Research Group

Molecular Immunology Group Head: Immanuel F. LUESCHER, Member E-mail: immanuel.luescher@isrec.unil.ch Phone # : 41-21-692.5988/5983 Fax # : 41-21-653.4474 > Research Interests > Current Projects > Selected publications

Research Interests

CD8+ Cytotoxic T lymphocytes (CTL) play a crucial role in the eradication of cells that are transformed (e.g. cancer) or infected with intracellular pathogen (e.g. virus). Activation of these cells involves engagement of their TCR by cognate MHC-peptide complexes on target cells. The coreceptor CD8 plays a crucial role in the positive selection and activation of CD8+ T cells. On one hand, by binding to non-cognate MHC on the APC it acts as an adhesion molecule in conjugate formation. On the other hand, by binding to TCR associate MHC-peptide complexes it strengthens TCR ligand binding and brings CD8-associated p56lck (Lck) to TCR/CD3. The main interest concern a better understanding of the molecular mechanisms of TCR and CD8-mediated activation and inactivation of CD8+ thymocytes and T cells.

Current Projects

Molecular basis of CD8 coreceptor function. We found previously that the cytoplasmic tail of CD8b is palmitolyated, which allows CD8 partitioning in lipid rafts, where it associates efficiently with lck. On the other hand, CD8b mediates association of CD8 with TCR/CD3. As a result of this dual function of CD8b, a fraction of TCR/CD3 is raft-associated, which is important for TCR signaling, as rafts are privileged sites for phosphorylation events. So far we know that CD8b associates with CD3d, but it remains to be elucidated what portion of the molecules are involved and in what way. To clarify this, mice will be generated that express as transgene the T1 TCR, which is specific for Kd and the PbCS peptide 252-260 (SYIPSAEKI) modified with photoreactive 4-azidobenzoic acid (ABA) on K259 (PbCS(ABA)). This system allows assessment of TCR ligand interactions by TCR photoaffinity labeling. By crossing with CD8b -/- and CD3d -/- mice, T1TCR+, CD8b- and T1TCR+, CD3d- lines will be obtained in which engineered CD8b and CD3d genes, respectively, will be introduced. In addition to conventional examination of positive selection and activation of CD8+ T cells, the T1 system offers unique and powerful possibilities to vigorously analyze molecular mechanism. For example, using TCR photoaffinity labeling with fluorescent labeled Kd-PbCS(ABA) complexes the contribution of CD8 in TCR-ligand binding can be measured and the distribution, aggregation or association of TCR with other molecules can be directly assessed by confocal microscopy, fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). These studies should also clarify in what way changes in CD8 glycosylation and phosphorylation may affect CD8 coreceptor function.

Cooperative TCR aggregation. The extraordinary sensitivity of CD8+ CTL to recognize target cells expressing only a few cognate MHC class I peptide complexes requires CD8 and lipid raft. Since TCR typically have low affinity for MHC-peptide and fast TCR-ligand complex dissociation, an outstanding question is how scarce MHC-peptide complexes can elicit CTL activation. On resting CTL, rafts contain CD8/lck and CD8/LAT and exclude TCR/CD3, CD45 and MHC class I molecules. Coordinate binding of cognate pMHC to CD8 and TCR strengthens their binding and promotes docking of TCR/CD3 to raft resident CD8/lck complexes. In this project we investigate whether upon appropriate cross-linking, these TCR/CD8 adducts undergo cooperative aggregation. We contend that this aggregation dramatically increases the size of rafts, which in turn increases their exclusion of CD45 and MHC class I molecules, which contributes to activation of Src kinases, mainly cross-linking-mediated activation of CD8-associated lck.

To study TCR aggregation we will use various soluble mono-, di-, tetra- and octameric Kd-PbCS(ABA) complexes that contain spacers of variable and defined length and that can or cannot co-engage CD8. The prediction is that MHC-peptide complexes containing appropriate MHC-peptide distances elicit TCR aggregation, whereas complexes with too long spacers will impede it. By using fluorescent labeled MHC-peptide complexes and TCR photoaffinity labeling TCR aggregation is examined by confocal microscopy, FRET and FCS.

The role of cell adhesion for CTL activation. Recognition by CD8+ CTL of antigenic peptides bound to MHC class I molecules on target leads to sustained calcium mobilization and CTL degranulation, resulting in perforin-dependent killing. Here we show that ß1 and ß3 integrin-mediated adhesion provides crucial co-stimulation for this CTL activation. Spontaneous adhesion of CTL to fibronectin induces phosphorylation of Lck, Fyn, the focal adhesion kinase Pyk2, and the cytoskeleton linker paxillin, as well as redistribution of TCR/CD3 and other signaling molecules to rafts. The convergence of integrin- and TCR/CD8-mediated signals results in the formation of large cytoskeleton-associated rafts, that are able to integrate and amplify both signals, thus eliciting CTL degranulation.

It is well established that the ß2 integrin LFA1 plays an essential role in conjugate formation and in CTL-mediated cytotoxicity. It will be therefore of interest to elucidate also the molecular basis of LFA1-mediated costimulation. The expectation is that LFA1 functions in a similar way as b1 (and b3) integrins, but that other focal adhesion kinases, cytoskeletal linkers and adaptors are involved. Moreover, we will investigate how adhesion mediated association of TCR with focal adhesion kinases affects TCR signaling.

Inhibition of CTL mediated cytotoxicity. T cell activation by antigen is a sensitive process, which involves the detection of low density epitopes on the target cell surface. We showed previously that certain mutations in the peptide or the CDR3b loop decrease the TCR-ligand dissociation and that this is detrimental for effector function of CD8+ T cells. This project is aimed to elucidate the mechanism by which "excessive" TCR signaling inhibits CTL activation. The hypothesis is that initial over-activation leads to the recruitment of cytosolic phosphatases (e.g. SHP1, SHP2 or PEST) to signalosomes (i.e. immunological synapse), namely to adaptors like LAT, and that this impedes phosphorylation reactions and hence cell activation. Because the recruitment of cytosolic phosphatases is of high selectivity, it is assumed that they are recruited via a specific adaptor, such as CTLA4 or PAG.

Selected publications

Naeher, D., Luescher, I.F. and Palmer, E. 2002. A role for the a-chain connecting peptide motif in mediating TCR/CD8 co-operation. J. Immunol. 169:2964-2970.

Arcaro, A., Grégoire, C., Bakker, T.R., Baldi, L., Jordan, M., Goffin, L., Boucheron, N., Wurm, F., van der Merwe, P.A., Malissen, B. and Luescher, I.F. 2001. CD8b endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56lck complexes. J. Exp. Med. 194:1485-1495.

Doucey, M.-A., Legler, D.F., Boucheron, N., Cerottini, J.-C., Bron, C. and Luescher, I.F. 2001. CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts. Eur. J. Immunol. 31:1561-1570.

Kalergis, A.M., Boucheron, N., Doucey, M.-A., Palmieri, E., Goyarts, E.C., Vegh, Z., Luescher, I.F. and Nathenson, S.G. 2001. Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex. Nat. Immunol. 2:229-234.

Arcaro, A., Grégoire, C., Boucheron, N., Stotz, S., Palmer, E., Malissen, B. and Luescher, I.F. 2000. Essential role of CD8 palmitoylation in CD8 coreceptor function. J. Immunol. 165:2068-2076.

Hudrisier, D., Kessler, B., Valitutti, S., Horvath, C., Cerottini, J.-C. and Luescher, I.F. 1998. The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement. J. Immunol. 161:553-562.

Kessler, B.M., Bassanini, P., Cerottini, J.-C. and Luescher, I.F. 1997. Effects of epitope modification on T cell receptor-ligand-binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. J. Exp. Med. 185:629-640.

Luescher, I.F., Anjuère, F., Peitsch, M.C., Jongeneel, C.V., Cerottini, J.-C. and Romero, P. 1995. Structural analysis of TCR-ligand interactions studied on H-2Kd- restricted cloned CTL specific for a photoreactive peptide derivative. Immunity 3:51-63.

Luescher, I.F., Vivier, E., Layer, A., Mahiou, J. Godeau, F., Malissen, B. and Romero, P. 1995. CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes. Nature 373:353-356.