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Keywords:

  • Human embryonic stem cells;
  • Induced pluripotent stem cells;
  • Natural killer cells;
  • HIV-1 infection inhibition;
  • In vitro;
  • In vivo

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

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. Stem Cells 2014;32:1021–1031


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

Highly active antiretroviral therapy (HAART) has significantly decreased the morbidity and mortality of human immunodeficiency virus (HIV)/AIDS, but latent virus still persists in cellular reservoirs. Restoration of cellular immunity in treated patients proceeds slowly and may never return to preinfection status [1, 2]. Cell-based therapy for HIV has gained more attention for its potential of long-term virus control or cure (reviewed in ref. [ [3, 4]). Initially, studies were designed to inhibit HIV transcription or translation, which is less efficient in controlling the late steps of the viral life cycle [5]. More studies were then focused on early step inhibition before HIV integration into host genome. For instance, several groups have demonstrated resistance against R5-tropic HIV in vitro using shRNAs to knock down CCR5 in hematopoietic stem cells (HSCs) [6-8]. Holt et al. [9] used zinc finger nucleases to disrupt the CCR5 gene in human cord blood and fetal liver CD34+ cells, which protected reconstituted NSG mice from R5 HIV infection. The use of a CCR5−/− donor for a HSC transplant of an HIV-infected patient with acute myelogenous leukemia has provided a novel impetus for other potential cell-based curative approaches for patents with HIV/AIDS [10, 11].

Alternatively, there are also efforts to help redirect specific immune cell subsets to target and kill HIV. Studies have engineered immune cells with “chimeric antigen receptors” (CAR) to provide enhanced antigen recognition and cellular activation. By fusing the antigen-specific portion of an antibody to intracellular signaling domains of the T-cell signaling framework, several groups have shown promising results in different cancer clinical trials [12-15]. A similar strategy has also been used on HIV treatment by modifying peripheral T-cells with a molecularly cloned T-cell receptor (TCR) to redirect cells to HIV targets [16-18]. Using a lentiviral approach, Kitchen et al. successfully expressed an HIV-specific TCR into HSCs and developed CD8 T-cell with response to HIV in vivo [19]. Other groups have also demonstrated anti-HIV activity by expressing a functional neutralizing antibody in B cells derived from human HSCs in vitro [20] and in vivo [21].

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are becoming an alternative promising source for gene or immunotherapy [22]. Several groups, including our own, have reported that hESCs and iPSCs can give rise to different lymphoid and myeloid lineages [23-29]. Some studies have also demonstrated that hESC- and/or iPSC-derived immune cells are either susceptible to HIV [25, 30] or capable of targeting HIV-infected cells [31]. More recently, TCR-specific T-cells derived from T-iPS cells [28] further demonstrated the potential of using either hESC or iPSCs-based gene/immunotherapy. We have previously shown that natural killer (NK) cells derived from hESCs and iPSCs have potent anti-HIV activity [31] but these innate immune cells do not possess any antigen-specific recognition receptors [1, 32, 33]. Therefore, we hypothesized that engineering hESC- and iPSC-derived NK cells with chimeric receptors would enhance their anti-HIV activity. In addition, we have recently demonstrated large-scale production of hESC/iPSC-derived NK cells, which could provide an unlimited cellular therapeutic for off-the-shelf use [34, 35]. We now advance these studies using the CAR strategy to direct NK cell effector function to HIV-infected cells [36].

As the CD4 protein is an absolute requirement for HIV entry, it is plausible to use this as an effective “antigen recognition” domain independent of human leukocyte antigen restriction. Several groups pioneered this approach to both basic research and clinical trials [36-40]; however, this had varying efficacy in vivo when transduced into patients autologous T-cells. Here, we modified both hESCs and iPSCs with a CD4ζ construct to generate NK cells that express the specific HIV CD4ζ chimeric receptor. We then tested these NK cells for HIV suppression both in vitro and found that both CD4ζ expressing hESC (CD4ζ-hESC)- and iPSC (CD4ζ-iPSC)-NK cells were able to suppress HIV replication more efficiently than their unmodified counterparts. We also determined CD4ζ -hESC-/iPSC-NK cells mediate in vivo anti-HIV activity in a peripheral blood lymphocyte NSG (PBL-NSG) mouse xenograft model. We found that CD4ζ-hESC- and CD4ζ-iPSC-NK cells were able to inhibit HIV replication and prevent CD4 T-cell depletion but no difference compared with regular hESC- and iPSC-NK cells. These studies establish a novel system to understand and direct innate immunity against HIV-1 infection. Eventually, hESC- or iPSC-based immune therapy could be used as a unique resource for HIV/AIDS treatment.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

Maintenance of hESCs/iPSCs and Generation of CD4ζ-hESC and CD4ζ-iPSC NK Cells

hESCs (H9) and iPSCs (UCBiPS7, derived from umbilical cord blood CD34+ cells) were maintained on mouse embryonic fibroblasts as described previously [41]. The plasmid pCCL.PPT.hPGK 1.9.IRES.eGFP containing the CD4ζ chimeric receptor was kindly provided by Drs. Scott Kitchen and Otto Yang from University of California, Los Angeles [36, 38]. Lentiviral production was produced in 293T cells using the Invitrogen ViraPower Lentiviral Expression System (Invitrogen). hESC and iPSC cell lines were then infected with CD4ζ lentivirus and fluorescence-activated cell sorting (FACS) sorted for GFP+ cells. These two lenti CD4ζ-modified cell lines were used in all in vitro studies. Because the expression of CD4ζ/GFP in hESCs and iPSCs was readily silenced during differentiation, we generated more stable CD4ζ-hESC and CD4ζ-iPSC lines using the sleeping beauty system [42, 43] and used these NK cells for all in vivo experiments.

NK Cell Derivation from CD4ζ-hESC and CD4ζ-iPSC Cells

We have previously used stromal-based systems and stroma-free embryoid body (EB)-based systems for hematopoietic differentiation of hESCs and iPSCs. Here, we use “Spin-EBs” for hematopoietic differentiation of hESCs and iPSCs [34, 44-46]. Briefly, 3,000 single cells were seeded per well of 96-well round-bottom plates in Bovine Serum Albumin (BSA) Polyvnylalchohol Essential Lipids (BPEL) media with stem cell factor (40 ng/ml, PeproTech, Rocky Hill, NJ, http://www.peprotech.com), vascular endothelial growth factor (20 ng/ml), and bone morphogenic protein 4 (20 ng/ml). BPEL media was made in 200 ml volumes and contained Iscove's Modified Dulbecco's Medium (86 ml, Invitrogen, Carlsbad, CA, http://www.invitrogen.com), F12 Nutrient Mixture with Glutmax I (86 ml, Invitrogen), 10% deionized bovine serum albumin (5 ml, Sigma), 5% polyvinyl alcohol (10 ml, Sigma), linolenic acid (20 µl of 1 g/ml solution, Sigma), linoleic acid (20 µl of 1 g/ml solution, Sigma), Synthecol 500X solution (Sigma), a-monothioglyceral (Sigma, 3.9 µl/100 ml), protein-free hybridoma mix II (Invitrogen), ascorbic acid (5 mg/ml, Sigma), glutamax I (Invitrogen), insulin–transferrin–selenium 100× solution (Invitrogen), and penicillin/streptomycin (Invitrogen). At day 11 of hematopoietic differentiation, spin EBs were directly transferred into 24-well plates with or without EL08-1D2 stromal cells in NK media supplied with cytokines [34]. After 4–5 weeks of culture, single cell suspensions were stained with Allophycocyanin (APC)-, Phycobiliproteins (PE)-, Fluorescein Isothiocyanate (FITC)-, and PerCP-cy5.5-coupled IgG or specific antibodies against human blood surface antigens: CD45-PE, CD56-APC, CD56-PE, CD16-PerCP-cy5.5, NKG2D-PE, NKp44-PE, NKp46-PE, CD158b-FITC, CD158e1/2-FITC (all from BD Pharmingen), CD158a/h-PE, and CD158i-PE (Beckman Coulter, Brea, CA, http://www.beckmancoulter.com) as shown in Figure 1. All analyses were performed with a FACS Calibur (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR). NK cells isolated from peripheral blood (PB-NK) using an NK negative selection kit (Miltenyi Biotech, Bergisch Bladbach, Germany) were used as controls for phenotyping characterization and all following experiments.

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Figure 1. Expression of CD4ζ chimeric receptor in human embryonic stem cells (hESCs) and induced pluripotent stem cells. (A): Diagram of CD4ζ cloned in lentiviral vector or Sleeping Beauty transposon vector. (B): Transduced hESC cells were analyzed by flow cytometry for expression green fluorescent protein (GFP) and CD4ζ receptor (upper and middle lanes). Both GFP and CD4ζ expression getting silenced during culture maintain (lower lane). (C): SB-transduced hESCs stably express GFP-CD4ζ. Abbreviations: eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; hESC, human embryonic stem cells; TCR, T-cell receptor; P-#-T, Number of passages in TrypLE.

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NK Cell Stimulation

CD4ζ-hESC-, CD4ζ-iPSC-NK cells, and GFP only controls were starved in RPMI 1640 media overnight. As described previously [36], cells were spun down and stimulated with anti-CD4 mAb (OKT4A) for 15 minutes at 4°C, washed and crossed linked by goat anti-mouse IgG F(ab′)2 fragments (Jackson Immunoresearch, West Grove, PA) for 3 minutes at 37°C. Cells were then fixed and stained for the tyrosine phosphorylation using mouse anti-human mAb4G10 (Millipore, Billerica, MA, http://www.millipore.com) and PE-donkey anti-mouse IgG (Jackson Immunoresearch) following the instruction of BD phosflow kit (BD Biosciences).

CD4ζ-hESC and CD4ζ-iPSC NK Cell Anti-HIV Activity In Vitro

As in our previous studies, CEM (a human T cell lymphoblast-like cell line) -GFP cells infected with HIV-1 NL4-3 were used as targets to test the suppression of HIV of CD4ζ-hESC- and CD4ζ-iPSC-NK cells by comparison with their unmodified hESC- and iPSC-NK cells. Briefly, CEM-GFP cells were infected with HIV-1 NL4-3 (Multiplicity of infection (MOI) = 0.1) for 4 hours at 37°C and then washed twice with fresh medium. Approximately 1 × 105 cells were plated with CD4ζ-hESC-, hESC-, CD4ζ-iPSC-, and iPSC-NK cell at effector: target ratios 1:1 and 5:1 or alone for 14 days in the presence of 100 IU/ml interleukin 2. Cells were collected on days 4, 7, 11, and 14 for GFP expression by flow cytometry. Loss of GFP expression in the CEM-GFP cells indicates suppression of HIV replication. To detect the anti-HIV activity of CD4ζ-hESC- and CD4ζ-iPSC-NK cells to HIV–infected primary human cells, CD4+ T-cells enriched from PB were stimulated with phytohemagglutinin (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) in RMPI 1640 with 10% FBS, 2 mM l-glutamine supplemented with 100 IU/ml IL-2 for 48–72 hours. At day 3, expanded CD4+ T-cells were infected with a laboratory-adapted strain HIV-1 SF2 (X4R5, MOI = 0.05) (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) [47]. At day 10, 1 × 105 of HIV-1-infected CD4 T-cells were, respectively, mixed with same number of CD4ζ-hESC-, hESC-, CD4ζ-iPS-, and iPSC-NK cells at 37°C for 5 hours, the activation of NK cells was evaluated by CD107a surface expression. PB-NK cells were used as positive controls for all experiments.

Generation of Human PBL-NSG Mouse

NSG mice aged 8- to 10-week-old were reconstituted with 1 × 107 freshly isolated human PBLs [48]. After 2 weeks, blood from the retro orbital venous plexus was collected to evaluate for engraftment prior to HIV NL4-3 infection.

HIV Infection and NK Cell Treatment

HIV-1 NL4-3 virus was grown in 293 T-cells. Virus infectivity was determined by limiting dilution titration on 293 T-cells. HIV-1 NL4-3 stocks were prepared as described [49]. Two weeks after PBL reconstitution, mice were infected by intraperitoneal (IP) injection of 100 µl cell-free HIV stocks containing 30,000 50% tissue culture infectious doses (TCID50) [50]. Mice were then IP injected with 2 × 106 hESC-, CD4ζ-hESC-, iPSC-, CD4ζ-iPSC-, or PB-NK cells, the day after HIV-1 NL4-3 infection. As demonstrated previously in our laboratory, mice received IP injection of Interleukin (IL)-15 and IL-2 every day for the first 7 days following NK cell treatment and then IL-2 every other day for another week to enhance NK cell proliferation and function [51]. Days 6, 9, and 12 after NK cell injection, blood was collected for human CD4 T-cell levels, HIV gag protein p24, viral RNA, and proviral DNA detection. Day 13 after NK cell treatment (day 14 of HIV infection), mice were sacrificed and cells were recovered from the spleen and peritoneal cavity for proviral DNA and intracellular p24 detection.

Measurement of Human CD4 T-Cell Levels in PBL-NSG Mice

The levels of hCD4 in PB were monitored every 3 days after NK cell treatment. Whole blood was collected in EDTA-coated tubes and red blood cells were lysed by ammonium chloride twice, 5 minutes each time if necessary. Cells were then stained for hCD45-APC, hCD3-PECy7, and hCD4-PE (BD Pharmingin). CD4+ T-cell levels were determined as a ratio of CD4+CD3+/CD4CD3+. To establish baseline CD4+CD3+/CD4CD3+ratios, all mice were analyzed before HIV infection. To detect hESC-, iPSC-, and PB-NK cells in PB, peritoneal cavity, and spleen, cells were stained with hCD45-PE and hCD56-APC (BD Pharmingin) after treatment.

Measurement of HIV Viral Load

Mouse PB was collected by facial bleeding in accordance with the University of Minnesota Institutional Review Board. Plasma was separated by spin at 400 rpm/minute for 10 minutes and frozen at −80°C for viral RNA isolation. Viral RNA was extracted from less than 50 µl of EDTA-treated plasma with the QIAamp Viral RNA kit (Qiagen, Valencia, CA). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed using the Taqman one-step RT-PCR Master Mix Reagents kit (Applied Biosystems, Branchburg, NJ) with a set of primers specific for the HIV long terminal repeat (LTR) sequence and an LTR-specific probe as described [52-54]. Viral RNA was expressed as the number of HIVRNA copies per milliliter plasma. To detect integrated provirus, cellular DNA was extracted from PB, peritoneal cavity, and spleens using the high pure PCR template preparation kit (Roche, Mannheim, Germany) and subjected to qPCR with the same set of primers above and SYBR Green PCR Master Mix (Applied Biosystems).

Statistical Analysis

Experiments were analyzed with Prism 5 software using the Student's t-test or the Wilcoxon rank sum test. Results are shown as means and SD and the value of p < .05 was determined as significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

Expression of CD4ζ in hESCs and iPSCs Using Lentivirus or Sleeping Beauty Transgenesis

The CD4ζ construct contains the fused extracellular and transmembrane domains of CD4 and the cytoplasmic domain of TCR CD3ζ chain, linked to enhanced green fluorescent protein (eGFP) by a 2A self-cleaving peptide (Fig. 1A). The CD4ζ lentivirus was made in 293 T-cells, collected and transduced into hESCs or iPSCs. We then performed flow cytometry sorting for GFP-positive cells to purify CD4ζ expressing hESCs and iPSCs (CD4ζ-hESCs or CD4ζ-iPSCs). hESCs and iPSCs expressing GFP-only were used as controls (Fig. 1B). During the maintenance of CD4ζ-hESCs and CD4ζ-iPSCs, we found that the expression of GFP and CD4ζ was commonly silenced after 10–15 passages (Fig. 1B). Our previous studies have demonstrated that the Sleeping Beauty (SB) transposon system is a more stable means for transferring genetic information to hESCs [42, 43]. We then retransduced the CD4ζ-GFP fused protein into hESCs and iPSCs using the SB system with puromicin antibiotic selection and did not find CD4ζ-GFP silencing untill passage 37 (Fig. 1C). We therefore used NK cells derived from CD4ζ-GFP-SB transduced-hESCs or iPSCs all in vivo studies.

NK Cells Derived from CD4ζ-hESCs and CD4ζ-iPSCs

Previous studies by our group to derive NK cells from both hESCs and iPSCs have used stromal-based systems [31, 51]. More recently, we shifted to use of defined serum-free conditions that can be effectively scaled to produce potentially clinical-scale quantities of NK cells [44, 45, 55]. Briefly, in this system, undifferentiated hESCs or iPSCs are dissociated as single cell suspension and seeded into 96-well round-bottom plates by brief centrifugation EBs. After 11 days of culture in serum-free media with defined cytokines, differentiated spin EBs containing hematopoietic progenitors (CD34+/CD45+) were transferred to NK cell differentiation media supplemented with a combination of cytokines with or without EL08-1D2 stromal cells routinely generates a lymphocyte population where more than 90% of the cells are CD45+CD56+ (Fig. 2A). Both CD4ζ-hESC- and CD4ζ-iPSC-derived CD45+CD56+ populations expressed the CD4 receptor and GFP. Similar to unmodified hESC-, iPSC-, or PB-NK cells [31, 51], these CD45+CD56+ cell populations are mostly CD117CD94+, which has been demonstrated to be a more cytotoxic subset of NK cells [51, 56, 57]. We have previously demonstrated extensive phenotypic analysis of hESC and iPSC-derived NK cells expressing similar surface makers including the Fc receptor CD16, killer immunoglobulin receptors (KIRs), NKG2A, NKG2D, NKp44, and NKp46 as PB-NK cells [31]. CD4ζ-hESC- and CD4ζ-iPSC-NK cells also had a similar phenotype as their unmodified counterparts and PB-NKs (Fig. 2B). We then examined chemokine receptors expression on CD4ζ-hESC- or CD4ζ-iPSC-NK cells. CCR5 and CXCR4, also known as HIV coreceptors [58] were not observed to be expressed on either CD4ζ-modified hESC- or iPSC-NK cells (Fig. 2B). The chemokine receptors CXCR3, CCR7, and adhesion molecule CD62L are all involved in NK cell homing to secondary lymphoid tissues [59]. We found that CD4ζ-hESC or iPSC-NK cells expressed similar levels of CXCR3 as PB-NKs, but less CCR7 and CD62L (Fig. 2B). Next, to evaluate the function of the CD4ζ chimeric receptor in hESC- and iPSC-NK cells, addition of anti-CD4 mAb OKT4A followed by goat F(ab)′ anti-mouse IgG was used to cross-link and stimulate cells. Stimulation of effector function through the CD4 chimeric receptor is dependent on tyrosine phosphorylation [60], which can be determined by phosphoflow cytometry (Fig. 2C). We found tyrosine phosphorylation is rapidly induced in both CD4ζ-hESC- and CD4ζ-iPSC-NK cells by cross-linking of the CD4ζ chimeric receptors (Fig. 2D), indicating this chimeric receptor is functionally active following differentiation of pluripotent stem cells into NK cells.

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Figure 2. Generation of natural killer (NK) cells from CD4ζ expressing human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). (A): Flow cytometric analysis of CD56+CD45+ NK cells derived from hESC, CD4ζ-hESC, iPSC, and CD4ζ-iPSC. Expression of lymphocyte activating receptors and homing receptors on NK cells as indicated. These cells are compared with NK cells isolated from peripheral blood (PB-NK). (B): CD56+ NK cell from hESC, CD4ζ-hESC, iPSCs, and CD4ζ-iPSCs are all CD3- as are PB-NKs. Expression of surface marker CD16, killer immunoglobulin receptors, NKG2A, NKG2D, NKp44, NKp46, HIV coreceptors CCR5, CXCR4, and homing receptors CXCR3, CCR7, and CD62L. (C): Activity of CD4ζ in NK cells derived from CD4ζ-hESCs and CD4ζ-iPSCs. Both CD4ζ-hESC- and CD4ζ-iPSC-NK cells were stimulated with ([squlf]) or without ([squlf]) anti-CD4 and goat anti-mouse IgG F(ab′)2 to initiate receptor cross-linking. Cells were then intracellularly stained by tyrosine phosphorylation Ab 4G10 followed by PE-anti-mouse IgG. Cross-linked cells stained with mouse IgG and PE-anti-mouse IgG were used as isotype controls ([squlf]). Flow cytometry plots represented one of the three independent experiments. (D): Tyrosine phosphorylation measured by flow cytometry for mean fluorescent intensity. The solid lines represent mean ± SD. Abbreviations: FSC, Forward scatter; SSC, Side scatter; GFP, green fluorescent protein; PB, peripheral blood; NK, natural killer; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell.

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CD4ζ-hESC- and CD4ζ-iPSC-NK Cell Inhibition of HIV Replication In Vitro

Our previous studies demonstrated that both hESC- and iPSC-NK cells have potent ability to inhibit HIV infection [31]. The CEM-GFP T-cell line infected with HIV-1 NL4-3 leads to GFP expression, which provides accurate and reliable quantification of HIV infection and the effects of our NK cell-based inhibition to HIV replication [61]. To determine if the expression of CD4ζ enhanced anti-HIV activity, CD4ζ-hESC- or CD4ζ-iPSC-NK cells, and their unmodified counterparts were cocultured with NL4-3-infected CEM-GFP cells at different effector–target (E/T) ratios and monitored for HIV replication for 2 weeks [31, 62]. As we have demonstrated previously, unmodified hESC- and iPSC-NK cells both inhibit HIV replication in a dose-dependent manner (Fig. 3A, 3B). Notably, CD4ζ modified hESC- and iPSC-NK cells lead to 90% inhibition of HIV replication, significantly greater than unmodified hESC- and iPSC-NK cells (p < .05). These studies indicate CD4ζ expression on hESC- and iPSC-derived NK cells effectively directs NK cells to HIV-infected cells.

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Figure 3. CD4ζ-human embryonic stem cell (hESC)- and CD4ζ-induced pluripotent stem cells (iPSC)-natural killer (NK) cells inhibit the replication of HIV in vitro. (A, B): CEM-green fluorescent protein (GFP) cells were incubated with HIVNL4-3 virus for 4 hours. Cells were then cocultured with CD4ζ-modified hESC-, CD4ζ-iPSC-, or peripheral blood (PB)-NK cells for 14 days. HIV infection was assessed by flow cytometry for GFP expression. Activity of HIV-1 was measured by the percentage GFP+ of CEM-GFP cells cocultured with (A) PB-, hESC-, and CD4ζ-hESC-NK cells or (B) PB-, iPSC-, and CD4ζ-iPSC-NK cells at day 11 with E:T ratios of 1:1([squlf]) and 5:1 ([squlf]). Cells were CEM gated. The error bars represent mean ± SD. Statistical comparison of percentage GFP+ between CD4ζ-hESC-/iPSC-NK versus hESC-/iPSC-NK cells was performed using the Student's t-test. (C–F): NK cells function against HIV-1-infected human CD4+ primary T-cells. (C, D) NK cells were cocultured with SF2-infected CD4+ T-cells at E:T rations of 5:1 for two weeks. HIV infection was evaluated by intracellular staining for gag p24 in all CD4 T-cells. The percentage of p24+ CD4+ in the cocultures of (C) no NKs, PB-, hESC-, and CD4ζ-hESC-NK cells or (D) no NKs PB-, iPSC-, and CD4ζ-iPSC-NK cells with HIV-infected CD4 T-cells at day 11. Cells were CD56 gated. (C, D): These demonstrate statistically lower percentage p24+ in CD4ζ-hESC-/iPSC-NK culture compared with hESC-/iPSC-NK cells, respectively. (E): NK cells were evaluated for HIV infection in all CD56+ cells at day 11 of coculture. Either CD4ζ-hESC-NKs or hESC-NKs were negative for p24 staining. hESC-NKs with no HIV as negative controls. (F, G): Surface expression of CD107a was evaluated to measure NK cell cytolytic activity. Flow cytometric analyses of CD107a expression on (F) hESC- and CD4ζ-hESC-NK cells or (G) iPSC- and CD4ζ-iPSC-NK cells following stimulation with HIV-1-infected CD4+ T-cells for 5 hours. Uninfected CD4+ T-cells were used as controls. Cells were all CD56+ gated. Both CD4ζ-hESC- and CD4ζ-iPSC-NK cell populations stimulated by HIV-1-infected CD4+ T-cells show significantly increased CD107a expression compared with hESC- and iPSC-NK cells (p < .05). The data represent one of the three independent experiments. Abbreviations: hESC, human embryonic stem cells; iPSC, induced pluripotent stem cells; NK, natural killer; PB, peripheral blood.

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As a more rigorous in vitro test, we next studied the ability of each cell population to limit infection of primary CD4+ T-cells. To compare the anti-HIV activity of CD4ζ -hESC or CD4ζ -iPSC-NK cells versus unmodified hESC- or iPSC-NK cells, primary CD4+ T-cells infected with HIV-1SF2 (X4R5) [47] were used as targets. Again, HIV-infected CD4+ T-cells were cocultured with or without NK cells for 2 weeks. HIV infection was quantified by intracellular staining of CD4+T-cells for the viral gag protein p24 every 3–4 days over time. Lower percentages of p24+ T-cells were observed in cultures with all populations of NK cells compared with controls (Fig. 3C, 3D). Additionally, the percentage of p24+ T-cells were significantly lower in the coculture with CD4ζ-hESC- (p = .034, n = 3) or CD4ζ-iPSC-NK cells (p = .029, n = 3) compared with unmodified hESC- and iPSC-NK cells, respectively. These findings again demonstrate CD4ζ-modified hESC- and iPSC-NK cells inhibited HIV infection more effectively than unmodified hESC- and iPSC-cells. To further confirm that the inhibition of HIV infection is due to NK cell activation, we monitored NK cell degranulation during coculture with HIV-1-infected CD4+ T-cells. Expression of CD107a has been used as a measure of NK cell killing following stimulation [62, 63]. Here, we found CD107a was significantly increased on CD4ζ-hESC- (p = .045, n = 3) and CD4ζ-iPSC-NK cells (p = .041, n = 3) than unmodified hESC- or iPSC-NK cells after cocultured with HIV-1-infected CD4+ T-cells at d7 (Fig. 3E, 3F). Overall, these data demonstrate that both CD4ζ-hESC- and CD4ζ-iPSC-NK cells inhibit HIV replication more efficiently than their unmodified counterparts.

In Vivo Anti-HIV Activity of CD4ζ-hESC-NK Cells

To evaluate hESC-/iPSC-NK cell anti-HIV activity in vivo, we used the human PBL (hPBL)-NSG mouse model of HIV-1 infection [48]. In these studies, 107 PBL are injected intraperitoneally into NSG mice. After 2 weeks, the mice were then infected with HIV NL4-3 at 30,000 TCID50 [50]. This leads to productive HIV infection as demonstrated by loss of CD4+ cells and production of virions [64]. Then, we injected two million CD4ζ-hESC-, hESC-, or PB-NK cells in the day following HIV infection. At days 6, 9, and 12 post-NK cell treatment, blood samples were collected and examined for CD4 T-cell levels determined by human CD4+CD3+/CD4CD3+ ratios and HIV infection determined by the percentage p24+ T-cells. A general pattern of higher CD4 T-cell level was observed in HIV-infected mice with NK cell treatment (Fig. 4), demonstrating suppression of HIV activity [19, 65]. CD4ζ-hESC-NKs cells, illustrated by their expression of CD45, CD56, and CD4 and negative for CD3, were detected in PB early as day 6 after treatment (Fig. 4A). At this time point, CD4+CD3+/CD4CD3+ ratios were seen to decrease less in mice with NK cell treatment than controls (Fig. 4B, 4C, left panel). By day 12 of NK cell treatment (day 13 of HIV infection), there was a statistically significant difference in CD4+CD3+/CD4CD3+ ratios between mice treated with CD4ζ-hESC- (p = .024, n = 3), hESC- (p = .028, n = 3), PB-NK (p = .029, n = 3) cells, and controls. (Fig. 4C, right panel). As CD4 receptor on surface of HIV-infected cells is decreased after HIV infection, we used CD3+CD8 instead of CD3+CD4+ to evaluate HIV infection of CD4 T-cells. No significant levels of HIV-infected p24+CD3+CD8 cells were observed (less than 1%) at day 6, but by day 9 of NK cell injection, the percentages of p24+CD3+ CD8 cells were dramatically increased in control mice than those treated with either CD4ζ-hESC-, hESC-, or PB-NKs (Fig. 4D). Notably, we did not observe any statistical difference between CD4ζ-hESC-NK cells and unmodified hESC-NK cells on suppression of HIV infection and retaining CD4 T-cell level, but these data indicate that both CD4ζ-modified and unmodified hESC-derived NK cells can inhibit HIV replication and prevent CD4+ T-cell depletion in vivo.

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Figure 4. Human embryonic stem cells (hESC)-natural killer (NK) cells and CD4ζ-hESC-NK cells suppress HIV replication in peripheral blood (PB). Two weeks after PB lymphocyte (PBL) reconstitution, mice were infected with HIV NL4-3 and treated with NK cells next day. PB was then collected at days 6, 9, and 12 with or without NK cell treatment. HIV infection was evaluated by CD4+ T-cell depletion and HIV+ cell percentage in PB. CD4+ T-cell level was determined by flow cytometry for CD4+CD3+/CD4CD3+ ratios. HIV-infected human cells were evaluated by intracellular staining for gag p24+. (A): CD45+CD3CD4+ cells that were CD56+ and green fluorescent protein (GFP+) detected in PB of mice treated with CD4-hESC-NK cells after day 6. (B): Human CD45+ cells that express CD3 and CD4 were assessed in PB of HIV-1-infected NSG mice treated with or without NK cells at day 6. All cells were hCD45+ gated. Flow cytometry plots are representative of one mouse of each condition in at least three independent experiments with a minimum of three mice in each experimental group. (C): CD4+ T-cell levels in PB of HIV-infected mice determined by CD4+CD3+/CD4CD3+ ratios at day 6 (left panel) and day 12 (right panel) of NK cell treatment. All mice were analyzed before HIV infection to set up baseline CD4+CD3+/CD4CD3+ratios. (D): Suppression of HIV infection was evaluated by the percentages of p24+ cells in all hCD3+CD8 from PB at day 9 of NK cell treatment. Data in (C) and (D) represent the average of the three separate experiments with at least three mice in each group, the error bars indicate mean ± SD. Statistical comparison of CD4+ T-cell level and percentage p24+CD3+CD8 in NK cell treated mice to untreated mice was performed using Student's t-test. p values are provided for each indicated comparison. Abbreviations: hESC, human embryonic stem cells; NK, natural killer; PB, peripheral blood.

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To further determine whether the NK cell treatment could suppress HIV replication, the levels of viral RNA in mouse PB plasma and proviral DNA in different tissues was evaluated. We isolated viral RNA from plasma collected on day 12 of NK cell treatment and performed quantitative reverse PCR to measure the RNA level of the LTR sequence [54, 65]. Standard LTR cDNA was used to calculate RNA copy numbers [19, 65]. The viral RNA in HIV-infected mice had approximately 2,000 copies/ml, whereas the RNA levels were significantly lower in all mice treated with either CD4ζ-hESC-, hESC-, or PB-NK cells at day 12 (Fig. 5A). This confirms NK cell-mediated suppression of HIV replication in vivo. To test integrated proviral DNA, PB was collected from mice at days 6, 9, and 12 post-NK cell treatments. DNA was extracted and proviral elements were detected by specific amplification of HIV-1. We were not able to detect proviral DNA from blood samples by qPCR until day 9 (Fig. 5B). Similar to the viral RNA level, the proviral DNA was decreased in PB in mice receiving NK cells. We did not observe detectable DNA levels in PB at day 12, which might be due to low levels of CD4+ T-cells. To test proviral DNA in other peripheral tissues/organs, mice were then sacrificed on day 13 of NK cell treatment (day 14 of HIV infection) when mouse peritoneal washes and spleens were collected for LTR analysis. Again, HIV DNA were found to be significantly lower in the peritoneal cavity (Fig. 5C) and spleen (Fig. 5D) of HIV-1-infected mice treated with CD4ζ-hESC-NK cells, hESC-NK cells, or PB-NK cells when compared with control mice. Thus, these findings suggest that NK cell treatment resulted in significant suppression of HIV replication in several tissues/organs in this hPBL NSG mouse model.

image

Figure 5. CD4ζ-modified and -unmodified human embryonic stem cells (hESC)-natural killer (NK) cells suppress HIV replication in the plasma and tissues of peripheral blood lymphocyte (PBL)-NSG mice. (A): Blood plasma from HIV-infected mice was collected 12 days after NK cell treatment. Viral RNA levels per sample were determined by quantitative reverse transcription polymerase chain reaction (PCR) and results were calculated based on the standard LTR cDNA copy numbers. The points represent the copies of HIV RNA per milliliter of blood and the solid line represents mean per group. HIV proviral DNA was quantitatively assessed in human cells from peripheral blood collected on day 6, 9, 12-day after NK cell treatment. The DNA level was detected by qPCR at day 9 (B). Mice were killed at day 13 of NK cell treatment and cells were collected from spleen (C) and peritoneal fluid (D) for qPCR. The points represent the copies of HIV proviral DNA per 106 human CD45+ cells and the solid line represents mean per group. Statistical comparison was performed Prism 5 between NK cell-treated groups versus nontreated group. The solid lines represent mean ± SD. The data are representative of one of the three experiments with at least three mice each group. Abbreviations: hESC, human embryonic stem cells; NK, natural killer; PB, peripheral blood.

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In Vivo Anti-HIV Activity of CD4ζ-iPSC-NK Cells

Next, we investigated in vivo anti-HIV activities of both CD4ζ-modified and -unmodified iPSC-NK cells. Using the PBL-NSG mouse model, we treated HIV-infected mice with CD4ζ-iPSC-, iPSC- or autologous PB-NK cells as above. CD4+ T-cell levels (measured as CD4+CD3+/CD4CD3+ ratios again) were examined at days 6, 9, and 12 post NK cell treatment (Fig. 6A). Consistent with CD4ζ-modified and -unmodified hESC-NK cells, CD4ζ-iPSC-, and iPSC-NK cells were able to maintain CD4+CD3+/CD4CD3+ ratios at higher levels in mice up to two weeks after HIV infection (Fig. 6B). The viral load was suppressed to a significantly lower level in the plasma of mice treated with all NK cells compared with controls (Fig. 6C). These results suggest that NK cells derived from iPSCs are also capable of inhibiting HIV replication in vivo.

image

Figure 6. Induced pluripotent stem cells (iPSC)- and CD4ζ expressing natural killer (NK) cells suppress HIV replication in peripheral blood (PB) in HIV-infected mice. PB was collected from HIV-infected NSG mice after NK cell treatment. CD4+ T-cell levels were determined by flow cytometry for CD4+CD3+/CD4CD3+. HIV-infected human cells were evaluated by gag p24+. (A): Human CD45+ cells that express CD3 and CD4 were assessed in PB of HIV-1-infected NSG mice treated with or without NK cells at day 12. All cells were hCD45+ gated. The flow cytometry plots are representative of one mouse of each group in at least three independent experiments with a minimum of three mice. (B): CD4+ T-cell levels in PB of HIV-infected mice determined by CD4+CD3+/CD4CD3+ ratios after NK cell treatment at day 12. (C): HIV infection was evaluated by the percentages of p24+ cells in CD3+CD8. Data in (B) and (C) represent the average of one of the three separated experiments with at least three mice in each group, the error bars indicate mean ± SD. Abbreviations: iPSC, induced pluripotent stem cells; NK, natural killer; PB, peripheral blood; PBS, phosphate buffered saline.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

Recent studies based on genetic modification of HSCs or mature lymphocytes have begun to use combined gene and immune therapy against HIV/AIDS. However, this approach typically needs to be carried out on a patient-specific basis, which could be challenging to apply in a broad clinical setting. In contrast, hESCs and iPSCs derived cells could provides a novel “universal” and “off-the-shelf” cell population for anti-HIV treatment without concerns for immune rejection. In fact, studies have demonstrated that hESCs and iPSCs are able to differentiate into different hematopoietic lineages including TCR specific-T-cells derived from direct reprogramming or T-iPSCs [23-29], indicating the potential of hESC- or iPSC-based innate and adaptive immunotherapy for HIV.

CARs have been designed and targeted to a wide-array of cancers and virally infected targets. This system has been suggested as a highly effective means to increase immunity in cancer therapy [12-15]. Investigators have also adapted this strategy to HIV treatment by genetic engineering primary T/NK cells with specific anti-HIV TCR or HIV CD4 receptor (reviewed in ref. [4]). The CD4ζ construct has been extensively tested in multiple systems such primary T and NK cells against HIV [36-40]. Some of these trials transduced CD4ζ into primary T-cells of HIV-infected patients with varying effects [37, 39]. Here, we combined the unique advantages of the hESCs/iPSCs system and the specificity and efficacy of CARs together to genetically modify hESCs/iPSCs. We initially expressed CD4ζ in hESCs and iPSCs by lentivirus, which was commonly silenced after 10–15 passages (Fig. 1C). Our previous studies demonstrated that the Sleeping Beauty (SB) transposon system is a more stable means to transfer genetic information to hESCs [42, 43]. Therefore, we used the SB system to achieve more stable CD4ζ expressing hESC and iPSC cell lines for our in vivo studies.

By using the “spin EB” system, we were able to develop NK cells from CD4ζ-modified hESCs and iPSCs expressing high levels of CD4 and a panel of surface markers as unmodified hESC-, iPSC-, and PB-NKs (Fig. 2). We found that both CD4ζ-modified hESC-NK and iPSC-NK cells are able to inhibit HIV replication more efficiently in vitro than their unmodified counterparts respectively (Fig. 3). Although KIR expression is associated with NK licensing through interaction with HLA class I molecules, the activation of NK cells is ultimately determined by the balance between inhibitory and activating receptors including but not limited to KIRs [66]. Thus, higher KIR expression may not necessarily lead increased functional responses at a single cell level [67]. Here, we noticed that CD4ζ-iPSC-NKs express higher levels of KIRs (Fig. 2B), but their response (measured by CD107a) to HIV-infected targets was similar to CD4ζ-hESC-NKs that had lower KIRs expression (Fig. 3B). However, fewer infected T-cells (as evidenced by p24+CD4+) in CD4ζ-hESC-NKs and HIV coculture (Fig. 3C) might be secondary to other mechanisms such as increased apoptosis through TNF-related apoptosis-inducing ligand (TRAIL) pathway [68].

During the in vivo assays, we also observed both CD4ζ-modified hESC- and iPSC-NK cells, and their unmodified cells were able to suppress HIV infection in PBL-NSG mice (Figs. 4-6). However, we did not find a significant difference in HIV inhibition from CD4ζ-modified NK cells as in vitro. In this case, we treated HIV-infected mice with 2 × 106 NK cells, which may be insufficient cell to induce significant inhibition on viral infection in vivo. The lack of additional costimulatory domains present on the intracellular portion ζ chain may also limit the activity [69]. As has been demonstrated in several preclinical cancer models, CARs containing additional signaling molecules (second and third generation CARs) have enhanced in vivo persistence and activity [70]. Additionally, other investigators have found that addition of membrane bound cytokines can also enhance the in vivo activity of CAR containing effectors [67, 71]. Collectively, the CD4ζ receptor may not be the best choice for HIV therapy as its varying effect has been suggested in preclinical studies [37, 39]. Recently, a broadly neutralizing anti-HIV antibody b12, which recognizes gp120 CD4 binding site, has been successfully engineered into human HSCs and produced functional IgG in culture system [20]. Interestingly, several neutralizing Abs have been identified and characterized (reviewed in ref. [72]). It may be beneficial to engineer them as CARs and test their in vivo efficacy.

Studies by Bernstein et al. showed that stimulated CD4+ NK cells had a low percentage of p24+ when infected by HIV-1-Bal (R5) [73]. As we expressed HIV CD4 receptor in hESCs and iPSCs, it is reasonable to question whether the CD4 expression would make hESC- and iPSCs-NK cells susceptible to HIV infection. Fortunately, these hESC- or iPSC-NK cells did not express the HIV coreceptor CXCR4, which is required for HIV-1 X4 or R5 entry into targets [74] (Fig. 2B). Although a small proportion of CD4ζ-hESC-NK cells are positive for another coreceptor CCR5 (Fig. 2B), NK cells were not infected when they were cocultured with HIV-1 (SF2, X4R5)-infected CD4 T-cells (Fig. 3E).

CXCR3, CCR7, and CD62L are three major receptors involved in NK cells homing to secondary lymphoid organs [59]. We examined their expression on hESC- or iPSC-NK cells (Fig. 2B) and found that CXCR3 was comparably expressed on both CD4ζ modified and unmodified hESC- and iPSC-NK cells as PB-NKs. However, CD62L and CCR7 are expressed at a lower level on hESC- and iPSC-NK cells, which may explain why hESC- or iPSC-NK cells were not detected in secondary lymphoid organs such as the spleen in NSG mice after injection (data not shown). It is also pertinent to note that the mouse model does not recapitulate the extent of human physiology and could alter the in vivo trafficking of the infused cells. Subsequent work will aim to improve the homing and trafficking activity of hESC- or iPSC-derived NK cells by enforced expression of defined homing receptors [67, 71], which could also potentially help to clear viral reservoirs.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

CD4ζ-hESC- and - iPSC-derived NK cells are capable of suppressing HIV replication in vitro with higher efficacy than their unmodified counterparts. Both CD4ζ- modified and -unmodified hESC- and iPSC-NK cells demonstrate their inhibition of HIV inhibition in vivo, indicating the feasibility of using hESC/iPSCs as a cellular source for combined immune/gene therapy of HIV treatment. These studies also provide a foundation and a model system to further investigate innate immune responses during viral infection.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

We thank Mike Depp and Dr. Louis Mansky (University of Minnesota) for providing HIV-1 NL 4-3 virus, Dr. Scott Kitchen (University of California, Los Angeles) for providing CD4ζ construct, technical support, and suggestions, as well as Dr. Ramesh Akkina (Colorado State University) for providing standard long terminal repeat cDNA and RNA and technical support. We also thank Melinda Hexum, Allison Bock, and Mike Lepley for technical assistance and David Hermanson for proofreading. This work was supported by NIH grant HL77923 and a Grand Challenges Exploration grant from the Bill and Melinda Gates Foundation (to D.S.K.) and a fellowship grant from amfAR-The Foundation for AIDS Research (to Z.N.).

Author Contribution

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References

Z.N.: designed and performed experiments and wrote the manuscript; D.A.K.: performed experiments and wrote the manuscript; L.B. and J.A.: performed experiments; D.S.K.: designed experiments, wrote and edited the manuscript.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Author Contribution
  10. Disclosure of Potential Conflicts of Interest
  11. References
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