MEDICINE 2013;2:274 -283

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.

No abstract

Cell-based immunotherapy has been gaining interest as an improved means to treat 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-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 to 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 utilized as a unique resource to target HIV/AIDS.

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34 ؉ cells, studies of T-and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34 ؉ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34 ؉ cells also did not produce B cells in vitro. In contrast, CD34 ؉ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, find- IntroductionHuman embryonic stem cells (hESCs) provide an important model system to define the mechanisms that mediate cellular development. hESC-derived hematopoietic progenitor cells efficiently produce erythroid, myeloid, and lymphoid lineage cells in vitro. [1][2][3][4] We previously defined an in vitro culture system to generate natural killer (NK) cells from hESCs. 5 hESC-derived NK cells express surface receptors characteristic of primary NK cells, kill tumor target cells, and produce interferon-␥ when stimulated with cytokines. These results suggest that hESC-derived progenitors may also readily commit to the T-cell lineage in vitro, since T and NK lymphocytes are developmentally closely related. 6,7 One study has used an in vivo model to examine the T-cell potential of hESCs. 8 Galic et al injected hESC-derived hematopoietic progenitor cells into human thymus/fetal liver (Thy/Liv) grafts in severe combined immunodeficient-human (SCID-hu) mice. This study demonstrated T-cell development after 3 to 5 weeks in vivo, although in a less efficient manner than what has been observed with hematopoietic progenitor cells from human fetal liver (FL), bone marrow (BM), or umbilical cord blood (UCB) 9-11 evaluated in SCID-hu mice. Although useful, SCID-hu mice are not optimal to evaluate development of specific phenotypic cell populations over time, and the effects of specific molecular signaling pathways are difficult to quantify via this SCID-hu system. Therefore, in vitro models of lymphocyte development are needed, although despite the considerable interest in hematopoietic development of hESCs, in vitro studies have not provided significant evidence of functional T and B lymphocyte maturation of hESC-derived hematopoietic progenitors. Al...