CD95 ligand (CD95L) in normal human lymphoid tissues: a subset of plasma cells are prominent producers of CD95L.

CD95(Fas/APO-1)-ligand (CD95L) mediates apoptosis by trimerization of the CD95 receptor on the surface of sensitive cells. In vitro studies have shown CD95L expression mainly by activated T cells and suggested a role for CD95L in the regulation of immune responses. Little is known, however, about the cellular distribution of CD95L in situ in the normal human immune system. We investigated CD95L expression in tissue sections of the thymus, lymph node, spleen, tonsil, and gastrointestinal tract using in situ hybridization and two monoclonal antibodies. In all these organs, cells expressing CD95L message and protein were scarce and comprised scattered lymphocytes, rare nonlymphoid cells, and a subset of epithelioid endothelial cells. Surprisingly, a subset of plasma cells turned out to be the most prominent producers of CD95L, matching the reports on CD95L in myeloma cells. CD95L+ plasma cells were most numerous in the mucosa-associated lymphoid tissue. This also applied to acquired mucosa-associated lymphoid tissue in chronic gastritis in which CD95L+ plasma cells were found scattered in the lamina propria. Our data suggest that plasma cells as yet may be neglected modulators of immune responses.

Involvement of the CD95 (APO-1/Fas) receptor and ligand system in Helicobacter pylori-induced gastric epithelial apoptosis.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulceration, and gastric carcinoma. The potential role of CD95-mediated apoptosis was investigated in a panel of gastric biopsies obtained from patients with H. pylori-associated chronic gastritis (n = 29) and with noninfected normal mucosa (n = 10). Immunohistochemistry revealed increased CD95 receptor expression in epithelial and lamina propria cells in chronic gastritis. By in situ hybridization, CD95 ligand mRNA was absent or low in normal mucosa but expressed at high levels in lamina propria lymphocytes and, unexpectedly, in epithelial cells in chronic gastritis. Apoptotic cells were rare in normal mucosa but were observed regularly in chronic gastritis in close proximity to CD95 ligand mRNA expression throughout the epithelial and lamina propria cells. In a functional analysis gastric epithelial cell lines were incubated with supernatants of H. pylori. Treatment with the cytotoxic isolate H. pylori 60190 but not with the noncytotoxic isolate Tx30a upregulated CD95 in up to 50% of gastric epithelial cells and induced apoptosis in these cells. H. pylori-induced apoptosis was partially prevented by blocking CD95, demonstrating the functional role of the CD95 system. These findings suggest that H. pylori-associated chronic gastritis involves apoptosis of gastric epithelial cells by activation of the CD95 receptor and ligand system.

Hepatic failure and liver cell damage in acute Wilson's disease involve CD95 (APO-1/Fas) mediated apoptosis.

Wilson's disease can result in fulminant liver failure due to hepatic copper overload. The CD95 system mediates apoptosis and has been demonstrated to be involved in liver disease. In this study CD95 mediated apoptosis was investigated in patients with fulminant hepatic failure in the course of Wilson's disease and in an in vitro model of copper treated human hepatoma cells. In patients, hepatic expression of CD95 and CD95L mRNA and apoptosis were detected. Copper overload in vitro resulted in hepatocytic apoptosis which could be reduced with a neutralizing anti-CD95L antibody. Copper treatment of hepatocytes results in activation of the CD95 system and induction of apoptosis which is operative during the course of hepatic failure in acute Wilson's disease.

Surface expression of TRAIL/Apo-2 ligand in activated mouse T and B cells.

Like other members of the TNF family, TRAIL/Apo-2 ligand induces apoptosis in sensitive target cells in a caspase-dependent fashion. We recently found that TRAIL may be constitutively expressed on the surface of mouse and human tumor cells of T and B origin. To define the pattern of TRAIL expression in normal immune cells, freshly isolated splenocytes, Concanavalin A/IL-2-activated T cells and lipopolysaccharide-activated B cells were analyzed by surface staining with or without secondary stimulation. Activated, but not resting, CD3+ cells expressed TRAIL in an activation-dependent fashion. Conversely, freshly isolated B220+ cells displayed surface TRAIL and CD95L that were retained following activation. Restimulation with the protein kinase C activator phorbol 12-myristate 13-acetate and the calcium ionophore ionomycin or an agonistic anti-CD3 monoclonal antibody induced significant up-regulation of surface TRAIL and CD95L in CD3+, TCRalphabeta cells with CD4+ or CD8+ phenotype. Similarly to CD95L, TRAIL up-regulation was protein synthesis dependent and cyclosporin A sensitive. These results indicate that both TRAIL and CD95L are displayed on the cell surface of activated immune cells and may thus represent complementary effector pathways in the regulatory functions of T and B cells.

Differential regulation of TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte lineage.

TRAIL (APO-2 ligand) and CD95L (CD95/APO-1/Fas ligand) share the highest homology among the TNF family members and the ability to induce apoptosis. These similarities raise the issue of a potential functional redundancy between the two ligands. We have previously shown that CD95L-resistant cells may be sensitive to TRAIL, even though apoptosis induced by both ligands is blocked by caspase inhibitors. Here we investigated TRAIL protein expression in cells of T and B origin and compared its regulation of expression with that of CD95L. A rabbit antibody (Ab) to a peptide sequence in the extracellular region of TRAIL identified recombinant TRAIL (rTRAIL) produced by Sf9 cells as a protein of approximately 32-33 kDa and soluble rTRAIL as a 19-20-kDa protein. In human and mouse cells, the Ab identified a 33-34-kDa and an additional 19-20-kDa protein only in human cells. Both transformed cells of the T and B lymphocyte lineage were found to react with the anti-TRAIL Ab by immunoblot analysis and surface staining. The majority of the cells analyzed co-expressed TRAIL and CD95L. Two cell lines showed a mirror-pattern, one being TRAILhigh CD95Llow and the other TRAILlow CD95Lhigh, thus suggesting the existence of a cell type-specific regulation of expression of the two ligands. Differently from CD95L, surface TRAIL was not up-regulated by any of the metalloprotease inhibitors tested, independently of the cell type analyzed. Conversely, reactivity with the anti-TRAIL but not with the anti-CD95L Ab was enhanced by cysteine protease inhibitors. An in vitro cleavage assay showed that generation of soluble rTRAIL was dependent on the functional activity of cysteine proteases, as it was blocked by leupeptin and E64 but not by the metalloprotease inhibitor 1,10-phenanthroline. Thus, even though TRAIL and CD95L share structural and functional properties, they have unique properties as they differ in their regulatory pathways, i.e. cell-type-dependent expression and sensitivity to protease inhibitors.

Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells.

The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNF-family and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1beta-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO. These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

The CD95 system and the death of a lymphocyte.

Since the discovery of the CD95 receptor in 1989 and its ligand in 1993 as major initiators of apoptosis, extensive progress has been made in understanding their functions in programmed cell death. This review will summarize the cellular, molecular and biochemical requirements that play an important role in the CD95 system. Of importance in understanding the biology of CD95 are naturally occurring mutations in the mouse, the lpr and gld mutations, in which the expression of either the receptor or its ligand is disturbed. This review will discuss the roles of the CD95 receptor/ligand system in negative selection of thymocytes and in the peripheral T- and B-lymphocyte compartment. Emphasis is put on its role in cell-mediated cytotoxicity, transplant rejection and on its pathophysiological role in organ damage. Finally, recent data are presented to give an overview of CD95-associated signalling proteins.

Detection of active infection of Sf9 insect cells by recombinant baculoviruses.

Production of different recombinant proteins in baculovirus AcMNPV (BV)-infected cells may be facilitated by the availability of immunoassays to monitor active infection of Sf9 insect cells. To this end, two hybridomas secreting mouse monoclonal antibodies (mAbs) were established to different BV-related products. The proteins recognized by mAb SM22 and SM62 were easily detectable by immunoblotting and immunostaining in Sf9 cells infected with recombinant BV (rBV), but not in non-infected cells. Their production paralleled that of the recombinant proteins analyzed but was independent of the type of recombinant protein expressed. Thus, immunoassays with these mAbs allow: (1) daily monitoring of the infection occurring in small and large scale cultures of Sf9 cells using a defined rBV; (2) preliminary assessment of active rBV infection in the absence of a specific reagent for the recombinant protein and (3) single-reagent comparison of the infection achieved in Sf9 cells exposed to rBVs expressing different recombinant proteins.

Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells--a mechanism of immune evasion?

The CD95 (APO-1/Fas) system is an important mediator of T-cell cytotoxicity. We investigated this system in 22 hepatocellular carcinomas (HCCs) from patients. All HCCs had partially or completely lost the expression of the CD95 receptor constitutively expressed by normal liver cells and might thus evade CD95-mediated killing. We also considered a new mechanism of immune evasion, namely, the active destruction of T-lymphocytes by tumor cells expressing CD95 ligand (CD95L). CD95L messenger RNA and protein could be detected in the HCCs. In coculture experiments, HepG2 hepatoblastoma cells, expressing CD95L mRNA after treatment with cytostatic drugs, killed CD95+ Jurkat lymphocytes. Our data suggest that tumor cells can evade immune attack by down-regulation of the CD95 receptor and killing of lymphocytes through expression of CD95L.

Expression of biologically active mouse and human CD95/APO-1/Fas ligand in the baculovirus system.

CD95L (CD95/APO-1/Fas ligand) is a type II transmembrane glycoprotein that induces apoptosis in sensitive target cells. CD95L can be proteolytically cleaved from the membrane by a metalloprotease and occurs in a soluble form. Thus CD95L may act as a cytotoxic effector molecule at a distance from the producer cell. In order to develop an expression system yielding large quantities of CD95L, we expressed mouse and human CD95L tagged with a FLAG sequence in insect cells (Sf9) infected with recombinant baculovirus. CD95L expressed by Sf9 cells was detected with rabbit antibodies directed against the carboxy-terminal region of CD95L (which is highly conserved between mouse and human CD95L) and with an anti-FLAG monoclonal antibody. Immunoblotting showed that recombinant mouse and human CD95L expression was associated with the presence of 40 kDa and 32-33 kDa proteins. CD95L released into the supernatant of infected Sf9 cells specifically induced apoptosis in sensitive target cells, thus indicating that recombinant mouse and human CD95L were functional. The presence of the amino-terminal FLAG sequence did not modify this biological activity. Infection of Sf9 cells with recombinant baculoviruses may thus provide an efficient system for the expression of biologically active recombinant CD95L.

Clonal deletion of major histocompatibility complex class I-restricted CD4+CD8+ thymocytes in vitro is independent of the CD95 (APO-1/Fas) ligand.

The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca(2+)-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33-41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95+ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.

Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo.

Fas/APO-1 (CD95) is a cell surface receptor which mediates apoptosis when ligated by specific antibodies or by its recently cloned natural ligand, FasL. We have studied the cytotoxic potential of FasL in vivo using Fas/APO-1-expressing Yac-1 cells as targets. Supernatant harvested from Neuro-2a cells transfected with the murine FasL cDNA contains FasL and transduces a potent apoptotic signal to Yac-1 cells in vitro. Specificity of FasL-mediated cytotoxicity was confirmed by competition assays using soluble Fas or anti-Fas/APO-1 F(ab')2 fragments which specifically interfere with FasL-Fas/APO-1 interactions. Intraperitoneal injection of FasL-containing supernatant efficiently killed Yac-1 target cells which had been implanted in capsules into the peritoneal cavity of mice. Analysis of the target cells revealed DNA fragmentation and nuclear changes typical of apoptosis. As previously shown, intraperitoneal injection of anti-Fas/APO-1 antibodies caused liver failure (Ogasawara, J., Watanabe, F.R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S., Nature 1993. 364:806) and was observed at doses which did not reduce Yac-1 cell viability. In contrast, FasL did not induce histopathology in the liver when applied intraperitoneally at doses cytotoxic for Yac-1 cells. However, intravenous administration of FasL induced lethal liver hemorrhages and hepatocyte apoptosis. Thus, locally applied FasL kills tumor cells very efficiently without systemic toxicity and may therefore represent a candidate for local tumor treatment.

Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases.

APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca2(+)-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40 kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a molecular mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn2+, but not Ca2+, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn2(+)-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO-1L.

The APO-1/Fas (CD95) receptor is expressed in homozygous MRL/lpr mice.

APO-1/Fas (CD95) is a transmembrane receptor that transduces apoptotic signals within various cells including T and B cells. The APO-1 gene was found to be defective in lpr mice. In these mice insertion of a retrotransposon gives rise to transcription of an abnormal mRNA and only a fraction of wild-type APO-1 mRNA. It is not clear if lpr mice still express wild-type APO-1 protein. To address this question, we prepared rabbit anti-APO-1 antibodies (Ab) with a peptide representing the extracellular sequence corresponding to residues 5-23 of APO-1. The rabbit Ab reacted with thymocytes from different mouse strains and the extent of binding was correlated with the two known APO-1 alleles. In addition, the Ab reacted with mouse cell lines expressing mouse APO-1 mRNA but not with human cell lines. Binding of the Ab to MRL and BALB/c thymocytes was completely blocked by the immunizing peptide. Immunofluorescence analysis of MRL/lpr thymocytes showed that they still express APO-1 protein at approximately one tenth of the wild-type expression level on their surface. In addition, in lpr as in wild-type mice we found a decrease of APO-1 expression in the more mature thymic compartment. Western blot analysis of whole cell lysates from lpr and wild-type thymocytes showed that the Ab recognized APO-1 in both cell types. Approximately 50% of CD3+ splenocytes and 80% of in vitro activated CD3+ cells from wild-type mice reacted with the Ab, but to a lower extent than thymocytes. The same differential reactivity was found in lpr CD3+ splenocytes. lpr T cells, however, showed a substantially lower level of APO-1. Thus, the differential expression of APO-1 on thymic versus peripheral lpr T cells might influence their sensitivity towards APO-1-mediated apoptosis.

Preferential selection of a subset of anti-HLA-DR monoclonal antibodies by a syngeneic antiidiotypic monoclonal antibody.

In spite of the potential role of antiidiotypic (anti-id) antibodies in the immune response to mismatched HLA antigens and in the outcome of renal allografts, the idiotypic cascade in the HLA system has been characterized to a limited extent. For instance, it is not known whether anti-id antibodies defining idiotopes coexpressed in the combining site of the original anti-HLA antibody selectively stimulate distinct subsets of B cell clones. Since this information contributes to our understanding of the role of anti-id antibodies in the generation of diversity in anti-HLA immune response, we have determined whether a preferential recognition of the immunizing anti-id mAb is displayed by the two subsets of anti-HLA-DR mAb elicited with anti-id mAb F5-444 and F5-830. The latter two mAb recognize idiotopes coexpressed in the antigen-combining site of the immunizing anti-HLA-DR1,4,w14,w8,9 mAb AC1.59. All the anti-HLA-DR mAb elicited with mAb F5-830 displayed a higher functional affinity for the immunizing anti-id mAb than the anti-HLA-DR mAb elicited with mAb F5-444. This finding reflects the higher reactivity of the subset of anti-HLA-DR mAb elicited with mAb F5-830 with the light chain of the immunizing mAb. The present study, therefore, shows for the first time that distinct anti-id mAb recognizing idiotopes coexpressed in the combining site of anti-HLA-DR mAb stimulate different subsets of idiotope positive B cell clones in a nonrandom fashion. This preferential selection by anti-id mAb affects the characteristics of the immune response, as the two subsets of anti-HLA-DR mAb display differences in their fine specificity. These results are consistent with the possibility that anti-id antibodies may play a role in the changes in the specificity of anti-HLA antibodies that may occur in the course of an immune response to mismatched HLA alloantigens.

Serological and molecular characterization of mouse anti-idiotypic monoclonal antibodies elicited with the syngeneic anti-HLA-A2,28 monoclonal antibody CR11-351.

The molecular basis of the differential specificity of seven mouse anti-id mAb elicited with the syngeneic anti-HLA-A2,28 mAb CR11-351 was analyzed by comparing their specificity with their heavy and light chain variable region sequences. Of the six mAb recognizing idiotopes within the antigen combining site of mAb CR11-351, mAb T10-352, T10-440 and T10-505 recognize the same (or spatially close) idiotope(s) since they cross-inhibit each other in their binding to mAb CR11-351 and elicit syngeneic anti-anti-id antibodies with similar specificity. On the other hand, mAb T10-421, T10-649 and T10-938 appear to recognize spatially close but distinct idiotopes since they cross-inhibit each other, but elicit anti-anti-id antibodies which inhibit only the binding of the respective immunizing anti-id mAb to mAb CR11-351. mAb T8-203 is the only anti-id mAb which recognizes an idiotope outside the antigen combining site of mAb CR11-351 since it does not inhibit the binding of the latter to target cells and binds to mAb CR11-351 coated B lymphoid cells. In addition, mAb T8-203 defines an idiotope which is shared by seven anti-HLA mAb, while the remaining six anti-id mAb recognize idiotopes which are not detectable on the panel of anti-HLA mAb. mAb T10-352, T10-440 and T10-505 are highly homologous in their VH and VL regions and in their V(D)J junctions suggesting that they may be clonally related. On the other hand, mAb T8-203, T10-649 and T10-938 share some degree of homology in their VH region as all of them use J558 VH genes but differ considerably in their VL regions. Finally, mAb T10-421 is the most unrelated mAb as it utilizes VH, D, JH, VK and JK gene segments different from those of all the other anti-id mAb. These findings indicate that in the HLA-A antigenic system defined by mAb CR11-351 the main mechanism underlying the differential target specificity of syngeneic anti-id mAb is the combinatorial diversity together with the differential pairing of heavy and light chains.

Heterogeneity in the anti-HLA-DR immune response elicited by the antiidiotypic monoclonal antibody F5-444. Analysis at the clonal level.

A total of 630 hybridomas were generated from a BALB/c mouse immunized with the anti-id mAb F5-444, which binds an idiotope within the antigen-combining site of mAb AC1.59. The latter recognizes a determinant shared by HLA-DR1, DRw8, DR9 antigens and subtypes of HLA-DR4 and DR6 that is poorly expressed by HLA-DRw16 and DRw17 antigens. Eight anti-anti-id mAb were shown with serological and immunochemical assays to react with HLA-DR antigens. Detailed analysis of these anti-HLA-DR anti-anti-id mAb showed that they differ from the original anti-HLA-DR mAb AC1.59 and among themselves in either isotype, fine specificity, extent of reactivity with the nominal antigen, differential reactivity with soluble or cell-bound antigen, and/or idiotype profile. These observations emphasize the need to characterize a panel of antigen-binding anti-anti-id mAb in order to evaluate the degree of similarity existing between antigen-binding antibodies induced by an anti-id mAb and the original antigen-binding mAb.

Diversity of anti-HLA-DR antibodies elicited by distinct anti-idiotypic monoclonal antibodies recognizing idiotopes co-expressed by the immunizing monoclonal antibody.

Analysis at the clonal level of the idiotypic network has identified differences in fine specificity between antigen-binding anti-anti-idiotypic (anti-anti-id) monoclonal antibody (mAb) and the original mAb as well as among antigen-binding anti-anti-idiotypic (anti-id) mAb. However, the diversity of humoral immune responses elicited by anti-id mAb recognizing idiotopes co-expressed on the immunizing mAb has not been analysed. Since this information may contribute to our understanding of the role of anti-id antibodies in the generation of diversity in the course of an immune response, we have compared the fine specificity and idiotype profile of two subsets of anti-HLA-DR mAb generated with the anti-id mAb F5-444 and F5-830. The latter mAb recognize idiotopes co-expressed in the antigen-combining site of the immunizing anti-HLA-DR1,4,w14,w8,9 mAb AC1.59. These investigations showed that: (1) the two subsets of anti-HLA-DR mAb overlap only partially in their reactivity patterns with HLA-DR+ cells; (2) both subsets of anti-HLA-DR mAb recognize spatially close epitopes; (3) each subset of anti-HLA-DR mAb has unique reactivity patterns with soluble HLA-DRw16 and DRw17 antigens; and (4) each subset of anti-anti-id mAb displays a distinct idiotype profile. The subtle differences in the fine specificity and idiotype profile of the two subsets of anti-HLA-DR mAb suggest that anti-id antibodies may play a role in the generation of diversity in the course of a humoral immune response.

Molecular analysis of anti-idiotypic monoclonal antibodies in the HLA-DR antigenic system.

The structural organization of anti-idiotypic (id) antibodies has been investigated mostly in haptenic systems. No information is available about the structural characteristics of anti-id antibodies in major histocompatibility complex (MHC) antigenic systems, although these data may contribute to our understanding of the molecular basis of their functional role in the immune response. Therefore, we have determined the nucleotide and derived amino acid sequence of the VH and VL regions of the anti-id monoclonal antibodies (mAb) F5-444, F5-830, F5-963, F5-1126, F5-1336 and F5-1419, which had been elicited with the syngeneic anti-HLA-DR1, 4, w14, w8, 9 mAb AC1.59. The six anti-id mAb are heterogenous in their VH and VL region gene usage. This structural heterogeneity is not correlated with their target specificity and with their ability to elicit anti-HLA-DR antibodies. The latter characteristic is markedly influenced by a limited number of amino acid substitutions, since mAb F5-444, which induces anti-HLA-DR antibodies, differs only in two residues in complementarity-determining regions and in five residues in framework regions from mAb F5-1126, which does not induce anti-HLA-DR antibodies. The heterogeneity in VH and VL region gene usage by the six anti-id mAb in the HLA-DR system is at variance with the restricted VH and VL region gene usage by syngeneic anti-id mAb in several haptenic systems. Furthermore, at variance with haptenic systems, the primary structure of the D segments of the anti-id mAb is not correlated with their ability to induce anti-HLA-DR antibodies. On the other hand, the frequency of D-D fusion events underlying the derivation of the D segments of the six anti-id mAb in the HLA-DR system and their average length are similar to those found in anti-id mAb in haptenic systems. In addition, like in the latter systems, somatic mutations appear to contribute to the generation of diversity of anti-id mAb in the HLA-DR system.