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. 2015 Feb;23(2):387-95.
doi: 10.1038/mt.2014.207. Epub 2014 Nov 4.

Broadly-specific cytotoxic T cells targeting multiple HIV antigens are expanded from HIV+ patients: implications for immunotherapy

Sharon Lam  1 , Julia Sung  2 , Conrad Cruz  3 , Paul Castillo-Caro  4 , Minhtran Ngo  5 , Carolina Garrido  2 , Joann Kuruc  2 , Nancie Archin  2 , Cliona Rooney  5 , David Margolis  2 , Catherine Bollard  3
Affiliations

Broadly-specific cytotoxic T cells targeting multiple HIV antigens are expanded from HIV+ patients: implications for immunotherapy

Sharon Lam et al. Mol Ther. 2015 Feb.

Abstract

Antiretroviral therapy (ART) is unable to eradicate human immunodeficiency virus type 1 (HIV-1) infection. Therefore, there is an urgent need to develop novel therapies for this disease to augment anti-HIV immunity. T cell therapy is appealing in this regard as T cells have the ability to proliferate, migrate, and their antigen specificity reduces the possibility of off-target effects. However, past human studies in HIV-1 infection that administered T cells with limited specificity failed to provide ART-independent, long-term viral control. In this study, we sought to expand functional, broadly-specific cytotoxic T cells (HXTCs) from HIV-infected patients on suppressive ART as a first step toward developing cellular therapies for implementation in future HIV eradication protocols. Blood samples from seven HIV+ patients on suppressive ART were used to derive HXTCs. Multiantigen specificity was achieved by coculturing T cells with antigen-presenting cells pulsed with peptides representing Gag, Pol, and Nef. All but two lines were multispecific for all three antigens. HXTCs demonstrated efficacy as shown by release of proinflammatory cytokines, specific lysis of antigen-pulsed targets, and the ability to suppress HIV replication in vitro. In conclusion, we are able to generate broadly-specific cytotoxic T cell lines that simultaneously target multiple HIV antigens and show robust antiviral function.

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Figures

Figure 1
Figure 1
HXTC manufacturing process. Peripheral blood mononuclear cells (PBMCs) are isolated from 60–100 ml of whole blood samples from aviremic HIV+ patients. Monocytes are separated using plastic adherence and used to generate dendritic cells. Dendritic cells are pulsed with PepMixes spanning Gag, Pol, and Nef before being used to stimulate the nonadherent PBMC fraction which contains T cells. T cells undergo two more weekly stimulations by coculture with autologous, PepMix-pulsed PHA-blasts. During the third stimulation, irradiated K562 cells that have been genetically modified to express costimulatory molecules CD80, CD83, CD86, and 41-BBL are cocultured with the T cells in addition to the PepMix-pulsed PHA-blasts.
Figure 2
Figure 2
CD8, effector memory HIV-specific T cells expand in response to Gag, Pol, and Nef stimulation. (a) 1 × 106 T cells were stimulated with Gag, Pol, and Nef PepMixes on day 0. Expansion is shown in absolute cell counts and was measured 10 days after the third stimulation. (b) Nonadherent PBMCS containing T cells (n = 3, error bars defined by SEM) and (c) HXTCs were phenotyped using extracellular flow cytometry for T cell markers (CD3, CD4, and CD8), NK cell markers (CD56+), and memory subsets (CD45RA and CD62L) 7–10 days after the third stimulation (n = 5, error bars defined as the SEM). (d) PD-1 expression on HXTCs or EBV-specific CTL generated from healthy, HIV-negative donors measured 7–10 days after third stimulation (representative of n = 3). (e) Comparison of percentage of PD-1+ T cells found in HXTCs versus EBV-specific CTLs generated from healthy, HIV-negative donors (n = 3, error bars defined by SEM).
Figure 3
Figure 3
HXTCs have CD8 and CD4-mediated responses to multiple HIV antigens. (a) Nonadherent PBMCs on day 0 (n = 3, error bars defined by SEM) and (b) HXTCs (n = 7, error bars defined by the SEM) 10 days after the third stimulation were tested for IFN-γ release as a marker of T cell activation using IFN-γ ELIspot assay against media as a negative control, irrelevant antigens actin or Oct4 PepMixes as negative controls, HIV PepMixes, or staphylococcus enterotoxin B (SEB) as a positive control. (c) HXTCs were pretreated for 1 hour with no antibodies (black bar), MHC class I (solid gray bar), and class II (striped bar) blocking antibodies before being plated in an IFN-γ ELIspot assay against Gag, Pol, and Nef PepMixes (representative example of n = 3, error bars defined by the SD). (d,e) Intracellular IFN-γ staining in HXTCs after 12-hour exposure to HIV PepMixes, SEB (positive control), or no antigen (media, negative control) (n = 4, error bars defined by SD). (f) Summary of the number of antigens each HXTC line simultaneously recognized based on IFN-γ ELIspot assay. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.
Figure 4
Figure 4
HXTCs release multiple proinflammatory cytokines and are polyclonal. (a) IL-2 and (b) TNF-a release by HXTCs in response to no antigen (neg), Gag, Pol, and Nef PepMix was measured by ELISA as described in the Materials and Methods section (representative of n = 3, error bars represent SD). (c) Vβ usage of HXTCs was analyzed using a flow cytometry–based assay, IOTest Beta Mark TCR V Kit (BD Biosciences). (d) HXTCs were tested against the Nef group M consensus region broken up into peptide pools containing seven to eight peptides each in an IFN-γ ELIspot assay. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.
Figure 5
Figure 5
HXTCs are cytotoxic and inhibit viral outgrowth in infected cultures. (a,b) Ability of HXTCs to lyse antigen-expressing targets was measured using an 8-hour 51Chromium-release assay. Target cells consisted of autologous PHA-blasts that were pulsed with either media or Gag, Pol, or Nef PepMixes and were loaded with chromium. HXTCs were cultured with target cells pulsed with only the antigens they were specific for, which was predetermined by IFN-γ ELIspot assay. (c) Autologous CD8 depleted PBMCs were infected with HIV strain, JR-CSF by spinoculation. HXTCs or unexpanded CD8 T cells were cocultured with the infected target cells for 7 days before p24 release was measured using ELISA. *P ≤ 0.05. All error bars represent the SD.
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