Elz, A. | bioRxiv Science

bioRxiv2025

Genital herpes shedding episodes associate with alterations in the spatial organization and activation of mucosal immune cells

Herpes Simplex Virus 2 (HSV-2) infection results in variable rates of local viral shedding in anogenital skin. The impact of episodic viral exposures on immune cells in adjacent mucosal tissues, including the genital tract, is unknown. However, any immune responses at this site could impact protective mucosal immunity, tissue homeostasis, and adverse health outcomes. To investigate the impact of HSV-2 on cervicovaginal tract immunity, we applied flow cytometry, immunofluorescent imaging, analysis of soluble immune factors, and spatial transcriptomics to cervicovaginal tissue and blood samples provided by a total of 232 HSV-2 seropositive and seronegative participants, with genital HSV-2 shedding evaluated at the time of biopsy. This unique dataset was used to define and spatially map immune cell subsets and localized gene expression via spatial transcriptomics. HSV-2 seropositivity alone was associated with minimal differences in cervicovaginal and circulating T cell phenotypes. However, the vaginal mucosa during active HSV-2 shedding was associated with alterations in T cell, macrophage, and dendritic cell localization and gene expression consistent with increased immune surveillance, with immune activating and suppressing signals potentially reinforcing mucosal tissue homeostasis. SummaryIn context of episodic HSV-2 shedding, immune cells mobilize and co-localize in the vaginal epithelium, expressing cytotoxic and inflammatory genes and immunoregulatory genes that collectively may promote tissue homeostasis in settings of episodic viral shedding to limit damage.

immunology↗

bioRxiv2025

Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1+Tcf1+ stem-like reservoir and overcomes resistance to PD-1 axis blockade

PD-1+Tcf1+ stem-like cells are critical mediators of endogenous T cell responses to PD-1/PD-L1 blockade and are maintained by MHC-dependent interactions with professional antigen-presenting cells (APCs). Unlike conventional T cells, CAR-T cells are activated by intact antigen expressed on tumors, not by peptide/MHC expressed on APCs, restricting their activation to the hostile tumor microenvironment (TME) that may impair preservation of this critical stem-like subset. Indeed, in an autochthonous model of ROR1+ lung cancer that we developed, CAR-T cells targeting the tumor-associated antigen ROR1 were uniformly Tcf1+ prior to infusion but rapidly downregulated Tcf1 in vivo and became terminally exhausted, similar to observations in patients, resulting in faster attrition and no enhancement in response to PD-L1 blockade. We hypothesized that overexpression of AP-1 family transcription factors, which can regulate T cell exhaustion, could enable CAR-Ts to maintain this critical PD-1+Tcf1+ subset within tumors independently of APCs and sensitize them to PD-1/PD-L1 blockade. Overexpression of the AP-1 TF c-Jun, but not BATF, improved preservation of PD-1+Tcf1+ CAR-T cells within tumors in a cell-intrinsic manner that correlated with increased persistence deeper within tumors. Notably, c-Jun overexpression alone was insufficient to prevent CAR-T exhaustion in the lung TME, in contrast to prior work in xenograft models, with progressive CAR-T dysfunction correlated with PD-1-dependent downregulation of c-Jun. However, c-Jun overexpression dramatically sensitized CAR-Ts to PD-L1 blockade, which restored c-Jun levels in CAR-Ts, drove log-fold expansion of CAR-Ts within tumors, and induced nearly complete eradication of ROR1+ tumor in highly aggressive models of lung cancer. Altogether, our data show that combination with PD-L1 blockade is necessary to unleash the full potential of c-Jun-overexpressing CAR-T cells in aggressive solid tumors like lung cancer and suggest that strategies to enhance formation of intratumoral PD-1+Tcf1+ reservoirs can overcome CAR-T resistance to PD-1 blockade.

immunology↗

bioRxiv2023

snPATHO-seq: unlocking the pathology archives

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low DNA and RNA quality. In this study, leveraging recent single-cell genomic technology advances, we introduce a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples.

molecular biology↗