Véronique Thomas-Vaslin

The past, present, and future of immune repertoire biology - the rise of next-generation repertoire analysis

T and B cell repertoires are collections of lymphocytes, each characterized by its antigen-specific receptor. We review here classical technologies and analysis strategies developed to assess immunoglobulin (IG) and T cell receptor (TR) repertoire diversity, and describe recent advances in the field. First, we describe the broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations: global overview of the diversity by flow cytometry, IG repertoire descriptions at the protein level for the identification of IG reactivities, IG/TR CDR3 spectratyping strategies, and related molecular quantification or dynamics of T/B cell differentiation. Additionally, we introduce the recent technological advances in molecular biology tools allowing deeper analysis of IG/TR diversity by next-generation sequencing (NGS), offering systematic and comprehensive sequencing of IG/TR transcripts in a short amount of time. NGS provides several angles of analysis such as clonotype frequency, CDR3 diversity, CDR3 sequence analysis, V allele identification with a quantitative dimension, therefore requiring high-throughput analysis tools development. In this line, we discuss the recent efforts made for nomenclature standardization and ontology development. We then present the variety of available statistical analysis and modeling approaches developed with regards to the various levels of diversity analysis, and reveal the increasing sophistication of those modeling approaches. To conclude, we provide some examples of recent mathematical modeling strategies and perspectives that illustrate the active rise of a “next-generation” of repertoire analysis.

Comprehensive Assessment and Mathematical Modeling of T Cell Population Dynamics and Homeostasis

Our current view of T cell differentiation and population dynamics is assembled from pieces of data obtained from separate experimental systems and is thus patchy. We reassessed homeostasis and dynamics of T cells 1) by generating a mathematical model describing the spatiotemporal features of T cell differentiation, and 2) by fitting this model to experimental data generated by disturbing T cell differentiation through transient depletion of dividing T cells in mice. This specific depletion was obtained by administration of ganciclovir to mice expressing the conditional thymidine kinase suicide gene in T cells. With this experimental approach, we could derive quantitative parameters describing the cell fluxes, residence times, and rates of import, export, proliferation, and death across cell compartments for thymocytes and recent thymic emigrants (RTEs). Among other parameters, we show that 93% of thymocytes produced before single-positive stages are eliminated through the selection process. Then, a postselection peripheral expansion of naive T cells contributes three times more to naive T cell production than the thymus, with half of the naive T cells consisting of dividing RTEs. Altogether, this work provides a quantitative population dynamical framework of thymocyte development, RTEs, and naive T cells.

In vivo correction of ZAP-70 immunodeficiency by intrathymic gene transfer

SCID patients have been successfully treated by administration of ex vivo gene-corrected stem cells. However, despite its proven efficacy, such treatment carries specific risks and difficulties. We hypothesized that some of these drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. Indeed, in vivo intrathymic transfer of a gene that provides a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny, allowing long-term immune reconstitution. We assessed the feasibility of this approach in a murine model of ZAP-70-deficient SCID. A T cell-specific ZAP-70-expressing lentiviral vector was injected into thymi of adult ZAP-70-/- mice without prior conditioning. This resulted in the long-term differentiation of mature TCR-alphabeta+ thymocytes, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70-expressing T cells demonstrated a partially diversified receptor repertoire and were responsive to alloantigens in vitro and in vivo. Improved treatment efficacy was achieved in infant ZAP-70-/- mice, in which the thymus is proportionately larger and a higher percentage of prothymocytes are in cycle. Thus, intrathymic injection of a lentiviral vector could represent a simplified and potentially safer alternative to ex vivo gene-modified hematopoietic stem cell transplantation for gene therapy of T cell immunodeficiencies.

Suicide Gene-Mediated Modulation of Graft-Versus-Host Disease

The development of suicide genes and progress in retroviral gene transfer to T-cells open new perspectives for the treatment of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) for leukemia and lymphoma. Indeed, suicide genes that metabolize inactive prodrugs into compounds toxic for dividing cells provide a powerful means for the pharmacogenetic control of T-cell reactivity. Here, we demonstrate the selective destruction of activated TK-transgenic T-cells in vivo and develop two new transgenic lines which should be useful for preclinical studies of suicide gene therapy strategies for GVHD.

Turning immunological memory into amnesia by depletion of dividing T cells

Immunological memory, defined as more efficient immune responses on antigen reexposure, can last for decades. The current paradigm is that memory is maintained by antigen-experienced “memory T cells” that can be long-lived quiescent or dividing. The contribution of T cell division to memory maintenance is poorly known and has important clinical implications. In this study, we directly addressed the role of dividing T cells in immunological memory maintenance by evaluating the consequences of their elimination. The specific ablation of dividing T cells was obtained by administration of ganciclovir to immune mice expressing the herpes simplex type 1 thymidine kinase suicide gene in T cells. We show that depletion of dividing T cells for 5 or 2 weeks suffices to abolish in vitro and in vivo memory responses against the male H-Y transplantation alloantigen or against lymphocytic choriomeningitis virus antigens, respectively. Similar results were obtained after the nonspecific elimination of all dividing cells by using hydroxyurea, a cytostatic toxic agent commonly used for cancer chemotherapy. This immune amnesia occurred in otherwise immunocompetent mice and despite the persistence of functional quiescent T cells displaying a “memory” phenotype. Thus, division of antigen-experienced T cells is an absolute requirement for immunological memory maintenance and the current concept of memory T cells is challenged.

Systems biology in vaccine design

Vaccines are the most effective tools to prevent infectious diseases and to minimize their impact on humans or animals. Despite the successful development of vaccines that are able to elicit potent and protective immune responses, the majority of vaccines have been so far developed empirically and mechanistic events leading to protective immune responses are often poorly understood. This hampers the development of new prophylactic as well as therapeutic vaccines for infectious diseases and cancer. Biological correlates of immune‐mediated protection are currently based on standard readout such as antibody titres and ELISPOT assays. The development of successful vaccines for difficult settings, such as infectious agents leading to chronic infection (HIV, HCV. . .) or cancer, calls for novel ‘readout systems’ or ‘correlates’ of immune‐mediated protection that would reliably predict immune responses to novel vaccines in vivo. Systems biology offers a new approach to vaccine design that is based upon understanding the molecular network mobilized by vaccination. Systems vaccinology approaches investigate more global correlates of successful vaccination, beyond the specific immune response to the antigens administered, providing new methods for measuring early vaccine efficacy and ultimately generating hypotheses for understanding the mechanisms that underlie successful immunogenicity. Using functional genomics, specific molecular signatures of individual vaccine can be identified and used as predictors of vaccination efficiency. The immune response to vaccination involves the coordinated induction of master transcription factors that leads to the development of a broad, polyfunctional and persistent immune response integrating all effector cells of the immune systems.

Transient Depletion of Dividing T Lymphocytes in Mice Induces the Emergence of Regulatory T Cells and Dominant Tolerance to Islet Allografts

We previously showed that transient depletion of dividing T cells at the time of an allogeneic transplantation induces long‐term tolerance to the allograft. Here we investigated the role of homeostatic perturbation and regulatory T cells (Treg) in such tolerance. Transient depletion of dividing T cells was induced at the time of an allogeneic pancreatic islets graft, by administration of ganciclovir for 14 days, into diabetic transgenic mice expressing a thymidine kinase (TK) conditional suicide gene in T cells. Allograft tolerance was obtained in 63% of treated mice. It was not due to global immunosuppression, permanent deletion or anergy of donor‐alloantigens specific T cells but to a dominant tolerance process since lymphocytes from tolerant mice could transfer tolerance to naïve allografted recipients. The transient depletion of dividing T cells induces a 2‐ to 3‐fold increase in the proportion of CD4+CD25+Foxp3+ Treg, within 3 weeks that persisted only in allograft‐bearing mice but not in nongrafted mice. Tolerance with similar increased proportion of Treg cells was also obtained after a cytostatic hydroxyurea treatment in normal mice. Thus, the transient depletion of dividing T cells represents a novel means of immuno‐intervention based on disturbance of T‐cell homeostasis and subsequent increase in Treg proportion.

State-Transition Diagrams for Biologists

It is clearly in the tradition of biologists to conceptualize the dynamical evolution of biological systems in terms of state-transitions of biological objects. This paper is mainly concerned with (but obviously not limited too) the immunological branch of biology and shows how the adoption of UML (Unified Modeling Language) state-transition diagrams can ease the modeling, the understanding, the coding, the manipulation or the documentation of population-based immune software model generally defined as a set of ordinary differential equations (ODE), describing the evolution in time of populations of various biological objects. Moreover, that same UML adoption naturally entails a far from negligible representational economy since one graphical item of the diagram might have to be repeated in various places of the mathematical model. First, the main graphical elements of the UML state-transition diagram and how they can be mapped onto a corresponding ODE mathematical model are presented. Then, two already published immune models of thymocyte behavior and time evolution in the thymus, the first one originally conceived as an ODE population-based model whereas the second one as an agent-based one, are refactored and expressed in a state-transition form so as to make them much easier to understand and their respective code easier to access, to modify and run. As an illustrative proof, for any immunologist, it should be possible to understand faithfully enough what the two software models are supposed to reproduce and how they execute with no need to plunge into the Java or Fortran lines.

Dynamical and Mechanistic Reconstructive Approaches of T Lymphocyte Dynamics: Using Visual Modeling Languages to Bridge the Gap between Immunologists, Theoreticians, and Programmers.

Dynamic modeling of lymphocyte behavior has primarily been based on populations based differential equations or on cellular agents moving in space and interacting each other. The final steps of this modeling effort are expressed in a code written in a programing language. On account of the complete lack of standardization of the different steps to proceed, we have to deplore poor communication and sharing between experimentalists, theoreticians and programmers. The adoption of diagrammatic visual computer language should however greatly help the immunologists to better communicate, to more easily identify the models similarities and facilitate the reuse and extension of existing software models. Since immunologists often conceptualize the dynamical evolution of immune systems in terms of “state-transitions” of biological objects, we promote the use of unified modeling language (UML) state-transition diagram. To demonstrate the feasibility of this approach, we present a UML refactoring of two published models on thymocyte differentiation. Originally built with different modeling strategies, a mathematical ordinary differential equation-based model and a cellular automata model, the two models are now in the same visual formalism and can be compared.

Tolerance induced without immunosuppression in a t-lymphocyte suicide-gene therapy cardiac allograft model in mice

BACKGROUND Life-long immunosuppression is a major cause of mortality and morbidity in transplant recipients. Gene therapy could provide new ways to obtain tolerance and avoid indefinite immunosuppression. EpTK mice are derived from the FVB/N strain (H2q) and express the thymidine kinase gene of herpesvirus in all mature T cells. Thus any mature dividing T cell can be killed in the presence of ganciclovir. We investigated the survival of alloincompatible C57B1/6 (H2b) hearts heterotopically transplanted into EpTK mice given only ganciclovir from day 0 to day 7 or 14. METHODS Abdominal cardiac transplantations were performed in 22 control mice (untreated FVB [n = 15], ganciclovir-treated FVB [n = 5], and untreated EpTK mice [n = 2]) and in 28 EpTK mice given ganciclovir from day 0 to day 7 (n = 15) or day 14 (n = 13). Rejection was defined as complete cessation of cardiac beat. Histologic examination of the grafts was performed at rejection, at day 7, or at day 100. Lymphocyte proliferation assays (concanavalin A stimulation or mixed lymphocyte reaction) were performed at day 7 and at day 100. RESULTS All control animals rejected transplants in 7 days (range, 5-9 days), whereas indefinite survival (>100 days) was observed in 89% of the ganciclovir-treated EpTK group, irrespective of the duration of ganciclovir treatment. Graft histology showed extensive cellular infiltrates with myocyte necrosis and arteritis in the control animals but only a mild infiltrate without necrosis or arteritis in the ganciclovir-treated EpTK group. The proliferative responses of the tolerant mice at day 100 were identical to those of naive mice, including a preserved proliferation against the donors lymphocytes in mixed lymphocyte reaction. CONCLUSION Functional transplantation tolerance of a fully incompatible heart can be achieved without immunosuppressive drugs in this model of suicide gene therapy.