Allogeneic Recipients of Ex-Vivo Manipulated Donor T Cells Have Altered Plasma Analyte Profiles Compared to Recipients of Unmanipulated T Cells.
Authors: Rettig Michael P. , Ritchey Julie K. , DiPersio John F. 
Published: October 2019

Abstract
Background: Maintaining T cell function and survival after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously demonstrated that murine T cells activated and expanded ex-vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) exhibit reduced GVHD-inducing potential compared to naïve (unmanipulated) T cells in a murine allogeneic BMT model. Blood diagnostics are commonly used to help identify patterns of biomarkers in multiple disease states.
Objective: Determine if plasma profiles of 59 different analytes are altered in murine allogeneic BMT recipients who receive ex-vivo activated, suicide gene transduced and selected (Td) T cells compared with animals that received naïve suicide gene expressing T cells.
Methods: Murine T cells were Td with a chimeric CD34-thymidine kinase (CD34-TK) fusion suicide gene. High efficiency (70%) gene transfer of CD34-TK to C57BL/6 (B6) murine T cells was accomplished 24 h after CD3/CD28 bead activation and gene-modified cells were purified to > 96% by CD34 immunomagnetic selection 2 days post-infection. Naïve B6 CD34-TK-expressing T cells were purified from the spleens of CD34-TK transgenic mice the day of BMT. To induce GVHD, lethally irradiated BALB/c allogeneic recipients were given T cell depleted (TCD) B6 BM supplemented with or without B6 CD34-TK purified naïve or Td T cells. Animals were bled 1 week after BMT and EDTA plasma samples were prepared and frozen. Quantitative measurements of 59 analytes were obtained using a rodent multi-analyte profile test (MAP test; Charles River Lab) on the plasma of 2 mice that received naïve T cells and died from GVHD on days 16 and 21 after BMT, 2 mice that received Td T cells and died from GVHD on day 34 after BMT, and 2 mice that received TCD BM only and survived > 100 days.
Results: As before, we found that ex vivo activation of the donor T cells before BMT significantly prolonged the survival of mice transplanted with the Td T cells compared with mice receiving naïve T cells (p = 0.0028). Eighteen analytes, including IFN-γ, IL-2, IL-3, IL-4, IL-7, IL-11, and TNF-α were below the detection limits of the rodent MAP test for all samples analyzed. Twenty-six analytes, including VEGF, SCF, MIP-2, MIP-1α, MIP-3β, MCP-5, IL-1β, IL-18 and GM-CSF were detectable but not significantly different than the BM only controls for either the naïve or Td T cell groups. Nine analytes, including eotaxin, MIP-1γ, MCP-3, MCP-1, and IL-10 were similarly increased 2- to 3-fold in both the Td and naïve T cell groups compared to the BM only control. Interestingly, recipients of naive T cells exhibited increased plasma levels of growth hormone (≥14-fold), MIP-1β (4.6-fold), IP-10 (2.3-fold), and lymphotactin (2-fold), as well as decreased levels of leptin (≥14-fold), compared to both the BM only and Td T cell groups. This extreme modulation of growth hormone and leptin levels is particularly interesting given the fact that leptin influences local growth hormone secretion from lymphocytes and that both of these analytes have multiple biologic effects on T cells. Finally, only a single analyte, monocyte derived chemokine (MDC), was increased (4-fold) in mice that received Td T cells compared to the naïve T cell recipients.
Conclusion: These results indicate that several plasma analytes with important immunological functions are altered when donor T cells are manipulated ex-vivo. These alterations may account for the decreased GVHD-inducing potential of ex vivo manipulated T cells after allogeneic BMT.