Zhenya Ni

Adoptive transfer of antitumor lymphocytes has gained intense interest in the field of cancer therapeutics over the past two decades. Human natural killer (NK) cells are a promising source of lymphocytes for anticancer immunotherapy. NK cells are part of the innate immune system and exhibit potent antitumor activity without need for human leukocyte antigen matching and without prior antigen exposure. Moreover, the derivation of NK cells from pluripotent stem cells could provide an unlimited source of lymphocytes for off-the-shelf therapy. To date, most studies on hematopoietic cell development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have used incompletely defined conditions and been on a limited scale. Here, we have used a two-stage culture system to efficiently produce NK cells from hESCs and iPSCs in the absence of cell sorting and without need for xenogeneic stromal cells. This novel combination of embryoid body formation using defined conditions and membrane-bound interleukin 21-expressing artificial antigen-presenting cells allows production of mature and functional NK cells from several different hESC and iPSC lines. Although different hESC and iPSC lines had varying efficiencies in hematopoietic development, all cell lines tested could produce functional NK cells. These methods can be used to generate enough cytotoxic NK cells to treat a single patient from fewer than 250,000 input hESCs/iPSCs. Additionally, this strategy provides a genetically amenable platform to study normal NK cell development and education in vitro.

CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1' positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.

Cell-based immunotherapy has been gaining interest as an improved means to treat human immunodeficiency virus (HIV)/AIDS. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) could become a potential resource. Our previous studies have shown hESC and iPSC-derived natural killer (NK) cells can inhibit HIV-infected targets in vitro. Here, we advance those studies by expressing a HIV chimeric receptor combining the extracellular portion of CD4 to the CD3ζ intracellular signaling chain. We hypothesized that expression of this CD4ζ receptor would more efficiently direct hESC- and iPSC-derived NK cells to target HIV-infected cells. In vitro studies showed the CD4ζ expressing hESC- and iPSC-NK cells inhibited HIV replication in CD4+ T-cells more efficiently than their unmodified counterparts. We then evaluated CD4ζ expressing hESC (CD4ζ-hESC)- and iPSC-NK cells in vivo anti-HIV activity using a humanized mouse model. We demonstrated significant suppression of HIV replication in mice treated with both CD4ζ-modified and -unmodified hESC-/iPSC-NK cells compared with control mice. However, we did not observe significantly increased efficacy of CD4ζ expression in suppression of HIV infection. These studies indicate that hESC/iPSC-based immunotherapy can be used as a unique resource to target HIV/AIDS.

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication, their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly, both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis, antibody-dependent cellular cytotoxicity, and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS.