Engineering human peripheral blood stem cell grafts that are depleted of naïve T cells and retain functional pathogen-specific memory T cells

Abstract

Graft-versus-host disease (GVHD) is a frequent major complication of allogeneic hematopoietic cell transplantation (HCT). Approaches that selectively deplete T cells that cause GVHD from allogeneic stem cell grafts and preserve T cells specific for pathogens may improve HCT outcomes. It has been hypothesized that the majority of T cells that can cause GVHD reside within the naïve T cell (TN) subset, and previous studies performed in mouse models and with human cells in vitro support this hypothesis. As a prelude to translating these findings to the clinic, we developed and evaluated a novel 2-step clinically compliant procedure for manipulating peripheral blood stem cells (PBSC) to remove TN, preserve CD34(+) hematopoietic stem cells, and provide for a fixed dose of memory T cells (TM) that includes T cells with specificity for common opportunistic pathogens encountered after HCT. Our studies demonstrate effective and reproducible performance of the immunomagnetic cell selection procedure for depleting TN. Moreover, after cell processing, the CD45RA-depleted PBSC products are enriched for CD4(+) and CD8(+) TM with a central memory phenotype and contain TM cells that are capable of proliferating and producing effector cytokines in response to opportunistic pathogens.

Keywords: Graft engineering; Graft-versus-host disease; Immune reconstitution; Memory T cells; Naïve T cells; Selective T cell depletion.

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 T_N 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.

Figure 2. Cell selection procedure

The flow diagram depicts the cell processing procedure to deplete T_N from G-PBSC, preserve CD34⁺ stem cells and deliver a fixed dose of T_M cells.

Figure 3. Results of cell selection procedures

A) T_N (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⁺ T_N 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⁺ T_M content in the CD45RA⁻ fraction to 10⁷ cells/kg. C) Total CD34⁺ cell content and D) CD3⁺ and T_N CD3⁺CD45RA⁺ content of the infused cell products. E) T_N (CD3⁺CD45RA⁺) content in the CD34⁺ fraction and in the CD34⁻, CD45RA-depleted cell fraction.

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⁺ T_CM (CD3⁺CD4⁺CD45RO⁺ CD45RA⁻CCR7⁺ CD27⁺ CD28⁺) F) CD8⁺ T_CM (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. * indicates P<0.05 (Student’s paired t test)

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’s paired t test.

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 one of three 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 is 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 (<0.1%) are considered negative. Analyzed by Student’s paired t test.

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 following re-stimulation 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).

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 is 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 CMV (i), ADV (ii), HSV (iii), VZV (iv), and influenza. The stimulation index (SI) shows proliferation relative to negative control wells. A SI <3 is considered negative. Analyzed by Student’s paired t test.