Human transporters associated with antigen processing (TAPs) select epitope precursor peptides for processing in the endoplasmic reticulum and presentation to T cells.

Antigen presentation by major histocompatibility complex (MHC) class I molecules requires peptide supply by the transporters associated with antigen processing (TAPs), which select substrates in a species- and, in the rat, allele-specific manner. Conflicts between TAPs and MHC preferences for COOH-terminal peptide residues in rodent cells strongly reduce the efficiency of MHC class I antigen presentation. Although human TAP is relatively permissive, some peptide ligands for human histocompatibility leukocyte antigen class I molecules are known to possess very low TAP affinities; the significance of these in vitro findings for cellular antigen presentation is not known. We studied two naturally immunodominant viral epitopes presented by HLA-A2 that display very low affinities for human TAP. Low TAP affinities preclude minimal epitope access to the endoplasmic reticulum (ER) and assembly with HLA-A2 in vitro, as well as presentation by minigene-expressing cells to cytotoxic T lymphocytes. However, NH(2)-terminally but not COOH-terminally extended epitope variants with higher TAP affinities assemble in vitro and are presented to cytotoxic T lymphocytes with high efficiency. Thus, human TAP can influence epitope selection and restrict access to the ER to epitope precursors. Analysis of TAP affinities of a panel of viral epitopes suggests that TAP selection of precursors may be a common phenomenon for HLA-A2-presented epitopes. We also analyzed HLA-A2-eluted peptides from minigene-expressing cells and show that an NH(2)-terminally extended variant with low A2 binding affinity undergoes ER processing, whereas another with high affinity is presented unmodified. Therefore, the previously reported aminopeptidase activity in the ER can also act on TAP-translocated peptides.

CDR3 size analysis of T cell receptor V beta transcripts: follow-up study in a patient with T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is generally considered to be a clonal disorder arising as an expansion of committed lymphoid precursors. The generation of functional T cell receptor (TCR) genes involves DNA rearrangement processes. This predisposes immature lymphoid cells to abnormal rearrangements. Previous analysis of the TCR genes strongly suggested the clonal origin of human T-ALL. We present a sensitive clonal analysis of bone marrow TCR V beta transcripts in a case of T-ALL. This study allows the analysis not only of TCR V beta transcripts in tumor cells but also the temporal modification of the global TCR repertoire in the follow-up of clinical specimens from bone marrow aspirates. Moreover, we used clone-specific junctional region oligonucleotide probes corresponding to the clonal leukemic population for the molecular monitoring of the malignant clone throughout the clinical course of the disease. This molecular fingerprint appears to be a sensitive method to detect minimal residual disease. It shows that junctional regions of rearranged TCR beta genes corresponding to the tumor cells can also be detected at the time of the complete remission.

Partially modified retro-inverso pseudopeptides as non-natural ligands for the human class I histocompatibility molecule HLA-A2.

Syntheses of a series of partially modified retro-inverso analogues of the antigenic peptide M58-66 derived from the influenza virus matrix protein are reported. The retro-inverso modification phi(NH-CO) was obtained by replacement of two successive amino acid residues with a 2-substituted malonate derivative and gem-diaminoalkyl residue. The resulting compounds 1-8 were tested for their binding to the human histocompatibility class I molecule HLA-A2 in an assembly assay using lysates of peptide transporter-deficient cells T2. Specific peptide-dependent HLA-A2 assembly was revealed by an enzyme-linked immunosorbent assay. Significant HLA-A2 assembly was detected in the presence of analogues [gGly58-(S)mLeu59]-M58-66 (1a), [gGly61-(R,S)mPhe62]M58-66 (4), [gVal63-(R,S)mPhe64]M58-66 (6), and [gPhe64-(R,S)mAla65]M58-66 (7). The introduction of the retro-inverso modification between P2-P3, P3-P4, P5-P6, and P8-P9 (compounds 2, 3, 5, and 8, respectively) however led to a dramatic reduction in peptide binding to HLA-A2. Interestingly, compound 1a which contains modification between P1-P2 was found to be the most potent analogue, being able to retain the original HLA-A2 binding profile of the parent peptide M58-66. Taken together, these results and recent binding data obtained in the context of murine MHC class I molecule H-2Kd suggest that the incorporation of peptide bond surrogates in MHC class I-restricted epitopes is a useful approach to design molecules having both increased stability and high MHC-binding capacity. Depending on their agonist or antagonist effects at the T-cell receptor, such non-natural MHC ligands are likely to find many applications in the development of peptide-based vaccines or as potential therapeutic agents in the treatment of allergies and autoimmune diseases.

[Tumor cell antigenicity: cancers and vaccines]. / Antigénicité des cellules tumorales: cancers et vaccins.

Tumor-specific immunity is partly linked to the observation that cancerous cells express some proteins that are not present in normal cells. In the past, one of the major problems for the development of immunotherapy approaches has been linked to the difficulty to identify the antigens which are the targets of the tumor-specific T cells. Recent progress in fundamental immunology, particularly concerning the understanding of the molecular basis of cellular immunity, has facilitated the identification of tumor-specific antigens. These results allow to study new therapeutic approaches today.

Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes.

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210(bcr-abl) fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210(bcr-abl) is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210(bcr-abl) breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.

Generation of Melan-A/MART-1-specific CD8+ cytotoxic T lymphocytes from human naive precursors: helper effect requirement for efficient primary cytotoxic T lymphocyte induction in vitro.

This study investigates the generation of primary melanoma cell-specific cytotoxic T lymphocytes (CTLs) in vitro. Induction of peptide-specific CTLs from unfractionated naive peripheral blood mononuclear cells from HLA-A2 healthy donors was assessed using 2 recently described 9-mer epitopes from the melanoma tumor antigen Melan-A/MART-1. The need for help from CD4+ T lymphocytes for the long-lasting induction of CTLs and the capacity of the peptide-induced CTL lines to recognize many melanoma cells were evaluated. CTL lines were obtained reproducibly when CD4+ T-lymphocyte help was provided during the primary stimulation either in an autologous way, in the case of tetanus toxoid antigen (TT) responder donors, or with allogeneic TT-activated T-helper cells, separated by an insert well, in the case of tetanus toxoid non-responder donors. We also investigated helper T-cell-derived factors that are produced by TT-activated lymphocytes. Our results strongly suggest that a complex network of cytokines like interleukin-2 (IL-2), interferon-gamma, IL-6 and IL-1 exerts stimulatory effects for the initiation process of CTLs. In contrast, cytokine-like IL-4 might inhibit generation of cytolytic activity if provided by TT-activated T cells at early stages of induction. Our approach can be used to generate CTLs of a desired specificity for clinical use in adoptive immunotherapy protocols.

Dendritic cells transfected with the nef genes of HIV-1 primary isolates specifically activate cytotoxic T lymphocytes from seropositive subjects.

The HIV-1 Nef protein down-modulates surface expression of MHC class I proteins. Primary infected T lymphocytes thus escape lysis by cytotoxic T lymphocytes (CTL). In contrast, during HIV-1 infection there are strong CTL responses to several HIV proteins, and there is mounting evidence that CTL are critical for controlling the virus. The present study was carried out to assess Nef protein-cell interaction as it occurs in naturally infected antigen-presenting cells. To evaluate the presentation of peptides derived from viral antigen to CTL, we transfected nef genes obtained from peripheral blood mononuclear cells of HIV-1-seropositive subjects into dendritic cells isolated from monocytes of healthy donors. We demonstrate that expression and subsequent processing of Nef by transfected dendritic cells did not alter the presentation of an immunodominant epitope of Nef to CTL of HIV+ subjects. However, mutations in nef gene sequences from primary isolates may abolish this presentation by a mechanism that probably interferes with protein processing.

Membrane-bound Fas (Apo-1/CD95) ligand on leukemic cells: A mechanism of tumor immune escape in leukemia patients.

There is evidence from bone marrow transplantation that T cells may be involved in the immunologic control of leukemia. But many patients relapse despite a potent graft-versus-leukemia effect mediated by allogeneic T cells. The expression of the FasL protein has been suggested as a mechanism of tumor immune escape. We, therefore, evaluated the capacity of leukemic cells from patients with acute or chronic myelogenous leukemia to escape the allogeneic or autologous immune response by bearing the FasL molecule. Although almost all leukemic cells express the 37-kD form of FasL, only 54% of acute myeloblastic leukemia and 27% of chronic myeloid leukemia (CML) cells bore a FasL with killing properties, as assessed by the ability of leukemic cells to cause the apoptosis of a Fas-sensitive target cell line or autologous activated T cells in 3 tested leukemic cases. Experiments with a recombinant Fas-Fc molecule confirmed the role of Fas/FasL in leukemic-mediated cell death. Only CML leukemic cells from certain individuals contained the 26-kD truncated form of FasL. Thus, myeloid leukemic cells from some, but not all patients can set up a mechanism of immune escape involving the Fas/FasL pathway. This leukemic escape may have implications for patients eligible for adoptive cellular immunotherapy.

HLA class II antigens and the HIV envelope glycoprotein gp120 bind to the same face of CD4.

Although the HIV gp120 binding site of CD4 is well characterized, its interaction site with HLA class II molecules is still controversial. Sixty-seven mutations within the four extracellular domains of CD4 were examined for their effects on cell surface expression and conformational changes and for adhesion of HLA class II-expressing B lymphocytes and HIV gp120 binding to CD4-transfected COS cells. Mutations within the gp120 binding site affected both assays similarly, indicating that the two sites fully overlap. A few additional substitutions of residues mapping on the same face of domains 1 and 2 induced decreased rosette formation. Molecular modeling studies indicated that this is likely to be the consequence of conformational changes induced by the mutations. Thus, CD4 appears to interact with HLA class II molecules mainly through the HIV gp120 binding site and possibly through a second minor interaction site mapping on the same face of the molecule.

Antileukemic HLA-restricted T-cell clones generated with naturally processed peptides eluted from acute myeloblastic leukemia blasts.

Recent studies have shown that transfusions of HLA-compatible donor lymphocytes may induce complete remission in marrow-grafted patients with relapses of acute myeloblastic leukemia (AML). We investigated the in vitro generation of antileukemia T-cell clones obtained from the peripheral blood mononuclear cells of a partially HLA-compatible donor (HLA-A2 and B7 molecules in common with the leukemic blasts) after stimulation with a pool of naturally processed peptides extracted from leukemic blast cells collected at diagnosis from a patient with hyperleucocytosis AML. We recovered a significant quantity of peptides that bound to the HLA-A2 or HLA-B7 molecules that were able to induce cytolytic T-lymphocyte (CTL) lines and clones specific for the eluted AML peptides and restricted to the HLA-A2 or B7 molecules. Such CTL line did not recognize the patient's nonleukemic cells, and one clone was able to interact with the leukemic blasts from which the naturally processed peptides had been eluted. Such T-cell clones might provide a rationale for the development of adoptive immunotherapy and could be used to improve the efficiency of HLA-compatible T-lymphocyte transfusions and the graft-versus-leukemia response in patients with AML.

A partially modified retro-inverso pseudopeptide modulates the cytokine profile of CTL specific for an influenza virus epitope.

There is considerable evidence that peptides corresponding to MHC class I-restricted epitopes can be used as immunogens or immunomodulators. Pseudopeptides containing isosteric replacements of the amide bond provide more stable analogues, which may even have enhanced biologic activity. But there have been very few studies on the use of pseudopeptides to initiate or modulate the cellular immune response. This study describes the immunogenicity of a partially modified retro-inverso pseudopeptide of an influenza virus epitope and shows that this pseudopeptide modulates the cytokine profile expressed by CD8+ CTL generated from primed precursors. Moreover, the pseudopeptide is much more efficient at low concentration than the wild-type epitope to stimulate IFN-gamma secretion by CD8+ T effector cells. These results are analyzed with reference to changes in the conformation of the MHC molecule/peptide complex deduced from molecular modeling. The findings support the idea that partially modified retro-inverso analogues can be used as altered peptide ligands to enhance the stimulation of natural epitope-specific CTL and to modify their functional properties. Hence, pseudopeptide ligands might be promising tools for use in immunotherapy.