Biology subjects

Connolly, K. A.

Publications and source records attributed to Connolly, K. A..

7 recordsLinked to original sources

bioRxiv2026

A standardized method for T cell receptor (TCR) replacement through CRISPR-Cas9 mediated editing and retroviral transduction of primary murine naïve CD8 T cells

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.

immunology↗

bioRxiv2026

Spatiotemporal profiling reveals the role of inflammatory niche in driving prostate cancer

Prostate cancer (PCa) is a lethal malignancy that displays profound resistance to immune checkpoint blockade (ICB), via mechanisms that are poorly understood. Here, we investigate the causes of CD8 T cell exhaustion and mechanisms of tumor progression in a PCa animal model, by single cell and spatial profiling, along a time course, following orthotopic transplantation of RB1/TP53/PTEN-deficient mouse organoids, competent to express neoantigens. The resulting tumors were castration resistant, consisting of largely basal and L2 malignant cells with upregulated inflammatory gene programs, and a specific spatial distribution of macrophages, cancer associated fibroblast (CAF) subtypes, and CD8 T-cells that was not previously reported. Using Zman-seq, we demonstrate that the effector function of tumor-infiltrating CD8 T cells was rapidly impaired as early as 24hrs after their infiltration, likely driven by signals from proinflammatory macrophages, Ccl2-Jak2+ inflammatory CAFs, and malignant basal cells, thus driving resistance to ICB. Interestingly, dual blockade of JAK1/2 and PD1 induced potent anti-tumor effects in tumor epithelial cells, decreased malignant epithelial cells and pro-inflammatory macrophages, and increased the proportion of normal (Pi16+) fibroblasts in the TME. Our results underscore the therapeutic potential of targeting JAK1/2 to enhance the efficacy of ICB, providing a rationale for clinical investigation of this combination in PCa.

cancer biology↗

bioRxiv2025

CD8 T cells mediate immunosurveillance for neoantigen+ epithelial stem cells in the colon

Epithelial cells in the colon accumulate substantial numbers of somatic mutations, some of which can be recognised as neoantigens. The ability of CD8 T cells to survey for neoantigen+ cells in the healthy colon would provide an early detection mechanism to prevent cancer, but it is unclear whether neoantigen-specific CD8 T cells can mediate this process of immunosurveillance without becoming tolerant. To address this question, we used a genetically engineered mouse model to express a neoantigen in the epithelial cells of the adult proximal colon. Induction of neoantigen expression led to rapid elimination of neoantigen+ epithelial cells from the colon in a CD8 T cell-dependent manner. Neoantigen-specific CD8 T cells acquired cytolytic function within the colon tissue under steady-state conditions, which was required for elimination of the neoantigen+ epithelial cells. Despite the elimination of [~]25% of their epithelial cells over a two-day period, the colons looked histologically normal. Immunofluorescence and single-cell transcriptomic analyses revealed that neoantigen-specific CD8+ T cells specifically target neoantigen+ stem cells at the crypt base, which was associated with Ki67 in the crypt wall and abundance of neoantigen-negative stem cells. Infiltrating neoantigen-specific CD8 T cells made IFNg and expressed PD-1, raising the question of why PD-1-dependent suppression did not prevent the acquisition of effector functions by these neoantigen-specific CD8 T cells. Despite an increased signature of interferon-stimulated genes in colonic epithelial cells, PD-L1 expression was surprisingly absent. Moreover, we found that colonic epithelial stem cells also did not express PD-L1 under conditions of chronic inflammation, such as ulcerative colitis, immune checkpoint-induced colitis, and ageing, or when directly stimulated with IFN-{gamma} in vitro. Analyses of the PD-L1 gene promoter across humans and mice showed hypermethylation at sites associated with PD-L1 repression in cancer. Thus, our data support a model in which the acquisition of neoantigens by colonic epithelial cells triggers CD8 T cell-mediated immunosurveillance. This results in the elimination of PD-L1-negative neoantigen+ stem cells by effector CD8 T cells and simultaneous repair of the colon by neoantigen-negative epithelial cells to prevent immunopathology.

immunology↗

bioRxiv2022

WNK kinases sense molecular crowding and rescue cell volume via phase separation

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

cell biology↗

bioRxiv2021

A reservoir of stem-like CD8 T cells in the tumor-draining lymph node maintains the ongoing anti-tumor immune response

"Stem-like" TCF1+ CD8+ T cells (TSL) are necessary for long-term maintenance of T cell responses and the efficacy of immunotherapy but, as tumors contain signals that should drive T-cell terminal-differentiation, how these cells are maintained in tumors remains unclear. We found that a small number of TCF1+ tumor-specific CD8+ T cells were present in tumors throughout development. Yet, most intratumoral T cells differentiated as tumors progressed, corresponding with an immunologic shift in the tumor microenvironment (TME) from "hot" to "cold". By contrast, most tumor-specific CD8+ T cells in tumor-draining lymph nodes (dLNs) had functions and gene expression signatures similar to TSL from chronic LCMV infection and this population was stable over time, despite the changes in the TME. dLN T cells were the precursors of their more-differentiated intratumoral counterparts, and maintenance of TCF1 by intratumoral T cells required continuous migration from dLNs. Finally, TSL CD8 T cells were also present in LNs from lung adenocarcinoma patients, suggesting this population is also relevant in human disease. Thus, we propose that the dLN TSL reservoir has a critical function during tumor development in sustaining antitumor T cells during tumor development and protecting them from the terminal differentiation that occurs in the TME.

immunology↗

bioRxiv2020

Neoantigen-driven B cell and CD4+ T follicular helper cell collaboration promotes robust anti-tumor CD8+ T cell responses

CD4+ T follicular helper (TFH) cells provide help to B cells, which is critical for germinal center (GC) formation, but the importance of TFH-B cell interactions in cancer is unclear. We found TFH cells correlated with GC B cells and with prolonged survival of lung adenocarcinoma (LUAD) patients. To investigate further, we developed an LUAD model, in which tumor cells expressed B-cell- and T-cell-recognized neoantigens. Interactions between tumor-specific TFH and GC B cells were necessary for tumor control, as were effector CD8+ T cells. The latter were reduced in the absence of T cell-B cell interactions or the IL-21 receptor. IL-21 was produced primarily by TFH cells, development of which required B cells. Moreover, development of tumor-specific TFH cell-responses was also reliant upon tumors that expressed B-cell-recognized neoantigens. Thus, tumor-neoantigens themselves can control the fate decisions of tumor-specific CD4+ T cells by facilitating interactions with tumor-specific B cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/424168v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f31b57org.highwire.dtl.DTLVardef@881959org.highwire.dtl.DTLVardef@1385ccdorg.highwire.dtl.DTLVardef@154ba3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

NINJA: an inducible genetic model for creating neoantigens in vivo

Mouse models with inducible neoantigens have been historically difficult to generate because of leaky expression of antigens in the thymus, which causes central tolerance in developing CD8 and CD4 T cells. Attempts to resolve this problem using existing genetic tools have been unsuccessful. We developed the iNversion INducible Joined neoAntigen (NINJA) mouse model that uses RNA splicing, DNA recombination, and three levels of regulation to prevent neoantigen leakiness and allow tight control over the induction of neoantigen expression. We describe the development of these genetic tools and their use for obtaining tumor cell lines with inducible neoantigen expression. Moreover, we show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cells responses upon neoantigen induction in peripheral tissues. Thus, NINJA fills a long-standing gap in the field and will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, autoimmune diseases, and developing tumors.

immunology↗