The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the beta cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.     

The T cell surface protein, CD28

The cluster of differentiation (CD) antigen CD28 is a 44-kDa, disulphide-bonded, homodimeric glycoprotein, which is constitutively expressed on the surface of all murine T cells and the majority of human T cells. Ligation of CD28 by its counter receptor, B7, expressed on the surface of antigen presenting cells, has been shown to induce signals that, in synergy with those derived from engagement of the T cell receptor by an antigen bound to a major histocompatibility complex, enhance proliferation and cytokine production. Manipulation of this interaction can have dramatic effects on the outcome of T cell activation. Blocking CD28/B7 interactions may be useful in preventing unwanted activation in allergy and autoimmune diseases, whereas enhancing this interaction can promote tumour rejection. Thus, CD28 and its signalling pathways may prove to be useful targets in the development of new therapeutic treatments.     

SPC3, a V3 loop-derived synthetic peptide inhibitor of HIV-1 infection, binds to cell surface glycosphingolipids

Synthetic multibranched peptides derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120 inhibit HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms: competitive inhibition of HIV-1 binding to CD4-/GalCer+ colon cells and postbinding inhibition of HIV-1 fusion with CD4+ lymphocytes. In the present study, we have characterized the cellular binding sites for the V3 peptide SPC3, which possesses eight V3 consensus motifs GPGRAF radially branched on a neutral polyLys core matrix. These binding sites are glycosphingolipids that share a common structural determinant, i.e., a terminal galactose residue with a free hydroxyl group in position 4: GalCer/sulfatide on CD4-/GalCer+ colon cells; LacCer and its sialosyl derivatives GM3 and GD3 on CD4+ human lymphocytes. These data suggest that the V3 peptide binds to the GalCer/sulfatide receptor for HIV-1 gp120 on HT-29 cells and thus acts as a competitive inhibitor of virus binding to these CD4- cells, in full agreement with previously published virological data. In contrast, SPC3 does not bind to the CD4 receptor, in agreement with the data showing that the peptide inhibits HIV-1 infection of CD4+ cells by acting at a postattachment step. The binding of SPC3 to LacCer, GM3, and GD3, expressed by CD4+ lymphocytes, suggests a role for these glycosphingolipids in the fusion process between the viral envelope and the plasma membrane of CD4+ cells. Since the multivalent peptide can theoretically bind to several of these glycosphingolipids, we hypothesize that the resulting cross-linking of membrane components may affect the fluidity of the plasma membrane and/or membrane curvature, altering the virus-cell fusion mechanism.     

Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development

The T cell coreceptors CD4 and CD8 enhance T cell responses to TCR signals by participating in complexes containing TCR, coreceptor, and MHC molecules. These ternary complexes are also hypothesized to play a seminal role during T cell development, although the precise timing, frequency, and consequences of TCR-coreceptor-MHC interactions during positive selection and lineage commitment remain unclear. To address these issues, we designed transgenic mice expressing mutant I-Ek molecules with reduced CD4-binding capability. These transgenic lines were crossed to three different lines of I-Ek-specific TCR transgenic mice, and the efficiency of production of CD4+ lineage cells in the doubly transgenic progeny was assessed. Surprisingly, replacing wild-type I-Ek molecules with these mutant molecules did not affect the production of CD4+CD8- thymocytes or CD4+ peripheral T cells expressing any of the three TCRs examined. These data, when considered together with other experiments addressing the role of coreceptor during development, suggest that not all MHC class II-specific thymocytes require optimal and simultaneous TCR-CD4-MHC interactions to mature. Alternatively, it is possible that these particular alterations of I-Ek do not disrupt the CD4-MHC interaction adequately, potentially indicating functional differences between I-A and I-E MHC class II molecules.     

A peptide binding weakly to the major histocompatibility molecule augments T cell responses

An I-A(d)-derived peptide PB1 was found to enhance the reactivity of I-A(d)-restricted T cells. The augmentative effect was not due to the cross-reactivity of PB1 peptide with antigens. PB1 had no effect on T cells specific for I-A(b) and I-E(k), nor did PB1 increase the T cell responses to concanavalin A and staphylococcal enterotoxin B. The strict I-A(d) specificity suggests that PB1 enhances the recognition of antigen-I-A(d) complex by T cell receptor. PB1 bound to I-A(d) weakly. The augmentative effect could be found on other I-A(d)-binding peptides in appropriate conditions; however, PB1 was distinct in its prominently augmentative effect on all the I-A(d)-restricted T cells analyzed. A similar enhancing activity was demonstrated on a synthetic transferrin receptor peptide with minimum affinity for I-A(d). The unusual enhancing activity of PB1 may thus be attributed to the low I-A(d) binding affinity. It was postulated that the binding of low-affinity PB1 would not only stabilize I-A(d) structure, but also enhance the binding of other peptides. This was supported by the increased binding of OVA 323-339 and cI 84-98 to I-A(d) in the presence of PB1. The inclusion of PB1 in the immunization mixture also enhanced T cell responses in vivo, suggesting the possibility of using low-affinity peptide to promote specific immunity.     

The CD7- T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA- T cells

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7- T cells express the CD4CD45RO+CD45RA- memory phenotype. We analysed CD4+CD45RA- peripheral blood lymphocytes that were separated into CD7+ and CD7- for their in vitro cytokine secretion in response to different stimuli. The CD4+CD7- subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+CD7- subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-gamma) secretion was not significantly different in CD7+ and CD7- T cells upon stimulation in vitro. The data indicate that the CD4+CD7- T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA- memory T cells. Since CD4+CD7- T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7- T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.     

Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface

Tissue factor pathway inhibitor (TFPI) is one of the main regulators of the tissue factor (TF) pathway of coagulation. To tether human TFPI to the cell surface, full length or truncated TFPI lacking the third Kunitz domain were fused with domains three and four and the carboxy-terminal sequence of human CD4. Constructs were transfected into a mouse fibroblast cell line and individual clones were checked for expression using monoclonal antibodies directed against the first two TFPI Kunitz domains and against CD4. Specific human FXa binding was detected by flow cytometry using an anti-FX polyclonal antibody, and inhibition of FXa proteolytic activity was verified by chromogenic substrate assay using S-2765. In addition, TFPI-CD4-expressing cells, preincubated with FXa, specifically bound human TF-FVIIa complexes as revealed with an anti-human TF polyclonal antibody. No functional difference was observed between full length or truncated TFPI-CD4. These results demonstrate that functionally intact TFPI can be tethered to the cell surface. Genetic manipulation of, for example, endothelial cells leading to the stable expression of TFPI may inhibit the development of coronary artery heart disease following cardiac allotransplantation, and may inhibit thrombosis in the context of xenotransplantation.     

Structure-activity studies of peptide YY(22-36): N-alpha-Ac-[Phe27]PYY(22-36), a potent antisecretory peptide in rat jejunum

Peptide YY (PYY) and its homologous peptide, neuropeptide Y (NPY), are known to exhibit potent antisecretory effects in the intestine. To determine the structural requirements to elicit antisecretory effects, we have synthesized several analogs of the PYY active site, PYY(22-36), and compared their binding affinities and antisecretory potencies in rat jejunum. These investigations revealed that the hydroxyl groups of Ser23 and Thr32, as well as the imidazole group of His26, are important for activity in the intestine. N-alpha-acetylation of PYY(22-36) increased both the binding affinity and antisecretory potency. Structure-activity studies with N-alpha-Ac-PYY(22-36) showed that substitution of His26 with parachlorophenylalanine (pCl-Phe) or Tyr36 with N-Me-Tyr reduced receptor affinity, while replacement of Tyr27 with Phe increased the activity substantially. Furthermore, acylation of the alpha-NH2 group with hydrophobic groups, myristic and naphthaleneacetic acids, substantially reduced the antisecretory potencies but not the binding affinities. Further modification of N-alpha-Ac-[Phe27]PYY(22-36) may lead to the development of more potent agonist compounds, which may provide a framework for the design of a new class of antidiarrheal drugs.     

Production of [Asn1, Val5] angiotensin II and [Asp1, Val5] angiotensin II in kallikrein-treated trout plasma (T60K)

Incubation of heat-denatured plasma from the rainbow trout Oncorhynchus mykiss with porcine pancreatic kallikrein generates, in addition to bradykinin-related peptides, previously uncharacterized peptides that contract mammalian and amphibian vascular smooth muscle. Using rings of vascular smooth muscle from the bullfrog systemic arch as bioassay, we have isolated two myotropic peptides whose primary structures were established as: Asn-Arg-Val-Tyr-Val-His-Pro-Phe ([Asn1, Val5]angiotensin II) and Asp-Arg-Val-Tyr-Val-His-Pro-Phe ([Asp1, Val5]angiotensin II). These peptides are the same as those generated in salmon plasma by an extract of kidney. The data raise the possibility that activation of the kallikrein-kinin system in trout generates both bradykinin-related and angiotensin II-related peptides that may act synergistically in the regulation of blood pressure.     

Requirement of CD4 T cells for skin graft rejection against thymus leukemia (TL) antigen and multiple epitopes on the TL molecule recognized by CD4 T cells

Two HPLC assays were developed and validated for simultaneous quantitation of two sulfate metabolites, PD 163637 (VI) and PD 163639 (VIII), of an investigational antipsychotic drug CI-1007 (I) in monkey plasma and urine. VI and VIII were identified as major metabolites in monkey plasma, and both were excreted in urine. Monkey plasma samples were directly injected after deproteinization, and urine samples were analyzed after a clean-up procedure using methyl-tert.-butyl ether. Liquid chromatographic separation was achieved on a Zorbax RX C8 analytical column using gradient elution. Column effluent was monitored using fluorescence detection with excitation and emission wavelengths of 254 and 330 nm, respectively. Minimum quantitation limit was 50 ng/ml in plasma and 100 ng/ml in urine. Linearity was demonstrated up to 3000 ng/ml in plasma and urine. Recoveries of the analytes from plasma and urine were greater than 85%. The assay has been applied to the determination of VI and VIII in plasma and urine samples from monkeys receiving oral administration of I.     

CD8 inhibits signal transduction through the T cell receptor in CD4-CD8- thymocytes from T cell receptor transgenic mice reconstituted with a transgenic CD8 alpha molecule

T cell repertoire selection processes involve intracellular signaling events generated through the TCR. The CD4 and CD8 coreceptor molecules can act as positive regulators of TCR signal transduction during these developmental processes. In this report, we have used TCR transgenic mice to determine whether TCR signaling can be modulated by the CD8 coreceptor molecule. These mice express on the majority of their T cells a TCR specific for the male (H-Y) Ag presented by the H-2Db MHC class I molecule. We show that CD4-CD8-, but not CD4-CD8+, thymocytes expressing the H-Y TCR responded with high intracellular calcium fluxes to TCR/CD3 stimulation without extensive receptor cross-linking. To examine the effects of CD8 expression on intracellular signaling responses in the CD4-CD8- cells, the H-Y TCR transgenic mice were mated with transgenic mice that constitutively expressed the CD8 alpha molecule on all T cells. The expression of the CD8 alpha alpha homodimer in the CD4-CD8-thymocytes led to impaired intracellular calcium responses and less efficient protein tyrosine phosphorylation of substrates after TCR engagement. In male H-2b H-Y transgenic mice, the majority of thymocytes have been deleted with the surviving cells expressing a high density of the transgenic TCR and exhibiting either a CD4-CD8- or CD4-CD8lo phenotype. It has been postulated that these cells escaped deletion by down-regulating the CD8 molecule. In the H-Y TCR/CD8 alpha double transgenic male mice, the CD4-CD8lo cells were completely eliminated as a result of CD8 alpha expression. However, the CD4-CD8- T cells were not deleted despite normal levels of the CD8 alpha transgene expression. These results suggest that the CD4-CD8- thymocytes may not be susceptible to the same deletional mechanisms as other thymocytes expressing TCR-alpha beta.     

Pocket 4 of the HLA-DR(alpha,beta 1*0401) molecule is a major determinant of T cells recognition of peptide

To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alpha,beta 1*0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1*0401 chain or 19 positions of the DR alpha chain were used as antigen-presenting cells for five T cell clones specific for the influenza hemagglutinin peptide, HA307-19. Of the six polymorphic positions in the DR beta floor that were examined, mutations at only two positions eliminated T cell recognition: positions 13 (four clones) and 28 (one clone). In contrast, individual mutations at DR beta positions 70, 71, 78, and 86 on the alpha helix eliminated recognition by each of the clones, and mutations at positions 74 and 67 eliminated recognition by four and two clones, respectively. Most of the DR alpha mutations had minimal or no effect on most of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants. Mutants that abrogated recognition by all of the clones were assessed for peptide binding, and only the beta 86 mutation drastically decreased peptide binding. Single amino acid substitutions at polymorphic positions in the central part of the DR beta alpha helix disrupted T cell recognition much more frequently than substitutions in the floor, suggesting that DR beta residues on the alpha helix make relatively greater contributions than those in the floor to the ability of the DR(alpha,beta 1*0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in pocket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues.     

The orientation of a T cell receptor to its MHC class II:peptide ligands

The TCR found on CD4 T cells recognizes peptides bound to self MHC class II molecules as well as non-self MHC class II molecules. We have used the receptor on a cloned T cell line called D10.G4.1 (D10) to perform a structure-function analysis of this interaction. The D10 T cell clone recognizes not only a peptide from conalbumin (CA-wt) bound to syngeneic I-Ak against which it was raised, but also the allogeneic MHC molecules I-A(b,v,p,q,d). In the present study, we show that residue 30 in complementarity-determining region 1 (CDR1) of the TCR alpha-chain interacts with the I-A alpha-chain at hvr2 (residues 52, 53, and 55). We also show that residue 51 in CDR2 of the TCR alpha-chain interacts with the peptide at peptide residue 2. Finally, we show that residue 29 in CDR1 of the TCR beta-chain affects recognition of the glutamic acid at residue 66 in the I-A beta-chain. These data suggest an orientation of TCR relative to its peptide:MHC class II ligands. We argue that this orientation will be shared by all CD4 TCRs, and that it is only subtly different from the common orientation proposed for receptors binding to MHC class I.     

The recognition of the nonclassical major histocompatibility complex (MHC) class I molecule, T10, by the gammadelta T cell, G8

Recent studies have shown that many nonclassical major histocompatibility complex (MHC) (class 1b) molecules have distinct antigen-binding capabilities, including the binding of nonpeptide moieties and the binding of peptides that are different from those bound to classical MHC molecules. Here, we show that one of the H-2T region-encoded molecules, T10, when produced in Escherichia coli, can be folded in vitro with beta2-microglobulin (beta2m) to form a stable heterodimer in the absence of peptide or nonpeptide moieties. This heterodimer can be recognized by specific antibodies and is stimulatory to the gammadelta T cell clone, G8. Circular dichroism analysis indicates that T10/beta2m has structural features distinct from those of classical MHC class I molecules. These results suggest a new way for MHC-like molecules to adopt a peptide-free structure and to function in the immune system.     

The interaction between CD4 and MHC class II molecules and its effect on T cell function

During T cell activation, CD4 and CD8 form a 'bridge' between the T cell receptor (TCR) and major histocompatibility complex (MHC) class II and class I molecules, respectively. Due to this intimate association, CD4 and CD8 are now termed co-receptors and considered an integral part of this multimolecular complex. In addition, interest in CD4 has been heightened by the discovery that it is, in part, the receptor for HIV. Although CD4 and CD8 appear to perform similar immune functions, they are structurally diverse suggesting that their mode of interaction with the TCR and MHC molecules may differ. This review will focus primarily on a series of studies which have attempted to map the residues which mediate CD4:MHC class II interaction. These data will be evaluated in light of our current understanding of CD8:MHC class I, and CD4:TCR interactions. In addition, a model to explain the structural and functional differences between CD4 and CD8 will be presented. Finally, the potential effect of these multiple interactions on T cell function will be discussed.     

2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48

2B4 is a cell surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T lymphocyte function. A recombinant protein containing the extracellular region of mouse (m)2B4 attached to avidin-coated fluorescent beads bound to rodent cells, and binding was completely blocked by CD48 monoclonal antibodies (mAbs). Using surface plasmon resonance, we showed that purified soluble mCD48 bound m2B4 with a six- to ninefold higher affinity (Kd approximately 16 microM at 37 degreesC) than its other ligand, CD2. Human CD48 bound human 2B4 with a similar affinity (Kd approximately 8 microM). The finding of an additional ligand for CD48 provides an explanation for distinct functional effects observed on perturbing CD2 and CD48 with mAbs or by genetic manipulation.     

Following antigen challenge, T cells up-regulate cell surface expression of CD4 in vitro and in vivo

The low precursor frequency of Ag-specific T cells has raised significant barriers to studying the T cell response in vivo. We demonstrate that T cells up-regulate the cell surface expression of CD4 following Ag recognition, which identifies Ag-specific T cells in vitro and in vivo and allows their characterization. The CD4high cell subpopulation contains the Ag-specific population as indicated by Ag-induced proliferation and limiting dilution analyses. The use of the CD4high marker will allow analysis of the dynamics of the T cell immune response in vivo, the study of the suboptimal T cell response to Ag, and the identification of T cells which are reactive to known and unknown autoantigens.     

Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE)

T cell receptor (TCR)-recognizing regulatory cells, induced after vaccination with self-reactive T cells or TCR peptides, have been shown to prevent autoimmunity. We have asked whether this regulation is involved in the maintenance of peripheral tolerance to myelin basic protein (MBP) in an autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). Antigen-induced EAE in (SJL x B10.PL)F1 mice is transient in that most animals recover permanently from the disease. Most of the initial encephalitogenic T cells recognize MBP Ac1-9 and predominantly use the TCR V beta 8.2 gene segment. In mice recovering from MBP-induced EAE, regulatory CD4+ T cells (Treg) specific for a single immunodominant TCR peptide B5 (76-101) from framework region 3 of the V beta 8.2 chain, become primed. We have earlier shown that cloned B5-reactive Treg can specifically downregulate responses to Ac1-9 and also protect mice from EAE. These CD4 Treg clones predominantly use the TCR V beta 14 or V beta 3 gene segments. Here we have directly tested whether deletion/blocking of the Treg from the peripheral repertoire affects the spontaneous recovery from EAE. Treatment of F1 mice with appropriate V beta-specific monoclonal antibodies resulted in an increase in the severity and duration of the disease; even relapses were seen in one-third to one-half of the Treg-deleted mice. Interestingly, chronic disease in treated mice appears to be due to the presence of Ac1-9-specific T cells. Thus, once self-tolerance to MBP is broken by immunization with the antigen in strong adjuvant, TCR peptide-specific CD4 Treg cells participate in reestablishing peripheral tolerance. Thus, a failure to generate Treg may be implicated in chronic autoimmune conditions.