Expression of chimeric receptor CD4ζ by natural killer cells derived from human pluripotent stem cells improves in vitro activity but does not enhance suppression of HIV infection in vivo

Abstract

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.

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

Figure 1. Expression of CD4ζ chimeric receptor in hESCs and iPSCs

(A) Diagram of CD4ζ cloned in lentiviral vector or Sleeping Beauty transposon vector. (B) Transduced hESC cells were analyzed by flow cytometry for expression 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.

Figure 2. Generation of NK cells from CD4ζ-hESCs and CD4ζ-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 to NK cells isolated from peripheral blood (PB-NK). (B) CD56⁺ NK cell from hESC, CD4ζ-hESC, iPSCs, CD4ζ-iPSCs are all CD3- as are PB-NKs. Expression of surface marker CD16, KIRs, NKG2A, NKG2D, NKp44, NKp46, HIV co-receptor CCR5, CXCR4 and homing receptor 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 () or without () anti-CD4 and goat anti-mouse IgG F(ab’)₂ to initiate receptor cross-linking. Cells were then intracellular stained by tyrosine phosphorylation Ab 4G10 followed by PE- anti-mouse IgG. Cross-linked cells were stained with mouse IgG and PE- anti-mouse IgG were used as isotype controls (). Flow cytometry plots represented 1 of at least 3 independent experiments. (D) Trysine phosphorylation measured by flow cytometry for the mean fluorescent intensity (MFI). The solid lines represent the mean +/− the SD.

Figure 3. CD4ζ-hESC- and CD4ζ-iPSC-NK cells inhibit the replication of HIV in vitro

(A to B) CEM-GFP cells were incubated with HIVNL4-3 virus for 4 hours. Cells were then co-cultured with CD4ζ-modified hESC-, CD4ζ-iPSC- or PB-NK cells for 14 days. HIV infection was assessed by flow cytometry for GFP expression. Activity of HIV-1 was measured by the percent GFP⁺ of CEM-GFP cells co-cultured 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() and 5:1 (). Cells were CEM gated. The error bars represent the mean +/− the standard deviation (SD). Statistical comparison of % GFP⁺ between CD4ζ-hESC-/iPSC-NK vs. 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 and D) NK cells were co-cultured 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 co-cultures 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) and (D) demonstrate statistically lower % p24⁺ in CD4ζ-hESC-/iPSC-NK culture compared to hESC-/iPSC-NK cells respectively. (E) NK cells were evaluated for HIV infection in all CD56⁺ cells at day 11 of co-culture. Either CD4ζ-hESC-NKs or hESC-NKs were negative for p24 staining. hESC-NKs with no HIV as negative controls. (F and 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-NKs or (G) iPSC- and CD4ζ-iPSC-NKs 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 cells populations stimulated by HIV-1-infected CD4⁺ T cells show significantly increased CD107a expression compared to hESC- and iPSC-NK cells (P<0.05). The data represent one of at least 3 independent experiments.

Figure 4. hESC-NK cells and CD4ζ-hESC-NK cells suppress HIV replication in peripheral blood

Two weeks after PBL reconstitution, mice were infected with HIV NL4-3 and treated with NK cells next day. Peripheral blood was then collected at day6, 9 and 12 with or without NK cell treatment. HIV infection was evaluated by CD4⁺ T cell depletion and HIV⁺ cell percentage in peripheral blood. CD4⁺ T cell level was determined by flow cytometry for CD4⁺CD3⁺/CD4⁻CD3⁺ ratios. HIV infected human cells were evaluated by intracellular staining for gag p24⁺. (A) CD45⁺CD3⁻CD4⁺ cells that were CD56⁺ and GFP⁺ detected in peripheral blood of mice treated with CD4-hESC-NK cells after day 6. (B) Human CD45⁺ cells that express CD3 and CD4 were assessed in peripheral blood 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 1 mouse of each condition in at least 3 independent experiments with a minimum of 3 mice in each experimental group. (C) CD4⁺ T cell levels in peripheral blood of HIV infected mice determined by CD4⁺CD3⁺/CD4⁻CD3⁺ ratios at day 6 (left panel) and day 12 (right panel) of NK cell treatment. All mice were analyzed prior to HIV infection to set up baseline CD4⁺CD3⁺/CD4⁻CD3⁺ratios. (D) Suppression of HIV infection was evaluated by the percentages of p24⁺ cells in all hCD3⁺CD8⁻ from peripheral blood at day 9 of NK cell treatment. Data in (C) and (D) represent the average of one of three separated experiments with at least 3 mice in each group, the error bars indicate the mean +/− the SD. Statistical comparison of CD4⁺ T cell level and % p24⁺CD3⁺CD8⁻ in NK treated mice to untreated mice was performed using Student's t test. P values are provided for each indicated comparison.

Figure 5. CD4ζ- modified and unmodified hESC-NK cells suppress HIV replication in the plasma and tissues of 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 transcriptase (Q-RT)-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 Q-PCR at day 9 (B). Mice were sacrificed at day 13 of NK cell treatment and cells were collected from spleen (C) and peritoneal fluid (D) for Q-PCR. 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 using prism 5 between NK cell treated groups vs. non-treated group. The solid lines represent the mean +/− the SD. The data are representative of one of 3 experiments with at least 3 mice each group.

Figure 6. iPSC- and CD4ζ- expressing NK cells suppress HIV replication in peripheral blood in HIV-infected mice

Peripheral blood was collected from HIV-infected NSG mice after NK cell treatment. CD4⁺ T cell levels were determined by flow cytometry for CD4⁺CD3⁺/CD4⁻CD3⁺. HIV infected human cells were evaluated by gag p24⁺. (A) Human CD45⁺ cells that express CD3 and CD4 were assessed in peripheral blood of HIV-1-infected NSG mice treat with or without NK cells at day 12. All cells were hCD45⁺ gated. The flow cytometry plots are representative of 1 mouse of each group in at least 3 independent experiments with a minimum of 3 mice. (B) CD4⁺ T cell levels in peripheral blood of HIV infected mice determined by CD4⁺CD3⁺/CD4⁻CD3⁺ 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 three separated experiments with at least 3 mice in each group, the error bars indicate the mean +/− the SD.