H2-M3 major histocompatibility complex class Ib-restricted CD8 T cells induced by Salmonella enterica serovar Typhimurium infection recognize proteins released by Salmonella serovar Typhimurium.

Salmonella enterica serovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection against S. enterica serovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived from S. enterica serovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected with S. enterica serovar Typhimurium or pulsed with proteins from S. enterica serovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected with S. enterica serovar Typhimurium SL3261 or treated with Salmonella culture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known for Salmonella antigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.

Functional analysis of the molecular factors controlling Qa1-mediated protection of target cells from NK lysis.

CD94/NKG2 receptors on mouse NK cells recognize the nonclassical class I molecule Qa1 and can deliver inhibitory signals that prevent NK cells from lysing Qa1-expressing cells. However, the exact circumstances under which Qa1 protects cells from NK lysis and, in particular, the role of the dominant Qa1-associated peptide, Qdm, are unclear. In this study, we examined in detail the lysis of Qa1-expressing cells by fetal NK cells that express CD94/NKG2 receptors for Qa1 but that lack receptors for classical class I molecules. Whereas mouse L cells and human C1R cells transfected with Qa1 were resistant to lysis by these effectors, Qa1-transfected TAP-deficient human T2 cells showed no resistance despite expressing high levels of surface Qa1. However, these cells could be efficiently protected by exposure to low concentrations of Qdm peptide or certain Qdm-related peptides. By contrast, even prolonged exposure of TAP-deficient RMA/S cells to high doses of Qdm peptide failed to induce levels of surface Qa1 detectable with a Qa1-specific mAb or to protect them from NK lysis, although such treatment induced sensitivity to lysis by Qa1-specific CTL. Collectively, these findings indicate that high surface expression of Qa1 is necessary but not sufficient for protection, and that effective protection requires the expression of sufficient levels of suitable Qa1-peptide complexes to overcome activatory signals. Results obtained with a series of substituted Qdm peptides suggest that residues at positions 3, 4, 5, and 8 of the Qdm sequence, AMAPRTLLL, are important for recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors.

The mouse tumor cell lines EL4 and RMA display mosaic expression of NK-related and certain other surface molecules and appear to have a common origin.

As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of mouse tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3zeta determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRbeta genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling.

Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.

MHC class I expression protects target cells from lysis by Ly49-deficient fetal NK cells.

Using appropriate conditions natural killer (NK) cells can be cultured from the liver and thymus of day 14 fetal mice. These fetal NK cells are phenotypically and functionally indistinguishable from adult NK cells with the exception that they lack measurable expression of all of the Ly49 molecules that can currently be detected with antibodies. Despite this, they preferentially kill tumor cells and blast cells deficient in the expression of major histocompatibility complex class I molecules, although the degree of discrimination is usually weaker than that shown by adult NK cells and varies depending on the particular combination of effector and target cells used. Polymerase chain reaction analysis revealed that although fetal NK cells are severely deficient in the expression of mRNA for Ly49A, B, C, D, G, H, and I they express high levels of Ly49E mRNA, raising the possibility that Ly49E may have an important and special function in the early development of the NK lineage.

Fetal mouse NK cell clones are deficient in Ly49 expression, share a common broad lytic specificity, and undergo continuous and extensive diversification in vitro.

NK cells obtained by exposing mouse fetal thymocytes to appropriate combinations of IL-4, IL-2, and PMA are phenotypically indistinguishable from cultured adult splenic NK cells with the exception that they generally lack measurable expression of all of the inhibitory Ly49 molecules that can currently be detected with Abs (Ly49A, -C, -G, and -I) and of the activating molecule Ly49D. Despite this deficiency, fetal NK cells have a similar specificity to Ly49-expressing adult splenic NK cells. Individual fetal NK cell clones display an essentially invariant and broad specificity similar to that of polyclonal populations of fetal or adult NK cells, although significant differences in the fine specificity of clones can occasionally be detected. Most remarkably, cloned fetal NK cell lines display heterogeneous expression of a restricted set of surface molecules that includes 10A7, Ly6C, 3C2, CD8, certain isoforms of CD45, and also, occasionally, Ly49 molecules. This heterogeneity is not related to the cell cycle or activation status of the cells, and micromanipulation recloning demonstrates unambiguously that it is not due to a lack of a single cell origin. Diversity is generated rapidly and the capacity for diversification appears to persist indefinitely in vitro. The expression of individual variable Ags is independent and stochastic, resulting in fetal NK "clones" being potentially composed of hundreds of phenotypically distinct cells. We hypothesize that fetal NK cells behave as progenitor cells that are undergoing a process of rapid, extensive, and continuous diversification and that are individually capable of generating and regenerating a complex NK cell repertoire.

Vehicle travel speeds and the incidence of fatal pedestrian crashes.

The aim of this study was to estimate the likely effect of reduced travel speeds on the incidence of pedestrian fatalities in Adelaide, Australia. The study was based on the results of detailed investigations of 176 fatal pedestrian crashes in the Adelaide area between 1983 and 1991. The method developed to estimate the effect of reduced travelling speed is described and supported by references to the published literature. A reduction in the speed limit from 60 to 50 km/h was one of four speed reduction scenarios considered. The smallest estimated reduction in fatal pedestrian collisions in the selection presented was 13%, for a scenario in which all drivers obeyed the existing speed limit. The largest estimated reduction was 48% for a scenario in which all drivers were travelling 10 km/h slower. The estimated reductions in fatalities obtained in this study are compared with those observed in places where the urban area speed limit has been lowered.

Patterns of lymphokine secretion amongst mouse gamma delta T cell clones.

Although the patterns of lymphokine (LK) secretion by CD4 and CD8 alpha beta T cells have been extensively studied, the question of whether gamma delta T cells display patterns of restricted LK production and whether these patterns are the same as seen in conventional alpha beta T cells has not been previously addressed. In this study we generated panels of gamma delta T cell clones from normal C57BL/6 and BALB/c mice using a lectin-driven system and compared their patterns of secretion of nine LK with those of CD4 and CD8 alpha beta T cell clones generated in the same system. The results showed that gamma delta T cell clones displayed nonrandom patterns of highly restricted LK production with a strong bias towards the production of type 1 LK. The dominant pattern was one of high level secretion of interferon-gamma and tumor necrosis factor (TNF), with variable production of interleukin (IL)-2, and little or none of the type 2 LK IL-4, IL-5, IL-6, and IL-10. This pattern differed significantly from that of CD4 Th1 clones in that gamma delta clones showed a striking deficiency in the production of IL-3 and granulocyte/macrophage colony-stimulating factor. A small subset of gamma delta clones displayed a novel pattern, in which the only LK produced in substantial quantity were TNF and variable amounts of IL-2. The bias of gamma delta T cells towards type 1 LK production was not an artefact associated with cloning because bulk populations of splenic gamma delta T cells behaved in the same way, even when activated in the presence of high concentrations of IL-4.

Phenotypic and functional characterization of long-lived NK cell lines of different maturational status obtained from mouse fetal liver.

Culture of day 14 mouse fetal liver (FL) cells in high dose IL-2, together with appropriate combinations of IL-4 and PMA, resulted in the generation of cell lines, termed FL-A lines, that were phenotypically and functionally indistinguishable from cultured adult splenic NK cell populations with the single important exception that no Ly49-expressing cells were present. By contrast, when FL cells were cultured in low-dose IL-2 alone, a second population of slow-growing NK-like cells, termed FL-B cells, emerged. These cells expressed the NK markers asialoGM1, 10A7, 2B4, and Fc gammaRII/III but differed from FL-A and splenic NK cells in expressing IL-2R alpha and stem cell factor receptor (SCFR) but no B220. Most lines derived in this manner had minimal or no cytolytic activity and only very low levels of NK1.1. However, they could secrete substantial quantities of several lymphokines including IL-3, granulocyte-macrophage (GM)-CSF, TNF-alpha, and, most surprisingly, IL-2. A minority of FL-B lines, typified by line 903, displayed marked cytolytic activity, moderate levels of NK1.1, reduced production of IL-2, and the capacity for accelerated growth in high-dose IL-2. FL-B lines generally expressed mRNA for CD3gamma but not for other CD3 chains, whereas FL-A and fetal thymic (FT) NK lines often expressed mRNA for all four CD3 chains. Despite many similarities to pro-T cells, FL-B cells showed no capacity to differentiate into mature T cells. Taken together, our results suggest that NK lines of different maturity can be obtained from fetal liver, with FL-B lines being the most immature, FL-A lines the most mature, and lines such as FL-B 903 representing an intermediate state of differentiation.

Analysis of the functional potential of mouse CD4+ T cells using a high-efficiency cloning system.

In order to examine the functional potential of individual mouse CD4+ T cells selected, as far as possible, in a random manner, a high-efficiency cloning system driven by Con A was utilized. Under optimal conditions, cloning efficiencies of CD4+ cells of about 50% were regularly attained. Although the relative proportion of different TH subsets varied depending on the cloning conditions, the high cloning efficiency, coupled with the analysis of over 100 clones, allowed important conclusions to be drawn regarding the general construction of the mouse CD4+ T cell repertoire. (1) At least 50% of all mouse splenic CD4+ T cells have the potential to produce IL4, supporting the view that TH subsets arise by an instructional or regulatory mechanism, rather than by selection. (2) TH0 clones produce amounts of IL2 and IL4 similar to those produced by TH1 and TH2 clones, respectively, but secrete much lower quantities of IFN than TH1 clones. (3) A large proportion of TH2 clones secrete measurable amounts of IFN. (4) Lymphokine secretion patterns among CD4+ T cells are clearly not determined at random, since IL2 production is always accompanied by IFN production. (5) At least 50% of all mouse splenic CD4+ T cells have cytolytic capacity as shown by killing in a 20-hr assay, but only a proportion can also kill in 4-hr assays. Killing in 4-hr assays was strongly correlated with the ability to secrete IL2, regardless of whether IL4 was also secreted.

The majority of immature fetal thymocytes can be induced to proliferate to IL-2 and differentiate into cells indistinguishable from mature natural killer cells.

Although immature thymocytes from day 14 embryonic mice (FTC) express IL-2R, they have generally been considered to be unresponsive to IL-2. We show here that they can in fact undergo substantial and prolonged growth in vitro provided that high doses of IL-2 are present. The ability of FTC to grow in IL-2 could be enhanced slightly by PMA and also by IL-4, but dramatically by the combination of IL-4 + PMA with IL-2. Pretreatment of FTC with IL-4 + PMA for as little as 24 h primed FTC for rapid and prolonged responsiveness to IL-2, permitting the establishment of long term lines. Kinetic and clonal analysis revealed that most individual FTC could grow under these conditions. Although proliferating cells expressed functional IL-2R of only very low affinity, and IL-2R alpha chains were undetectable by immunofluorescence, blocking experiments showed unambiguously that both IL-2R alpha and IL-2R beta were involved in signal transmission. FTC lines and clones developed in this manner lacked lineage-specific markers of mature T cells, B cells, and myeloid cells, but expressed the NK cell markers NK1.1 and asialo-GM1. They displayed potent cytolytic activity against NK-sensitive targets, and, when stimulated with PMA+ionomycin, secreted IL-3 and IFN-gamma, but not IL-2 or IL-4. After intrathymic injection they showed no evidence of growth or differentiation. These results demonstrate that most, if not all, immature thymocytes have the capacity to differentiate into cells which appear to be indistinguishable from mature NK cells. They suggest that T cells and NK cells derive from a common precursor which in the thymic environment differentiates into T cells and in the extrathymic environment into NK cells.

Palmitic acid conjugation of a protein antigen enhances major histocompatibility complex class II-restricted presentation to T cells.

The effect on antigenicity of covalent attachment of lipid groups to a protein antigen was investigated. Coupling of palmitic acid to ovalbumin (OVA) enhanced major histocompatibility complex (MHC) class II-restricted presentation to most OVA-specific murine T-cell clones in vitro. The enhanced antigenicity of palmitoylated antigen was localized to the level of presentation of the synthetic peptide epitope, OVA 323-339. T-cell responses to palmitoylated antigen were more difficult to block with anti-MHC class II antibodies than responses to native antigen. However, T-cell proliferation to palmitoyl (p)-OVA and native (n)-OVA were blocked equally by anti-CD4 antibodies. Taken together, the results suggest that lipid conjugation of a protein antigen leads to the formation of a lipopeptide T-cell epitope with increased affinity of binding to MHC class II and/or T-cell receptor (TcR). These results have implications for the design of synthetic peptide vaccines.

The effect of transfected MHC class I genes on sensitivity to natural killer cells.

To test the hypothesis that major histocompatibility complex (MHC) molecules protect target cells from lysis by natural killer cells (NKC), we transfected the MHC- B16 melanoma line F10 with the class I genes encoding Dd, Kb, and Kk. Only low levels of Dd expression could be obtained and there was no protection against NKC. By contrast, Kb and Kk transfectants were obtained which displayed significant resistance to NKC, and with the latter transfectants resistance was clearly related to the level of transgene expression. Various mutants of the F10 line with altered patterns of MHC expression were also obtained. These mutant lines provided evidence that (i) the Db molecule is also capable of inducing resistance to NKC and (ii) high MHC class I expression does not by itself guarantee lowered susceptibility to NKC.

Electric shock devices and their effects on the human body.

Stun guns, shock batons and cattle prods are electric shock devices which can be used as weapons against the human body. Stun guns cause temporary incapacitation of the body whereas the other devices do not. The electrical outputs of examples of each type of device were measured using a digital storage oscilloscope at the moment when the device was discharged across resistors chosen to simulate the resistance of the human body. The outputs from the stun guns and the non-incapacitating devices have characteristic waveform shapes and magnitudes: significantly, the peak current from the stun guns is two orders of magnitude greater than from the other devices. To understand the possible hazardous effects of these outputs on the human body, the output parameters were related to the available information on the effects of electric currents on the human body and on the electro-immobilization of farm animals.

IL-3 and IL-4 affect thymocyte differentiation in organ culture.

The ability of lymphokines to affect the development and differentiation of mouse thymocytes in vitro was evaluated in a carefully controlled 3-day organ culture system. Concanavalin A (Con A)-induced supernatant (SN) from the T-cell clone D10.G4, which contains high concentrations of interleukin-3 (IL-3), IL-4 and IL-5, but lacks IL-1, IL-2 and interferon (IFN), markedly increased the proportion of CD4+CD8- cells, and decreased the proportion of CD4+CD8+ cells. These effects were unaffected by dialysing the SN, showing them to be caused by macromolecular factors. Highly purified recombinant IL-3 and IL-4 could exert similar effects, rIL-3 and rIL-4 both increasing the proportion of CD4+CD8- cells, and rIL4 in addition reducing the proportion of CD4+CD8+ cells. In conjunction with the findings of other investigators, these results indicate that at least four lymphokines (IL-1, IL-2, IL-3 and IL-4) can control T-cell development in the thymus.

Cell surface molecules involved in NK recognition by cloned cytotoxic T lymphocytes.

Short-term treatment of cloned mouse cytotoxic T lymphocytes (CTL) with interferon (IFN) induces lytic activity for natural killer- (NK) sensitive targets. Extended culture of CTL in high concentrations of interleukin 2 induces promiscuous lytic activity in which state both NK-sensitive and NK-resistant target cells are lysed. Cold-target competition analysis showed that the development of NK activity was associated with the acquisition of binding activity for NK-sensitive but not for NK-resistant targets, whereas the development of promiscuous lytic activity was associated with the acquisition of binding activity for both types of target. Antigen-specific cytolysis was inhibited by antibodies to Ly-2, Ly-5, LFA-1 and to the V region of the T cell antigen receptor (TCR), whereas NK and promiscuous lytic activity in the same cells was resistant to inhibition by anti-Ly-2 and anti-TCR. NK activity was expressed normally against a variant NK-sensitive cell line lacking all MHC antigens. These results show that, in contrast to antigen-specific recognition, the NK and promiscuous lytic activities of CTL are expressed without participation of effector cell Ly-2 and TCR molecules or target cell MHC molecules, and are most likely mediated through novel and distinct receptor systems.

T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments.

Both cDNA and genomic clones of the T cell receptor (TCR) alpha- and beta-chain genes of the alloreactive cytotoxic T lymphocyte (CTL) clone F3 were examined. Two distinct rearrangement events, one functional and one non-functional, were found for both the alpha and beta loci. Thus only a single functional TCR alpha beta heterodimer could be defined, consistent with allelic exclusion in the TCR genes. The V alpha gene employed by F3 is part of a six-member V alpha subfamily. Genomic clones containing each member of this subfamily were isolated and the V alpha nucleotide sequences determined. Five of these six genes are functional; these genes differ from each other by 7-14% at the amino acid level. A single dominant hypervariable region was defined within this subfamily, in contrast to the pattern of variability seen between V alpha genes in general.