Death from drought in tropical forests is triggered by hydraulics not carbon starvation.

Drought threatens tropical rainforests over seasonal to decadal timescales, but the drivers of tree mortality following drought remain poorly understood. It has been suggested that reduced availability of non-structural carbohydrates (NSC) critically increases mortality risk through insufficient carbon supply to metabolism ('carbon starvation'). However, little is known about how NSC stores are affected by drought, especially over the long term, and whether they are more important than hydraulic processes in determining drought-induced mortality. Using data from the world's longest-running experimental drought study in tropical rainforest (in the Brazilian Amazon), we test whether carbon starvation or deterioration of the water-conducting pathways from soil to leaf trigger tree mortality. Biomass loss from mortality in the experimentally droughted forest increased substantially after >10 years of reduced soil moisture availability. The mortality signal was dominated by the death of large trees, which were at a much greater risk of hydraulic deterioration than smaller trees. However, we find no evidence that the droughted trees suffered carbon starvation, as their NSC concentrations were similar to those of non-droughted trees, and growth rates did not decline in either living or dying trees. Our results indicate that hydraulics, rather than carbon starvation, triggers tree death from drought in tropical rainforest.

The open reading frames in the 3' long terminal repeats of several mouse mammary tumor virus integrants encode V beta 3-specific superantigens.

Mice expressing the minor lymphocyte stimulation antigens, Mls-1a, -2a, or -3a, singly on the B10.BR background have been generated. Mls phenotypes correlate with the integration of mouse mammary tumor viruses (MTV) in the mouse genome. The open reading frames within the 3' long terminal repeats of the integrated MTVs 1, 3, 6, and 13 encode V beta 3-specific superantigens. Sequence data for these viral superantigens is presented, indicating that it is the COOH-terminal portion of the viral superantigen that interacts with the T cell receptor V beta element.

Analysis of the interaction site for the self superantigen Mls-1a on T cell receptor V beta.

Superantigen bound to major histocompatibility complex (MHC) products have been shown to stimulate T cells in a V beta-specific manner. Mouse T cells bearing V beta 8.1 usually respond to the self superantigen, Mls-1a, whereas T cells bearing V beta 8.2a do not. Previously, using site-directed mutational analysis, we identified the residues of natural variants of T cell receptor (TCR) V beta 8.2 that conferred Mls-1a reactivity. These residues are predicted to lie on a beta-pleated sheet of the TCR V beta element, well away from the expected binding site for antigen and MHC proteins. This study was undertaken to determine the effect of glycosylation on this beta-pleated sheet on Mls-1a reactivity and to map the extent of the interaction site on V beta 8.2 for Mls-1a. to Mls-1a, as well as to peptides derived from the conventional protein antigen, chicken ovalbumin. Here we demonstrate that first, N-linked carbohydrate on the lateral surface of V beta blocks the interaction of the TCR V beta with the self superantigen, Mls-1a, but has no effect on the TCR interaction with peptide antigen and MHC, second, that the interaction site for Mls-1a extends over the surface of the solvent-exposed beta-pleated sheet on the side of the TCR, and third, that mutations which affect both superantigen and peptide antigen reactivity lie at the beginning of the first complementarity determining region of V beta, consistent with models of the trimolecular complex of TCR-peptide-MHC.

Surprisingly uneven distribution of the T cell receptor V beta repertoire in wild mice.

We have examined TCR V beta expression in a collection of wild mice. Many of the mice were homozygous for a large deletion at the V beta locus, and many animals also suppressed expression of several V betas using self superantigens. Expression of V beta 8.2 was unexpectedly suppressed by a self superantigen in some wild mice, which was due to the presence in these animals of a variant V beta 8.2 gene. The amino acid changes in this gene product suggest contact sites between V beta and the superantigen. Although all V betas are expressed within each wild mouse population, individual mice have a limited and variable V beta repertoire. The independent origin of multiple V beta deletions and the presence of polymorphic self superantigens suggest that this variation may be maintained by balancing selection.

Citrate synthase: an immunochemical investigation of interspecies diversity.

Rabbit antibodies have been raised to pig heart citrate synthase. Using purified IgG, competitive enzyme-linked immunoassays and assays of citrate synthase activity indicate the presence of antibodies to a number of antigenic sites on the enzyme, only some of which are essential for catalytic activity. From a comparison of citrate synthases from prokaryotic and eukaryotic organisms, the degree of interaction between antibody and enzyme was in the order: pig heart greater than pigeon breast greater than Bacillus megaterium greater than Escherichia coli. These findings are discussed in terms of the known interspecies diversity of the enzyme.

"Auto-reactive" T-cell hybridomas and the role of foetal calf serum.

A panel of "auto-reactive" T-cell hybridomas has been generated by fusing Sendai virus-primed, Peyer's patch T cells from CBA mice with BW 5147. The hybridomas produce Il-2 in response to stimulation by Class II-positive syngeneic cells in the absence of added Sendai virus. The stimulator genes for two hybridoma clones have been mapped using recombinant mouse strains and the restriction has been confirmed by inhibition with monoclonal anti-Class II antibodies. Hybridomas grown in serum-free medium do not respond to syngeneic cells in the absence of foetal calf serum (FCS) and the response is restored by the addition of FCS, but not bovine serum albumin (BSA), normal mouse serum or Sendai virus. The component of FCS which is required for stimulation of the hybridomas has been partially purified and characterised. Its mode of action has been investigated and it acts on the stimulator cells and not on the hybridomas.

Fetal calf serum stimulates 'autoreactive' T-cell hybridomas.

During attempts to generate Sendai virus-specific T-cell hybridomas, a number of autoreactive T-cell hybridomas were produced. These hybrids secrete IL-2 in response to class II-positive syngeneic cells in the absence of added Sendai virus. Class II molecules on the stimulator cells are involved since monoclonal anti-class II antibodies block the reaction, and mapping studies using recombinant mouse strains show the response is class II restricted. Hybridomas adapted to grow in serum-free medium do not respond to syngeneic cells in the absence of serum; however, addition of fetal calf serum (FCS) restores the response. The component of FCS responsible for the stimulation of the hybridomas has been partially purified and characterized, and its mode of action investigated. These findings suggest that the putative 'autoreactive' T-cell hybridomas reported by others may be specific for a component of the culture medium rather than being truly self-reactive.

Receptors on T cells escaping superantigen-mediated deletion lack special beta-chain junctional region structural characteristics.

The TCR V beta element is pivotal for superantigen recognition; however, not all T cells bearing a particular V beta element respond to an individual superantigen. Recent evidence has indicated that the TCR V alpha element also contributes to recognition of superantigen/MHC class II complexes. To determine whether the TCR beta-chain junctional regions influence recognition of a superantigen encoded by mouse mammary tumor virus (MMTV) proviral integrant Mtv-1, we have analyzed these regions in T cells that have survived superantigen-mediated negative selection in B10.BR-Mtv-1 mice. Our data indicate: 1) no TCR J beta skewing, 2) no difference in the length of the third complementarity-determining region (CDR3), and 3) no outstanding structural features that are shared among the junctional regions of the V beta 3+ T cells that escape thymic clonal elimination in superantigen-expressing mice. Several possible models for TCR engagement of viral superantigen/MHC class II complexes are discussed.

The T-cell repertoire is heavily influenced by tolerance to polymorphic self-antigens.

T cells with V beta 3+ alpha beta receptors are deleted by self-tolerance in mice with particular major histocompatibility complex/self-antigen combinations. This also occurs for other V beta elements. Polymorphism in the major histocompatibility complex and/or the self-antigens that cause massive deletion of T cells using particular V beta elements may be maintained by the need to balance the advantage of a diverse T-cell repertoire against the potential involvement of those elements in autoimmune disease.

Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes.

V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.

Distinct methylation states of the CD8 beta gene in peripheral T cells and intraepithelial lymphocytes.

The CD8 coreceptor is expressed on both immature and mature T cells as either an alphabeta heterodimer or an alpha alpha homodimer. Thymocytes and peripheral T cells express CD8 alphabeta, whereas TCR alphabeta+ intraepithelial lymphocytes (IEL) express CD8 alpha alpha or CD8 alphabeta, and the majority of TCR gammadelta+ IEL bear CD8 alpha alpha. The presence of CD8 beta enhances the signaling and adhesion properties of the CD8 alphabeta coreceptor and is necessary for efficient T cell development in the thymus, but is not required for the extrathymic maturation of CD8 alpha alpha+ IEL. To address whether CD8 alpha alpha+ IEL express CD8 beta during their development, we examined the methylation state of cytosines in the CD8 beta gene 5' regulatory region to identify those for which the methylation state inversely correlates with expression of the CD8 beta protein. We identified four such cytosines that were demethylated in CD8 beta-expressing thymocytes and T cells. Interestingly, these cytosines were also demethylated in CD4+ lymph node T cells that had transiently expressed CD8 beta during their development. The methylation state of these cytosines was examined in DNA purified from TCR alphabeta+ CD8 alpha alpha+ and TCR alphabeta+ CD8 alphabeta+ IEL, as well as from TCR gammadelta+ CD8 alpha alpha+ and CD3- CD8 alpha alpha+ IEL. The methylation pattern for TCR alphabeta+ CD8 alpha alpha+ IEL DNA was distinct from that seen for DNA from CD4+ lymph node cells, suggesting that TCR alphabeta+ CD8 alpha alpha+ IEL have not previously expressed CD8 beta. Analysis of DNA from CD3- CD8 alpha alpha+ IEL indicated that the unique methylation pattern of the CD8 beta gene in TCR alphabeta+ CD8 alpha alpha+ IEL DNA was not due to transcription of the CD8 alpha gene or the influence of the gut microenvironment.

Mouse mammary tumor virus superantigens require N-linked glycosylation for effective presentation to T cells.

Mouse mammary tumor viruses (MMTVs) encode superantigens that associate with major histocompatibility complex class II products on antigen-presenting cells and stimulate T cells in a V beta-specific manner. This T cell activation is critical for completion of the viral life cycle and vertical transmission to the next generation. To investigate the functional significance of extensive viral superantigen (Sag) glycosylation, we disrupted the six potential sites for N-linked carbohydrate addition in the Sag encoded by proviral integrant Mtv-1. Shifts in the apparent molecular mass of these mutant glycoproteins suggested that wild-type Mtv-1 Sag is glycosylated on four of its six sites. Intracellular and cell surface staining of the panel of mutants indicated that any decrease in glycosylation resulted in reduced levels of intracellular protein and undetectable surface expression, suggesting that decreased glycosylation leads to rapid Sag degradation and abates trafficking to the plasma membrane. Nevertheless, several mutants with intermediate levels of glycosylation expressed enough Sag on the B cell surface to potently stimulate reactive T cell hybrids. We show there is no specific site bearing N-linked glycosylation that is essential for activity, but at least one carbohydrate addition is necessary for effective B cell presentation of MMTV superantigens to T cells.

Superantigens: mechanism of T-cell stimulation and role in immune responses.

Superantigens combine with MHC class-II molecules to form the ligands that stimulate T cells via the V beta element of the T-cell receptor. Two groups of superantigens have been described so far: first, endogenous murine products that include the Mls determinants, and second, bacterial products such as the Staphylococcal enterotoxins. Here, we review studies that address the interactions between the foreign superantigens and MHC class-II molecules, the mechanism of T-cell stimulation, and the role that tolerance to self-superantigens plays in shaping the T-cell repertoire. We speculate on the possible evolutionary significance of superantigens.

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection.

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.

Transposon-mediated random insertions and site-directed mutagenesis prevent the trafficking of a mouse mammary tumor virus superantigen.

Mouse mammary tumor viruses (MMTVs) encode superantigens (Sags) which are critical to the life cycle of infectious virus and can mediate extensive deletion of T lymphocytes when expressed by endogenous proviruses. Little is known about the structure, intracellular trafficking, or nature of Sag association with major histocompatibility (MHC) class II products. In order to gain a better understanding of Sag structure-function relationships, we extensively mutagenized this type II glycoprotein using two different approaches: transposon-mediated random in-frame insertion mutagenesis and site-directed mutagenesis targeting clusters of charged residues. We find that 31 codon insertions are infrequently tolerated in Mtv-7 Sag, with just 1 of 14 insertion mutants functionally presented on the surface of B cells. Surprisingly, similar effects were observed with Sag mutants with substitutions at pairs of charged residues; only 2 of 6 mutants trafficked to the plasma membrane and stimulated T cells, 1 with a temperature-sensitive phenotype. The data suggest that the nonfunctional Mtv-7 Sag mutants are stringently retained in the endoplasmic reticulum due to conformational defects rather than disrupted interactions with MHC class II, thus identifying charged amino acids critical to the structural stability of viral superantigens.

Mtv-1 superantigen trafficks independently of major histocompatibility complex class II directly to the B-cell surface by the exocytic pathway.

Presentation of the Mtv-1 superantigen (vSag1) to specific Vbeta-bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II alphabeta dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells.

Identification of the region of T cell receptor beta chain that interacts with the self-superantigen MIs-1a.

Superantigen-MHC complexes are known to stimulate T cells primarily via the V beta element of the T cell receptor. In this paper we identify a number of amino acid residues that define the region of a particular V beta element interacting with one of the self-superantigens, MIs-1a. These residues are predicted to lie on a beta-pleated sheet of the T cell receptor, away from the complementarity determining regions of the receptor, which are thought to interact with complexes of conventional peptide antigens and MHC. In support of this prediction, mutations affecting MIs-1a activity have no effect on the response to conventional antigen and MHC.

The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice.

Staphylococcal enterotoxin B is known to be a powerful T cell stimulant in mouse and man. In this paper we show that, for mice, this is because the protein in association with major histocompatibility complex class II molecules stimulates virtually all T cells bearing V beta 3 and V beta 8.1, 8.2, and 8.3, and few others. Neonatal mice given the enterotoxin eliminate all mature, and some immature, T cells bearing these V beta s, demonstrating that tolerance to exogenously administered antigen can be caused by clonal deletion of reactive T cells. The enterotoxin shares these "superantigenic" properties with known self-antigens in mice, Mls-1a and Mls-2a, and a B cell-derived product, a shared property that is unlikely to be coincidental or inconsequential.

Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.

Transgenic mice expressing a T cell receptor heterodimer specific for a fragment of pigeon cytochrome c plus an MHC class II molecule (I-Ek) have been made. We find that H-2k alpha beta transgenic mice have an overall increase in the number of T cells and express a 10-fold higher fraction of cytochrome c-reactive cells than H-2b mice. Surface staining of thymocytes indicates that in H-2b mice, T cell development is arrested at an intermediate stage of differentiation (CD4+8+, CD310). Analyses of mice carrying these T cell receptor genes and MHC class II I-E alpha constructs indicate that his developmental block can be reversed in H-2b mice by I-E expression on cortical epithelial cells of the thymus. These data suggest that a direct T cell receptor-MHC interaction occurs in the thymus in the absence of nominal antigen and results in the enhanced export of T cells, consistent with the concept of "positive selection".

Differential contribution of Lck and Fyn protein tyrosine kinases to intraepithelial lymphocyte development.

The developmental stages and the role of protein tyrosine kinases (PTK) in the maturation of CD3+CD8 alpha alpha+ intraepithelial lymphocytes (IEL) have not been extensively characterized. However, comparisons of thymic and extrathymic T cell development indicate that these processes involve some distinct signaling and selection events. We used mice deficient in Lck, Fyn, or both Lck and Fyn to analyze the role that these src-family PTK play in IEL development. In contrast to thymocyte development, we found that all IEL subsets develop in mice deficient for either kinase alone. However, lck-/- animals exhibited reduced numbers of TcR alphabeta+ CD8alpha alpha+ IEL, indicating that Lck is important in the development of these cells. Mice which lack both Lck and Fyn fail to generate TcR alphabeta+ IEL, suggesting that signaling through the preTcR, mediated by Lck and, to a lesser extent Fyn, is required for maturation of all TcR alphabeta+ IEL lineages. Interestingly, a small population of TcR gammadelta+ CD8 alpha alpha+ cells are apparent in lck-/-fyn-/- animals, demonstrating that TcR alphabeta+ CD8 alpha alpha+ and TcR gammadelta+ CD8alpha alpha+ IEL have distinct PTK requirements for their development or expansion. CD3-CD8alpha- CD44+ and CD3-CD8alpha alpha+ CD16/32+ B220+ cells comprise the majority of IEL in both lck-/- fyn-/- and rag -/- mice, while they are poorly represented in wildtype controls. Comparison of the cell surface phenotype of these putative precursor IEL in lck-/- fyn-/- and rag-/- animals suggests that IEL maturation in these animals is arrested at an equivalent developmental stage. Overall, the data presented demonstrate that signals mediated by Lck or Fyn direct TcR alphabeta+ CD8alpha alpha+ IEL maturation but are dispensable for the development of TcR gammadelta+ CD8 alpha alpha+ IEL.