A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors

Abstract

Several obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy).

T-Cell Progenitors As A New Immunotherapy to Bypass Hurdles of Allogeneic Hematopoietic Stem Cell Transplantation

“…If this hypothesis is confirmed, the injection of T-cell progenitors could improve thymic function, which would be particularly useful in patients whose thymus has been altered by chemotherapy, irradiation, infections, GvHD, or inflammation, as well as in elderly patients whose thymuses have undergone thymic involution. Moreover, increased recovery of other thymus-dependent lineages such as gd T cells, NKT cells, or MAIT cells (111)(112)(113) could be speculated on the basis of preclinical results (72). These properties of T-cell progenitors are under investigation in ongoing clinical trials in adults treated for malignant hemopathies.…”

“…We have further improved this 7-day culture system by a combination of cytokines crucial for stem cell survival and T-cell commitment, allowing the production of a large number of CD34 - CD7 + progenitors. In particular, the addition of TNFα in the culture process resulted in significant improvement in terms of both purity and harvest yield of the final T-cell progenitor product ( 72 ). These cells express TCF7, BCL11b, GATA3, and CD3ε; have not yet started their TCR rearrangement; and possess enhanced T-cell potential in vitro and in vivo , leading to the fast production of mature and polyclonal TCRαβ and γδ T cells upon transplantation in NSG mice ( 72 ), similarly to T-cell progenitors produced in 14 days with hDLL4-Fc, IL-7, SCF, and VCAM-1 ( 73 ).…”

“…Notably, this culture process can be applied to all sources of HSC (such as umbilical cord blood and blood of mobilized donors) ( 72 , 74 ). It was translated to the clinic and is currently being tested in pediatric and adult patients undergoing HSCT for hemopathies or severe combined immune deficiencies (NCT04707300, NCT03879876, and NCT04959903).…”

“…These data paved the way for the development of an off-the shelf T-cell progenitor strategy as a universal immunotherapy. Cryopreserved cord blood unit (CBU) cells provide the ideal platform for the generation of these cellular therapy products due to its immediate availability, high proliferation yield during differentiation ( 72 ), and the possibility of covering the majority of the population using a limited number of units. By creating an off-the-shelf T-cell progenitor bank from umbilical cord blood CD34 + cells, the therapeutic cell product would become immediately available to patients in need, independently of the donor, even when allogeneic HSCT is not possible (e.g., following lymphodepleting chemotherapy, in aging patients as protection against viral infections, or in the context of immunosuppressive therapy).…”

Modeling of human T cell developmentin vitroas a read-out for hematopoietic stem cell multipotency

“…Upon injection in immunodeficient mice, these pro-T cells home to the thymus and differentiate into functional SP8 T cells. Also specific cytokines such as TNFα, combined with coated DLL4, have been shown to promote the development of early human T cell precursors from both UCB and mobilized PBL (mPBL) sources [ 85 , 86 ]. More recently, a novel method has been designed for the generation of clinical-grade T cells by incubating HSPCs with DLL4-coated microbeads in suspension which significantly enhances the scalability of T cell precursor generation out of HSPCs from either ESCs, UCB and mobilized peripheral blood (mPBL) and the therapeutic applicability [ 87 ].…”