Experimental models to investigate the function of dendritic cell subsets: challenges and implications.

The dendritic cell (DC) lineage is remarkably heterogeneous. It has been postulated that specialized DC subsets have evolved in order to select and support the multitude of possible T cell differentiation pathways. However, defining the function of individual DC subsets has proven remarkably difficult, and DC subset control of key T cell fates such as tolerance, T helper cell commitment and regulatory T cell induction is still not well understood. While the difficulty in assigning unique functions to particular DC subsets may be due to sharing of functions, it may also reflect a lack of appropriate physiological in-vivo models for studying DC function. In this paper we review the limitations associated with many of the current DC models and highlight some of the underlying difficulties involved in studying the function of murine DC subsets.

Antigen-pulsed CD8alpha+ dendritic cells generate an immune response after subcutaneous injection without homing to the draining lymph node.

Two subsets of murine splenic dendritic cells, derived from distinct precursors, can be distinguished by surface expression of CD8alpha homodimers. The functions of the two subsets remain controversial, although it has been suggested that the lymphoid-derived (CD8alpha+) subset induces tolerance, whereas the myeloid-derived (CD8alpha-) subset has been shown to prime naive T cells and to generate memory responses. To study their capacity to prime or tolerize naive CD4(+) T cells in vivo, purified CD8alpha+ or CD8alpha- dendritic cells were injected subcutaneously into normal mice. In contrast to CD8alpha- dendritic cells, the CD8alpha+ fraction failed to traffic to the draining lymph node and did not generate responses to intravenous peptide. However, after in vitro pulsing with peptide, strong in vivo T cell responses to purified CD8alpha+ dendritic cells could be detected. Such responses may have been initiated via transfer of peptide-major histocompatibility complex complexes to migratory host CD8alpha- dendritic cells after injection. These data suggest that correlation of T helper cell type 1 (Th1) and Th2 priming with injection of CD8alpha+ and CD8alpha- dendritic cells, respectively, may not result from direct T cell activation by lymphoid versus myeloid dendritic cells, but rather from indirect modification of the response to immunogenic CD8alpha- dendritic cells by CD8alpha+ dendritic cells.

Outer periarteriolar lymphoid sheath arrest and subsequent differentiation of both naive and tolerant immunoglobulin transgenic B cells is determined by B cell receptor occupancy.

T-dependent B cell responses in the spleen are initiated in the outer periarteriolar lymphoid sheath (PALS) and culminate in the generation of proliferative foci and germinal center reactions. By pulsing anti-hen egg lysozyme (HEL) immunoglobulin transgenic (IgTg) B cells with various concentrations of HEL in vitro before adoptive transfer into normal recipients, it was shown that a critical number of B cell receptors (BCRs) must be ligated for B cells to undergo arrest in the outer PALS. T cell help was manipulated independently of the BCR stimulus by incubating B cells expressing the appropriate major histocompatibility complex class II antigen with a peptide recognized by CD4(+) TCR Tg T cells. B cells which either failed to arrest in the outer PALS due to a subthreshold BCR stimulus, or arrested only transiently due to the brevity of the BCR stimulus, underwent an abortive response within the follicles when provided with T cell help. In contrast, naive B cells stimulated by a sustained, suprathreshold concentration of either foreign or self-antigen and given T cell help, proliferated in the outer PALS and then differentiated. Outer PALS arrest was not influenced by the nature of the B cells occupying the follicle, but appeared to be determined solely by the magnitude of BCR stimulation. Thus antigen-pulsed B cells arrested in the outer PALS in an identical manner irrespective of whether the follicles comprised a population of normal B cells with multiple specificities, a monoclonal naive population, or a monoclonal population of tolerant B cells. In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding. Similarly, when anti-HEL Tg mice were crossed with a second HEL Tg strain expressing a higher concentration of soluble HEL, the tolerant anti-HEL Tg B cells were located constitutively in the outer PALS. Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen. A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.

The avidity spectrum of T cell receptor interactions accounts for T cell anergy in a double transgenic model.

The mechanism of self-tolerance in the CD4(+) T cell compartment was examined in a double transgenic (Tg) model in which T cell receptor (TCR)-alpha/beta Tg mice with specificity for the COOH-terminal peptide of moth cytochrome c in association with I-Ek were crossed with antigen Tg mice. Partial deletion of cytochrome-reactive T cells in the thymus allowed some self-specific CD4(+) T cells to be selected into the peripheral T cell pool. Upon restimulation with peptide in vitro, these cells upregulated interleukin (IL)-2 receptor but showed substantially lower cytokine production and proliferation than cells from TCR Tg controls. Proliferation and cytokine production were restored to control levels by addition of saturating concentrations of IL-2, consistent with the original in vitro definition of T cell anergy. However, the response of double Tg cells to superantigen stimulation in the absence of exogenous IL-2 was indistinguishable from that of TCR Tg controls, indicating that these self-reactive cells were not intrinsically hyporesponsive. Measurement of surface expression of Tg-encoded TCR alpha and beta chains revealed that cells from double Tg mice expressed the same amount of TCR-beta as cells from TCR Tg controls, but only 50% of TCR-alpha, implying expression of more than one alpha chain. Naive CD4(+) T cells expressing both Tg-encoded and endogenous alpha chains also manifested an anergic phenotype upon primary stimulation with cytochrome c in vitro, suggesting that low avidity for antigen can produce an anergic phenotype in naive cells. The carboxyfluorescein diacetate succinimidyl ester cell division profiles in response to titered peptide +/- IL-2 indicated that expression of IL-2 receptor correlated with peptide concentration but not TCR level, whereas IL-2 production was profoundly affected by the twofold decrease in specific TCR expression. Addition of exogenous IL-2 recruited double Tg cells into division, resulting in a pattern of cell division indistinguishable from that of controls. Thus, in this experimental model, cells expressing more than one alpha chain escaped negative selection to a soluble self-protein in the thymus and had an anergic phenotype indistinguishable from that of low avidity naive cells. The data are consistent with the notion that avidity-mediated selection for self-reactivity in the thymus may lead to the appearance of anergy within the peripheral, self-reactive T cell repertoire, without invoking the induction of hyporesponsiveness to TCR-mediated signals.

Suppressor T cell memory. II. The role of memory suppressor T cells in tolerance to human gamma globulin.

The transient presence of suppressor T cell (Ts) activity in high-dose tolerance to human gamma globulin (HGG), and its (apparent) absence in low-dose tolerance, have been advanced as strong evidence against the concept that Ts play an important role in maintenance of immunological unresponsiveness. To analyze this question, CBA mice were exposed to high or low doses of deaggregated HGG (dHGG) and later challenged with HGG in immunogenic form (aHGG); their capacity to mount a primary or secondary suppressive response was assessed in an adoptive hapten-carrier system. Primary suppression reached a maximum 7 d after high-dose tolerance induction and gradually waned thereafter, being no longer detectable by day 30-35. Subsequent challenge of tolerant mice with aHGG, however, led to a rapid reactivation of suppression that bore the hallmarks of an anamnestic secondary response, and this effect was still demonstrable 135 d after tolerance induction. It was also shown that a single low dose of dHGG was capable of generating memory for suppression despite the absence of detectable primary suppression, indicating that the latter is not a prerequisite for induction of memory cells. The results were interpreted as indicating that tolerance, like immunity, is a manifestation of specific immunological memory. If tolerance to self-antigens is maintained by a similar mechanism, it would be expected that memory Ts could be induced during the early stages of fetal development. Mice were therefore exposed to tolerogen in utero by injection of their mothers with dHGG at day 7 of gestation, and were assessed at various times after birth for the capacity to exhibit primary or secondary suppression in adoptive transfer. Nonspecific suppression masked any specific effects during the first 5 wk of life. Antigen-specific, primary suppression was demonstrable subsequently until 10-12 wk of age, and if the animals were challenged with aHGG before transfer an anamnestic secondary suppressive response could be elicited up to 6 mo of age. These observations are consistent with the notion that memory Ts may play an important role in the maintenance of self-tolerance.

The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice.

To study the factors that determine whether CD4+ T cells produce interleukin 4 (IL-4) or interferon gamma (IFN-gamma) upon stimulation we used a system allowing naive T cells to be primed in vitro by specific antigen. Dense CD4+ T cells were purified from mice that expressed transgenes encoding a T cell receptor specific for pigeon cytochrome C peptide 88-104 in association with I-Ek. These T cells produced very limited amounts of IL-4 and IFN-gamma upon immediate challenge with 88-104 and antigen-presenting cells (APC). However, after an initial "priming" culture in which they were incubated for 4 d in the presence of 88-104, APC, and 1,000 U/ml IL-4, the T cells acquired the capacity to produce substantial amounts of IL-4 upon rechallenge but made very little IFN-gamma. Cells primed in the absence of IL-4 produced IFN-gamma upon rechallenge but virtually no IL-4. The inhibitory effect of IL-4 on IFN-gamma production did not appear to be mediated by the induction of IL-10 production since IL-10 addition to initial cultures did not suppress priming for IFN-gamma production, nor did anti-IL-10 block the inhibitory effect of IL-4. IFN-gamma itself did not increase priming for IFN-gamma production, nor did anti-IFN-gamma reduce such priming. IFN-gamma did, however, diminish priming for IL-4 production when limiting amounts of IL-4 (100 U/ml) were used in the initial culture. The dominant effect of IL-4 in determining the lymphokine-producing phenotype of primed cells was observed with dendritic cells (DC), activated B cells, and I-Ek-transfected fibroblasts as APC. However, the different APC did vary in their potency, with DC being superior to activated B cells, which were superior to transfected fibroblasts.

The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help.

Self-reactive B cells from tolerant double-transgenic (Dbl-Tg) mice coexpressing hen egg lysozyme (HEL) and rearranged anti-HEL immunoglobulin genes have a relatively short life span when compared to normal B cells, irrespective of whether they are exposed to antigen in multivalent membrane-bound form (mHEL-Dbl-Tg mice) or soluble form (sHEL-Dbl-Tg mice). The factors responsible for determining the fate of these B cells after encounter with self-antigen were investigated using a cell-tracking technique in which anti-HEL Ig-Tg spleen cells were labeled with the intracellular dye 5-carboxyfluorescein diacetate-succinimidyl ester (CFSE) and injected either into non-Tg recipients or a variety of HEL-Tg hosts. In non-Tg recipients, HEL-binding B cells persisted in the circulation and could be detected in the follicles of the spleen for at least 5 d. On transfer into either mHEL-Tg or sHEL-Tg hosts, they underwent activation and then rapidly disappeared from the blood and spleen over the next 3 d, consistent with the short life span reported previously. Immunohistology of spleens from sHEL-Tg recipients indicated that the transferred B cells had migrated to the outer margins of the periarteriolar lymphoid sheath (PALS), where they were detectable for 24 h before being lost. The positioning of B cells in the outer PALS depended on a critical threshold of Ig receptor binding corresponding to a serum HEL concentration between 0.5 and 15 ng/ml, but was not restricted to endogenously expressed HEL in that the same migratory pattern was observed after transfer into non-Tg recipients given exogenous (foreign) HEL. Moreover, bone marrow-derived immature Ig-Tg B cells homed to the outer PALS of sHEL-Tg mice and then disappeared at the same rate as mature B cells, indicating that the stage of maturation did not influence the fate of self-reactive B cells in a tolerant environment. On the other hand, HEL-binding B cells transferred into sHEL-Dbl-Tg recipients persisted over the 3-d period of study, apparently due to insufficient availability of antigen, as indicated by the fact that the degree of Ig receptor downregulation on the transferred B cells was much less than in sHEL-Tg recipients. If T cell help was provided to Ig-Tg B cells at the time of transfer into sHEL-Tg recipients in the form of preactivated CD4+ T cells specific for major histocompatibility complex-peptide complexes on the B cell surface, HEL-binding B cells migrated through the outer PALS of the spleen to the follicle, where they formed germinal centers, or to adjacent red pulp, where they formed proliferative foci and secreted significant amounts of anti-HEL antibody. Taken together, these results indicated that the outcome of the interaction between self-antigen and B cells is largely determined by a combination of the degree of receptor engagement and availability of T cell help.

Low affinity interaction of peptide-MHC complexes with T cell receptors.

The interaction of antigen-specific T cell receptors (TCRs) with their ligands, peptides bound to molecules of the major histocompatibility complex (MHC), is central to most immune responses, yet little is known about its chemical characteristics. The binding to T cells of a labeled monoclonal antibody to the TCR was inhibited by soluble class II MHC heterodimers complexed to different peptides. Inhibition was both peptide- and TCR-specific and of low affinity, with a KD = 4 x 10(-5) to 6 x 10(-5) M, orders of magnitude weaker than comparable antibody-antigen interactions. This finding is consistent with the scanning nature of T cell recognition and suggests that antigen-independent adhesion precedes TCR engagement.

Abelson virus transformation of an interleukin 2-dependent antigen-specific T-cell line.

Abelson murine leukemia virus (A-MuLV) carries the gene v-abl, one of a group of oncogenes with structural and functional (tyrosine kinase) homology to three growth factor receptors. Work in this and other laboratories has shown that A-MuLV infection can render myeloid and lymphoid cells independent of the growth factors interleukin 3 and granulocyte-macrophage colony-stimulating factor. We have now shown that v-abl can also relieve interleukin 2 (IL-2) dependence in T cells. We infected a cloned IL-2-dependent antigen-specific cell line. Transformed cells were generated which were factor independent and tumorigenic. The transformants each bore unique v-abl DNA inserts and expressed v-abl mRNA. No elevation of expression of either IL-2 or its receptor could be detected in these cells. Thus, A-MuLV can short-circuit the dependence of hematopoietic cells on IL-2, IL-3, and possibly granulocyte-macrophage colony-stimulating factor, none of whose receptors are known to be of the tyrosine kinase type.

Expression of T-cell receptor alpha-chain genes in transgenic mice.

To examine the influences responsible for shaping the T-cell repertoire in vivo, we have introduced T-cell receptors of defined specificity into mice. In this report, we analyze transgenic mice carrying a T-cell receptor alpha-chain gene from a pigeon cytochrome c-reactive T-cell line. A variant of this construct, which has the immunoglobulin heavy-chain enhancer inserted into the JC intron, was also introduced into mice. Addition of the enhancer increased the steady-state level of transgene-encoded mRNA three- to fivefold in cultured T cells, leading to a two- to threefold increase in surface expression. In vivo, the difference between these two constructs was even more significant, increasing the number of transgene-positive cells from approximately 5 to 70% and the T-cell receptor surface density two- to threefold. Surprisingly, while surface expression of either type of transgene was limited to T cells, we found little tissue specificity with respect to transcription. In T cells expressing the alpha chain from the enhancer-containing construct, immunoprecipitation with a 2B4 alpha-specific monoclonal antibody revealed the expected disulfide-linked dimer. Costaining of these T cells with the 2B4 alpha-specific monoclonal antibody versus anti-CD3 indicated that expression of the transgene-encoded alpha chain precludes expression of endogenous alpha chains on the majority of cells; in contrast, 2B4 alpha-chain expression from the construct lacking the enhancer is inefficient at suppressing endogenous alpha-chain expression. In mice of the enhancer lineage, Southern blot analysis indicated suppression of endogenous alpha-chain rearrangements in T-cell populations, consistent with the observed allelic exclusion at the cellular level. Interestingly, newborn, but not adult, mice of this lineage also showed an increase in retention of unrearranged delta-chain loci in thymocyte DNA, presumably resulting from the suppression of alpha-chain rearrangements. This observation indicates that at least a fraction of alpha:beta-positive T cells have never attempted to produce functional delta rearrangements, thus suggesting that alpha:beta and gamma:delta T cells may be derived from different T-cell compartments (at least during the early phases of T-cell differentiation).

The role of T cells in the regulation of B cell tolerance.

The study of conventional models of B cell tolerance has suggested that self-tolerance is imposed on B cells at an early stage in their development due to a peculiar sensitivity of immature B cells to tolerance induction. While this concept accounts for some aspects of central B cell tolerance, it is inconsistent with recent reports of tolerance induction in mature splenic B cells from immunoglobulin transgenic mice. We present an alternative model, the hierarchical model (Aust. N. Z. J. Med. 25, 761-767, 1995), in which regulation of naive B cell reactivity is a function of antigen signal strength and availability of T cell help, but is independent of B cell maturation stage. In turn, the development of tolerance or memory in the T cell compartment is dependent on a combination of antigen-MHC recognition by T cells and antigen-nonspecific signalling by antigen-presenting cells. Using a transgenic model of T-B collaboration, we have shown that both immature and mature self-reactive B cells can be rescued and induced to secrete auto-antibody if the B cell determinant is linked to a carrier protein bearing a foreign T cell determinant.

DCs and peripheral T cell tolerance.

Tolerance is a state in which the immune system as a whole fails to make an active response to antigen. Three mutually exclusive mechanisms appear to account for the fate of antigen-specific peripheral T cells within a tolerant animal: maintenance of naive status, deletion after responding to antigen, and long term survival after responding to antigen, a mechanism that should probably be considered part of the spectrum of memory responses. The types and functional status of the DCs that present antigen in each case remain controversial. This review will summarize the indirect evidence that underlies some of the hypotheses that account for peripheral T cell tolerance.

Regulation of the immune response--lessons from transgenic models.

Generation of the immune antigen receptor repertoire by means of semi-random recombination requires a mechanism for ensuring that self reactivity is constrained. A model is presented which accounts not only for the generation of self tolerance during ontogeny but also for regulation of tolerance and immunity in peripheral immune responses. The model proposes a hierarchy of immune regulation in which antigen presenting cells (APCs) determine the responses of T cells and T cells those of B cells. APCs respond to environmental triggers such as microbes, particulate antigen and tissue injury by becoming highly immunogenic for T cells. At other times, APCs can either be non-stimulatory or tolerogenic, depending on their environment. The model suggests that during ontogeny T and B cells mature in a tolerogenic environment in the thymus and bone marrow, thus ensuring that immunological tolerance results from early contact with self antigen.

The evolution of self-tolerance: a new cell arises to meet the challenge of self-reactivity.

Naive T cells can become either tolerant or immune as a result of their first encounter with antigen. It has been suggested that lymphoid and myeloid dendritic cells, respectively, control such decisions. Here, Barbara Fazekas de St Groth discusses evolutionary aspects of the functional distinction between these two types of dendritic cells.

Nature versus nurture: contributions of developmental programming and the microenvironment to B cell tolerance.

The original Burnet Lederberg and Bretscher Cohn models of immunological tolerance are essentially incompatible, one considering tolerance to be the obligatory outcome of antigen recognition by an immature lymphocyte and the other considering it as one of two possible responses to antigen, the crucial determinant being interaction with a second antigen-reactive cell. The early experimental evidence was confusing, in that it appeared to support both theories. In response to this situation, a hybrid model retaining some of the features of the original models was proposed. In particular, immature B cells were regarded as 'hypersensitive to tolerance induction', but could also make a positive response to antigen under some circumstances. More recent data from B cell transgenic mice have challenged even these hybrid models, stimulating renewed interest in the question of how B cell tolerance is regulated in vivo. This article presents a new interpretation of the data, in which the increased resistance of mature B cells to tolerance induction is postulated to result from partial receptor desensitization in response to environmental antigen, rather than from a developmentally programmed change in B cell signalling. Thus, it is suggested that Burnet's 'window of tolerance induction' is determined by the environment rather than developmental pre-programming. If this postulate is accepted, induction of B cell self-tolerance in both the bone marrow and periphery follows the simple and elegant rules originally laid down by Bretscher and Cohn.

Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody.

The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4) was hapten-specific (anti-ABA + Iak), the other (4.35F2) was alloreactive (anti-Iak). Activation of these clones by antigen, concanavalin A (Con A) or by the F23.1 antibody was studied by assaying the production of interleukin 3 (IL 3). Both soluble and solid phase-coupled F23.1 induced T cell activation in the complete absence of class II MHC, immobilized antibody (either Sepharose-coupled or plastic-adsorbed) being more effective. The induction of IL 3 production by suboptimal doses of either Con A or plastic-adsorbed F23.1 was inhibited by the anti-L3T4 antibody GK1.5, as was the response to F23.1 coupled to Sepharose-4B beads. However, the responses to optimal or superoptimal doses of these stimuli were not inhibited. In contrast, weak responses to non-TCR cross-linking stimuli such as phorbol myristate acetate (PMA) or low concentrations of soluble F23.1 were not inhibited by GK1.5 (the latter response was usually slightly enhanced). These results show that anti-L3T4 antibodies are not inherently inhibitory, but require both ligation and cross-linking of the TCR for their effect. We propose a model whereby L3T4 interacts with the TCR during T cell activation. Anti-L3T4 antibodies sterically hinder the formation of TCR complexes and so prevent activation. However, by increasing the epitope density of the activating ligand, the avidity of the T cell/ligand interaction can be increased sufficiently to prevent this disruption.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of memory and effector suppressor T cells by perinatal exposure to antigen.

The development of tolerance and suppression after perinatal exposure to antigen was studied to clarify the role of suppressor cells in self tolerance. Deaggregated human gamma globulin (dHGG) was administered transplacentally to fetal mice by injecting their mothers at various stages of pregnancy. Other mice were exposed to dHGG present in the colostrum of foster mothers previously injected with dHGG. Nonspecific suppression was seen on adoptive transfer of spleen cells from all donors less than six weeks of age. After that time, HGG-specific primary suppression by Ly-1-2,3+Ia+ T cells was detected in all animals exposed to dHGG either pre- or postnatally and persisted for up to three months. In addition, recall of specific memory suppression by antigen challenge was demonstrable for up to six months. Tolerance, on the other hand, was generated only by higher doses of dHGG in utero and was relatively short-lived when compared to suppression. The short duration of tolerance could not be attributed to the generation of T helper cells, since no significant HGG-specific helper activity was detected after perinatal exposure to HGG; rather it was thought to be due to the failure of small doses of dHGG to generate sufficient effector T suppressor (Ts) cells to inactivate the continually increasing number of HGG-specific B cells being generated during ontogeny. Since perinatal tolerance to HGG is known to be prolonged by repeated exposure to antigen, this model implies that the maintenance of natural self tolerance is Ts cell dependent and requires the continued presence of antigen.

Involvement of Lyt-2 and L3T4 in activation of hapten-specific Lyt-2+ L3T4+ T-cell clones.

The murine T-cell surface molecules Lyt-2 and L3T4 play a role in the activation of antigen-specific T cells. The currently accepted model for the function of these molecules proposes that Lyt-2 and L3T4 increase the overall avidity of the interaction between the T-cell antigen receptor and antigen in association with the major histocompatibility complex (MHC) molecules on the antigen-presenting cell. We have used two unusual Lyt-2+ L3T4+ class II MHC-restricted T-cell clones to test whether Lyt-2 can substitute for L3T4 when the T-cell antigen receptor is class II MHC-restricted. Monoclonal antibodies against L3T4 profoundly inhibited antigen-induced lymphokine production by both T-cell clones. Anti-Lyt-2 monoclonal antibody had no effect. These results strongly suggest that L3T4 and the class II-restricted T-cell antigen receptors are physically close during antigen recognition, probably as part of a multimolecular complex from which Lyt-2 is excluded. The ability of L3T4 but not Lyt-2 to participate in such a complex with class II-restricted T-cell antigen receptors may explain the striking correlation between class II restriction and L3T4 expression in the peripheral T-cell pool.