that included lymphocytes, granulocytes, and/or monocytes/macrophages. Limits for the quadrant markers were always set based on negative populations and isotype control antibodies. Cells were acquired with a BD FACSCanto II cytometer and analyzed using FlowJo 7.5.3 software. The frequency (percentage) of the analyzed population in the total acquired events was used in the construction of the graphs.

Rezende B.M., Athayde R.M., Gonçalves W.A., Resende C.B., Teles de Tolêdo Bernardes P., Perez D.A., Esper L., Reis A.C., Rachid M.A., Castor M.G., Cunha T.M., Machado F.S., Teixeira M.M, & Pinho V. (2017). Inhibition of 5-lipoxygenase alleviates graft-versus-host disease. The Journal of Experimental Medicine, 214(11), 3399-3415.

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Multiparametric Analysis of Antigen-Specific T Cells

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Peripheral mouse blood cells and the splenocytes (fresh or stimulated) were stained with indicated combinations of antibodies plus H-2D^b/AFP₂₁₂ or H-2K^b-D^b/AFP₄₉₉ tetramers. For tetramer staining after re-stimulation with peptide in the media, the stimulated cells need 12 hours rest to recover the TCR. DAPI was added to exclude dead cells. Intracellular staining of IFNγ was conducted as described (24 (link)) after the cells were re-stimulated with peptides in the presence of GolgiStop for 3.5 hours. Events were collected on the LSR II and the data was analyzed using FCS Express III software. The antibodies used in this study include antibodies to mouse CD8 (Clone: 53-6.7, Biolegend), CD45.1 (Clone: A20, Biolegend), CD45.2 (Clone: 104, BD Bioscience), CD44 (Clone: 1M7, Biolegend), CD62L (Clone: MEL-14, Biolegend), CD127 (IL-7Ra) (Clone: A7R34, Biolegend), PD-1 (Clone: 29F.1A12, Biolegend), CCR7 (Clone: 4B12, Biolegend), CD122 (IL-2Rβ) (Clone: TM-β1, Biolegend), Sca-1 (Clone: D7, Biolegend,), CD95 (Clone: SA367H8, Biolegend), IFN-γ (Clone: XMG1.2, Biolegend), Bcl-2 (Clone: BCL/10C4, Biolegend), H-2^K b/H-2D^b (Clone: 28-8-6, Biolegend), TCRβ (Clone: H57-597, BD Bioscience). The Annexin-V and DAPI (4,6-diamidino-2 phenylinole dilactate) were from Biolegend.

Wu S., Zhu W., Peng Y., Wang L., Hong Y., Huang L., Dong D., Xie J., Merchen T., Kruse E., Guo Z.S., Bartlett D., Fu N, & He Y. (2017). The antitumor effects of vaccine-activated CD8+ T cells associate with weak TCR signaling and induction of stem-like memory T cells. Cancer immunology research, 5(10), 908-919.

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Comprehensive Lymphocyte Profiling by Flow Cytometry

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Cells were stained either with a panel of lymphocyte-associated antibodies against CD8a (BioLegend, Cat# 100705; Clone: 53–6.7 RRID:AB_312744; 1:100), CD4 (BioLegend, Cat# 116023; Clone: RM4–4; RRID:AB_2800579; 1:100), CD45 (BioLegend, Cat# 103155; Clone: 30-F11; RRID:AB_2650656; 1:200), and LIVE/DEAD™(Thermo Fisher Scientific, Cat# L34957; 1:1000), Granzyme B (BioLegend, Cat# 515403; Clone: GB11; RRID:AB_2114575; 1:100), IFN-γ (BioLegend, Cat# 505808; Clone: XMG1.2; RRID:AB_315402; 1:100), TNF-α (BioLegend, Cat# 506321; Clone: MP6-XT22; RRID:AB_961435; 1:100), H-2Kb/H-2Db (BioLegend Cat# 114608, RRID:AB_313599).

Wu M.J., Shi L., Dubrot J., Merritt J., Vijay V., Wei T.Y., Kessler E., Olander K.E., Adil R., Pankaj A., Tummala K.S., Weeresekara V., Zhen Y., Wu Q., Luo M., Shen W., García-Beccaria M., Fernández-Vaquero M., Hudson C., Ronseaux S., Sun Y., Saad-Berreta R., Jenkins R.W., Wang T., Heikenwälder M., Ferrone C.R., Goyal L., Nicolay B., Deshpande V., Kohli R.M., Zheng H., Manguso R.T, & Bardeesy N. (2022). Mutant-IDH inhibits Interferon-TET2 signaling to promote immunoevasion and tumor maintenance in cholangiocarcinoma. Cancer discovery, 12(3), 812-835.

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Immunological Profiling of Tumor Microenvironment

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Recombinant human interferon gamma (IFNγ; catalog no. 570206) and antibodies to human HLA-ABC (catalog no. 311404) and PD-L1 (catalog no. 329706) were obtained from Biolegend. The antibody for intracellular calreticulin was obtained from R&D Systems (catalog no. IC3898R), and the antibodies to TAP1, TAP2, LMP2, ERp57 were obtained from Abcam (catalog numbers ab83817, ab180611, ab190350, and ab13506, respectively). Pharmaceutical grade cisplatin was obtained from the veterinary pharmacy at NIH. Antibodies for in vivo mouse treatments specific for PD-L1 (clone 10F.9G2), PD-1 (clone RMP1-14), NK1.1 (clone PK136) and CD8 (clone YTS 169.4) were from BioXCell. Fluorescent-conjugated flow cytometry antibodies for mouse tumor experiments were obtained from eBioscience (CD137/41BB, catalog no. 46-1371-80) or Biolegend: CD8 (catalog no. 100712), CD45.2 (catalog no. 109806), CD80 (catalog no. 104721), CD11b (catalog no. 101211), CD11c (catalog no. 117307), CD107a (catalog no. 121619), Ly6G (catalog no. 127623), Ly6C (catalog no. 128017), and H-2Kb/H-2Db (catalog no. 114611).

Tran L., Allen C.T., Xiao R., Moore E., Davis R., Park S.J., Spielbauer K., Van Waes C, & Schmitt N.C. (2017). Cisplatin Alters Antitumor Immunity and Synergizes with PD-1/PD-L1 Inhibition in Head and Neck Squamous Cell Carcinoma. Cancer immunology research, 5(12), 1141-1151.

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ASTX660 Immunotherapy Protocol

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ASTX660 was acquired from Astex Pharmaceuticals through a cooperative research and development agreement (CRADA) with the National Institute on Deafness and Other Communication Disorders (NIDCD). Pharmaceutical grade mitoxantrone (MTX) was obtained from the National Institutes of Health veterinary pharmacy. Recombinant human IFN-γ, and human and mouse TNFα were obtained from BioLegend. Fluorescent-conjugated antibodies for mouse tumor flow cytometry were obtained from eBioscience (CD3) and BioLegend (CD45.2, CD8a, H-2Kb/H-2Db, CD11b, CD11c, CD80, CD86, I-A/I-E). Viability dyes were obtained from BD Biosciences (7AAD) and Biolegend (Zombie Yellow, Zombie NIR). Unconjugated antibodies for flow cytometry in human cell lines were obtained from LSBio (TAP1) and Abcam (TAP2, LMP2) with corresponding secondary antibodies obtained from BioLegend. Fluorescent-conjugated antibodies for flow cytometry in human cell lines were obtained from Abcam (CRT, HSP70, HMGB1, and ERp57) and Biolegend (HLA-A,B,C). The in vivo anti-mouse MHC class I antibody used for ex vivo T cell impedance assays was from BioXCell (clone M1/42.3.9.8). Antibodies and concentrations used for ICD and APM flow panels are detailed in Supplemental Methods.

Ye W., Gunti S., Allen C.T., Hong Y., Clavijo P.E., Van Waes C, & Schmitt N.C. (2020). ASTX660, an antagonist of cIAP1/2 and XIAP, increases antigen processing machinery and can enhance radiation-induced immunogenic cell death in preclinical models of head and neck cancer. Oncoimmunology, 9(1), 1710398.

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