Superantigen recognition by gammadelta T cells: SEA recognition site for human Vgamma2 T cell receptors.

Human gammadelta T cells expressing the Vgamma2Vdelta2 antigen receptors recognize nonpeptide prenyl pyrophosphate and alkylamine antigens. We find that they also recognize staphylococcal enterotoxin A superantigens in a manner distinct from the recognition of nonpeptide antigens. Using chimeric and mutant toxins, SEA amino acid residues 20-27 were shown to be required for gammadelta TCR recognition of SEA. Residues at 200-207 that are critical for specific alphabeta TCR recognition of SEA do not affect gammadelta TCR recognition. SEA residues 20-27 are located in an area contiguous with the binding site of V beta chains. This study defines a superantigen recognition site for a gammadelta T cell receptor and demonstrates the differences between Vgamma2Vdelta2+ T cell recognition of superantigens and nonpeptide antigens.

Exogenous peptides enter the endoplasmic reticulum of TAP-deficient cells and induce the maturation of nascent MHC class I molecules.

MHC class I molecules assemble within the endoplasmic reticulum (ER) in complexes that include beta2-microglobulin (beta(2)m), the transporter associated with antigen processing (TAP)and several additional chaperones. Release of class I complexes from the ER is thought to require the binding of an appropriate endogenous peptide, predominantly delivered from the cytosol to the ER by TAP. It was recently demonstrated that exogenous synthetic peptide could 'directly' enter the ER of intact cells, independently of TAP function, and bind to the class I molecule H-2K(b).In TAP-deficient cells, we show that nascent K(b) or K(b)-L(d) chimeric molecules have a high trafficking background; 50-80% of these class I molecules are released from the ER independently of TAP function or the addition of exogenous peptide. The addition of exogenous K(b) cognate peptides enhanced the release of these class I molecules only slightly over the high background. The chimeric class I-b molecule, M3-L(d), differs from K(b)-L(d) only in its peptide binding domains, and M3-L(d) preferentially binds N-formylated peptides, which are rare in eukaryotic cells. Release of M3-L(d) from the ER in the absence of exogenous peptide was negligible. Addition of exogenous formylated peptides induced significant trafficking and surface expression of M3-L(d). These observations suggest that peptide binding is necessary for class I release from the ER even in TAP-deficient cells. These results demonstrate that exogenous peptide not only enters the ER of intact cells independently of TAP but also functionally induces class I antigen presentation.

Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling.

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related serine/threonine protein kinase, is a hematopoietic-specific upstream activator of the c-Jun N-terminal kinase. Here, we provide evidence to demonstrate the involvement of HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and tyrosine-phosphorylated with similar kinetics following TCR/CD3 or pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck and Zap70, with HPK1 led to HPK1 activation and tyrosine phosphorylation in transfected mammalian cells. Upon TCR/CD3 stimulation, HPK1 formed inducible complexes with the adapters Nck and Crk with different kinetics, whereas it constitutively interacted with the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also inducibly associated with linker for activation of T cells (LAT) through its proline-rich motif and translocated into glycolipid-enriched microdomains (also called lipid rafts) following TCR/CD3 stimulation, suggesting a critical role for LAT in the regulation of HPK1. Together, these results identify HPK1 as a new component of TCR signaling. T cell-specific signaling molecules Lck, Zap70, and LAT play roles in the regulation of HPK1 during TCR signaling. Differential complex formation between HPK1 and adapters highlights the possible involvement of HPK1 in multiple signaling pathways in T cells.

Immunization with f-Met peptides induces immune reactivity against Mycobacterium tuberculosis.

OBJECTIVE: To determine whether synthetic peptides containing an amino terminal formyl-methionine residue and corresponding to the sequence of several proteins produced by Mycobacterium tuberculosis, would elicit an immune response in mice. DESIGN: Peptides corresponding to the amino termini of 8 M. tuberculosis proteins and initiating with formyl methionine residues were synthesized. The ability of these peptides to bind to the mouse non-classical MHC class I molecule H-2M3a was determined by flow microfluorimetry. These peptides were used to pulse dendritic cells that were then injected into normal mice. These mice were subsequently challenged with aerosolized M. tuberculosis and, 30 days later, the number of viable bacteria in the lungs was determined. RESULTS: Four of the 8 synthetic peptides bound to H-2M3a and stabilized its expression on the cell surface. Injection of mice with dendritic cells pulsed with H-2M3a binding peptides elicited non-MHC restricted cytotoxic T lymphocytes that killed peptide pulsed target cells and macrophages infected with M. tuberculosis. Immunization of mice with syngeneic dendritic cells pulsed in vitro with 2 of these peptides led to retardation of the growth of M. tuberculosis following aerosol challenge. CONCLUSION: Peptides that bind to non-polymorphic class I molecules can elicit immune reactivity directed towards M. tuberculosis.

The future of allergy practice: an academician's perspective.

The subspecialty of allergy and immunology, like all medical specialties, has been dramatically impacted by the managed care revolution. Many of the changes that have been imposed by our environment are likely to persist, including increased emphasis on efficiency of practice and cost-effectiveness of treatment modalities. It is predicted that these changes will decrease the involvement of allergists and immunologists in the primary treatment of patients with allergic rhinitis and mild asthma, in favor of management by generalists with subspecialty consultation. Conversely, outcomes studies demonstrate the cost-effectiveness of management of moderate to severe asthma by an allergy and immunology subspecialist. It is thought probable that HMOs will recognize this fact and implement it as a pattern of practice. The allergist and immunologist will continue to offer, uniquely, expertise in allergic history taking, patient education, environmental control, and management of allergic inflammation. He or she will also be afforded an opportunity for practice expansion, particularly as an expert consultant, into other areas of immune inflammation, such as autoimmunity and graft rejection. Potentially new and increasingly specific products of the pharmaceutical and biotechnology industries will enhance these opportunities for practice expansion by physicians who combine intellectual understanding with practical expertise in patient management. Realization of these new opportunities will require us to work together as teachers and role models to communicate the excitement of our subspecialty to new physicians. Allergy and immunology is a subspecialty with a bright future, provided that we have the will and the insight to deal effectively with our challenges and to master opportunities that our science presents to us.

Binding specificity of a class II-restricted hepatitis B epitope by DR molecules from responder and nonresponder vaccine recipients.

A small but significant proportion of people who receive the hepatitis B vaccine do not produce anti-hepatitis B antibodies, a phenomenon associated with certain human leukocyte antigen (HLA) class II haplotypes. We were interested in determining whether natural allelic differences between two HLA-DR4 molecules associated with responder versus nonresponder subtypes differed with respect to binding of an immunodominant hepatitis B surface antigen (HBsAg) peptide as measured using a resonant mirror biosensor. In contrast to our original hypothesis, we found a ten-fold difference in the affinity in favor of the nonresponder DRB1*0401 allele, with a KD of 6.89 x 10(-8) M versus a KD of 6.71 x 10(-7) M for the responder DRB1*0404 allele. Half-times of dissociation were 1.3 min and 7.7 min, respectively, although association rate constants for both HLA class II molecules were similar (approximately 10(4) M(-1)s(-1)). Of particular interest was the observation of different on-rates during the association phase, suggesting that stoichiometry of binding was not 1:1 or that different structural forms of the HLA-peptide complex exist. Our observations indicate that whereas HBsAg peptide binding to HLA class II molecules is influenced by HLA polymorphism, the nonresponse to hepatitis B vaccine associated with this HLA-DR4 subtype is not a result of failure of processed HBsAg to bind HLA class II molecules.

Intercellular adhesion molecule-1 and leukocyte function-associated antigen-3 provide costimulation for superantigen-induced T lymphocyte proliferation in the absence of a specific presenting molecule.

Bacterial superantigens can bind TCR in the absence of MHC class II molecules and activate T lymphocytes when cocultured with certain class II-deficient accessory cells. It has not been determined, however, whether these accessory cells provide direct costimulation to the T cell or serve to present superantigens via a nonconventional ligand. We have identified a human adenocarcinoma cell line, SW480, that assists in the activation of human T cells by the staphylococcal enterotoxins B (SEB), C1 (SEC1), and D (SED), but not SEA, SEC2, SEC3, or SEE. SW480 cells did not express class II molecules, and anti-class II mAbs did not inhibit T cell proliferation, supporting the hypothesis that class II is not absolutely required for enterotoxin-mediated T cell activation. The TCR Vbeta profile of T cells stimulated by SEB plus SW480 cells was similar to that of T cells stimulated by SEB plus class II+ APC, indicating that TCR-SEB interactions were preserved in the absence of class II molecules. Binding studies failed to detect specific association of SEB with SW480 cells, suggesting that SW480 cells do not express receptors for enterotoxin. SEB coupled to beads, however, stimulated T cell proliferation, but only in the presence of SW480 cells. SW480 cells express both ICAM-1 and LFA-3 molecules, and the addition of Abs to these receptors inhibited T cell proliferation. These findings support a model in which certain enterotoxins engage the TCR independent of MHC class II or other specific presenting molecules and induce T cell proliferation with signals provided by nonconventional accessory cells.

Structural dichotomy of staphylococcal enterotoxin C superantigens leading to MHC class II-independent activation of T lymphocytes.

We have recently characterized an MHC class II-deficient human cell line, SW480, that supports the proliferation of purified human T cells in the presence of the staphylococcal enterotoxin and superantigen SEC1, but not the closely related isotypes SEC2 or SEC3. We now investigate the structural basis of this dichotomy and explore possible mechanisms that may account for it. Differences in activity between SEC1 and SEC2 were not attributable to differences in biochemical modification, to differences in Vbeta specificity, or to the potential to induce anergy. SEC2 inhibited SEC1-mediated T cell activation in the presence of SW480 cells, suggesting that SEC2 could compete with SEC1 for binding to the TCR but was unable to productively signal through the TCR. Utilizing a panel of hybrid enterotoxins we identified specific amino acids near the NH2-terminus of SEC1 that abrogated MHC class II-independent T cell activation, yet did not alter potency in the presence of class II+ APC. These residues mapped to the putative TCR binding domain of SEC1, and suggest that subtle differences in TCR binding affinity or the topology of the SEC1-TCR interaction can compensate for the lack of MHC class II and hence promote T cell proliferation.

Altered orientation of streptococcal superantigen (SSA) on HLA-DR1 allows unconventional regions to contribute to SSA Vbeta specificity.

Crystallographic studies reveal even distantly related bacterial superantigens (SAg) to adopt a common structural topology. Mutational analyses confirm that this shared folding pattern often confers a conserved function to analogous residues in different SAg, albeit with specificities for particular TCR or MHC class II molecules. It was thus surprising that the streptococcal SAg SSA differed from related SAg in the location of its Vbeta-determining residues. Because it seemed unlikely that SSA would deviate significantly from an SAg-like topology, we hypothesized that variations in SSA Vbeta-determining regions might result from differences in SSA-MHC class II interactions relative to other SAg during SSA presentation to the TCR. Comparison of the DR1-binding properties of SSA with its closest homologue SEB found different amino acid positions within SAg primary sequences to contribute to SSA-DR1 and SEB-DR1 interactions, and suggested that SSA bound DR1 with an altered orientation relative to SEB. The common involvement of DR1 alpha39K, however, predicted that the two SAg bound overlapping sites on DR1. Nevertheless, SSA and SEB did not effectively cross-compete for DR1 binding and had opposite patterns of DR1-binding affinity in the presence of distinct DR1-expressing cell lines. The data thus suggest that SSA and SEB bind not only with different orientations on DR1, but may bind preferentially to distinct DR1 subsets delineated by cell-specific factors. Differences in orientation of SSA on DR1 and/or interaction of SSA with particular DR1 subsets may explain why unconventional regions influence SSA TCR Vbeta specificity.

Species-dependent post-translational modification and position 2 allelism: effects on streptococcal superantigen SSA structure and V beta specificity.

Epidemiologic and molecular population genetic analyses support a role for superantigens (SAg) in the pathogenesis of severe staphylococcal and streptococcal infections. To investigate how variations in SAg structure influence immunomodulatory activity, we examined the biochemical and functional properties of two allelic variants of streptococcal SAg SSA that differ at position 2. Mass spectrometry revealed both recombinant (Escherichia coli) and native (Streptococcus pyogenes) SSA allelic variants to have significantly larger molecular masses than predicted by primary sequence alone and provided evidence that the proteins were modified by the addition of biochemical moieties, a phenomenon that has not been described for related SAg. Furthermore, the molecular masses of native and recombinant SSA were not the same; SSA was differentially post-translationally modified by the two bacterial genera. The substitution of E. coli-dependent processing for that of S. pyogenes altered both protease digestion and V beta specificity, suggesting that recombinant SAg from E. coli may not accurately represent the native toxin. In addition, the observation that SSA allelic variants differed in V beta specificity supports a role for position 2 in SSA-TCR interactions. That SSA position 2 contributes to V beta specificity could not have been predicted from functional or crystallographic studies of other SAg and suggests that SSA may adopt unique interactions with TCR and/or MHC class II molecules. Determining the structural basis for these differences should offer additional clues to the manner in which SAg exert their effects on the immune system during infection and may allow the designing of SAg mutants with specific quantitative and qualitative immunomodulatory properties.

Phylogenetic distribution of streptococcal superantigen SSA allelic variants provides evidence for horizontal transfer of ssa within Streptococcus pyogenes.

Phylogenetic analyses recently found the gene encoding the streptococcal superantigen SSA of Streptococcus pyogenes to occur in several well-differentiated clones comprising 10 (12.5%) of 80 clonal lineages examined. To determine if distinct clonal lineages carried the same ssa coding sequence or harbored a group of allelic variants, ssa was sequenced from 23 S. pyogenes strains representing the 10 clones identified by multilocus enzyme electrophoresis. Three alleles of ssa were found in natural populations of S. pyogenes. ssa-1 and ssa-3 differed by a single synonymous substitution in codon 94; both encoded SSA-1. Each of these alleles was present in phylogenetically diverse clones that had not shared a recent common ancestor. ssa-2 was present in a single clonal lineage. It was identical to ssa-3 at codon 94 but had a nonsynonymous substitution at codon 28 that changed the second amino acid of the mature protein from serine to arginine. This substitution altered the predicted isoelectric point and affected the apparent molecular mass during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite sequence variation both upstream of and within the ssa locus, all ssa-positive lineages expressed either SSA-1 or SSA-2. The observed patterns of ssa allele-clone distribution provide evidence for individual incidences of horizontal transfer and recombination of ssa among distinct group A streptococcal lineages. Although the extensive homology of SSA to the staphylococcal superantigen SEB raises the possibility of intergeneric gene transfer, a search for ssa in 68 genetically diverse clones of Staphylococcus aureus did not identify the gene. Moreover, the absence of ssa among 119 representative strains of Lancefield group B, C, or G streptococci suggests that ssa is confined to S. pyogenes.

Practice parameters for the diagnosis and management of immunodeficiency. The Clinical and Laboratory Immunology Committee of the American Academy of Allergy, Asthma, and Immunology (CLIC-AAAAI)

In this brief review, only the most useful immunologic tests available for defining host defects that lead to susceptibility to infection have been emphasized. It should be pointed out that those evaluations and tests ordered by the physician will rule out the vast majority of the currently recognized defects. Finally, it is important that any patients identified as abnormal by these screening tests be characterized as fully as possible in centers specializing in these diseases before therapy is initiated, since what may appear to be a simple diagnosis on the surface may be an indicator of more complex underlying problems.

Residues near the amino and carboxyl termini of staphylococcal enterotoxin E independently mediate TCR V beta-specific interactions.

Previous studies identified three COOH-terminal residues in staphylococcal enterotoxin E (SEE; Asp200, Pro206, and Asp207) that in part mediate TCR V beta recognition. We have identified an additional three residues near the NH2-terminus of SEE (Arg20, Asn21, and Ser24 that are needed in conjunction with these COOH-terminal residues to fully restore native levels of V beta-specific T cell proliferation. A staphylococcal enterotoxin A SEA-SEE hybrid molecule containing the NH2-terminal V beta determinants of SEE to activate alone exhibited V beta specificities of both SEA and SEE, indicating that these residues of SEE independently contribute to V beta recognition and do not obscure the native V beta determinants of SEA. These findings suggest that the ability of SEE to activate certain V beta-specific T cell subsets may result from multiple interactions with a single TCR beta-chain or perhaps by cross-linking two TCR. High affinity binding to HLA-DR1, a property of native SEA, was not altered in the SEA-SEE hybrid enterotoxins containing amino acid substitutions in regions 20 to 24 and 200 to 207, indicating that residues comprising the V beta determinants of SEE are separate from residues that contribute to HLA-DR1 binding affinity. Computer models of the predicted structure of SEE revealed that the V beta determinants of SEE are located on two adjacent solvent-exposed loops. Thus, the residues of SEE that mediate V beta recognition may coalesce to form a TCR binding site with specificities for multiple TCR beta-chains.

H2-M3wt-restricted, Listeria monocytogenes-specific CD8 T cells recognize a novel, hydrophobic, protease-resistant, periodate-sensitive antigen.

Mice infected with Listeria monocytogenes (LM) generate H2-M3wt-restricted CD8 effectors which recognize a heat-killed LM-associated antigen (HAA) presented by macrophages. To characterize HAA, we extracted a bioactive component from LM using SDS or NaOH. Extracted HAA aggregated in hydrophilic solvents but dissociated in the presence of SDS into a smaller subunit which migrated in Sephadex G-200 between chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). HAA bioactivity and size was unaffected by proteinase K under conditions which degraded virtually all detectable protein. HAA was also unaffected by other proteases, RNase and DNase, but HAA bioactivity was destroyed by periodate, an agent that degrades carbohydrates. These studies demonstrate that H2-M3wt can present a hydrophobic, non-peptide, microbial antigen, probably glycolipid in origin, to CD8 T cells.

Clear cell adenocarcinoma arising from a urethral diverticulum.

Carcinoma of the urethra is rare with only about 1,500 cases in the literature. Even more rare is carcinoma arising from a urethral diverticulum with about 100 cases reported to date. We report a case of clear cell adenocarcinoma arising from a urethral diverticulum.

Peptides from the amino-terminus of mouse mitochondrially encoded NADH dehydrogenase subunit 1 are potent chemoattractants.

Binding of N-formylated chemotactic peptides to specific cell surface receptors on polymorphonuclear leukocytes initiates a wide range of biological responses including migration of inflammatory cells, superoxide release, lysosomal enzyme secretion, calcium mobilization, and cellular activation. We previously established that the mouse MHC class I-b molecule H-2M3a binds peptides from the NH2-terminus of the mitochondrially encoded NADH dehydrogenase subunit 1 (ND1). Inasmuch as the N-formyl group is essential for peptide binding both to the chemotactic peptide receptor and to H-2M3a, we sought to test whether ND1 peptides can induce chemotaxis. We now show that fND1(1-12), fND1(1-8), fND1(1-5), fND1(1-4) and fND1(1-3) trigger the chemotactic receptor. Although all tested ND1 peptide derivatives were chemotactic, we found an inverse relationship between peptide length and chemotactic potency (ED50). Our data establish that mitochondrially derived peptides are potent chemotactic ligands. The release of N-formylated peptides from disintegrating mitochondria may play an important role in the inflammatory response resulting from tissue injury. By attracting the host phagocytic cells to sites of tissue breakdown, these peptides could mediate an essential first step in tissue repair and healing.

H-2M3a violates the paradigm for major histocompatibility complex class I peptide binding.

The major histocompatibility (MHC) class I-b molecule H-2M3a binds and presents N-formylated peptides to cytotoxic T lymphocytes. This requirement potentially places severe constraints on the number of peptides that M3a can present to the immune system. Consistent with this idea, the M3a-Ld MHC class I chimera is expressed at very low levels on the cell surface, but can be induced significantly by the addition of specific peptides at 27 degrees C. Using this assay, we show that M3a binds many very short N-formyl peptides, including N-formyl chemotactic peptides and canonical octapeptides. This observation is in sharp contrast to the paradigmatic size range of peptides of 8-10 amino acids binding to most class I-a molecules and the class I-b molecule Qa-2. Stabilization by fMLF-benzyl amide could be detected at peptide concentrations as low as 100 nM. While N-formyl peptides as short as two amino acids in length stabilized expression of M3a-Ld, increasing the length of these peptides added to the stability of peptide-MHC complexes as determined by 27-37 degrees C temperature shift experiments. We propose that relaxation of the length rule may represent a compensatory adaptation to maximize the number of peptides that can be presented by H-2M3a.