A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes.

MHC class II molecules have a crucial role in thymic selection and in generating Ag-specific T cell responses. There is extensive evidence for second messenger generation via MHC class II molecules, which can lead to apoptosis of B lymphocytes. We have examined HLA class II-mediated apoptosis in both normal and tumoral human B lymphocytes. Phosphatidylserine exposure and DNA fragmentation were observed in B cells within 24 h of stimulation via HLA class II. In marked comparison with Fas, the cell-permeable and irreversible caspase inhibitors zVAD-fmk and DEVD-fmk failed to inhibit HLA-DR-mediated apoptosis. No direct activation of caspase 3 was detected, and cleavage of pro-caspase 3 was not observed. Cleavage of poly(ADP-ribose) polymerase was detected via Fas but not via HLA class II. Although phosphatidylinositol-3-kinase has been implicated in HLA class I-mediated apoptosis, neither wortmannin nor LY294002 affected HLA class II-mediated apoptosis. CD95-sensitive cells were used to reveal that death occurred independently of CD95-CD95 ligand interactions. Overall, these data reveal a pathway of HLA-DR-mediated apoptosis that neither requires nor involves caspases. Moreover, it is phosphatidylinositol-3-kinase independent and Fas/CD95 independent. This pathway of HLA class II-mediated apoptosis could have an important role in the regulation of APC populations or in the control of malignant B lymphocyte proliferations.

HLA class II-induced translocation of PKC alpha and PKC beta II isoforms is abrogated following truncation of DR beta cytoplasmic domains.

Several studies have attempted to identify regions of the MHC class II molecule that participate in signal transduction. The importance of intact murine I-A cytoplasmic domains, either to tether the class II molecule to the cytoskeleton or to recruit signal-transducing proteins, is now well established. Recent data have also suggested that residues of the I-A beta transmembrane are involved in a second distinct signaling pathway. In the light of data suggesting that the second messengers activated on ligation of human and mouse class II molecules may differ, we set out to investigate whether the structural requirements for signaling for the human DR molecule are similar to those already described for the murine I-A molecule. We show that mutant DR class II molecules lacking 12 amino acids of the beta-chain cytoplasmic tail fail to translocate the protein kinase C alpha (PKC alpha) and PKC beta II isoenzymes following stimulation with an anti-class II mAb. In contrast, truncation of either or both cytoplasmic domains of the DR molecule has no effect on class II-induced tyrosine kinase activity. PKC translocation following class II stimulation has been observed in both human and murine B lymphocytes, whereas tyrosine kinase activation is present in human B lymphocytes but absent in resting murine B lymphocytes. Therefore, we conclude that the DR beta cytoplasmic tail is requisite for the principal signaling pathway initiated via MHC class II. These data suggest that the signaling pathways seen in resting vs primed murine B cells may also operate in human APCs.

HLA matching in unrelated donor bone marrow transplantation.

The availability of an HLA-matched sibling donor in only 30% to 35% of patients requiring allogeneic bone marrow transplantation (BMT) has led to the proposal of unrelated donors as an alternative source of bone marrow. The greater HLA incompatibility, which, although present, was undetected until recently in many unrelated donor BMT cases, has resulted in a higher rate of posttransplant complications and impaired acturial survival when compared with HLA-matched sibling BMT. Molecular HLA typing enables us to evaluate the impact of incompatibility at each locus in the outcome of unrelated donor BMT. The overall retrospective data would recommend that HLA-A, -B and -C allelic molecular matching should be implemented in addition to HLA-DR allelic matching. Further retrospective analysis is needed in order to assess which incompatibility or combinations are better tolerated than others. Only the definitive knowledge at the sequence level of the donor and the recipient HLA allelic diversity involved in controlling the allogeneic immune response will allow us to understand the precise biologic rationale of the graft-versus-host disease. Knowledge and control of the HLA incompatibilities should allow us to offset the detrimental effects of histoincompatibility while developing strategies to take advantage of the beneficial graft-versus-leukemia effect. Also the role of minor histocompatibility antigens remains largely unknown and will require careful evaluation before minor antigens can be used as a selection criterion in BMT. Carefully designed prospective studies will enable us to test the impact of each HLA locus. HLA typing and BMT represent a successful example of productive cooperation between basic and clinical sciences that should be pursued for the improvement of the clinical outcome of unrelated donor BMT.

Conformation of human leukocyte antigen class II molecules. Evidence for superdimers and empty molecules on human antigen presenting cells.

Subpopulations of human leukocyte antigen (HLA) class II molecules were studied in antigen presenting cells. We present evidence for double dimers or "superdimers" of HLA class II molecules that were stable in an SDS solution at room temperature but dissociated when heated to 50 degrees C into 60-kDa alphabeta heterodimers. Development of an immunofluorescence assay allowed us to quantify the expression of HLA antigens as reflected by the number of bound isotype-specific monoclonal antibodies per cell. The total expression of class II (DR, DQ, and DP) augmented 6-fold after a 36-h interferon-gamma (IFNgamma) treatment of freshly isolated monocytes. Next, we used a recombinant and fluorescein-conjugated form of the class II-associated invariant chain as a quantitative probe for empty peptide-binding sites. The fraction of empty class II molecules was 0.73-2.9% in resting monocytes but was reduced to 0. 12-0.5% of the total after IFNgamma treatment. The fraction of empty sites in B lymphocytes was 0.09-0.36%. The mean number of empty sites per cell were: 6.3 x 10(3) (monocytes), 7.2 x 10(3) (IFNgamma-activated monocytes), 5.2 x 10(2) (B lymphocytes), and 3.6 x 10(3) (Raji B cells). A minor population (4.3-7.4% of total cells), which expressed a much higher number of empty sites, was consistently present in all cell types studied.

Prokaryotic production of the human T cell receptor Vbeta2 chain and binding to toxic-shock syndrome toxin-1.

The human T-cell receptor Vbeta2-, D- and J-encoding domains were PCR-amplified from MOLT-4 total cDNA and subcloned in Escherichia coli. The V/D/J fragment was subsequently transferred to a prokaryotic expression vector in frame with a polyhistidine-encoding prosequence which enabled us to affinity-purify the fusion protein with IMAC (immobilized metal-ion affinity chromatography [correction of chromatorgraphy]). Since the recombinant (re-) human T-cell receptor Vbeta2 fusion protein (Vbeta2 sol) produced in E.coli was found to be insoluble, purification was carried out under denaturating conditions. The purified and renatured re-protein, Vbeta2 sol, was immunoreactive with an anti-Vbeta2 monoclonal antibody in an ELISA assay. The specificity of Vbeta2 sol was shown by its binding in vitro to the staphylococcal superantigen TSST-1, but not to the Staphylococcus aureus exotoxin-1 (SEA).

Mutually exclusive binding of peptide and invariant chain to major histocompatibility complex class II antigens.

The invariant chain is a membrane protein associated with the major histocompatibility complex class II antigens both intra- and extracellularly. The extracellular portion of the human invariant chain (Ii) was expressed in Escherichia coli as a fusion protein with a polyhistidine tail and purified by metal affinity chromatography. The recombinant Ii was used as a ligand to probe binding to the cell surface of Chinese hamster ovary cells stably transfected with human class II alpha and beta genes of the DR4 isotype. We show that recombinant Ii inhibits peptide loading on class II polypeptides and also the converse; the presence of peptide in the antigen groove prevents binding of fluorescein-conjugated Ii. Moreover, blocking of Ii binding by peptide did not require a transition of the class II dimers to an SDS-stable state. A monoclonal antibody, L243, known to bind to (or close to) the peptide pocket of the class II molecule likewise blocked Ii-fluorescein binding. Further, we investigated whether or not the Ii, a variety of bacterial superantigens or the CD4 molecule, have overlapping binding sites on the class II heterodimer. Of the class II ligands tested, reduced binding was detected for the Staphylococcus superantigen type SEB on cells precincubated with soluble Ii while the binding of the other ligands was either unchanged or marginally changed. These data clarify by a direct biochemical approach the binding characteristics of Ii in comparison with other class II ligands.

Bacterial superantigen signaling via HLA class II on human B lymphocytes.

Staphylococcus enterotoxins and toxic shock syndrome toxin-1 (TSST-1) are members of the family of staphylococcal exoproteins (SE) which binds specifically to HLA class II molecules and certain V beta T cell receptor phenotypes. These bacterial products have been termed "superantigens" due to their capacity to stimulate a greater proportion of T lymphocytes than peptide antigens without a requirement for antigen processing. The SE stimulate monocytes to secrete IL-1 and TNF-alpha and affect B lymphocyte proliferation in response to anti-human IgM and Ig production by PBMC. The current study concerns the transmission of signals in human B lymphocytes following fixation of TSST-1. Activation of both PLC and PKC are observed while intracellular calcium levels remain unchanged. Levels of HLA class II mRNA were increased suggesting that a pathway leading to activation was triggered. This study therefore identifies some of the second messengers involved after SE fixation on HLA class II molecules and suggests that the signals transmitted via class II antigens as well as those via the TCR may have a role in the physiological responses to bacterial superantigens.

Lymphocyte programmed cell death is mediated via HLA class II DR.

HLA class II molecules are constitutively expressed on human B lymphocytes and are induced on human T lymphocytes after activation, through which signal transduction via these molecules has been extensively described. We have observed cell death of as many as 60% after stimulation of lymphocytes via HLA class II DR molecules, but not via DP or DQ. Propidium iodide fluorescence, DNA fragmentation and morphology of the dead cells were examined. The reported cell death was very rapid, and independent of Fc receptors and of complement. A morphologically distinct sub-population of activated B cells was sensitive to HLA-DR mediated death, while resting B lymphocytes from the same donor did not die as a result of HLA class II mediated stimulation. Death via HLA-DR was distinguishable from necrosis. Cytoskeletal integrity, serine/threonine phosphatase activity and endonucleases were required for the pathway leading to HLA-DR mediated death. The 'ladder' pattern of DNA fragmentation which typically characterizes apoptosis was not observed, despite the observation of cell and nuclear shrinkage normally associated with apoptosis. These data suggest that HLA class II mediated death is a means of rapidly removing either T or B lymphocytes which have already served their role in the immune response, thereby avoiding the inflammatory responses associated with necrosis and concentrating the ligands for new TCR and/or CD4 interactions.

Modulation of lipopolysaccharide-induced cytokine gene expression in mouse bone marrow-derived macrophages by muramyl dipeptide.

Previous studies have shown that an i.v. injection of muramyl dipeptide (MDP) before a LPS challenge strongly potentiated serum TNF and IL-6 release in mice. Therefore the direct action of MDP was examined on TNF-producing cells, namely in macrophages stimulated or not by LPS. The level of TNF-alpha, IL-1 alpha, and IL-6 mRNA was determined in bone marrow-derived macrophages (BMM). A marked TNF-alpha mRNA accumulation was found between 1 and 6 h after stimulation with MDP or LPS. LPS-induced IL-1 alpha mRNA transcript was delayed (3 h) than those after MDP induction (1 h). Conversely, kinetic induction of the IL-6 mRNA transcript was delayed in MDP-treated BMM as compared with LPS-stimulated cells. MDP pretreatment of BMM for 3 h not only enhanced the total level of LPS-induced TNF-alpha, IL-1 alpha, and IL-6 mRNA (respectively 2.9-, 1.6-, and 2.4-fold increase), but it also delayed the kinetics of IL-1 alpha and IL-6 species accumulation. The enhancement induced by MDP pretreatment at the level of cytokine mRNA accumulation was correlated with an increase in LPS-induced TNF and IL-6 biologic activity production in supernatant fluids. In addition, in BMM from C3H/Hej mice MDP pretreatment enhanced the weak effect of LPS on TNF mRNA transcript accumulation and was required to produce LPS-induced TNF bioactivity. Our results suggest that MDP and LPS could act through distinct pathway(s) to induce cytokine gene expression. Moreover, the priming effect displayed by MDP could result in modulation of the LPS-induced cytokine gene expression at the transcriptional and/or post-transcriptional level.

New aspects of HLA: perspectives for rheumatoid arthritis.

The association of several HLA class II alleles with genetic susceptibility to rheumatoid arthritis (RA) suggests a model in which a three-dimensional conformation of the DR molecule represents the key disease susceptibility element. However, recently found new genes of the HLA-D region may contribute additionally to the susceptibility in particular of genes coding for proteases and transporter channels. Since expression of the class II molecule is a common feature of the autoimmunity process, regulatory polymorphisms of the class II genes may influence the antigen-presenting capacity of the cells. The role of the invariant chain, a 31 kD protein associated with the alpha beta complex, is emphasized in antigen presentation and may provide new insights into the pathogenesis and therapy of autoimmune rheumatoid arthritis.

Shear stress affects expression of major histocompatibility complex antigens on human endothelial cells.

Endothelialization of the inner face of a prosthesis appears to improve patency of small caliber arterial substitutes. The importance of understanding the factors that affect human endothelial cell behavior is highlighted by failure of vascular prosthetic grafts to endothelialize when implanted in man. In the present study, endothelial cells isolated from microvasculature are used for their ability to be easily selected from human adult fat, their proliferative capacity, and for their immunologic properties relevant to human pathology: allograft implantation, vessel injury or atherosclerosis. The system described provides a tool for assessing the individual roles of shear stress in modulating endothelial cell morphology and major histocompatibility complex (MHC) antigen expression. Using indirect immunofluorescent staining, initial results showed a homogenous increase of class I and appearance of class II expression after an exposure for 30 hr to physiologic arterial values. Significantly increased staining intensity was observed following exposure to supraphysiologic values. Moreover, precoating of substrate with fibronectin instead of poly-L-Lysine enhanced MHC straining intensity. Scanning electron microscopy (SEM) confirmed the activated morphology of stained cells. This provides a model to study involvement of MHC expression in endothelial cell activation under physical constraints. It may contribute to the development of biomaterial for implantation.

Glycosylation of human leukocyte locus A molecules is dependent on the cell type.

Peripheral blood monocytes and B cells were isolated from a normal donor, and a portion of the B cells was transformed by the Epstein-Barr virus (EBV). Human leukocyte locus A (HLA) class-I and class-II molecules were immunoprecipitated by specific monoclonal antibodies after cell labeling with [3H]mannose. Glycopeptides of HLA molecules were obtained by pronase digestion and were analysed by lectin-affinity chromatography. Complex structures were hydrazinolysed, and their sialic acid content was analysed by ion-exchange chromatography, whereas the high-mannose structures were separated by HPLC. In normal cells, class-I antigens bear principally fucosylated biantennary structures while HLA-DR class-II antigens bear bi-, tri- and tetra-antennary structures and high-mannose structures. HLA antigens are more sialylated on normal B cells than on normal monocytes. An EBV cell line had a very different pattern of HLA-antigen glycosylation when compared with the original B cells. In the transformed cells, the fractions containing biantennary structures are largely decreased. In contrast, an increase of the tri- and tetra-antennary structure fractions is noticed, particularly in class-II molecules, while both triantennary and high-mannose structures are increased in class-I molecules. Moreover, when compared to normal B cells, the complex structures of class-I antigens in the EBV-transformed B-cell line are undersialylated while they are oversialylated in the case of the class-II molecules.

Epitopic analysis of HLA DR molecule expression on alloreactive T-cell clones.

The expression of HLA-DR epitopes, recognized by a set of anti-DR MoAbs clustered into four groups according to immunochemical studies, was analyzed at the surface of in vivo-sensitized alloreactive T-cell clones derived from a rejected kidney allograft and on the autologous B lymphoblastoid cell line (BLCL). Although no clear-cut differences were noted between T cells themselves and T cells vs BLCL in the relative expression of most of the DR epitopes studied (D1.12, BT2/9, VI.15C, 135, 206, 141, BM50), relative and absolute expression of one DR epitope (BMMag8) was strikingly higher on autologous BLCL than on T-cell clones. Moreover, the absolute expression of DR epitopes was heterogeneous among T cells. Such differences could not be explained either by T-cell activation level (assessed by IL2 receptor or transferrin receptor expressions) or by differences in time kinetics expression. In other tests, anti-DR MoAbs were assayed for their ability to block cytotoxic activity of several T-cell clones directed against DR allospecificities. Each T-cell clone showed distinct inhibition patterns. By such analysis, it was possible to define several groups of epitopes not always identical to those defined by immunochemical studies. Finally, analysis of the ability of MoAb to specifically block cytotoxicity mediated by 2 anti-DRw8 T-cell clones at the target level allowed precise epitopic study of polymorphic DRw8 determinants recognized by these cells, in this case borne by a single DR alpha/beta heterodimer.

[Demonstration of new class I antigens in man]. / Mise en évidence de nouveaux antigènes de classe I chez l'homme.

The hypothesis of new class I antigens has been postulated in man, and several antigen systems have been proposed: HT (Gazit), TC (TCA, TCB) (Van Leeuwen). The present study describes a new class I antigenic marker system, expressed selectively on PHA-activated T lymphocytes and on lymphoblastoid B cell lines. These markers correlated at the cellular activation stage, have been called: human activation or HA markers. 7% of the sera from multiparous women present anti-HA antibodies. The definition of class I molecule (dimer 41K - 12K) has been established by a structural analysis of the molecule; the responsible gene, located on the 6th chromosome could be close to the HLA-A gene. The equivalence with the mouse Qa markers is postulated, but remains to be totally demonstrated.