Research projects

P1. Autoreactivity and cancer (Project leader: David Klatzmann, Bruno M. Colombo)

State of the art.
There are numerous observations showing that beyond immune ignorance or immune surveillance, tumors induce immune tolerance that is largely mediated by natural regulatory T cells (Tregs). In numerous tumors models, in different genetic backgrounds and with tumors of different origins, the ablation of Tregs before tumor implantation often leads to tumor eradication. Similarly, we showed that Treg ablation leads to fetus rejection (16). Our previous results indicated that the subset of antigen-experienced “activated/memory” Tregs (amTregs), which are involved in the permanent suppression of immune responses against self-antigens (42-44), are the main culprit for the induction of tolerance to emerging self-tumors. However, the specificity of these Tregs is still to discover. Moreover, we showed that the respective activation status, and thus activation speeds of “self-specific amTregs” and “anti-tumor Teffs” at tumor emergence dictate tumor outcome. Our work also highlighted that Treg very rapidly induce a state of resistance of the tumor to activated Teffs. The molecular events associated with these phenomenon – induction of tolerance at tumor cell emergence and later induction of resistance to effector T cells are not known. We hypothesized that they are similar in the tumor and fetus implantation settings.

Objectives.
Our main objectives are:

to characterize the specificities of the Tregs that control anti-tumor and anti-fetus effector responses at tumor emergence
to define the molecular and immune signatures that characterize the early phase of active tolerance induction in the tumor and fetus implantation settings
to define molecular and immune signatures that characterize the established tolerance, as defined by a state of resistance to memory effectors T cells, in the tumor and fetus implantation settings
to analyze the similarities and differences between the molecular and immune signatures of tolerance to tumors and to fetuses
to design means of breaking tolerance to tumors
to visualize the early immune response to tumor or fetus implantation

P2. Alloreactivity and cancer (Project leader: José Cohen)

State of the art.
Allogeneic hematopoietic stem cell transplantation (HSCT) is a treatment of choice for several hematological disorders, including leukemia and lymphoma. After a myeloreductive conditioning, patients receive hematopoietic stem cells (HSC) from a healthy donor, which have the potential to durably reconstitute hematopoïesis and lymphopoïesis. The role of donor T cells also present in the graft is essential since they (i) promote engraftment, (ii) support peripheral T cell reconstitution and (iii) provide a graft-versus-leukemia effect, but is also deleterious since they induce the life-threatening graft-versus-host disease (GVHD). In order to prevent GVHD, grafted patients receive an immunosuppressive regimen, but this treatment is only partially effective.

We and others observed that Treg are present in HSC transplants and that their depletion in the T cell inoculum prior to grafting yielded significantly accelerated GVHD in different models of allogeneic bone marrow transplantation in mice(14, 32, 46). T cell adoptive transfers further support a prominent role for Tregs in GVHD and suggest that donor Treg might be used as new therapeutics. To summarize, we have developed a procedure of culture to generate high numbers of recipient-specific (rs) Treg (37) and identified the effect of these rsTreg on conventional T cells (36) during the control of GVHD in mice. Finally, in HSCT, some studies analyzing the Treg content of the transplant and/or the blood Treg counts in grafted patients, suggested that Treg content negatively correlates with GVHD (for review see (47), reinforcing the rational to use Treg to prevent GVHD in humans.
Since high Treg numbers will be required for obtaining clinical efficacy against GVHD, a central issue is to determine how they can be purified and eventually expanded. Indeed, using GMP immuno-magnetic CD25 T cell sorting, is only possible to date to reach ~50% purity of Treg with currently available methods (48). Non-Treg may thus expose to a risk for grafted patients of developing GVHD.. A specific program to optimize the methods for sorting and expand human rsTregs is described in the “translational section”.

The general objective of this program is to realize a global analysis of the immunomodulation mediated by Treg in the context of allogeneic HSCT and to improve their clinical effect in this setting. For this we will pose two questions. First, how could Treg be reactivated in vivo to improve their clinical effect. Since we still studied the Treg/T cells interaction in vivo, this requires now a better understanding of the Treg/Dcs interaction to have a global cellular view on the mode of action of Treg in allo-HSCT. Second, does rsTreg act by bystander effect on conventional T cells activated by different antigens than those used to activate rsTreg? If this is the case, we will develop alternative strategy to generate and reactivate Treg in vivo by antigens which can not activate GVHD-effector T cells. This could permit to circumvent the problem to sort Treg to purity for clinical purposes.
For all this program, we will also take advantage of the identification/characterization and cloning of TcR of Treg specific for autoantigens (see A2C-P1).

Objectives.
1. To identify interactions between Treg and recipient’s APC, and evaluate the immune reconstitution and GVL effect when GVHD is controlled by Treg
2. To study the potential bystander effect of Treg and evaluate its implication for clinical strategy

P3. Manipulating alloreactivity in leukemia and solid tumors (Project leaders: José Cohen & David Klatzmann, François Lemoine)

State of the art.
Cord blood can be used as a source of allogeneic HSCT in patients with various hematological disorders. One advantage of this source of stem cells is a lower rate of induced GVHD as compared to other sources, likely due to the immaturity of T cells at birth and the presence of a high frequency of Treg in cord blood. Conversely, this lack of alloreactivity could be associated with partial loss of GVL effect, and lower rate of engraftment. This last point is potentially due to the low number of HSC present in cord blood, which does not permit to graft an adult patient with a single donor. This strongly limits the use of cord blood HSC which otherwise would be the perfect source of HSC.
This problem of engraftment is not restricted to the use of cord blood as source of HSC, but also concerns patients who receive reduced-intensity conditioning to avoid the side effects of intensive conditioning. This reduced regimen cannot be used in haplo-mismatched transplantation, which requires for each patient intensive conditioning for optimal engraftment.
We thus aimed to develop a new approach that will permit (i) to reduce the conditioning of patients before grafting, (ii) to reduce the number of injected cells, (iii) to increase engraftment and GVL effect, (iv) without GVHD. A possibility to reach the (i-iii) goals would be the use alloreactive effector T cells. However, this carries an increase risk of GVHD in the absence of a known procedure to eliminate dangerous cells after grafting. This safety issue is, currently, the limiting obstacle opposing the development of alloreactive T cells.

Objectives
To use TK-T cells to improve engraftment and anti-leukemic effect.

P4. Immuno-intervention et cancer (Project leader: François Lemoine)

Objectifs du projet.
Mise en place de stratégies d'immuno-intervention du cancer reposant sur le transfert de gène et le ciblage des cellules dendritiques avec des vecteurs codant pour des antigènes associés aux tumoraux. Cette stratégie est developpé dans deux modèles de cancers:
- les cancers des voies aéro-supérieures digestives (VADS) induits notamment par le virus HPV16,
- la leucémie myéloïde chronique (LMC) en situation de maladie résiduelle minime.