Comparison of the Division Rate and Proliferative Capacity of Naive and Ex Vivo Activated T Cells after Allogeneic Bone Marrow Transplantation.
Authors: Rettig Michael P. , Ritchey Julie K. , Nervi Bruno , Bonyhadi Mark L. , DiPersio John F. 
Published: October 2019

Abstract
Maintaining T cell function after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously showed that murine T cells activated ex vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) retain greater GVHD-inducing potential than cells activated with either soluble anti-CD3 antibody alone or plate bound anti-CD3 and anti-CD28 antibodies after allogeneic BMT. However, CD3/CD28 bead activated T cells still exhibit reduced GVHD-inducing potential compared to naïve T cells. In this study, we used CFSE and congenic mice to monitor the proliferative kinetics of naïve and CD3/CD28 bead activated, transduced, and selected T cells in the same mouse after allogeneic and syngeneic BMT. High efficiency (>50%) gene transfer of our chimeric suicide gene, CD34/TK, into C57BL/6 (B6) murine T cells was achieved 24 h after CD3/CD28 bead activation and gene-modified cells were purified to >98% by CD34 immunomagnetic selection 48 h later. CD34/TK+ T cells were then rested for 3 days in medium containing 10 U/mL IL-2. Purified CD34/TK+ (CD45.1+) and naïve (CD45.2+) T cells from B6 mice were then labeled with CFSE, mixed 1/1 (3e6 total T cells), and injected, along with T cell depleted B6 (CD45.1+) BM, into lethally irradiated allogeneic (BALB/c, CD45.2+) or syngeneic (B6, CD45.2+) recipients. Mice were sacrificed daily up to 6 days after BMT to assess donor T cell engraftment, division, and phenotype by five color flow cytometry. The CD34/TK+ donor T cells (both CD4+ and CD8+ T cells) underwent 1–2 rounds of division within 24 h after infusion. In contrast, <5% of the naïve T cells divided during the first 24 h after infusion. Thereafter, the CD34/TK+ and naïve CD4+ and CD8+ T cells exhibited similar division kinetics between days 1 and 4 after BMT. At 3 days after BMT, both CD34/TK+ and naïve CD4+ and CD8+ T cells were detected in the 8 cell divisions discernible by CFSE, with approximately equal percentages (5%–15%) of cells in each division cycle. However, virtually all of the CD34/TK+ and naïve CD4+ and CD8+ T cells had divided more than 7 times by day 4 after allogeneic BMT. In contrast, <10% of the CD34/TK+ or naïve CD4+ and CD8+ T cells had undergone more than 7 cell divisions in the syngeneic recipients. Interestingly, the CD34/TK+ T cells exhibited a dramatic decrease in expansion compared with the naïve T cells between days 4 and 6 after allogeneic BMT. Although ~ equal percentages of CD34/TK+ and naïve T cells were observed 4 days after infusion, >6-fold and 10-fold more naïve CD4+ and CD8+ T cells, respectively, were detected in the allogeneic recipients at day 6 after BMT. This effect was specific to the allogeneic response, because we observed no difference in expansion between the CD34/TK+ and naïve CD4+ and CD8+ T cells in the syngeneic recipients. Phenotypically, both the CD34/TK+ and naïve CD4+ and CD8+ cells upregulated CD25 expression after 4 divisions, upregulated CD69 and CD44 expression after 1 to 2 divisions, and downregulated CD62L expression. In summary, CD3/CD28 bead activated, transduced, and selected T cells exhibit decreased expansion compared to naïve T cells after injection into allogeneic recipients. Ongoing studies are evaluating whether this decrease in expansion is caused by activation induced cell death, altered trafficking, or a decrease in the proliferative capacity of the ex vivo manipulated cells.