Ex Vivo Expanded Multi-Specific Cytotoxic T Lymphocytes Derived From HIV+ Patients and HIV Negative Donors Using GMP Compliant Methodologies Recognize Multiple HIV Antigens and Suppress HIV Replication
Authors: Lam Sharon , Sung Julia Marsh , Cruz Conrad RY , Castillo-Caro Paul , Ngo Minh T. , Garrido Carolina , Kuruc Joann , Rooney Cliona M. , Margolis David , Bollard Catherine M. 
Published: October 2019

Abstract
Antiretroviral therapy (ART) does not eliminate HIV from latently infected reservoirs, has long-term toxicities and fails to fully prevent immune attenuation. Therefore there is a need for alternative therapies that will decrease dependency on ART. Previous studies have demonstrated the safety and feasibility of infusing single-epitope specific CD8 T cells or artificial T cell receptor- transduced T cells to HIV+ patients. However, these T cells were restricted to a single HLA restricted epitope and had limited persistence in vivo. Hence, we hypothesized that broadly HIV-specific T cells could be expanded from patients on ART and HIV negative individuals to effectively target HIV infection using a non-HLA restricted, GMP-compliant approach. We developed a method by which PBMCs from patients on ART were stimulated with autologous dendritic cells (DCs) pulsed with gag, pol, and nef peptide libraries (pepmixes) in the presence of IL-7, IL-12, and IL-15, followed by a second stimulation with pepmix-pulsed PHA blasts and IL-15 and a third stimulation with pepmix-pulsed PHA blasts, co-stimulatory K562 cells, and IL-2. Starting from 60 to 100 mL of blood, T cells expanded to clinically relevant numbers (Mean=1.62e8 cells, Range (3.72e7, 2.87e8 cells), n=7) using co-stimulatory K-562 cells and gas-permeable tissue culture devices in the presence of antiretrovirals to prevent possible viral spread during expansion. The majority of the expanded T cells had an effector memory phenotype (CD3+CD45RO+CD62L-) with approximately 10% suggestive of a central memory phenotype (CD3+CD45RO+CD62L+) which is important for long-term persistence of T cells in vivo. After 3 stimulations, 5 of 7 patient sample lines showed specific activity to all 3 HIV antigens in IFNY ELISPOT assays, with the remaining 2 showing specificity to 1 of 3 antigens. The T cell lines were broadly specific to gag (mean=99.33 SFC/10e5 cells), pol (mean=131.11 SFC/10e5 cells) and nef (mean=337.26 SFC/10e5 cells), and polyclonal as shown by flow-based Vbeta usage analysis (mean usage= 14.67 of the 24 Vbetas analyzed). In addition, due to the association of gag-specific T cell responses with viral control in long-term nonprogressors we also determined whether gag-specific T cells could be derived from seronegative donors for potential third party use. Using the same methodology developed for HIV+ patients, but using only the gag pepmix, gag-specific T cells were expanded from HIV seronegative individuals. These T cells released IFNγ in response to gag pepmix (163.79 SFC/1e5 cells, n=9) but not an irrelevant antigen (mean=7.0 SFC/1e5 cells). Importantly, T cells expanded from both ART patients and HIV seronegative individuals were cytotoxic, as expanded T cells lysed antigen loaded autologous PHA blasts (mean=67.55% specific lysis at 10:1 effector:target ratio) but not PHA blasts alone (mean=0.46% specific lysis at 10:1 effector target ratio) in chromium release assays. Expanded T cells from ART patients also showed a greater ability to suppress HIV outgrowth in vitro compared to unexpanded CD8 T cells when co-cultured with reactivated resting CD4+ T cells from ART-suppressed HIV+ patients, the authentic latently infected cells that define viral reservoirs in treated patients. In 5 patients on ART a statistically lower recovery of virus from resting CD4+ cells was seen in the presence of CTLs as compared to no effectors (p<0.006 by Mann Whitney), while the unexpanded autologous CD8 cells showed only a modest trend towards decreased recovery that was not statistically significant (p>0.9). Similarly, HIV-specific T cells derived from HIV seronegative individuals were able to suppress HIV replication more than unexpanded CD8 T cells when co-cultured with autologous CD4 T cells infected with HIVSF162 (HIV only condition p24=681.95 pg/mL, nonspecific CD8 T cells=448.80 pg/mL, expanded CTL=145.82 pg/mL). We have developed robust GMP-compliant methodologies for expanding functional HIV-specific T cells from both HIV+ patients and HIV negative donors for autologous and third-party use, respectively. We now plan to translate our approach to the clinical setting where we will test HIV-polyspecific T cell products as a part of a strategy to fully eradicate HIV infection.

Disclosures:
No relevant conflicts of interest to declare.