Human pluripotent stem cells produce natural killer cells that mediate anti-HIV-1 activity by utilizing diverse cellular mechanisms

Abstract

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.

FIG. 1.

Hematopoietic development from hESCs and iPSCs cocultured with the stromal cell line M210-B4. hESCs and iPSCs were first allowed to differentiate on M210-B4 stromal cells for 19 to 21 days to develop hematopoietic progenitor cells. These hematopoietic progenitors were enriched based on CD34 and CD45 surface expression using magnetic sorting methods and then cultured on AFT024 cells with defined cytokines for NK cell derivation. Shown are data from flow cytometric analyses of unsorted, CD34⁺-sorted, and then CD34⁺ CD45⁺-sorted hESCs (top row) or iPSCs (bottom row).

FIG. 2.

Generation of NK cells from hESCs and iPSCs. (A) Flow cytometric analysis of CD56⁺CD45⁺ NK cells derived from hESCs, iPSCs, and UCB progenitor cells cultured under NK cell conditions for 5 weeks. These cells were compared to NK cells isolated from peripheral blood (PB-NK). Both hESC- and iPSC-derived CD56⁺CD45⁺ cells are a uniform population of CD117⁻CD94⁺ cells, similar to PB-NK cells, whereas UCB-derived CD56⁺CD45⁺ cells are a mixture of CD117⁻CD94⁺ and CD117⁺CD94⁻ populations. (B) Expression of activating and inhibitory receptors on hESC-NK, iPSC-NK, UCB-NK, and PB-NK cells, as indicated. (C) Four-hour ⁵¹Cr release NK cell-mediated cytotoxicity assay. Both hESC-NK (blue) and iPSC-NK (green) cells, similar to PB-NK cells (black), show higher cytolytic activities against K562 cells than do UCB-NK cells (red) at the indicated effector-to-target cell (E:T) ratios (n = 3) (P < 0.05).

FIG. 3.

hESC- and iPSC-derived NK cells inhibit the replication of HIV-1 in the CEM-GFP T-cell line. CEM-GFP cells were incubated with HIV-1 NL4-3 for 4 h. Cells were then cocultured with hESC-NK, iPSC-NK, UCB-NK, or PB-NK cells for 14 days. In these studies, HIV-1 infection was assessed by flow cytometry for GFP expression. (A) Histogram representing GFP expression in CEM cells cocultured with hESC-NK or UCB-NK cells at E:T ratios of 10:1 (green line), 1:1 (blue line), or 0:1 (red line) at day 11. (B) Activity of HIV-1 measured by the inhibition of GFP⁺ cells in cocultures of hESC-NK or UCB-NK cells with CEM-GFP cells at day 11 with E:T ratios of 1:1 (dark bars) and 10:1 (light bars). (C) Histogram of GFP expression in CEM cells cocultured with iPSC-NK or PB-NK cells at an E:T ratio of 5:1 (green line), 1:1 (blue line), or 0:1 (red line) at day 11. (D) Activity of HIV-1 in cocultures of iPSC-NK or PB-NK cells with CEM-GFP cells at day 11 at E:T ratios of 1:1 (dark bars) and 5:1 (light bars). All data in B and D show statistically decreased HIV-1 activity (P < 0.01). (E and F) Quantification of supernatant for p24 Gag protein in cocultures of CEM-GFP cells with NK cells. (E) HIV-infected CEM-GFP cells with no NK cell treatment (red), UCB-NK cells (maroon), and hESC-NK cells (blue) at 1:1 E:T ratios and UCB-NK cells (dark green) and hESC-NK cells (lilac) at 10:1 E:T ratios. (F) iPSC-NK cells (dark blue) and PB-NK cells (light green) at a 1:1 E:T ratio and iPSC-NK cells (orange) and PB-NK cells (purple) at a 5:1 E:T ratio. For statistical analysis, shown are data for UCB-NK cells and hESC-NK cells at 1:1 E:T ratios versus HIV-infected CEM-GFP cells (P < 0.05 for each) and UCB-NK cells and hESC-NK cells at 10:1 E:T ratios versus HIV-infected CEM-GFP cells (P < 0.01 for each) (E) and iPSC-NK cells at a 1:1 E:T ratio, iPSC-NK cells at a 5:1 E:T ratio, PB-NK cells at a 1:1 E:T ratio, and PB-NK cells at a 5:1 E:T ratio versus HIV-infected CEM-GFP cells (all P < 0.05) (F). Data in B, D, and E represent the means of data from three experiments and standard deviations, and data in F represent the means of data from two experiments and standard deviations. These results demonstrate that all hESC-NK, UCB-NK, iPSC-NK, and PB-NK cells significantly inhibit HIV replication over this 14-day time course.

FIG. 4.

hESC-NK and iPSC-NK cell activation against HIV-1-infected human CD4⁺ primary T cells. Surface expression of CD107a provides a measure of NK cell cytolytic activation. Shown are data from flow cytometric analyses of CD107a⁺ NK cells following stimulation with HIV-1-infected CD4⁺ T cells. Uninfected CD4⁺ T cells were used as controls. (A) Representative analysis of surface CD107a expression on hESC-NK, iPSC-NK, UCB-NK, and PB-NK cells stimulated with HIV-1-infected cells (right) versus uninfected control cells (left). All NK cell populations stimulated by HIV-1-infected CD4⁺ T cells show significantly increased CD107a expression compared to CD107a expression after stimulation by uninfected T cells (P < 0.01 for both hESC-NK and iPSC-NK cell groups and P < 0.05 for UCB-NK and PB-NK cell groups [n = 4]). (B) Expression of CD107a on hESC-NK and iPSC-NK cells upon stimulation with HIV-1-infected CD4⁺ T cells treated with either anti-gp41 Ab or IgG isotype to evaluate for increased anti-HIV activity via ADCC. Again, all three NK cell populations demonstrated increased CD107a surface expression after the addition of anti-gp41 compared to the isotype control (P < 0.01 for hESC-NK or iPSC-NK cells treated with HIV-1-infected CD4⁺ T cells plus anti-gp41 versus isotype IgG-treated HIV-1-infected CD4⁺ T cells and P < 0.05 for UCB-NK cells treated with HIV-1-infected CD4⁺ T cells plus anti-gp41 versus isotype IgG-treated HIV-1-infected CD4⁺ T cells [n = 3 for all studies]).

FIG. 5.

Anti-HIV chemokine and cytokine production by hESC-NK and iPSC-NK cells. Shown are data for intracellular staining measured by flow cytometry analysis for the production of CCL4 in hESC-NK, iPSC-NK, UCB-NK, and PB-NK cells upon stimulation with HIV-1-infected CD4⁺ T cells or control CD4⁺ T cells (P < 0.01 for both hESC-NK and iPSC-NK cells treated with HIV-1-infected CD4⁺ T cells versus noninfected CD4⁺ T cells and P < 0.05 for UCB-NK cells treated with HIV-1-infected CD4⁺ T cells versus noninfected CD4⁺ T cells [n = 4]) (A) and IFN-γ production in hESC-NK, UCB-NK, and PB-NK cells upon stimulation with HIV-1-infected CD4⁺ T cells or control CD4⁺ T cells (n = 3) (B).