Gohl, Daryl M.

Background Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. Methods We developed transgenic mice carrying D oxycycline (Dox)- i nducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8 + T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 + T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. Results Under chronic stimulation, SB -mutagenized CD8 + T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Conclusions Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

Background. Preterm Premature Rupture of Membranes (PPROM) is a major leading cause of preterm births. While the cause for PPROM remains unidentified, it is anticipated to be due to subclinical infection, since a large proportion of PPROM patients display signs of chorioamnionitis. Since subclinical infections can be facilitated by dysbiosis, our goal was to characterize the vaginal microbiome and amniotic fluid discharge upon PPROM, through latency antibiotic treatment, and until delivery, to detect the presence of pathogens, microbiota alteration, and microbial response to treatment. Methods. Enrolled subjects (15) underwent routine institutional antenatal care for PPROM, including the administration of latency antibiotics. Serial vaginal swabs were obtained from diagnosis of PPROM through delivery and the sequencing of the V3–V5 region of the 16S rRNA gene was performed for all collected samples. Results. The results show that Lactobacilli species were markedly decreased when compared to vaginal swabs collected from uncomplicated pregnancy subjects with a matched gestational time. Prevotella and Peptoniphilus were the most prevalent taxa in PPROM subjects at presentation. The vaginal microbiome of the PPROM subjects varied substantially intra- and inter-subjects. Several taxa were found to be significantly reduced during and after the antibiotic treatment: Weeksella , Lachnospira , Achromobacter , and Pediococcus . In contrast, Peptostreptococcus and Tissierellaceae ph2 displayed a significant increase after the antibiotic treatment. However, the relative abundance of Lactobacillus , Prevotella , and Peptoniphilus was not substantially impacted during the hospitalization of the PPROM subjects. The deficiency of Lactobacillus , and constancy of known pathogenic species, such as Prevotella and Peptoniphilus during and after antibiotics, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches. Discussion. PPROM is responsible for one third of all preterm births. It is thought that subclinical infection is a crucial factor in the pathophysiology of PPROM because 25–40% of patients present signs of chorioamnionitis on amniocentesis. Here we sought to directly assess the bacterial content of the vagina and leaking amniotic fluid of subjects at presentation, throughout treatment and up until delivery, in order to search for common pathogens, microbiota changes, and microbial response to latency antibiotic treatment. We have found that the vaginal microbiome of PPROM subjects is highly variable and displays significant changes to treatment. However, the unchanging deficiency of Lactobacillus , and persistence of known pathogenic species, such as Prevotella and Peptoniphilus from presentation, through antibiotic treatment and up until delivery, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches.