S. Valitutti

  Molecular dynamics of lymphocyte interactions

 

 

 

Our research project at a glance

Immune responses are based on multiple and dynamic interactions among cells of the immune system resulting in the formation of specialized signaling areas, named immunological synapses. Our team applies a combination of cutting-edge approaches, including rapid live-cell imaging and super-resolution microscopy (on isolated cells and human tissue slices) together with mathematical modeling, to visualize and interpret the molecular dynamics occurring at immunological synapses. Deciphering intercellular communication at immunological synapses may contribute to design innovative therapeutic strategies aiming at modulating immune responses in human disease.

Our two major lines of research:

Research Axis 1: On the fight between cytotoxic T lymphocytes and cancer cells at the immunological synapse

We dissect the architecture and dynamics of the lytic synapses formed at the contact site between cytotoxic T lymphocytes (CTL) and tumor target cells.

We have recently shown that CTL-mediated cytotoxicity is composed of two-phases:

  1. an extremely rapid killing phase occurring within seconds after the encounter of the target cell and preceding immunological synapse formation (Fig. 1),

    Figure 1. Millisecond-scale microtubule reorganization at the CTL/target cell lytic synapse.
    A CTL loaded with Tubulin Tracker Green (to visualize microtubules) is shown during interaction with a cognate target cell previously loaded with Fluo-4 AM (to detect [Ca2+]i increase upon perforin-mediated pore formation). Panels show the rapid re-organization of tubulin cytoskeleton upon CTL/target cell contact that is accompanied by the rapid entry of Ca2+ into target cell cytosol. Images were collected using a spinning-disk confocal microscope. The pseudo color scale indicates fluorescence intensity.

  2. a late sustained killing phase allowing a few CTL to emerge as super-killers and therefore annihilate multiple target cells.

We have also shown that clinically aggressive cancers such as melanomas “speak back” to CTL at the lytic synapse by deploying defense mechanisms that neutralize the CTL lytic attack (Fig. 2).

Figure 2. Melanoma cell late endosome/lysosome compartment (LLE) vesicles are enriched at the lytic synapse.
Melanoma cells expressing CD107a-GFP were monitored during interaction with two antigen-specific CTLs using a spinning-disk confocal microscope. Snap shots depict the CD107a-GFP+ LLE localization before conjugation and after conjugation. The pseudo color scale indicates fluorescence intensity (from R. Khazen et al, Nat Com 2016).

We set up a “CTL-centered” multidimensional phenotypic analysis in cancer patients based on multicolor flow-cytometry, associated to mathematical analysis of complex data  (collaboration with S. Gadat and M. Costa at the Institute of Mathematics of Toulouse). Our aim is to establish patient-related “personalized CTL functional fingerprints” that might open the road to new approaches to stratify patients and guide immunotherapy.

In collaboration with S. Cussat-Blanc at the Institute of Informatics of Toulouse (IRIT) we generated an agent-based model describing long-term outcomes of tumor nodule/CTL confrontation (Fig. 3).

Figure 3. Reverse engineering allows to realistically reproduce cytotoxicity assays and to predict long-time outcomes of cellular interactions

Research Axis 2: Bidirectional functional cooperation between human mast cells and T lymphocytes

Our main goal is to study the functional interaction of mast cells with other cells of the immune system via the formation of immunological synapses or via the exchange of soluble factors.

We have shown that mast cells can serve as unconventional antigen presenting cells for helper T lymphocytes by forming immunological synapses where bi-directional cell-cell cooperation occurs. In addition, we demonstrated that mast cells form a specialized area of polarized granule release when interacting with antibody-targeted cells. We named this process ADDS (antibody dependent degranulatory synapse). More recently, we have shown that different stimuli involving either antibody receptors (FcR) or G-protein coupled receptors (GPCR) induce different degranulation modalities (Fig. 4).

Figure 4. Mast cell degranulation strategies and control
(1) GPCRs trigger the degranulation of secretory granule units with short lived in vivo effects, while FcRs trigger the degranulation of large-sized secretory granules with prolonged stored mediator relaase. (2) Localized engagement of FcRs with cell-bound antigens induces the formation of a degranulatory synapse allowing dedicated secretion. (3) IL-33 augments the magnitude of MC degranulation and allows the emergence of high responder MCs. (from E. Espinosa and S. Valitutti, Curr Opin Immunol 2018).

Objectives

The aim of our work is to employ experimental and computational tools to decipher intercellular communication at the immunological synapse among cells of the human immune system. Our final goal is to provide a better understanding of immune responses in cancer patients and, in perspective, to contribute to ameliorate immunotherapeutic strategies against cancer.

Research Axis 1: On the fight between cytotoxic T lymphocytes and cancer cells at the immunological synapse

Our general objective for the coming years is to deepen the dissection of the molecular key events taking place at the lytic synapse to shed new light on the fundamental aspects of CTL efficacy when facing tumor cells and on the molecular mechanisms by which tumor cells “speak back” to CTL at the lytic synapse. In particular we are developing ultra-fast methods for 4-D (3-D plus time) analysis of CTL/target cell interaction to dissect, with an extremely high time/space resolution, the “hits” occurring during cell-cell fight at the lytic synapse. We are also applying methods based on Imaging Flow Cytometry to dissect, within individual members of polyclonal and monoclonal CTL populations, the molecular mechanism allowing super-killer cells to emerge.

Moreover, we will profit from our expertise in monitoring human CTL/target cell confrontation and of established collaboration with several members of the IUCT medical community to develop innovative metrics of CTL function and/or failure in cancer patients. In particular, we will extend our search for patient-related “personalized CTL functional fingerprints” into their 3-D tumor microenvironment using adapted multiplexed immunofluorescence techniques.

Research Axis 2: Bidirectional functional cooperation between human mast cells and T lymphocytes

Our current aim is to unravel the complex network of soluble and cell surface signals exchanged between mast cells and T lymphocytes during antigen presentation and to define its impact on the plasticity of both mast cells and T lymphocyte responses. We also aim at defining how microenvironment-derived signals might shape mast cell function and, in turn, T cell responses. We will also investigate the role played by mast cells in the tumor microenvironment and how these cells influence helper T cell and/or CTL biological function in the context of anti-tumor defense.

Key words
  • Cytotoxic T lymphocytes
  • Immunological synapse
  • Tumor immunology
  • Imaging techniques
  • Mast cells
  • Multiplexed tissue staining
  • Multidimensional analysis
  • Mathematical modeling

Selected publications


2017

Joulia, R; L'Faqihi, F E; Valitutti, S; Espinosa, E

IL-33 fine tunes mast cell degranulation and chemokine production at the single-cell level Journal Article

J Allergy Clin Immunol, 140 (2), pp. 497-509 e10, 2017, ISSN: 1097-6825 (Electronic) 0091-6749 (Linking).

Links | BibTeX

2016

Khazen, R; Muller, S; Gaudenzio, N; Espinosa, E; Puissegur, M P; Valitutti, S

Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse Journal Article

Nat Commun, 7 , pp. 10823, 2016, ISSN: 2041-1723 (Electronic) 2041-1723 (Linking).

Links | BibTeX

2015

Joulia, R; Gaudenzio, N; Rodrigues, M; Lopez, J; Blanchard, N; Valitutti, S; Espinosa, E

Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence Journal Article

Nat Commun, 6 , pp. 6174, 2015, ISSN: 2041-1723 (Electronic) 2041-1723 (Linking).

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Vasconcelos, Z; Muller, S; Guipouy, D; Yu, W; Christophe, C; Gadat, S; Valitutti, S; Dupre, L

Individual Human Cytotoxic T Lymphocytes Exhibit Intraclonal Heterogeneity during Sustained Killing Journal Article

Cell Rep, 11 (9), pp. 1474-85, 2015, ISSN: 2211-1247 (Electronic).

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2013

Bertrand, F; Muller, S; Roh, K H; Laurent, C; Dupre, L; Valitutti, S

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse Journal Article

Proc Natl Acad Sci U S A, 110 (15), pp. 6073-8, 2013, ISSN: 1091-6490 (Electronic) 0027-8424 (Linking).

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2011

Laurent, C; Muller, S; Do, C; Al-Saati, T; Allart, S; Larocca, L M; Hohaus, S; Duchez, S; Quillet-Mary, A; Laurent, G; Brousset, P; Valitutti, S

Distribution, function, and prognostic value of cytotoxic T lymphocytes in follicular lymphoma: a 3-D tissue-imaging study Journal Article

Blood, 118 (20), pp. 5371-9, 2011, ISSN: 1528-0020 (Electronic) 0006-4971 (Linking).

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2006

Wiedemann, A; Depoil, D; Faroudi, M; Valitutti, S

Cytotoxic T lymphocytes kill multiple targets simultaneously via spatiotemporal uncoupling of lytic and stimulatory synapses Journal Article

Proc Natl Acad Sci U S A, 103 (29), pp. 10985-90, 2006, ISSN: 0027-8424 (Print) 0027-8424 (Linking).

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2005

Depoil, D; Zaru, R; Guiraud, M; Chauveau, A; Harriague, J; Bismuth, G; Utzny, C; Muller, S; Valitutti, S

Immunological synapses are versatile structures enabling selective T cell polarization Journal Article

Immunity, 22 (2), pp. 185-94, 2005, ISSN: 1074-7613 (Print) 1074-7613 (Linking).

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2003

Faroudi, M; Utzny, C; Salio, M; Cerundolo, V; Guiraud, M; Muller, S; Valitutti, S

Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold Journal Article

Proc Natl Acad Sci U S A, 100 (24), pp. 14145-50, 2003, ISSN: 0027-8424 (Print) 0027-8424 (Linking).

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2000

Leupin, O; Zaru, R; Laroche, T; Muller, S; Valitutti, S

Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes Journal Article

Curr Biol, 10 (5), pp. 277-80, 2000, ISSN: 0960-9822 (Print) 0960-9822 (Linking).

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