Rabbit Anti-B2M monoclonal antibody, clone TV46-16

PURPOSE. Antitumor T cells need expression of HLA class I molecules but can be inhibited by ligands such as programmed death ligand 1 (PD-L1). We determined expression and regulation of these molecules in human conjunctival melanoma (CM) samples, cell lines, and murine xenografts. METHODS. Immunofluorescence staining was performed to examine the expression of HLA-A, HLA-B/C, and beta-2-microglobulin (B2M) in 23 primary CM samples. HLA class I expression was compared with clinicopathologic characteristics, the presence of tumor-infiltrating leukocytes, and PD-L1/PD-1 status. The effect of interferon gamma (IFN-gamma) on HLA class I expression was tested on three CM cell lines using quantitative PCR and flow cytometry. Furthermore, HLA class I expression was determined in CM cell line-derived murine xenografts. RESULTS. One third of tumors had positive HLA-A, HLA-B/C, and B2M expression. A positive expression was especially seen in thin and epibulbar tumors but was not associated with recurrences. HLA class I expression was correlated with M2 macrophage density and tended to associate with CD8+ T-cell density but was independent of PD-L1 or PD-1 expression. IFN-gamma upregulated HLA class I expression and genes involved in HLA transcription and transportation on CM cell lines. Murine xenografts showed a comparable HLA class I expression as their respective cell lines. CONCLUSIONS. Our data indicate that subsets of CM have positive HLA class I expression, and HLA class I and PD-L1/ PD-1 are expressed independently. When one considers immunotherapy, one should also analyze HLA class I expression, whose downregulation can limit the efficacy of T cell-mediated therapies.

Background Despite major improvements in pig-to-primate xenotransplantation, long-term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. Methods In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta-2-microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M-KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. Results Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. Conclusion Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta-2-microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.