Immune escape induced by stem cells is partly due to reduced expression of several cell surface molecules of immune activation on stem cell surfaces. For example, reduced alloreactivity caused by lack of MHC II and reduced MHC I expression on ESCs has been reported . Others have reported that stem cells induced T-cell apoptosis by Fas–FasL interaction, which had been observed to be one of the immune escape mechanisms on T cells [5, 30].
CD4+ T-cell apoptosis seemed to continuously occur even 24 hours after the co-culture, because the number of propidium iodide-positive cells continuously increased even after the number of Annexin V-positive cells reached its peak at 24 hours. However, it has not been clearly demonstrated whether this reaction was caused by direct interaction between the FasL expressed on the surface of NSCs and Fas on CD4+ T cells. Similar to other researchers' observations of human ESCs in various stages of differentiation , we could not detect either FasL mRNA or surface protein by FACS in NSCs. FasL expression on HB1.F3 was examined using FACS analysis and immunocytochemistry. Both results showed an absence of FasL on HB1.F3, which was similar to the previous results of a study performed with ESCs . Direct Fas–FasL interactions between NSC and CD4+ T cells are therefore less likely to occur (see Additional file 3). NSCs must therefore have provoked Fas–FasL-mediated CD4+ T-cell apoptosis through an indirect pathway.
In addition, transplant rejection occurs through recognition of foreign antigens presented by MHC molecules expressed on antigen-presenting cells, a process that can be mimicked by triggering T cells with CD3-specific mAb . We used anti-CD3 (OKT3) and anti-CD28 (CD28.2) mAbs, and recombinant mouse CD27L (Minneapolis, MN, USA) to stimulate T cells in a manner that partially mimics stimulation by antigen-presenting cells. We then checked FasL expression and apoptosis of CD4+ T cells. Our previous data showed an increase of FasL-positive cells and apoptotic CD4+ T cells by antigen presentation by T-cell receptor and co-stimulatory signals. However, the median fluorescence intensity of FasL and the apoptosis level varied by blood donor, incubation time, and the combinations of antibodies and recombinant protein (see Additional file 4).
In this paper, we demonstrated that CD70–CD27 interaction was involved in NSCs-induced CD4+ T cell apoptosis. Originally, CD27 is a cell surface glycoprotein belonging to the TNF receptor superfamily, which can provide stimulatory signals for both cell growth and apoptosis. CD70–CD27 interactions between most immune cells produce T cell expansion and the development of effector cytotoxic or memory T cells . On the other hand, it has been reported that CD27 can bind apoptosis-inducing factor (Siva 1), an intracellular mediator of apoptosis [33, 34], but the role of this interaction for the function of CD27 is yet to be resolved, because ligation of CD27 generally does not limit but rather contributes to the expansion of activated lymphocytes .
CD70 expressed on malignant cells showed novel function in immune escape . Human brain tumor cells, such as malignant glioma like or glioblastoma multiforme, express CD70 , and this CD70 – but not TNFα or FasL – initiated T-cell death through the receptor-dependent pathway . However, the exact mechanism of this process is not yet fully understood.
Levels of inhibition by both antibodies were otherwise incomplete, implicating the existence of other causes for apoptosis in addition to CD70–CD27 interaction. In CD70-mediated T-cell apoptosis, the role of Siva, a pro-apoptotic molecule, was identified, as well as soluble mediators such as transforming growth factor beta [32, 39]. We evaluated the expression of Siva protein using western blotting. Siva was increased at 48 hours in CD4+ T cells and the NSC co-culture system. We therefore think that, at least in our system, apoptosis was mainly induced by the FasL upregulation by CD27–CD70 ligation between NSCs and CD4+ T cells in the early phase (see Additional file 5).
We then examined cytokine profiles from the supernatant at various co-culture times (1, 3, 6, 12, 18, and 24 hours). Several cytokines (such as IL-1β, TNFα, IL-4, IL-10, IFNγ, IL-5, and IL-13) were shown to be negative in the co-culture periods. Moreover, IL-6 increased in a time-dependent manner. However, the IL-6 level is similar in the NSC-only culture group. In addition, the supernatant of the CD4+ T-cell-only culture group was shown to be negative for IL-6. Therefore, IL-6 might require NSC self-renewal and progenitor cell division and differentiation [40–42]. As in ESCs, lack of co-stimulation [9, 43] or participation of soluble mediators could play a role in T-cell immune escape. However, activation-induced cell death was less likely to play a role  since CD4+ T-cell apoptosis in our experiments reached its peak in 24 hours.
Finally, we have demonstrated that FasL expression on CD4+ T cells was significantly increased as a consequence of CD70–CD27 ligation. Antibody blocking experiments also confirmed that FasL expression on CD4+ T cells was CD70–CD27 dependent. NSCs therefore probably induce CD4+ T-cell apoptosis in two stages: CD70–CD27 ligation between NSCs and CD4+ T cells, which induces FasL expression on some CD4+ T cells, followed by Fas–FasL-mediated CD4+ T-cell apoptosis.