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

The therapeutic use of T cells has long been studied to boost HIV-specific T-cell immunity in HIV+ individuals. However, clinical efficacy has been relatively modest.  Infusion of single-epitope specific CD8 T cells showed safety but could not provide lasting viral control, most likely due to their restricted specificity. Recently, the development of HIV entry resistant cells by other groups (eg. CCR5 deletion) has allowed T cell therapy to be even more feasible for HIV. We hypothesized that broadly HIV-specific T cells could be expanded from patients on antiretrovirals (ARVs) as well as HIV negative individuals to effectively target HIV infection using a non-HLA restricted approach for patients receiving an autologous or allogeneic HSCT for HIV-associated hematologic diseases. PBMCs from healthy donors or HIV+ patients were stimulated with gag, pol, and nef peptide libraries (pepmixes) in the presence of co-stimulatory and growth factors. T cells expanded to clinically relevant numbers (Mean=1.62e8 cells, Range=3.72e7-2.87e8 cells, n=7) even in the presence of ARVs. 5 of 7 patient sample lines showed specific activity to all 3 HIV antigens in IFNY ELISPOT assays. The T cells were broadly specific to gag (99.33 SFC/10e5 cells), pol (131.11 SFC) and nef (337.26 SFC). HIV-specific T cells were also expanded from 9 healthy (HIV negative) donors. Expanded T cells released IFNγ in response to gag (163.79 SFC, n=9) and nef (291.25 SFC, n=6) but not an irrelevant antigen (7.0 SFC). Importantly, T cells expanded from both HIV+ and HIVneg were cytotoxic, as expanded T cells lysed HIV antigen loaded autologous PHA blasts (mean=67.55% specific lysis at 10:1 effector:target ratio) but not PHA blasts alone (0.46% specific lysis at 10:1 effector target ratio). Expanded T cells from HIV+ patients also showed a greater ability to suppress HIV outgrowth in vitro compared to unexpanded CD8+ T cells when co-cultured with autologous, reactivated resting CD4+ T cells, the authentic latent reservoirs. In 5 patients, a 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 non-specific CD8+ T-cells showed only a modest trend towards decreased recovery (p>0.9). Similarly, HIV-specific T cells derived from HIVneg donors were able to suppress HIV replication more than non-specific CD8+ T-cells when co-cultured with autologous CD4+ T cells infected with HIV_SF162 (HIV only p24=681.95 pg/mL, nonspecific CD8+ T-cells=448.80 pg/mL, expanded CTL=145.82 pg/mL). In summary, we have developed robust GMP-compliant methodology for expanding functional HIV-specific T cells from both HIV+ and HIVneg donors for use after autologous and allogeneic HSCT, 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 after HSCT.