Metal-specific CD4+ T-cell responses induced by beryllium exposure in HLA-DP2 transgenic mice.

Chronic beryllium disease (CBD) is a granulomatous lung disorder that is associated with the accumulation of beryllium (Be)-specific CD4(+) T cells into the lung. Genetic susceptibility is linked to HLA-DPB1 alleles that possess a glutamic acid at position 69 (ßGlu69), and HLA-DPB1*02:01 is the most prevalent ßGlu69-containing allele. Using HLA-DP2 transgenic (Tg) mice, we developed a model of CBD that replicates the major features of the human disease. Here we characterized the T-cell receptor (TCR) repertoire of Be-responsive CD4(+) T cells derived from the lungs of Be oxide-exposed HLA-DP2 Tg mice. The majority of Be-specific T-cell hybridomas expressed TCR Vß6, and a subset of these hybridomas expressed identical or nearly identical ß-chains that were paired with different α-chains. We delineated mimotopes that bind to HLA-DP2 and form a complex recognized by Be-specific CD4(+) T cells in the absence of Be. These Be-independent peptides possess an arginine at p5 and a tryptophan at p7 that surround the Be-binding site within the HLA-DP2 acidic pocket and likely induce charge and conformational changes that mimic those induced by the Be(2+) cation. Collectively, these data highlight the interplay between peptides and Be in the generation of an adaptive immune response in metal-induced hypersensitivity.

Elucidation of some Bax conformational changes through crystallization of an antibody-peptide complex.

The Bcl-2 family member Bax plays a critical role in apoptosis. In healthy resting cells, Bax resides in the cytoplasm and loosely attached to the mitochondrial membrane. Apoptotic stimuli induce Bax activation, which is characterized by translocation and multimerization on the mitochondrial membrane surface resulting in exposure of an amino terminal epitope recognized by the monoclonal antibody 6A7. To understand the structural changes that occur during Bax activation, we determined the crystal structure of a Bax peptide bound to the 6A7 Fab fragment to a resolution of 2.3 A. The structure reveals the conformation of the 6A7 peptide epitope on Bax in the activated form and elucidates the extensive structural changes that Bax must undergo during the conversion from its native to its activated conformation.

A novel family of retroviral vectors for the rapid production of complex stable cell lines.

The production of stable cell lines is an important technique in cell biology, and it is often the rate-limiting step in studies involving the characterization of the function of novel genes or gene mutations. To facilitate this process, a novel family of retroviral vectors, the pE vector family, has been generated. The retroviral sequences in the pE vectors have been taken from the Moloney murine leukemia virus (MMLV) vector pMFG, which has been shown to express cDNA inserts more consistently and at higher levels than earlier generations of MMLV vectors. These vectors contain four different internal ribosome entry site-selectable markers, allowing high-efficiency selection of transductants expressing the desired cDNA. The pE vectors have an episomal design to allow long-term production of high-titer virus without the need for subcloning the producer line. Using a strategy of combinatorial infection followed by combinatorial drug selection, we demonstrate that the pE vectors can be used to generate stable, polyclonal cell lines expressing at least three novel cDNAs in less than 2 weeks. The use of these vectors will thus dramatically accelerate the production of complex stable cell lines.

Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo.

In order to track hematopoetic cells of all lineages unambiguously at all stages of development, we have developed C57BL/6 mice that express a transgene coding for green fluorescent protein (GFP) under control of the human ubiquitin C promoter. These mice, called UBI-GFP/BL6, express GFP in all tissues examined, with high levels of GFP expression observed in hematopoetic cells. UBI-GFP/BL6 mice are unique in that B cells, T cells, and dendritic cells have distinct levels of GFP fluorescence. In cell transfer experiments, leukocytes from UBI-GFP/BL6 mice are readily identified by FACS or fluorescence microscopy. We demonstrate that transplanted UBI-GFP/BL6 dendritic cells are easily identified in secondary lymphoid tissues. Direct interactions between individual dendritic cells and multiple naïve CD8+ T cells are observed in lymph nodes within 12 h of cell transfer and require loading of the dendritic cells with the appropriate peptide antigen. Dendritic cells undergo specific morphologic changes following interactions with antigen-specific T cells.

Activation-induced inhibition of interleukin 6-mediated T cell survival and signal transducer and activator of transcription 1 signaling.

The cytokines interleukin (IL)-2, IL-4, IL-6, IL-7, and IL-15 have all previously been shown to inhibit resting T cell death in vitro. We have found a difference in the response of T cells to IL-6, depending on the activation status of the cells. IL-6 inhibited the death of naive T cells, but had no effect on the death of either superantigen-activated T cells, or T cells bearing memory markers. This was true even when the resting and activated T cells were isolated from the same animal; thus, the determining factor for IL-6 insensitivity was the activation status or activation history of the cell, and not the milieu in the animal from which the cells were isolated. Activated T cells expressed lower levels of IL-6 receptors on their surfaces, yet there were sufficient levels of receptors for signaling, as we observed similar levels of signal transducer and activator of transcription (Stat)3 phosphorylation in resting and activated T cells treated with IL-6. However, there was profound inhibition of IL-6-induced Stat1 phosphorylation in activated T cells compared with resting T cells. These data suggest that there is activation-induced inhibition of IL-6 receptor signaling in T cells. This inhibition appears to be specific for some but not all of the IL-6-mediated signaling cascades in these cells.

Live cell fluorescence imaging of T cell MEKK2: redistribution and activation in response to antigen stimulation of the T cell receptor.

T cell activation requires engagement of the T cell receptor (TCR) at the interface of conjugates formed with antigen-presenting cells. TCR engagement is accompanied by a redistribution of specific signaling molecules to the cytoplasmic region of the TCR complex. In this study, immunocytochemistry and live cell fluorescence imaging demonstrate that T cell MEK kinase 2 (MEKK2) is translocated to the T cell/antigen-presenting cell interface in response to antigen activation. MEKK2 translocation occurs more rapidly as the antigen concentration is increased. Biochemical activation of MEKK2 follows TCR stimulation, and expression of a dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK and p38 MAP kinase phosphorylation. Live cell fluorescence imaging thus enables characterization of signal transducers that are dynamically translocated following TCR engagement.

Influence of the NH2-terminal amino acid of the T cell receptor alpha chain on major histocompatibility complex (MHC) class II + peptide recognition.

The alpha/beta T cell receptor (TCR) recognizes peptide fragments bound in the groove of major histocompatibility complex (MHC) molecules. We modified the TCR alpha chain from a mouse T cell hybridoma and tested its ability to reconstitute TCR expression and function in an alpha chain-deficient variant of the hybridoma. The modified alpha chain differed from wild type only in its leader peptide and mature NH2-terminal amino acid. Reconstituted cell surface TCR complexes reacted normally with anti-TCR and anti-CD3 antibodies. Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not. We demonstrated that this phenotype could be reproduced simply by substituting the glutamic acid (E) at the mature NH2 terminus of the wild type TCR alpha chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble alpha/beta-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma. These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.

IL-6 rescues resting mouse T cells from apoptosis.

It has previously been demonstrated that mature mouse T cells live for many weeks in vivo. In contrast, explanted lymph node or splenic T cells undergo spontaneous death within days, suggesting that survival factors supplied in vivo are not present in normal tissue culture medium. We discovered that IL-6 can rescue resting T cells from apoptosis in vitro. We show that recombinant mouse IL-6 as well as IL-6 in endothelial cell supernatants are sufficient to rescue T cells from death in the absence of additional cytokines. We show that CD4+ T cells express Bcl-2 immediately following isolation from the mouse, but after 24 h in culture Bcl-2 is undetectable. If during this time period the T cells are incubated with rIL-6, Bcl-2 expression is not down-regulated. It is, therefore, possible that IL-6 rescue from death is mediated by maintenance or induction of Bcl-2 expression. Addition of rIL-6 does not by itself induce blastogenesis or proliferation, and therefore, this cytokine appears to be a true survival factor rather than a mitogenic factor for resting T cells. Together, these results support a potential role for IL-6 as one of the factors important for prolonging resting T cell survival in vivo.

CD28 engagement and proinflammatory cytokines contribute to T cell expansion and long-term survival in vivo.

To mount a productive response to Ag, CD4+ T cells in mice must divide, differentiate, and survive at least until the Ag has been eliminated. It has been suggested that to accomplish this, T cells must receive two signals, one through their TCRs and a second through CD28. The second signal through CD28 has been thought to fulfill two roles, to stimulate T cell proliferation and to promote T cell survival. In this paper we confirm that CD28 engagement can contribute to vigorous T cell expansion in mice injected with superantigens. However, CD28 engagement does not protect T cells produced during a superantigen-specific proliferative response from undergoing subsequent deletion. Even if CD28 is bound, 4 days after superantigen exposure, the majority of T cells produced in response to superantigen exposure are eliminated in vivo. In contrast, this loss of superantigen-stimulated T cells can be prevented by the inflammatory stimuli created by injection of bacterial LPS. This protection does not require engagement of CD28 by its ligands, B7-1 and B7-2. These data suggest that productive T cell responses in mice involve a number of signals, including those initiated through TCR and CD28, which are primarily involved in the activation and expansion of T cells, and others delivered by proinflammatory cytokines that protect an activated T cell from subsequent deletion.

Transcytosis of staphylococcal superantigen toxins.

Staphylococcus aureus produces a set of proteins (e.g., staphylococcal enterotoxin A [SEA], SEB, toxic shock syndrome toxin 1 [TSST-1]) which act both as superantigens (SAgs) and toxins. Although their mode of action as SAgs is well understood, little is known about how they enter the body via the intestine and cause food poisoning. To examine this problem we used an in vitro culture system to study the capacity of class II MHC-negative human intestinal epithelial cells (Caco-2) to transcytose several staphylococcal toxins. We found that Caco-2 cells are capable of dose-dependent, facilitated transcytosis of SEB and TSST-1, but not SEA. We extended these studies in vivo in mice by showing that ingested SEB appears in the blood more efficiently than SEA. Our data suggest that these toxins can cross the epithelium in an immunologically intact form. These results may have important implications for the pathogenesis of food poisoning.

Allergic airway sensitization induces T cell activation but not airway hyperresponsiveness in B cell-deficient mice.

B cells play an important role in the allergic response by producing allergen-specific Igs as well as by serving as antigen-presenting cells. We studied the involvement of B cells in the development of responses in a murine model of allergic airway sensitization. Normal and B cell-deficient (muMt-/-) B10.BR mice were sensitized via the airways to ovalbumin; Ig production, cytokine elaboration from local lymph node cells, development of airway hyperresponsiveness, and histological changes in the airways were evaluated. Both strains of mice had increased production of T helper 2-like cytokines and developed an accumulation of eosinophils in the bronchial tissue after airway sensitization. However, only wild-type mice produced allergen-specific antibodies and exhibited altered airway function. B cell-deficient mice reconstituted with anti-ovalbumin IgE during the course of sensitization developed increases in airway responsiveness. These results indicated that neither B cells nor IgE were necessary for the induction of a T helper 2-type cytokine response or eosinophil infiltration of the airways after allergic sensitization but that IgE was required as a second signal for the development of airway hyperresponsiveness in this model of airway sensitization.

Development and function of T cells in T cell antigen receptor/CD3 zeta knockout mice reconstituted with Fc epsilon RI gamma.

Engagement of alpha-beta T cell receptors (TCRs) induces many events in the T cells bearing them. The proteins that transduce these signals to the inside of cells are the TCR-associated CD3 polypeptides and zeta-zeta or zeta-eta dimers. Previous experiments using knockout (KO) mice that lacked zeta (zeta KO) showed that zeta is required for good surface expression of TCRs on almost all T cells and for normal T cell development. Surprisingly, however, in zeta KO mice, a subset of T cells in the gut of both zeta KO and normal mice bore nearly normal levels of TCR on its surface. This was because zeta was replaced by the Fc epsilon RI gamma (FcR gamma). These cells were relatively nonreactive to stimuli via their TCRs. In addition, a previous report showed that zeta replacement by the FcR gamma chain also might occur on T cells in mice bearing tumors long term. Again, these T cells were nonreactive. To understand the consequences of zeta substitution by FcR gamma for T cell development and function in vivo, we produced zeta KO mice expressing FcR gamma in all of their T cells (FcR gamma TG zeta KO mice). In these mice, TCR expression on immature thymocytes was only slightly reduced compared with controls, and thymocyte selection occurred normally and gave rise to functional, mature T cells. Therefore, the nonreactivity of the FcR gamma + lymphocytes in the gut or in tumor-bearing mice must be caused by some other phenomenon. Unexpectedly, the TCR levels of mature T cells in FcR gamma TG zeta KO mice were lower than those of controls. This was particularly true for the CD4+ T cells. We conclude that FcR gamma can replace the functions of zeta in T cell development in vivo but that TCR/CD3 complexes associated with FcR gamma rather than zeta are less well expressed on cells. Also, these results revealed a difference in the regulation of expression of the TCR/CD3 complex on CD4+ and CD8+ T cells.

Thymocytes can become mature T cells without passing through the CD4+ CD8+, double-positive stage.

T cells bearing the class II-restricted, DO-T cell receptor (TCR) are CD4+ if their thymocyte precursors are positively selected on the class II protein, IAd, but they are almost all CD4- after positive selection on a class II for which they have higher avidity, IAb. DO-TCR+ T cells mature in H-2b mice lacking CD4. CD4- DO-TCR+ T cells appear in H-2b mice at the same rate as their CD4+ counterparts appear in H-2d animals, suggesting that the CD4- cells are not the product of some minor pathway of thymocyte development and selection. In H-2b CD4 knock out mice expressing human CD2 under the control of the mouse CD4 promoter, mature DO-TCR+ cells did not express human CD2. These results suggest that the CD4-CD8-, DO-TCR+ mature T cells have developed without ever passing through the equivalent of a CD4+,CD8+ stage. The early expression of alpha/beta receptors (TCRs) on thymocytes in TCR transgenic mice may allow maturation of this type. Passage through the equivalent of the CD4+ CD8+, double-positive stage is not essential for differentiation of thymocytes into mature T cells.

B cells are not essential for peripheral T-cell tolerance.

Some self-reactive T cells avoid thymic tolerance and become mature peripheral cells. Nevertheless, these cells do not usually attack their hosts because T cells can be inactivated or killed, even after they are mature, by various means. The details of these processes are not fully understood; however, a number of experiments have suggested that peripheral tolerance may be induced in mature mouse T cells by exposure to antigen on resting B cells, cells that can express antigen bound to major histocompatibility complex proteins but that lack critical costimulatory molecules such as B7-1 and B7-2. Conversely, previous experiments have indicated that mature T cells can be stimulated by exposure to antigen on cells such as dendritic cells, cells that are thought to express the essential costimulatory molecules. We tested this idea in vivo by using mice that lack B cells. Unexpectedly, T-cell tolerance and antigen-induced T-cell death occurred normally in mice free of B cells. On the other hand, antigen-specific T-cell expansion in the spleens of such mice was impaired. Finally, we have recently shown that T-cell death in mice can be prevented by exposure to antigen and an inflammatory agent such as bacterial lipopolysaccharide. This was also true in mice that lacked B cells. Overall, these data show that mature T cells can be tolerized and rescued from tolerance in the absence of B cells.

Lipopolysaccharide interferes with the induction of peripheral T cell death.

In mice injected with superantigens, T cells specific for that antigen proliferate and then die. It has been suggested that the target cells die because they encounter superantigen on the surfaces of nonprofessional presenting cells, such as B cells, which cannot deliver costimulatory signals to T cells. A number of reagents that induce costimulatory molecules on B cells were tested. Lipopolysaccharide very effectively prevented T cell death driven by superantigen. Perhaps surprisingly, the action of lipopolysaccharide was not mediated through the expected costimulatory molecule, B7. Rather, the effects of lipopolysaccharide involved the production of inflammatory cytokines, in particular TNF alpha. The rescued cells survived in vitro culture and were resistant to Fas-induced killing. These data demonstrate that LPS can block antigen-induced T cell death perhaps by interfering with Fas signaling.

Monoclonal antibodies defining functional sites on the toxin superantigen staphylococcal enterotoxin B.

Four monoclonal antibodies (mAbs) were produced binding to four nonoverlapping epitopes on the superantigen staphylococcal enterotoxin B (SEB). The mAbs were tested for their ability to detect SEB bound to major histocompatibility complex (MHC) class II, to inhibit SEB binding to MHC class II, to inhibit SEB stimulation of T cell hybridomas, to bind to various nonfunctional mutants of SEB, and to capture and present SEB and its mutants to T cells in the absence of MHC class II. We concluded that two mAbs, B344 and B327, bound to epitopes not required for superantigen function, one mAb, 2B33, blocked an MHC interaction site on SEB, and the fourth mAb, B87, blocked the T cell recognition site on SEB. Moreover, two mAbs (B344 and 2B33) were capable of presenting SEB, although much less efficiently than APC, to CD4- but not CD4+ T cell hybridomas. The results confirm the functional domains on SEB originally defined by mutation and show that MHC class II is not always an essential component of the superantigen ligand.

Genetic analysis of low V beta 3 expression in humans.

While studying the T cell receptor (TCR) repertoire of normal individuals, we found that more than 20% of adults have low levels of circulating V beta 3.1+ T cells in both CD4 and CD8 populations. A similar frequency was found in fetal cord blood samples, suggesting that in most cases, the V beta 3.1low phenotype is inherited. In support of this conclusion, children expressing low levels were only found in families where one of the parents expressed this phenotype. In two large families, genetic studies showed that low expression was a recessive trait and dependent on inheritance of particular TCR VB gene complexes. Family members with the low phenotype, however, expressed VB3.1 genes with normal sequences and expressed normal levels of receptor per cell. Results from these families suggest that up to 50% of normal individuals may carry a VB3.1 allele that is defective in its ability to rearrange effectively. In another large family, low expression in one individual was shown not to be determined by genes within the TCR VB gene or major histocompatibility complexes, suggesting a different mechanism for low V beta 3.1+ T cells. Overall, our results describe novel mechanisms that result in low levels of V beta 3.1+ T cells in a relatively large subset of the normal human population.

Processing and major histocompatibility complex binding of the MTV7 superantigen.

Mouse mammary tumor viruses produce superantigens (vSAGs) which interact with class II major histocompatibility complex (MHC) proteins and stimulate T cells. vSAGs are synthesized as Type II membrane proteins, but at least one of these proteins (vSAG7) is found on the cell surface in a proteolytically processed form. Monoclonal antibodies (MAbs) were used to characterize vSAG7 and its binding to class II molecules. vSAG7 is synthesized in the endoplasmic reticulum (ER) as a 45 kd glycoprotein containing N-asparagine-linked oligomannosyl carbohydrates. vSAG7 transits the golgi complex, where it is modified by the addition of complex-type glycans and proteolysed at three positions. After proteolysis, the amino and carboxyl termini remain noncovalently associated. The ER, golgi, and surface forms of vSAG7 are stably bound to class II, but one of the proteolysed forms comprises the majority of the class II-bound material.

Thymic epithelial cell lines that mediate positive selection can also induce thymocyte clonal deletion.

Negative selection of potentially autoreactive thymocytes occurs mainly in the thymus and is thought to be induced primarily by interaction with bone marrow-derived cells. However, some studies have also reported a role for radioresistant thymic cells, which are probably epithelial in origin, in the deletion of thymocytes reacting to endogenous superantigens. We have previously demonstrated that thymic epithelial cell lines could induce thymocyte-positive selection in vivo. In this study, we assessed the potential of these cells to delete thymocytes reacting to the staphylococcal enterotoxin A or B superantigens in vitro. In the presence of staphylococcal enterotoxin A or B we found that all thymic epithelial cell lines used in this study were capable of activating T cell hybrids or deleting CD4+CD8+ thymocytes expressing an appropriate TCR. The extent of superantigen-mediated thymocyte deletion mediated by thymic epithelial cell lines was comparable to that mediated by a thymic macrophage cell line. Similar results were obtained with three phenotypically distinct thymic cell lines, suggesting that the ability to induce thymocyte deletion might be a general feature of various subsets of thymic epithelium. The observations provided in this study, combined with our previous demonstration that the same thymic epithelial cell lines can participate in positive selection, suggest that a given stromal cell population might be capable of taking part both in positive and negative selection of thymocytes.

Identification of two V beta 7-specific viral superantigens.

The commonly used strains of laboratory mice have mouse mammary tumor viruses (MTV) integrated at various locations in their DNA. The number and position of these integrants varies from one strain of mouse to another. It has recently been shown that the genomes of many of the MTV code for superantigens. The predicted amino acid sequences of these superantigens and their specificity for TCR V beta differs for each MTV integrant. This study contains the predicted amino acid sequence and V beta specificity of two MTV superantigens that had not previously been analyzed. The results show that both of these MTV superantigens are specific for TCR that bear V beta 7, but unlike the MTV7 superantigen not for receptors bearing V beta 6 or V beta 8.1. The data also support the conclusion of previous studies that the COOH-terminal sequence of these proteins is a major factor in controlling their V beta reactivity.