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. 2018 Nov 5:9:2552.
doi: 10.3389/fimmu.2018.02552. eCollection 2018.

In vitro Evidence That Combination Therapy With CD16-Bearing NK-92 Cells and FDA-Approved Alefacept Can Selectively Target the Latent HIV Reservoir in CD4+ CD2hi Memory T Cells

Amanda G Tomalka  1 , Ivelisse Resto-Garay  1 , Kerry S Campbell  2 , Daniel L Popkin  1
Affiliations

In vitro Evidence That Combination Therapy With CD16-Bearing NK-92 Cells and FDA-Approved Alefacept Can Selectively Target the Latent HIV Reservoir in CD4+ CD2hi Memory T Cells

Amanda G Tomalka et al. Front Immunol. .

Abstract

Elimination of the latent HIV reservoir remains the biggest hurdle to achieve HIV cure. In order to specifically eliminate HIV infected cells they must be distinguishable from uninfected cells. CD2 was recently identified as a potential marker enriched in the HIV-1 reservoir on CD4+ T cells, the largest, longest-lived and best-characterized constituent of the HIV reservoir. We previously proposed to repurpose FDA-approved alefacept, a humanized α-CD2 fusion protein, to reduce the HIV reservoir in CD2hi CD4+ memory T cells. Here, we show the first evidence that alefacept can specifically target and reduce CD2hi HIV infected cells in vitro. We explore a variety of natural killer (NK) cells as mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) including primary NK cells, expanded NK cells as well as the CD16 transduced NK-92 cell line which is currently under study in clinical trials as a treatment for cancer. We demonstrate that CD16.NK-92 has a natural preference to kill CD2hi CD45RA- memory T cells, specifically CD45RA- CD27+ central memory/transitional memory (TCM/TM) subset in both healthy and HIV+ patient samples as well as to reduce HIV DNA from HIV+ samples from donors well controlled on antiretroviral therapy. Lastly, alefacept can combine with CD16.NK-92 to decrease HIV DNA in some patient samples and thus may yield value as part of a strategy toward sustained HIV remission.

Keywords: ADCC; CD16; CD2; FDA; HIV; NK; NK92; alefacept.

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Figures

Figure 1
Figure 1
Primary NK cells selectively target HIV+ 3C9 Jurkat for killing by ADCC with alefacept more than HIV− E6.1 in Jurkat mix. (A) Flow cytometry plots of Jurkat mix. Deep red cell tracker labeled HIV+ 3C9 was diluted 1:100 with CFSE labeled uninfected E6.1 parent Jurkat cell line. 3C9 Jurkat was either unstimulated or pre-stimulated with 1 ng/mL TNFα before mixing with E6.1 Jurkat to induce expression of GFP-HIV. (B) Experimental representative of quantification of CD2 MFI on Jurkat cells and n = 6 primary NK cell donors. (C) Flow cytometry plots of representative donor primary NK cells and Jurkat mix killing assay with 10 μg/mL IgG1 or alefacept. (D) Percent cytotoxicity of 3C9, E6.1 and primary NK cells with IgG1 control antibody or alefacept after co-culture as measured by absolute cell count flow cytometry with n = 6 primary NK cell donors with E:T = 0.1:1, after 19 h in co-culture Mean and SEM shown for 6 primary NK cell donors, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Statistical significance between IgG1 and alefacept conditions are indicated with black bars, statistical significance between cell types with IgG1 are indicated with gray bars and statistical significance between cell types with alefacept are indicated with red bars. Absolute CD2 MFI and relative MFI between cell types was consistent across experiments. One representative of 5 similar experiments is shown as the Jurkat cell lines are clonal.
Figure 2
Figure 2
Expanded NK cells exhibit enhanced spontaneous killing and reduced ADCC compared to primary NK cells; ADCC can be rescued by engaging CD2. (A) Primary NK cells from healthy donors were expanded on K562 C9 feeder cells with membrane bound IL-21 with recombinant human IL-2 ± BD FastImmuneTM α-CD2. (B) Percent positive of total population surface markers CD2, CD16, and CD56 throughout NK cell expansion as measured by flow cytometry. Statistical significance between CD2 across conditions are indicated in red. (C) CD2 MFI of cell types in killing assay where primary NK cells (day 0) or expanded NK cells (day 21) are co-cultured with CD4+ T cells from the same donor (self;auto) or mismatched donor (non-self;allo) for 19 h with E:T = 1:1 and 10 μg/mL alefacept or IgG1 control antibody. (D) Percent cytotoxicity of self (auto) or non-self (allo) CD4+ T cells after co-culture with primary NK cells (day 0) or expanded NK cells (day 21) as measured by absolute count flow cytometry (E) Percent cytotoxicity of self (auto) or non-self (allo) NK cells (NK cell death) after co-culture with CD4+ T cells as measured by absolute count flow cytometry (F) Representative flow cytometry plots of healthy donor day 0 primary NK cells or day 21 expanded NK cells killing self (auto) CD4+ T cells with CD4 vs. CD45RA. (G) Percent cytotoxicity of CD4+ T cell subsets CD45RA+ and CD45RA– of bulk CD4+ T cell population shown in (D). Mean and SEM shown for 4 healthy donors, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. For percent cytotoxicity graphs, statistical significance between IgG1 and alefacept conditions are indicated with black bars and statistical significance between cell types with alefacept are indicated with red bars.
Figure 3
Figure 3
CD16.NK-92 has natural preference to kill CD45RA– CD4+ memory T cells and that is enhanced by alefacept in healthy donors. (A) Representative healthy donor flow cytometry plots from killing assay with CD16.NK-92 and healthy donor CD4+ T cells 19 h in co-culture with CD45RA subsetting shown with no treatment, with CD16.NK-92 and 10 μg/mL alefacept or IgG1 control antibody. (B) CD2 MFI of CD16.NK-92, and CD4+ T cells. (C) Percent cytotoxicity of CD4+ T cells in killing assay with CD16.NK-92 after 19 h in co-culture with 10 μg/mL alefacept or IgG1 control antibody and E:T ratio dose response, as measured by absolute count flow cytometry. (D) Percent cytotoxicity of CD16.NK-92 in killing assay 19 h in co-culture with 10 μg/mL alefacept or IgG1 control antibody and E:T ratio dose response, as measured by absolute count flow cytometry. (E) Percent cytotoxicity of CD45RA+ and CD45RA– subsets of CD4+ T cells (as shown in C) at E:T = 0.5:1 after 19 h co-culture with CD16.NK-92 and 10 μg/mL alefacept or IgG1 control antibody, as measured by absolute count flow cytometry. Mean and SEM shown for 6 healthy donors, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Statistical significance between IgG1 and alefacept conditions are indicated with black bars, statistical significance between cell types with IgG1 are indicated with gray bars and statistical significance between cell types with alefacept are indicated with red bars.
Figure 4
Figure 4
CD45RA– CD27+ Central Memory/Transitional Memory (TCM/TM) CD4+ T cell subset is significantly depleted by CD16.NK-92 and alefacept in healthy donors. (A) Representative flow cytometry plots of CD16.NK-92 killing of CD4+ T cell subsets in healthy donor at E:T = 0.5:1, 19 h co-culture with 10 μg/mL alefacept or IgG1 control antibody. (B) CD2 MFI of CD4+ T cell subsets. (C) Percent cytotoxicity of CD4+ T cell subsets (same CD4+ T cell donors from Figure 3) co-cultured with CD16.NK-92 for 19 h at E:T = 0.5:1 with 10 μg/mL alefacept or IgG1 control antibody, as measured by absolute count flow cytometry. (D) Percent of whole representations of CD4+ T cell subsets by donor with and without treatment. (E) Absolute count of CD27+ CD45RA+ TCM/TM subset with no treatment and with CD16.NK-92 at E:T = 0.5:1 and 10 μg/mL alefacept or IgG1 control antibody after 19 h in co-culture. Mean and SEM are shown for 6 healthy donors, **P ≤ 0.01, ***P ≤ 0.001. For percent cytotoxicity graph, statistical significance between IgG1 and alefacept conditions are indicated with black bars and statistical significance between cell subsets with alefacept are indicated with red bars.
Figure 5
Figure 5
CD16.NK-92 and alefacept also target CD45RA– CD27+ T(CM/TM) CD4+ T cell subset in HIV+ donors. (A) Representative flow cytometry plots of HIV+ CD4+ T cell donor with memory subsetting at 19 h (1 day) and 3 days with no treatment, with CD16.NK-92 and 10 μg/mL alefacept or IgG1 control antibody at E:T = 0.5:1. (B) CD2 MFI of all cell types and subsets in co-culture at days 1 and 3. (C) Percent cytotoxicity of CD16.NK-92, CD4+ T cells, and CD4+ T cell subsets in HIV+ donors after co-culture for 19 h (1 day) with 10 μg/mL alefacept or IgG1 control antibody at E:T = 0.5:1 as measured by absolute count flow cytometry. (D) Percent cytotoxicity of CD16.NK-92, CD4+ T cells, and CD4+ T cell subsets after co-culture for 3 days with 10 μg/mL alefacept or IgG1 control antibody at E:T = 0.5:1 as measured by absolute count flow cytometry. Mean and SEM of 6 HIV+ donors shown, *P ≤ 0.05, **P ≤ 0.01. Statistical significance CD2 MFI at days 1 and 3 of the same cell type are indicated with black bars, statistical significance between cell types and subtypes of CD2 MFI at day 1 are indicated with blue bars and at day 3 by green bars. Statistical significance between IgG1 and alefacept conditions are indicated with black bars, statistical significance between cell types with IgG1 are indicated with gray bars and statistical significance between cell types with alefacept are indicated with red bars.
Figure 6
Figure 6
CD16.NK-92 and alefacept significantly reduce HIV DNA in vitro. (A) Percent of whole representation of CD4+ T cell memory subsets by HIV+ donor with no treatment, after 3 day co-culture with CD16.NK-92 and 10 μg/mL alefacept or IgG1 control antibody. (B) Absolute counts of CD27+ CD45RA– T(CM/TM) subset in HIV+ donors after 3 days in co-culture with CD16.NK-92 and 10 μg/mL alefacept or IgG1 control antibody. (C) HIV DNA copies per million CD4+ T cells as measured by quantitative PCR, with no treatment or after 3 day co-culture with CD16.NK-92 and 10 μg/mL alefacept or IgG1 control antibody. Samples in which HIV gag DNA was not detectable are indicated with N.D. Dotted line represents the limit of detection (L.O.D.) of 2 copies of HIV gag DNA copies/106 CD4+ T cells. Mean and SEM shown for 3 qPCR reactions of same sample and 6 HIV+ donors, *P ≤ 0.05, **P ≤ 0.01.
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