HLA class II molecule-mediated signal transduction mechanism responsible for the expression of interleukin-1β and tumor necrosis factor-α genes induced by a staphylococcal superantigen

Superantigens including staphylococcal enterotoxins (SE) bind to major histocompatibility complex class II molecules and interact with T cells bearing particular vβ chains. SEB was shown to induce the expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α genes in human peripheral blood monocytes bearing HLA class II molecules. Monoclonal antibodies directed against HLA-DR and -DQ abolished the SEB-induced expression of both the EL-1β and TNF-α genes, suggesting that the HLA class II molecules mediated the gene expression. Therefore, we investigated the signal transduction mechanism responsible for the expression of IL-1β and TNF-α genes induced by binding of SEB to the HLA class II molecules. Three protein tyrosine kinase (PTK) inhibitors, genistein, herbimycin A., and tyrphostin, each of which has a different mechanism of action, strongly inhibited the expression of the monokine mRNA induced by SEB. Analyses of PTK activity revealed that SEB induced a rapid increase of membrane-associated PTK activity and this was blocked by tyrphostin. Furthermore, H-7 inhibited the expression of the monokine mRNA induced by SEB, suggesting the involvement of protein kinase C (PKC) in the signaling pathway. The involvement of PKC was confirmed by the observations that phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, induced the expression of the monokine mRNA and that SEB evoked the activation of membrane-associated PKC. Both activation of PKC and expression of the monokine mRNA induced by SEB appeared to be inhibited by tyrphostin, but those induced by PMA were not. Taken together, these findings indicate that both PTK and PKC play essential roles in HLA class II molecule-mediated signal transduction elicited by SEB and that PTK activation may precede PKC activation in the signaling pathway.     

Signal transduction by HLA-DR is mediated by tyrosine kinase(s) and regulated by CD45 in activated T cells.

Recently, it was shown that HLA class II molecules on B cells and activated human T cells can transmit signals involving tyrosine phosphorylation of specific proteins, activation of the inositol phospholipid pathway, and release of cytosolic free Ca2+(Ca2+)i. The regulation of class II induced signals is poorly understood, however, and it remained unknown whether these pathways were coupled or activated independently. Here we show that a specific inhibitor of protein tyrosine kinases (PTK), herbimycin, abrogated DR-induced elevation of (Ca2+)i in activated human T cells. Genistein, belonging to another family of PTK inhibitors, had weaker but significant inhibitory effects on DR-induced (Ca2+)i responses. CD45 crosslinking with DR almost completely abrogated DR-induced (Ca2+)i responses and profoundly changed the PTK profiles. In contrast, CD4 crosslinking with DR enhanced the (Ca2+)i responses, but the inhibitory effect of CD45 dominated over the enhancing effect of CD4. These data indicate that PTK activation is obligatory for DR-induced (Ca2+)i responses, suggesting a linkage between these pathways in class II signal transduction. This conclusion is consistent with our observation that in activated human T cells, class II signals are up regulated by CD4, which is associated with p56lck, and down regulated by CD45, which is a tyrosine phosphatase.     

MHC class II isotype-specific signaling complex on human B cells

The highly polymorphic human major histocompatibility complex (HLA) class II molecules are acknowledged as signaling receptors although their coupling to signaling pathways is not yet fully elucidated. In this study, we investigated how HLA class II can be coupled to protein tyrosine kinase (PTK) signaling pathway in B cells and whether there might be differences depending on HLA class II isotype. Using the human B cell line Ramos, we demonstrate that CD19 and CD20 are two HLA class II-associated receptors that couple HLA class II to PTK signaling pathway where CD20 appears to be amajor component of HLA class II-mediated activation of Src kinases. Both HLA-DR and HLA-DP co-immunoprecipitate tyrosine-phosphorylated proteins (p-Tyr) whereas only activation through HLA-DR increases the tyrosine phosphorylation of these proteins. Indeed, in contrast to HLA-DR, cross-linking HLA-DP induces neither tyrosine phosphorylation nor homotypic adhesion, and induces ERK1/2 activation. Differential association of these isotypes with CD20 appears to be one of the mechanisms underlying their differential signaling. We provide an experimental evidence for a mechanism by which HLA class II molecules can be coupled to PTK signaling pathway and, underscores their isotypes differential signaling. Further investigation of these mechanisms is likely to provide new insights into how isotype specific MHC class II signaling can contribute to the regulation of the immune response.     

Targeting of human cytotoxic T lymphocytes to MHC class II-expressing cells by staphylococcal enterotoxins.

The staphylococcal enterotoxins (SE) comprise a family of structurally related phage-encoded bacterial proteins, which are the most potent mitogens known for murine and human T lymphocytes. In this report we describe a novel cytotoxic mechanism, where SE directs human CD3+ T lymphocytes to mediate strong cytotoxicity against target cells of irrelevant nominal specificity. The SE-dependent cellular cytotoxicity (SDCC) occurred at picomolar concentrations of SE and involved the initial binding of the SE to the target cells and subsequent triggering of the cytotoxic T cells. SDCC was induced by SEA, SEB, SEC1 and SED, which indicates that this is a common property conserved among all SE. Certain antibodies to the HLA-DR molecule efficiently blocked SDCC. Major histocompatibility complex (MHC) class II+ RAJI cells and HLA-DR-transfected murine L cells were sensitive to SDCC, whereas the MHC class II- RJ.2.2.5 RAJI cell mutant and untransfected L cells were completely resistant to SDCC. These results demonstrate that the MHC class II antigen is the target molecule in SDCC. HLA-DR molecules acted as receptors for SE and the complex was recognized by T lymphocytes in a polyclonal fashion. SDCC was mediated by allospecific cytotoxic CD8+ T cells, by cloned CD8+ T cells and by fresh human peripheral blood mononuclear cells. The SDCC phenomenon provides a rapid, potent and specific mechanism for elimination of HLA-DR+ target cells. We suggest that SDCC is an important combat strategy, employed by the bacteria to avoid specific MHC class II antigen-dependent immune recognition, by inducing T-cell dependent autologous lysis of MHC class II-expressing cells.     

Presentation of superantigen by human T cell clones: a model of T-T cell interaction.

Superantigens (SAg) interact with T lymphocytes bearing particular V beta sequences as part of their T cell receptor (TcR). The interaction, however, requires the presence of major histocompatibility complex (MHC) class II molecules on antigen-presenting cell (APC). In peculiar circumstances, MHC class II+ T cell clones (TCC) have been shown to present peptides and selected antigens interacting with antigen-specific TCC in the absence of APC. In this report we studied the capacity of SAg to mediate a T-T cell interaction, investigating the TCC ability to present a panel of staphylococcal enteroxins (SE) independently of the presence of added APC. Upon exposure to a broad range of SE concentrations, MHC class II+ TCC showed an intense proliferative response even in the absence of professional APC. Diverse SE optimally stimulated responder TCC at different concentrations. The proliferation was inhibited by anti-DR monoclonal antibodies, both in the presence and in the absence of APC. The SE activation of TCC in the absence of APC induced the same series of phenotypic variations as that observed following the TCC stimulation with APC. Irradiated TCC efficiently presented membrane-bound SE to responder TCC as well as professional APC. These results show that a single cell of a given clone effectively presents the SE to other cells of the same clone, and provide evidence that SAg can efficiently mediate T-T cell interaction. In addition, the possibility also exists that one cell of the clone can actually undergo an auto-stimulation via SAg-mediated interactions between its own TcR and MHC class II molecule. It has recently been suggested that the V beta-selective depletion of T cells observed in acquired immunodeficiency syndrome (AIDS) patients might be a consequence of the interaction between a human immunodeficiency virus (HIV)-encoded SAg and T cells expressing a SAg complementary V beta. We suggest that the hypothesized HIV-encoded SAg might mediate T-T cell interactions that could play a relevant role in the V beta-selective depletion of T lymphocytes observed in HIV-infected patients.     

Role of the CD1a molecule in the superantigen-induced activation of MHC class II negative human thymocytes

Bacterial superantigens (Sag) are potent activators of T cells. This T-cell activation has been described as an MHC class II dependent phenomenon. We have observed that human thymocytes depleted of MHC class II positive cells are still able to proliferate in response to the staphylococcal enterotoxin A (SEA). This proliferation was clearly inhibited by the addition of monoclonal antibodies directed against the CD1a molecule. In contrast, monoclonal antibodies directed against the CD1b and CD1c molecules have no effect on the Sag-induced activation of the CD2 (+) MHC class II (-) thymocytes. We next examined the ability of the CD1a molecule to transmit transmembrane signals. Results obtained indicate that CD1a ligation on these thymocytes induced tyrosine phosphorylation of the p56(lck) tyrosine kinase. Signal transduction via CD1a is further confirmed by the observation of a significant intracellular calcium flux (Ca(i)(++)) in thymocytes following CD1a engagement. These data demonstrate that CD1a ligation induces a signal transduction pathway which has a potential role in the bacterial superantigen-induced activation of human CD2 (+) MHC class II (-) thymocytes.     

Different staphylococcal enterotoxins bind preferentially to distinct major histocompatibility complex class II isotypes

The stimulation of T cells by staphylococcal enterotoxins (SE) is strictly dependent on major histocompatibility complex (MHC) class II-bearing cells. The interaction between SE and MHC class II molecules was studied on the human B cell lymphoma Raji and its MHC class II-negative variant RJ 2.2.5. Affinity purification with SEA and SEB matrix allowed the isolation of HLA-DR-like molecules from detergent lysates of 125I surface-labeled Raji cells, but not from RJ 2.2.5 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also revealed preferences in the binding of other SE such as SED, SEE and toxic shock syndrome toxin 1 to DR-like molecules, SEC2 to HLA-DQ-like molecules and SEC3 to DR- and DQ-like molecules. Preadsorption of the different MHC class II MHC isotypes confirmed the preferential binding of SEA to DR and of SEC2 to DQ. The implications of these findings for the understanding of SE-induced T cell activation and the potency of SE as a tool in the study of MHC class II antigens are discussed.     

Human major histocompatibility complex class II-negative colon carcinoma cells present staphylococcal superantigens to cytotoxic T lymphocytes: evidence for a novel enterotoxin receptor.

The staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules on target cells and activate T cells expressing particular T cell receptor V beta sequences. In this report we demonstrate that SE bind to the MHC class II- SW620, Colo320DM and WiDr human colon carcinoma cell lines and direct cytotoxic T lymphocytes (CTL) to mediate strong target cell killing. Flow cytometry analysis, immunoprecipitation and Northern blotting experiments failed to demonstrate any surface expression of HLA-DR, HLA-DP and HLA-DQ isotypes on the SW620 colon carcinoma cell line, whereas abundant expression of these isotypes was seen on Raji cells, SEB and SEC1 were efficiently presented at picomolar concentration by the MHC class II- colon carcinoma cells and MHC class II+ Raji cells, whereas SEA and SED were preferentially presented on the MHC class II+ Raji cells. An anti-HLA-DR monoclonal antibody inhibited SEB-induced CTL targeting to Raji, but did not influence the killing of SW620 cells. Our data suggests the existence of functionally active SE-binding structures on human colon carcinoma cells which are distinct from the conventional MHC class II molecules. The possibility that these putative new SE receptors play a role in the enterotoxin action of SE must be considered.     

Tyrosine phosphorylation participates in peripheral T-cell activation and programmed cell death in vivo.

T-cell antigen receptor (TCR), which is not itself a protein tyrosine kinase (PTK), is thought to be associated with at least two SRC-like PTKs, P59fyn and ZAP-70. Activation of these PTKs is required for T-cell signal transduction. The aim of the present study was to determine the roles of PTKs in peripheral T-cell activation, induced by in vivo bacterial superantigen administration. We demonstrated that in vivo staphylococcal enterotoxin B (SEB) administration induced an enhanced tyrosine phosphorylation in peripheral spleen T cells undergoing a programmed cell death. In vitro immunecomplex kinase assay using antibody against P59fyn showed increased fyn kinase activity in SEB-stimulated spleen T cells. We examined the effect of PTK-specific inhibitors on DNA fragmentation and programmed cell death of V beta 8 positive T cells following in vitro culture of SEB-primed spleen T cells. Our results indicated that pretreatment of SEB-activated T cells with PTK inhibitors reduced DNA fragmentation and programmed cell death of V beta 8 positive T cells. These findings suggest that PTK plays an important role in activation and apoptosis of peripheral T cells induced by in vivo SEB administration.     

HLA class-II-mediated homotypic aggregation: involvement of a protein tyrosine kinase and protein kinase C.

Homotypic aggregation of B-lymphocytes, B-cell lines and class-II-positive T cells via HLA class II molecules was examined. Signaling via DR antigens induced rapid aggregation in a dose- and time-dependent manner, maximum and stable aggregation was induced within 20 minutes. On the contrary, rapid signaling via DP or DQ required prestimulation with either PMA or anti-sIg. Aggregation was temperature and energy dependent. [Ca2+] and [Mg2+] concentrations and an intact cytoskeleton were required while neither mRNA or protein synthesis were required. Furthermore, FACS analysis revealed that aggregation was not directly correlated with cell surface expression of HLA class II molecules. Our results demonstrate that aggregation was mediated through a protein tyrosine kinase (PTK)-dependent pathway that preceded activation of protein kinase C (PKC) and failure to generate either the PTK signal or the PKC signal prevented aggregation. The contribution of a tyrosine kinase was further demonstrated by the total inhibition of aggregation following treatment with an anti-CD45 mAb.     

The protein tyrosine kinase inhibitor herbimycin A, but not genistein, specifically inhibits signal transduction by the T cell antigen receptor.

Several lines of evidence implicate a regulatory tyrosine phosphorylation in the activation of phospholipase C (PLC) by the T cell antigen receptor (TCR). These include studies using inhibitors of protein tyrosine kinases (PTKs). In Jurkat T cells expressing the heterologous human muscarinic receptor (HM1), PLC activity can be induced by either the TCR or HM1. HM1 activates PLC via a guanine nucleotide binding protein. We have studied the selectivity of the effects of the PTK inhibitors, herbimycin A and genistein, in this system. The results indicate that these inhibitors have different mechanisms of action, and suggest that herbimycin A, but not genistein, is a specific inhibitor of PTKs in T cells. Herbimycin A markedly inhibited both the resting and induced levels of phosphotyrosine-containing proteins, including the gamma 1 isozyme of PLC and the zeta chain of the TCR, and prevented activation of PLC by anti-TCR mAb. Herbimycin A did not inhibit activation of PLC by HM1. Genistein had a much less pronounced effect than herbimycin A on the appearance of tyrosine phosphoproteins. Moreover, genistein inhibited activation of PLC by both the TCR and HM1, and inhibition was only partial. Genistein was cytotoxic and markedly inhibited protein synthesis in both Jurkat cells and human peripheral lymphocytes. Herbimycin A was not cytotoxic. These findings confirm the role of a regulatory tyrosine phosphorylation in activation of PLC by the TCR. Herbimycin A was a selective inhibitor of a subclass of PTKs in Jurkat cells. In contrast, inhibition of signal transduction and later events in T cells by genistein may be due to effects other than direct inhibition of PTK activity.     

Detection of low-frequency human antigen-specific CD4(+) T cells using MHC class II multimer bead sorting and immunoscope analysis

MHC class II tetramers are attractive tools to study antigen-specific CD4(+) T cell responses in various clinical situations in humans. HLA-DRA1*0101/DRB1*0401 MHC class II heterodimers were produced as empty molecules using the Drosophila melanogaster expression system. Peptide binding experiments revealed that these molecules could be loaded efficiently with appropriate MHC class II tumor epitopes. Interestingly, MHC class II tetramer staining was influenced by modifications in membrane lipid rafts, and could in itself induce activation changes of stained CD4(+) T cells at 37 degrees C. In order to increase the threshold of detection of poorly represented peripheral antigen-specific CD4(+) T cells, we combined cell sorting using MHC class II multimer beads together with TCR analysis using the immunoscope technology. This strategy greatly increased the sensitivity of detection of specific CD4(+) T cells to frequencies as low as 4 x 10(-6) among peripheral blood mononuclear cells. Such a combined approach may have promising applications in the immunomonitoring of patients under vaccination protocols to tightly follow induced antigen-specific CD4(+) T cells expressing previously identified TCR.     

The response of gamma delta T cells to Plasmodium falciparum is dependent on activated CD4+ T cells and the recognition of MHC class I molecules.

Peripheral blood gamma delta T cells from non-exposed individuals respond to antigens of the malaria parasite, Plasmodium falciparum, in vitro. This response, largely caused by T cells bearing the V gamma 9+ chain of the T-cell receptor, is stimulated by components of the parasite expressed on the schizont stage and released at schizont rupture. The response of V gamma 9+ T cells to parasite components is inhibited by antibodies to major histocompatibility complex (MHC) class I and class II. However, the inhibition by anti-MHC class II antibodies can be overcome by the addition of interleukin-2 (IL-2) to the cultures, suggesting that gamma delta T cells themselves do not recognize MHC class II molecules but require an MHC class II-dependent response taking place in the culture. In contrast, the inhibition by anti-class I antibodies cannot be reversed by addition of IL-2. Since an accompanying CD4+ T-cell response occurred in peripheral blood mononuclear cells cultured with P falciparum antigens, it was considered that these cells provide the cytokines necessary for the subsequent activation and expansion of V gamma 9+ T cells recognizing components of the parasite and MHC class I molecules. This was confirmed by reconstituting the response of enriched gamma delta T cells to P falciparum schizont extract by addition of purified CD4+ T cells.     

CD40 associates with the MHC class II molecules on human B cells

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross-linking of MHC class II or CD40 molecules leads to the association as well as the co-association of both molecules to the NP-49-insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.     

Control of self-reactive cytotoxic T lymphocytes expressing γδ T cell receptors by natural killer inhibitory receptors

The majority of peripheral blood γδ T cells in human adults expresses T cell receptors (TCR) with identical V regions (Vγ9 and Vδ2). These Vγ9Vδ2 T cells recognize the major histocompatibility complex (MHC) class I-deficient B cell line Daudi and broadly distributed nonpeptidic antigens present in bacteria and parasites. Here we show that unlike αβ or Vγ9^? γδ T cells, the majority of Vγ9Vδ2T cells harbor natural killer inhibitory receptors (KIR) (mainly CD94/NKG2A heterodimers), which are known to deliver inhibitory signals upon interaction with MHC class I molecules. Within Vγ9δ2 T cells, KIR were mainly expressed by clones exhibiting a strong lytic activity against Daudi cells. In stark contrast, almost all Vγ9Vδ2 T cell clones devoid of killing activity were KIR^?, thus suggesting a coordinate acquisition of KIR and cytotoxic activity within Vγ9Vδ2 T cells. In functional terms, KIR inhibited lysis of MHC class I-positive tumor B cell lines by Vγ9Vδ2 cytotoxic T lymphocytes (CTL) and raised their threshold of activation by microbial antigens presented by MHC class I-positive cells. Furthermore, masking KIR or MHC class I molecules revealed a TCR-dependent recognition by Vγ9Vδ2 CTL of ligands expressed by activated T lymphocytes, including the effector cells themselves. Taken together, these results suggest a general implication of Vγ9Vδ2 T cells in immune response regulation and a central role of KIR in the control of self-reactive γδ CTL.     

Induction of mouse syngeneic MLR by in vivo xenogeneic immunization with HLA-DR antigens

To study in mice the effects of in vivo xenogeneic immunization with human major histocompatibility complex (MHC) class II antigens, the animals were injected with HLA-DR antigens and their proliferative responses tested in vitro. The results showed that small amounts of HLA-DR proteins, acting as nominal antigens, were not only able to prime mice for a secondary in vitro xenogeneic mixed lymphocyte reaction but also induced a syngeneic mixed lymphocyte reaction. In contrast, allogeneic or syngeneic immunization of mice with soluble MHC class II molecules failed to stimulate an autoreactive response. The syngeneic mixed lymphocyte reaction was primarily directed against syngeneic MHC class II molecules since the murine T lymphocytes reacted against MHC class II-positive dendritic spleen cells and MHC class II-transfected mouse fibroblasts. A self-reactive T-cell line induced under these experimental conditions did not react in xenogeneic and allogeneic mixed lymphocyte reactions. However, these T lymphocytes proliferated when human peripheral blood lymphocytes of various haplotypes were presented in the context of syngeneic mouse antigen presenting cells.     

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8+ CD45RC- subset and lyse MHC class II+ tumor cells.

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II+) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II+ tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II+ human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50-250 micrograms SEA per animal with a peak in response 48-72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCR alpha beta+ CD4- CD8+ CD45RC- cell population. MHC class II- colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-gamma (IFN-gamma) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II+ colon carcinomas revealed a peak in sensitivity to lysis at 10-100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarian and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.     

Interactions of T lymphocytes with human vascular endothelial cells: role of endothelial cells surface antigens

We have studied the interactions of peripheral blood T lymphocytes with cultured human vascular endothelial cells, focusing upon endothelial cell surface antigens important for T cell recognition. Under standard culture conditions endothelial cells express class I but not class II major histocompatibility complex (MHC) antigens. However, class II antigens may be induced by activated T cells or T cell products, including the lymphokine immune interferon. Immune interferon concomitantly increases class I antigen expression and causes a change in cell shape. In addition to vascular endothelial cells, we have found that vascular smooth muscle cells and human dermal fibroblasts may also be induced by immune interferon to express class II antigens. All known human class II antigens are induced (i.e. HLA-DR, DC and SB) as is the associated invariant chain. Induced antigen expression in these cells is stable over several days, although mRNA levels decline rapidly upon withdrawal of interferon. Vascular and stromal cell class II antigens are functional, in that they can be recognized by cytolytic and helper T cell clones. Several non-MHC antigens are also involved in the recognition of endothelial and stromal cells by T cells. We propose a model for the role of inducible class II molecules on endothelium and stromal cells in vivo: The induction of class II MHC antigens on endothelial cells, locally mediated by activated T cells, enables endothelium to present an immunogenic cell surface structure, comprised of antigen plus self class II polymorphic determinants, which in turn, serves to recruit additional antigen-specific T cells from the circulation into the site of a developing cell mediated immune response. Class II molecules on stromal cells, also induced locally at the site of a developing response, confers immune accessory function on these cells and may serve to augment and sustain a T cell response.     

Anti-adhesive signals are mediated via major histocompatibility complex class II molecules in normal and neoplastic human B cells: Correlation with B cell differentiation stage

We show that major histocompatibility complex (MHC) class II molecules on B cells transmit signals which regulate adhesion in a negative manner. Engagement of MHC class II molecules with antibodies results in detachment of B cells previously bound to interferon-γ-activated human umbilical cord venous endothelial cells. This process depends on metabolic energy, active signaling and protein tyrosine kinase activity. The adhesion pathway influenced by this signaling event is neuraminidase sensitive. The anti-adhesive signaling program is activated in B cell lines with a mature phenotype, e.g. normal B cells from spleen and tonsil. In contrast, cell lines with a pre-B cell phenotype and normal B cells from peripheral blood are refractory to MHC class II-mediated regulation of adhesion. These results extend to neoplastic cells from patients with lymphopro-liferative diseases representing different stages of B cell maturation. These results suggest that MHC class II-mediated signals regulate B cell adhesion in a developmentally programmed fashion; this might have implications for clinical behavior of B cell malignancies.     

Superantigens initiate cognate CD4+ T cell/ B cell interactions leading to early activation and proliferation of B cells

Dimerization or even multimerization of various receptors is commonly required for signal transduction. We report here that clustering of major histocompatibility complex class II molecules in human B cells by biotinylated staphylococcal enterotoxin A (SEA) cross-linked with avidin induces an increase in the level of intracellular calcium. This response was abolished by prior treatment with protein tyrosine kinase (PTK) inhibitors, suggesting that SEA-triggered calcium mobilization in B cells is probably dependent on the activation of PTK. The implication of PTK in SEA-induced early B cell activation was then confirmed by demonstrating that cross-linked SEA induces a significant increase in the level of tyrosine phosphorylation in B cells. The requirement of biotinavidin cross-linking in SEA-induced calcium mobilization in B cells can be fulfilled by the addition CD4⁺ T cells, suggesting a role for CD4 molecules. Using the murine CD4^? T cell hybridoma 3DT, or its derivative 11B3 transfected with human CD4 that both express SEA-specific TCR, we confirmed the CD4 requirement for B cell calcium mobilization and that both specific TCR and CD4 molecules are required in early events of B cell activation induced by SEA. The role of CD4 in SEA-induced B cell proliferation was then investigated. SEA-stimulated B cells proliferated in the presence of CD4⁺ T cells, whereas no response was observed in the presence of CD8⁺ T cells. The addition of clone 11B3 CD4⁺ T cells failed to fulfill the requirement of CD4⁺ T cells in SEA-induced B cell proliferation, indicating the possible involvement of other CD4⁺ T cell surface molecules in this response. This issue is currently under investigation.