Jean Charles Cerottini

HLA photoaffinity labeling reveals overlapping binding of homologous melanoma-associated gene peptides by HLA-A1, HLA-A29, and HLA-B44

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8 cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPTGHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibly also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to assess the ability of the homologous peptides derived from MAGE-1, −2, −3, −4a, −4b, −6, and −12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

Differential Roles of T Cell Receptor α and β Chains in Ligand Binding Among H-2Kd-restricted Cytolytic T Lymphocyte Clones Specific for a Photoreactive Plasmodium berghei Circumsporozoite Peptide Derivative

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nγ-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded β chains along with specific complementary determining region (CDR) 3β regions but diverse α chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the α chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Vα C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Vα/Jα C-, F-, and G-strands and adjacent CDR3α residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

T cell recognition of hapten. Anatomy of T cell receptor binding of a H-2kd-associated photoreactive peptide derivative

To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium bergheicircumsporozoite peptide 252–260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2β (CDR2β). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3α and CDR2β. We conclude that TCR expressing selective Vβ and CDR3α sequences form a binding domain between CDR3α and CDR2β that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.

Construction and characterization of a recombinant adenovirus directing expression of the MAGE-1 tumor-specific antigen

The finding that many human melanomas express distinct antigens that can be recognised by specific cytolytic T lymphocytes (CTL) implies that immunotherapeutic strategies against this cancer might prove effective. The ex vivo delivery of a tumour‐associated antigen to autologous cells and the subsequent re‐administration of these cells to the patient might prove effective in boosting the T cell immune response. Recombinant human adenoviral vectors provide an efficient delivery system and have many advantages over other viral and non‐viral delivery vehicles. Infection of a panel of human melanoma cell lines by AdCMVMAGE‐1, a novel recombinant adenovirus which incorporates the full‐length MAGE‐1 cDNA, was shown to induce production of high levels of MAGE‐1 protein. Incubation of transduced HLA‐A1 expressing melanoma cell lines with 2 anti‐MAGE‐1.A1 CTL clones resulted in specific recognition and lysis of target cells, indicating that the exogenous MAGE‐1 protein was processed and presented in a normal manner. Furthermore, quantitative analyses demonstrated a correlation between the efficiency of transduction and the proportion of cells lysed. Importantly for future clinical trials, stimulation of peripheral blood lymphocytes (PBLs) from a melanoma patient by AdCMVMAGE‐1‐transduced autologous cells resulted in the generation of specific CTLs against the MAGE‐1 antigen. Together, our data emphasize the utility of adenoviruses as vaccination vehicles and highlight the potential efficacy of this approach for the treatment of melanoma. Int. J. Cancer 72:1045–1055, 1997.

Relationship between expression of Fcγ receptors or la antigens and cytolytic activities of alloactivated human T cells

Abstract The relationship between expression of Ia antigens or Fcγreceptors (FcγR) and different cytolytic activities of mixed leukocyte culture (MLC)-activated T-cell populations was studied. Lymphocytes mediating specific lysis of target cells bearing the stimulating alloantigens (CTL) could clearly be distinguished from cells mediating antibody-dependent cellular cytotoxicity (ADCC) in that CTL were FcγR − while cells mediating ADCC were FcγR + . In contrast, MLC-generated natural killer (NK) cells were distributed in both the FcγR − and FcγR + cell fractions. The presence or absence of Ia antigens did not correlate with any of the cytolytic activities studied. In addition, FcγR − and not FcγR + cells responded in the secondary MLC. Thus, although FcγR + cells are generated in large numbers in MLC, they do not appear to play a direct role in specific cytotoxicity nor do they proliferate in response to secondary stimulation. However, their ability, following MLC generation, to mediate NK activity and ADCC, both of which may contribute to in vitro and in vivo lysis of allogeneic cells, might explain their appearance following allogeneic stimulation.

A Monoclonal Antibody Directed Against HLA-DR Molecules Inhibits the Interleukin-2-Dependent T Cell Proliferation in Humans

Publisher Summary This chapter discusses the mechanism of action of a monoclonal antibody (Mabs) that is directed against HLA-DR molecules. In a first set of experiments 10 4 human MLC - T cells were cultured with IL2 containing medium in the presence or the absence of D1/12 or 4F2 Mabs. In the presence of D1/12 Mab, IL2 induced T cell proliferation was markedly inhibited. Dose response analysis showed that 90% inhibition of cell proliferation was obtained in cultures containing 1.25μg of D1/12 Mab. In contrast, no inhibitory effect was observed in cultures containing 4F2 Mab or parent myeloma PX63. Similar results were obtained in 11 additional experiments involving different responder-stimulator combinations. Because human IL2 has been demonstrated to be active on activated murine T cells, one has used long-term C57BL/6 anti-DBA/2 MLC cells as a source of IL2 responding cells. These cells do not react with D1/12 Mab as assessed by quantitative immunofluorescence and complement dependent lysis.