Mesenchymal Stem Cells (MSC) Are Not Intrinsically Immune Privileged: Direct Demonstration in TCR Transgenic Mice and by Imaging of Allogeneic Luciferase+ MSC in Immune Competent Vs Immune Deficient Mice
Authors: Zangi Lior , Beilhack Andreas , Negrin Robert , Margalit Raanan , Reisner Yair 
Published: April 2020

Abstract
Mesenchymal stem cells (MSC) can induce a broad array of immunomodulating mechanisms. Furthermore, several studies have advocated that MSC can be transplanted across allogeneic barriers without eliciting an immune response. This notion was based on clinical case reports or animal studies using highly sensitive techniques such as polymerase chain reaction, fluorescent in-situ hybridization or enhanced green fluorescent protein, enabling detection of rare cells in different tissues. However, a recent study comparing syngeneic and allogeneic MSC demonstrated that while the former cells induced tolerance to allogeneic bone marrow (BM) the use of donor type allogeneic MSC was counteractive leading to enhanced rejection of the BM cells. Thus it was indicated for the first time that allogeneic MSC might induce immune memory rather than tolerance to donor type cells. In the present study we directly addressed this possibility by infusing intravenously MSC isolated from H2db (C57BLxBalb) F1 donors, into TCR transgenic mice (the 2C model, C57BL/6 background), in which CD8+ T cells express a TCR transgene against H2d. Mice in the control group were infused with phosphate buffered saline (PBS). Thirty days after the first immunization, the mice were re-challenged with MSC and 5 days later peripheral blood CD8+ T cells were examined by FACS for the acquisition of a memory phenotype (CD122+, CD44+ and CD62Llow). This assay revealed a significantly elevated level of memory CD8 T cells (6.7±0.45 %) in the re-challenged mice compared to that found in the control group of naïve mice (0.4 ± 0.5 %, P&lt;0.01). Further evidence for induction of immune memory by MSC was directly demonstrated by non-invasive imaging of bone marrow derived MSC isolated from Luciferase+ (Luc+) transgenic FVB-L2G85 mice (MSC-Luc+). Thus, while MSC (0.9–1.8 *108 MSC/Kg) infused intravenously or intraperitonealy into immune competent Balb/c mice survived longer (27% survival at 35 days) compared to adult fibroblast (Fib-Luc+ ) (9% survival at 15 days, p&lt;0.01), this prolonged survival of MSC is significantly shorter compared to that exhibited in immune deficient Balb-Nude and NOD-SCID recipients (100% survival at 120 days, p&lt;0.01), indicating that the MSC cannot evade immune rejection although capable of delaying it. The enhanced survival of MSC in Balb-Nude mice strongly indicates that rejection of these cells in normal Balb/c mice is mediated by T cells. Remarkably, rejection was found upon infusion of about 100- fold more MSC, compared to the cell number, which can currently be generated ex-vivo for transplantation in humans (around 1*106/Kg). Infusion of a lower number of MSC (4 *107 MSC/Kg) was found to be even less effective (9% survival at 15 days, p&lt;0.01). To define whether the allogeneic rejection of MSC-Luc+ or Fib-Luc+ is associated with induction of immune memory, we re-challenged mice previously rejecting 2*106 Fib-Luc+ or MSC-Luc+ cells, with Fib-Luc+ cells. Thus, 30 days after rejection of the first inoculums the recipients were implanted with a second transplant of 2*106 Fib-Luc+ cells. Our data reveals that graft rejection was significantly more rapid in re-transplanted Balb/c mice. While a significant density of Fib-Luc+ cells can be detected in all transplanted recipients at day two, survival at day 5 was reduced to 27% or 18% in mice primed with Fib or MSC, respectively, compared to 81% in naive recipients (p&lt;0.01). Survival of Fib-Luc+ cells in re-challenged mice was further reduced at day 9 (9% or 0% in mice previously receiving Fib or MSC, respectively) compared to 72% survival in naive recipients, p&lt;0.01). Collectively, these results demonstrate that MSC are not intrinsically immune privileged and under allogeneic settings these cells induce rejection, which is followed by an immune memory. Considering that the use of allogeneic or even a third party (‘off the shelf’) MSC is commonly advocated for a variety of clinical applications, our results strongly suggest that long term survival of allogeneic MSC likely represents a major challenge. Further studies attempting to overcome rejection of donor MSC in the context of hematopoietic stem cell transplantation or in conjunction with co-stimulatory blockade are warranted.