Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens. Evidence from studies of CTL-target cell binding.

The present study examines the potential role of the T4 molecule in functional cell-cell interactions between target cells and human cytotoxic T lymphocyte (CTL) clones that are specific for HLA class II alloantigens encoded by the SB locus. There were marked differences (greater than 30-fold) between the seven SB-specific clones studied with respect to their susceptibility to inhibition by anti-T4 as well as anti-T3 antibodies. We wished to test the hypothesis that such variation among the clones would be due to differences in clonal "affinity" for antigen. To quantitate differences among the CTL clones in the tightness with which they bind target cells, the clones were analyzed using a previously published assay of susceptibility of CTL-target cell conjugates to dissociation in the presence of unlabeled targets. The results revealed that the clones that were most susceptible to inhibition by anti-T4 and anti-T3 were the weakest target cell binders, and vice versa. Anti-T4 antibody could partially induce dissociation of functional CTL-target cell conjugates in the absence of any added cold targets. For the "highest affinity" clone such anti-T4 antibody-induced dissociation could be observed at 4 degrees C but not 23 degrees C. These results indicate that the T4 molecule is functionally involved in target cell binding by CTL, and raise the possibility that although it is easiest to demonstrate the function of the T4 molecule in "low affinity" clones, that function may also be operative in the "high affinity" clones.

Possible involvement of the OKT4 molecule in T cell recognition of class II HLA antigens. Evidence from studies of cytotoxic T lymphocytes specific for SB antigens.

A recently described HLA gene, SB, which maps between GLO and HLA-DR, codes for Ia-like molecules that are similar to but distinct from HLA-DR molecules. Cytotoxic T lymphocytes (CTL) specific for SB1, SB2, SB3, and SB4 were compared with HLA-A2-specific CTL with respect to their surface expression of the T cell differentiation antigens OKT3, OKT4, and OKT8. All CTL activity was eliminated by treatment with OKT3 and C'. The SB-specific cytotoxicity was eliminated by OKT4 plus C' but not by OKT8 plus C'. In contrast, HLA-A2-specific killing was completely susceptible to treatment with OKT8 plus C' but not with OKT4 plus C'. Cytotoxicity was analyzed in the presence of OKT8 and a series of monoclonal antibodies (OKT4A, 4B, 4C, and 4D) that react with distinct epitopes on the OKT4 molecule. SB1-, SB3-, and SB4-specific CTL were partially inhibited by OKT4A and 4B (45-75%), whereas HLA-A2-specific CTL were partially inhibited by OKT8 (48-63%) but not by OKT4. SB2-specific CTL were not inhibited (less than 26%) by OKT8 or by any of the OKT4-related antibodies. These results suggest that the OKT4 marker may be expressed on most T cells that recognize allogeneic Ia or self Ia plus foreign antigens; OKT4+ cells do not appear to be functionally homogeneous in that they can act both as helper/inducer and cytotoxic cells. Models are proposed for the functional involvement of the OKT4 molecule in T cell-Ia antigen interactions.

Cell membrane perturbation of resting T cells and thymocytes causes display of activation antigens.

Three human lymphocyte antigens recognized by monoclonal antibodies OKIa1, OKT9, and OKT10 were found to be minimally represented on resting peripheral T cells (all three) and thymocytes (OKIa1 and OKT9). These antigens, which are present on "activated" T cells, were promptly displayed on "resting" T cells or thymocytes following cross-linking of surface-bound monoclonal antibody by horse alpha-mouse IgG. These experiments suggested that membrane perturbations may induce the expression of certain antigens that are normally present in an unexpressed form in resting cells.

Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein.

The human immunodeficiency virus type 1 (HIV-1) uses the CD4 protein as a receptor for infection of susceptible cells. A candidate structure for the HIV-1 binding site on the CD4 protein was identified by epitope mapping with a family of eight functionally distinct CD4-specific monoclonal antibodies in conjunction with a panel of large CD4-derived synthetic peptides. All of the seven epitopes that were located reside within two immunoglobulin-like disulfide loops situated between residues 1 and 168 of the CD4 protein. The CD4-specific monoclonal antibody OKT4A, a potent inhibitor of HIV-1 binding, recognized a site between residues 32 and 47 on the CD4 protein. By analogy to other members of the immunoglobulin superfamily of proteins, this particular region has been predicted to exist as a protruding loop. A synthetic analog of this loop (residues 25 to 58) showed a concentration-dependent inhibition of HIV-1-induced cell fusion. It is proposed that a loop extending from residues 37 to 53 of the CD4 protein is a binding site for the AIDS virus.

OKT3E, an anti-CD3 antibody that does not elicit side effects or antiidiotype responses in chimpanzees.

Chimpanzees were injected with OKT3 and two other anti-CD3 antibodies, OKT3D and OKT3E. Both of the new antibodies were of the mouse IgG2b isotype. Administration of the antibodies was identical to the clinical regimen used for OKT3 in humans: 5 mg i.v., daily for 10 consecutive days. All animals were monitored for fever during administration of the antibodies, and blood samples were taken throughout the treatment period for monitoring the effects of the antibodies on peripheral lymphocyte subsets and the appearance of circulating cytokines. OKT3 produced similar clinical effects to those observed in humans; fever (2/3), as well as elevations in cytokines were observed. As in humans, peripheral T cells were cleared with the first dose and remained absent or modulated of their T cell receptor molecules throughout treatment. OKT3D, IgG2b also produced fevers (2/3) and elevations of cytokines. Although it also cleared circulating T cells with the first dose and T cell counts remained low throughout treatment, remaining circulating T cells were coated with administered antibody and were able to reexpress the CD3 antigen. OKT3E, IgG2b produced no temperature elevations and no elevations in cytokines. Although it cleared the circulation of T cells with the first does, cells reappeared during treatment, modulated of their CD3 antigens or coated with the administered antibody. All three antibodies raised antimouse antibodies, and OKT3 and OKT3D also produced blocking antiidiotype antibodies. OKT3E treatment did not result in anti-OKT3E blocking antibodies.

Identification of anti-CD3 antibodies that do not modulate antigen but induce mitogenesis and block the mixed lymphocyte reaction.

Using a panel of anti-CD3-TCR monoclonal antibodies (OKT3 A-E), it appears possible to separate the ability to cause surface antigen modulation from inhibition of MLR or induction of mitosis. OKT3D, an antibody that recognizes the CD3 antigen at a site that can be differentiated from the epitopes recognized by other members of this panel by competition binding, does not cause antigen modulation when incubated with human T cells for up to 3 days. Despite this, OKT3D is mitogenic and is capable of blocking MLR. Two different isotypes were produced from the OKT3D clone, IgG1 and IgG2b. The IgG2b isotype of OKT3D blocked MLR even in individuals unable to respond mitogenically to this antibody. Use of members of this panel may now permit dissection of the types of signals delivered by the CD3-TCR complex inducing mitosis, receptor modulation, and other T-cell responses.

Distinct epitopes on the T8 molecule are differentially involved in cytotoxic T cell function.

The present report attempts to determine if there are distinct epitopes on the T8 molecule that are involved in class I-restricted cytotoxic T lymphocyte (CTL) function. A panel of 9 monoclonal antibodies (OKT8A,B,C,E,F,G,H,I, and OKT5) was produced and all antibodies were shown to bind to the T8 molecule. This panel of antibodies was employed to characterize the distribution of distinct epitopes on the T8 molecule and to block the activity of class I-specific influenza virus-immune and allo-immune CTL effector function. Significant differences in the ability of the anti-T8 antibodies to block CTL function were observed: OKT8C and T8F blocked best (49 and 55% respectively); OKT8A,E,G,H,I, and OKT5 blocked less well (24-31%); and OKT8B blocked marginally (11%). There was no correlation between the capacity of the antibodies to block CTL function and their heavy chain isotype. Competitive binding of the different OKT8 antibodies to the cell surface and differential trypsin sensitivity of the epitopes recognized by the antibodies indicated that OKT8C and T8F were located on topographically distinct regions of the T8 molecule. These results indicate that specific epitopes on the T8 molecule are involved in CTL function, and that there could be more than one functional site on the molecule.

CD4 changes conformation upon ligand binding.

Aurintricarboxylic acid (ATA) has been shown to block the binding site for both HIV gp120 and mAb anti-Leu 3a on CD4. We have unexpectedly found that brief treatment with > or = 1 micrograms/ml ATA rapidly disengages another mAb, OKT4E, after it has been bound to CD4 on human PBL. OKT4E is specific for a discontinuous epitope overlapping the MHC class II-binding region in the N-terminal CD4 domain. Interestingly, among 10 other mAb tested, only anti-Leu 8, specific for a leukocyte homing receptor is also quickly released from the cells by ATA treatment. Disengagement of the OKT4E mAb is also seen on a CD4-positive cell line (HPB-ALL) and with recombinant soluble CD4 (sCD4) bound to immobilized OKT4E. In all of these cases, disengagement is prevented if OKT4E is cross-linked, or the Leu 3a site is blocked by the mAb, but not by gp120. Photobleaching fluorescence resonance energy transfer (pFRET) measurements suggest that OKT4E is released as an indirect consequence of ATA-evoked conformational changes of CD4. Similar changes were detected as a result of gp120 binding to PBL. These data raise the possibility of a novel type of immunomodulation: induced disengagement of a bound ligand from its Ag.

Cross-linking of CD4 in a TCR/CD3-juxtaposed inhibitory state: a pFRET study.

Instances when T cell activation via the T cell receptor/CD3 complex is suppressed by anti-CD4 Abs are generally attributed either to the topological separation of CD4-p56lck from CD3, or their improper apposition. Photobleaching fluorescence resonance energy transfer measurements permitted direct analysis of these alternatives on human peripheral blood lymphocytes. Distinction between changes of relative antigen densities or positioning was made possible by simultaneously recording donor and acceptor fluorescence in the energy transfer experiment performed on homogeneous populations of flow-sorted cells. We show here that CD4 stays in the molecular vicinity of CD3, while anti-CD3 stimulation is suppressed by anti-CD4 or cross-linked HIV gp120. Our data suggest that cross-linking of CD4 through particular epitopes is capable of inhibiting activation driven by Abs binding to specific sites on CD3 without major topological sequestration of the Ags, in such a way that additional positive signals will also be affected. Thus, these and other related cases of negative signaling via CD4 may be interpreted in terms of functional uncoupling rather than a wide physical separation of CD4 from the T cell receptor-CD3 complex.

Membrane glycoproteins involved in cell--substratum adhesion.

A combination of immunological and biochemical methods were used to identify surface membrane components involved in cell-substratum adhesion. Broad-spectrum antiserum, prepared against surface membranes from hamster cells, induced reversible rounding and detachment of hamster fibroblasts from a substratum in vitro. This phenomenon was inhibited by Nonidet P-40 extracts of hamster cells. Therefore, an antibody neutralization assay was developed to detect the presence of antigen during the fractionation of Nonidet P-40 extracts of cells. After two differential precipitation steps, anion exchange chromatography, and sequential lectin affinity chromatography, a fraction greatly enriched in ability to block antiserum-induced changes in cell adhesion and appearance was isolated. Analysis of this fraction by NaDodSO4/polyacrylamide gel electrophoresis revealed a highly restricted group of glycoproteins with Mr approximately 140,000. A lectin-purified glycoprotein fraction was used to raise a higher titer antiserum that was able to induce reversible rounding and detachment of cells from a substratum and, when immobilized on an antibody affinity column, was able to bind and release material capable of blocking antiserum-induced cell rounding. These methods have allowed us to focus attention on a restricted group of glycoproteins that are integral constituents of the surface membrane and which play some as yet undetermined role in the process of cell--substratum adhesion.

Five epitopes of a differentiation antigen on human inducer T cells distinguished by monoclonal antibodies.

A series of mouse monoclonal antibodies reacting with human T cells of the helper/inducer subclass, OKT4, 4A, 4B, 4C, and 4D, have been reported. Using double-fluorescent staining and complement-mediated depletion, it was shown that the antigen(s) recognized by OKT4, 4A, 4B, 4C, and 4D antibodies are present on the same cell. Using FITC-labeled OKT4, 4A, 4B, 4C and 4D, a lack of competition between the antibodies for their epitopes was shown. Immunoprecipitation of the antigen recognized by each antibody yielded a molecule of approximately 60,000-62,000 Da. Sequential precipitation with several antibodies resulted in a minimum of additional precipitated antigen following removal of the first antigen. Capping of cell surface antigen with OKT4, followed by staining with OKT4, 4A, 4B, 4C, or 4D, indicated that the epitopes for all five antibodies co-cap. A sandwich ELISA assay using OKT4 and the other antibodies showed that molecules binding to OKT4A, 4B, 4C, and 4D also bound OKT4. It can therefore be concluded that monoclonal antibodies OKT4, 4A, 4B, 4C, and 4D recognize distinct epitopes present on a molecule of approximately 60-62,000 Da on human helper/inducer T cells.

Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens: evidence from studies of proliferative responses to SB antigens.

The T4 molecule has been identified as a marker of human T cell differentiation, but the function of this molecule remains to be defined. We have investigated its possible functional involvement in T cell proliferative responses to class II HLA antigens encoded by the recently described SB locus. The responses of SB-primed cells (specific for each of four different SB antigens) were studied with the use of two proliferation-inducing stimuli, SB antigen or TCGF. The proliferative responses to both stimuli were found to be mediated by T4+, T8- cells. Monoclonal antibodies against some epitopes on the T4 molecule (OKT4A and OKT4B) substantially blocked antigen-stimulated proliferative responses; antibodies against other epitopes of the T4 molecule (OKT4, T4C, T4D) blocked less well. Inhibition of SB-specific proliferation by antibodies to the T4 molecule was maximal only when the antibodies were incubated with the responder cells before the addition of stimulator cells. Proliferative responses of SB-primed cells stimulated with TCGF alone were not inhibited by any of the OKT4-related antibodies, but were completely inhibited by the anti-Tac monoclonal antibody, which reacts with the TCGF receptor. These results lend further support for the hypothesis that the T4 molecule is involved in T cell recognition of and/or activation by class II HLA antigens. We suggest that 1) the T4 molecule binds a nonpolymorphic epitope on class II HLA molecules, and 2) this interaction may facilitate, but not be an obligate requirement for, T cell activation by class II antigens.

Detection by ELISA of shed cell-surface molecules in serum.

Monoclonal antibodies OKT9 and OKT9A, which recognize distinct epitopes on the transferrin receptor, were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for this molecule. A 115,000 dalton molecule was detected in serum which probably represents a proteolytic cleavage fragment of the 186,000 dalton (dimer of two 93,000 chains) found on the cell surface of replicating cells. Serum levels of the shed transferrin receptor in human sera may be studied by means of this assay.

Early events in lymphocyte activation as defined by three new monoclonal antibodies.

Three new lymphocyte activation antigens are described whose kinetics of appearance place them very early in the activation pathway. The 78,000 dalton early antigen (Ea) 1 is present at low levels on resting lymphocytes, and its expression is enhanced twofold to threefold within 3 hr of stimulation. Ea2, a nondisulfide-bonded 86,000 and 73,000 dalton heterodimer, is first detectable 3 hr after activation and peaks by 9 hr. Its presence on all but a few cell lines, plus the variable association with a lower m.w. (28,000) structure, suggest that it may serve as a receptor for a growth factor. Neither Ea1 nor Ea2 are restricted to lymphocytes. The 31,000 dalton Ea3 antigen is induced only by PHA but not by other means of activation, and may pre-exist within the cell. The Ea3 antibody blocks PHA-induced but not OKT3-induced mitogenesis, suggesting differences in the pathways of activation by these two stimuli. These reagents, and OKT3, were used to define the cyclosporine A (CSA)-sensitive stage of lymphocyte activation. CSA blocks at a point before the biosynthesis of Ea1 and after that of T3/T cell receptor loss from the cell surface, at a point close to Ea2 biosynthesis.

OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2.

OKB7, an IgG2 mouse monoclonal antibody, binds to the C3d receptor (CR2) of human B lymphocytes in or near the active site of the receptor. OKB7 precipitates the same molecule as monoclonal antibodies B2 and HB-5 which have previously been reported to react with the C3d receptor at epitopes distant from the active site. The epitope recognized by OKB7 is distinct from those bound by B2 and HB-5 as shown in competitive binding experiments and is near the complement binding site since OKB7 blocks EAC3d rosetting at concentrations as low as 1 microgram/ml in the absence of cross-linking antibodies.

Direct involvement of the CDR3-like domain of CD4 in T helper cell activation.

The CD4 glycoprotein, a member of the Ig super-family, has long been known to play an important role in the immunologic activation of Th cells. The precise manner in which CD4 participates in this activation process is not yet understood. In an attempt to further define its role in Th cell activation, we modeled the D1 domain of the murine CD4 protein (L3T4) based on the experimentally determined high resolution structure of the human CD4 protein. Because the D1 domain of CD4 strongly resembles the V kappa chain of an antibody, we addressed the question of whether the CDR-like regions of CD4 are also involved in mediating protein-protein interactions. Consequently, we used the modeled L3T4 structure as a template in the design of conformational mimics of the CDR3-like region (residues 86-94). Only the analog designed to mimic both the sequence and conformation of this region exhibited highly specific inhibition of CD4-dependent responses. Because the inhibitory activity could be localized to the Th cell itself, it appears that this analog acts by uncoupling a CD4 association (independent of an APC) critical to generating a proliferative response.

Cytochalasin D modulates CD4 crosslinking sensitive mitogenic signal in T lymphocytes.

It has previously been shown that crosslinking of the CD4 molecule, either with anti-Leu3a mAb or with gp120 (the HIV coat protein) plus anti-gp120 mAb, suppresses activation induced by wt31, a TcR/CD3-specific mAb. This suppression was associated with hindrance of the necessary association of the p56lck kinase bearing CD4 molecule with the TcR/CD3 complex. In this paper we demonstrate that this crosslinking-induced suppression can be bypassed by perturbing the microfilament system of CD4+ cells by pretreatment with 1 microM cytochalasin D. Using the fluorescence resonance energy transfer method, we have shown that the cytochalasin D-affected increase of mitogenesis is not due to changes in the TcR/CD3 to CD4 distance. Likewise, other membrane biophysical parameters, membrane potential and lateral diffusion of surface receptors, cannot be associated with these cytochalasin D-affected mitogenic changes. Cytochalasin D treatment elevates intracellular Ca2+ levels induced by wt31 mAb plus crosslinking and generates a TcR/CD3-dependent signal which is cyclosporin sensitive.

The L-selectin (Leu8) molecule is associated with the TcR/CD3 receptor; fluorescence energy transfer measurements on live cells.

Several accessory molecules were shown to play important roles in T cell functions and be in close proximity to the T cell receptor (TcR/CD3). The L-selectin molecule (Leu8, LAM1-1, LECAM1) also plays an important role in lymphocyte homing and proliferation. We were interested in determining the proximity of this molecule to the TcR/CD3 complex on live peripheral human T cells. Using a fluorescence energy transfer method, designed to study individual cells, we could show that L-selectin is within 170 A of the TcR/CD3 complex. Monoclonal antibody directed against the LAM1-1 (Leu8) epitope of the L-selectin molecule suppressed the mitogenic activity of antibodies specific for various CD3 epitopes in vitro. Intracellular Ca2+ mobilization obtained with wt31 followed by cross-linking antibody or with anti-CD3 was not influenced by anti-Leu8 antibody. Also antibody directed against the LAM1-1 epitope did not influence the binding of the mitogenic antibodies, as shown by fluorescence-based flow cytometry. Therefore, we suggest that binding of TcR/CD3 bound mitogenic antibodies to accessory cell Fc receptors may be hindered by antibodies bound to the close proximity L-selectin molecules.

Generation and characterization of monoclonal antibodies reactive with human B lymphocytes.

Four novel monoclonal antibodies directed at determinants on human B lymphocytes were produced and characterized by flow cytometry using indirect immunofluorescence analysis. The antibodies, OKB1, OKB2, OKB4, and OKB7, define antigens not previously described on human B lymphocytes using B cell reactive monoclonal antibodies or standard B cell phenotypic markers. OKB4 [immunoglobulin M (IgM)] and OKB7 (IgG2a) were reactive with greater than 95% of all surface membrane immunoglobulin positive (Smlg+) lymphocytes and were restricted to the B cell lineage. OKB2 (IgG1) likewise reacted with greater than 95% of all Smlg+ lymphocytes; however, the antigen was also expressed on all granulocytes. In contrast to OKB2, OKB4, and OKB7, OKB1 reacted with a variable percentage of normal peripheral B cells (approximately 70 to 95%). Functional studies employing polyclonally activated B cells in a pokeweed mitogen (PWM) driven system and the reverse hemolytic plaque assay for the quantitation of Ig secreting plasma cells demonstrated that OKB1 and OKB2 inhibited the generation of plaque-forming cells (PFC) when added to cultures at day 0. OKB4 had a marginal inhibitory effect, whereas OKB7 consistently enhanced the PFC response. Immunoprecipitation studies with OKB1, OKB4, and OKB7 demonstrated that these antibodies precipitated antigens of approximately 168,000, 87,000, and 175,000 m.w., respectively. The OKB2 antigen is presently under study.

Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies.

A series of seven monoclonal antibodies directed at determinants on human peripheral blood monocytes were produced and characterized. The antibodies were separated into three groups based on cell distribution and percentages of monocytes bearing antigen. Hybridoma antibodies, termed OKM1, OKM9, and OKM10, recognized antigen(s) expressed on the majority of adherent monocytes, null cells, and granulocytes. The second group, comprising OKM5 and OKM8, reacted with most adherent monocytes and platelets. OKM3 and OKM6, comprising a third group of antibodies, reacted with a subpopulation of adherent monocytes and platelets. OKM antibodies were not expressed on lymphocytes, thymocytes, and lymphoblastoid cells, with the exception of OKM3 which reacted with three B-cell lines. SDS gels of immunoprecipitates formed with OKM antibodies yielded the following tentative molecular weight results: OKM1 and OKM9 antigens appeared to be 160,000 (nonreduced) and 170,000 (reduced); OKM10 precipitated two polypeptides of 170,000 and 115,000 (reduced); OKM5 and OKM8 precipitated a single polypeptide of 88,000 (reduced, nonreduced); OKM6 antigen appeared to be 116,000 (nonreduced) and 130,000 (reduced).