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.     

The orientation of a T cell receptor to its MHC class II:peptide ligands

The TCR found on CD4 T cells recognizes peptides bound to self MHC class II molecules as well as non-self MHC class II molecules. We have used the receptor on a cloned T cell line called D10.G4.1 (D10) to perform a structure-function analysis of this interaction. The D10 T cell clone recognizes not only a peptide from conalbumin (CA-wt) bound to syngeneic I-Ak against which it was raised, but also the allogeneic MHC molecules I-A(b,v,p,q,d). In the present study, we show that residue 30 in complementarity-determining region 1 (CDR1) of the TCR alpha-chain interacts with the I-A alpha-chain at hvr2 (residues 52, 53, and 55). We also show that residue 51 in CDR2 of the TCR alpha-chain interacts with the peptide at peptide residue 2. Finally, we show that residue 29 in CDR1 of the TCR beta-chain affects recognition of the glutamic acid at residue 66 in the I-A beta-chain. These data suggest an orientation of TCR relative to its peptide:MHC class II ligands. We argue that this orientation will be shared by all CD4 TCRs, and that it is only subtly different from the common orientation proposed for receptors binding to MHC class I.     

Cytotoxic T cell responses to DNA vaccination: dependence on antigen presentation via class II MHC

This study was designed to test whether cytotoxic T cell (CTL) responses to DNA vaccination are dependent upon MHC class II-restricted priming of CD4+ T cells. Because DNA vaccination may directly transfect dendritic cells, and dendritic cells may be capable of directly stimulating CD8+ T cell responses, such priming might be unnecessary. To test this hypothesis, C57BL/6 mice were immunized intramuscularly or intradermally with DNA encoding either whole OVA, a class I (Kb)-restricted peptide epitope of OVA (amino acids 257-264, SIINFEKL), or this class I-restricted epitope plus the adjacent class II (I-Ab)-restricted epitope of OVA (amino acids 265-280, TEWTSSNVMEERKIKV). Very low to negligible CTL responses were observed in mice vaccinated with the SIINFEKL construct, whereas mice vaccinated with the SIINFEKLTEWTSSNVMEERKIKV or with the complete OVA construct made equally robust CTL responses. These responses were sensitive to blocking by anti-CD8 mAb and were shown to be SIINFEKL-specific by using SIINFEKL peptide-pulsed EL-4 cells as targets. To ensure that the generation of these CTL responses was indeed dependent upon CD4+ T cell help, mice were depleted of either CD4+ or CD8+ cells before immunization. Depletion of CD4+ cells completely abrogated the CTL response to OVA DNA, as did depletion of CD8+ cells. Thus, we conclude that the CTL response to both intramuscular and intradermal DNA vaccination is highly dependent upon the generation of CD4+ T cell help via a class II MHC-dependent pathway. These results will be relevant for the construction of minimal-epitope vaccines for DNA immunization.     

The interaction between CD4 and MHC class II molecules and its effect on T cell function

During T cell activation, CD4 and CD8 form a 'bridge' between the T cell receptor (TCR) and major histocompatibility complex (MHC) class II and class I molecules, respectively. Due to this intimate association, CD4 and CD8 are now termed co-receptors and considered an integral part of this multimolecular complex. In addition, interest in CD4 has been heightened by the discovery that it is, in part, the receptor for HIV. Although CD4 and CD8 appear to perform similar immune functions, they are structurally diverse suggesting that their mode of interaction with the TCR and MHC molecules may differ. This review will focus primarily on a series of studies which have attempted to map the residues which mediate CD4:MHC class II interaction. These data will be evaluated in light of our current understanding of CD8:MHC class I, and CD4:TCR interactions. In addition, a model to explain the structural and functional differences between CD4 and CD8 will be presented. Finally, the potential effect of these multiple interactions on T cell function will be discussed.     

MHC class II-associated invariant chain peptide replacement by T cell epitopes: engineered invariant chain as a vehicle for directed and enhanced MHC class II antigen processing and presentation

Proteolysis of the invariant chain (li) leads to the generation of abundant MHC class II-associated invariant chain peptides (CLIP), which bind in the MHC class II binding groove via supermotifs in a manner similar to that of antigenic peptides. We have engineered an li vector with the capacity to express any antigenic peptide of interest instead of CLIP, for T cell stimulation. When peripheral blood mononuclear cells (PBMC) were pulsed with li hybrids encoding T cell epitopes of tetanus toxin or acetylcholine receptor, stimulation of T cells was dramatically enhanced compared to stimulation after priming with either the native or recombinant proteins. Site-specific insertion of antigenic sequences into the CLIP region promoted enhanced antigenicity of li hybrids which were shown to be processed intracellularly in a chloroquine-sensitive compartment. Naturally processed T helper epitopes were visualized directly on the surface of PBMC and identified as analogs of CLIP associated with MHC class II molecules. This novel li vector provides a flexible and efficient system for the delivery of defined peptide epitopes to T cells which might be useful in the development of specific vaccines and in the study of intracellular processing.     

Expression of functional MHC class II molecules by a mouse pro-B cell clone

OBJECTIVE: Pooled bronchoalveolar lavage fluid (BALF), the return of lavage, contains both bronchial and alveolar material which differ from each other. Artifacts may be created by filtering, centrifuging and washing cells before cytopreparation. This study presents reference values of healthy volunteers for the alveolar sample, ALF, cytopreparation being performed without filtration or centrifugation. METHODS: Eighteen healthy, non-smoking volunteers underwent a standard bronchoalveolar lavage using 10 aliquots of 20 ml of saline. Excluding the return of the first and second aliquots, the rest were pooled and examined cytologically, immunocytochemically and biochemically. The mean, standard deviation, and 95% confidence limits were calculated for the following variables: amount of return, estimated content of epithelial lining fluid (ELF), total and differential cell counts on filter and cytocentrifuge (CCF) preparations, computed cell counts per unit volume of ALF, distribution of lymphocyte subgroups CD3+CD2, CD4, CD8, CD19, CD25 and CD57, and the ratio of CD4 to CD8, the amounts of lymphocytes in the same subgroups per volume of ALF, and the concentrations of total protein, albumin, immunoglobulins A, G and M, hyaluronic acid, eosinophilic cationic protein (ECP), procollagen III aminoterminal propeptide (PCP) and beta 2-microglobulin in ALF and in ELF, as well as the ratios of the concentrations of the solutes in ALF to the same in serum. RESULTS: The 95% confidence limits of means for the most important variables were as follows: estimated ELF content 0.42-0.74%; total cells in ALF 76.6-143.0 x 10(6) l-1; distribution of inflammatory cells on filter and CCF slides: macrophages 74.9-83.6 and 81.4-90.1%, lymphocytes 13.1-22.5 and 8.1-16.4%, and neutrophils 1.0-4.1 and 0.7-2.7%, respectively; distribution of lymphocyte subsets: CD3+CD2 85.6-90.6%, CD4 44.3-53.1%, CD8 26.9-35.8%; concentration of solutes in ALF: total protein 44.8-61.3 mg l-1, albumin 15.4-22.2 mg l-1, IgA 1.8-3.4 mg l-1, IgG 3.1-6.1 mg l-1, IgM 0.05-0.26 mg l-1, hyaluronic acid 8.8-11.1 micrograms l-1, ECP 0.19-0.77 micrograms l-1, PCP 0.005-0.58 micrograms l-1, beta 2-microglobulin 62.2-81.5 micrograms l-1. CONCLUSIONS: Our results show that excluding the bronchial sample from ALF of volunteer subjects and omitting filtering and washing before cytopreparation produces cytologic, immunocytochemical and biochemical reference values with reasonable 95% confidence limits to be used in clinical settings.     

Dual MHC class I and class II restriction of a single T cell receptor: distinct modes of tolerance induction by two classes of autoantigens

In the final stages of thymic development, immature T cells undergo three distinct processes (positive selection, negative selection, and lineage commitment) that all depend on interactions of thymocyte TCRs with MHC molecules. It is currently thought that TCRs are preferentially restricted by either MHC class I or class II molecules. In this report, we present direct evidence that the TCR previously described as H-Y/H-2Db specific cross-reacts with H-2IAb if expressed in CD4+ cells. We also demonstrate an increase in thymocyte numbers in H-Y TCR-trangenic mice deficient in MHC class II, suggesting a relatively discrete form of negative selection by MHC class II compared with that induced by H-Y/H-2Db. We propose that inability to generate CD4+ T cells expressing H-Y TCR in different experimental settings may be due to tolerance to self-MHC class II. These results, therefore, support an intriguing possibility that tolerance to self may influence and/or interfere with the outcome of the lineage commitment.     

Neuronal control of MHC class II inducibility in rat astrocytes and microglia

We analysed the inducibility of major histocompatibility complex (MHC) class II molecules of astrocytes and microglia in organotypic hippocampus slice cultures of Lewis rats. Treatment with interferon-gamma (IFN-gamma) resulted in the induction of MHC class II molecules on microglia preferentially in the injured marginal zones of the slice culture, but only sporadically in areas containing intact neuronal architecture. In astrocytes, inducibility of MHC class II molecules was even more strictly controlled. IFN-gamma treatment induced MHC class II expression only in the slice culture zones containing degenerated neurons, and not in the presence of functional neurons. After suppression of spontaneous neuronal activity of the slice culture by the sodium channel blocker tetrodotoxin, MHC class II molecules on astrocytes could be induced by IFN-gamma in areas with intact neuronal architecture, and microglia cells exhibited a higher level of expression. These data suggest that loss of neurons could result in MHC class II inducibility of glial cells, and thus in increased immune reactivity of nervous tissue.     

Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA

Retrovirus-mediated gene transfer was used to restore expression to MHC class II-negative patient cells from complementation group A(II) of MHC class II immunodeficiency or bare lymphocyte syndrome (BLS). The cells of these patients do not transcribe MHC class II genes due to a defect in the trans-acting factor, CIITA. We constructed a vector, pGAG/Ii-CIITA, with the MHC class II-associated invariant chain promoter driving CIITA expression. Cocultivation with the virus producer line was consistently shown to be the optimal method for infection of all cell types. The induction of MHC class II expression after virus infection was rapid, and high levels of expression were achieved in cell lines within 1 wk of infection. In addition, expression was easily detectable even in peripheral blood cells of a BLS patient within a few days. Cell lines maintained in vitro for several months remained positive, and the proportion of cells with surface expression of DR was correlated with the number of integrated proviruses. Moreover, transduced B lymphoblastoid cell lines readily established tumors in CB17-scid/scid mice, and the MHC class II-positive cells demonstrated a clear competitive advantage in vivo. Ultimately, we hope to use this transduction system to restore normal immune function to a BLS patient for which no other therapeutic option currently exists.     

Coreceptor-independent T cell activation in mice expressing MHC class II molecules mutated in the CD4 binding domain

We have previously reported that efficient selection of the mature CD4+ T cell repertoire requires a functional interaction between the CD4 coreceptor on the developing thymocyte and the MHC class II molecule on the thymic epithelium. Mice expressing a class II protein carrying the EA137/VA142 double mutation in the CD4 binding domain develop fewer than one-third the number of CD4+ T cells found in wild-type mice. In this report we describe the functional characteristics of this population of CD4+ T cells. CD4+ T cells that develop under these conditions are predicted to be a CD4-independent subset of T cells, bearing TCRs of sufficient affinity for the class II ligand to undergo selection despite the absence of accessory class II-CD4 interactions. We show that CD4+ T cells from the class II mutant mice are indeed CD4 independent in their peripheral activation requirements. Surprisingly, we find that CD4+ T cells from the class II mutant mice, having been selected in the absence of a productive class II-CD4 interaction, fail to functionally engage CD4 even when subsequently provided with a wild-type class II ligand. Nevertheless, CD4+ T cells from EA137/VA142 class II mutant mice can respond to T-dependent Ags and support Ig isotype switching.     

Suppression of MHC class II expression by human class II trans-activator constructs lacking the N-terminal domain

The class II trans-activator (CIITA) is a bi- or multi-functional domain protein which plays a critical role in the expression of MHC class II genes. We report that removal of the N-terminal 151 amino acids, encompassing all of the acidic domain but leaving intact the proline/serine/threonine-rich domain, results in a mutant protein with potent suppressive properties for MHC class II expression. HeLa cells stably or transiently transfected with mutant CIITA constructs showed up to 99% suppression of MHC class II antigen induction by IFN-gamma and marked suppression of HLA-DRA mRNA expression. Transient transfection of a B lymphoma line resulted in up to 89% reduction of constitutive MHC class II expression within 5 days and suppression of HLA-DRA mRNA synthesis.     

Killing of rat adenocarcinoma 13762 in situ by adoptive transfer of CD4+ anti-tumor T cells requires tumor expression of cell surface MHC class II molecules

Cerebrovascular disease exemplifies the poor regenerative capacity of the CNS. While there are methods to prevent cerebral infarction, there is no effective therapy available to ameliorate the anatomical, neurochemical and behavioral deficits which follow cerebral ischemia. Focal and transient occlusion of the middle cerebral artery (MCA) in rodents has been reported to result in neuropathology similar to that seen in clinical cerebral ischemia. Using specific techniques, this MCA occlusion can result in a well-localized infarct of the striatum. This review article will provide data accumulated from animal studies using the MCA occlusion technique in rodents to examine whether neural transplantation can ameliorate behavioral and morphological deficits associated with cerebral infarction. Recent advances in neural transplantation as a treatment modality for neurodegenerative disorders such as Parkinson's disease, have revealed that fetal tissue transplantation may produce neurobehavioral recovery. Accordingly, fetal tissue transplantation may provide a potential therapy for cerebral infarction. Preliminary findings in rodents subjected to unilateral MCA occlusion, and subsequently transplanted with fetal striatal tissue into the infarcted striatum have produced encouraging results. Transplanted fetal tissue, assessed immunohistochemically, has been demonstrated to survive and integrate with the host tissue, and, more importantly, ameliorate the ischemia-related behavioral deficits, at least in the short term. Although, this review will focus primarily on cerebral ischemia, characterized by a localized CNS lesion within the striatum, it is envisioned that this baseline data may be extrapolated and applied to cerebral infarction in other brain areas.     

Generation of an MHC class II-restricted T cell epitope by extracellular processing of hepatitis delta antigen

Hepatitis delta virus is a human pathogen that is responsible for a severe form of hepatitis affecting hepatitis B envelope Ag carriers. We have previously identified a series of hepatitis delta Ag (HDAg) epitopes that are recognized by CD4+ T cell clones isolated from infected subjects. Herein, we show that the presentation of soluble HDAg to CD4+ T cell clones that are specific for the HDAg(106-121) epitope was exceptionally unaffected by the inhibition of the APC-processing machinery when APCs were fixed with glutaraldehyde before Ag pulsing or treated with chloroquine or brefeldin A. Interestingly, 5 h of pulsing was strictly required for the efficient presentation of the HDAg(106-21) epitope by fixed APCs, suggesting that some form of extracellular processing had occurred. Indeed, fixed APCs were able to present HDAg after only 1 h of pulsing when HDAg was preincubated with serum for 5 h. More important, presentation was completely abrogated when Ag was previously incubated in medium containing serum in the presence of a potent inhibitor of trypsin activity such as the secretory leukoprotease inhibitor. These results show that HDAg may undergo extracellular processing and suggest that the generation of immunogenic epitopes directly by serum proteases could play a role in the immune response against hepatitis delta virus during infection.     

Retroviral-mediated expression of an MHC class I-restricted T cell receptor in the CD8 T cell compartment of bone marrow-reconstituted mice

The introduction of cloned T cell receptor (TCR) genes into bone marrow cells could provide a way to increase the frequency of tumor- or pathogen-specific cytotoxic T lymphocyte (CTL) precursors. We demonstrate here the ability of a retroviral vector to direct expression of a Valpha15/Vbeta13 MHC class I-restricted TCR in lethally irradiated mice reconstituted with transduced bone marrow cells. We have detected retroviral-mediated TCR expression by flow cytometry 6-19 weeks after transplantation in C57L (Vbeta13(-/-)) and Rag1(-/-) bone marrow-reconstituted mice, and in C57BL/6 hosts reconstituted with transduced C57BL/6-Rag1(-/-) bone marrow. Southern analysis confirmed the presence of integrated provirus and revealed that the frequency of transduction is greater than the frequency of cell surface TCR expression. Although TCR expression on Vbeta13+ transduced cells is lower than endogenous TCR levels, it is largely confined to CD4+CD8+ (thymus) and CD8+ (thymus and spleen) T cells. In Rag1(-/-) mice, which display a developmental arrest of thymocytes at the immature CD4-CD8- stage, retrovirus-mediated TCR expression selectively rescues CD4+CD8+ and CD8+ populations. These results indicate that the ectopically expressed TCR is functional during T cell development. Furthermore, we have observed Vbeta13+ TCR expression by up to 13% of peripheral CD8+ T cells in C57L and C57BL/6 hosts. This represents a substantial increase relative to total Vbeta13 frequency in normal C57BL/6 mice (3-5%), and an even greater increase over the estimated frequency of CTL precursors of a defined specificity (10(-5)-10(-4)). Our findings indicate that TCR gene transfer can be used to develop new approaches to immunotherapy, and provide the basis for further studies examining the contribution of retrovirus-mediated TCR expression to an antigen-specific CTL response.     

Topology of T cell receptor-peptide/class I MHC interaction defined by charge reversal complementation and functional analysis

The molecular interactions between the CD8 co-receptor dependent N15 and N26 T cell receptors (TCRs) and their common ligand, the vesicular stomatitis virus octapeptide (VSV8) bound to H-2Kb, were studied to define the docking orientation(s) of MHC class I restricted TCRs during immune recognition. Guided by the molecular surfaces of the crystallographically defined peptide/MHC and modeled TCRs, a series of mutations in exposed residues likely contacting the TCR ligand were analyzed for their ability to alter peptide-triggered IL-2 production in T cell transfectants. Critical residues which diminished antigen recognition by 1000 to 10,000-fold in molar terms were identified in both N15 Valpha (alphaE94A or alphaE94R, Y98A and K99) and Vbeta (betaR96A, betaW97A and betaD99A) CDR3 loops. Mutational analysis indicated that the Rp1 residue of VSV8 is critical for antigen recognition of N15 TCR, but R62 of H-2Kb is less critical. More importantly, the alphaE94R mutant could be fully complemented by a reciprocal charge reversal at Kb R62 (R62E). This result suggests a direct interaction between N15 TCR Valpha E94R and Kb R62E residues. As Rp1 of VSV8 is adjacent to R62 in the VSV8/Kb complex and essential for T cell activation, this orientation implies that the N15 Valpha CDR3 loop interacts with the N-terminal residues of VSV8 with the Valpha domain docking to the Kb alpha2 helix while the N15 Vbeta CDR3 loop interacts with the more C-terminal peptide residues and the Vbeta domain overlies the Kb alpha1 helix. An equivalent orientation is suggested for N26, a second VSV8/Kb specific TCR. Given that genetic analysis of two different class II MHC-restricted TCRs and two crystallographic studies of class I restricted TCRs offers a similar overall orientation of V domains relative to alpha-helices, these data raise the possibility of a common docking mode between TCRs and their ligands regardless of MHC restriction.     

Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development

The T cell coreceptors CD4 and CD8 enhance T cell responses to TCR signals by participating in complexes containing TCR, coreceptor, and MHC molecules. These ternary complexes are also hypothesized to play a seminal role during T cell development, although the precise timing, frequency, and consequences of TCR-coreceptor-MHC interactions during positive selection and lineage commitment remain unclear. To address these issues, we designed transgenic mice expressing mutant I-Ek molecules with reduced CD4-binding capability. These transgenic lines were crossed to three different lines of I-Ek-specific TCR transgenic mice, and the efficiency of production of CD4+ lineage cells in the doubly transgenic progeny was assessed. Surprisingly, replacing wild-type I-Ek molecules with these mutant molecules did not affect the production of CD4+CD8- thymocytes or CD4+ peripheral T cells expressing any of the three TCRs examined. These data, when considered together with other experiments addressing the role of coreceptor during development, suggest that not all MHC class II-specific thymocytes require optimal and simultaneous TCR-CD4-MHC interactions to mature. Alternatively, it is possible that these particular alterations of I-Ek do not disrupt the CD4-MHC interaction adequately, potentially indicating functional differences between I-A and I-E MHC class II molecules.     

Selective cytotoxicity of two rodent T cell lymphomas to rat yolk sac tumours involves a retroviral envelope protein expressed by the lymphoma

OBJECTIVE: To test a model for the study of inequalities in hospitalizations in the city of Ribeirao Preto (SP), understanding them to be due both to the social position of inpatients and also to health care policies in Brazil. MATERIAL AND METHOD: Using a hospital information system in existence for more than 25 years in the city of Ribeirao Preto-SP, 56.293 hospitalizations of municipal inhabitants occurring in some of the 12 general hospitals in 1993, were studied. Using the Brazilian occupancy classification for mortality, these inpatients were grouped on 6 occupational levels, as in the British classification: professional, intermediate, qualified non manual, qualified manual, partially qualified and unqualified. RESULTS AND CONCLUSIONS: Two-thirds of the inpatients had no place in the i.e. did not belong to the economically active population--and consisted of housewives, pensioners, children and students--and one third had some economic activity and thus belonged to the economically the active population. A close association was found between social strata and the classification of the hospital financing system into private, private group clinic and public health system patients. There were differences in hospital parameters as well as in morbidity patterns between these groups. The inequalities relating to average age, average age of hospital deaths, mean lengths of stay, hospital mortality, re-internment and frequency of diseases are discussed. This model allows the social position of the inpatient to be estimated using the hospital financing system, including also those patients with no economic activity, which covers the majority of the population. Social mechanisms created to compensate for inequalities in the welfare state do not cancel out the social differences.