A New Immortalized Human Lacrimal Gland Cell Line.

The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells' capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.