Hierarchical Epstein-Barr Virus (EBV)-specific T cell responses – a model of T cell memory establishment.

International context and working hypothesis 

Herpes viruses are carried asymptomatically by the majority of the world’s human inhabitants. They therefore offer a unique opportunity to study the generation and maintenance of memory T cell responses to chronic infections. In this context I searched for the biological parameters responsible for the size and shape of the memory T cell responses. My principal interests were memory T cell induction and maintenance.

Objectives

To determine the association between memory T cell responses and A) the interaction between the T cell receptor (TCRs) and its cognate peptide-Major Histocompatibility Complex (MHC) ligand and B) receptors controlling T cell faith through antigen independent mechanisms.

Results

A. Antigen load and memory T cell induction

T cell responses are induced by the encounter between antigen-specific T cells and antigen presenting cells (APCs). Peptide titration assays of T cell clones demonstrate that the strength of this interaction is on the one hand defined by the affinity of the T cell receptor and on the other hand by the density of antigen presented on the APCs. Measuring the natural antigen density on APCs is challenging and was first explored using highly sensitive mass spectrometry of peptide elution samples from bulk amounts of in vitro cultured APCs. Since 1990 an emerging new technology based on the generation of high-affinity TCR-like antibodies by immunization and later antibody phage display has enabled direct single-cell detection of antigenic peptides presented by MHCs on the surface of APCs. To pursue this approach I transferred my expertise on antibody phage display technology to the laboratory of Pr. Rickinson along with the needed tools for molecular cloning, recombinant antibody expression and purification. I then initiated the isolation and characterization of antibodies specific for viral antigens that would serve as tools to precisely monitor antigen density on APCs. I successfully obtained 15 TCR-like antibodies binding 8 different virus derived peptide-MHC molecules. The antibodies displayed high target specificity when screened against a panel of 20+ peptide-MHC molecules that I generated in-house. To my great disappointment, although the antibodies had nanomolar affinity to their targets (measured by surface plasmon resonance), they failed to detect naturally presented antigens on APCs. This may be due to lack of post-translational modifications in the bacterial expression system employed for in vitro generation of peptide-MHC targets.

B. Receptors implicated in memory T cell induction and preservation.

In order to understand which determinants were important for the generation and maintenance of antigen-specific T cell memory responses, I studied the role of homeostatic, co-stimulatory and co-inhibitory pathways observed in individuals infected by herpes viruses either asymptomatically or manifesting as infectious mononucleosis. These works were realized in close collaboration with Dr. Sauce and Dr. Taylor. Importantly, my panel of peptide-MHC molecules (tetramers) gave me the ability to identify and specifically study the genotype, phenotype and functional capacity of virus-specific T cells ex vivo and in vitro. The studies demonstrated close association between the capacity of T cells to respond to cytokines and the establishment of anti-viral memory T cell responses.Indeed, the size of anti-viral T cell responses is tightly linked with anatomic location and inversely correlated with the frequency of T cells expressing IL-7 receptor. Surprisingly, we discovered an irreversible functional IL-15 receptor knock-out on not only virus-specific, but on all peripheral T cells from individuals having suffered from EBV-induced infectious mononucleosis (IM). A particular lytic EBV epitope encoded by BMRF1 was present transitionally in patients with IM but not in asymptomatic virus carriers. To understand the loss of T cell memory I therefore investigated if BMRF1-specific T cells displayed alterations in phenotypic markers related to cell fate. Interestingly, BMRF1 responses in the last bleed before their disappearance showed displayed elevated IL-7Rα expression levels and very high Programmed Death Receptor 1 (PD-1) expression levels. This suggests that homeostatic regulation alone is insufficient to maintain long-lived memory T cell responses.

My post doc has resulted in the following publications:

• Sauce D, Larsen M et al. Blood. 2006; 108 (1) :11-18 (Plenary).

• Taylor GS, Long HM, Haigh TA, Larsen M et al. JI. 2006; 177 : 3746-3756.

• Sauce D, Larsen M et al. Journal of Infectious Diseases. 2007; 195 (2): 268-278.

• Sauce D, Larsen M et al. J Virol. 2009; 83 (18): 9068-9078.

A. T cell plasticity and autoimmunity.

International context and working hypothesis

A high priority goal of the team of Pr Gorochov has continuously been the study of the balance between pro-inflammatory and regulatory T cells in the context of autoimmunity. The comprehension of CD4⁺ T cell responses have over the past decade gone from a fairly simple combination of Th1 and Th2 responses, to a much more complex cellular network of regulatory and pro-inflammatory T cell subsets. FoxP3⁺ CD4⁺ regulatory T (Treg) cells, involved in the maintenance of self-tolerance are subdivided in naturally occurring (nTregs) and inducible (iTregs) Treg cells. Conversely, pro-inflammatory IL-17- and IL-22-producing T cell subsets, involved in anti-microbial immunity and autoimmune inflammation, are characterized by the expression of IL-17, IL-22, CCR6, CD161 and the RORγt transcription factor. Whereas the number of phenotypically distinguishable CD4⁺ T cell subsets increases every year, our knowledge about their ontogeny remained elusive, in particular in humans.

Objective

The aim of my study was to determine the ontogeneic relation between phenotypically distinct T cell subsets.

Results

Whereas mice models utilizing cell trackers like GFP enable sequential studies of T cell fate, human studies can only trace T cells by sequencing of their TCR, which is difficult to combine with the measure of single-cell T cell phenotypes ex vivo. I therefore supervised a master student in the establishment of CD4⁺ T cell clones from psoriatic lesions and then phenotyped them by measuring clonal cytokine secretion patterns. I finally sequenced their TCRαβ chains to elucidate if one T cell can adopt multiple phenotypes either by multiple initial programming events defined by local micro-environments or as a reprogramming event of an already fully programmed T cell. Using T cells from psoriatic skin lesions I identified Th1, Th2, Th17, Th22 and Tr1 T cell clones and demonstrated a large clonal plasticity across most of these subsets, including between Th1 and Th17 T cells. I have also been recurrently involved in projects developed by Post Docs and PhD students within the group aiming at characterizing inflammatory responses in various systemic disorders.

B. T cell polyfunctionality - quality rather than quantity.

International context and working hypothesis

Recent works have highlighted the importance of T cell quality as defined by the capacity to execute multiple functions simultaneously. Indeed, polyfunctional T cell responses are positively associated with control of viral replication and thus vaccine efficacy. Data analysis of polyfunctional T cell responses has been central to all the projects described in paragraphs A and B above. I have frequently been confronted with one general problem concerning this type of analysis, which is the lacking capacity to quantify polyfunctionality and thus to properly associate polyfunctionality with clinical and biological measures in a statistical manner.

Objective

My aim was to create a method enabling direct quantification of cellular polyfunctionality.

Results

The projected resulted in the invention of a novel mathematical algorithm, coined “Polyfunctionality Index”, which quantifies polyfunctionality as a continuous but normalized variable compatible with comparative and associative statistics. The algorithm has been patented by Inserm-Transfert and license negotiations are ongoing to include the algorithm in software for polyfunctionality data analysis.(www.funkycells.com) The algorithm and associated statistics have rapidly created a demand from 4 teams within our unit (Teams lead by Pr B. Autran, Dr V. Viellard, Dr A. Morris and Dr. V. Appay) and 3 external collaborators at the NIH, Washington (Dr. D. Douek, Dr. R. Seder and Dr. M. Roederer), IMI Bruxelles, Belgium (Dr. A. Marchant) and CFRI Vancouver, Canada (Dr. T. Kollmann). 

Since my arrival in the laboratory of Pr. Gorochov, within the infectious immunology unit (Inserm U1135), I have been in charge of the projects described above, but also engaged in numerous collaborative projects with the teams headed by Pr. Autran and Dr. Appay. Overall these studies have materialized in multiple research articles.

My work as scientist in Paris has resulted in the following publications:

• Sauce D, Almeida JR, Larsen M et al. AIDS. 2007; 21 : 2005-2013.

• Appay V and Larsen M. Progress in Inflammation Research. 2008; 59-70.

• Sauce D, Larsen M et al. J Clin Invest. 2009; 119 : 3070-3078.

• Almeida JR, Sauce D, Price D, Papagno L, Shin SY, Morris A, Larsen M et al. Blood. 2009; 113 : 6351-6360.

• Xie J, Lu W, Samri A, Costagliola D, Schnuriger A, da Silva BC, Blanc C. Larsen M et al. AIDS. 2010; 24 : 2323-2329.

• Arnaud L, Gorochov G, Charlotte F, Lvovschi V, Parizot C, Larsen M et al. Blood. 2011; 117 : 2783-2790.

• Larsen M et al. EJI. 2011; 41 : 2596-2605.

• Arnaud L, Larsen M et al. N Eng J Med. 2011; 365 : 1450-1452.

• Sauce D, Larsen M et al. Blood. 2011; 117 : 5142-5151.

• Larsen M et al. PLoS Pathogens. 2011; 7 : e1002328

• Papagno L, Alter G, Assoumou L. Murphy RL, Garcia F, Clotet B, Larsen M et al. AIDS. 2011; 25 : 27-36.

• Larsen M et al. Patent. 2012.

• Larsen M et al. PLoS One. 2012; 7 : e42403.

• Mathian A, Parizot C, Dorgham K, Trad S, Arnaud L, Larsen M et al. Rheum Dis. 2012; 71 : 1227-1234.

• Sauce D, Larsen M  et al. JI. 2012; 189 : 5541-8

• Lvovschi V, Arnaud L, Parizot C, Freund Y, Juillien G, Ghillani-Dalbin P, Bouberima M, Larsen M et al. PLoS One. 2012; 6 : e28870.

• Roux A, Mourin G, Fastenackels S, Almeida JR, Iglesias MC, Boyd A, Gostick E, Larsen M et al. Clin Immunol. 2013;148 : 16-26.

• Roux A, Mourin G, Larsen M et al. J Immunol. 2013;191 : 1300-6

• Larsen M et al. Med Sci (Paris). 2013;29 : 1080-2