Anergy in peripheral memory CD4(+) T cells induced by low avidity engagement of T cell receptor.

Induction of tolerance in self-reactive memory T cells is an important process in the prevention of autoimmune responses against peripheral self-antigens in autoimmune diseases. Although naive T cells can readily be tolerized, memory T cells are less susceptible to tolerance induction. Recently, we demonstrated that low avidity engagement of T cell receptor (TCR) by low densities of agonist peptides induced anergy in T cell clones. Since memory T cells are more responsive to lower antigenic stimulation, we hypothesized that a low avidity TCR engagement may induce tolerance in memory T cells. We have explored two antigenic systems in two transgenic mouse models, and have tracked specific T cells that are primed and show memory phenotype. We demonstrate that memory CD4(+) T cells can be rendered anergic by presentation of low densities of agonist peptide-major histocompatibility complex complexes in vivo. We rule out other commonly accepted mechanisms for induction of T cell tolerance in vivo, such as deletion, ignorance, or immunosuppression. Anergy is the most likely mechanism because addition of interleukin 2-reversed anergy in specific T cells. Moreover, cytotoxic T lymphocyte antigen (CTLA)-4 plays a critical role in the induction of anergy because we observed that there was increased surface expression of CTLA-4 on anergized T cells, and that injection of anti-CTLA-4 blocking antibody restored anergy in vivo.

HLA-DM recognizes the flexible conformation of major histocompatibility complex class II.

DM facilitates formation of high affinity complexes of peptide-major histocompatibility complex (MHC) by release of class II MHC-associated invariant chain peptide (CLIP). This has been proposed to occur through discrimination of complex stability. By probing kinetic and conformational intermediates of the wild-type and mutant human histocompatibility leukocyte antigen (HLA)-DR1-peptide complexes, and examining their reactivities with DM, we propose that DM interacts with the flexible hydrophobic pocket 1 of DR1 and converts the molecule into a conformation that is highly peptide receptive. A more rigid conformation, generated upon filling of pocket 1, is less susceptible to DM effects. Thus, DM edits peptide-MHC by recognition of the flexibility rather than stability of the complex.

Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1.

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the beta subunit designed to fill the P1 pocket from within the protein (Gly(86) to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.

Induction of T cell anergy by low numbers of agonist ligands.

Engagement of TCR by its ligand, the MHC/peptide complex, causes T cell activation. T cells respond positively to stimulation with agonists, and are inhibited by antagonist MHC/peptide ligands. Failure to induce proper conformational changes in the TCR or fast TCR/MHC dissociation are the leading models proposed to explain anergy induction by antagonist ligands. In this study, we demonstrate that presentation of between 1 and 10 complexes of agonist/MHC II by unfixed APC induces T cell anergy that persists up to 7 days and has characteristics similar to anergy induced by antagonist ligand or TCR occupancy without costimulation. Furthermore, anergy-inducing doses of hemagglutinin 306-318 peptide led to the engagement of less than 1000 TCR/CD3 complexes. Thus, engagement of a subthreshold number of TCR by either a low density of agonist/MHC or a 2-3 orders of magnitude higher density of antagonist/MHC causes anergy. Moreover, we show that anergy induced by low agonist concentrations is inhibited in the presence of IL-2 or cyclosporin A, suggesting involvement of the calcineurin signaling pathway.

Stable peptide binding to MHC class II molecule is rapid and is determined by a receptive conformation shaped by prior association with low affinity peptides.

Formation of stable class II MHC/peptide complex involves conformational changes and proceeds via an intermediate. Although this intermediate complex forms and dissociates in minutes, its conversion to a stable complex is a very slow process, taking up to a few days to reach completion. Here, we investigate the different steps of this binding and demonstrate that the conformational changes necessary to generate a receptive molecule is the rate-determining slow step in the process, while formation of the stable MHC/peptide complex is very rapid. With HLA-DR1 as our model class II molecule, we first used low affinity variants of hemagglutinin peptide (HA306-318), which lack the principal anchor, to shape the conformation of the MHC and then studied the kinetics of stable binding of HA306-318 to such an induced conformation. We found that the apparent association rate of HA306-318 is equivalent to the dissociation rate of the low affinity peptide. A 4- to 18-fold enhancement in the binding rates of HA306-318 was observed depending on the dissociation rates of the low affinity peptides. These results establish that 1) formation of stable MHC/peptide complexes is very rapid and 2) prior binding of low affinity peptide induces a receptive conformation in MHC for efficient stable peptide binding. Furthermore, in the absence of any free peptide, this receptive molecule rapidly reverts to slow binding behavior toward the subsequently offered peptide. These results have important implications for the roles of low affinity MHC/peptide complexes in Ag presentation.

Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1.

Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens.

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.

Invariant chain made in Escherichia coli has an exposed N-terminal segment that blocks antigen binding to HLA-DR1 and a trimeric C-terminal segment that binds empty HLA-DR1.

Invariant chain (Ii), a membrane glycoprotein, binds class II major histocompatibility complex (MHC) glycoproteins, probably via its class II-associated Ii peptide (CLIP) segment, and escorts them toward antigen-containing endosomal compartments. We find that a soluble, trimeric ectodomain of Ii expressed and purified from Escherichia coli blocks peptide binding to soluble HLA-DR1. Proteolysis indicates that Ii contains two structural domains. The C-terminal two-thirds forms an alpha-helical domain that trimerizes and interacts with empty HLA-DR1 molecules, augmenting rather than blocking peptide binding. The N-terminal one-third, which inhibits peptide binding, is proteolytically susceptible over its entire length. In the trimer, the N-terminal domains act independently with each CLIP segment exposed and free to bind an MHC class II molecule, while the C-terminal domains act as a trimeric unit.

Accuracy of a structural homology model for a class II histocompatibility protein, HLA-DR1: comparison to the crystal structure.

Structural homology modeling is used to test the accuracy by which a Class I major histocompatibility complex (MHC) could be used to model a Class II MHC. The crystal structure of HLA-aw68 served as a reference molecule to model HLA-DR1. The resulting model was compared to the recently released crystal structure by Brown et al. (Nature, Vol. 364, p. 33-39 (1993)). The overall tertiary structure motif (two alpha-helices and a beta-sheet forming a peptide binding cleft) was maintained. However, significant deviations in the secondary structure elements were found between the model and the DR1 crystal structure. These deviations were consistent with the differences between Class I and Class II crystal structures. In regions where the model and DR1 crystals structures are most similar, side chain orientations are also similar. Specific peptide-MHC interactions are discussed and compared with the crystal structure results.

MHC class II function preserved by low-affinity peptide interactions preceding stable binding.

Major histocompatibility complex class II molecules and their peptide ligands show unusual interaction kinetics, with slow association and dissociation rates that yield an apparent equilibrium constant of approximately 10(-6)-10(-8) M (refs 1-5). However, there is evidence for a specific, rapidly formed, short-lived complex. The altered migration on SDS-polyacrylamide gel electrophoresis of class II molecules upon stable peptide binding has led to the hypothesis that the two kinetically distinguishable types of class II-peptide complexes correspond to different structures. In accord with this model, we demonstrate here that insect cell-derived HLA-DR1 class II molecules show fast, almost stoichiometric occupancy with rapidly dissociating peptide while remaining sensitive to SDS-induced chain dissociation. The same DR1 molecules slowly and quantitatively form long-lived complexes resistant to SDS-induced denaturation. Surprisingly, low-affinity interaction with peptide protects class II from denaturation at physiological temperature, a finding that has implications for understanding the role of invariant chain in the intracellular behaviour of class II molecules.

Use of bispecific heteroconjugated antibodies (anti-T cell antigen receptor x anti-MHC class II) to study activation of T cells with a full length or truncated antigen receptor zeta-chain.

Ligand-induced activation of T cells involves recognition of monovalent peptide Ag complexed with a cell surface MHC-encoded molecule. In contrast, antibody-induced activation of T cells typically requires external cross-linking of the TCR. To examine the mechanisms that underlie the ability of these different stimuli to signal, we have created bispecific chimeric antibody molecules (BA) that mimic Ag in several important aspects. Anti-TCR-alpha, -beta, or anti-CD3-epsilon Fab fragments were covalently coupled to an anti-MHC class II Fab fragment. These BA elicited IL-2 production or proliferation from Ag-specific T cell hybridoma cells or splenic T cells, respectively, in the presence, but not the absence, of accessory cells expressing the appropriate MHC class II molecule. This response was prevented by soluble blocking antibodies against the TCR or MHC class II. When "presented" by MHC class II-bearing accessory cells, anti-TCR x anti-MHC class II BA, like cell surface Ag, elicited IL-2 production from T cell transfectants expressing full length TCR zeta-chain but not from otherwise identical cells expressing truncated zeta; when immobilized on a plastic surface these BA were potent stimulators that induced equal amounts of IL-2 from the same cells. Purified Ag/MHC complexes immobilized on plastic were able to induce IL-2 production from T cells expressing the full length, but not the truncated, form of zeta. We hypothesize that TCR-mediated T cell activation requires stable aggregation of the TCR. In this model, activation by mobile cell surface Ag/MHC or BA occurs in two steps, occupancy-induced TCR clustering followed by stable aggregation facilitated by the presence of a full length zeta-chain. Immobilized high affinity anti-TCR antibodies, but not low affinity Ag/MHC complexes, directly promote stable receptor aggregates, and thus would not require a full length zeta-chain.

The importance of dominant negative effects of amino acid side chain substitution in peptide-MHC molecule interactions and T cell recognition.

Previous studies on the role of specific residues of the peptide or MHC molecule in Ag presentation have revealed the sensitivity of this complex system to even small changes in structure. In our study, we have analyzed the effect of amino acid substitution in a major CD4+ T cell determinant (T1) of HIV-1 gp160 on binding and recognition in the context of various E alpha E beta MHC class II molecules. Individual alanine substitutions at all but three positions had little or no negative effect on either MHC binding or recognition by a specific T hybridoma, whereas substitutions with larger side chains often diminished reactivity. A poly-alanine peptide containing only four of the original residues was an effective MHC class II binder and in vivo immunogen, although lacking the ability to stimulate the hybridoma. Replacement of a glutamic acid in T1 with alanine or a size-conservative, uncharged glutamine, but not a negatively charged aspartic acid produced a peptide at least 100-fold more potent than the parent peptide, indicating an inhibitory effect of the negative charge. Conversely, substitution of a glutamic acid for valine at position 29 in the floor of the peptide binding site of the E alpha E beta molecule decreased functional presentation of this peptide by more than 2 logs. However, these two effects of glutamic acid were not complementary and were mediated by distinct mechanisms, as the change in the peptide altered the extent of binding to class II, but the change in the MHC molecule decreased recognition without inhibiting peptide binding. Taken together, the data all suggest the conclusion that changes in side-chains of peptides and MHC molecules affect Ag presentation and T cell stimulation most often by introducing dominant negative or interfering groups that prevent or alter the pattern of binding events primarily mediated by a very limited number of other residues in the Ag or presenting molecule. These results have important implications for understanding the biochemistry of peptide-MHC-TCR interactions and for the possible design of vaccines both more potent and less subject to allele-specific limitations on immunogenicity.

How MHC class II molecules work: peptide-dependent completion of protein folding.

It is now clear that peptides play a key role in stabilizing the structure of MHC class II molecules. Here, Scheherazade Sadegh-Nasseri and Ron Germain propose that newly synthesized MHC class II, and indeed class I, molecules behave like partially folded proteins, with peptides acting as a surrogate portion of the MHC polypeptide structure that is necessary for completion of conformational maturation.

A role for peptide in determining MHC class II structure.

T lymphocytes recognize antigen-derived peptides associated with major histocompatibility complex (MHC) class I or class II proteins. Peptide is critical in class I heavy-chain folding and/or stable association with beta 2-microglobulin. Although data exist suggesting a relationship between class II structure and peptide association, no equivalent positive contribution of peptide to the folding state or stability of class II dimers has yet been demonstrated. We report here that most purified E alpha k E beta k molecules leaving low pH in the absence of specific peptide lack a compact, stable dimeric structure. Brief exposure to the appropriate peptide just before and during neutralization promotes this specific conformation in proportion to stably bound peptide, indicating that peptide is important in determining class II MHC structure. Our results also indicate that efficient generation of long-lived peptide-class II complexes involves two stages: initial peptide binding in an acidic environment, which enhances the ability of class II to enter a conformation, from which stabilization upon neutralization results in high-affinity binding of previously associated peptide.

A kinetic intermediate in the reaction of an antigenic peptide and I-Ek.

Helper T cells are triggered by molecular complexes of antigenic peptides and cell surface glycoproteins of the MHC (gene products of the major histocompatibility complex) on antigen-presenting cells. There is now a lot of evidence that the complexes between isolated class II MHC molecules and selected peptides have long half-lives of approximately one day. The reported equilibrium binding constants between antigenic peptides and class II MHC molecules however, are only micromolar, suggesting that the association rate constants are very low. The only reported association rate constant is for a chicken ovalbumin peptide (OVA323-339) binding to I-Ad, and is indeed remarkably low, about 1 litre per mole per second. Prompted by these unusual data, we have used the pigeon cytochrome-c peptide pCytc(88-104) and I-E reconstituted in planar lipid bilayers on glass slides to investigate further the kinetics of peptide-MHC reactions. We report the formation of two IEk-pCytc peptide complexes. One complex has slow apparent association and dissociation kinetics, very similar to those reported previously for the chicken ovalbumin peptide and I-Ad. The second complex forms and dissociates about a hundred times more rapidly. The short-lived complex shows peptide-MHC specificity and is a kinetic intermediate in the formation of the long-lived complex; the long-lived complex is recognized by specific T-helper cells.

Immunogenic presentation of an immunoglobulin "tolerogen" requires interleukin 2.

Previously it has been reported that hapten-specific B cell unresponsiveness could be elicited by macrophages pulsed with the tolerogen deaggregated fluoresceinated sheep gamma globulin (FL-SGG; R. P. Phipps and D. W. Scott, J. Immunol. 1983. 131:2122). In contrast, FL-SGG-pulsed P388AD.2, a lymphoid dendritic cell-like tumor line, presents this signal as an immunogenic one leading to augmented anti-FL antibody responses. In the present study, we examined the role of T cells and their secreted lymphokines in the immunogenic presentation of FL-SGG by P388AD.2. Lymphocytes and FL-SGG-pulsed P388AD.2 form large clusters in vitro. In order to examine whether the clustered lymphocytes were responsible for the augmented antibody responses, cultures of P388AD.2 and lymphocytes were separated into P388AD.2 adherent (clustered) and nonadherent fractions. Interestingly, the clustered fraction was entirely responsible for the augmented responses and contained Ly-1+ T cells and B cells primed by FL-SGG on P388AD.2. Moreover, a requirement for T cells in the presentation of a normally tolerogenic signal as an immunogenic one was demonstrated as responses of T-depleted spleen cells could be reconstituted by addition of normal T cells or by an autoreactive T cells clone. Furthermore, the requirement for T cells could be bypassed using supernatant from concanavalin A-stimulated spleen cells or by culture supernatant from AOFS, an IL 2-secreting T cell hybridoma. This suggested that a cognate interaction between T and B cells was not required to induce B cell priming and the augmented anti-FL antibody responses. Further studies revealed that doses of recombinant IL 2 greater than 12.5 units/ml, in conjunction with FL-SGG-pulsed P388AD.2, replaced the need for T cells. Overall, our data suggest that one mechanism of presentation of FL-SGG in an immunogenic fashion involves T cell secretion of IL2 by autoreactive T cells triggered by close association with P388AD.2.

Selective reversal of H-2-linked genetic unresponsiveness to lysozymes. II. Alteration in the T helper/T suppressor balance, owing to gene(s) linked to Ir-2, leads to responsiveness in BALB.B.

Immune responsiveness to lysozyme in H-2b mice is under the control of H-2-linked Ir genes, with T suppressor (Ts) cells playing a dominant role in strains such as C57BL/6 (B6), C57BL/10 and A.BY. However, non-H-2-genes were found to be capable of specific reversal of the effect of the H-2-linked genes in responsiveness to chicken lysozyme (HEL), but not to human lysozyme (HUL). Therefore, studies were performed to identify any lesion in the suppressor circuit in BALB.B. It was known that HUL-induced suppressor cells could cross-suppress the anti-HEL response in B10.Q mice, which are responsive to HEL but nonresponsive to HUL. Similarly, BALB.B Ts cells were able to suppress the anti-HEL response, using as T helper (Th) source a T cell line (BB-1), derived from HEL-primed BALB.B periaortic and inguinal lymph node cells. A protocol designed to examine the in vivo suppression by the use of HUL-induced suppressor cells also demonstrated a significant suppression of the anti-HEL response. Since the suppressive circuitry seemed intact in the BALB.B, the possibility was examined that a step in T-B cell collaboration was more efficient in this strain than in the B6 nonresponder. With a B6-derived HEL-specific T cell line, BO1H, the B cell and antigen-presenting (B/APC) populations from B6 required addition of concanavalin A supernatant for anti-HEL antibody formation, whereas BALB.B B/APC were capable of responding to HEL in culture without the addition of concanavalin A supernatant. In agreement with this finding, when B/APC cell populations from BALB.B and B6 were compared for their extent of anti-HEL responsiveness, as measured with BB-1 Th cells, BALB.B B/APC populations responded significantly higher than B6 populations when the responses were activated by picogram/nanogram amounts of HEL. The response level of (BALB.B X B6)F1 B/APC measured in the same assay resembled that of B6. However, when HEL was used at the microgram level, both B6 and BALB.B strains responded equivalently. The above data are consistent with the expression of the reversing non-H-2 Ir gene(s) resulting from the balance of antigen presentation to Th and Ts cells in the H-2b mouse. In the B6, processing and handling of antigen may be inefficient in activating response-enhancing Th, and more effective in triggering Ts cells, while the reverse may be true for the BALB.B.

Selective reversal of H-2 linked genetic unresponsiveness to lysozymes. I. Non-H-2 gene(s) closely linked to the Ir-2 locus on chromosome 2 permit(s) an antilysozyme response in H-2b mice.

Genes outside of the mouse major histocompatibility complex (H-2) were found to be capable of specifically reversing the previously described nonresponsiveness to hen egg-white lysozyme (HEL) owing to H-2b immune response (Ir) genes. C3H.SW, BALB.B, and C57L, all of the H-2b haplotype, showed responsiveness to HEL, but not to human lysozyme (HUL). Mapping of the reversing gene(s) was attempted by testing H-2b recombinant inbred (RI) strains of mice carrying C3H, BALB, and C57L non-H-2 genes. Analysis of the strain distribution pattern of responsiveness with both CXB and BXH RI strains was consistent with the location of the responsible site within the H-3 region on chromosome 2. The anti-HEL proliferative responsiveness in two H-3 congenic strains of mice, B10.C(28NX)SN and B10.C-H-3cH-3a, that have BALB/c genes within the H-3 region confirmed the mapping, as well as localized the reversing gene(s) near the Ir-2 gene. The data are discussed with regard to the site of expression of the reversing gene(s) and its mechanism of action.