Human Splenic Fibroblasts (HSF)

Collagen I Level in HSF Is Down-Regulated During rPvTRAg23 Stimulation

The spleen is pivotal in Plasmodium infection, contributing to immune functions and filtration of aging cells, with splenic fibroblasts (SFs) playing a critical role. Type I collagen, secreted by SFs, forms part of the extracellular matrix crucial for immune response. During infections, the SFs become abnormally activated, impacting collagen I and allowing Plasmodium to evade immune responses.

Zhang's team evaluate the protein PvTRAg23, identified from spleen-dependent PvTRAgs of the Pv-fam-a gene family, to determine its effects on splenic fibroblasts and collagen I production. To evaluate PvTRAg23's effect on HSFs, cells were exposed to various concentrations of recombinant protein for 48 hours, with collagen I levels monitored. Increasing rPvTRAg23 concentrations reduced collagen I expression in HSFs, unlike the control rPvTRAg26 (Fig. 1A), showing that rPvTRAg23 affected the collagen I content in HSFs specifically and in a concentration-dependent manner. Similar decreases were seen in COL1A1 and COL1A2 mRNA levels (Fig. 1B). ELISA confirmed reduced collagen I in the HSF culture medium's supernatant after 48 hours of protein stimulation (Fig. 1C), indicating rPvTRAg23 reduced HSF collagen I production and secretion.

Fig. 1. The levels of collagen I secreted by HSFs after stimulated by recombinant proteins (Zhang H, Shen F, et al., 2022).

Human Splenic Fibroblasts Alter BCL-2 Dependence

T-cell ALL is a malignant disease, causing immature T cells to spread to multiple areas, including the spleen. ETP-ALL is a subtype with poor outcomes, relying heavily on BCL-2 for cell survival. While ABT-199 shows promise against it, acquired resistance remains a critical hurdle.

Grande et al. assessed if the human splenic microenvironment affects BCL-2 dependence. They used human splenic fibroblasts (HSF) to create an ex vivo coculture model (Fig. 2A). LOUCY cells cocultured with HSF for 48 hours showed reduced cytochrome c release after BAD BH3 peptide and ABT-199 treatment, indicating decreased BCL-2 dependence (Fig. 2A-B). This was linked to reduced sensitivity to ABT-199 but not to other BH3 mimetics (Fig. 2C, Fig. 3A-C). BCL-2 expression decreased, while BCL-XL increased, without impacting WEHI-539 sensitivity (Fig. 2D-E). Additionally, using a transwell migration assay, LOUCY cells migrated more toward HSF-conditioned media than HS-5 media, unlike typical T-ALL cells (Fig. 2F, Fig. 3D). Furthermore, they examined if HSF coculture with an ETP-ALL sample could change the BH3 profile. Although ETP-ALL samples are hard to grow in mice, ETP-5 showed high BCL-2 expression from patient-derived pediatric tumor models. ETP-5 was confirmed as BCL-2 dependent, being highly sensitive to BH3 mimetics ABT-199 and ABT-263 after 6 hours in vitro (Fig. 2G). Coculturing ETP-5 with HSF for 16 hours reduced the BAD BH3 peptide response by 20%, indicating decreased BCL-2 dependence (Fig. 2H). These results suggest HSF-conditioned media provides a strong migration signal and that HSF coculture can reduce BCL-2 dependence in LOUCY and ETP samples.

Fig. 2. Human splenic fibroblasts reduce BCL-2 dependence in ETP-ALL in vitro (Grande A D, Peirs S, et al., 2021).

Fig. 3. Human splenic fibroblasts do not change sensitivity to BCL-XL or MCL-1 BH3 mimetics (Grande A D, Peirs S, et al., 2021).