Engineering Human Peripheral Blood Stem Cell Grafts that Are Depleted of Naïve T Cells and Retain Functional Pathogen-Specific Memory T Cells Marie Bleakley,

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Engineering Human Peripheral Blood Stem Cell Grafts that Are Depleted of Naïve T Cells and Retain Functional Pathogen-Specific Memory T Cells Marie Bleakley, Shelly Heimfeld, Lori A. Jones, Cameron Turtle, Diane Krause, Stanley R. Riddell, Warren Shlomchik Biology of Blood and Marrow Transplantation Volume 20, Issue 5, Pages 705-716 (May 2014) DOI: 10.1016/j.bbmt.2014.01.032 Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Flow cytometric analysis of normal donor blood cells. Blood cells obtained from normal donors were analyzed by flow cytometry to assist in designing of a cell selection strategy to deplete TN from G-PBSC. (A) Stability of CD62L and CD45RA expression on CD4+ or CD8+ T cells in G-PBSC at 2 and 24 hours after apheresis collection (live CD3+ lymphocyte gate). (B) Expression of CD45RA and CD45RO on CD3+, CD4+, and CD8+ T cells in normal donors with the typical (panels 1 and 2) and variant patterns of CD45 expression (panels 3 and 4). Data is shown after gating on live lymphocytes. (C) CD45RA is expressed on a minor subset of CD34+ cells in samples of normal donor PBMC, or CD34+ cells isolated by positive immunomagnetic selection from G-PBSC. Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Cell selection procedure. The flow diagram depicts the cell processing procedure to deplete TN from G-PBSC, preserve CD34+ stem cells and deliver a fixed dose of TM cells. Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Results of cell selection procedures. (A) TN (CD45RA+CD45RO−CD3+) content of (CD34-depleted) G-PBSC product, CD45RA-depleted fraction, and CD45RA+ fraction from cell selection on G-PBSC from a representative donor (gated on live CD3+ T cells). (B) CD3+ TN content in the initial G-PBSC and in an aliquot prepared for infusion after depletion of CD45RA+ cells on 15 representative donors after adjusting the CD3+ TM content in the CD45RA− fraction to 107 cells/kg. (C) Total CD34+ cell content and (D) CD3+ and TN CD3+CD45RA+ content of the infused cell products. (E) TN (CD3+CD45RA+) content in the CD34+ fraction and in the CD34- and CD45RA-depleted cell fraction. Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Cell composition of G-PBSC before and after CD45RA depletion. Enumeration of cell content of G-PBSC before (circles) and after (squares) CD45RA depletion. (A) Total nucleated cells. (B) CD14+ monocytes. (C) CD3+ CD4+ T cells. (D) CD3+ CD8+ T cells. (E) CD4+ TCM (CD3+CD4+CD45RO+CD45RA−CCR7+ CD27+ CD28+). (F) CD8+ TCM (CD3+CD8+CD45RO+ CD45RA−CCR7+ CD27+CD28+). (G) T regulatory cells (CD3+ CD4+ CD25+ FOXP3+CD45RA+/−). (H) CD45RA+ T regulatory cells (CD3+CD4+CD25+FOXP3+CD45RA+). (I) CD56+CD3− NK cells. (J) CD19+ B cells. *P < .05 (Student paired t test) Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Virus-specific T cells in G-PBSC before and after CD45RA depletion. (A) Flow cytometry plots showing MHC tetramer staining for viral epitopes (CMV pp65 NLVPMVATV and EBV BMLF1 GLCTLVAML) of CD8+ T cells enriched from G-PBSC and CD45RA-depleted PBSC from a representative HLA-A2+ CMV+ EBV+ donor. (B) Tetramer evaluation of G-PBSC and CD45RA-depleted PBSC from 7 HLA-A2+ CMV+ EBV+ donors. (i) CMV NLV–specific T cells and (ii) EBV GLC–specific T cells as a proportion of CD8+ T cells; CD28 expression on (iii) CMV NLV CD8+ and (iv) EBV GLC CD8+ T cells; CCR7 expression on (v) CMV NLV CD8+ and (vi) EBV GLC CD8+ T cells. Analyzed by Student paired t-test. Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 ELISpot assays evaluating pathogen-specific IFNγ secretion by T cells after peptide stimulation of G-PBSC and CD45RA+-depleted G-PBSC. (A) Representative IFN-γ ELISpot showing response of T cells from G-PBSC and CD45RA-depleted G-PBSC to pp65 and control (NYBR1) peptides. The figure shows 1 of 3 replicate wells for each cell concentration for each condition. (B) IFN-γ ELISpot assay of T cells derived from G-PBSC and CD45RA+-depleted G-PBSC from a CMV+ donor (i) and a CMV− donor (ii) to CMV pp65, CMV IE1, EBV BZLF, EBV EBNA1, EBV LMP2A, or adenovirus 15 mer peptide pools. Shown are the mean and standard deviation of the spot frequency in response to the viral or control peptide stimulation (PBSC + viral peptide = black; PBSC + control = dark grey; CD45RA-depleted PBSC + viral peptide = white; CD45RA-depleted + control = light grey). (C) IFN-γ response in donor PBSC (circles) and corresponding CD45RA-depleted PBSC (triangles): responses to CMV pp65 (i) and IE-1 (ii) peptides in CMV-seropositive donors (upper 5) and CMV-seronegative donors (lower 2), adenovirus (iii), EBV BZLF (iv), EBNA-1 (v), and LMP2A (vi). Responses to control peptide are subtracted from the virus-specific response. Spot frequencies of < 100/100,000 (<.1%) are considered negative. Analyzed by Student paired t-test. Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 7 Intracellular cytokine flow cytometry to evaluate virus-specific T cells derived from PBSC and CD45RA-depleted PBSC. (A) IFNγ and IL2 secretion by CD4+ or CD8+ T cells expanded from G-PBSC or CD45RA+-depleted G-PBSC after restimulation with EBNA1 peptide or moDC alone. (B&C) Frequency of CD4+ and CD8+ T cells from G-PBSC or CD45RA-depleted G-PBSC from representative donors that produce IFNg or both IFNg and IL-2 after restimulation with (B) EBNA-1 and (C) pp65 peptides. The frequency of T cells responding to the negative control is subtracted from the data shown in (B) and (C). Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 8 Lymphoproliferation assays to evaluate CD4+ T cell responses to opportunistic pathogens. (A) Proliferation of CD4+ T cells enriched from G-PBSC (striped) and CD45RA+-depleted G-PBSC (white) from representative CMV+ (left) and CMV− (right) donors after stimulation with protein antigen preparations from dengue (negative control), CMV, adenovirus, HSV, VZV, and influenza A viruses. Shown are the mean and standard deviation. (B) Proliferation of CD4-enriched PBSC (circles) and CD45RA-depleted CD4-enriched PBSC (squares) in response to antigen stimulation with (i) CMV, (ii) ADV, (iii) HSV, (iv) VZV, and (v) influenza. The stimulation index shows proliferation relative to negative control wells. A simulation index < 3 is considered negative. Analyzed by Student paired t-test. Biology of Blood and Marrow Transplantation 2014 20, 705-716DOI: (10.1016/j.bbmt.2014.01.032) Copyright © 2014 American Society for Blood and Marrow Transplantation Terms and Conditions