Activação dos Linfócitos

Interesse da Investigação

Adaptive immunity begins with the generation of antigen-specific effector T cells from their naïve precursors, upon interaction with professional Antigen Presenting Cells (APC). Once generated, effector T cells are the central actors of antigen specific immune responses and their function is strongly influenced by the environment where lymphocyte interaction take place. In particular, signaling through Pattern Recognition Receptors (PRR) by specific natural or synthetic agonists is known to modulate T cell activity. Our research team employs a combination of morphological, biochemical and functional approaches to investigate T lymphocyte activation and their communication with APC in the presence of PRR signaling.

Membros do Grupo

Projecto de Investigação

How Pattern Recognition Receptors signaling controls T lymphocytes activation.

The induction of CD4+ T cell responses results from TCR interaction with MHC-II expressed on dendritic cells (DC) and the outcome of this process is influenced by the engagement of Pattern Recognition Receptors (PRR), including Toll-like Receptors (TLR) and Nacht-Leucine Repeats (NLR). During activation, a CD4+ T cell can receive an instructive PRR signal indirectly (in trans) or directly (in cis). Activation in trans is provided by cytokines released by DC that have been stimulated through PRR. Trans-activating signals are known to induce T cell proliferation and differentiation into cytokine-producing T helper cells. However, T cells activated through the T cell receptor (TCR) complex also express functional TLR molecules and direct TLR stimulation on differentiating T cells enhances T cell proliferation and cytokines secretion, suggesting that TLR act as costimulatory receptors on T cells. Whether TLR activation in cis occurs during T cell-DC interaction and how T cells integrate trans- and cis-activating signals into their physiology remains poorly understood. We are currently addressing these issues using mouse and human models. In the human system employ Th1 and Th17 T cell clones reactive to bacterial enterotoxins to define possible differences existing in the capacity of these different effector T cells to communicate with antigen presentig cells, in the presence or absence of PRR stimuli. In the mouse system we are comparing the direct and indirect activation induced by TLR signaling in co-cultures of TLR2KO DC and naïve T cells from TLR4KO transgenic animals in the presence of specific TCR and TLR agonists.

Funding

PTDC/SAU-MII/69796/2006 - E. Padovan
Fundação para Ciencia e a Tecnologia

Colaboradores

Instituto Gulbenkian de Ciência
Jocelyne Demengeot

Projecto de Investigação

Delayed killing of melanoma cells by CTL: multiple hits must be provided for tumor cell annihilation

It is well established that CTL are triggered to kill target cells offering a very small number of specific pMHC complexes. It is also known that lethal hit delivery is a very rapid response that occurs within a few minutes after cell-cell contact. Whether cytotoxicity is efficient and rapid in the context of CTL interaction with tumor target cells is still elusive. We addressed this question by visualizing the dynamics of human CTL interaction with melanoma cells and their efficiency in eliciting cytotoxicity. Our results show that in spite of CTL activation to lethal hit delivery, killing of melanoma cells was inefficient. Time-lapse microscopy showed that individual CTL rapidly polarized their lytic machinery towards target cells, yet the apoptotic process in melanoma cells was delayed as compared to conventional target cells, since it required a prolonged time after receiving lytic granules. These results indicate that while CTL activation to lethal hit delivery can be viewed as a “digital” phenomenon rapidly triggered by a few ligands, melanoma cell annihilation is an “analog” response requiring multiple hits and prolonged contact time. Our next aim is to explore novel strategies to enhance CTL activity against tumor cells that may be relevant for tumor vaccine implementation. Thus, we are making use of our experience on TLR immunobiology to assess the effect of TLR signaling on CTL/target conjugate formation and cytolytic function and explore whether TLR signaling during CTL/target interaction may favor the establishment of a functional stimulatory synapse at low antigen concentrations and prolong CTL survival.

Funding

SFRH/ BPD/ 23661/2005 - I. Caramalho
Fundação para Ciencia e a Tecnologia

Colaboradores

Max Planck Institute for Immunebiology, Freiburg, D
Markus Simon
Marina Freudenberg

Projecto de Investigação

Adjuvanticity of microbial-derived particles and synthetic analogs in vitro.

Engagement of Toll-Like Receptors (TLR) on Antigen Presenting Cells (APC) by molecular patterns expressed by natural pathogens or live/attenuated vaccines is crucial for the development of long-term immuneprotection. On the contrary, antigen formulations that lack the capacity to signal through TLR are poorly immunogenic. These observations have generated a strong demand for the rational design of synthetic analogs of TLR agonists suitable for complementing the activity of subunit vaccines. As new compounds become available, there is need to develop applicable screening methods predicting adjuvanticity and safety of those molecules. We have developed a systematic study that provides the rational for the screening and subsequent development of adjuvants suitable for use in human vaccines based on responses observed on human T cells and APC. Our results will help to refine most current methods for adjuvant selection that are based on in vivo studies using a variety of animal models not necessarily representative of human reactivity.
By monitoring proliferation and differentiation of CD4+ T cells, as well as APC responses in short-term in vitro cultures, in the presence of standard TLR2, TLR4 and TLR3 we have define molecular signatures of adjuvanticity and pyrogenicity, restricted to human DC and monocyte, respectively. Based on our observations we propose to use pre-screening tests assessing the production of TNF- and/or CXCL10 by human DC, and IL-1 release by CD14+ monocytes in order to narrow down the number of compounds that need to be assessed for enhanced immune protection and lack of toxicity in vivo. When applied to large-scale chemical libraries this method can facilitate the selection of candidate adjuvants prior in vivo testing, thus reducing the need of costly, demanding and potentially irrelevant animal studies.

Funding

Project 92/04 - E. Padovan
3R Research Foundation Switzerland

Colaboradores

Instituto Gulbenkian de Ciência
Jorge Carneiro

Max Planck Institute for Immunebiology, Freiburg, D
Markus Simon
Marina Freudenberg

Department of Research, University Hospital, Basel, CH
Gennaro De Libero
Regine Landmann

Publicações

Caramalho I, Faroudi M, Müller S, Padovan E and Valitutti S. (2008). Visualizing the dynamics of CTL/melanoma cell interaction: multiple hits must be provided for tumor cell annihilation. Submitted

Kamgang RK, Ramos I. Rodrigues-Duarte L, Ghielmetti M, Freudenberg M, Dahinden C and Padovan, E. (2008). Using distinct molecular signatures of human monocytes and dendritic cells to predict adjuvant activity and pyrogenicity of TLR agonists Med Microbiol Immunol DOI 10.1007/s00430-008-0081-6

Padovan, E., Landmann RM and De Libero G. (2007). How pattern recognition receptor triggering influences T cell responses: a new look into the system. Trends Immunol 28 :308-314

Carneiro J, Duarte L and Padovan, E. (2007). Limiting Dilution Analysis of Antigen-specific T cells. In: T cell Protocols Editor: Gennaro De Libero; Human Press Inc.

Wiedemann, A., Depoil, D., Faroudi, M., Valitutti, S. (2006). Cytotoxic T lymphocytes kill multiple targets simultaneously via spatiotemporal uncoupling of lytic and stimulatory synapses. Proc Natl Acad Sci USA 103 :10985-90

Depoil, D., Zaru, R., Guiraud, M., Chauveau, A., Harriague, J., Bismuth, G., Utzny, C., Muller, S., Valitutti, S. (2005). Immunological synapses are versatile structures enabling selective T cell polarization Immunity 22 :185-94

Utzny, C., Faroudi, M., Valitutti, S. (2005). Frequency encoding of T-cell receptor engagement dynamics in calcium time series. Biophys J 88 :1-14

Ghielmetti, M., Zwicker, M., Ghielmetti, T., Simon, M.M., Villiger, P.M. and Padovan, E. (2005). Synthetic bacterial lipopeptide analogs facilitate naive CD4+ T cell differentiation and enhance antigen-specific HLA-II restricted responses. Eur J Immunol 35 :2434-2442