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S. Vasileiou et al.
cated unless patients are at least six months post-HSCT.12 Aerosolized ribavirin (RBV) has been approved by the US Food and Drug Administration (FDA) for the treatment of RSV, but it is extremely costly (5-day course = $149,756) and logistically difficult to administer, requiring a specialized nebulization device that connects to an aerosol tent sur- rounding the patient.13-16 Thus, the lack of approved antiviral agents for many clinically problematic CARV, and the high cost and complexity of administering aerosolized RBV, underscores the need for alternative treatment strategies.
Our group has previously demonstrated that the adop- tive transfer of in vitro-expanded virus-specific T cells (VST) can safely and effectively prevent and treat infec- tions associated with both latent [Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), human her- pesvirus 6 (HHV6)] and lytic [adenovirus (AdV)] viruses in allogeneic HSCT recipients.17,18 Given that susceptibility to CARV is associated with underlying cellular immune deficiency,1,5,6 in the current study, we explored the feasi- bility of extending the therapeutic range of VST therapy to include Influenza, RSV, hMPV and PIV-3.
We here describe a mechanism by which we can rapidly generate a single preparation of polyclonal (CD4+ and CD8+) VST with specificity for 12 immunodominant anti- gens derived from our four target viruses using Good Manufacturing Practices (GMP)-compliant manufacturing methodologies. The viral proteins used for stimulation were chosen on the basis of both their immunogenicity to T cells and their sequence conservation.19-21 The expanded cells are Th1-polarized, polyfunctional and selectively able to react to and kill, viral antigen-expressing target cells with no activity against non-infected autologous or allo- geneic targets, attesting to both their selectivity for viral targets and their safety for clinical use. We anticipate such multi-respiratory virus-targeted cells (multi-R-VST) will provide broad spectrum benefit to immunocompromised individuals with uncontrolled CARV infections.
Methods
Donors and cell lines
Peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers and HSCT recipients with informed consent using Baylor College of Medicine institutional review board- approved protocols (H-7634, H-7666) and were used to generate phytohemagglutinin (PHA) blasts and multi-R-VST. PHA blasts were generated as previously reported20 and cultured in VST medi- um [45% RPMI 1640 (HyClone Laboratories, Logan, UT, USA), 45% Click’s medium (Irvine Scientific, Santa Ana, CA, USA), 2 mM GlutaMAX TM-I (Life Technologies, Grand Island, New York, NY, USA), and 10% human AB serum (Valley Biomedical, Winchester, VA, USA)] supplemented with interleukin 2 (IL2) (100 U/mL; NIH, Bethesda, MD, USA), which was replenished every two days.
Multi-respiratory virus-targeted cell generation
Pepmixes
Peripheral blood mononuclear cells were stimulated with peptide libraries (15mers overlapping by 11aa) spanning Influenza A (NP1, MP1), RSV (N, F), hMPV (F, N, M2-1, M) (JPT Peptide Technologies, Berlin, Germany) and PIV-3 antigens (M, HN, N, F) (Genemed Synthesis, San Antonio, TX, USA). Lyophilized pep- mixes were reconstituted in dimethyl sulfoxide (DMSO) (Sigma- Aldrich) and stored at -80°C.
Generation of virus-specific T cells
To generate multi-R-VST, PBMC (2.5x107) were transferred to a G- Rex10 (Wilson Wolf Manufacturing Corporation, St. Paul, MN, USA) with 100 mL of VST medium supplemented with IL7 (20 ng/mL), IL4 (800 U/mL) (R&D Systems, Minneapolis, MN, USA) and pepmixes (2 ng/peptide/mL) and cultured for 10-13 days at 37°C, 5% CO2.
Flow cytometry
Immunophenotyping
Multi-R-VST were surface-stained with monoclonal antibodies to: CD3, CD25, CD28, CD45RO, CD279 (PD-1) [Becton Dickinson (BD), Franklin Lakes, NJ, USA], CD4, CD8, CD16, CD62L, CD69 (Beckman Coulter, Brea, CA, USA) and CD366 (TIM-3) (BioLegend, San Diego, CA, USA). Cells were acquired on a GalliosTM Flow Cytometer and analyzed with Kaluza® Flow Analysis Software (Beckman Coulter). See Online Supplementary Appendix for details.
Intracellular cytokine staining
Multi-R-VST were harvested, resuspended in VST medium (2x106/mL) and 200 mL added per well of a 96-well plate. Cells were incubated overnight with 200 ng of individual test or control (irrelevant non-viral, e.g. SURVIVIN, WT1) pepmixes, along with Brefeldin A (1 mg/mL), monensin (1 mg/mL), CD28 and CD49d (1 mg/mL) (BD). Intracellular cytokine staining (ICS) for IFNγ and TNFα was performed as described in the Online Supplementary Appendix.
FoxP3 staining
FoxP3 staining was performed using the eBioscience FoxP3 kit (Thermo Fisher Scientific, Waltham, MA, USA), per manufactur- ers’ instructions and as detailed in the Online Supplementary Appendix.
Functional studies
Enzyme-linked immunospot
Enzyme-linked immunospot (ELIspot) analysis was used to quantitate the frequency of IFNγ and Granzyme B-secreting cells. PBMC and multi-R-VST were resuspended at 5x106 and 2x106 cells/mL, respectively, in VST medium and 100 mL of cells added to each ELIspot well. Antigen-specific activity was measured as previously described after direct stimulation (500 ng/peptide/mL) with the individual test or control pepmixes. See the Online Supplementary Appendix for more details.
Multiplex
The multi-R-VST cytokine profile was evaluated using the MIL- LIPLEX High Sensitivity Human Cytokine Panel (Millipore, Billerica, MA, USA), per manufacturer’s instructions (see the Online Supplementary Appendix).
Chromium release assay
A standard 4-hour chromium (Cr51) release assay was used to measure the specific cytolytic activity of multi-R-VST with autologous antigen-loaded PHA blasts as targets (20 ng/pep- mix/1x106 target cells). Effector:Target (E:T) ratios of 40:1, 20:1, 10:1, and 5:1 were used to analyze specific lysis. The percent- age of specific lysis was calculated [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. In order to measure the autoreactive and alloreactive potential of multi-R-VST lines, autologous and allogeneic PHA blasts alone were used as targets.
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haematologica | 2020; 105(1)