IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3.

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.

Spatial differences in active caspase-8 defines its role in T-cell activation versus cell death.

Caspase-8, a cysteine-protease, initiates apoptosis when activated by death receptors. Caspase-8 is also essential for initiating T lymphocyte proliferation following T-cell antigen receptor (TCR) signaling. Given these disparate functions of caspase-8, we sought to determine whether this represented only a difference in the magnitude of caspase-8 activation, or different intracellular locations of active caspase-8. We demonstrate by high-resolution multicolor confocal laser scanning microscopy an aggregation of active caspase-8 within membrane lipid rafts in T cells stimulated with anti-CD3. This suggests that following TCR stimulation active caspase-8 physically interacts with lipid raft proteins, possibly to form a signaling platform. In contrast, Fas stimulation of T cells resulted in a much more profound activation of caspase-8 that was exclusively cytosolic. These confocal microscopic findings were confirmed using discontinuous sucrose gradient ultracentrifugation to isolate lipid raft versus cytosolic components. This sequestration model of caspase-8 activation was further supported by the observation that a classic caspase-8 substrate, BID, was not cleaved in CD3-stimulated T cells, but was cleaved after Fas engagement. Our data support a model that the location of active caspase-8 may profoundly influence its functional capacity as a regulator of either cell cycling or cell death.

Liver damage by infiltrating CD8+ T cells is Fas dependent.

Ag stimulation of CD8+ lymphocytes in vivo results in their migration to various tissues as well as the activation of a cytolytic program involving perforin, TNF-alpha, and Fas ligand. The liver is one of the main sites for infiltration by activated CD8+ T cells, and this is followed by the death of hepatocytes. The contribution of the various cytolytic components to this process is unclear. Hepatocyte damage by CD8+ T cells was studied using the MHC class I-restricted OVA-specific TCR transgenic mouse (OT-1) to examine the contribution of Fas to hepatocyte death. Activated CD8+ T cells from both OT-1 and Fas-deficient OT-1lpr mice migrated to the liver in similar numbers after OVA administration, but only in OT-1 mice was there evidence of significant hepatocyte damage histologically and by elevation of serum aspartate transaminase. These differences were not the result of inefficient induction of cytolytic activity in OT-1lpr liver T cells, since they were as cytolytic in vitro as OT-1 liver T cells. This was supported by findings of similar high levels of message for perforin, TNF-alpha, and Fas ligand in liver lymphocytes from both mice. These findings demonstrate that following Ag activation, infiltrating liver CD8+ T lymphocytes induce hepatocyte damage in a Fas-dependent manner.

A model for the origin of TCR-alphabeta+ CD4-CD8- B220+ cells based on high affinity TCR signals.

The origin of TCR-alphabeta+ CD4-CD8- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-alphabeta+ CD4-CD8- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-alphabeta+ CD4-CD8- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rbeta and the absence of surface CD2. The CD4-CD8- B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-alphabeta+ CD2- CD4-CD8- B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.

Liver damage preferentially results from CD8(+) T cells triggered by high affinity peptide antigens.

Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)-restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4(-)CD8(-)B220(+) T cells occurs more frequently from a CD8(+) precursor than from CD4(+) T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8(+) cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC-restricted TCR. The findings show that CD4(-)CD8(-)B220(+) T cells preferentially derive from a CD8(+) precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.

Anti-CD40L accelerates renal disease and adenopathy in MRL-lpr mice in parallel with decreased thymocyte apoptosis.

The CD40/CD40L (CD40 ligand) axis regulates several interactions between T cells and B cells. Blocking of CD40 engagement by CD40L inhibits Ig class switch by B cells as well as diminishes T cell response to an immunizing Ag. For these reasons, disruption of CD40/CD40L interactions by anti-CD40L administration or by genetic disruption of CD40L has ameliorated a variety of autoimmune conditions. More recent findings suggest that a direct signal can be transmitted to T cells via their expressed CD40L, which can costimulate proliferation with CD3 or promote germinal center formation. It is therefore possible that treatment with anti-CD40L Ab might produce a different outcome than observed in genetically CD40L-deficient mice. In this regard, we observe that in contrast to the genetic deletion of CD40L in MRL-lpr mice, which diminishes autoimmune disease but has little effect on adenopathy, administration of anti-CD40L to MRL-lpr mice accelerates both of these parameters. This difference appears to result from anti-CD40L actively delivering a signal that inhibits T cell apoptosis in lpr mice. This was confirmed by in vitro studies demonstrating that CD40L cross-linking on lpr thymocytes inhibited apoptosis and surface TCR down-modulation induced by CD3 ligation.

Down-modulation of CD2 delays deletion of superantigen-responsive T cells.

CD2 is a cell surface glycoprotein expressed on most T lymphocytes that is generally viewed as a cell adhesion molecule and, in this capacity, contributes to T cell receptor (TCR) signaling. CD2 has a relatively long cytoplasmic tail which associates with the src family tyrosine kinases, p56(lck) and p59(fyn), and could potentially signal directly. Down-modulation of CD2 on T cells has been shown to result in diminished proliferative capacity and interleukin (IL)-2 production. Furthermore, re-expression of CD2 can result in the restoration of these functions. This suggests that CD2 can influence the intensity of TCR signaling. As TCR signal intensity is pivotal to the induction of T cell apoptosis, we considered the hypothesis that the level of CD2 on the T cell surface may influence its propensity toward apoptosis. Using an anti-CD2 antibody, CD2 was down-modulated in vivo on mouse T lymphocytes without affecting the levels of surface CD3, TCR alphabeta, CD4 or CD8. Deletion of superantigen-responsive T cells was delayed in mice with down-modulated CD2 following the administration of staphylococcal enterotoxin B (SEB). This was paralleled by diminished apoptosis of SEB-responsive cells. The findings suggest a model whereby the level of CD2 expression influences the intensity of TCR signaling and the ability to undergo apoptosis.

Transgene-encoded human CD2 acts in a dominant negative fashion to modify thymocyte selection signals in mice.

CD2 is a cell surface glycoprotein present on all T cells which has been shown to function as an adhesion and signaling molecule. Expressed early in T cell development, human CD2 (HCD2) has been suggested to play a role during thymopoiesis. However, the relevance of CD2 in T cell development has been called into question recently, as neither disruption of the CD2 gene nor anti-CD2 antibody treatment of fetal thymic organ cultures in mouse were shown to have any discernible consequences. We have expressed HCD2 at high levels in transgenic mice and found a profound effect of the transgene on thymocyte differentiation. Transgenic thymuses are considerably reduced in cell number as a consequence of increased apoptosis of double-positive (DP) thymocytes in the cortex. The remaining DP cells have up-regulated levels of T cell receptor (TCR) and are resistant to apoptosis mediated by administration of antigen. These effects are dependent on the cytoplasmic domain of HCD2, as mice expressing comparable levels of a tailless HCD2 transgene have a normal phenotype. The HCD2 cytoplasmic domain contains several regions of identity with mouse CD2 and can interact effciently with mouse intracellular signaling machinery. These results suggest there is considerable cross-talk between CD2 and TCR on developing thymocytes with consequences for the stimulation threshold of mature T cells.

Decreased CD4-CD8- TCR-alpha beta + cells in lpr/lpr mice lacking beta 2-microglobulin.

The CD4-CD8- T cells that accumulate in lpr/lpr mice have previously expressed CD8, on the basis of studies of CD8 alpha gene demethylation. The actual requirement for CD8 interaction with class I MHC molecules to promote the appearance of CD4-CD8- T cells in lpr/lpr mice has also been suggested. To examine this point in more detail, the lpr mutation was bred onto a beta 2-microglobulin-deficient background (beta 2-m-/-). C57BL/6 (B6) mice homozygous for both the lpr mutation of the fas gene and inactivation of the beta 2-m gene (beta 2-m-/- lpr/lpr) develop less alpha beta T cell lymphadenopathy than the parental B6 lpr/lpr strain. This is caused by the near absence of CD8+ T cells and a considerable reduction in CD4-CD8- T cells, revealing an important role for positive selection on class I MHC molecules during the ontogeny of lpr CD4-CD8- T cells. Although absolute numbers of peripheral T cells are decreased in beta 2-m-/- lpr/lpr mice, they manifest a B cell lymphadenopathy with age. beta 2-m-/- lpr/lpr mice display only subtle indications of autoimmune disease with age, compared with parental B6 (beta 2-m+/+)lpr/lpr mice. These include limited histopathologic stages of kidney disease and lack of proteinuria, despite the presence of serum anti-DNA Abs. Thus, absence of class I MHC-positive selection of CD8+ and CD4-CD8- TCR-alpha beta + cells limits the autoimmune diathesis observed in beta 2-m+/+ lpr/lpr mice.

Delayed kinetics of T lymphocyte anergy and deletion in lpr mice.

The Fas/APO-1 (Fas) antigen is a cell surface protein that mediates apoptosis and belongs to the tumor necrosis factor receptor family. The lymphoproliferative (lpr) anomaly in mice results from a retroviral disruption within the fas gene. Mice that are homozygous for the lpr mutation accumulate large numbers of T lymphocytes and exhibit an autoimmune syndrome resembling systemic lupus erythematosus. A possible explanation for this process is that in the absence of Fas antigen, lpr T cells may be resistant to normal peripheral deletional signals. The bacterial superantigen staphylococcal enterotoxin B (SEB) rapidly induces anergy and deletion by apoptosis of reactive T lymphocytes in normal mice. Administration of SEB to adult lpr mice results in the delayed induction of both unresponsiveness and deletion of V beta 8+ lymph node cells. This is not due merely to an increased thymic output in lpr mice; the delayed induction of tolerance and elimination of reactive lpr T cells by superantigens are intrinsic properties of the cells. The progressive lymphadenopathy in lpr mice may reflect a process of lymphoaccumulation rather than lymphoproliferation. A delay in tolerance induction and elimination of self-reactive T cells could have profound influence on the autoimmune diathesis of lpr mice.

Prominent T lymphocyte response to Borrelia burgdorferi from peripheral blood of unexposed donors.

The proliferative response of peripheral blood T cells to the spirochete, Borrelia burgdorferi, can be as pronounced in unexposed normal individuals as it is in Lyme disease patients. This finding was observed using three geographically distinct isolates of B. burgdorferi. The response is not due to a lipopolysaccharide effect of the spirochete, is sensitive to Proteinase K, and requires antigen processing. It does not result from cross-reactivity of memory T cells that may be reactive to another antigen; the proliferative response to B. burgdorferi is equally distributed between naive (CD29-, CD45RO-) and memory (CD29+, CD45RO+) T cells, whereas the tetanus response is confined to the memory subset. In support of this notion, cord blood specimens that contain almost entirely naive T cells, respond as vigorously to B. burgdorferi as T cells from normal adult peripheral blood. A large panel of CD4+ T cell clones has been derived that are specific for B. burgdorferi. The majority of these clones are reactive to B. burgdorferi in the presence only of autologous HLA-DR molecules. Collectively, these data suggest that the T cell response from normal individuals is more likely due to multiple antigenic epitopes within Borrelial proteins than a superantigen response.

Laboratory review of reference strains of Corynebacterium diphtheriae indicates mistyped intermedius strains.

All biotyped strains of Corynebacterium diphtheriae from the American Type Culture Collection (ATCC) were compared for morphology and biochemical reactions. Biotypes of all gravis strains and most mitis strains were confirmed, but intermedius strains were found to be misclassified. New lipid-dependent intermedius strains have been deposited with the ATCC.

CD2-CD4-CD8- lymph node T lymphocytes in MRL lpr/lpr mice are derived from a CD2+CD4+CD8+ thymic precursor.

MRL lpr/lpr (lymphoproliferative, lpr) mice demonstrate an age-dependent lymphoproliferation and development of autoimmunity. Characteristic of the lymphoproliferation in these mice is the accumulation of large numbers of CD4-CD8-(CD4-8-),CD3+ T lymphocytes in their lymph nodes. The development of the CD4-8- cells, which also aberrantly express B220 and CD44 (Pgp-1) but are CD2-, has been shown to be thymus dependent. An unusual feature of lpr CD4-8-T lymphocytes is that although they appear unresponsive to stimulation, as defined by proliferation and IL-2 production, they have undergone thymic negative selection. As thymic deletion normally occurs at the CD4+CD8+ (CD4+8+) stage, this raises the dilemma that lpr CD4-8- T lymphocytes have either previously been CD4+8+, or they are able to undergo thymic selection as CD4-8- cells. We have addressed this question by examining the methylation status of the CD8 gene in MRL lpr CD4-8- lymph node cells. Demethylation of the CD8 gene has been shown to be an indicator of previous CD8 expression. We find that the CD8 gene in lpr CD4-8- lymph node cells, as well as in the abnormal B220+ CD4-8- lpr thymocytes, is demethylated, suggesting that these cells have previously expressed CD8. In addition, we find that the lpr CD4+8+ thymocyte population contains an increased percentage of atypical B220+, CD44+ cells that are virtually all CD2+. Taken together, these data are consistent with the lpr CD2-CD4-8- population of LNC having arisen from a CD2+ CD4+8+ thymic stage of differentiation.

CD2 expression correlates with proliferative capacity of alpha beta + or gamma delta + CD4-CD8- T cells in lpr mice.

The T lymphocytes that accumulate in vast numbers in the lymphoid tissues of lpr/lpr (lpr) mice express a TCR-alpha beta that is polyclonally rearranged, and yet is devoid of surface CD4 or CD8 (CD4-8-) as well as CD2. lpr CD2- alpha beta + CD4-8- T cells exhibit an apparent block in signal transduction, in that when activated they produce little or no IL-2 and proliferate minimally in the absence of exogenous IL-2. In contrast to the predominant hyporesponsive alpha beta + CD4-8- T cells, we observe that a minor subset (1 to 2%) of lpr lymph node CD4-8- cells expresses a TCR-gamma delta and can proliferate upon activation with PMA and ionomycin in the absence of exogenous IL-2. Furthermore, these responsive gamma delta T cells express surface CD2. The functional and phenotypic distinctions of lpr gamma delta T cells led us to identify an analogous minor (4 to 10%) subset of alpha beta + CD4-8- cells in lpr thymus and lymph nodes that does express CD2. Similar to the gamma delta subset, these CD2+ alpha beta + CD4-8- cells are also capable of proliferation and IL-2 production. Thus the capacity for IL-2 production and proliferation by a small proportion of lpr CD4-8- T cells, either alpha beta + or gamma delta +, correlates with their expression of surface CD2. This correlation is supported by the observation that the lpr liver contains actively cycling alpha beta + CD4-8- lymphocytes that are strikingly enriched for CD2 expression. Consequently, unlike the vast proportion of abnormal lpr CD2- CD3+ CD4-8- cells, the CD2+ CD3+ CD4-8- T cells may not express the basic lpr defect, or else are not affected by its presence. These studies suggest that expression of the lpr abnormality may be restricted to a particular T cell lineage. This functional correlation with CD2 expression may be more broadly applicable to phenotypically similar subsets of normal thymocytes, and possibly peripheral tolerized T lymphocytes.

The molecular epidemiology of three biotypes of Corynebacterium diphtheriae in the Seattle outbreak, 1972-1982.

Between 1972 and 1982 Seattle experienced a diphtheria outbreak involving 1,100 cases, primarily adults with cutaneous lesions. Biotyping revealed three consecutive overlapping outbreaks including 433 toxinogenic intermedius cases. Isolates from each quarter year were examined for DNA restriction fragment patterns and hybridization patterns with three DNA probes. All intermedius outbreak isolates appeared identical in all analyses and most stock cultures had the outbreak DNA fragment pattern, but DNA probes detected six patterns within 11 intermedius stock cultures. The mitis outbreak was heterogeneous, involving at least five restriction fragment patterns. In contrast, DNA restriction fragment analyses indicated that 22 of 25 gravis outbreak isolates belonged to a single strain, whereas there were seven strains within eight gravis stock cultures. DNA probe analyses of gravis outbreak isolates detected seven different patterns, five involving copy numbers of the toxB fragment and attB sites that presumably reflected lysogenic events that occurred during the outbreak.

Oral toxicity of ferric dimethyl-dithiocarbamate (ferbam) and tetramethylthiuram disulfide (thiram) in rodents.

Single oral doses of ferbam, thiram, zineb, or maneb produced central nervous system stimulation followed by depression and alopecia. Ferbam and thiram were more toxic on the basis of weight than zineb; maneb was relatively nontoxic. There was no species difference in acute toxicity between rats and mice. In 13- and 80-wk feeding studies, the toxic effects of ferbam and thiram in rats were similar; however, thiram was more toxic on the basis of weight than ferbam. During the 80-wk feeding study, weight gain was reduced in ferbam-treated rats starting at daily doses of 8 mg/kg in males and 37 mg/kg in females and in thiram-treated rats staring at daily doses of 5 mg/kg in males and 26 mg/kg in females. Food consumption was reduced in proportion to the reduced weight gain. Death occurred in males fed 109 or 331 mg/kg.d ferbam and in males fed 58 or 132 mg/kg.d thiram. Female rats fed 96 mg/kg.d ferbam or 67 mg/kg.d thiram developed alopecia and ataxia, which led to paralysis of the hind limbs. Male rats fed ferbam or thiram had a more severe incidence of squamous metaplasia in the thyroid and fatty infiltration in the pancreas than control males. Ferbam or thiram reduced the incidence of spontaneous nephritis in both males and females. The male rats that were fed 109 or 331 mg/kg.d ferbam and died betweeen 1 and 5 wk had golden pigment in the reticuloendothelial cells of the spleen and in the enlarged mesenteric lymph nodes, associated with hemosiderosis. Moderate tubular degeneration of the testes with atypical spermatids in the epididymis occured in some rats fed 132 mg/kg.d thiram for 13 wk but not in rats fed up to 52 mg/kg.d for 80 wk. Periodic hematologic examination and terminal clinical blood tests did not reveal any severe changes. Ferbam and thiram did not alter the occurrence or latent period of the spontaneous tumors seen in control rats.