The T cell lineage is generally divided into two distinct subpopulations on the basis of their function, phenotype, and genetic restriction (1, 2). In the mouse, helper/inducer T cells are L3T4 +, Lyt-2-, class II-restricted, and secret multiple lymphokines. CTL are L3T4-, Lyt-2 ÷, class I-restricted, and lyse target cells via direct cell-cell contact. Protective immunity against intracellular bacteria, including Listeria monocytogenes, is mediated by specific T cells and expressed by activated macrophages (2). Class II-restricted, Lyt 2-L. monocytogenes-specific T lymphocytes produce many lymphokines in vitro, and adoptive protection against listeriosis has been found to be class II-restricted (3-6). These findings led to the concept that Th are crucial for cellular antibacterial immunity. This concept, however, requires verification because it was also found that successful adoptive protection against listeriosis depends on Lyt-2 + T cells and on class I compatibility between T cells and recipients (7-9). Whether L. monocytogenesspecific Lyt-2 + T cells reside in the CTL set has not been addressed by these studies. To approach this question we have established Lyt-2 + T cell clones from L. monocytogenes-infected mice and studied their cytotoxic potential against infected phagocytes. Materials and MethodsMice. Male C57BL/6, DBA/2, B6.C-H2 b'l, and B6.C-H2 bmL2 mice, 8-12 wk old, were used. Mice were raised under specific pathogen-free conditions at the Max-PlanckInstitute, Freiburg.Establishment of Lyt-2 +, L. monocytogenes-reactive T cell clones. C57BL/6 mice were intravenously infected with 5 x 104 live L. monocytogenes EGD organisms (originally obtained from G. B. Makaness, Saranac Lake, NY) and 6 d later splenic T cells were enriched by passage over nylon wool columns, as described previously (7). T cells were analyzed or sorted at a flow rate of 2,000 cells/s using a Cytofluorograph 50 H (Ortho Diagnostic Systems Inc., Raritan, N J) as described (8). After washing, positively selected Lyt-2 ÷ T cells (2 × 104/0.2 ml) were cultured in Iscove's modified Dulbecco's medium (IMDM, Gibco) supplemented with 10% FCS, antibiotics, 1 mM glutamine, and 2 × 10 -5 M 2-ME in the presence of 2 × 105/0.2 ml L. monocytogenes-infected stimulator cells and 10% crude IL-2 in round-bottomed microculture plates (Nunc, Roskylde, Denmark)at 37°C, 7% CO2. Irradiated (2,200 rad) spleen cells from mice that had been infected G. De Libero is a recipient of the EMBO (European Molecular Biology Organization) long-term fellowship.
Selected L3T4-and Lyt 2-T-cell subpopulations from Listeria monocytogenes-infected mice were transferred into syngenic recipients, and their capacity to adoptively mediate protection against L. monocytogenes and delayed-type hypersensitivity to listerial antigens was determined. Both functions were markedly reduced by pretreatment of cells with either anti-L3T4 or anti-Lyt 2.2 antibodies plus complement, but they could be restored by admixture of the two selected T-cell subsets. Thus, after systemic cell transfer effective protection against L. monocytogenes and delayed-type hypersensitivity to listerial antigens depend on cooperation between specific L3T4+ and Lyt 2+ T cells.
Mice were infected with the intracellular bacterium, Listeria monocytogenes, and T cell clones from spleens, lymph nodes and peritoneal exudates were established. The capacity of L3T4+, Lyt2- T-cell clones to specifically lyse L. monocytogenes-infected macrophages was analyzed. As a source of target cells, bone marrow macrophages (BMM phi) after 9 days of culture in hydrophobic teflon bags were used. These BMM phi were totally Ia-; however, significant Ia-expression could be induced by interferon-gamma (IFN-gamma). IFN-gamma-stimulated BMM phi, after priming with live or killed L. monocytogenes organisms were effectively lysed by the vast majority of L3T4+ T cell clones. In the absence of either IFN-gamma stimulation or antigen priming, no lysis occurred. Cytolysis was demonstrable in a conventional 4-h 51Cr-release assay and in an 18-h neutral red uptake assay and was antigen specific and class II restricted. Native T cells from L. monocytogenes-infected mice failed to lyse stimulated, L. monocytogenes-primed BMM phi and gained their cytolytic activity after antigenic restimulation in vitro. These data demonstrate that L. monocytogenes-specific L3T4+ T cells could lyse M phi presenting listerial antigens provided that Ia antigen expression had been induced. L3T4+ T cell clones produced IFN-phi after restimulation with antigen plus accessory cells in vitro and IFN-gamma secretion could be increased by costimulation with recombinant IL 2. These T cell clones conferred significant protection upon recipient mice which was more pronounced in the liver. The possible relevance of lysis by L3T4+ T cells of infected M phi to protection against and pathogenesis of intracellular bacterial infections is discussed.
Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.
Variant lines expressing high and low surface densities of the accessory molecule CD4 have been developed by repeated preparative flow cytometric cell sortings from the murine Th cell clone D10.G4.1 (D10). The high CD4 variant line (D10H) fully maintained the original I-Ak restricted specificity for conalbumin of wild-type D10 cells. In contrast, the low CD4 variant line (D10L) showed a strong autoreactivity to I-Ak carrying stimulator cells alone which was only slightly augmented by addition of conalbumin. Cell surface molecules other than CD4, including TCR, CD3, CD11a, CD2, CD45, CD44, and MHC class I, remained identical on D10H and D10L sublines as on D10 wild-type cells. The possibility that D10L cells had suffered alterations of their TCR-alpha beta was excluded by demonstrating their reactivity with a panel of eight different anti-clonotypic mAb specific for various epitopes of the D10 TCR. By limiting dilution analysis we show that the majority of responding cells of D10L sublines were autoreactive. Although the reactivity for allogeneic I-A also increased as compared with D10H cells, a clear preference for self-I-Ak was maintained so that a true autoreactive phenotype was evident. The results indicate that the surface concentration of CD4 has a decisive influence on self-non-self discrimination of MHC class II-restricted Th cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.