Immunogenicity of autologous and allogeneic human primary cholangiocyte organoids

Primary human cells cultured in 3D organoid format have great promise as potential regenerative cellular therapies, but their immunogenicity has not yet been fully characterized. In this study, we use in vitro co-cultures and in vivo humanized mouse experimental models to examine the human immune response to autologous and allogeneic primary cholangiocyte organoids (PCOs). Our data demonstrate that PCOs upregulate the expression of HLA-I and HLA-II in inflammatory conditions. The immune response to allogeneic PCOs is driven by both HLA-I and HLA-II and is substantially ameliorated by donor-recipient HLA matching. Autologous PCOs induce a low-level immune infiltration into the graft site, while allogeneic cells display evolving stages of immune rejection in vivo. Our findings have important implications for the design and clinical translation of autologous and allogeneic organoid cellular therapies., One representative mouse per group was selected, PCO and matrigel-only kidneys were sectioned (5mm) and slides sent to Nanostring for analysis. The slide preparation for WTA followed standard procedure. Briefly, for WTA assay, after baking for 3 hours in 65°C, slides were processed on Leica automation platform with a protocol includes three major steps: 1) slide baking and dewax, 2) Antigen Retrieval for 20 min at 100°C, 3) 1µg/ml Proteinase K treatment for 20 min. Slides were then incubated with GeoMx WTA assay probe cocktail overnight. The following day, slides were washed and incubated with morphology markers before loading onto the GeoMx machine. The conjugated antibodies used were: KRT7 (1:200, abcam, ab215855, clone [KRT7/760]), CD3 (1:100, Origene, UM000048BF, clone [UMAB54]), CD45 (1:200, CST, 13917BF, clone [D9M8I]), in addition to nuclear staining (Syto 83, ThermoFisher Scientific, S11364). 
On the GeoMx machine, slides were scanned for fluorescence and Region of Interest (ROI..., , # Immunogenicity of autologous and allogeneic human primary cholangiocyte organoids

#### ABSTRACT

Primary human cells cultured in 3D organoid format have great promise as potential regenerative cellular therapies, but their immunogenicity has not yet been fully characterized. In this study, we use *in vitro* co-cultures and *in vivo* humanized mouse experimental models to examine the human immune response to autologous and allogeneic primary cholangiocyte organoids (PCOs). Our data demonstrate that PCOs upregulate the expression of HLA-I and HLA-II in inflammatory conditions. The immune response to allogeneic PCOs is driven by both HLA-I and HLA-II and is substantially ameliorated by donor-recipient HLA matching. Autologous PCOs induce a low-level immune infiltration into the graft site, while allogeneic cells display evolving stages of immune rejection *in vivo*. Our findings have important implications for the design and clinical translation of autologous and allogeneic organoid cel...