Functionally Active HIV-Specific T Cells that Target Gag and Nef Can Be Expanded from Virus-Naïve Donors and Target a Range of Viral Epitopes: Implications for a Cure Strategy after Allogeneic Hematopoietic Stem Cell Transplantation  Shabnum Patel, Sharon Lam, Conrad Russell Cruz, Kaylor Wright, Christina Cochran, Richard F. Ambinder, Catherine M. Bollard  Biology of Blood and Marrow Transplantation  Volume 22, Issue 3, Pages 536-541 (March 2016) DOI: 10.1016/j.bbmt.2015.12.007 Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Biology of Blood and Marrow Transplantation 2016 22, 536-541DOI: (10 Biology of Blood and Marrow Transplantation 2016 22, 536-541DOI: (10.1016/j.bbmt.2015.12.007) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Priming and expansion of HIV-specific T cells from HIV-naïve donors (dHXTCs), characterization, and viral inhibition studies. (A) Nonadherent PBMCs underwent 3 rounds of stimulation with gag and nef peptide libraries. On day 7 after the third stimulation, the expanded cultures were counted (n = 8). Each seronegative (SN) donor is labeled from SN1 to SN8. (B) Phenotype of expanded T cells. (C) CTLs were tested for specificity using IFNγ ELISPOT assay 7 days after the third stimulation. No antigen (media) and actin (irrelevant) were used as negative controls and staphylococcus enterotoxin B superantigen or PHA (phytohemagglutinin) was used as a positive control. 62LnegCD45RAneg cells were considered effector memory cells, and 62L+CD45RA− were considered central memory cells. (D, E) CD8-depleted cells from the donor were infected with a laboratory R5 strain of HIV, SF162. Statistical significance was determined using 2-way ANOVA with multiple comparisons correction (Dunnet's). In (D), unexpanded CD8 T cells were cultured overnight in IL-2 or HIV-specific CTLs were cocultured at a 1:2 effector to target ratio (n = 4). In (E), infected CD8-depleted PBMCs were cocultured under multiple conditions at a 20:1 effector to target ratio (n = 3). P24 was measured using ELISA on day 5. Biology of Blood and Marrow Transplantation 2016 22, 536-541DOI: (10.1016/j.bbmt.2015.12.007) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Polyfunctional characterization and epitope mapping of expanded HIV-specific T cells (dHXTCs). (A) dHXTCs were stimulated with irrelevant peptide or gag/nef and cytokine responses were measured in cell supernatant after 24 hours by multiplex assay (P < .05, n = 3). (B) Perforin ELISA performed with cell supernatant from 2A (P < .05, n = 3). (C) Peptides spanning the consensus regions for gag (A) and nef (B) (provided by the NIH AIDS Reagent Program) were pooled according to matrices (Supplemental Figure 1 A,B). Peptide pools are shown as “p1, p2 … pn” in the gray and comprised of all the peptides listed in the same column or row. Expanded T cells were then tested for reactivity against these peptide pools using ELISPOT. Epitope specificity was the peptides shared by both vertical and horizontal peptide pools. The dotted line represents the level of background in each assay. (D) Identified peptides from 2C or entire antigens gag/nef were tested for HLA specificity in a blocking assay performed with ELISPOT (n = 3). Biology of Blood and Marrow Transplantation 2016 22, 536-541DOI: (10.1016/j.bbmt.2015.12.007) Copyright © 2016 American Society for Blood and Marrow Transplantation Terms and Conditions