Sources/Clones
TIA-1: Coulter (2G9)
Granzyme B: Coulter (GB7), Sanbio/Monosan (GrB7), clone GB9 (Dr Kummer, Amsterdam, The Netherlands).
Perforin: Kaimya (KM583), Sumitomo Denko, Osaka, Japan (1B4), T cell Diagnostics (polyclonal).
Fixation/Preparation
All antibodies against cytotoxic molecules can be used in formalin-fixed, paraffin-embedded tissues. Microwave or high-pressure cooking pretreatment in citrate buffer is essential for antigen retrieval.
Background
Natural killer cells and cytotoxic T lymphocytes are characterized by their inclusion of cytoplasmic granules that are released in response to target cell recognition. Among the wealth of cytotoxic molecules found in cytotoxic cells, perforin and granzyme B are two well-characterized proteins involved in one major pathway leading to apoptosis in target cells (Smyth & Trapani, 1995; Liu et al, 1995). Perforin allows for the entry of granzyme molecules into the target cells, which then activate the apoptotic protease CPP32 (Darmon et al, 1995). The genes for perforin (Lichtenheld and Podack, 1989) and granzyme B (Smyth & Trapani, 1995) have been cloned and antibodies directed against these molecules have been generated (Kummer et al, 1993). T cell-restricted intracellular antigen (TIA-1), another molecule found in cytotoxic cells, is recognized by the antibody 2G9 (Anderson et al, 1990). The exact function of TIA-1 has not been elucidated. Since it induces DNA fragmentation of digitonin-permeabilized thymocytes (Tian et al, 1991), it may be implicated in the killing induced by cytotoxic lymphocytes. TIA-1 has been demonstrated in many intestinal intraepithelial lymphocytes of normal proximal small intestine and a corresponding increase of TIA-1 positive cells in active celiac disease (Russell et al, 1993).
Applications
The expression of all three above-mentioned molecules appears to be largely restricted to cytotoxic cells. In addition, in vitro findings have also suggested that, with rare exceptions, expression of perforin and granzyme B is restricted to cytotoxic cells, including natural killer cells and cytotoxic T cells (Liu et al, 1995; Smyth & Trapani, 1995). Analysis of these antigens in conjunction with other marker molecules can therefore further specify the cellular origin of lymphocytes and lymphoid malignancies (Daums et al,
1997). In this regard, granzyme B, TIA-1 and perforin have been demonstrated in the majority of intestinal T-cell lymphomas but not in intestinal B-cell lymphomas and CD8- peripheral nodal T-cell lymphomas (Daums et al, 1997). Antibody 2G9, which recognizes TIA-1, proved to be the most sensitive immunohistological marker, being demonstrated in the highest number of cases and also in high numbers of neoplastic cells in positive cases (Daums et al, 1997). Hence, the cytotoxic differentiation in intestinal T-cell lymphoma was clearly shown, supporting derivation from intraepithelial cytotoxic T lymphocytes. Anaplastic large cell lymphomas of T cell (T-ALCL) have also been shown to express cytotoxic molecules with antibody molecules with antibody GB9 to granzyme B, whilst being absent in B-cell anaplastic large cell lymphomas, proving that T-ALCL are derived from activated cytotoxic T cells (Foss et al, 1996). Granzyme B+ T-cell lymphomas have also been mainly found in mucosa-associated lymphoid tissue, being more often associated with angioinvasion: nasal, gastrointestinal tract and lung (De Bruin et al, 1994). It has also been shown that immunohistochemical staining with anti-TIA-1 can be used to identify cytolytic T lymphocytes in epidermal lesions of human graft-versus-host disease (Sale et al, 1992). Recent work with antibodies to cytotoxic molecules has shown that the predominant mechanism of cellular destruction in Kikuchi's lymphadenitis was apoptosis mediated by cytolytic lymphocytes (Takakuwa et al, 1996; Felgar et al, 1997).
Comments
Until recently, it was impossible to differentiate most functional T-cell subsets, e.g. suppressor and cytotoxic T cell, by membrane characteristics on paraffin-embedded tissue. The production of monoclonal antibodies against cytotoxic molecules has enabled the identification of the major components of the cytotoxic granules found in the cytoplasm of activated cytotoxic and natural killer cells. Intestinal T-cell lymphomas provide an ideal positive control for antibodies to cytotoxic molecules.
References
•Anderson P, Nagler-Anderson C, O'Brien C, et al 1990. A monoclonal antibody reactive with a 15-kDa cytoplasmic granule associated protein defines a subpopulation of CD8+ T lymphocytes. Journal of Immunology, 144: 574-582.
•Darmon AJ, Nicholson DW, Bleackley RC 1995. Activation of apoptotic protease CPP32 by cytotoxic T-cell derived granzyme B. Nature 377: 446-448.
•Daums S, Foss H-D, Anagnostopoulos I et al 1997. Expression of cytotoxic molecules in intestinal T-cell lymphomas. Journal of Pathology 182: 311-317.
•De Bruin PC, Kummer JA, Van Der Valk P, et al 1994. Granzyme B-expressing peripheral T-cell lymphomas: neoplastic equivalents of activated cytotoxic T cells with preference for mucosa-associated lymphoid tissue localization. Blood 84: 3785-3791.
•Felgar RE, Furth EE, Wasik MA, Gluckman SJ, Salhany KE 1997 Histiocytic necrotizing lymphadenitis (Kikuchi's Disease): in situ labeling, immunohistochemical, and serologic evidence supporting cytotoxic lymphocyte-mediated apoptotic cell death. Modern Pathology 10:231-241.
•Foss HD, Anagnostopoulos I, Araujo I et al 1996. Anaplastic large cell lymphoma of T-cell and null-cell phenotype express cytotoxic molecules. Blood 88: 4005-4011.
•Kummer JA, Kamp A, Van Katwijk M, et al 1993. Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins. Journal of Immunological Methods 163: 77-83.
•Lichtenheld MG, Podack ER 1989 Structure of the human perforin gene. A simple gene organization with interesting potential regulatory sequences. Journal of Immunology 143: 4267-4274.
•Liu C-C, Walsh CM, Young JD-E 1995 Perforin: structure and function. Immunology Today 16: 194-201.
•Russell GJ, Nagler-Anderson C, Anderson P, Bhan AK 1993 Cytotoxic potential of intraepithelial lymphocytes (IELs): presence of TIA-1, the cytolytic granule associated protein in human IELs in normal and diseased intestine. American Journal of Pathology 143: 350-354.
•Sale GE, Anderson P, Browne M, Myerson D 1992 Evidence of cytotoxic T-cell destruction of epidermal cells in human graft-vs-host disease: immunohistology with monoclonal antibody TIA-1. Archives of Pathology and Laboratory Medicine 116: 622-625. epidermal cells in human graft-vs-host disease: immunohistology with monoclonal antibody TIA-1. Archives of Pathology and Laboratory Medicine 116: 622-625.
•Smyth MJ, Trapani JA 1995 Granzymes: exogenous proteinases that induce target cell apoptosis. Immunology Today 16:202-206.
•Takakuwa T, Ohnuma S, Koike J, Hoshikawa M, Koizumi H 1996. Involvement of cell-mediated killing in apoptosis in histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto disease). Histopathology 28: 41-48.
•Tian Q, Streuli M, Saito H, Schlossman SF, Anderson P 1991 A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induced DNA fragmentation in target cells. 1991 67:629-639.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
TIA-1: Coulter (2G9)
Granzyme B: Coulter (GB7), Sanbio/Monosan (GrB7), clone GB9 (Dr Kummer, Amsterdam, The Netherlands).
Perforin: Kaimya (KM583), Sumitomo Denko, Osaka, Japan (1B4), T cell Diagnostics (polyclonal).
Fixation/Preparation
All antibodies against cytotoxic molecules can be used in formalin-fixed, paraffin-embedded tissues. Microwave or high-pressure cooking pretreatment in citrate buffer is essential for antigen retrieval.
Background
Natural killer cells and cytotoxic T lymphocytes are characterized by their inclusion of cytoplasmic granules that are released in response to target cell recognition. Among the wealth of cytotoxic molecules found in cytotoxic cells, perforin and granzyme B are two well-characterized proteins involved in one major pathway leading to apoptosis in target cells (Smyth & Trapani, 1995; Liu et al, 1995). Perforin allows for the entry of granzyme molecules into the target cells, which then activate the apoptotic protease CPP32 (Darmon et al, 1995). The genes for perforin (Lichtenheld and Podack, 1989) and granzyme B (Smyth & Trapani, 1995) have been cloned and antibodies directed against these molecules have been generated (Kummer et al, 1993). T cell-restricted intracellular antigen (TIA-1), another molecule found in cytotoxic cells, is recognized by the antibody 2G9 (Anderson et al, 1990). The exact function of TIA-1 has not been elucidated. Since it induces DNA fragmentation of digitonin-permeabilized thymocytes (Tian et al, 1991), it may be implicated in the killing induced by cytotoxic lymphocytes. TIA-1 has been demonstrated in many intestinal intraepithelial lymphocytes of normal proximal small intestine and a corresponding increase of TIA-1 positive cells in active celiac disease (Russell et al, 1993).
Applications
The expression of all three above-mentioned molecules appears to be largely restricted to cytotoxic cells. In addition, in vitro findings have also suggested that, with rare exceptions, expression of perforin and granzyme B is restricted to cytotoxic cells, including natural killer cells and cytotoxic T cells (Liu et al, 1995; Smyth & Trapani, 1995). Analysis of these antigens in conjunction with other marker molecules can therefore further specify the cellular origin of lymphocytes and lymphoid malignancies (Daums et al,
1997). In this regard, granzyme B, TIA-1 and perforin have been demonstrated in the majority of intestinal T-cell lymphomas but not in intestinal B-cell lymphomas and CD8- peripheral nodal T-cell lymphomas (Daums et al, 1997). Antibody 2G9, which recognizes TIA-1, proved to be the most sensitive immunohistological marker, being demonstrated in the highest number of cases and also in high numbers of neoplastic cells in positive cases (Daums et al, 1997). Hence, the cytotoxic differentiation in intestinal T-cell lymphoma was clearly shown, supporting derivation from intraepithelial cytotoxic T lymphocytes. Anaplastic large cell lymphomas of T cell (T-ALCL) have also been shown to express cytotoxic molecules with antibody molecules with antibody GB9 to granzyme B, whilst being absent in B-cell anaplastic large cell lymphomas, proving that T-ALCL are derived from activated cytotoxic T cells (Foss et al, 1996). Granzyme B+ T-cell lymphomas have also been mainly found in mucosa-associated lymphoid tissue, being more often associated with angioinvasion: nasal, gastrointestinal tract and lung (De Bruin et al, 1994). It has also been shown that immunohistochemical staining with anti-TIA-1 can be used to identify cytolytic T lymphocytes in epidermal lesions of human graft-versus-host disease (Sale et al, 1992). Recent work with antibodies to cytotoxic molecules has shown that the predominant mechanism of cellular destruction in Kikuchi's lymphadenitis was apoptosis mediated by cytolytic lymphocytes (Takakuwa et al, 1996; Felgar et al, 1997).
Comments
Until recently, it was impossible to differentiate most functional T-cell subsets, e.g. suppressor and cytotoxic T cell, by membrane characteristics on paraffin-embedded tissue. The production of monoclonal antibodies against cytotoxic molecules has enabled the identification of the major components of the cytotoxic granules found in the cytoplasm of activated cytotoxic and natural killer cells. Intestinal T-cell lymphomas provide an ideal positive control for antibodies to cytotoxic molecules.
References
•Anderson P, Nagler-Anderson C, O'Brien C, et al 1990. A monoclonal antibody reactive with a 15-kDa cytoplasmic granule associated protein defines a subpopulation of CD8+ T lymphocytes. Journal of Immunology, 144: 574-582.
•Darmon AJ, Nicholson DW, Bleackley RC 1995. Activation of apoptotic protease CPP32 by cytotoxic T-cell derived granzyme B. Nature 377: 446-448.
•Daums S, Foss H-D, Anagnostopoulos I et al 1997. Expression of cytotoxic molecules in intestinal T-cell lymphomas. Journal of Pathology 182: 311-317.
•De Bruin PC, Kummer JA, Van Der Valk P, et al 1994. Granzyme B-expressing peripheral T-cell lymphomas: neoplastic equivalents of activated cytotoxic T cells with preference for mucosa-associated lymphoid tissue localization. Blood 84: 3785-3791.
•Felgar RE, Furth EE, Wasik MA, Gluckman SJ, Salhany KE 1997 Histiocytic necrotizing lymphadenitis (Kikuchi's Disease): in situ labeling, immunohistochemical, and serologic evidence supporting cytotoxic lymphocyte-mediated apoptotic cell death. Modern Pathology 10:231-241.
•Foss HD, Anagnostopoulos I, Araujo I et al 1996. Anaplastic large cell lymphoma of T-cell and null-cell phenotype express cytotoxic molecules. Blood 88: 4005-4011.
•Kummer JA, Kamp A, Van Katwijk M, et al 1993. Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins. Journal of Immunological Methods 163: 77-83.
•Lichtenheld MG, Podack ER 1989 Structure of the human perforin gene. A simple gene organization with interesting potential regulatory sequences. Journal of Immunology 143: 4267-4274.
•Liu C-C, Walsh CM, Young JD-E 1995 Perforin: structure and function. Immunology Today 16: 194-201.
•Russell GJ, Nagler-Anderson C, Anderson P, Bhan AK 1993 Cytotoxic potential of intraepithelial lymphocytes (IELs): presence of TIA-1, the cytolytic granule associated protein in human IELs in normal and diseased intestine. American Journal of Pathology 143: 350-354.
•Sale GE, Anderson P, Browne M, Myerson D 1992 Evidence of cytotoxic T-cell destruction of epidermal cells in human graft-vs-host disease: immunohistology with monoclonal antibody TIA-1. Archives of Pathology and Laboratory Medicine 116: 622-625. epidermal cells in human graft-vs-host disease: immunohistology with monoclonal antibody TIA-1. Archives of Pathology and Laboratory Medicine 116: 622-625.
•Smyth MJ, Trapani JA 1995 Granzymes: exogenous proteinases that induce target cell apoptosis. Immunology Today 16:202-206.
•Takakuwa T, Ohnuma S, Koike J, Hoshikawa M, Koizumi H 1996. Involvement of cell-mediated killing in apoptosis in histiocytic necrotizing lymphadenitis (Kikuchi-Fujimoto disease). Histopathology 28: 41-48.
•Tian Q, Streuli M, Saito H, Schlossman SF, Anderson P 1991 A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induced DNA fragmentation in target cells. 1991 67:629-639.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
