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Tumor-specific neo-antigens that arise as a consequence of mutations (1,2) are thought to be important for the therapeutic efficacy of cancer immunotherapies (3-5). Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral [CD8.sup.+] T cells (3-7), but it is unclear whether neoantigen-specific [CD4.sup.+] T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific [CD4.sup.+] T-cell responses in tumor control (8-10), we addressed whether neo-antigen-specific [CD4.sup.+] T-cell reactivity is a common property in human melanoma., Here, we exploit oncogene-immortalized autologous B cells to measure the occurrence of [CD4.sup.+] T-cell responses against melanoma mutanomes identified by tumor exome sequencing. Using this approach, we detected neo-antigen-reactive [CD4.sup.+] [...]

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to [...]

[CD8.sup.+] T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern [...]

The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology., Most human tumor-associated antigens that are shared between individuals consist of nonmutated self antigens (1). Consequently, the endogenous T cell repertoire that reacts to these antigens will generally be small [...]

The magnitude of antigen-specific [CD8.sup.+] T cell responses is not fixed but correlates with the severity of infection. Although by definition T cell response size is the product of both the capacity to recruit naive T cells (clonal selection) and their subsequent proliferation (clonal expansion), it remains undefined how these two factors regulate antigen-specific T cell responses. We determined the relative contribution of recruitment and expansion by labeling naive T cells with unique genetic tags and transferring them into mice. Under disparate infection conditions with different pathogens and doses, recruitment of antigen-specific T cells was near constant and dose to complete. Thus, naive T cell recruitment is highly efficient, and the magnitude of antigen-specific [CD8.sup.+] T cell responses is primarily controlled by clonal expansion.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic [CD8.sup.+] T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals. epitope | T cell | CD8

Activated CD[8.sup.+] T cells detect virally infected cells and tumor cells by recognition of major histocompatibility complex class I-bound peptides derived from degraded, endogenously produced proteins. In contrast, CD[8.sup.+] T cell activation often occurs through interaction with specialized antigen-presenting ceils displaying peptides acquired from an exogenous cellular source, a process termed cross-priming. Here, we observed a marked inefficiency in exogenous presentation of epitopes derived from signal sequences in mouse models. These data indicate that certain virus- and tumor-associated antigens may not be detected by CD[8.sup.+] T cells because of impaired cross-priming. Such differences in the ability to cross-present antigens should form important considerations in vaccine design., Cytotoxic CD[8.sup.+] T cells recognize peptides that are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. Two cellular pathways lead to this form of presentation. [...]

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD[8.sup.+] CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-[gamma] mRNAs in CD[8.sup.+] populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-[gamma] in activated CD[8.sup.+] cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD[8.sup.+] cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-[gamma] mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD[8.sup.+] T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.

Protein-protein interactions are often mediated by the recognition of short continuous amino acid stretches on target proteins by specific binding domains. Affinity-based selection strategies have successfully been used to define recognition motifs for a large series of such protein domains. However, in many biological systems specificity of interaction may be of equal or greater importance than affinity. To address this issue we have developed a peptide library screening technology that can be used to directly define ligands for protein domains based on both affinity and specificity of interaction. We demonstrate the value of this approach by the selection of peptide ligands that are either highly specific for the Grb2 Src homology 2 (SH2) domain or that are cross-reactive between a group of related SH2 domains. Examination of previously identified physiological ligands for the Grb2 SH2 domain suggests that for these ligands regulation of the specificity of interaction may be an important factor for in vivo ligand selection.

Author(s): John B.A.G. Haanen [1]; Monique G.C.T. van Oijen [1]; Felicia Tirion [1]; Lauran C.J.M. Oomen [2]; Ada M. Kruisbeek [1]; Florry A. Vyth-Dreese [1, 3]; Ton N.M. Schumacher (corresponding [...]

CD40 activation has been found to overcome peptide-induced peripheral cytotoxic T-cell tolerance in vivo in mice. It also adds to anti-tumor vaccine efficacy. Whether injection of the tolerizing peptides E1A and E1B with triggering of CD40 in vivo would stop induction of peripheral CTL tolerance and thereby lead to tumor-specific CTL immunity was studied as was triggering of CD40 and its possible enhancement of therapeutic action of a vaccine shown to confer protective anti-tumor immunity.

Genetically encoded libraries of peptides and oligonucleotides are well suited for the identification of ligands for many macromolecules. A major drawback of these techniques is that the resultant ligands are subject to degradation by naturally occurring enzymes. Here, a method is described that uses a biologically encoded library for the identification of D-peptide ligands, which should be resistant to proteolytic degradation. In this approach, a protein is synthesized in the D-amino acid configuration and used to select peptides from a phage display library expressing random L-amino acid peptides. For reasons of symmetry, the mirror images of these phage-displayed peptides interact with the target protein of the natural handedness. The value of this approach was demonstrated by the identification of a cyclic D-peptide that interacts with the Src homology 3 domain of c-Src. Nuclear magnetic resonance studies indicate that the binding site for this D-peptide partially overlaps the site for the physiological ligands of this domain., For the purposes of drug discovery, there are potential advantages in the use of genetically encoded libraries, such as phage display (1, 2), 'peptide on plasmid' (3), and in vitro [...]

Analysis of the influence of TAP polymorphism on the peptide specificity and ATP dependence of TAP-regulated peptide transport in mouse reveals that TAP gene polymorphism has no affect on the function of the protein. Thus, TAP polymorphism does not interfere in the onset and progression of autoimmune diseases. The peptide specificity of the TAP transporter in mouse is similar to that of the F pocket of major histocompatibility class I molecules.

Major histocompatibility complex (MHC) class I molecules associate with peptides that are delivered from the cytosol to the lumen of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Liver microsomes of SHR and Lewis rats, which express different alleles of TAP ([cim.sup.b] and [cim.sup.a], respectively), accumulate different sets of peptides. Use of MHC congenic rats assigned this difference to the MHC, independent of the class I products expressed. Both the [cim.sup.a] and [cim.sup.b] TAP complexes translocate peptides with a hydrophobic carboxyl terminus, but translocation of peptides with a carboxyl-terminal His, Lys, or Arg residue is unique to [cim.sup.a]. Thus, the specificity of the TAP peptide translocator restricts the peptides available for antigen presentation., MHC class I molecules present peptides to antigen-specific receptors on[CD8.sup.+] T lymphocytes. These peptides are eight to ten residues in length and are acquired by newly synthesized MHC class I [...]

Foreign protein peptide recognizing by T cells enables impairment prevention. This was predominantly observed in proteins attached to the major histocompatibility complex (MHC) class 1 molecules, which are responsible for peptide-endoplasmic reticulum binding. Two MHC class II locus genes encoding proteins are essential for a substantial peptides presence. Three genes are considered transporters associated with antigen processing (TAP 1 and TAP 2), and belong to the membrane translocating ATP-binding family.

The residues involved in the interaction with TCRS were altered resulting in the conversion of a nonselecting peptide into a mixture of selecting peptides. Self-peptides were derived from C57 BL/6 and employed in the experiment which revealed that self-peptides influence the properties of CD8+ T cells. Mice lacking in the specific gene which encodes the peptide dealing with antigen processing have been found to have lower levels of MH4 class I molecular and fewer CD8+ T cells.

Cytotoxic T-cells may be activated by the presentation of a specific antigen. This presentation takes place when a competent cell degrades a foreign protein into peptide fragments, which are then bound by molecules of the major histocompatibility complex (MHC). In humans, molecules of the MHC are often called HLA antigens, or human leukocyte antigen. Molecules of the MHC are then brought to the cell surface along with their peptide fragment. The 'cytotoxic' set of T-cells are only capable of recognizing an antigen when it is properly presented by a class I histocompatibility molecule. However, an unresolved question has been whether class I molecules can be expressed on the surface of cells without the presence of a peptide antigen fragment. Researchers have shown that when mouse lymphoma cells are cultured at reduced temperatures, between 19 and 33 degrees Centigrade, class I molecules not binding antigen fragments were detected on the cell surface. However, when the temperature of these cells is raised to 37 degrees, close to body temperature, the class I molecules quickly disappeared. These results suggest that the antigen fragment has a stabilizing effect on the class I molecular complex. Without this action, the complex is unstable at body temperature, and is undetected. At lower temperatures, the complex is more stable. The use of temperature to control the expression of class I molecules will likely aid in the study of these molecules and to a better understanding of their role in the immune response. (Consumer Summary produced by Reliance Medical Information, Inc.)

Photocaged class I major histocompability complex (MHC) ligands are designed that can be cleaved by an ultraviolet (UV) light trigger in the MHC bound state under conditions that has not affected the integrity of the MHC structure. The method has allowed loading of MHCs with defined peptides in cell culture devices, thus permitting antigen-specific T cell expansion and purification for cell therapy, and it has helped in developing miniaturized assay systems for performing high-throughput screens for natural and unnatural MHC ligands.

An induced replacement strategy is designed for facilitating high-throughput structure determination of protein-ligand complexes. The approach is exemplified for the MHC class I proteins and a UV-sensitive peptide liagand is developed whose UV-induced cleavage in the crystals has led to the exchange of the low-affinity lytic fragments for full-length peptides.