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C Pinilla

Publications and source records attributed to C Pinilla.

At least 19 recordsLinked to original sources
PubMed2001

Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands.

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

Amino Acid Motifs↗
PubMed2001

Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor-reactive cytolytic T lymphocytes.

The recent identification of molecularly defined human tumor antigens recognized by autologous CTLs has opened new opportunities for the development of antigen-specific cancer vaccines. Despite extensive work, however, the number of CTL-defined tumor antigens that are suitable targets for generic vaccination of cancer patients is still limited, mostly because of the painstaking and lengthy nature of the procedures currently used for their identification. A novel approach is based on the combined use of combinatorial peptide libraries in positional scanning format (positional scanning synthetic combinatorial peptide libraries, PS-SCLs) and tumor-reactive CTL clones. To validate this approach, we herein analyzed in detail the recognition of PS-SCLs by Melan-A-specific CTL clones. Our results indicate that, at least for some clones, most of the amino acids composing the native antigenic peptide can be identified through the use of PS-SCLs. Interestingly, this analysis also allowed the identification of peptide analogues with increased antigenic activity as well as agonist peptides containing multiple amino-acid substitutions. In addition, biometrical analysis of the data generated by PS-SCL screening allowed the identification of the native ligand in a public database. Overall, these data demonstrate the successful use of PS-SCLs for the identification and optimization of tumor-associated CTL epitopes.

Amino Acid Sequence↗
PubMed2001

Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries.

The pp65(495-503) cytotoxic T-lymphocyte (CTL) epitope from cytomegalovirus (CMV) is universally recognized among CMV+ individuals who express an allele of the human leukocyte antigen A (HLA-A*0201). The relative binding affinity of the epitope to HLA-A*0201 is moderate, and its increased activity might prove beneficial in its use as a CTL epitope vaccine. A new approach to enhance the activity of T-cell epitopes is the use of positional scanning synthetic combinatorial libraries (PS-SCLs). Using a nonamer PS-SCL, the pp65(495-503) epitope was modified after screening a CMV-specific T-cell clone (TCC) (3-3F4) from which the native peptide sequence was derived. Two peptides with amino acid substitutions at P1, P3, P7, and P8 are between 10(3) and 10(4) more active than the native epitope. Although the native CTL epitope terminates as a free acid, both tetrasubstituted peptides only function as CTL epitopes when the carboxyl terminus is amidated. Selective substitution of the native sequence based on PS-SCL screening results identified 3 amidated monosubstituted and disubstituted peptides that are better recognized than the native epitope by TCCs from a cohort expressing HLA-A*0201. In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV-infected targets among a high percentage of CMV+ individuals. Binding studies of peptide analogs with HLA-Ig (immunoglobulin) dimers and 2 different TCCs correlated with in vitro lysis results. These data suggest that increasing the activity of CTL epitopes while maintaining broad recognition is possible, which holds promise for vaccine development in infectious disease and cancer.

Antigens, Viral↗
PubMed2001

Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet beta cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528-539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC(50) > 100 microM), a larger 16-mer fragment, 526-541, shows activity in the low micromolar range (EC(50) = 2.3 microM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.

Adoptive Transfer↗
PubMed2001

Molecular mimicry and antigen-specific T cell responses in multiple sclerosis and chronic CNS Lyme disease.

The concept of molecular mimicry provides and elegant framework as to how cross-reactivity between antigens from a foreign agent with self proteins may trigger autoimmune diseases. While it was previously thought that sequence and structural homology between foreign and self proteins or the sharing of T cell receptor (TCR) and MHC-binding motifs are required for molecular mimicry to occur, we have shown that even completely unrelated peptide sequences may lead to cross-recognition by T cells. The use of synthetic combinatorial peptide libraries in the positional scanning format (PS-SCL) together with novel biometric prediction approaches has allowed us to describe the recognition profiles of individual autoreactive T cell clones (TCC) with unprecedented accuracy. Through studies of myelin-specific TCC as well as clones from the nervous system of patients suffering from chronic central nervous (CNS) Lyme disease it has become clear that at least some T cells are more degenerate than previously anticipated. These data will not only help us to redefine what constitutes specific T cell recognition, but also allow us to study in more detail the biological role of molecular mimicry. A recent clinical trial with an altered peptide ligand (APL) of one of the candidate myelin basic protein (MBP) epitopes in MS (amino acids 83-99) has shown that such a modified MBP peptide may not only have therapeutic efficacy, but also bears the potential to exacerbate disease. Thus, we provide firm evidence that the basic principles of cross-recognition and their pathogenetic significance are relevant in MS.

Amino Acid Sequence↗
PubMed2000

Dissection of bacteriophage lambda site-specific recombination using synthetic peptide combinatorial libraries.

A wide variety of tools have been used to dissect biochemical pathways, inhibitors being chief among them. Combinatorial approaches have made the search for inhibitors much more efficient. We have applied such an approach to identify hexapeptides which inhibit different steps in a site-specific recombination reaction mediated by the bacteriophage lambda integrase protein. Integrase's mechanism is still incompletely understood, in large part because several pathway intermediates remain hard to isolate. Integrase-catalyzed recombination is very efficient, but if blocked, it is highly reversible to substrates; this combination makes some intermediates exceedingly transient. We have used synthetic peptide combinatorial libraries to screen for hexapeptides that affect the recombination pathway at different stages, and have identified two families of peptides: one probably blocks DNA cleavage, the other may stabilize the Holliday junction intermediates. These peptides do not resemble parts of integrase or any of the other helper functions in the pathway. The deconvolution of hexapeptide libraries based both on inhibition of an enzymatic reaction as well as on accumulation of reaction intermediates is a novel approach to finding useful tools for dissecting a biochemical pathway.

Alanine↗
PubMed2000

Contribution of individual amino acids within MHC molecule or antigenic peptide to TCR ligand potency.

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.

Amino Acids↗
PubMed2000

Drug discovery and vaccine development using mixture-based synthetic combinatorial libraries.

The approaches and concepts that encompass combinatorial chemistry represent a paradigm shift in drug discovery and basic research. Viewed initially as a curiosity by the pharmaceutical industry, combinatorial chemistry approaches are now recognized as essential drug discovery tools that decrease the time taken for discovery and increase the throughput of chemical screening by as much as 1000-fold. Although the use of mixture-based synthetic combinatorial libraries was one of the first approaches presented, its inherent strengths are only recently being recognized. Numerous mixture-based libraries of peptides, peptidomimetics and heterocycles have been synthesized and deconvoluted using the positional scanning approach. Mixture-based library approaches for drug discovery and vaccine development will be reviewed herein.

Journal Article↗
PubMed2000

Minimal peptide length requirements for CD4(+) T cell clones--implications for molecular mimicry and T cell survival.

CD4(+) T lymphocytes usually recognize peptides of 12-16 amino acids in the context of HLA class II molecules. We have recently used synthetic peptide combinatorial libraries to dissect in detail antigen recognition by autoreactive CD4(+) T cell clones (TCC). The results of these studies demonstrated that antigen recognition by T cells is highly degenerate and that many cross-reactive ligands can be defined, some of which much more potent than the selecting autoantigen. Based on these observations, we examined the response of a myelin basic protein-specific HLA class II-restricted CD4(+) TCC to truncation variants of optimal ligands. Surprisingly, pentapeptides, tetrapeptides and even tripeptides derived from different segments of the optimal ligands were recognized by the TCC, and some were even more potent than the selecting autoantigen. In addition, these peptides enhanced the survival of the TCC at low concentration. The relevance of this finding was supported by the generation of pentapeptide-specific CD4(+) TCC from peripheral blood lymphocytes. These observations not only change existing views on the length requirements for activation of CD4(+) HLA class II-restricted T cells, but also extend our knowledge about the flexibility of TCR recognition and the potential for cross-reactivity in the immune system.

Antigen Presentation↗
PubMed1999

Immunogenicity. I. Use of peptide libraries to identify epitopes that activate clonotypic CD4+ T cells and induce T cell responses to native peptide ligands.

Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.

Amino Acid Sequence↗
PubMed1999

Exploring immunological specificity using synthetic peptide combinatorial libraries.

The definition of epitopes for human B and T cells is fundamental for the understanding of the immune response mechanism and its role in the prevention and cause of human disease. This understanding can be applied to the design of diagnostics and synthetic vaccines. In recent years, the understanding of the specificity of B and T cells has been advanced significantly by the development and use of combinatorial libraries made up of thousands to millions of synthetic peptides. The use of this approach has had four major effects: first, the definition of high affinity ligands both for T cells and antibodies; second, the application of alternative means for identifying immunologically relevant peptides for use as potential preventive and therapeutic vaccines; third, a new appreciation of the requirements for TCR interactions with peptide-MHC complexes in immunogenicity; fourth, the establishment of new principles regarding the level of cross-reactivity in immunological recognition.

Animals↗
PubMed1999

Identification of candidate T-cell epitopes and molecular mimics in chronic Lyme disease.

Elucidating the cellular immune response to infectious agents is a prerequisite for understanding disease pathogenesis and designing effective vaccines. In the identification of microbial T-cell epitopes, the availability of purified or recombinant bacterial proteins has been a chief limiting factor. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. Here, we describe a new method to effectively identify both microbial epitopes and candidate autoantigens. The approach combines data acquisition by positional scanning peptide combinatorial libraries and biometric data analysis by generation of scoring matrices. In a patient with chronic neuroborreliosis, we show that this strategy leads to the identification of potentially relevant T-cell targets derived from both Borrelia burgdorferi and the host. We also found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T-cell recognition does not preclude specificity. This approach has potential applications in the identification of ligands in infectious diseases, tumors and autoimmune diseases.

Adult↗
PubMed1998

All-D peptides recognized by an anti-carbohydrate antibody identified from a positional scanning library.

Monoclonal antibodies recognize antigens with high affinity and specificity, but the structural basis for molecular mimicry remains unclear. It is often assumed that cross-reactive antigens share some structural similarity that is specifically recognized by a monoclonal antibody. Recent studies using combinatorial libraries, which are composed of millions of sequences, have examined antibody cross-reactivity in a manner entirely different from traditional epitope mapping approaches. Here, peptide libraries were screened against an anti-carbohydrate monoclonal antibody for the identification of peptide mimics. Positional scanning libraries composed of all-l or all-d hexapeptides were screened for inhibition of monoclonal antibody HGAC 39.G3 binding to an antigen displaying N-acetyl-d-glucosamine (GlcNAc) residues on a polyrhamnose backbone. Inhibitory activity by mixtures from the all-d hexapeptide library was greater than the activity from the all-l libraries. The most active d-amino acid residues defined in each of the six positions of the library were selected to prepare 27 different individual hexapeptides. The sequence Ac-yryygl-NH2 was specifically recognized by mAb HGAC 39.G3 with a relative affinity of 300 nM when measured in a competitive binding assay. The contributions to overall specificity of the residues of the all-d peptide (Ac-yryygl-NH2) in binding to mAb HGAC 39.G3 were examined with a series of truncation, l and d-amino acid substitution, and retro analogs. Dimeric forms of the all-d peptide were recognized with tenfold to 100-fold greater affinities relative to the monomer. The all-d peptide was found to inhibit mAb HGAC 39.G3 binding to an anti-idiotype antibody with approximately 1000-fold greater affinity than GlcNAc. As demonstrated here, the study of immune recognition using combinatorial chemistry may offer new insights into the molecular basis of cross-reactivity.

Amino Acid Sequence↗
PubMed1998

Predictable TCR antigen recognition based on peptide scans leads to the identification of agonist ligands with no sequence homology.

The potential of CD4+ T cells for cross-recognition of self and foreign Ags has important implications for the understanding of thymic selection, lymphocyte survival, and the occurrence of autoimmune diseases. Here, we define the extensive flexibility of Ag recognition for three human CD4+ autoreactive T cell clones (TCC) by using ligands with single and multiple amino acid (aa) substitutions. Our results demonstrate that the spectrum of tolerated ligands and the resulting stimulatory potency of peptides for a TCC can be predicted by the relative influence of each aa. Using this approach, we have identified stimulatory ligands not sharing a single aa in corresponding positions with the Ag used to establish the TCC. These results argue for an independent contribution of each aa in the peptide sequence to the affinity of the MHC/peptide complex to the TCR.

Amino Acid Sequence↗
PubMed1998

Probing degeneracy in T-cell recognition using peptide combinatorial libraries.

Recent studies have demonstrated flexibility of the T-cell receptor (TCR) with respect to recognition of peptide bound to self major histocompatibility complex (MHC). With the introduction of peptide combinatorial libraries, it has become possible to dissect the extent of degeneracy in T-cell recognition. On the basis of these novel findings, Bernhard Hemmer and colleagues propose a conceptual framework for lymphocyte selection and survival and the occurrence of autoimmunity.

Humans↗
PubMed1998

The use of soluble synthetic peptide combinatorial libraries to determine antigen recognition of T cells.

T cells identify by their T-cell receptor (TCR) short peptides in the context of major histocompatibility complex (MHC) molecules. The interaction of the trimolecular complex composed of the TCR and MHC bound peptide was extensively studied using substitution analogs of the original peptide ligands to define those residues important for T-cell recognition in the peptide chain. This approach has led to the observation that T-cell recognition is highly flexible and that many different peptides can be recognized by an individual TCR. Others and we have recently introduced synthetic peptide combinatorial libraries (SCL) to investigate T-cell recognition. Here we review the SCL-based approaches and describe our current techniques for mapping TCR motifs for CD4+ T cells. The implications of our findings for the understanding of T-cell recognition, as well as for future applications to study T-cell responses in infectious diseases, autoimmune disorders and cancer are discussed.

Amino Acid Sequence↗
PubMed1998

Characterization of antigen-antibody interactions using single substitution analogs and mixture-based synthetic combinatorial libraries.

In an effort to use monoclonal antibodies (mAbs) as selective probes for early detection of breast cancer, the specificities of a number of antipeptide mAbs have been studied at the individual amino acid level using single substitution peptide analogs and peptide combinatorial libraries. In this study, the mapping results are presented for mAb172-12A4, which was raised against the haptenic peptide LGSGAFGTIYKG(C), corresponding to residues 138-149 of the oncogene v-erbB. This peptide is homologous with a region in epidermal growth factor receptor (EGFR) and human oncogene c-erbB-2, and contains the ATP binding motif that is common among protein kinases. The substitution profile of this interaction correlated well with the results from the screening of hexa- and decapeptide positional scanning libraries. Based on the results of this mAb's specificity for the antigenic determinant (-AFGTIYK-), proteins that have sequence homology were found from a database search of human sequences. Thirty-two unique peptide sequences, a majority of which was from protein kinases, were synthesized and tested for recognition by mAb 172-12A4. Eleven peptides had activities that differed from the original peptide by less than an order of magnitude, and the activities for 29 of the 32 (90%) could be accurately predicted based on the individual substitution analog results. While both epitope mapping approaches address the amino acid level of mAb specificity, positional scanning libraries offer an advantage of identifying the positional importance of each antigenic determinant residue without any prior knowledge of the mAb's specificity. The fine specificity mapping of peptide-specific mAbs using the synthetic tools illustrated here will be useful for the development of immunodiagnostics that detect cancer-related proteins in clinical samples.

Amino Acid Substitution↗