Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

Internalization of Ia molecules by antigen-presenting B cells is limited

Our data demonstrate that the uptake of surface Ia into an intracellular compartment of B lymphoma or normal spleen cells is limited to about 20% after 2 to 3 h. The extent of internalization does not vary with several types of stimulation, including LPS, phorbol esters, anti-Ig-plus phorbol ester-stimulated EL-4 T cell supernatant, and Con A supernatant. Resting and activated B cells had similar rates of internalization. The rate and extent of uptake of surface Ia molecules into an intracellular compartment was monitored quantitatively through the use of a mAb radiolabeled with 125I. The internalization of Ia molecules was compared to that of transferrin receptor, a receptor that undergoes rapid internalization and recycling and accumulates in a intracellular pool that can be trapped by monensin. The internalization of Ia was not affected by monensin, although its synthetic pathway is disturbed by this drug. The potential use of internalized Ia for formation of T cell-triggering complexes of Ia and Ag fragments is not ruled out by these data, but it appears unlikely that internalization provides the major mechanism permitting Ia interaction with Ag.     

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.     

Artificial T cell:B cell conjugation. A unique approach to analyze weak cell-cell interactions

An antibody response against a thymic-dependent Ag requires cognate recognition of the Ag by B and T cells. Functional T-B cell (T-B) interaction involves binding of Ag by B cell surface Ig, internalization and processing of Ag, expression of an Ag fragment in the context of Ia, binding of Ag/Ia by the TCR and binding of T cell-derived lymphokines by B cell lymphokine receptors. It is becoming increasingly evident that B and T cell accessory molecules also are involved in T-B interactions. To determine the role of accessory molecules in T-B collaboration, we have designed a system in which T-B interaction was artificially induced in the absence of carrier protein. TNP-modified, turkey gamma-globulin-specific, Th cells were allowed to form conjugates with TNP-specific B cells in the absence of hapten-carrier complex. Both B and T cells were induced to proliferate and B cells partially differentiated into antibody-secreting cells when B cells were cultured with TNP-modified but not unmodified T cells. The activation of B cells by TNP-modified T cells was not MHC restricted but was blocked by anti-Ia antibodies, suggesting a role for Ia distinct from Ag presentation. Furthermore, B cell proliferation was also inhibited by antibodies to L3T4 and LFA-1, suggesting a functional accessory role for these molecules in induction of B cell proliferation/differentiation.     

T cell recognition of QA-1b antigens on cells lacking a functional Tap-2 transporter.

MHC class Ia H chains and beta 2-microglobulin assemble with appropriate peptides to form stable cell surface molecules that serve as targets for Ag-specific CTL. The structural similarities of class Ia and the less polymorphic Q/T/M (class Ib) molecules suggest that class Ib molecules also play a role in antigen presentation, although the origin of the peptides they present remains mostly unclear. The cell line RMA-S has a defect in class I Ag presentation, presumably due to a mutation in a peptide transporter gene. This defect can be overcome by transfection of RMA-S cells with the Tap-2 gene (formerly Ham-2) that encodes an ATP-binding transporter protein. We now show that a substantial portion of alloreactive CTL specific for Qa-1 class Ib molecules recognize Qa-1b on RMA-S cells and thus differ from most class Ia specific CTL. Those anti-Qa-1b CTL that do not recognize untransfected RMA-S do lyse RMA-S transfected with Tap-2. We also examine the effects of Qdm, a gene that maps to the D region and alters recognition of Qa-1. Qdm(k) strains lack an epitope(s) recognized by some (Qdm dependent) anti-Qa-1 CTL whereas Qdm+ strains express this epitope. Thus, Qdm-dependent CTL do not recognize Qa-1 on Qdm(k) targets whereas Qdm-independent CTL recognize Qa-1 epitopes in all strains. Although Qdm-independent CTL varied as to whether they recognized RMA-S vs RMA, all nine Qdm-dependent clones only recognized Qa-1b on RMA and not RMA-S. This result is consistent with Qdm encoding a peptide dependent upon the TAP transporter for cell membrane expression.     

The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes

The MHC class Ib molecule Qa-1 is the primary ligand for mouse CD94/NKG2A inhibitory receptors expressed on NK cells, in addition to presenting Ags to a subpopulation of T cells. CD94/NKG2A receptors specifically recognize Qa-1 bound to the MHC class Ia leader sequence-derived peptide Qdm. Qdm is the dominant peptide loaded onto Qa-1 under physiological conditions and this peptide has an optimal sequence for binding to Qa-1. Peptide dissociation experiments demonstrated that Qdm dissociates from soluble or cell surface Qa-1(b) molecules with a t(1/2) of approximately 1.5 h at 37 degrees C. In comparison, complexes of an optimal peptide (SIINFEKL) bound to the MHC class Ia molecule H-2K(b) dissociated with a t(1/2) in the range from 11 to 31 h. In contrast to K(b), the stability of cell surface Qa-1(b) molecules was independent of bound peptides, and several observations suggested that empty cell surface Qa-1(b) molecules might be unusually stable. Consistent with the rapid dissociation rate of Qdm from Qa-1(b), cells become susceptible to lysis by CD94/NKG2A(+) NK cells under conditions in which new Qa-1(b)/Qdm complexes cannot be continuously generated at the cell surface. These results support the hypothesis that Qa-1 has been selected as a specialized MHC molecule that is unable to form highly stable peptide complexes. We propose that the CD94/NKG2A-Qa-1/Qdm recognition system has evolved as a rapid sensor of the integrity of the MHC class I biosynthesis and Ag presentation pathway.     

Regulation of macrophage activation by IL-3. I. IL-3 functions as a macrophage-activating factor with unique properties, inducing Ia and lymphocyte function-associated antigen-1 but not cytotoxicity

Here we report that IL-3 (also referred to as multi-CSF because of its colony-stimulating activity on a variety of hemopoietic cell lineages) can function as a macrophage-activating factor (MAF). IL-3 was able to regulate the expression of class II MHC Ag and the cellular interaction molecule lymphocyte function-associated Ag-1 on the surface of murine peritoneal exudate cells. The kinetics of IL-3-induced Ia expression appeared to be distinct from that induced by either IFN-gamma, IL-4, or granulocyte-macrophage-CSF. IL-3 was also distinguished from these factors by the finding that it did not induce macrophage tumoricidal activity. In addition to its inherent MAF activities, IL-3 also showed a marked synergy with low doses of LPS (0.05 to 0.5 ng/ml) as well as IFN-gamma in Ia induction. When lymphocyte function-associated Ag-1 expression was evaluated, the effects of these stimuli appeared to be only additive. Although LPS has been shown to inhibit IFN-gamma-induced Ia expression, in our experiments this property of LPS is manifest only when present at doses greater than or equal to 50 ng/ml. At lower concentrations, LPS potentiated both IL-3- and IFN-gamma-induced class II MHC Ag expression. Data presented here also suggest that the synergistic interactions between low doses of LPS and IL-3 are not mediated by known LPS-inducible cytokines of macrophage origin, because rIL-1, TNF-alpha, or IL-6 did not enhance the response to IL-3. Because IL-3 can also participate in the regulation of IL-1 expression, it appears that IL-3 can function as a MAF which selectively regulates the accessory cell characteristics required for Ag presentation, as opposed to the cytolytic functions of the macrophage.     

Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation.

Although the physiologic function of gangliosides is unknown, evidence suggests they play a role in the regulation of cell growth. The binding of ganglioside GM1 by recombinant B subunit of cholera toxin (rCT-B) inhibited mitogen-stimulated B cell proliferation without elevating intracellular cAMP. CT-B paradoxically enhanced the expression of MHC class II (Ia) molecules and minor lymphocyte-stimulating determinants without altering the expression of some other immunologically relevant B cell surface Ag. Increased expression of Ia was not detected until 4 h after stimulation, kinetics similar to those seen when B cells are stimulated with anti-Ig antibody or IL-4, suggesting that the enhancement was not the result of redistribution of existing cell surface markers but rather the result of a new metabolic event. Both the inhibitory and stimulatory effects of CT-B could be blocked by incubation of CT-B with ganglioside GM1. Furthermore, enhancement of the CT-B-mediated effect was seen when additional ganglioside GM1 was incorporated into the B cell membrane. rCT-B with a mutation that interfered with its binding to ganglioside GM1 did not enhance Ia expression. Taken together, these results indicate that the observed effects of CT-B were most likely mediated through the binding of cell surface ganglioside GM1. CT-B-mediated stimulation of Ia expression provides a potential explanation for the previously described ability of CT-B to act as an immunoadjuvant. These results suggest that the binding of ganglioside GM1 has multiple B cell growth-regulating effects.     

The role of Ia in the formation of a T cell antigen-recognition complex

We have evaluated the ability of a peptide-specific, I-Ak-restricted murine T hybridoma to bind its Ag in the presence and absence of class II MHC molecules. The restricting Ia molecule, when supplied as a plasma membrane preparation of I-Ak-expressing APC, specifically increases the avidity of the Ag-binding complex by lengthening its t1/2, without affecting the rate at which the complex is formed. Experiments using mutated I-Ak molecules indicate that the ability of a mutant Ia species to present Ag is distinct from its ability to stabilize the Ag-recognition complex, suggesting that T cell stimulation depends not only upon stabilization of Ag-TCR-Ia complexes, but also upon distinct Ia-influenced conformational signals.     

Possible role of neuraminidase in activated T cells in the recognition of allogeneic Ia

In a primary MLR, predominant stimulators in spleen cells are adherent cells and not B cells, although B cells are one of the cell types expressing a large amount of Ia molecules. Our previous experiments showed that T cells treated with neuraminidase (Nase) responded to an allogeneic Ia on B cells. In our experiments, the relationship between the responsiveness to the allogeneic Ia molecules on B cells and Nase activity of T cells was examined. The results showed that T cells increased in Nase activity with the acquisition of the reactivity to Ia on B cells. T cells from normal mice increased in Nase activity after the incubation for 3 days or more in MLR, and these T cells responded to allogeneic Ia on B cells. However, T cells from mice genetically deficient in Nase responded poorly to the Ia on allogeneic B cells even after the incubation in MLR for 3 days. T cells incubated for 3 days in MLR decreased in electrophoretic mobility, indicating the decrease of net negative charge of the cells, and increased in their binding of peanut agglutinin which has been reported to bind to galactosyl residues exposed on T cell surface by removing sialic acids. These results suggest that Nase in T cells was activated by the cultivation in MLR for 3 days, and sialic acids of some molecules on T cell surface were removed by the enzyme and, in turn, T cells acquired the responsiveness to allogeneic B cells in a secondary MLR. Thus, Nase was suggested to play a regulatory role in the recognition of Ia molecules in T cells.     

Direct induction of Ia antigen on murine thyroid-derived epithelial cells by reovirus

The ability of thyroid follicular epithelial cells (TFEC) to act as APC is linked to the expression of class II (Ia) molecules of the MHC. The cloned murine thyroid-derived epithelial cell line M.5 was used to demonstrate the potential effects of virus in the direct induction of Ia molecules on TFEC. Membrane binding and replication of reovirus type 1 in TFEC was demonstrated using fluorescein-labeled antireovirus antibody and fluorescence microscopy. One consequence of the interaction between reovirus and M.5 cells was the induction of Ia Ag and augmented class I molecule expression in M.5 cells. The levels of Ia expression at three days after reovirus binding were amplified 17.3-fold over controls and were 2-fold less than that seen upon treatment of M.5 cells with IFN-gamma. Supernatant transfer experiments showed that the induction of Ia expression was directly linked to the binding of virus to M.5 cells, and was not dependent upon virus replication or the presence of IFN. These results indicate that early events of reovirus binding or receptor internalization on TFEC initiate a signaling process which results in the induction of class II and augmentation of class I MHC protein levels on the cell surface.     

Effect of prostaglandin E2 on the gamma-interferon induction of antigen-presenting ability in P388D1 cells and on IL-2 production by T-cell hybridomas

The effect of prostaglandin E2 on the gamma-interferon (IFN-gamma)-mediated induction of Ia expression and antigen-presenting activity in macrophage cell lines was studied. Using a lymphokine preparation obtained from the T-cell hybridoma FS7-20.6.18 (known to produce interferon) to induce the expression of Ia in P388D1 cells, the influence of PGE2 on this phenomenon was studied. Screening of the cell cultures by indirect immunofluorescence using an anti-I-Ad monoclonal antibody confirmed the inhibitory effect of PGE2 in the induction of I-Ad. However, the inhibition of the antigen-presenting ability of these cells, as measured by their capacity to stimulate interleukin 2 (IL-2) production by antigen-specific, I-region-restricted (Ag/I) T-cell hybridomas, was more difficult to demonstrate and was only evident when using low concentrations of Ia-inducing lymphokines or when using "low avidity" T-cell hybridomas. The latter were distinguished by the limited response (in the form of IL-2 production) that was observed when they were tested with P388D1 cells that had been induced with IFN-gamma. By contrast, PGE2 had profound inhibitory effects on the ability of T-cell hybridomas to secrete IL-2 in response to Ag/I or concanavalin A. These results suggest that although PGE2 inhibits the full induction of Ia on macrophages, it has little effect on the induction of Ag/I presentation by the same cells, probably because most T cells require relatively low levels of Ia on the surface of presenting cells. T-cell responses to Ag/I are inhibited, however, because of the effects of PGE2 on the T cells themselves.     

Molecular cloning and characterization of a novel CD1 gene from the pig.

Much effort is underway to define the immunological functions of the CD1 multigene family, which encodes a separate lineage of Ag presentation molecules capable of presenting lipid and glycolipid Ags. To identify porcine CD1 homologues, a cosmid library was constructed and screened with a degenerate CD1 alpha3 domain probe. One porcine CD1 gene (pCD1.1) was isolated and fully characterized. The pCD1.1 gene is organized similarly to MHC class I and other CD1 genes and contains an open reading frame of 1020 bp encoding 339 amino acids. Expression of pCD1.1 mRNA was observed in CD3- thymocytes, B lymphocytes, and tissue macrophages and dendritic cells. The pCD1.1 cDNA was transfected into Chinese hamster ovary cells, and subsequent FACS analysis demonstrated that mAb 76-7-4, previously suggested to be a pig CD1 mAb, recognizes cell surface pCD1.1. Structurally, the pCD1.1 alpha1 and alpha2 domains are relatively dissimilar to those of other CD1 molecules, whereas the alpha3 domain is conserved. Overall, pCD1.1 bears the highest similarity with human CD1a, and the ectodomain sequences characteristically encode a hydrophobic Ag-binding pocket. Distinct from other CD1 molecules, pCD1.1 contains a putative serine phosphorylation motif similar to that found in human, pig, and mouse MHC class Ia molecules and to that found in rodent, but not human, MHC class-I related (MR1) cytoplasmic tail sequences. Thus, pCD1.1 encodes a molecule with a conventional CD1 ectodomain and an MHC class I-like cytoplasmic tail. The unique features of pCD1.1 provoke intriguing questions about the immunologic functions of CD1 and the evolution of Ag presentation gene families.     

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

Induction and functional characterization of class II MHC (Ia) antigens on murine keratinocytes

To induce Ia molecules on the surface of murine keratinocytes (KC), healthy mice were treated daily with i.p. injections of rIFN-gamma at a dose of 50,000 U/day for 6 days. This resulted in strong Ia expression by KC as determined by immunofluorescence of epidermal sheets or cell suspensions with anti-class II mAb. To obtain a population of Ia-bearing KC devoid of Langerhans cells, a method of depleting Langerhans cells from such suspensions was developed. Although Ia+ KC were unable to stimulate allogeneic T cells in a primary epidermal cell-lymphocyte reaction (less than 5% control), they did induce a proliferative response in an allospecific T cell line. Ia+ KC were unable to present native peptide molecules to class II restricted, Ag-specific T cell hybridomas. However, Ia+ KC were able to present a peptide fragment of pigeon cytochrome c to a hybridoma, suggesting that although these cells cannot process native protein Ag, they can present antigenic peptides. Ia+ (but not Ia-) KC also served as targets for class II restricted cytolytic T cell clones. These data indicate that the Ia expressed by KC is a functional molecule, and that Ia+ KC can participate in some immunologic reactions.     

The role of the Ia-invariant chain complex in the posttranslational processing and transport of Ia and invariant chain glycoproteins

The invariant chain (Ii) is a nonpolymorphic glycoprotein that associates with the Ia alpha- and beta-chains of MHC class II Ag during their transport to the cell surface. Although surface expression of Ia can occur in the absence of Ii, it has not been shown whether the intracellular association of Ia and Ii affects the biosynthetic rate or specificity of posttranslational modifications to the individual molecules. Analysis of transfected cell lines carrying either Ia, Ii, or both Ia and Ii demonstrated efficient assembly of alpha-beta whether or not Ii was present. Pulse-chase studies and two-dimensional gel electrophoresis showed that Ii did not affect the addition of Ia N-linked oligosaccharide chains, or the extent or rate of their conversion to the complex form, nor did it affect the sulfation of alpha and beta glycoproteins. Ii also did not affect the rate of Ia synthesis or appearance of Ia at the cell surface. In contrast, Ia dramatically affected the posttranslational modification of Ii. Although invariant chain was modified by addition of fatty acid, N-linked oligosaccharide, and glycosaminoglycan in the absence of Ia, the processing of Ii-linked oligosaccharide into more acidic, terminally glycosylated forms was significantly less when Ia was absent, and although conversion to proteoglycan did occur, the glycosaminoglycan chains were significantly shorter than normal. The disappearance of radiolabel from the 31,000 Da form of Ii was faster when Ia was present, and the processing of Ii was more rapid when it was associated with Ia. Thus, the rate and manner in which Ii enters and passes through the Golgi is critically affected by Ia.     

Constitutive competition by self proteins for antigen presentation can be overcome by receptor-enhanced uptake

The cells recognize a bimolecular ligand composed of a self Ia molecule and a fragment of foreign Ag that has been processed by an APC. The effect of self proteins on the processing and presentation of foreign Ag was examined in order to ascertain the mechanisms for competition between foreign and self Ag. How this competition can be overcome to allow an efficient immune response was also examined. Normal mouse serum proteins (NMS) compete for the processing and presentation of the foreign Ag bovine RNase by APC. This competition could have occurred at any of three levels in the APC: 1) Ag uptake, 2) Ag processing, or 3) the binding of Ag to an Ia molecule. No competition for either the uptake or the processing of RNase by self proteins could be demonstrated. However, self peptides do compete with foreign Ag by binding directly to Ia molecules, as has been shown previously. Thus, the observed inhibition by NMS of Ag presentation occurred because of competition for binding to the Ia molecule. We hypothesized that during the generation of an immune response this competition is overcome by enhanced uptake of foreign Ag. To test this, we compared the ability of NMS to compete for the presentation of RNase when it entered the APC via fluid-phase pinocytosis or through receptor-mediated uptake via the mannose receptor. When the RNase entered the APC through the mannose receptor, the ability of NMS to compete was dramatically reduced. Thus, self proteins constitutively compete for the presentation of foreign Ag at the level of binding to an Ia molecule, and this competition can be overcome by receptor-mediated uptake of the Ag.     

Characterization of cell surface molecules involved in the recognition of antigen-presenting cells by cloned helper T-cell lines

The recognition of antigen-presenting cells (APCs) by T helper (TH) cells occurs in an antigen (Ag)-specific, MHC-restricted manner. Recent evidence, however, suggests that other interaction molecules may also be involved in TH:APC interaction in addition to the T-cell receptor (Ti) and class II or la antigens. We chose, therefore, to examine the role of various interaction molecules (Ia, Ti, L3T4, and LFA-1) in Ag presentation using several TH clones with distinct recognition patterns (self-Ia, self-Ia/Ag, and allogenic Ia). We describe here the use of a rapid clustering assay to study the initial binding events that occur between TH cells and APCs of various types. In all combinations of TH cells and APCs, conjugate formation was both Ag-specific and MHC-restricted. Moreover, with one exception cell clustering was prevented by the addition of monoclonal antibodies (mAb) against either the T-cell receptor or class II MHC molecules. In contrast, mAb to L3T4 and LFA-1 generally failed to inhibit cluster formation even though T-cell proliferation was profoundly inhibited. The relative importance of these interaction molecules in conjugate formation appeared to depend on the APC type as well as on the T-cell clone used. The implications of these findings for the mechanisms of Ag presentation and T-cell activation are discussed.