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. Author manuscript; available in PMC: 2022 Jan 12.
Published in final edited form as: Immunity. 2020 Dec 2;54(1):116–131.e10. doi: 10.1016/j.immuni.2020.11.002

Figure 4. Specificity of MHC regulation and the role of SUGT1 in antigen presentation.

Figure 4.

(A) Opposing effects of genetic deletions on surface MHC-I of different tumor lines measured by flow cytometry. (B) Top performing sgRNAs for the loss of surface HLA-DR, quantified cumulatively across different tumor cells. GCBs orange; ABCs blue. Each gene is also classified with its MHC-I regulator status (bottom boxes). (C) Same as B, but for cumulative gains in HLA-DR. (D) Effects of RFX5 or SUGT1 loss on the surface expression of MHC-I and MHC-II in TMD8. (E) (left) Schematic of T cell co-culture assay. HLA-A2+ HBL1 cells, which natively express the cancer testis antigen NY-ESO-1, were co-cultured with primary human T-cells transduced with a TCR recognizing the NY-ESO-1 peptide 157–165 restricted by HLA-A2. (right) T-cells were monitored for activation by 4–1BB upregulation after 12–14 hours with the indicated HBL1 cells. NT, non-targeting sgRNA. Peptide, exogenously added SLLMWITQV. T cells grown without target cells were manually set to 0% (1.14% average donor 1, 5.03% average donor 2). (F) NLRC5 or SUGT1 were deleted in HBL1 or TMD8 cells, and whole cell lysates were subjected to immunoblot analysis. Arrow, NLRC5. *, undetermined band from anti-NLRC5 antibody. See also Figure S2. (G) qPCR analysis of the indicated transcripts in TMD8 cells modified with NT or SUGT1 sgRNA. (H) Same as G, but with RFX5 deletion. For entire figure, bar graphs represent mean with standard deviations, minimum n = 3.