Print version ISSN 0716-9760
Biol. Res. vol.35 no.2 Santiago 2002
Biol Res 35: 133-137, 2002
New insights to the functional role of the T cell-Antigen
Presenting Cell immunological synapse
MUSTAPHA FAROUDI, ROSSANA ZARU, BENOIT FAVIER AND SALVATORE VALITUTTI*
INSERM U563. Institut Claude de Préval, CHU Purpan, 31059 Toulouse Cedex 3, France
Phone (33) 562 74 83 66. FAX (33) 562 74 83 86
Three innovative and complementary morphological approaches were employed to study the T cell/antigen presenting cell (APC) interaction: (i) high resolution three-dimensional confocal microscopy of the T cell-APC contact site; (ii) time lapse video recording in living T cells of [Ca2+]I and changes in distribution of various GFP fusion proteins with TCR/CD3-z complex associated- and other signaling components; (iii) measurement of lateral TCR mobility and that of recruited signaling components using techniques based on fluorescence recovery after photo-bleaching. These approaches were combined with biochemical and functional experiments to investigate two principal issues: (A) Recruitment and the three-dimensional arrangement of receptors and signaling components at the contact site between human CD4+ T lymphocytes and APCs, (B) Structure of the immunological synapse formed at the contact site between cytotoxic T lymphocytes (CTLs) and target cells. We discuss evidence indicating that TCR engagement and triggering can occur in the absence of large-scale molecular segregation into the T cell-APC contact site. Taken together our results indicate that although not required for TCR engagement and triggering, formation of the IS is important to reinforce TCR-mediated signal transduction and achieve full T cell activation.
Key terms: T cell antigen receptor, Immunological synapse, T cell antigen receptor, signal transduction.
T lymphocytes play a crucial role in the immune-surveillance against pathogens and neoplastic cells. The recognition of foreign antigens by helper or cytotoxic T lymphocytes initiates a complex cascade of events, eventually leading to the production of specific antibodies by B cells, or to the destruction of infected or neoplastic cells.
T lymphocytes are activated by the engagement of their antigen receptors with peptide-MHC complexes displayed on the surface of the antigen presenting cells (APCs). The T cell antigen receptor (TCR) is a multimeric protein complex composed of six different subunits, a, b, g, d, e and z. The ab heterodimer is responsible for antigen recognition, while the associated
CD3 chains (g, d, e) and the z homodimer are necessary for the surface expression of the receptor complex and for signal transduction (Weiss and Littman, 1994).
Antigen recognition by the T cell is exquisitely sensitive. Indeed helper T cells proliferate and produce cytokines in response to APCs displaying as few as 50-100 specific peptide-MHC complexes on their surface. Even more strikingly, cytotoxic T cells reportedly kill target cells presenting an individual antigenic complex (Harding and Unanue, 1990; Sykulev et al., 1996). Such high sensitivity of T cell responses requires very strict control to avoid exaggerated T cell activation. A key element in such control is the rapid and long-lasting down-regulation of triggered TCRs. TCR down-regulation leads to the extinction of signaling in T-APC conjugates and blunts T cell responsiveness to further antigenic stimulation (Valitutti et al., 1995; 1997).
Recent studies have provided evidence for the existence of a specialized signaling domain at the T cell-APC contact site termed supramolecular activation cluster (SMAC) or immunological synapse (IS). This domain is characterized by aggregation of TCRs, as well as accumulation of accessory molecules and intracellular signaling components in the TCR vicinity (Van der Merwe et al., 2000; Grakoui et al., 1999; Monks et al., 1998). Such molecular congregation is commonly considered a consequence of productive TCR engagement rather than a pre-requisite to TCR/peptide-MHC interaction (Delon and Germain, 2000). This notion is based on the observation that the occurrence of large-scale molecular segregation into the mature IS occurs roughly 30 minutes following T cell-APC conjugation, hence within a time frame that is not compatible with a role for the IS in initiating TCR engagement and signaling (Grakoui et al., 1999; Monks et al., 1998; Leupin et al., 2000). Conversely, it has recently been shown that IS formation may occur in resting T cells upon interaction with dendritic cells, even in the absence of both antigenic peptide and MHC (Revy et al., 2001). This observation contrasts with previous results obtained using activated T cells, and raises the question whether re-organization of accessory molecules and signaling components may pre-dispose T cells to antigen recognition.
Another unresolved issue concerning molecular segregation into the T cell-APC contact site is that relating this process to T cell biology. Indeed, even though formation of a stable molecular array at the cell-cell contact site correlates temporally with T cell activation (Monks et al., 1998), the precise function of such cellular re-organization remains unclear. It has been proposed that the IS may be required to stabilize T cell/APC interaction and thereby to help sustain signaling for a period required to subsequently achieve complete T cell activation (Delon and Germain, 2000). Alternatively, the IS may be required to generate secondary signals in T cells and/or promote other specific T cell functions, such as secretion (Davis and Van der Merwe, 2001).
We investigated the functional role of the IS in CD4+ and CD8+ T lymphocytes. We discuss here evidence suggesting that large-scale segregation of surface molecules and signaling components into the IS are not required for TCR engagement and triggering; however, these events do appear to play a crucial role in quality control and determining the magnitude of T cell responses.
A) Analysis of the three-dimensional structure of the T cell-APC immunological synapse
Role of the IS in T cell-antigen recognition
Even though the formation of a specialized signaling domain at the T cell-APC contact site has been documented, its role in T cell activation remains to be defined. We investigated the functional role of large-scale molecular segregation into the T cell-APC contact site during T lymphocyte antigen recognition. Blocking CD2/CD58 interaction in T cell-APC conjugates with anti-CD58 antibodies had several consequences. CD2, CD2AP and PKCq recruitment to the T cell-APC contact site, as well as CD45 exclusion were prevented; calcium mobilization was inhibited and sensitivity of the T cell biological response to antigenic stimulation was reduced. However, this treatment did not affect TCR internalization, activation of ERK pathway and T cell polarization towards APCs. Our results indicate that the functional role of large-scale segregation of surface molecules and intracellular signaling components into the IS of antigen stimulated T cells is required to promote assembly of the downstream signal transduction cascade, rather than to favor TCR engagement and triggering per se. This supra-molecular structure appears to be a manifestation of the manner in which signals emanating from TCR engagement and signals derived from accessory molecules converge and synergize to achieve full T cell activation (Zaru et al., 2002).
In current work, different levels within the signaling cascade at which cross-talk between TCRs and accessory molecules occur are being identified. In this respect, the PLCg-1 pathway appears to be of particular interest. Indeed, preliminary results indicated that recruitment of PLCg-1 to the signaling area and phosphorylation are dependent on formation of the IS. In addition, our data suggest the existence of a direct correlation between large-scale segregation of surface-associated molecules to the IS and translocation of signaling components into the membrane raft compartment (see Magee et al., 2002; Bender et al., 2002).
Molecular dynamics at the IS in living T lymphocytes
To improve our understanding of TCR transport to the signaling domain during T cell activation, lateral mobility of GFP-tagged TCRs on the surface of living Jurkat cell was investigated by confocal microscopy and using FRAP techniques. We observed that surface-expressed TCRs displayed a relatively fast and actin cytoskeleton independent lateral mobility. This allows single TCR molecules to diffuse over the entire T cell surface within roughly 60 minutes. Furthermore, TCRs rapidly diffuse into the contact site between T cells and polystyrene beads coated with anti-TCR antibodies. Our results indicate that the lateral mobility per se of TCRs is sufficient to ensure TCR supply to the IS during sustained T cell activation (Favier et al., 2001).
We are now applying the above described techniques to investigate the rate of recruitment of different critical signaling components to the IS in living cells. Based on our initial findings showing that the pool of phosphoinositides is continuously replenished during T cell-APC cognate interaction, (Zaru et al., 2001), we are currently characterizing phosphoinositide turnover at the T cell-APC contact site using fluorescent cellular probes. For this purpose, T lymphocytes are being transfected with fusion proteins between GFP and different pleckstrin homology (PH) domains that allow visualization of lipid distribution by confocal microscopy as well as quantification of turnover using fluorescence recovery after photo-bleaching (FRAP) techniques (Magee et al., 2002).
B) Analysis of the three-dimensional structure of the CTL-target cell immunological synapse
Recognition of specific antigens by CTLs induces two principal biological responses.
On the one hand CTLs deliver a "lethal hit" to target cells that requires only brief interaction between CTLs and those cells. Alternatively, sustained interaction between CTLs and one or more target cells results in interleukin production and CTL proliferation. The relationship between these different biological responses and the molecular dynamics occurring at the CTL/target contact site is still unclear We are presently addressing related questions by studying the molecular dynamics at the contact site between CD8+ T lymphocytes and APCs.
Initially, the antigen concentration range leading to cytotoxicity in target cells is compared with parameters characteristic of CTL activation. In parallel experiments, we are studying the molecular organization at the CTL/target interface under conditions in which either only cytotoxicity or full T cell activation are observed. We visualize: a) morphological parameters characteristic of lethal hit delivery such as orientation in CTLs of microtubules and perforin granules towards the target cell; b) the recruitment of various surface molecules and signaling components to the contact site between the two cells. Our preliminary results indicate that the three-dimensional molecular organization at the contact site between CTLs and target cells changes according to the biological responses triggered. On the one hand, at antigen concentrations in which activation of interleukin production is achieved, formation of a «classical» immunological synapse is observed (see part A). On the other hand, at antigen concentrations inducing only cytotoxicity, polarization of the lytic machinery at the CTL/target cell contact site is observed.
If confirmed the above results would indicate that lethal hit delivery by CTLs is a response that can be triggered by low antigenic stimulation and that does not require elaborate molecular re-organization at the immunolgical synapse.
Taken together the results discussed above provide new insights to the role of large-scale molecular segregation in T cell activation. We propose that in antigen stimulated T lymphocytes large-scale molecular segregation into the signaling area is not required for TCR engagement and triggering but rather is needed for fine-tuning the extent and quality of T cell responses.
In the helper T cell model (part A) we discussed how some signaling pathways and biological T cell responses are triggered in the absence of a «classical» IS. Conversely, large-scale molecular segregation and IS formation is required for full T cell activation.
In the CTL model (part B) we reviewed complementary data. Our results suggest that at very low antigen concentrations morphological parameters of lethal hit delivery are observed in the absence of large-scale molecular segregation. This is consistent with the notion that CTLs can mount a response that will lead to killing of target cells under conditions where no other parameters of activation are detectable (Valitutti et al., 1996).
In our model, large-scale molecular segregation is proposed to serve a quality control function. Segregation concentrates various molecular players in the membrane and fine tunes the balance of signal mediators, thereby contributing to the final decision whether to trigger a full response or not. Although not essential for productive TCR engagement, the IS would, according to this model, represent an important consequence of TCR engagement and possibly constitutes the structural unit in which quality and extent of T cell responses are controlled.
The work reported in this manuscript was supported by la Ligue contre le Cancer, l' Association pour la Recherche sur le Cancer, la Fondation pour la Recherche Medicale, la Fondation Gabriella Giorgi-Cavaglieri.
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*To whom correspondence should be addressed: e-mail: email@example.com
Received: June 13, 2002. In revised form: August 06, 2002. Accepted: August 07, 2002