Selective stimulation of human T cells with streptococcal erythrogenic toxins A and B

Streptococcal exotoxins have been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Previous studies have demonstrated that these toxins are potent stimulators of human T cells and have structural homology to staphylococcal enterotoxins. In the current study, we investigated the mechanism by which streptococcal erythrogenic toxins type A (SPEA) and B (SPEB) activate T cells and compared it with anti-CD3 and the known "superantigen" staphylococcal enterotoxin B. SPEA was found to selectively activate T cells bearing V beta 8, V beta 12, and V beta 14, whereas SPEB selectively activated T cells bearing V beta 2 and V beta 8. Furthermore, fibroblasts transfected with MHC class II molecules were capable of presenting SPEA and SPEB to purified T cells. The T cell response to these toxins, however, was not MHC-restricted. Although the streptococcal exotoxins stimulated both CD4+ and CD8+ T cells, SPEA but not SPEB stimulated the CD4+ T cell subset proportionately more than the CD8+ T cell subset. Our results indicate that SPEA and SPEB, like the staphylococcal enterotoxins, are superantigens and suggest a mechanism by which they may mediate particular systemic syndromes associated with streptococcal infections.     

Activation of MHC class I-restricted CD8+ CTL by microbial T cell mitogens. Dependence upon MHC class II expression of the target cells and V beta usage of the responder T cells

The T cell response to microbial T cell mitogens (MTM) such as enterotoxins from Staphylococcus aureus (SE) and the soluble mitogen from Mycoplasma arthritidis, resemble the minor lymphocyte stimulatory locus (Mls) response in several aspects. An important feature of the Mls response is it restriction to CD4+ cells. This study demonstrates that in contrast to Mls, the MTM response includes both CD4+ and CD8+ subsets. Both CD4+ and CD8+ cells expanded in IL-2 after stimulation with SEB showed preferential expression of T cell receptors bearing V beta 8 domains. Mouse and human target cells could be lysed in the presence of MTM both by MTM-stimulated CD8+ lymphocytes and by MHC class I-restricted CTL clones of defined Ag specificity. MTM-induced lysis required the expression of MHC class II, but not class I Ag, on the target cells. Inhibition studies of SEB and Ag-dependent cytolysis by CTL clones underlined the crucial role of CD3 and LFA-1 in both instances, but showed CD8 dependence only for AG-dependent cytolysis. Together these findings suggest important differences between the putative MTM-mediated interaction of TCR with MHC molecules and the classical TCR/MHC interaction involved in MHC-restricted Ag recognition.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis (MAM). VIII. Selective activation of T cells expressing distinct V beta T cell receptors from various strains of mice by the "superantigen" MAM

Mycoplasma arthritidis T cell mitogen (MAM), in association with its MHC ligand, is recognized by T cells that express TCR-alpha/beta assembled with a product(s) of the V beta 8 gene family. We show here that lymphocytes from mice which fail to express V beta 8 products can also be activated by MAM and the resulting cultures exhibit a marked increase in V beta 6 TCR-bearing cells. Evidence was also obtained that MAM can activate T cells that express all three V beta 8 TCR. The mAb, F23.1, which recognizes all V beta 8 gene products, was strongly inhibitory for MAM-induced proliferation of CBA cells whose T cell repertoire for MAM consists of T cells that express V beta 8.2 and 8.3 TCR. In contrast, the F23.1 mAb was only weakly inhibitory for BALB/c splenocytes which express V beta 6 TCR in addition to all three V beta 8 TCR. Involvement of V beta 8.1, 8.2, 8.3, and V beta 6 in MAM-induced proliferation was confirmed by expanding lymphocyte cultures in the presence of MAM and phenotyping the activated cells for expression of individual V beta TCR. There was also evidence for a selective activation of T cells bearing specific V beta TCR because BALB/c T cell populations expanded with MAM were comprised of 46.2% V beta 8.2+ cells, 18.6% V beta 8.1+ cells, 7.6% V beta 8.3+ cells and 6.7% V beta 6+ cells. Recent studies suggest that the newly described "superantigens" including the staphylococcal enterotoxins and the self minor lymphocyte-stimulating Ag activate T cells in a manner similar to that described earlier for MAM. The discovery of shared recognition of these proteins by specific V beta TCR strongly suggests that MAM belongs to the superantigen protein family, the members of which may share cross-reactive epitopes. Inasmuch as MAM is produced by an organism which induces chronic joint disease, our findings provide the basis for a new model to study the role of superantigens in the development of chronic autoimmune type diseases.     

Control of T cell responses to staphylococcal enterotoxins by stimulator cell MHC class II polymorphism

The bacterial toxic mitogens or superantigens are a family of related proteins that elicit potent T cell proliferative responses. These responses require APC that express MHC class II proteins, but they are not MHC restricted and they do not depend on a processing step, presumably because these mitogens bind directly to MHC class II molecules. These mitogens stimulate T cells by interacting in an unknown way with the portion of the TCR encoded by certain V beta gene segments. In this paper, we explore the importance of MHC class II polymorphism in T cell responses to staphylococcal enterotoxins. We find that certain MHC molecules present SEB to V beta 8-bearing T cells far better than others. These data suggest that one route of host defence against bacterial toxic mitogens may be to alter MHC class II molecules so that stimulation is inhibited.     

Human CD4- CD8- alpha beta+ T cells express a functional T cell receptor and can be activated by superantigens.

The CD4 and CD8 molecules play an important role in the stimulation of T cells and in the process of thymic education. Most mature T cells express the alpha beta TCR and either CD4 or CD8; however, there is a small population of alpha beta+ TCR T cells that lack both CD4 and CD8. Little is known of the biology of the CD4- CD8- (double-negative) alpha beta+ TCR T cells or the nature of the Ag to which they may respond. These cells not only represent a novel population of T cells but also provide useful biologic tools to study the roles that CD4 and CD8 play in T cell activation. In this study we have addressed two questions. Firstly, whether CD4- CD8- alpha beta+ TCR T cells have functionally active TCR and, secondly, whether CD4 or CD8 is required for the activation of T cells by bacterial enterotoxins. Six double-negative alpha beta+ TCR T cell clones, propagated from two healthy donors, were challenged with a panel of nine bacterial enterotoxins. The V alpha and V beta usage of their TCR was determined by polymerase chain reaction. All of the CD4-CD8- clones proliferated in response to at least one of the enterotoxins, in a V beta-specific manner. The proliferative response of the CD4-CD8- alpha beta+ TCR T cell clones was similar in magnitude to that exhibited by CD4+ T cell clones of known V beta expression. These data clearly show that the CD4 and CD8 molecules are not required for the activation of untransformed human T cells by bacterial enterotoxins. Furthermore, these results indicate that CD4-CD8- alpha beta+ TCR T cells, normally present in all individuals, are not functionally silent, because they can be stimulated via their TCR. Their physiologic role, like that of gamma delta T cells, remains to be elucidated.     

Mutation of the disulfide loop in staphylococcal enterotoxin A. Consequences for T cell recognition.

The hallmark of T cell responses to staphylococcal enterotoxins (SE) and other super-Ag is a selective stimulation of cells expressing particular TCR-V beta segments. Our previous studies suggested that the disulfide loop in SE is critical for their interaction with the TCR. To investigate this concept in further detail we constructed disulfide loop mutants of staphylococcal enterotoxin A (SEA), and examined these altered toxins for mitogenicity, class II MHC binding, and V beta specificity. We found that substitutions of either Cys-96 or Cys-106 decreased mitogenicity by 100-fold without significantly affecting class II binding or resistance of the molecule to proteolysis. Several mutants lost the capacity to stimulate V beta 11+ cells, except a Cys-106----Gln mutant for which V beta 11-stimulatory activity was increased. By contrast, mutants containing Cys----Ala substitutions acquired the capacity to stimulate V beta 6+ cells. Despite these effects of V beta specificity, all mutants retained the predominant preference of SEA for V beta 3+ cells. Neither exchange of regions flanking the loop in SEA with corresponding residues in SEB, nor conversion of the entire loop region of SEA to that of SEE, were associated with transfers of V beta specificity. Our results suggest that the disulfide loop in SEA contributes to toxin avidity for the TCR, rather than specificity for particular V beta.     

In vivo induction of apoptosis in immature thymocytes by staphylococcal enterotoxin B.

Staphylococcal enterotoxins are potent T cell mitogens. Recent studies have shown that the binding of these toxins to class II MHC molecules on accessory cells is essential for the stimulation of T cells which bear specific V beta segment of TCR. In the present study we show that i.v. administration of staphylococcal enterotoxin B (SEB) results in an enlargement of spleen and lymph nodes but causes thymus atrophy. Elimination of CD4+CD8+ cells predominantly accounted for the shrinkage of thymus, and the lowest level of this cell population was reached 4 days after SEB injection. Furthermore, this decrease in CD4+CD8+ cells was accompanied by a relative increase in the percentages of CD4+CD8-, CD4-CD8+ and CD4-CD8- cells, whereas their absolute numbers actually reduced on day 4. The thymus shrinkage involved apoptosis which was characterized by DNA fragmentation and morphologic changes. The depletion of Thy-1 high, TCR-alpha beta low and TCR-alpha beta intermediate cells also occurred with a kinetic correlated to the reduction of CD4+CD8+ cells. Our results further showed that the percentages of V beta 8+ cells reduced 12 h post SEB injection, increased after 2 days, and decreased again thereafter. SEB thus causes both apoptotic and stimulative effects in the thymus. Apparently, the tremendous loss of double-positive cells (greater than 90% in cell number on day 4) is not simply due to the reduction of V beta 8+ cells, the possible modulatory effect of other factors or hormones which may play a role in the cell death is discussed.     

Clonal heterogeneity of superantigen reactivity in human V beta 6+ T cell clones. Limited contributions of V beta sequence polymorphisms.

Superantigens encoded in the genome or released by bacteria have been identified as potent modulators of the murine immune system. High frequencies of mature or immature T cells are activated or intrathymically deleted when superantigens cross-link MHC class II molecules and the V beta element of the TCR. The V beta specificity discriminates superantigens from polyclonal T cell stimulators as well as specific Ag and determines the immunomodulatory role in shaping the T cell repertoire. A similar regulatory function of superantigens in the human immune system is less well established. Here, we have studied a series of human T cell clones sharing the TCR V beta 6 element and describe a surprising heterogeneity in their responsiveness to staphylococcal exotoxins. The V beta 6 gene segment had the ability to respond to all staphylococcal enterotoxins (SE); however, for individual T cell clones, there was a clear predominance of SE C3 reactivity compared to SE B and SE C2. The clonal heterogeneity of SE responsiveness did not correlate to sequence polymorphisms in the fourth hypervariable region of the V beta 6 segment, the presumptive binding site for superantigens. Superantigen reactivity was crucially influenced by the presenting HLA-DR molecule, especially when the superantigen served as a coligand, enhancing or suppressing the Ag-specific activation of the TCR. These data suggest that the correlation between human TCR V beta gene segments and superantigen responses is not stringent. Potential intrathymic deletion mechanisms controlled by superantigens may be less selective in humans and may result in a leakiness influenced by the host HLA-DR molecules.     

Stimulation of PIP2 hydrolysis by aluminum fluoride in resting T cell subsets of normal and autoimmune-prone lpr mice

T cells of autoimmune-prone mice homozygous for the lpr mutation respond poorly to mitogens in terms of proliferation and of IL-2 production. In a previous study, we have correlated this deficient activation with the inability of mitogens to stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate in lpr T cells, although these cells bind mitogen and express the TCR/CD3 complex. In order to determine whether activation-deficient lpr T cells contain functional GTP-binding (G) protein(s) and phospholipase C, we examined the effects of the G protein activating agent sodium fluoride plus Al+3 (AlF-4). AlF-4 stimulated phosphatidylinositol turnover, a response characteristic of TCR/CD3 occupancy, in mature L3T4+ and Ly2+ T cells. Second, and more important, AlF-4 stimulated the same biochemical events in L3T4-, Ly2- (double-negative) T cells from the normal thymus or from the enlarged lymph nodes of autoimmune-prone mice homozygous for the lpr mutation. However, these double-negative T cells were unresponsive to receptor-active ligands such as T cell mitogens or anti-CD3-epsilon mAb, despite their ability to bind these ligands. These findings suggest that activation-deficient double negative T cells express the receptors, G protein(s) and effector enzymes necessary for second messenger formation and further suggest that the failure of these cells to generate the relevant second messengers in response to mitogens or anti-CD3-epsilon antibody may be due to inefficient coupling of the TCR/CD3 complex to G proteins.     

Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.

The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.     

Staphylococcal exotoxin activation of T cells. Role of exotoxin-MHC class II binding affinity and class II isotype

Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 bind directly to class II molecules of the MHC and stimulate T cells based predominantly on the V beta segment used by the TCR. We investigated the relationship between the class II binding affinities of four of these exotoxins, SEA, SEB, SEC1, and toxic shock syndrome toxin-1 and their T cell signaling capabilities. Although the toxins stimulated T cells at concentrations that ranged over more than two orders of magnitude, their affinities for class II (DR1) differed by less than sixfold. The affinities of the toxins predicted their capacity to stimulate resting T cells to proliferate. The binding affinities of the toxins for class II molecules indicated that at concentrations required for T cell stimulation, as few as 0.1% of the class II molecules are complexed with toxin. Finally, the isotype of class II molecules affected the ability of the toxins to bind and use these MHC Ag to stimulate T cells. These data thus demonstrate that of the staphylococcal exotoxins studied, both their potency as T cell mitogens and their ability to function in the presence of single class II isotypes can be attributed in part to their characteristic abilities to bind class II molecules.     

Stimulator cell-dependent requirement for CD2- and LFA-1-mediated adhesions in T lymphocyte activation by superantigenic toxins.

The staphylococcal enterotoxins and related microbial T cell mitogens stimulate T cells by cross-linking variable parts of the T cell receptor (TCR) with MHC class II molecules on accessory or target cells. We have used cloned human T cells and defined tumor cells as accessory cells (AC) to study the requirements for T cell activation by these toxins. On AC expressing high levels of CD54 (intercellular adhesion molecule-1, ICAM-1) and CD58 (lymphocyte function-associated antigen-3, LFA-3), mAb to CD2 were relatively ineffective in inhibiting the response to the toxins and antibodies to the lymphocyte function-associated antigen-1 (LFA-1) did not inhibit at all. If added together, however, these mAb inhibited the response completely. Similar results were obtained using antibodies to the target structures of CD2 and LFA-1. In contrast, on cells expressing low levels of LFA-3, mAb to LFA-1 but not to CD2 were strongly inhibitory. The same pattern of inhibition was found when these same cells were used as presenters of specific antigen to the T cells. These data show that adhesions via CD2 or LFA-1 are alternatively required for the stimulation of the T cells by superantigenic toxins and demonstrate another similarity between T cell stimulation by superantigens and by specific antigen recognition.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

The use of transfected fibroblasts and transgenic mice establishes that stimulation of T cells by the Mycoplasma arthritidis mitogen is mediated by E alpha

Mycoplasma arthritidis produces a soluble protein which is active for murine and human lymphocytes when presented by Ia-bearing accessory cells. By using fibroblasts transfected in vitro with various class II Ag, we demonstrated that presentation of the M. arthritidis mitogen (MAM) to T cells was mediated by E alpha-containing molecules. We also showed that splenocytes from transgenic mice expressing E alpha heterozygously (B10.TRG E alpha+) or homozygously (B10.E alpha TG +/+) underwent a similar proliferation in response to MAM as compared with the failure of control B10.TRG E alpha- splenocytes to respond to MAM. Although splenocytes from inbred C3H and CBA mice exhibited much higher proliferative responses to MAM than did those from B10.TRG.E alpha+ or B10.E alpha TG +/+ mice, flow cytometry showed similar levels of E alpha expression. Furthermore, gamma-irradiated splenocytes from B10.TRG E alpha + mice presented MAM to T hybridoma cells with a similar efficacy as did splenocytes from C3H mice. The lesser response to MAM of lymphocytes from the E alpha transgenic mice as compared with those from C3H and B10.K mice was likewise not due to differential expression of their V beta TCR. We conclude that presentation of MAM to T cells is accomplished by E alpha-containing molecules. The studies also suggest that the conserved, nonpolymorphic regions of class II molecules may play an important role in host immune response to microbial products.     

Stimulation of B10.BR T cells with superantigenic staphylococcal toxins

The Staphylococcus aureus enterotoxins are known to be potent T cell activators, stimulating cell proliferation and lymphokine production. Two additional S. aureus proteins, exfoliating toxin and toxic shock syndrome toxin, share these properties. Recently these molecules have been termed "super-antigens" because of their ability to bind to class II MHC molecules and thus form ligands that interact with TCR in an unconventional manner. In this paper we show that each toxin stimulates mouse T cells bearing receptors that include particular V beta regions, almost regardless of the other variable receptor components. In addition, different toxins have different specificities for V beta.     

Crystal structures of T cell receptor (beta) chains related to rheumatoid arthritis

The crystal structures of the Vbeta17+ beta chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1(F104-->Y/C187-->S)) forms, respectively. These TCR beta chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 Vbeta17 closely resemble those of a Vbeta17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three Vbeta17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the Vbeta regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 Vbeta17, implying that there is considerable variability in the structures of very similar beta chains. Structural alignments also reveal a considerable variation in the Vbeta-Cbeta associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the Vbeta domains of known TCR structures indicate that there are significant similarities among Vbeta regions that are MAM-reactive, whereas there appear to be significant structural differences among those Vbeta regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.     

Staphylococcal enterotoxin-mediated human T-T cell interactions.

Staphylococcal enterotoxins (SE) are known to stimulate a large proportion of T cells. SE bind to MHC-class II molecules on APC and a particular segment of certain TCR V beta and V gamma gene products. Resting human T cells do not express HLA class II Ag and therefore cannot present SE to T cells. Activated human T cells, however, do express HLA-DR, -DP, and -DQ Ag and could consequently serve as APC for SE. As such, local immune responses to SE might be regulated and/or abrogated by SE-mediated T-T cell interactions leading to T cell destruction. We have investigated if such SE-mediated T-T cell interactions can occur in vitro using human cytolytic TCR-alpha beta+ and TCR-gamma delta+ T cell clones. We demonstrate that the TCR-alpha beta+ T cell clones can efficiently present staphylococcal enterotoxin A (SEA) to each other: T cell clones coated with SEA are lysed by SEA-reactive T cell clones but not by a SEA-nonreactive T cell clone. Furthermore, the SEA-reactive TCR-alpha beta+ clones (but not the SEA-nonreactive clone) destruct themselves in the presence of SEA at low concentrations of SEA (less than 0.01 microgram/ml). Also, SEA-coated T cell clones can induce proliferative responses although such responses are much weaker than those induced when B cells are used as stimulator cells. In contrast, the SEA-reactive TCR-gamma delta+ T cell clones are resistant to autokilling in the presence of SEA and they do not lyse SEA-coated TCR-gamma delta+ targets. However, such targets can be lysed by TCR-alpha beta+ effector cells. These results indicate that TCR-gamma delta+ cells are relatively resistant to lysis and that during local nonspecific immune responses triggered by SE, which induces HLA-class II expression by the responding T cells, SE-mediated T-T cell interactions may play a role in the regulation and/or abrogation of these immune responses.     

T lymphocyte activation by staphylococcal enterotoxins: role of class II molecules and T cell surface structures

The enterotoxins produced by Staphylococcus aureus (SE) are the most potent mitogens known. Triggering of proliferation or cytotoxicity by SE requires the presence of MHC class II molecules on accessory or target cells. In this study we have investigated the role of HLA class II molecules in the activation of human T cells by SE and the nature of the target structure on the responding T lymphocyte for SE. This dependence on class II molecules is not due to an immunological "recognition" of SE since there is no restriction by polymorphic determinants of HLA molecules and since even xenogeneic class II molecules can reconstitute the human T cell response to SE. Furthermore, HLA class II-positive but not -negative cells absorb the mitogenic activity from SE solutions and significant binding of 125I-labeled SE can be demonstrated to class II-positive but not to class II-negative cells. Enterotoxin molecules react directly with T cells since they cause an increase in cytosolic Ca2+ concentration similar to anti-CD3 mAb. This increase is abrogated by prior modulation of the TCR/CD3 complex. Antibodies to CD2, CD3 and the TCR that block antigen-specific activation also block T cell activation by SE. Moreover, preincubation of purified resting accessory cell-free T cells with SE leads to modulation of the TCR/CD3 complex. Taken together these data indicate that SE interact selectively with HLA class II molecules on accessory or target cells and with a TCR-associated structure on the T cell.     

Abnormal T cell development in CD3-zeta-/- mutant mice and identification of a novel T cell population in the intestine.

The T cell antigen receptor (TCR)-associated invariable membrane proteins (CD3-gamma, -delta, -epsilon and -zeta) are critical to the assembly and cell surface expression of the TCR/CD3 complex and to signal transduction upon engagement of TCR with antigen. Disruption of the CD3-zeta gene by homologous recombination resulted in a structurally abnormal thymus which primarily contained CD4- CD8- and TCR/CD3very lowCD4+CD8+ cells. Spleen and lymph nodes of CD3-zeta-/- mutant mice contained a normal number and ratio of CD4+ and CD8+ single positive cells that were TCR/CD3very low. These splenocytes did not respond to antibody cross-linking or mitogenic triggering. The V beta genes of CD4-CD8- and CD4+CD8+ thymocytes and splenic T cells were productively rearranged. These data demonstrated that (i) in the absence of the CD3-zeta chain, the CD4- CD8- thymocytes could differentiate to CD4+CD8+ TCR/CD3very low thymocytes, (ii) that thymic selection might have occurred, (iii) but that the transition to CD4+CD8- and CD4-CD8+ cells took place at a very low rate. Most strikingly, intraepithelial lymphocytes (IELs) isolated from the small intestine or the colon expressed normal levels of TCR/CD3 complexes on their surface which contained Fc epsilon RI gamma homodimers. In contrast to CD3-zeta containing IELs, these cells failed to proliferate after triggering with antibody cross-linking or mitogen. In comparison to thymus-derived peripheral T cells in the spleen and lymph nodes, the preferential expression of normal levels of TCR/CD3 in intestinal IELs suggested they mature via an independent extrathymic pathway.     

Crystal structure of a complete ternary complex of TCR, superantigen and peptide-MHC

'Superantigens' (SAgs) trigger the massive activation of T cells by simultaneous interactions with MHC and TCR receptors, leading to human diseases. Here we present the first crystal structure, at 2.5-A resolution, of a complete ternary complex between a SAg and its two receptors, HLA-DR1/HA and TCR. The most striking finding is that the SAg Mycoplasma arthritidis mitogen, unlike others, has direct contacts not only with TCR Vbeta but with TCR Valpha.