Alteration of CD1 expression in multiple sclerosis.

Studies of multiple sclerosis (MS) have concentrated mainly on antigen presentation of peptides derived from the myelin sheath, while the implication of lipid antigen has been less explored in this pathology. As the extracellular environment regulates expression of the lipid antigen-presenting molecule CD1, we have examined whether sera from patients alters CD1 surface expression in monocyte-derived dendritic cells. We have shown that: (i) CD1 group 1 proteins were highly expressed in the presence of MS sera; (ii) sera from MS patients differentially regulated CD1 group 1 versus CD1 group 2 molecular expression; and (iii) CD1 was expressed strongly in monocytes from MS patients under immunosuppressive treatment. Overall, these results reveal that CD1 expression is modified in MS and provide novel information on the regulation of lipid antigen presentation in myeloid cells.

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.

CD38 expressed on human monocytes: a coaccessory molecule in the superantigen-induced proliferation.

This work analyzes the hypothesis that human CD38 may cooperate with MHC Class II by acting as coreceptor in a superantigen-induced activation. The initial evidence is that CD38 ligation by specific monoclonal antibodies inhibits superantigen-induced T lymphocyte proliferation. Inhibitory effects become apparent after engagement of CD38 expressed by monocytes, whereas ligation of CD38 expressed by T lymphocytes does not apparently affect activation. The inhibition requires a cell-to-cell interaction, followed by the relevant transmembrane signaling that is reproduced by CD38 ligation. Indeed, CD38 ligation on monocytes induces tyrosine phosphorylation of several intracellular proteins including the protooncogene product c-cbl and the fgr and hck tyrosine kinases. The receptorial nature of the CD38-mediated events is confirmed by the observation of an intracellular calcium flux in monocytes secondary to CD38 ligation. These effects are additive with the similar events elicited by MHC Class II ligation, a likely indication that CD38 and MHC Class II share a common activation pathway. This conclusion is strengthened by results of comodulation experiments, indicating that CD38 and MHC Class II display lateral associations on monocytes. These results attribute to CD38 expressed by monocytes a role in the transduction of signal(s) involved in superantigen-induced activation, operating in synergy with MHC Class II.

Human CD1a molecule expressed on monocytes plays an accessory role in the superantigen-induced activation of T lymphocytes.

The CD1 molecules exhibit characteristics of the MHC class I and class II molecules. They are expressed on cortical thymocytes and, similarly to MHC class II molecules, on antigen-presenting cells. In the present study, we investigated the role of the CD1 molecules in the T-cell response to bacterial superantigens. Indeed, we have observed that CD1 molecules could be detected on the CD14-positive population of some healthy donors (14% of donors tested). The CD1 expression on monocytes is correlated with an activation state of the donors as demonstrated by the increased expression of the CD25, CD38, CD45R0, and MHC class II molecules on their lymphocytes. On these donors, CD1a mAbs induced a clear inhibition (65%) of lymphocyte proliferation induced by either staphylococcal enterotoxin A or toxic shock syndrome toxin-1, whereas this proliferation was constantly unaffected by the addition of mAbs directed against CD1b or CD1c. Moreover, an intracellular calcium flux was induced in monocytes following CD1a engagement, and this calcium flux was partially inhibited by preincubation of these cells with the superantigen. These results attribute to the CD1a molecule expressed by monocytes a role in the transduction of signal(s) involved in superantigen-induced activation.

Effects of superantigenic stimulation on the cord blood alphabeta T cell repertoire.

Umbilical cord blood (CB) constitutes a promising alternative to bone marrow for allogeneic transplantation and is most remarkable for the reduced severity of GVHD compared with bone marrow. We have shown that although naive the TCR beta-chain repertoire appears fully constituted at birth in terms of mean size of the complementarity-determining region 3 (CDR3) and of the usage of V and J gene segments. Its ability to respond to exogenous stimuli was tested with staphylococcal superantigens TSST-1 and SEA (toxin at 1 ng/ml for 4 days). The amount of TCR transcripts was quantified and the percentage of representation of each BV family was calculated. TSST-1 induced BV2 expansion in both adult and CB samples. SEA activation gave a more variable pattern among individuals (adults n = 6; CB n = 6). BV6, BV18, BV22 and BV24 were the most frequently expanded families. We did not observe notable differences in either the modification of the TCRBV repertoire or the kinetics of the response to SEA superantigen between adults and newborns. These data suggest that although naive, CB lymphocytes are as equally capable as adult lymphocytes of responding to superantigen stimulation.

The umbilical cord blood alphabeta T-cell repertoire: characteristics of a polyclonal and naive but completely formed repertoire.

Umbilical cord blood (CB) constitutes a promising alternative to bone marrow for allogeneic transplantation and is increasingly used because of the reduced severity of graft-versus-host disease after CB transplantation. We have compared the T-cell receptor beta chain (TCRB) diversity of CB lymphocytes with that of adult lymphocytes by analyzing the complementarity determining region 3 (CDR3) size heterogeneity. In marked contrast to adult samples, we observed bell-shaped profiles in all of the 22 functional beta-chain variable (BV) subfamilies that reflect the lack of prior antigenic stimulation in CB samples. However, the mean CDR3 size and BV usage were comparable between CB and adult samples. BJ2 (65%) segments were used preferentially to BJ1 (35%), especially BJ2S7, BJ2S5, BJ2S3, and BJ2S1, in both CB and in adult lymphocytes. We therefore conclude that although naive as reflected by the heterogeneity of the CDR3 size, the TCRBV repertoire appears fully constituted at birth. The ability to expand TCRB subfamilies was confirmed by stimulation with staphylococcal superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. This study provides the basis for future analysis of the T-cell repertoire reconstitution following umbilical CB transplantation.

Antibodies to the CD5 molecule in normal human immunoglobulins for therapeutic use (intravenous immunoglobulins, IVIg).

IVIg are increasingly used for the treatment of autoimmune diseases. In the present study, we show that IVIg contain antibodies directed against CD5, a cell surface molecule of T cells which is also a marker of the autoantibody-producing CD20+ ('B-1') subset of B lymphocytes. Antibodies to the CD5 molecule were demonstrated in IVIg by the ability of therapeutic preparations of IVIg to inhibit the binding of labelled CD5 MoAb to the CD5-expressing human T cell line H9. Preincubation of H9 cells with IVIg or with F(ab')2 fragments prepared from IVIg resulted in dose-dependent inhibition of the binding of CD5 antibody. The presence in IVIg of antibodies to the CD5 molecule was further confirmed by the binding of IVIg to mouse L cells that expressed human CD5 molecules following a stable transfection with CD5 cDNA. Human CD5 antibodies in IVIg provide therapeutic immunoglobulin preparations with the potential of modulating T cell functions through CD5, and of regulating the expression of B cell subsets expressing CD5. This may have implications for the treatment of autoimmune diseases.

The CD39 molecule defines distinct cytotoxic subsets within alloactivated human CD8-positive cells.

Lymphocyte activation induces or increases the expression of several surface structures, none of which is characteristic of an activated cell subset. In particular, structures such as CD45RO, CD25, CD26, CD49b, CD54, CD71 are expressed by the vast majority of lymphocytes at various times following in vitro activation. CD39 molecules were originally identified on activated B lymphocytes and have recently been described on activated T cell clones. In the present report, we have characterized phenotypically and functionally defined cell subsets generated during an in vitro allostimulation. Results indicated that the percentage of CD39+ cells reached a maximum at day 6 and remained stable thereafter. We demonstrate that CD39 expression allows the identification within the allosensitized CD8+ cytotoxic cells of distinct subsets of cells mediating allo cytotoxic T lymphocyte or natural killer (NK)-like reactivity. More precisely, CD8+CD39+ alloactivated cells mainly mediate specific killer activity, whereas CD8+CD39- alloactivated cells predominantly exhibit NK-like reactivity. Further, we show a high functional correlation associated with the lack of CD39 expression on NK-like alloactivated CD8+ cells, while there is no association with CD56 or CD57 NK-associated structures.

The urge for an AIDS vaccine: perspectives from a developing country.

AIDS is inexorably involving all parts of the country and all strata of society, with 10% of the urban and 3% of the rural population infected with HIV. It is increasingly a disease of women and children. The major cofactors for transmission are also sexually transmitted. For most developing countries, in spite of all education efforts, the "silent epidemic" of AIDS continues. AIDS is known but not understood; counselling modifies behavior in only 10-20% of at-risk persons. Under optimal conditions, HIV discordant females have seroconversion rates of 4.7% per year and pregnancy rates of 10.4% per year. The recent political unrest in Zaire and Haiti will further enhance the spread of AIDS in these countries. Despite these difficult periods, the work can and must continue. After all, during our 10th year of collaboration with a Haitian private research group, the Haitian government and Cornell University, Haiti has known seven different political rulers. Finally, I want to make a pledge on behalf of the millions of people who face a certain death from HIV infection and AIDS and who will never make the front page of any newspaper. For these people, you can make a difference. You must give us the tools to carry on this fight. The clinical trials must be done where they are most needed: the developing countries. Vaccines represent the only viable alternative despite the recognized obstacles of viral heterogeneity, immunogenicity, and delivery.

The distribution of the CDW52 molecule on blood cells and characterization of its involvement in T cell activation.

The O97 mouse mAb was used to define, together with the CAMPATH-1 mAb series, the CDw52 in the course of the Fourth International Workshop on Human Leucocyte Differentiation Antigens. O97 and CAMPATH-1M produce full competitive inhibition and react with an epitope dependent on O-linked sugar residues. The two mAb, however, display significant differences in reactivity with leukocyte populations--in fact the reactivity of O97, which also exerts a powerful cytotoxic effect with human C', is similar to mAb from the CAM-PATH-1 series having the broadest one. Noteworthy, O97 spares PBL, NK, and LAK precursors, permitting an easy purification of these cells; O97 is able to kill long-term colony-forming cells in bone marrow culture and characteristically reacts, in contrast to CAMPATH-1M, with erythrocytes. Western blotting has revealed a strikingly different molecular size on red cells, since CDw52 mAb revealed a broad band ranging from 6-16 kDa, instead of the 21-28 kDa revealed from leukocyte extracts. In agreement with immunofluorescence data, O97 revealed abundant material from red cells in Western blotting, while reactivity of CAMPATH-1M was very faint. Overall, these results show that distinct molecular forms of the CDw52 molecules are present on different blood cells. We have also characterized an activation signal that can be delivered to T cells via the CDw52 molecule by CAMPATH-1M but not by O97. This is an accessory signal that can complement a primary activation signal delivered via the CD2 pathway but not via the CD3-TcR pathway. This is similar to the effects obtained with the CD45R (2H4) mAb, 2H4 and CAMPATH-1M mAb having additive effects on T cell activation via CD2.

Isoforms of the E2 molecule: D44 monoclonal antibody defines an epitope on E2 and reacts differentially with T cell subsets.

Human T cell rosetting with erythrocytes is clearly dependent on the CD2-CD58 interaction. We have previously demonstrated that other T cell molecules are involved in the rosette phenomenon, including the E2 molecule, a 32-kDa transmembrane glycoprotein. In this report we show that the D44 monoclonal antibody (mAb), previously shown to subdivide T cells into subpopulations with distinct functional repertoires and to identify 70% of lymphocytes from bronchoalveolar lavage from HIV+ patients, defined a new epitope on the E2 molecule. This was illustrated by the reactivity of the D44 mAb in Western blot experiments performed with the immunoaffinity purified E2 molecule. Moreover, double-labeling experiments showed that the E2 molecule exhibited varying epitopes when expressed in different cell types. The D44 and 12E7 epitopes were restricted to distinct subpopulations of T cells and, more importantly, the D44 expression was limited to the CD29+ population including the memory subset. The O662 and L129 epitopes are present on all T cells. Thus, the E2 molecule has both common and variable epitopes in its extracellular domain, and only the common epitopes seem to be involved in T cell adhesion processes.

Identification of CD3 Associated T Cell Receptor as a Diagnostic Tool in T Cell Acute Lymphoblastic Lymphoma or Leukemia.

By using several monoclonal antibodies (mAb) reacting either with the constant or variable regions of the T cell receptors (TcR) αß and γδ or various CD molecules, differences between two clinically related entities e.g. T cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LL) have been demonstrated. We studied a panel of fifteen T-ALL and fifteen T-LL because of their cell surface expression of the CD3-TcR molecules. The results indicated that TcR γδ is more frequently expressed in T-ALL (10 out of the 15 patients tested) than TcR αß. This is in contrast to the results obtained with T-LL where the vast majority showed TcR αß (13 out of the 15 patients). We discuss the significance of these findings which may imply that the leukemic cells are of a different origin in these two diseases. In addition analysis of TcR variable regions expressed by the leukemic blasts showed that in most cases they had rearranged functional Vδ1 gene to Jδ1 or Jδ2 segments (8 out of 11 patients) whereas in a unique case Vδ2 gene segment was evident. Taken together these results and those showing that T-ALL cells coexpress the CD1a, b and c molecules strengthen the possibility that despite the fact that these leukemic cells express the CD3-TcR complex at their surface their normal counterparts are not found in peripheral blood.

Modulation of lymphokine release and cytolytic activities by activating peripheral blood lymphocytes via CD2.

We had previously shown that the signal of activation delivered via CD2 varies according to the mitogenic pair of CD2 mAb used. We had selected two typical mAb pairs, D66 + T11(1) and GT2 + T11(1), the former delivering the "richest" signal, the latter the poorest. Here we analyzed the cytolytic activities generated within PBL-stimulated by these two pairs. When purified CD2+,3- cells were cultured with either one of these two pairs, no significant lymphokine-activated killer (LAK) activity--namely the activity exerted on NK-resistant malignant cell lines or fresh tumor cells--was detected, thereby demonstrating the inability of CD2 mAb pairs to directly trigger the LAK precursors. By contrast, when purified CD2+,3+ cells were cultured, only D66 + T11(1) was able to trigger a potent CTL activity, as judged by targeting their activity, at the effector phase, with a bridging CD3 mAb on a FcR+ target cell or by using heteroaggregates on FcR- malignant cells. When whole PBL were used, a similar and moderate LAK activity was generated after culture with either one of the 2 CD2 mAb pairs. This, in fact, masked quite different events. The amounts of endogeneous IL-2 released in PBL cultures with GT2 + T11(1) was rather low, although it was sufficiently high in PBL cultures with D66 + T11(1) to generate a potent LAK activity. Yet, PBL stimulated with D66 + T11(1) released concomitantly a high amount of IL-4 which inhibited the development of the LAK activity, as demonstrated by unmasking this activity with an anti-IL4 antiserum and which did not inhibit the T CTL activity; this IL-4 secretion was not seen with GT2 + T11(1). Therefore, stimulation by these two typical CD2 mAb pairs induce a striking different pattern of IL synthesis.

The E2 antigen, a 32 kd glycoprotein involved in T-cell adhesion processes, is the MIC2 gene product.

E2 is a 32 kd human T-cell surface glycoprotein involved in spontaneous rosette formation with erythrocytes. A 1.11 kb cDNA was isolated from a lambda gt11 expression library by screening with monoclonal antibodies directed against E2. The primary structure of E2, deduced from the nucleotide sequence of its gene, comprises 185 amino acids and is devoid of N-linked glycosylation sites. The E2 protein is rich in proline residues and displays an organization typical of an integral membrane protein. Northern blotting showed a good correlation between mRNA abundance, E2 surface density and the level of T cell differentiation. In fact, nucleotide sequencing revealed that E2 is the MIC2 gene product, previously identified with the 12E7 Mab. Xg(a-) female individuals have no E2 molecule on the surface of their red cells, in contrast with Xg(a+) individuals, but have the molecule in their cytoplasm, in the form of the 28 kd precursor. These findings show that the E2 antigen, a cell surface molecule involved in T cell adhesion processes, is the product of the MIC2 gene, the only pseudoautosomal gene to be described in man.

The biochemical characterization of E2, a T cell surface molecule involved in rosettes.

We have previously identified a molecule on the T cell surface, which, in addition to CD2 is involved in the rosette phenomenon. This is a 32-kDa single polypeptide chain which we have termed E2. The studies reported here show striking patterns on the glycosylation status of E2. It is a heavily sialylated and glycosylated molecule, the sugar moieties accounting for almost half of its relative molecular mass (Mr). It carries no N-linked sugar residues, only O-linked oligosaccharides. Despite heavy glycosylation, the molecule appears to behave homogeneously on gel electrophoresis, both in terms of Mr and pI. Neuraminidase treatment of E2 lowered its Mr to 28 kDa; this was further decreased to 18 kDa after removal of O-linked sugar residues by treatment with O-glycanase. An identical reduction in size was observed after treatment with trifluoromethane sulfonic acid, showing that the molecule carries no detectable N-linked sugar residues. Moreover, endoglycosidase F and endoglycosidase H treatment of either the immunoprecipitates from 125I surface-labeled thymocytes, or of a purified preparation of E2, did not reduce the Mr of E2, nor did tunicamycin treatment of T cells. Two-dimensional gel electrophoresis revealed two discrete spots of acidic pI (4.4 and 4.6) that were still seen after neuraminidase treatment, though they had moderately shifted. Pulse-chase experiments revealed a single 28-kDa precursor form that could have been the unsialylated molecule. Finally, sequencing 14 amino acid residues of the N-terminal side revealed no homology with known proteins. Since the sugar moieties of adhesion protein could play an important role, the results obtained in this study will prove valuable to our understanding of the role exerted by the E2 molecule.

Control of cell proliferation within the human thymus. A very limited thymocyte subpopulation generates a suppressive activity.

It is shown here that a minor subpopulation of the human thymus, representing 2-3% of the whole thymocyte population, can suppress proliferation of syngeneic or allogeneic thymocytes to various stimuli. In contrast, this suppressor cell population has no influence on the proliferative response of peripheral blood lymphocytes. This subpopulation was defined by its cell surface phenotype as CD3+, CD1-, 4-, 8- cells. Its suppressive activity is generated after concanavalin A (Con A) stimulation, but not after stimulation with phytohemagglutinin. In addition, the suppressive activity is not modified by extensive washes of Con A-stimulated cells with 1-O-methyl-alpha-D-glucopyranoside, a specific sugar for Con A. This suppressor cell population does not exert any detectable cytotoxic activity, nor does it act by consuming available interleukin 2 (IL 2). Rather, it prevents IL 2 receptor expression on thymic target cells. Since Con A might activate T cells by binding to molecules physiologically involved in T cell activation, particularly the CD3-T cell receptor complex, these data could be relevant to the regulation of normal thymic differentiation.

T cell activation via CD2 [T, gp50] molecules: accessory cells are required to trigger T cell activation via CD2-D66 plus CD2-9.6/T11(1) epitopes.

Binding monoclonal antibodies (MAb) both to D66 and 9.6/T11(1) epitopes on the CD2 [T,gp50]-defined molecule produces a high level of T cell mitosis. This was observed with a battery of MAb of different isotypes. In contrast, none of the anti-D66 or anti-9.6/T11(1)Ab could trigger T cell proliferation in combination with anti-T11(3). Moreover, all anti-D66-9.6/T11(1) pairs of MAb tested required monocytes to activate T cells which were recruited through their Fc receptors. Variations among normal individuals were observed in the level of response to anti-D66-9.6/T11(1) pairs of Ab, 75% of a population of French Caucasians giving a high response. The level of response of a given individual was determined by his accessory cells. However, the level of response of an individual appeared to be minimally influenced by the isotype of a peculiar anti-D66 or anti-9.6/T11(1) Ab. The addition of exogeneous IL 2 could overcome the removal of accessory cells or the modulation of CD3 molecules. In contrast, IL 2 receptor appearance was not overcome by removal of monocytes. Thus, T cell activation via CD2 seems to be produced by "touching" several definite regions of this molecule which trigger a cascade of events similar to those produced by mitogenic lectins. One can assume that the appropriate conformational changes of the CD2 molecule induced by anti-D66-9.6/T11(1) pairs of Ab are solely produced when they are presented by accessory cells. This leaves open the question of whether accessory cells would also play a more active role.

The heterogeneity and functional capacities of human thymocyte subpopulations.

Analysis of human thymocytes with monoclonal antibodies belonging to five distinct clusters of differentiation (CD1, CD3-CD5, CD8) revealed a high degree of phenotypic heterogeneity. Six subpopulations could be defined in the thymic compartment characterized by the presence of CD1 antigens (cortical type); four subpopulations could be defined in the compartment characterized by the lack of CD1 but by the presence of CD5 antigens (medullary type); two subpopulations could be defined in the compartment characterized by the lack of both CD1 and CD5 antigens. Thymic samples could be categorized as either high responder or low responder to phytohemagglutinin alone. The defect of low responders was, to a large extent, attributable to a lack of interleukin 2 availability in the medullary type compartment. Yet, cortical-type subpopulations, both from high and low responders, were able to respond to phytohemagglutinin alone to the same extent. Undesirable cell contamination was excluded by limiting dilution analysis. Moreover, cortical-type cells were found to be able to respond to concanavalin A alone, while medullary-type cells and total populations did not respond to concanavalin A alone. Thus, the human thymus includes a number of cell subpopulations involved in complex functional interactions.