Contributions
Abstract: EP722
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by an abnormal inflammatory response against nuclear antigens due to a loss of the immune tolerance with consequent damage to healthy organs. Auto-reactive B cells with autoantibody production and immune complex formation have a fundamental role in the pathogenesis of the disease. Regulatory T cells (Tregs) are a subset of T lymphocytes endowed with immune suppressive capabilities. Tregs control the activation of the immune system, preventing unwanted inflammatory reactions and preserving the immune tolerance against self-antigens. An impaired Treg functionality is involved in SLE, where they do not manage to control the inflammatory response. Adoptive transfer of polyclonal Tregs has been employed to restore the immune tolerance in different autoimmune diseases, achieving unsatisfactory results due to a low frequency of antigen specific suppressive cells. Chimeric Antigen Receptors (CARs) are chimeric molecules capable of redirecting T cell specificity against target antigens, greatly enhancing T cell activity. CAR-Tregs proved effective in controlling the inflammation in pre-clinical mouse models of autoimmune diseases different from SLE.
Aims
The aim of the project is to employ the CAR technology to redirect and enhance Treg activity in order to exploit their immune suppressive capabilities to restore immune tolerance in Systemic Lupus Erythematosus.
Methods
We sorted CD4+CD25+ cells from peripheral blood of Healthy Donors using magnetic separation beads. Next, we expanded Tregs using anti-CD3/CD28 magnetic beads in the presence of IL-2 and rapamycin. Considering the role of B cells, we transduced CAR-Tregs with a Lentiviral Vector encoding for a second generation anti-CD19 CAR, constituted by the intracellular portion of both CD28 and CD3-zeta to provide a more physiological stimulation. GFP was employed as transduction marker. Starting from day +14 since the initial stimulation, we performed functional assays. Immune and suppression assays were evaluated with flow cytometry.
Results
We obtained a cell product highly enriched in Tregs and with a transduction efficiency of 50%. CAR-Tregs retained their immune suppressive capabilities and performed equal to untransduced Tregs in suppressing the proliferation of autologous lymphocytes upon polyclonal stimulation. Noticeably, CAR-Tregs, but not untransduced Tregs, suppressed autologous B cell proliferation, demonstrating an antigen-specific suppression capacity. When co-cultured with CD19+ tumor cells, CAR-Tregs did not display killing capacities, but rather an impressive proliferation activity. More strikingly, anti-CD19 CAR-Tregs suppressed conventional anti-CD19 CAR-T cell killing when co-cultured together in the presence of a CD19+ tumor cell line. Finally, we tested the immune suppressive capacities of anti-CD19 CAR-Tregs in a humanized mouse model of lupus. Engineered cells delayed the occurrence of leukopenia compare to untransduced regulatory lymphocytes.
Conclusion
In conclusion, we efficiently generated anti-CD19 CAR-Tregs and proved their efficacy both in vitro and in an in vivo humanized mouse model of lupus.
Keyword(s): Adoptive immunotherapy, Autoimmunity, CAR-T, Regulatory T cell
Abstract: EP722
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by an abnormal inflammatory response against nuclear antigens due to a loss of the immune tolerance with consequent damage to healthy organs. Auto-reactive B cells with autoantibody production and immune complex formation have a fundamental role in the pathogenesis of the disease. Regulatory T cells (Tregs) are a subset of T lymphocytes endowed with immune suppressive capabilities. Tregs control the activation of the immune system, preventing unwanted inflammatory reactions and preserving the immune tolerance against self-antigens. An impaired Treg functionality is involved in SLE, where they do not manage to control the inflammatory response. Adoptive transfer of polyclonal Tregs has been employed to restore the immune tolerance in different autoimmune diseases, achieving unsatisfactory results due to a low frequency of antigen specific suppressive cells. Chimeric Antigen Receptors (CARs) are chimeric molecules capable of redirecting T cell specificity against target antigens, greatly enhancing T cell activity. CAR-Tregs proved effective in controlling the inflammation in pre-clinical mouse models of autoimmune diseases different from SLE.
Aims
The aim of the project is to employ the CAR technology to redirect and enhance Treg activity in order to exploit their immune suppressive capabilities to restore immune tolerance in Systemic Lupus Erythematosus.
Methods
We sorted CD4+CD25+ cells from peripheral blood of Healthy Donors using magnetic separation beads. Next, we expanded Tregs using anti-CD3/CD28 magnetic beads in the presence of IL-2 and rapamycin. Considering the role of B cells, we transduced CAR-Tregs with a Lentiviral Vector encoding for a second generation anti-CD19 CAR, constituted by the intracellular portion of both CD28 and CD3-zeta to provide a more physiological stimulation. GFP was employed as transduction marker. Starting from day +14 since the initial stimulation, we performed functional assays. Immune and suppression assays were evaluated with flow cytometry.
Results
We obtained a cell product highly enriched in Tregs and with a transduction efficiency of 50%. CAR-Tregs retained their immune suppressive capabilities and performed equal to untransduced Tregs in suppressing the proliferation of autologous lymphocytes upon polyclonal stimulation. Noticeably, CAR-Tregs, but not untransduced Tregs, suppressed autologous B cell proliferation, demonstrating an antigen-specific suppression capacity. When co-cultured with CD19+ tumor cells, CAR-Tregs did not display killing capacities, but rather an impressive proliferation activity. More strikingly, anti-CD19 CAR-Tregs suppressed conventional anti-CD19 CAR-T cell killing when co-cultured together in the presence of a CD19+ tumor cell line. Finally, we tested the immune suppressive capacities of anti-CD19 CAR-Tregs in a humanized mouse model of lupus. Engineered cells delayed the occurrence of leukopenia compare to untransduced regulatory lymphocytes.
Conclusion
In conclusion, we efficiently generated anti-CD19 CAR-Tregs and proved their efficacy both in vitro and in an in vivo humanized mouse model of lupus.
Keyword(s): Adoptive immunotherapy, Autoimmunity, CAR-T, Regulatory T cell