BD FastImmune. Catalog No Tests 20 µl/test

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1 Monoclonal Antibodies Detecting Human Antigens BD FastImmune Anti-Hu IFN-γ/CD69/CD8/CD3 Catalog No Tests 20 µl/test RESEARCH APPLICATIONS BD FastImmune Anti-Hu-IFN-γ/CD69/CD8/CD3 is designed for the detection of intracellular IFN-γ and the activation marker CD69 in antigen-activated CD8 + T lymphocytes in whole blood. 1-6 Applications include studies of T-cell responses to antigens, such as herpes viruses, 7-12 human immunodeficiency virus (HIV), and tumor antigens. 17 DESCRIPTION Specificity Anti-Human Interferon-γ (Anti-Hu IFN-γ) recognizes a 20- to 25-kilodalton (kda) glycoprotein. 18 The CD69 antibody recognizes a very early human activation antigen. The CD69 antigen is a surface homodimer formed by the association of 28-kDa and 32-kDa chains that are held together by disulfide bridges. 19 The CD8 antibody recognizes an antigen expressed on the 32-kilodalton (kda) α subunit of a disulfide-linked bimolecular complex. 20 The cytoplasmic domain of the α subunit of the CD8 antigen is associated with the protein tyrosine kinase p56 lck. 21 The CD8 molecule interacts with class I major histocompatibility complex (MHC) molecules resulting in increased adhesion between the CD8 + T lymphocytes and the target cells. 22 Binding of the CD8 molecule to class I MHC molecules enhances the activation of resting T lymphocytes. 22 The CD3 antibody reacts with the epsilon chain of the CD3 antigen/t-cell antigen receptor (TCR) complex. 23 The antigen recognized by CD3 antibodies is noncovalently associated with either α/β or γ/δ TCR (70 to 90 kda). 24 Antigen distribution IFN-γ is produced, upon activation, by most CD8 + T lymphocytes, by the T H 1 and T H 0 subsets of CD4 + T lymphocytes, and by natural killer (NK) lymphocytes. 18,25-27 IFN-γ is a multifunctional immunomodulator with anti-tumor and anti-viral activity. 28,29 IFN-γ is a pleiotropic cytokine instrumental in the regulation of immune and inflammatory processes, and it has a role in the differentiation and function of monocytes. 32 The CD69 antigen is present on activated T, B, and NK lymphocytes 19 and platelets. 33 In normal peripheral blood, a variable percentage of lymphocytes express the CD69 antigen. Upon activation, CD69 antigen expression increases on lymphocytes; peak expression generally occurs within 18 hours, preceding the appearance of HLA-DR, interleukin-2 (IL-2) receptor (CD25 antigen), and transferrin receptor (CD71 antigen) CD69 and phorbol ester are comitogenic for T lymphocytes. 36 In thymus, the CD69 antigen is constitutively expressed on the bright CD3 + subset. 37 For Research Use Only. Not for use in diagnostic or therapeutic procedures. Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA USA bdbiosciences.com ResearchApplications@bd.com 02/

2 Clones Composition PROCEDURE Method for Intracellular Cytokine Detection The CD8 antigen is expressed on the human suppressor/cytotoxic T-lymphocyte subset (CD3 + CD8 + ), 38,39 as well as on a subset of NK lymphocytes. 40 The CD8 antigen is expressed on 19% to 48% of normal peripheral blood lymphocytes 41 and the majority of normal thymocytes. 42 The CD3 antigen is present on 61% to 85% of normal peripheral blood lymphocytes. 41 Anti-Hu IFN-γ, clone , is derived from the hybridization of P3X-63-Ag8.653 mouse myeloma cells with lymph node cells from BALB/c mice immunized with recombinant human IFN-γ. CD69, clone L78, is derived from hybridization of Sp2/0-Ag14 mouse myeloma cells with lymph node cells from BALB/c mice immunized with a CD8 + alloantigen-directed cytotoxic T-lymphocyte (CTL) cell line. 36 CD8, clone SK1, is derived from hybridization of NS-1 mouse myeloma cells with spleen cells from BALB/c mice immunized with human peripheral blood T lymphocytes. CD3, clone SK , is derived from hybridization of NS-1 mouse myeloma cells with spleen cells from BALB/c mice immunized with human thymocytes. Anti-Hu IFN-γ is composed of mouse IgG 2b heavy chains and kappa light chains. CD69, CD8, and CD3 are each composed of mouse IgG 1 heavy chains and kappa light chains. The BD FastImmune Anti-Hu IFN-γ/CD69/CD8/CD3 reagent is supplied as a combination of IFN-γ FITC, CD69 PE, and CD8 PerCP-Cy 5.5 and CD3 APC in 1.0 ml of phosphate-buffered saline (PBS) containing bovine serum albumin (BSA), betalactoglobulin, and 0.1% sodium azide. For complete activation and staining protocol and the appropriate application note, visit our website (bdbiosciences.com) or contact your local BD representative. Abbreviated Intracellular Staining Procedure 1. Add 1 ml of 1X BD FACS lysing solution (Cat. No ) to 100 µl of activated heparinized whole blood. 2. Incubate for 10 minutes at room temperature. 3. Centrifuge at 500 x g for 5 minutes; decant the supernatant. 4. Add 0.5 ml of 1X BD FACS Permeabilizing Solution 2 (Cat. No ). 5. Vortex and incubate for 10 minutes at room temperature. 6. Wash by adding PBS containing 0.5% BSA and 0.1% sodium azide, and centrifuge for 5 minutes. 7. Add 20 µl of Anti-Hu IFN-γ FITC/CD69 PE/CD8 PerCP-Cy5.5/CD3 APC. 8. Vortex and incubate for 30 minutes at room temperature in the dark. 9. Repeat wash step. 10. Resuspend cells in 1% paraformaldehyde in PBS Page 2

3 REPRESENTATIVE DATA Performed on cytomegalovirus (CMV) pp65 peptide mix activated whole blood. Laser excitation is at 488 nm and 635 nm. Figure 1 Representative data analyzed with a BD FACS brand flow cytometer 10 0 CD8 PerCP-Cy R CD3 APC SSC 1000 R2 0 FSC-H CD69 PE 10 4 R Anti IFN-γ FITC 10 4 HANDLING AND STORAGE WARNING CHARACTERIZATION WARRANTY Store vials at 2 C 8 C. Conjugated forms should not be frozen. Protect from exposure to light. Each reagent is stable until the expiration date shown on the bottle label when stored as directed. All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 47,48 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves. To ensure consistently high-quality reagents, each lot of antibody is tested for conformance with characteristics of a standard reagent. Representative flow cytometric data is included in this data sheet. Unless otherwise indicated in any applicable BD general conditions of sale for non-us customers, the following warranty applies to the purchase of these products. THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT. REFERENCES 1. Maino VC. Rapid assessment of antigen induced cytokine expression in memory T cells by flow cytometry. Vet Immunol Immunopathol. 1998;63: Maino VC, Picker LJ. Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. Cytometry. 1998;34: Nomura LE, Walker JM, Maecker HT. Optimization of whole blood antigen-specific cytokine assays for CD4 + T cells. Cytometry. 2000;40: Suni MA, Picker LJ, Maino VC. Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. J Immunol Methods. 1998;212: Kern F, Surel IP, Brock C, et al. T-cell epitope mapping by flow cytometry. Nat Med. 1998;4( ). 6. Maecker HT, Dunn HS, Suni MA, et al. Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J Immunol Methods. 2001;255: Kern F, Faulhaber N, Fruömmel C, et al. Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides. Eur J Immunol. 2000;30: Ghanekar SA, Nomura LE, Suni MA, Picker LJ, Maecker HT, Maino VC. Gamma interferon expression in CD8 + T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65. Clin Diagn Lab Immunol. 2001;8: Maecker HT, Ghanekar SZ, Suni MA, HE X-S, Picker LJ, Maino VC. Factors affecting the efficiency of CD8 + T cell cross-priming with exogenous antigens. J Immunol. 2001;166: Page

4 10. He XS, Rehermann B, Lopez-Labrador FX, et al. Quantitative analysis of hepatitis C virus-specific CD8 + T cells in peripheral blood and liver using peptide-mhc tetramers. Proc Natl Acad Sci USA. 1999;96: Asanuma H, Sharp M, Maecker HT, Maino VC, Arvin AM. Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus determined by intracellular detection of cytokine expression. J Infec Dis. 2000;181: Kuzushima K, Hoshino Y, Fufii K, et al. Rapid determination of Epstein-Barr specific CD8 + T-cell frequencies by flow cytometry. Blood. 1999;94: Schmitz JE, Kuroda MJ, Santra S, et al. Control of viremia in simian immunodeficiency virus infection by CD8 + lymphocytes. Science. 1999;238: Kaul R, Plummer FA, Kimani J, et al. HIV-1-specific mucosol CD8 + lymphocyte responses in the cervix of HIV-1-resistant prostitues in Nairobi. J Immunol. 2000;164: Kaul R, Rowland-Jones SL, Kimani J, et al. Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8+ responses. J Clin Invest. 2001;107: Kaul R, Dong T, Plummer FA, et al. CD8 + lymphocytes respond to different HIV epitopes in seronegative and infected subjects. J Clin Invest. 2001;107: Karanikas V, Lodding J, Maino VC, McKenzie IFC. Flow cytometric measurement of intracellular cytokines detects immune responses in MUCI immunotherapy. Clin Cancer Res. 2000;6: Aggarwal BB, Puri RK. Common and uncommon features of cytokines and cytokine receptors: an overview. In: Human Cytokines: Their Role in Disease and Therapy. Cambridge, MA: Blackwell Science; 1995: Schwarting R, Biedobitek G, Stein H. Cluster report: CD69. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Moebius U. Cluster report: CD8. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Rudd CE, Burgess KE, Barber EK, Schlossman SF. Monoclonal antibodies to the CD4 and CD8 antigens precipitate variable amounts of CD4/CD8-associated p56 lck activity. In: Knapp W, Dörken B, Gilks WR, et al, eds. Leucocyte Typing IV: White Cell Differentiation Antigens. New York, NY: Oxford University Press; 1989: Gallagher PF, Fazekas de St. Groth B, Miller JFAP. CD4 and CD8 molecules can physically associate with the same T-cell receptor. Proc Natl Acad Sci USA. 1989;86: van Dongen JJM, Krissansen GW, Wolvers-Tettero ILM, et al. Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies. Blood. 1988;71: Clevers H, Alarcón B, Wileman T, Terhorst C. The T cell receptor/cd3 complex: a dynamic protein ensemble. Annu Rev Immunol. 1988;6: Paliard X, Malefijt RDW, Yssel H, et al. Simultaneous production of IL-1, IL-4, and IFH-γ by activated human CD4 + and CD8 + T cell clones. J Immunol. 1988;141: Openshaw P, Murphy EE, Hosken NA, et al. Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations. J Exp Med. 1995;182: Street N, Mosmann T. Functional diversity of T lymphocytes due to secretion of different cytokine patterns. FASEB J. 1991;5: Hardy KJ, Sawada T. Human γ interferon strongly upregulates its own gene expression in peripheral blood lymphocytes. J Exp Med. 1989;170: Johnson HM, Bazer FW, Szente BE, Jarpe MA. How interferons fight disease. Scientific American. 1994;: ElGhazali GEB, Paulie S, Andersson G, et al. Number of interleukin-4 and interferon-γ secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation. Eur J Immunol. 1993;23: Romagnani S, Del Prete G, Maggi E, et al. Human T H 1 and T H 2 subsets. Int Arch Allergy Immunol. 1992;99: Page 4

5 32. Powrie F, Coffman RL. Cytokine regulation of T-cell function: potential for therapeutic intervention. Immunol Today. 1993;14: Testi R, Pulcinelli F, Frati L, Gazzaniga P, Santoni A. CD69 is expressed on platelets and mediates platelet activation and aggregation. J Exp Med. 1990;172: Chen JH, Prince H, Buck D, et al. Leu-23: an early activation antigen on human lymphocytes. Fed Proc. 1988;2:A Testi R, Philips J, Lanier LL. Constitutive expression of a phosphorylated activation (Leu-23) by CD3 bright thymocytes. J Immunol. 1988;141: Testi R, Philips JH, Lanier LL. Leu-23 induction as an early marker for functional CD3/T cell antigen receptor triggering: requirement for receptor cross-linking, prolonged elevation of intracellular (Ca ++ ), and stimulation of protein kinase C. J Immunol. 1989;142: Testi R, Philips J, Lanier LL. T cell activation via Leu-23 (CD69). J Immunol. 1989;143: Evans RL, Wall DW, Platsoucas CD, et al. Thymus-dependent membrane antigens in man: inhibition of cell-mediated lympholysis by monoclonal antibodies to the T H 2 antigen. Proc Natl Acad Sci USA. 1981;78: Kotzin BK, Benike CJ, Engleman EG. Induction of immunoglobulin secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man. J Immunol. 1981;127: Lanier LL, Le AM, Phillips JH, Warner NL, Babcock GF. Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol. 1983;131: Reichert T, DeBruyère M, Deneys V, et al. Lymphocyte subset reference ranges in adult Caucasians. Clin Immunol Immunopath. 1991;60: Wood GS, Warner NL, Warnke RA. Anti Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans lineage. J Immunol. 1983;131: Ledbetter JA, Evans RL, Lipinski M, Cunningham-Rundles C, Good RA, Herzenberg LA. Evolutionary conservation of surface molecules that distinguish T-lymphocyte helper/inducer and T cytotoxic/suppressor subpopulations in mouse and man. J Exp Med. 1981;153: Haynes BF. Summary of T-cell studies performed during the Second International Workshop and Conference on Human Leukocyte Differentiation Antigens. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human T Lymphocytes. New York, NY: Springer-Verlag; 1986;1: Kan EAR, Wang CY, Wang LC, Evans RL. Noncovalently bonded subunits of 22 and 28 kda are rapidly internalized by T cells reacted with Anti Leu-4 antibody. J Immunol. 1983;131: Knowles RW. Immunochemical analysis of the T-cell specific antigens. In: Reinherz EL, Haynes BF, Nadler LM, Bernstein ID, eds. Leukocyte Typing II: Human T Lymphocytes. New York, NY: Springer- Verlag; 1986;1: Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document M29-A Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37: PATENTS AND TRADEMARKS Cy is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one, return this material, unopened, to BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company BD Page