Measurement of MHC/peptide interactions by gel filtration.

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. The establishment of an MHC/peptide binding assay, and its subsequent use in determining the MHC binding capacities of peptide ligands, requires sufficient stocks of purified MHC and both labeled and unlabeled peptides. Accordingly, this unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. A support protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a 2-day incubation, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Two methods for separation by size-exclusion gel-filtration chromatography are described, as is data analysis.

T-cell receptor antagonists induce Vav phosphorylation by selective activation of Fyn kinase.

T cell receptor (TCR) antagonists inhibit antigen-induced T cell activation and by themselves fail to induce phenotypic changes associated with T cell activation. However, we have recently shown that TCR antagonists are inducers of antigen-presenting cell (APC)-T cell conjugates. The signaling pathway associated with this cytoskeleton-dependent event appears to involve tyrosine phosphorylation and activation of Vav. In this study, we investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in antagonist-induced signaling pathway. Antagonist stimulation increased tyrosine phosphorylation and kinase activity of Fyn severalfold, whereas little or no increase in Lck and ZAP-70 activity was observed. Second, TCR stimulation of Lck(-), Fyn(hi) Jurkat cells induced strong tyrosine phosphorylation of Vav. In contrast, minimal increase in tyrosine phosphorylation of Vav was observed in Lck(hi), Fyn(lo) Jurkat cells. Finally, study of T cells from a Fyn-deficient TCR transgenic mouse also showed that Fyn was required for tyrosine phosphorylation and activation of Vav induced by both antagonist and agonist peptides. The deficiency in Vav phosphorylation in Fyn-deficient T cells was associated with a defect in the formation of APC-T cell conjugates when T cells were stimulated with either agonist or antagonist peptide. We conclude from these results that Vav is a selective substrate for Fyn, especially under conditions of low-affinity TCR-mediated signaling, and that this signaling pathway involving Fyn, Vav, and Rac-1 is required for the cytoskeletal reorganization that leads to T cell-APC conjugates and the formation of the immunologic synapse.

Epitope affinity for MHC class I determines helper requirement for CTL priming.

We show here that priming and memory generation of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) does not require help if the immunogen binds major histocompatibility complex (MHC) class I molecules with high affinity. This conclusion was based on the study of three chemically distinct optimal length CTL epitopes with high affinity for the restriction element Kb. In contrast, when two subdominant epitopes with intermediate MHC binding affinity were studied, either a class II MHC-restricted T helper cell epitope or administration of antibody to CD40 was required to obtain significant CTL priming. Depending on the epitope, one source of help was much more efficient than the other.

TCR antagonist peptides induce formation of APC-T cell conjugates and activate a Rac signaling pathway.

T cell receptor antagonists inhibit T cell activation by antigen, and by themselves fail to induce phenotypic changes associated with T cell activation. However, they can induce limited tyrosine phosphorylation of TCRzeta chain. Here we show that TCR antagonists are potent inducers of APC-T cell conjugates, cytoskeletal reorganization, and capping of certain T cell proteins. These events are associated with a signaling pathway involving tyrosine phosphorylation of Vav and SLP-76, activation and capping of Rac-1, a protein previously linked with cytoskeletal reorganization, and activation of JNK. The finding that antagonist peptides stimulate this pathway, while failing to stimulate other TCR-mediated signaling pathways, indicates the presence in T cells of a hierarchy of signaling that is sensitive to the avidity of Ag / MHC-TCR interaction.

The stimulation of low-affinity, nontolerized clones by heteroclitic antigen analogues causes the breaking of tolerance established to an immunodominant T cell epitope.

H-2K mice injected, intravenously in saline or intraperitoneally in incomplete Freund's adjuvant, with large quantities of the immunodominant I-E(k)-restricted epitope from moth cytochrome c (MCC) 88-103 fail to respond to subsequent immunization with this epitope when administered in complete Freund's adjuvant. This state of tolerance can be broken by immunization with certain MCC 88-103 analogues that are heteroclitic antigens as assessed on representative MCC 88-103 specific T cell clones. In this paper, the mechanism of breaking tolerance by heteroclitic antigens was investigated. The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88-103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vbeta3(+)/Valpha11(+), none of the hybridomas derived from tolerance-broken animals expressed Vbeta3, although they were all Valpha11(+). Also, the Vbeta complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88-103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vbeta3(+)/Valpha11(+) transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance. Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy. Further support for this mechanism was the finding that after activation, T cells apparently have a lowered threshold with respect to the affinity of interaction with antigen required for stimulation.

Target cells for an immunosuppressive cytokine, glycosylation-inhibiting factor.

Receptors for bioactive glycosylation-inhibiting factor (GIF) were demonstrated using a bioactive mutant of recombinant human (rh) GIF, which is comparable to the suppressor T (Ts) cell-derived bioactive GIF in its affinity for the receptors on helper T (Th) hybridoma cells. Both naive T and B cells in normal mouse spleen lacked GIF receptors. However, presentation of specific antigen to naive T cells resulted in the expression of the receptors on activated T cells. Furthermore, activation of small resting B cells with F(ab')2 fragments of anti-mouse IgM plus IL-4, lipopolysaccharide (LPS) plus IL-4 or LPS plus dextran sulfate induced the expression of the receptors within 48 h of B cell stimulation. It was also found that NK T cells freshly isolated from mouse spleen, but not conventional NK cells, expressed receptors for GIF. CD4(+) and CD4(-) subpopulations of NK T cells showed a similar binding capability. Mature dendritic cells derived from bone marrow did not bear the receptors. The dissociation constant (Kd) of the interaction between the bioactive rhGIF mutant and the high-affinity receptors was 10-100 pM, whereas inactive wild-type rhGIF failed to bind to the receptors. A bioactive derivative of rhGIF suppressed both IgG1 and IgE synthesis by purified B cells activated by LPS and IL-4, indicating that the binding of bioactive GIF to its receptors on activated B cells results in suppression of their differentiation.

Fine specificity and MHC restriction of trinitrophenyl-specific CTL.

In this study, the fine specificity and MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was analyzed. Based on the structure of peptide/Kb complexes and ternary TCR/Ag/MHC complexes, four TNP peptides, two octamers, and two nonamers were chosen for eliciting anti-TNP CTL responses. Hapten was conjugated at position 4 in the octamers and at position 5 in the nonamers, positions which should allow engagement of the hapten by TCRs. Potent CTL activity for each of the TNP peptides was obtained that was highly hapten-specific; however, there were considerable differences in the extent of cross-reactivity with other TNP peptides, with the octamers generating more cross-reactive CTL than the nonamers. MHC restriction analysis suggested that anti-hapten responses were less dependent on MHC recognition than anti-peptide responses. This was evidenced by the relative ease of detecting cross-reactivity to haptenated peptides presented by allo-MHC and by the relative insensitivity of anti-hapten vs anti-peptide CTL to mutations in the Kb molecule at potential TCR interaction sites. One potential explanation for this insensitivity to MHC mutation was the finding that the anti-hapten response appeared to be of higher avidity, since a > 100-fold difference in the amount of Ag required to sensitize target cells was found between these two types of Ags.

Termination of peripheral tolerance to a T cell epitope by heteroclitic antigen analogues.

Treating mice with an immunodominant T cell epitope from moth cytochrome c (MCC(88-103)) can induce T cell unresponsiveness under certain conditions of administration. In this report, we determined whether T cell tolerance to MCC(88-103) in adult animals can be overcome by immunization with cross-reactive analogues of the tolerizing Ag. A panel of analogues of the tolerogen were tested for their capacity to terminate the tolerant state following in vivo immunization. As analyzed by their stimulatory capacity for a representative MCC(88-103)-specific T cell clone, this panel covered a wide range of cross-reactivity, including nonantigenic, antagonistic, weakly, and strongly antigenic peptides. Interestingly, only heteroclitic analogues, as measured in vitro by their enhanced antigenicity for the T cell clone that was specific for MCC(88-103), were capable of breaking tolerance. Thus, an immune response to the cross-reactive, heteroclitic analogues of tolerized self Ags may represent a mechanism by which Ag molecular mimicry operates.

Pools of lipidated HTL-CTL constructs prime for multiple HBV and HCV CTL epitope responses.

Various peptide-based approaches to simultaneous induction of multiple cytotoxic T lymphocyte (CTL) responses were evaluated as part of ongoing efforts to develop immunotherapeutic vaccines for use in humans. To this end, HLA (human histocompatibility leukocyte antigen)-A2-restricted epitopes from several specific viral proteins were tested in an HLA-A2 transgenic mouse model system, which mimics human CTL responses to these viral proteins. Multiple CTL responses were elicited by immunization with either peptides emulsified in incomplete Freund's adjuvant (IFA), or lipidated peptides administered in phosphate buffered saline (PBS). In the case of lipidated peptides, induction of CTL responses was crucially dependent on the presence of helper T lymphocyte (HTL) epitopes, and most efficient in the case of lipidated covalently linked HTL-CTL epitope constructs. CTL could also be induced by immunization with lipidated HTL epitopes simply mixed with CTL epitopes and formulated in PBS. However, this approach was highly dependent on the particular lipidated HTL/CTL combination utilized, and was marginally effective for simultaneous priming of multiple CTL responses. By contrast, all HTL/CTL combinations were potent immunogens when delivered as lipidated, covalently linked molecules. This was the most effective of the approaches analysed in terms of multi-epitope priming, as demonstrated by the induction of simultaneous CTL responses to a pool of five different epitopes.

The role of peptides in thymic positive selection of class II major histocompatibility complex-restricted T cells.

A thymic epithelial cell line transfected with I-Ek was used in reaggregate cultures to study the role of peptides in positive selection of T cell receptor transgenic thymocytes. In this system, positive selection of CD4 SP cells occurred only after the addition of exogenous peptide. Analysis of antigen analogs indicated an inverse relationship between the antigenicity for peripheral T cells and the concentration of peptide required for positive selection. These data are most consistent with an avidity (rather than an affinity) model of positive selection, in which ligand density and the affinity of T cell receptor act in concert to determine the fate of developing thymocytes.

Modulation of naive CD4 T cell activation with altered peptide ligands: the nature of the peptide and presentation in the context of costimulation are critical for a sustained response.

Altered peptide ligands containing single amino acid substitutions have the potential to be used for modulating immune function. Using a panel of moth cytochrome c peptides, we demonstrate that different phases of naive CD4 T cell response are alternately modulated depending on altered peptide ligand dose and accessory molecule expression by APC. Weak agonists presented at high concentration, and with costimulation, efficiently induced early phase naive T cell activation as assessed by IL-2R/CD69 expression, but could only promote sufficient IL-2 for a short-lived proliferative response. In contrast, strong agonists and heteroclitic peptides induced early phase T cell activation even at low concentrations with costimulation, and allowed sustained IL-2 secretion and proliferation. In the absence of accessory molecule help, early and late phase activation was impaired with weak agonists, whereas strong agonists partially compensated for a lack of costimulation for early phase activation, and also promoted enhanced IL-2 with sustained proliferation. These studies support the hypothesis that the naive T cell response will be determined by the balance between provision of accessory molecule help and the affinity of peptide/MHC complexes for individual TCRs, and suggest that extended IL-2 production is the main facet of naive CD4 activation that is affected by altering the nature of the peptide.

Several common HLA-DR types share largely overlapping peptide binding repertoires.

The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.

Derivation of HLA-A11/Kb transgenic mice: functional CTL repertoire and recognition of human A11-restricted CTL epitopes.

Transgenic mice expressing chimeric human (alpha1 and alpha2 HLA-A11 domains) and murine (alpha3, transmembrane, and cytoplasmic H-2Kb domains) class I molecules were derived. These mice were used as a model system to study the immunogenicity of human CTL epitopes and also to examine the aspects of Ag processing differences of mice vs man. Immunization of these mice with seven known HLA-A11-restricted CTL epitopes emulsified in IFA resulted in vigorous specific CTL responses. A larger panel of 45 A11-binding peptides was used to examine the relationship between immunogenicity in the HLA-A11/Kb transgenic mice and HLA-A11 binding capacity. Twenty-one of 28 (75%) peptides with high binding affinities (50% inhibitory concentration (IC50), 2-50 nM) and 7 of 13 (54%) intermediate binding peptides (IC50, 50-500 nM range) were immunogenic. In parallel, 19 of these peptides were used for in vitro primary immunizations of PBMC derived from HLA-A11 healthy human donors. It was found that 8 of 8 peptides that were able to elicit CTL in primary human in vitro cultures were also immunogenic in HLA-A11/Kb mice. Finally, HLA-A11/Kb transgenic mice were found to generate an A11/Kb restricted CTL response following immunization with influenza virus A/PR/8/34, suggesting that, at least to some extent, A11 epitopes are generated by transgenic mice as a result of natural in vivo processing and presentation.

Differential T cell signaling induced by antagonist peptide-MHC complexes and the associated phenotypic responses.

Certain changes in TCR contact residues have been shown to have profound effects on the capacity of a peptide Ag to stimulate a T cell response. Although some of these changes apparently lead to a complete loss of the ability to interact with the TCR, others result in partial agonist activity (e.g., cytokine production without proliferation) or antagonist activity (i.e., the capacity to inhibit the engagement to the TCR by Ag). We show MHC class II-restricted antagonist activity was associated with a differential pattern of early tyrosine phosphorylation events that was characterized by a preponderance of phosphorylation of low molecular mass TCRzeta and the failure to phosphorylate Zap-70. These early tyrosine phosphorylation patterns are the same as those previously described for partial agonists. Thus, a partial agonist phenotype such as anergy induction cannot be ascribed in a causal manner to this pattern of tyrosine phosphorylation. We further extend the studies of signal transduction elicited by agonist and antagonist peptides by characterizing differential recruitment of Zap-70 associated with TCRzeta isoforms and differential phosphorylation of p120 proto-oncogene c-Cbl. Another early event following TCR engagement by Ag, down-modulation of the TCR, was studied with antagonist peptides. We show that antagonist peptides do not cause TCR down-modulation. This failure may represent a mechanism by which antagonists inhibit antigen-mediated stimulation of T cells.

Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper epitopes (PADRE) for antibody responses in vivo.

Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development.

Comparison of cytotoxic T lymphocyte responses induced by peptide or DNA immunization: implications on immunogenicity and immunodominance.

To study the mechanisms that influence the immunogenicity and immunodominance of potential cytotoxic T lymphocyte (CTL) epitopes, we conducted a systematic analysis of the CTL response raised in HLA-A*0201/Kb (A2/Kb) transgenic mice against the viral antigen, hepatitis B virus polymerase (HBV pol). From a pool of 26 nonamer peptides containing the HLA-A*0201-binding motif, we selected A2-binding peptides, immunized A2/Kb animals, and tested the CTL raised against the peptide for recognition of HBV pol transfectants. Of nine immunogenic CTL epitopes, only four were recognized on HBV pol transfectants, whereas the other five were cryptic. Characterization of the peptide-specific CTL lines indicated that crypticity may result from either poor processing or low T cell receptor (TCR) avidity. To identify the immunodominant epitopes, we determined the CTL specificities induced in A2/Kb animals in response to priming with HBV pol cDNA. We obtained a response against three epitopes that were contained with the set of four epitopes recognized by peptide-specific CTL on HBV pol transfectants. Comparative analysis of cDNA priming and peptide priming revealed, therefore, the presence of a subdominant epitope. We conclude that for the HBV pol antigen, the repertoire of CTL specificities is shaped by major histocompatibility complex class I peptide binding capacity, antigen processing, and TCR availability.

Two distinct HLA-A*0101-specific submotifs illustrate alternative peptide binding modes.

Previous studies have defined two different peptide binding motifs specific for HLA-A*0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A*0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A*0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE3 submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A*0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A*0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A*0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A*0101-restricted peptide epitopes.

The minimal number of antigen-major histocompatibility complex class II complexes required for activation of naive and primed T cells.

Although previous studies have shown that 50-200 antigen-major histocompatibility complex complexes (Ag-MHC) are sufficient to stimulate significant secretion of interleukin (IL)-2 from MHC class II-restricted T cell hybridomas, there have been no studies of this nature on more physiologically relevant T cell populations. In this study we have analyzed the ligand requirements for stimulation of responses from naive and previously primed T cells derived from T cell receptor (TCR)-transgenic animals whose TCR is specific for the pigeon cytochrome c (PCC) 88-104 peptide presented by I-Ek. Primed T cells were as sensitive as the previously reported T cell hybridomas, requiring about 100 Ag-MHC complexes to synthesize readily detectable quantities of IL-2, whereas naive T cells required 15 times more ligand to produce equivalent quantities of IL-2. Similarly, primed T cells required about 40 Ag-MHC complexes to produce a significant proliferative response, whereas naive T cells required about 400 complexes. In contrast to these results, naive and primed T cells showed similar ligand requirements when early events in the T cell activation pathway were analyzed; i.e. TCR down-modulation, CD69 and CD25 expression, and blast transformation. A further analysis of IL-2 and IL-2R expression indicated: 1) The first synthesis of IL-2 was detected at the same ligand concentration in both primed and naive T cells, but primed T cells made much more IL-2 as the ligand concentrations increased; 2) primed T cells expressed about fivefold more IL-2 receptor (R) than naive T cells, despite the fact that the antigen dose-response curves with respect to the percentage of cells expressing IL-2R were identical. These results suggest that naive and primed T cells have the same threshold with respect to the number of Ag-MHC complexes required to initiate T cell activation, but that due to the inefficient expression of IL-2 and IL-2R, engagement of more complexes is needed to enable naive T cells to synthesize the necessary amounts of these two molecules to allow T cells to go through a complete cycle of replication.

Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules.

The HLA-B7-like binding supertype includes several different HLA-B molecules. Herein, the primary and secondary anchor specificities of the five most common HLA-B7-like molecules (B*0702, B*3501, B51, B*5301, and B*5401) were defined by the use of molecular binding assays, analogue peptides, and large sets of peptides corresponding to naturally occurring sequences. All five B7-like molecules analyzed preferentially bound 9-mers, with a stringent requirement for proline in position 2, while a variety of hydrophobic or aromatic residues were well tolerated at the C-terminal anchor position. Although most peptides bound in an allele-specific fashion, approximately 20% of the binders identified were degenerate and bound at least three of the five B7-like molecules analyzed with affinities of 500 nM or less. It was also noted that, in general, peptides that bind with high affinity to any given one B7-like molecule were also most frequently capable of degenerate binding. Prominent roles for secondary anchors in positions 1 and 3 were observed for most B7-like molecules, and secondary anchor motifs were utilized to derive an HLA-B7-like supermotif. The validity of this B7-like supermotif was tested by a blind prediction set. Finally, the B7-like supermotif was utilized to derive a general strategy for rationally engineering peptide analogues of naturally occurring sequences with greatly increased binding affinity and degeneracy. Such engineered supermotif binding peptides may be of significant utility in the development of peptide-based vaccines against chronic viral diseases and cancer.