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R4 4:15 Signalling responses of nonspecific cytotoxic cells (NCC) required for innate resistance to bacterial and protozoan pathogens
220 R3
Abstracts of the 7th Congress of the ISDCI: Session R 4:oo
MIX-GENERATEDCYTOTOXICCELLS
IN CHANNEL
CATFISH
Tor B. Stwe, Steven Yoshida, Gregory Chinchar, Norman Miller, L. William Clem, Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA
Moderate levels of allotarget lysis are observed with PBLs from nonimmune channel cattish. However, greatly increased (- 100 fold) cytotoxic activities are generated in mixed leukocyte cultures (MLC) by stimulation of catfish PBL with irradiated cells of allogeneic cloned B cell lines. This increased cytotoxicity was not simply due to cell proliferation since stimulation of cattish PBL with polyclonal mitogens did not result in increased target cell lysis. Somewhat surprisingly, the increased MLC-generated cytotoxicity did not exhibit allospecificity. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by “cold” target inhibition assays. However, autologous targets were not killed, demonstrating that MLCgenerated effecters could distinguish “self” from “nonself’ at the level of lysis/recognition. Efforts at establishing a possible role for alloantigens indicated that targets treated with alloantisera are somewhat less susceptible to lysis by allogeneic effecters; they are still resistant to lysis by autologous effecters. These preliminary results suggest CTL-like, rather than ADCClike or “missing self” NK-like, target recognition. Efforts at formally identifying the MLCgenerated effecters have been hampered by the lack of suitable reagents. However, based upon RT-PCR data, mRNAs for catfish TCR Q and /3 chains are present in the MLC; unfortunately the heterogeneity of such cultures renders interpretation equivocal at this time. Hence, although the nature of fish cytotoxic cells is unresolved, the MLC protocols described should facilitate definitive studies. R4 4:15 SIGNALLING RESPONSES OF NONSPECIFIC CYTOTOXIC CELLS (NCC) REQUIRED FOR INNATE RESISTANCE TO BACTERIAL AND PROTOZOAN PATHOGENS Donald L. Evans, John H. Leary III, Emmett B. Shot& and Liliana Jas~Priedmann. Microbiology, University of Georgia, Athens, GA. USA
Department of Medical
NCC may provide the tjrst barrier of defense against tumor growth and infectious agents. In this study, the in vivo relevance and a mechanism of action of NCC in disease resistance is described. The interaction of the NCC receptor (NCCRF-1) and the antigen which it recognizes on target cells is demonstrated. In vivo treatment of cat&& with the phosphatase inhibitor sodium orthovanadate (vanadate) produced resistance to challenge with the pathogen Edwur&eIlu ictaluri. Resistance to fblminant infection closely correlated with the heightened activation status of NCC as measured by increased cytotoxicity as well as a characteristic increased phosphorylation of NCC membrane signalling proteins. These effects were observed only in the vanadate treated fish at 48-96 hours post-infection and they were associated with 17%- 100% survival of the infected fish. In the absence of vanadate, infection produced 100% mortality within 6- 12 days. At the molecular level, we have sequenced a protozoan parasite target antigen (NKTag) that binds to NCC by a 34 kDa receptor protein, Binding to NKTag initiated pleiotropic signalling responses in NCC characterized by increased: cytotoxicity, NCCRP- 1 membrane expression, kinase phosphorylation, etc.. using synthetic peptides based on NKTag sequences, the (NCC) cognate antigenic determinant (i.e. 17mer) was mapped. Evidence for specific binding of NKTag to NCCRP-1 was obtained by two color flow cytometry demonstrating binding by biotinylated 17-mer and mab SC6 (anti-NCCRP-1) to the same NCC membrane protein; competition experiments were also conducted using these same two reagents; and chemical cross&king demonstrated that the cognate 17-mer bound to the 34 kDa NCCRP- 1 protein on NCC membranes.
Abstracts of the 7th Congress of the ISDCI: Session R 4:oo
MIX-GENERATEDCYTOTOXICCELLS
IN CHANNEL
CATFISH
Tor B. Stwe, Steven Yoshida, Gregory Chinchar, Norman Miller, L. William Clem, Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA
Moderate levels of allotarget lysis are observed with PBLs from nonimmune channel cattish. However, greatly increased (- 100 fold) cytotoxic activities are generated in mixed leukocyte cultures (MLC) by stimulation of catfish PBL with irradiated cells of allogeneic cloned B cell lines. This increased cytotoxicity was not simply due to cell proliferation since stimulation of cattish PBL with polyclonal mitogens did not result in increased target cell lysis. Somewhat surprisingly, the increased MLC-generated cytotoxicity did not exhibit allospecificity. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by “cold” target inhibition assays. However, autologous targets were not killed, demonstrating that MLCgenerated effecters could distinguish “self” from “nonself’ at the level of lysis/recognition. Efforts at establishing a possible role for alloantigens indicated that targets treated with alloantisera are somewhat less susceptible to lysis by allogeneic effecters; they are still resistant to lysis by autologous effecters. These preliminary results suggest CTL-like, rather than ADCClike or “missing self” NK-like, target recognition. Efforts at formally identifying the MLCgenerated effecters have been hampered by the lack of suitable reagents. However, based upon RT-PCR data, mRNAs for catfish TCR Q and /3 chains are present in the MLC; unfortunately the heterogeneity of such cultures renders interpretation equivocal at this time. Hence, although the nature of fish cytotoxic cells is unresolved, the MLC protocols described should facilitate definitive studies. R4 4:15 SIGNALLING RESPONSES OF NONSPECIFIC CYTOTOXIC CELLS (NCC) REQUIRED FOR INNATE RESISTANCE TO BACTERIAL AND PROTOZOAN PATHOGENS Donald L. Evans, John H. Leary III, Emmett B. Shot& and Liliana Jas~Priedmann. Microbiology, University of Georgia, Athens, GA. USA
Department of Medical
NCC may provide the tjrst barrier of defense against tumor growth and infectious agents. In this study, the in vivo relevance and a mechanism of action of NCC in disease resistance is described. The interaction of the NCC receptor (NCCRF-1) and the antigen which it recognizes on target cells is demonstrated. In vivo treatment of cat&& with the phosphatase inhibitor sodium orthovanadate (vanadate) produced resistance to challenge with the pathogen Edwur&eIlu ictaluri. Resistance to fblminant infection closely correlated with the heightened activation status of NCC as measured by increased cytotoxicity as well as a characteristic increased phosphorylation of NCC membrane signalling proteins. These effects were observed only in the vanadate treated fish at 48-96 hours post-infection and they were associated with 17%- 100% survival of the infected fish. In the absence of vanadate, infection produced 100% mortality within 6- 12 days. At the molecular level, we have sequenced a protozoan parasite target antigen (NKTag) that binds to NCC by a 34 kDa receptor protein, Binding to NKTag initiated pleiotropic signalling responses in NCC characterized by increased: cytotoxicity, NCCRP- 1 membrane expression, kinase phosphorylation, etc.. using synthetic peptides based on NKTag sequences, the (NCC) cognate antigenic determinant (i.e. 17mer) was mapped. Evidence for specific binding of NKTag to NCCRP-1 was obtained by two color flow cytometry demonstrating binding by biotinylated 17-mer and mab SC6 (anti-NCCRP-1) to the same NCC membrane protein; competition experiments were also conducted using these same two reagents; and chemical cross&king demonstrated that the cognate 17-mer bound to the 34 kDa NCCRP- 1 protein on NCC membranes.