Transmission of Coxiella burnetii by ingestion in mice.

Coxiella burnetii, the causative agent of Q fever, is widely present in dairy products around the world. It has been isolated from unpasteurised milk and cheese and can survive for extended periods of time under typical storage conditions for these products. Although consumption of contaminated dairy products has been suggested as a potential route for transmission, it remains controversial. Given the high prevalence of C. burnetii in dairy products, we sought to examine the feasibility of transmitting the major sequence types (ST16, ST8 and ST20) of C. burnetii circulating in the United States. We delivered three strains of C. burnetii, comprising each sequence type, directly into the stomachs of immunocompetent BALB/c mice via oral gavage (OG) and assessed them for clinical symptoms, serological response and bacterial dissemination. We found that mice receiving C. burnetii by OG had notable splenomegaly only after infection with ST16. A robust immune response and persistence in the stomach and mesenteric lymph nodes were observed in mice receiving ST16 and ST20 by OG, and dissemination of C. burnetii to peripheral tissues was observed in all OG infected mice. These findings support the oral route as a mode of transmission for C. burnetii.

Multiple strains of Coxiella burnetii are present in the environment of St. Paul Island, Alaska.

In 2010, Coxiella burnetii was identified at a high prevalence in the placentas of Northern fur seals (Callorhinus ursinus) collected at a single rookery on St. Paul Island Alaska; an area of the United States where the agent was not known to be present. As contamination was hypothesized as a potential cause of false positives, but nothing was known about environmental C. burnetii in the region, an environmental survey was conducted to look for the prevalence and distribution of the organism on the island. While environmental prevalence was low, two strains of the organism were identified using PCR targeting the COM1 and IS1111 genes. The two strains are consistent with the organism that has been increasingly identified in marine mammals as well as a strain type more commonly found in terrestrial environments and associated with disease in humans and terrestrial animals. Further work is needed to elucidate information regarding the ecology of this organism in this region, particularly in association with the coastal environment.

Structural and functional consequences of altering a peptide MHC anchor residue.

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.

Ligand-specific selection of MHC class II-restricted thymocytes in fetal thymic organ culture.

Positive and negative selection of thymocytes is determined by the specificity of the TCR and signaling through its associated molecules. We have studied selection of thymocytes bearing a MHC class II-restricted TCR using fetal thymic organ culture. This system allows the addition of peptides to the already diverse panoply of endogenous peptide ligands and is useful for analyzing ligand-specific negative selection of CD4 single positive (CD4SP) thymocytes. The data reveal that the ability of a given ligand to mediate negative selection is related to its dissociation rate from the TCR. We find that negative selection is very sensitive, and only the weakest ligand that we can identify fails to induce negative selection. None of the numerous peptides tested were able to induce an increase in CD4SP thymocytes. In addition, the ligands that induce negative selection of CD4SP thymocytes also cause an increase in numbers of CD8SP thymocytes bearing high levels of the class II-restricted TCR. Although these cells have a cell surface phenotype consistent with positive selection, they most likely represent cells in the process of negative selection. Further analysis reveals that these cells are not induced by these ligands in intact adult animals and that their induction is probably only revealed in the organ culture system.

Partially phosphorylated T cell receptor zeta molecules can inhibit T cell activation.

The T cell receptor complex (TCR) zeta chain is constitutively tyrosine phosphorylated specifically at two of the six zeta immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues in resting peripheral T cells. Further phosphorylation of zeta is induced by both agonist and antagonist ligands of the TCR, with agonists inducing complete phosphorylation of the zeta ITAM tyrosines. After antagonist stimulation, zeta phosphorylation is incomplete and generates discrete forms of partially phosphorylated ITAMs. Here, we mutate specific tyrosines in chimeric human CD8-zeta molecules to reflect phosphorylation in resting T cells as well as phosphorylation induced by agonist and antagonist ligands. We demonstrate that such partially phosphorylated TCR-zeta species can inhibit IL-2 production in T cell hybridomas and proliferation in T cell clones. This reveals a previously unrecognized, inhibitory function of partially phosphorylated ITAMs. These findings support the concept that TCR antagonism can arise through the generation of an inhibitory signal within the TCR complex and that constitutive zeta phosphorylation in resting T cells is an inhibitory signaling environment.

Cutting edge: the tyrosine phosphatase SHP-1 regulates thymocyte positive selection.

The binding kinetics of the TCR for its interacting ligand and the nature of the resulting signal transduction event determine the fate of a developing thymocyte. The intracellular tyrosine phosphatase SHP-1 is a potential regulator of the TCR signal transduction cascade and may affect thymocyte development. To assess the role of SHP-1 in thymocyte development, we generated T cell-transgenic mice that express a putative dominant negative form of SHP-1, in which a critical cysteine is mutated to serine (SHP-1 C453S). SHP-1 C453S mice that express the 3.L2 TCR transgene are increased in CD4 single positive cells in the thymus and are increased in cells that express the clonotypic TCR. These data suggest that the expression of SHP-1 C453S results in increased positive selection in 3.L2 TCR-transgenic mice and support a role for SHP-1 thymocyte development.

A kinetic threshold between negative and positive selection based on the longevity of the T cell receptor-ligand complex.

We have developed a unique in vivo system to determine the relationship between endogenous altered peptide ligands and the development of major histocompatibility complex class II- restricted T cells. Our studies use the 3.L2 T cell receptor (TCR) transgenic mouse, in which T cells are specific for Hb(64-76)/I-Ek and positively selected on I-Ek plus self-peptides. To this endogenous peptide repertoire, we have individually added one of six well-characterized 3.L2 ligands. This transgenic approach expands rather than constrains the repertoire of self-peptides. We find that a broad range of ligands produce negative selection of thymocytes in vivo. When compared with the in vitro TCR-ligand binding kinetics, we find that these negatively selecting ligands all have a half-life of 2 s or greater. Additionally, one of two ligands examined with no detectable binding to the 3.L2 TCR and no activity on mature 3.L2 T cells (Q72) enhances the positive selection of transgenic thymocytes in vivo. Together, these data establish a kinetic threshold between negative and positive selection based on the longevity of TCR-ligand complexes.

TCR transgenic mice in which usage of transgenic alpha- and beta-chains is highly dependent on the level of selecting ligand.

We have produced a TCR transgenic mouse that uses a TCR derived from a Th1 clone that is specific for residues 64 to 76 of the d allele of murine hemoglobin presented by I-Ek. Examination of these TCR transgenic mice on an H-2(k/k) background that expressed the nonstimulatory s allele of murine hemoglobin revealed that these mice express many endogenous TCR chains from both alpha and beta loci. We found that this transgenic TCR is also very inefficient at mediating beta selection, thereby showing a direct linkage between beta selection and allelic exclusion of TCR beta. We have also examined these mice on MHC backgrounds that have reduced levels of I-Ek and found that positive selection of cells with high levels of the transgenic TCR depends greatly on the ligand density. Decreasing the selecting ligand density is a means of reducing the number of available selecting niches, and the data reveal that the 3.L2 TCR is used sparingly for positive selection under conditions where the number of niches becomes limiting. The results, therefore, show a way that T cells may get to the periphery with two self-restricted TCRs: one that efficiently mediates positive selection, and another that is inefficient at positive selection with the available niches.

High- and low-potency ligands with similar affinities for the TCR: the importance of kinetics in TCR signaling.

We have examined binding characteristics for a single TCR interacting with five of its different peptide/MHC ligands using surface plasmon resonance. We find that very small structural changes produce ligands with similar equilibrium binding affinities (K(D)) for the TCR, but vastly different potencies for T cell activation. Ligands with similar K(D)s induce similar amounts of total phospho-zeta but distinct patterns of zeta phosphorylation. Lower potency ligands induce only incomplete phosphorylation of TCR zeta and generally have faster off-rates. Therefore, the potency of TCR ligands is primarily determined by the half-life of the TCR-ligand complex and the consequent ability to induce complete phosphorylation of zeta.

Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway.

The integrin-associated protein (IAP, CD47) is a 50-kD plasma membrane protein with a single extracellular immunoglobulin variable (IgV)-like domain, a multiply membrane-spanning segment, and alternatively spliced short cytoplasmic tails. On neutrophils, IAP has been shown to function in a signaling complex with beta 3 integrins. However, the function of IAP on T cells, which express little or no beta 3 integrin, is not yet defined. Here, we show that mAbs recognizing IAP can enhance proliferation of primary human T cells in the presence of low levels of anti-CD3, but have no effect on T cell proliferation on their own. Together with suboptimal concentrations of anti-CD3, engagement of IAP also enhances IL-2 production in Jurkat cells, an apparently integrin-independent function of IAP. Nonetheless, costimulation by IAP ligation requires cell adhesion. IAP costimulation does not require CD28. Furthermore, anti-IAP, but not anti-CD28, synergizes with suboptimal anti-CD3 to enhance tyrosine phosphorylation of the CD3 zeta chain and the T cell-specific tyrosine kinase Zap70. Ligation of human IAP transfected into the hemoglobin-specific 3.L2 murine T cell hybridoma costimulates activation for IL-2 secretion both with anti-CD3 and with antigenic peptides on antigen-presenting cells (APCs). Moreover, ligation of IAP but not CD28 can convert antagonist peptides into agonists in 3.L2 cells. Using costimulation by IAP ligation as an assay to analyze the structure-function relationships in IAP signaling, we find that both the extracellular and multiply membrane-spanning domains of IAP are necessary for synergy with the antigen receptor, but the alternatively spliced cytoplasmic tails are not. These data demonstrate that IAP ligation initiates an adhesion-dependent costimulatory pathway distinct from CD28. We hypothesize that anti-IAP generates the costimulatory signal because it modulates interactions of the IgV domain with other plasma membrane molecules; this in turn activates effector functions of the multiply membrane-spanning domain of IAP. This model may have general significance for how IAP functions in cell activation.

Structural basis for T cell recognition of altered peptide ligands: a single T cell receptor can productively recognize a large continuum of related ligands.

T cells recognize short linear peptides bound to major histocompatibility complex (MHC)-encoded molecules. Subtle molecular changes in peptide antigens produce altered peptide ligands (APLs), which induce different T cell responses from those induced by the antigenic ligand. A molecular basis for how these slight molecular variations lead to such different consequences for the T cell has not been described. To address this issue, we have made amino acid substitutions at the primary T cell receptor (TCR) contact residue of the murine hemoglobin determinant, Hb(64-76)/I-Ek and produced 12 peptides that interact with the TCR of the T cell clone 3.L2. The 3.L2 T cell responds to these peptides, which vary 1 million-fold in their activity, and enables them to be ranked according to their relative ability to signal through the 3.L2 TCR. Such a ranking reveals that the ability of the 3.L2 T cell to respond to these peptides depends on how well the structure of the side chain at the primary TCR contact site mimics that of the Asn residue present in the antigenic ligand. The reactivity of the 3.L2 T cell also depends on an MHC contact residue that is next to the primary TCR contact residue, suggesting that conformation of the Asn side chain is also important. By using nonnatural amino acids at a TCR contact residue, we have demonstrated that APLs can be rationally designed based on structure. These data are consistent with a model in which the affinity of a peptide-MHC complex for the TCR determines how the T cell will respond.

Essential flexibility in the T-cell recognition of antigen.

alpha beta T cells specifically recognize a ligand composed of a peptide bound to a self-major-histocompatibility-complex molecule, but the recognition of slightly altered ligands by T cells can lead to a partial activation. This flexibility is crucial for T-cell development and can have both beneficial and harmful effects on peripheral T cells.

Efficient maturation of alpha beta lineage thymocytes to the CD4+CD8+ stage in the absence of TCR-beta rearrangement.

We have produced transgenic mice that express rearranged T cell Ag receptor gamma and delta transgenes in the alpha beta lineage of thymocytes. Thymi in these mice contain normal numbers of CD4+CD8+ cells that express low levels of the TCR-gamma delta. Analysis of the delta locus in these thymi indicates that these cells are in the alpha beta lineage even though they express the TCR-gamma delta. This shows that expression of the TCR-gamma delta in early thymocytes can lead to all of the consequences that are normally mediated by the beta-chain. These consequences include maturation to the CD4+CD8+ stage, entry into the cell cycle, and cessation of beta rearrangement. Therefore, the data support a model in which formation of a functional CD3 complex on immature CD4-CD8- thymocytes leads to further development in the absence of extracellular ligand recognition. The data also show that the gamma delta vs alpha beta lineage decision is made in a manner that is independent of gamma and beta gene expression.

Role of TCR specificity in CD4 versus CD8 lineage commitment.

We have examined the role that TCR-alpha beta specificity plays in lineage commitment of thymocytes. Thymocytes in the process of selection have been distinguished by the expression of high levels of CD69 and CD5. The phenotype of most of these selected thymocytes is either CD4highCD8low or CD4lowCD8high, but some are CD4lowCD8low, a phenotype referred to as double-positive dull. Analysis of the phenotype of apoptotic thymocytes shows that double-positive dull cells make up many of the cells undergoing negative selection. Thymocytes from MHC class I- or class II-deficient mice and from MHC class I- and class II-restricted TCR transgenic mice have been analyzed for cells undergoing positive and negative selection. This analysis shows that the TCR specificity is a critical factor in the lineage determination of thymocytes, and any stochastic component to lineage commitment is minor.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.

The M2 delta transmembrane domain of the nicotinic cholinergic receptor forms ion channels in human erythrocyte membranes.

A synthetic peptide with the sequence of the M2 delta segment of the nicotinic acetylcholine receptor from Torpedo californica forms pores in human erythrocyte membranes as determined by hemoglobin and potassium release. This peptide forms a permeability pathway with an apparent cross-sectional diameter of 7-9 A. The M2 delta pore is oligomeric and a pentamer is the species that accounts for the properties of the permeation path. Peptides that mimic other identifiable segments of the Torpedo acetylcholine receptor, M1 delta and MIR, do not form channels in erythrocytes under the same conditions.