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 (2013)

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.