NZB cytotoxic lymphocyte responses. Kinetic analyses.

Abstract

Recently, we have been studying the regulation of cytotoxic lymphocyte (CTL) responses in NZB mice. These studies have demonstrated that NZB CTL were relatively resistant to suppressor signals (1). In the present paper, we studied the timecourse of generation and the kinetics of lysis of CTL from nonprimed BALB/c, DBA/ 2, NZB, and alloantigen-primed BALB/c and NZB spleen cells in allogeneic mixed leukocyte cultures. These investigations suggested that NZB spleen cells have an accelerated generation of primary CTL responses, which, in some respects, mimic secondary CTL responses of alloantigen-primed mice. These observations shed light on recent reports of abnormal recognition phenomena by NZB T cells (2-4). Materials and MethodsMice. Alloantigen Sensitization In Vivo. Mice were sensitized by injection of 1 × 107 B6 spleen cells, in 0.05 ml of Hanks' balanced salt solution (HBSS), into each hind footpad. After 14 d, sensitized mice were sacrificed by cervical dislocation, and their spleens were removed under aseptic conditions. Single spleen-cell suspensions were prepared by gentle teasing in HBSS. The spleen cells were either assayed for CTL activity or were used as primed responder cells in in vitro mixed leukocyte cultures (MLC). MLC.Single spleen-cell suspensions were prepared in HBSS from in vivo primed mice and from nonprimed mice. B6 spleen cells, to be used as in vitro stimulator cells, were exposed to 1,500 rad of gamma irradiation. Cells were cultured in modified Eagle's minimum essential medium (MEM) with 10% heat-inactivated, mycoplasma-screened fetal calf serum (FCS) (Grand Island Biological Co., Grand Island, N. Y.), in 16-mm, flat-bottom culture wells (Cost ar, Data Packaging,, Cambridge, Mass.). 6-24 replicate wells that contained 1 × 107 responder cells and 1 X 10 irradiated stimulator cells were cultured in a final vol of 1.5 ml. Cultures were incubated for the specified number of days in a humidified atmosphere of 10% CO2, 7% 02, and 83% N2 at 37°C.Preparation of Target Cells. EL-4 tumor cells were maintained by weekly intraperitoneal 7 passage in B6 female mice. 2 × i0 tumor cells were incubated with 100 #Ci of 51Cr (Na2[ Cr]Oa, 200-500 Ci/g Cr sp act; New England Nuclear, Boston, Mass.) in 1 ml of HBSS with 10% FCS for 30 min at 37°C. The labeled target cells were then washed three times through 2 ml of FCS at 250 g for 10 rain and diluted in MEM with 10% FCS to the desired concentration.Cell-mediated Lympholysis Assay. Cytotoxicity was assayed as previously described (1). Effector cells were washed and resuspended in MEM with 10% FCS to the desired concentration of viable cells, and mixed with equal volumes (0.1 ml) of 51Cr-labeled target cells in serologic tubes. Effector and target cells were incubated for indicated times up to 4 h at 37°C in a humidified atmosphere with 5% CO2; radioactivity released into the supernate was measured 748

T cell abnormalities in NZB mice occur independently of autoantibody production.

“…Cell-mediated Lympholysis after Primary Allogeneic Mixed Lymphocyte Culture. NZB mice develop an accelerated cytotoxic response to allogenic cells in primary in vitro culture (16). To test ZB.CBA/N mice for this property, NZB, ZB.CBA/N, DBA/2, and D2.CBA/N spleen cells were cultured for 4 d with irradiated C56BL/6 spleen cells, and subsequently assayed for ability to kill 51Cr-labeled EL-4 tumor cell targets.…”

“…Death due to hemolytic anemia, lymphoid malignancy, or immune complex nephritis occurs at a mean of 15-18 mo of age (1)(2)(3)(4). Both the B and T cell populations of NZB mice have been shown to manifest abnormal immune functions (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). However, NZB mice spontaneously produce IgM anti-T cell antibodies beginning early in life (19)(20)(21)(22).…”

Differences in maternal lineages of New Zealand Black mice defined by restriction endonuclease analysis of mitochondrial DNA and by expression of maternally transmitted antigen.

“…New Zealand Black (NZB) 1 mice are an autoimmune, hyperresponsive strain important in the study of autoimmunity and immunoregulation (1,2). Recently, attempts to characterize target antigens for cytolytic T lymphocyte (CTL) responses from NZB anti-H-2-identical mixed leukocyte cultures have led to the identification of a new aUoantigen, called maternally transmitted antigen (Mta) (3)(4)(5).…”