Figure 5.

Resistance of B2M-null hESCs and their derivatives to alloreactive CD8⁺ T cell-mediated killing in vitro. Histogram representation of the cytotoxic effects of CD8⁺ T cells on undifferentiated and differentiated B2M^−/− hESCs (A) and on IFN-γ-treated B2M^−/− hESCs (B) before and after differentiation with hESC control. The various ratios of CD8⁺ T cell to target cell are indicated (n = 5; ∗, p < .01 vs. differentiated hESCs with or without IFN-γ treatment; ∗∗, p < .0001 vs. undifferentiated or differentiated hESCs). (C): 4 × 10⁶ hESCs or B2M^−/− hESCs were mixed with 200 ml of growth factor-reduced Matrigel and then injected subcutaneously into 10-week-old immunocompetent mice. Implants were harvested after 48 hours and then fixed in 4% formalin, sectioned, and stained with mouse anti-B2M (1:100 dilution; Santa Cruz Biotechnology) and rabbit anti-CD8 antibody (1:100 dilution; Bioss Inc., Woburn, MA, http://www.biossusa.com) or with H&E. Representative immunofluorescence detection of numerous CD8⁺ T cells (red) infiltrating into B2M-positive (green) Matrigel-hESC implants shown (second image), but rare CD8⁺ T cells were present in the B2M-negative Matrigel-B2M^−/− hESC implants (fourth image). A magnified view of representative CD8⁺ T cells indicated by red arrow was inserted in each corresponding image. Scale bars = 100 μm. Abbreviations: B2M, β₂-microglobulin; DAPI, 4′,6-diamidino-2-phenylindole; hESC, human embryonic stem cell; IFN-γ, interferon-γ.