Sydney Gang

Enriched gene sets in HupT3 cells following TFAP4 knockout and overexpression, generated by the Enrichr search engine

Genome-scale CRISPR-Cas9 screens

Expression of Hallmark IFNγ/IFNα gene sets in HupT3 cells following TFAP4 knockout and overexpression

Catalog of qRT-PCR primers, CRISPR guide sequences, and antibodies used in this study

Differential gene expression in HupT3 cells following TFAP4 knockout and overexpression

Chimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non–B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy.Significance:The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.

H5N1 highly pathogenic avian influenza viruses have spread globally and pose a pandemic risk. Prior studies suggest that early life exposures to group 1 influenza viruses (H1N1 and H2N2) prime antibodies that cross-react to the hemagglutinin of H5N1, which is also a group 1 virus. However, less is known about how immune history affects antibody responses against the H5N1 neuraminidase (NA). We measured NA inhibition antibodies against multiple H5N1 viruses using sera from 155 individuals born between 1927 and 2016. Individuals likely primed in childhood with H1N1 viruses possessed higher levels of antibodies that cross-react with the NA of H5N1 viruses compared to those primed with H2N2 or H3N2 viruses. While young children rarely possessed cross-reactive N1 antibodies, childhood infections with contemporary H1N1, but not H3N2, viruses elicited them. We also measured antibodies against an H5N5 virus (A6 genotype) that recently caused a fatal infection in the United States. Consistent with the lack of circulation of N5 viruses in humans, we found low levels of antibodies against the N5 NA. Our data suggest that immune history greatly impacts the generation of cross-reactive NA antibodies, and that reassortment with other NAs may increase the risk of H5 infection of humans.

Catalog of qRT-PCR primers, CRISPR guide sequences, and antibodies used in this study

Differential gene expression in HupT3 cells following TFAP4 knockout and overexpression