Unit: Antiviral immunity, Biotherapy and Vaccines
Director: GOUGEON Marie-Lise
Our unit is involved in the study of T and B lymphocyte repertoire diversity and in the functional characterization of various lymphocyte subpopulations in physiological and pathological (cancer, infectious diseases, autoimmune diseases) situations. We are also involved in the study of the strategies developed by HIV to escape the immune system.
1- Analysis of central and effector memory T lymphocyte subsets in mice and humans (Cecile Bouneaud and Christophe Pannetier)
It is generally accepted that memory T cells can be subdivided into two subsets, central memory (TCM) and effector memory (TEM) cells. However, their differentiation pathways are not well understood. In a murine model allowing the analysis of CD8 TCM et TEM spécific for the male peptide Smcy3 in the context H2-Db , we analyzed the TCRa repertoire repertoire of the two memory subsets, either in various lymphoid organs at a given time, or at two time points in the spleen with or without antigenic re-stimulation. Repertoire studies show that two thirds of TCM et TEM clones originate from a common precursor. In addition TCM and TEM populations behave differently in vivo: TCM clones are stable in the absence of re-stimulation, but they strongly respond to an antigenic challenge and differentiate into both TCM et TEM, although a fraction of TCM generates TEM. In contrast, TEM do not persist in the absence of antigen, and they are unable to mount a secondary response, male cells being eliminated by a primary response from the host. These data provide new insights into the drastically different behaviors of TCM and TEM CD8 subsets in vivo and show that vaccinologists should be careful not to generate the maximum quantity of memory T cells, but rather the right quality of memory cells,
2- Detection of low-frequency human antigen-specific CD4(+) T cells using MHC class II multimer bead sorting and immunoscope analysis (Fabrice Lemaitre, Manuelle Viguier).<
MHC class II tetramers are attractive tools to study antigen-specific CD4(+) T cell responses in various clinical situations in humans. HLA-DRA1*0101/DRB1*0401 MHC class II heterodimers were produced as empty molecules using the Drosophila melanogaster expression system. Peptide binding experiments revealed that these molecules could be loaded efficiently with appropriate MHC class II tumor epitopes. Interestingly, MHC class II tetramer staining was influenced by modifications in membrane lipid rafts, and could in itself induce activation changes of stained CD4(+) T cells at 37°C. In order to increase the threshold of detection of poorly represented peripheral antigen-specific CD4(+) T cells, we combined cell sorting using MHC class II multimer beads together with TCR analysis using the immunoscope technology. This strategy greatly increased the sensitivity of detection of specific CD4(+) T cells to frequencies as low as 4 x 10(-6) among peripheral blood mononuclear cells. Such a combined approach may have promising applications in the immunomonitoring of patients under vaccination protocols to tightly follow induced antigen-specific CD4(+) T cells expressing previously identified TCR.
3- Influence of terminal deoxynucleotidyl transferase on Valpha and Vbeta public repertoires (Nicolas Fazilleau, Fabrice Lemaitre, Jean Kanellopoulos)
T cell repertoires observed in response to immunodominant and subdominant peptides include private, i.e., specific for each individual, as well as public, i.e., common to all mice or humans of the same MHC haplotype, Valpha-Jalpha and Vbeta-Dbeta-Jbeta rearrangements. To measure the impact of N-region diversity on public repertoires, we have characterized the alpha beta TCRs specific for several CD4 or CD8 epitopes of wild-type mice and of mice deficient in the enzyme TdT. We find that V, (D), J usage identified in public repertoires is strikingly conserved in TdT(o/o) mice, even for the CDR3 loops which are shorter than those found in TdT(+/+) animals. Moreover, the 10- to 20-fold decrease in alpha beta T cell diversity in TdT(o/o) mice did not prevent T cells from undergoing affinity maturation during secondary responses. A comparison of the CDR3beta in published public and private repertoires indicates significantly reduced N-region diversity in public CDR3beta. Our findings suggest that public repertoires are produced more efficiently than private ones by the recombination machinery. Alternatively, selection may be biased in favor of public repertoires in the context of the interactions between TCR and MHC peptide complexes and we hypothesize that MHC alpha helices are involved in the selection of public repertoires.
4- Immunoscope in clinical studies (Annick Lim, Brigitte Lemercier, Xavier Wertz, François Huetz)
Since its conception (1992), Immunoscope has been improved and adapted to the clinical follow-up of T cells in immunotherapy trials and in pathological situations. The improvements include quantitative analyses of T cell repertoire diversity, clonotypic Immunoscope analyses on peptide-specific T cells sorted on the basis of peptide-HLA class I and class II tetramers, and the design of protocoles adapted to a prospective and retrospective clinical follow-up. The recent development of Immunoscope for B cells will extend its application to clinical and vaccinal situations.
5- Role of regulatory T cells in metastatic melanoma (Manuelle Viguier, Fabrice Lemaitre, Laurent Ferradini).
Dominant tolerance is mediated by regulatory T cells (Treg) that control harmful autoimmune T cells in the periphery, We have investigated the implication of T(reg) in modulating infiltrating T lymphocytes in human metastatic melanoma. We found that CD4(+)CD25(high) T cells are overrepresented in metastatic lymph nodes (LNs) with a 2-fold increased frequency compared with both tumor-free LNs and autologous PBMCs. These cells express the Foxp3 transcription factor, display an activated phenotype, and display a polyclonal TCR Vbeta chain repertoire. They inhibit in vitro the proliferation and cytokine production of infiltrating CD4(+)CD25(-) and CD8(+) T cells (IL-2, IFN-gamma) through a cell-contact-dependent mechanism, thus behaving as Treg. In some cases, the presence of Treg type 1/Th3-like lymphocytes could also be demonstrated. Thus, Treg are a major component of the immunosuppressive microenvironment of metastatic melanoma LNs. This could explain the poor clinical response of cancer patients under immunotherapeutic protocols, and provides a new basis for future immunotherapeutic strategies counteracting in vivo Treg to reinforce local antitumor immune responses.
6- T cell dynamics and antiviral immunity in HIV+ patients receiving IL-2 (Béatrice Poirier-Baudoin, Peggy Masdehors-Taoui, Valérie Seffer, Sylvie Rouyre, en collaboration avec le Pr. Yves Levy, Hôpital Henri Mondor, Créteil)
Combined antiretroviral therapy inhibiting HIV reverse transcriptase and protease induces a clinical benefit in a large fraction of chronically HIV-infected patients, while reducing the plasmatic viral load to undetectable levels and restoring the level of CD4 T cells. However, because of severe adverse effects, including metabolic complications, new therapeutic strategies are required to spare antiretroviral regimens. For example, antiretroviral therapy has been associated with IL-2 immunotherapy to restore qualitatively and quantitatively the pool of CD4 T lymphocytes. With the aim to better understand the mechanisms whereby IL-2 induces a significant increase in CD4 T cells in HIV+ patients, we have developed several methodological approaches to detect ex-vivo recent thymic emigrants (TREC), to identify lymphocytes submitted either to homeostatic proliferation, or to programmed cell death by apoptosis. In parallel, HIV-specific immunity has been analyzed after stimulation with viral antigens and multiparametric analysis by FACS. In the context of three clinical trials ((ANRS 048, ANRS 079, Silcaat), we have shown that T cell dynamics is modified under the impact of IL-2, inducing increased survival and homeostatic proliferation and, in the long-term, IL-2 therapy can preserve effector T cells specific for HIV.
7- T cell homing in the gut mucosa (Delphine Guy-Grand, en collaboration avec Pierre Vassalli).
The gut epithelium comprises 4 T lymphocyte subpopulations: TCRb CD4+ T cells or CD8ab+ (type a), TCRb T cells without co-receptors or CD8aa+, and TCRgd T cells (type b). The expression of the integrin, a4b7, is responsible for the tropism in gut mucosa of type a circulating lymphocytes. The precursors of mucosa lymphocytes circulate in the lymph of thoracic duct. Lymph comprises 2.5% of a4b7 lymphocytes, shared out by the four subsets. The study of thymocytes and lymph from mice thymectomised at adult age shows that a4b7 expression can be acquired by type a lymphocytes in different sites (in lymphoid area associated to gut) and type b lymphocytes (on thymocytes). Mucosal lymphocytes which recirculate in the lymph are mostly central memory and effector memory subsets. Circulating effector memory cells (CD44+CD62L-) are the less differentiated in the cytotoxic pathway. In euthymic mice, all subsets originate from the thymus. These activated lymphocytes comprise memory cells that circulate through the lamina propria, and effector cells which don't circulate and acquire NK-like lytic activity. T cell precursors are not the only ones to circulate through thoracic duct before homing in the lamina propria. The small subset of diviving cells which circulate also comprises precursors from plasmocytes, mastocytes and dendritic cells. All these cells participate to the maintenance of epithelial integrity.
8- In vivo dynamics of T cell responses (Susanna Celli, Zacarias Garcia, Philippe Bousso).
This team is involved on the one hand in the study of the regulation of T lymphocyte (TL) - dendritic cell (DC) in vivo, and on the other hand in the analysis of how DC impact the activation program in TL. Two-photon imaging and confocal microscopy are used to analyze lymphocyte migration and cell interaction in the lymph nodes. The role of some parameters (such as TCR affinity, antigen density, the phenotype of TL) and the characteristics of TL-DC contacts (duration, stability, frequency) are studied. A murine experimental system has been established to follow the historic of individual encounters of T cells with the antigen and the functional consequences of multiple interactions with DC. We are also interested in the molecular dynamics of TL-DC interactions, and particularly in the in vivo formation of immunological synapse. Our objectives are the following: i) to determine if such a structure exists in vivo; ii) to define whether each type of contact leading to cell activation induces the same profil of immunological synapse. Finally, we use two-photon imaging to analyze cell interactions required for CD4 T cell help of cytotoxic responses, and to study the suppression of T cell responses by regulatory T cells CD4+ CD25+. These studies should contribute to determine how cells of the immune system move and interact in their native environments.
9- Proteomic analysis of ash allergens (Pascal Poncet, en collaboration avec Gabriel Peltre, ESPCI, et Jean-Michel Wal, INRA)
In Europe, sensitization to ash pollen is underestimated because of a lack of precise pollinisation calendar, lack of standardized extract for diagnosis and wide cross reactivities with other pollens. Ash is member of the Oleaceae family (olive, privet, forsythia ). In order to identify the panel of ash pollen allergens and to decipher the IgE response, 62 sera from patients diagnosed for ash, olive or grass allergies were screened by IgE immunoblot of an aquaeous extract of ash pollen separated by SDS-PAGE. Six sera were selected for 2D IgE immunoblot analysis followed by mass spectrometry for protein identification. We have shown that 92% of allergic patients to ash had IgE reactivity against Fra e, the major ash pollen allergen, and 46% react with profilin. The overlapped patterns determined 5 zones of IgE reactivity based on pI, defining the allergome of ash pollen. Protein identification by mass spectrometry and MALDI-TOF analysis revealed the existence of isoforms of Fra e 1, and 10 proteins did not exhibit IgE reactivity. This work contributes to a better delineation of ash pollen allergens and pattern of sensitization. The investigation of diagnostic and therapeutic relevance of these identified allergens may help to predict the evolution of symptoms and to refine an "a-la-carte" treatment of an allergy that results for 70% in asthma.
Keywords: TCR and BCR Répertoire, Immunoscope, regulatory T cells, mélanoma, immunotherapy, AIDS