miR-19-Mediated Inhibition of Transglutaminase-2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer.

UNLABELLED: Transglutaminase-2 (TG2) is a critical cross-linking enzyme in the extracellular matrix (ECM) and tumor microenvironment (TME). Although its expression has been linked to colorectal cancer, its functional role in the processes that drive disease appears to be context dependent. There is now considerable evidence of a role for microRNAs (miRNA) in the development and progression of cancer, including metastasis. A cell model of metastatic colon adenocarcinoma was used to investigate the contribution of miRNAs to the differential expression of TG2, and functional effects on inflammatory and invasive behavior. The impact of TG2 in colorectal cancer was analyzed in human colorectal tumor specimens and by manipulations in SW480 and SW620 cells. Effects on invasive behavior were measured using Transwell invasion assays, and cytokine production was assessed by ELISA. TG2 was identified as a target for miR-19 by in silico analysis, which was confirmed experimentally. Functional effects were evaluated by overexpression of pre-miR-19a in SW480 cells. Expression of TG2 correlated inversely with invasive behavior, with knockdown in SW480 cells leading to enhanced invasion, and overexpression in SW620 cells the opposite. TG2 expression was observed in colorectal cancer primary tumors but lost in liver metastases. Finally, miR-19 overexpression and subsequent decreased TG2 expression was linked to chromosome-13 amplification events, leading to altered invasive behavior in colorectal cancer cells. IMPLICATIONS: Chromosome-13 amplification in advanced colorectal cancer contributes to invasion and metastasis by upregulating miR-19, which targets TG2.

Abituzumab combined with cetuximab plus irinotecan versus cetuximab plus irinotecan alone for patients with KRAS wild-type metastatic colorectal cancer: the randomised phase I/II POSEIDON trial.

BACKGROUND: Integrins are involved in tumour progression and metastasis, and differentially expressed on colorectal cancer (CRC) cells. Abituzumab (EMD 525797), a humanised monoclonal antibody targeting integrin αν heterodimers, has demonstrated preclinical activity. This trial was designed to assess the tolerability of different doses of abituzumab in combination with cetuximab and irinotecan (phase I) and explore the efficacy and tolerability of the combination versus that of cetuximab and irinotecan in patients with metastatic CRC (mCRC) (phase II part). METHODS: Eligible patients had KRAS (exon 2) wild-type mCRC and had received prior oxaliplatin-containing therapy. The trial comprised an initial safety run-in using abituzumab doses up to 1000 mg combined with a standard of care (SoC: cetuximab plus irinotecan) and a phase II part in which patients were randomised 1 : 1 : 1 to receive abituzumab 500 mg (arm A) or 1000 mg (arm B) every 2 weeks combined with SoC, or SoC alone (arm C). The primary end point was investigator-assessed progression-free survival (PFS). Secondary end points included overall survival (OS), response rate (RR) and tolerability. Associations between tumour integrin expression and outcomes were also assessed. RESULTS: Phase I showed that abituzumab doses up to 1000 mg were well tolerated in combination with SoC. Seventy-three (arm A), 71 (arm B) and 72 (arm C) patients were randomised to the phase II part. Baseline characteristics were balanced. PFS was similar in the three arms: arm A versus SoC, hazard ratio (HR) 1.13 [95% confidence interval (CI) 0.78-1.64]; arm B versus SoC, HR 1.11 (95% CI 0.77-1.61). RRs were also similar. A trend toward improved OS was observed: arm A versus SoC, HR 0.83 (95% CI 0.54-1.28); arm B versus SoC, HR 0.80 (95% CI 0.52-1.25). Grade ≥3 treatment-emergent adverse events were observed in 72%, 78% and 67% of patients. High tumour integrin αvß6 expression was associated with longer OS in arms A [HR 0.55 (0.30-1.00)] and B [HR 0.41 (0.21-0.81)] than in arm C. CONCLUSION: The primary PFS end point was not met, although predefined exploratory biomarker analyses identified subgroups of patients in whom abituzumab may have benefit. The tolerability of abituzumab combined with cetuximab and irinotecan was acceptable. Further study is warranted. CLINICALTRIALS.GOV IDENTIFIER: NCT01008475.

Innate and adaptive immune responses to the major Parietaria allergen Par j 1 in healthy subjects.

In this study we wanted to analyse the pattern of the immune response to the Parietaria major allergen Par j 1 in freshly purified peripheral blood mononuclear cell (PBMC) from healthy subjects. We observed that Par j 1 was capable of inducing IFN-γ production by CD3⁻ and CD16⁺/CD56⁺ cells exclusively in healthy individuals. Furthermore, a multiparametric analysis allowed us a better definition of two IFN-γ-Par j 1 specific populations (IFN-γ(dim) and IFN-γ(high)) characterized by the presence of different proportions of NKT and NK cells. We also identified the concomitant presence of a subset of IL-10⁺ NK cells. Moreover, CFSE staining showed that the Par j 1 preferentially induced the proliferation of CD3⁻/CD56⁺/CD335⁺ cells. Finally, a subset of CD4⁺/CD25⁺/FoxP3⁺/IL-10⁻ T cells was identified. The result of this pilot study suggest that during a tolerogenic response, the major allergen of the Parietaria pollen works as an activator of both the innate and the adaptive human immune system.

Inhibition of p38 mitogen activated protein kinase controls airway inflammation in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) airways are characterised by chronic inflammation, increased interleukin (IL)-8 secretion, and neutrophil activation which are considered the principal factors of morbidity and mortality in CF patients. Optimising management of this chronic inflammatory response is therefore a key issue of basic and clinical CF research. Several reports have addressed ways to manage CF airways inflammation, and an attractive therapeutic strategy may be the inhibition of the p38-mitogen activated protein kinase (p38-MAP-k) pathway. METHODS: A new ex vivo model was used to study the mucosal inflammatory response to environmental airways stimuli. Nasal biopsy tissues from CF patients and controls were cultured ex vivo for 20 minutes, 4 hours, and 24 hours in the presence of lipopolysaccharide (LPS) from Pseudomonas aeruginosa (PA) with and without the p38-MAP-k inhibitor SB203580. Quantitative mRNA assessment, immunohistochemistry, and Western blots were used to detect the expression and modulation of inflammatory markers. RESULTS: PA-LPS challenge induced a time dependent mucosal inflammation indicated by rapid epithelial activation, IL-8 release, COX-2 upregulation, and neutrophil migration to the upper mucosal layers. Some of these LPS induced changes (IL-8 release and neutrophil migration) were specific to CF tissues. SB203580 significantly controlled all LPS induced mucosal changes in CF tissues. CONCLUSION: These findings provide a rationale and proof of principle for the potential use of p38-MAP-k inhibitors to control inflammation in patients with CF.

Celiac disease: a model autoimmune disease with gene therapy applications.

Gene therapy (GT) is still at the 'experimental' stage and some recent setbacks have cooled the potential use of this therapeutic tool even in life-threatening conditions. However, this therapeutic approach has a potential, which is not limited to disease for which we have not other option. There are increasing evidence that GT will be soon used in diseases that are not life threatening. One group of diseases that can benefit from GT is the autoimmune one. Several experimental animal models have indicated the efficacy (proof of principle) of GT. In the present review, we have addressed the possibility that even extremely benign autoimmune-like diseases such as Celiac Disease (CD) might one day profit from this type of therapy. We further point that in conditions such as CD, where the trigger is well known and the pathogenic cascade is relatively well defined, a situation not common in autoimmunity, we can even have a better situation where to explore and use GT to control disease initiation and progression. Once the risks that are still intrinsic to GT will have been reduced the therapeutic options we outline in the present review might not appear too far from reality.

IL-15 drives the specific migration of CD94+ and TCR-gammadelta+ intraepithelial lymphocytes in organ cultures of treated celiac patients.

OBJECTIVES: Celiac disease (CD) is an under-diagnosed but extremely frequent disease, triggered by the ingestion of gliadin. The pathogenic mechanisms of CD are still poorly understood, but intraepithelial lymphocytes are considered to have a key role. We intended to define the subsets of T lymphocytes migrating upon gliadin challenge in organ cultures of treated celiac patients and establish the type of factor(s) driving such an infiltration. METHODS: Duodenum biopsies from 10 treated celiacs and 7 controls were cultured in vitro with/without gliadin digest (1 mg/ml) or interleukin (IL)-15 (10 ng/ml). In 7 treated celiacs IL-7, IL-4, and IL-2 were similarly tested. Intraepithelial CD3, CD8, TCR-gammadelta, and CD94 were detected by immunohistochemistry and numbered per mm epithelium. RESULTS: IL-15 but not IL-7, IL-4, or IL-2 induced intraepithelial increase of CD3+ and CD8+ cells in celiac and control intestine (p < 0.001 vs cultures with medium). IL-15 induced increases in the number of intraepithelial TCR- gammadelta+ and CD94+ cells only in celiacs (p < 0.001). IL-7 was also effective in increasing intraepithelial TCR-gammadelta+ (but not CD94+) cells in celiac biopsies (p < 0.001). Gliadin induced intraepithelial migration of CD3+, CD8+ (p < 0.001), and CD94+ (p < 0.05) cells in celiacs, but not in controls. CONCLUSIONS: The results we describe in this report indicate that IL-15 might have a key role in modulating and driving intraepithelial infiltration and ultimately in the pathogenesis of CD.

FAS engagement drives apoptosis of enterocytes of coeliac patients.

BACKGROUND: Villus atrophy is the most distinctive sign of untreated coeliac disease (CD) and epithelial apoptosis is considered to be involved in this stage of the coeliac lesion. The extent of villus atrophy is, however, not homogeneous and patients with patchy or mild lesions have been described. AIMS: To address: (a) the degree of "patchiness" in untreated CD patients; and (b) to clarify if apoptosis, and eventually which trigger drives it, causes epithelial damage. PATIENTS: Twenty of 40 untreated, 14 treated coeliac patients, and 15 controls received five or more multiple duodenal biopsies; the remaining 20 untreated CD patients had no more than three biopsies. METHODS: All biopsies were analysed to monitor the presence of a "flat" mucosa. Biopsies of 14 untreated, 10 treated coeliacs, and seven controls were cultured with or without gliadin. DNA fragmentation was studied by terminal deoxynucleotidyl transferase (TdT) mediated dUTP digoxigenin nick end labelling (TUNEL), and FAS and Ki67 expression by immunohistochemistry. Antiendomysium antibodies (EMA) were surveyed in biopsy culture supernatants. RESULTS: A pattern of patchy duodenal lesions was observed in all untreated CD patients biopsied up to five times. High enterocyte FAS expression, and a high number of TUNEL+ and Ki67+ enterocytes were detected in areas with villus atrophy but not in those with a normal morphology (p<0.001). Conversely, EMA in culture supernatants and signs of immunological activation were present in all untreated CD biopsies. In vitro gliadin challenge increased the number of TUNEL+ and Ki67+ enterocytes (p<0.001 v cultures with medium alone) only in "flat" biopsies. Neutralising anti-FAS monoclonal antibodies were found to control gliadin induced enterocyte apoptosis (p>0.01) while agonist anti-FAS monoclonal antibody increased it (p<0.001). CONCLUSIONS: Patchy lesions are observed in untreated CD mucosa and epithelial FAS engagement is a key trigger in driving villus atrophy in CD.

Interleukin 15 mediates epithelial changes in celiac disease.

BACKGROUND & AIMS: Villous atrophy and crypt proliferation are key epithelial features of untreated celiac disease. We tried to define whether cytokines such as interleukin (IL)-15, IL-2, IL-4, and IL-7, which share chains of their receptors, could influence the epithelial modifications. METHODS: Duodenal biopsy specimens (14 treated and 13 untreated celiac patients, 7 controls) were cultured in vitro for 24 hours with or without gliadin (1 mg/mL), IL-15, IL-7, IL-4, or IL-2 (10 ng/mL). Tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were also used in some specimens of untreated celiacs. Epithelial expression of Ki67, FAS, and transferrin receptor (TFR) was detected by immunohistochemistry, and apoptosis by TUNEL technique (percentage of positive enterocytes). IL-15-positive cells were detected by immunohistochemistry in celiac disease and control biopsy specimens; presence of IL-15 was also determined by semiquantitative polymerase chain reaction. RESULTS: Only IL-15 induced enterocyte expression of Ki67, TFR, and FAS in treated celiac (P<0.01 vs. medium) and enterocyte apoptosis in untreated celiac disease specimens. Anti-IL-15 monoclonal antibodies neutralized gliadin-induced enterocyte TFR and FAS expression in treated celiac and enterocyte apoptosis in untreated celiac disease specimens (P<0.05 vs. gliadin). IL-15-positive cells were increased in untreated celiacs (P<0.001 vs. treated celiacs and controls). CONCLUSIONS: IL-15 is involved in the modulation of epithelial changes in celiac disease, indicating that this cytokine has an unforeseen role in the pathologic manifestations of celiac disease.

Fully competent dendritic cells as inducers of T cell anergy in autoimmunity.

Mature immunologically competent dendritic cells are the most efficient antigen-presenting cells that powerfully activate T cells and initiate and sustain immune responses. Indeed, dendritic cells are able to efficiently capture antigens, express high levels of costimulatory molecules, and produce the combination of cytokines required to create a powerful immune response. They are also considered to be important in initiating autoimmune disease by efficiently presenting autoantigens to self-reactive T cells that, in this case, will mount a pathogenic autoimmune reaction. Triggering T cells is not a simple on-off procedure, as T cell receptor responds to minor changes in ligand with gradations of T cell activation and effector functions. These "misfit" peptides have been called Altered Peptide Ligands, and have been shown to have important biological significance. Here, we show that fully capable dendritic cells may present, upon natural antigen processing, a self-epitope with Altered Peptide Ligands features that can unexpectedly induce anergy in a human autoreactive T cell clone. These results indicate that presentation of a self-epitope by immunologically competent dendritic cells does not always mean "danger" and show a mechanism involved in the fine balance between activation and tolerance induction in humans.

Blockage of T-cell costimulation inhibits T-cell action in celiac disease.

BACKGROUND & AIMS: Celiac disease is an exemplary model of T cell-mediated pathology. Therefore, therapeutic approaches that target T cells may successfully control this disease. CTLA-4 immunoglobulin (CTLA-4Ig) can inhibit T-cell activation by blocking the engagement of CD28. We took advantage of this tool to define the pathogenic role of gliadin-specific T cells in the induction of celiac disease. METHODS: Duodenal biopsy specimens from 7 treated celiac patients were challenged in vitro with gliadin and CTLA-4Ig or CD40-Ig. After 24 hours, the biopsy specimens were analyzed for the presence of characteristic modifications induced by gliadin challenge. RESULTS: CTLA-4Ig down-regulated the expression of CD25, intercellular adhesion molecule 1, interleukin 2, and interferon gamma (stained lamina propria mononuclear cells/mm2; P < 0.05) induced by gliadin challenge, caused apoptosis of gliadin-specific T cells (apoptotic T cells/mm2; P < 0.05), and inhibited the production of antiendomysial antibody (P < 0.01). However, it did not control intraepithelial T-cell migration (P = NS) and Fas expression by enterocytes. Conversely, CD40-Ig only controlled production of antiendomysial antibody. CONCLUSIONS: In an organ culture model, CTLA-4Ig controls many but not all of the immunologic features of celiac disease.

Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step.

Human CD1+ CD14- dendritic cells (DC) can be derived from CD14+ monocytes using granulocyte/monocyte colony-stimulating factor and interleukin (IL)-4. We have previously shown that IL-10 pre-treatment of such DC significantly inhibited their antigen-presenting capacity to CD4+ T cell clones. In this study, we further analyze how IL-10 influences antigen presentation. We first investigated whether IL-10 could alter the early stage of antigen presentation, the capture of antigen. This can be mediated by mannose receptor (MR)-mediated endocytosis and by fluid-phase uptake through macropinocytosis. IL-10-treated DC showed an enhancement of both mechanisms of antigen capture, as indicated by the increase of fluorescein isothiocyanate-dextran uptake through MR and lucifer yellow uptake. However, IL-10-treated DC, irradiated or glutaraldehyde-fixed, were less efficient than untreated DC in stimulating mixed leukocyte reaction as well as in inducing the activation of peptide-specific T cell clones, indicating that IL-10 achieves its effects mainly by modifying the cell surface phenotype of DC. HLA class I and II, as well as intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen-3, B7-1, B7-2 and ICAM-3 expression were either significantly increased or essentially unchanged, and the ability to bind the epitope recognized by the T cell clones was also unaffected regardless of IL-10 treatment. Our study also indicates that as-yet unidentified accessory molecules may play an essential role in T cell activation. Thus, the IL-10-treated DC possess an increased capacity to capture antigen, with a concomitant decreased stimulatory activity. Our study suggests that IL-10-treated DC have the characteristics of highly immature DC (high capture ability, low stimulatory potency) and may represent an early maturative step of human DC of monocytic origin.

Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fc epsilon receptor by a recombinant human autoantibody Fab converted to IgE.

Fc epsilon receptor (CD23)-mediated capture of IgE-antigen complexes by B cells provides a powerful antigen presenting system. Our goal was to develop a system using high affinity, human, organ-specific monoclonal autoantibodies for antigen capture by B cells. For this purpose, we converted a recombinant human autoantibody to TPO from a Fab (SP1.4) to an IgE molecule. Sera from all patients with autoimmune thyroid disease contain autoantibodies with the same epitope as SP1.4. The SP1.4 H and L chain V region genes were spliced by overlap PCR to a mammalian, non-immunoglobulin signal peptide and transferred to expression vectors for human IgG1 and kappa, respectively. After inserting the IgE constant region genes into the H chain vector, the kappa and IgE H chain vectors were expressed in SP2/0 cells. SP1.4-IgE retains its high affinity (Kd) for TPO (approximately 2 x 10(-10) M), recognizes the same epitope as Fab SP1.4 and, importantly binds to a different epitope than does Fab TR1.9. Binding of preformed complexes of SP1.4-IgE and biotinylated TPO to EB virus transformed B cells (EBVL) was weakly detectable by flow cytometry and was displaced by unlabeled TPO. SP1.4-IgE/125I-TPO complex binding to EBVL was much more clearly evident, was also inhibited by the addition of unlabeled TPO, and was greatly reduced by preincubation of the EBVL with anti-CD23. Further, autologous EBVL preincubated with SP1.4-IgE/TPO complexes stimulated proliferation of TPO-specific T cells. IgE autoantibody-mediated antigen focusing to B cells is unlikely to operate in vivo but is, instead, a powerful investigative tool. In conclusion, SP1.4-IgE is the first monoclonal human autoantibody to be developed for IgE-mediated antigen presentation to T cells by EBVL. Recombinant human autoantibodies converted to IgE, possibly in combinations if their epitopes permit simultaneous binding to the same molecule, provide a unique system to generate human T cell lines and clones specific for peptides naturally processed from internalized high affinity autoantibody/autoantigen complexes.

Human self-reactive T cell clones expressing identical T cell receptor beta chains differ in their ability to recognize a cryptic self-epitope.

Recognition of self-antigens by T lymphocytes is a central event in autoimmunity. Understanding of the molecular interactions between T cell receptors (TCR) and self-epitopes may explain how T cells escape thymic education and initiate an autoimmune reaction. We have studied five human in vivo activated T cell clones specific for the region 535-551 of human thyroid peroxidase (TPO) established from a Graves' patient. Three clones (37, 72, and 73) expressed identical TCR beta and alpha chains rearranging V beta 1.1 and V alpha 15.1, and were considered sister clones. Clone 43 differed from clone 37 and its sisters in the J alpha region only. Clone NP-7 expressed V beta 6.5 but rearranged two in-frame TCR alpha chain, both using the V alpha 22.1 segment. Fine epitope mapping using nested peptides showed that clones using identical TCR beta chains, identical V alpha, but a different J alpha recognized distinct, nonoverlapping epitopes in the TPO 535-551 region. This finding shows that a different J alpha region alone leads to a heterogeneous pattern of recognition. This indicates that the "restricted" TCR V region usage sometimes found in autoimmune diseases may not always correspond to identical epitope recognition. To confirm that clones 37 (and its sisters) and 43 recognize different epitopes, the T cell clones were stimulated with a TPO-transfected autologous Epstein-Barr virus (EBV) cell line (TPO-EBV) that presents TPO epitopes afer endogenous processing. Only clone 37 and its sisters recognizes the TPO-EBV cell line, suggesting that the epitope recognized by clone 43 is not presented upon endogenous processing. We have shown that thyroid epithelial cells (TEC), the only cells that produce TPO, express HLA class II molecules in Graves' disease and can act as an antigen-presenting cells, presenting TPO after endogenous processing to autoantigen-reactive T cell clones. We tested, therefore, whether autologous TEC induced the same pattern of stimulation as TPO-EBV; T cell clone 37 recognizes the TEC, whereas it is stimulated poorly by the TPO loaded to autologous peripheral blood mononuclear cells (PBMC). Clone 43, which fails to recognize the TPO-EBV, also fails to recognize the TEC, but is activated by exogenous TPO presented by autologous PBMC. These results show that exogenous versus endogenous processing in vivo generates a different TPO epitope repertoire, producing a "cryptic" epitope (epitope not always available for recognition). Our findings define a route by which human self-reactive T cells may escape thymic selection and become activated in vivo, thus possibly leading to autoimmunity.

Similar antigenic surfaces, rather than sequence homology, dictate T-cell epitope molecular mimicry.

Molecular mimicry, normally defined by the level of primary-sequence similarities between self and foreign antigens, has been considered a key element in the pathogenesis of autoimmunity. Here we describe an example of molecular mimicry between two overlapping peptides within a single self-antigen, both of which are recognized by the same human self-reactive T-cell clone. Two intervening peptides did not stimulate the T-cell clone, even though they share nine amino acids with the stimulatory peptides. Molecular modeling of major histocompatibility complex class II-peptide complexes suggests that both of the recognized peptides generate similar antigenic surfaces, although these are composed of different sets of amino acids. The molecular modeling of a peptide shifted one residue from the stimulatory peptide, which was recognized in the context of the same HLA molecule by another T-cell clone, generated a completely different antigenic surface. Functional studies using truncated peptides confirmed that the anchor residues of the two "mimicking" epitopes in the HLA groove differ. Our results show, for two natural epitopes, how molecular mimicry can occur and suggest that studies of potential antigenic surfaces, rather than sequence similarity, are necessary for analyzing suspected peptide mimicry.

Comparison of peptide and superantigen-induced anergy in a peptide-specific polyclonal human T cell line.

T cells recognizing tetanus toxin peptide 'p2' (sequence 830-844) raised in HLA DR6 individuals preferentially express V beta 2 in the TCR. A p2-specific T cell line (60% V beta 2+) was used to compare peptide and superantigen [toxic shock syndrome toxin-1 (TSST-1)]-induced clonal anergy. Many experiments consistently revealed that the degree of 'tolerance' or 'clonal anergy' induced by peptide was greater than with the superantigen TSST-1. These results are of interest in a number of contexts. First they suggest that using superantigens or anti-V beta to delete the majority population of T cells may not be sufficient to diminish an autoimmune response. Secondly, the results indicate that induction of anergy of a large proportion of peptide-specific T cells does not lead to a suppressive bystander effect on the remaining responsive T cells. These results emphasize the need to define the dominant autoantigenic epitopes in human autoimmune diseases, since peptide based therapy such as the use of peptide analogues to induce anergy or a change in cytokine profile, is possibly more effective in controlling undesired immune responses than the use of non-antigen, TCR-directed approaches such as superantigens.

Definition of unique traits of human CD4-CD8- alpha beta T cells.

We have studied the nature of human CD4-CD8- (double negative) alpha beta T cells to determine whether they possess unique characteristics which could further differentiate them from conventional CD4+ or CD8+ (single positive) T cells. We observed that double negative TCR alpha beta+ T cells differ from single positive T cells in the following respects: (i) their T cell receptor (TCR) repertoire is different, as revealed by the analysis of 47 clones derived from three individuals and by analysis of peripheral blood lymphocytes (PBL) prior to in vitro manipulation; (ii) their in vivo CD3:TCR expression is lower before in vitro manipulation and expansion; (iii) their direct proliferative response to IL-3, which is not mediated by secondary release of other T cell growth factors. These characteristics have also been recently ascribed to murine double negative alpha beta T cells, which develop extrathymically and are considered to be a distinct T cell lineage. Our data suggest that, like their murine counterparts, human double negative alpha beta T cells may represent a distinct T cell lineage which might develop extrathymically.

Expression of the identical V beta gene in human T-cell clones does not confer the same pattern of responsiveness to bacterial enterotoxins.

Superantigens are the most potent T-cell mitogens so far described, and are believed to activate virtually all the T lymphocytes bearing the appropriate V beta segment in their T-cell receptor (TcR). In order to determine whether the expression of the identical V beta gene confers the same pattern of responsiveness to bacterial superantigens, we have established a panel of 20 untransformed human T-cell clones expressing the V beta 6.7a gene in their TcR. The V beta usage was defined by immunostaining, using the V beta 6.7a-specific monoclonal antibody (mAb) OT145, and confirmed by DNA sequencing of the beta-chain. Although all the clones analysed expressed the same V beta gene, they had disparate patterns of proliferation to challenge with a panel of bacterial enterotoxins. This study demonstrates that the mere expression of the same V beta region by T lymphocytes does not grant an indistinguishable responsiveness to bacterial superantigens. Thus other, as yet undefined, T-lymphocyte components play a key role in the process of T-cell activation induced by bacterial superantigens, influencing the effects mediated by exogenous superantigens on human T cells.

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.