Cord-Derived Invariant Natural Killer T Cells As Novel Cell Therapy to Improve the Outcome of Hematopoietic Stem Cell Transplantation
Authors: Trujillo-Ocampo Abel , Grefe Maison , Borges Pamella , Clinton Jelita , He Hong , Li Dan , Yu Ling , Vaz de Freitas Martiela , St. John Lisa , Clise-Dwyer Karen , Alatrash Gheath , Amaral Antues Dinler , Ma Qing , Molldrem Jeffrey J. , Shpall Elizabeth J. , Im Jin S. 
Published: December 2024

Allogeneic stem cell transplantation (ASCT) is a curative immunotherapy for patients with hematologic malignancies and performed more than 7000 per year in the US. Here, patients receive the conditioning chemo-radiation to eradicate the residual blood cancer, followed by donor stem cells to reconstitute the donor immunity. However, only 30-40% patients with high-risk leukemia survive long-term due to the relapse of disease from the insufficient graft versus tumor (GVT) effects of donor T cells and treatment-related complications including Graft-Versus-Host-Disease (GVHD) due to dysregulated donor T cells and life-threatening infections from the poor graft function. Thus, there is an unmet need for the innovative strategy to mitigate the major causes of the treatment failure - the relapse, GVHD, infection, and poor graft function, leading to the better transplant outcome.

CD1d-restricted invariant Natural Killer (iNK) T cells are rare but powerful innate T cells that influence the adaptive immunity toward tumor surveillance or immune-tolerance via the production of Th1- or Th2-type cytokines and exert anti-tumor effects via iNK T cell receptor (TCR) or NK receptor (NKR)-mediated cytolysis or facilitating the cross-priming of antigen-specific T cells. Finally, iNK T cells can mediate antiviral immunity through NK-like properties and facilitate the immune-reconstitution in a humanized mouse model. Thus, we hypothesied that iNK T cells can play as a master-regulator to improve the outcome of ASCT by preventing GVHD while promoting GVT effects, anti-viral immunity, and donor immune-reconstitution in ASCT.

Here, we report an effective strategy to preferentially expand human iNK T cells from adult donor (AD) and cord blood (CB) to a clinically meaningful number in an extremely high purity via a single antigenic stimulation. CB-derived iNK T cells were highly enriched with IL-10 producing neuropilin1 (NRP1)+Th2+CD4+ iNK T cells (NKT10) and displayed superior in vitro and in vivo anti-GVH effects compared with AD-iNK T cells. Despite superb regulatory properties, CB-iNK T cells expressed a variety of NK receptors and exerted direct anti-tumor activity against CD1dhigh B lymphoblastic cells when pulsed with agonist glycolipid antigen. Further, the cytotoxicity of CB-iNK T cells can be re-directed against HLA-A2+ myeloid leukemia cells via an expression of 8F4CAR, chimeric antigen receptor targeting PR1/HLA-A2 myeloid antigen. Moreover, CB-iNK T cells facilitated the expansion of antigen-specific donor T cells in vitro when co-activated and enhanced donor GVT effects in xenogenic GVHD/GVT model. Lastly, CB-iNK T cells promoted the xenograft engraftment in a humanized murine model. Mechanistically, we demonstrated that NRP1, highly expressed on NKT10 subset, inhibited Th1 cytokine production by iNK T cells during antigenic stimulation leading to Th2 functional polarization.

In conclusion, NKT10-enriched CB-iNK T cells maintain a prototypical multi-functionality and are suitable candidate as off the shelf cell therapy platform engineered to prevent/treat GVHD and relapse, and to promote donor immune-reconstitution after ASCT, improving the transplantation outcome.