Transmembrane Adaptor Protein Scimp Promotes TLR Signaling to Regulate Hematopoietic Stem Cell Function

SLP65/SLP76, Csk-interacting membrane protein (SCIMP) is a transmembrane adaptor protein widely expressed in antigen presenting cells (APC) and hematopoietic stem cells (HSCs). It comprises a short extracellular domain, a long intracellular domain containing multiple tyrosine phosphorylation sites, a proline-rich motif and two palmitoylation sites. In APCs, SCIMP co-localizes with MHCII and tetraspanins, playing crucial role in MHCII signaling. SCIMP interacts with multiple toll like receptors (TLRs) to scaffold Lyn for TLR phosphorylation, and the promotion of ERK1/2 signaling. Additionally, it recruits Syk to help propagate TLR signaling and the production of proinflammatory cytokines. TLRs are expressed on HSCs and multiple studies have demonstrated the role of TLR signaling in regulation of both normal and malignant HSCs. TLR agonist treatment enhances HSC proliferation and differentiation towards myeloid population (Megias et.al, 2012), and TLR deficient HSCs have increased engraftment efficiency upon transplantation (Ichii et. al., 2010). Our previous studies demonstrated that TLR2 agonist treatment leads to expansion of bone marrow phenotypic HSCs but loss of BM repopulating activity (Hermam et.al., 2016). Here, we studied the role of SCIMP in TLR mediated signaling and repopulating activity of HSC.

To assess the role of SCIMP in the regulation of TLR signaling in HSCs, Scimp^-/- mice and their WT littermates were treated with TLR2 agonist PAM₂CSK₄ (chronic: 50ug/kg; 3 dose/week for 2 weeks and acute: 100ug/Kg, 24hrs) to induce inflammatory stress. While the frequency of HSCs in the bone marrow increased in WT mice upon chronic PAM₂CSK₄ treatment; this expansion was attenuated by 55% in the absence of Scimp despite normal TLR2 expression. Additionally, the cycling response to PAM₂CSK₄ was reduced by 17.76% in the Scimp^-/- HSCs compared to WT HSCs from bone marrow. Notably, the mobilization of HSCs to the spleen was similar in both groups. A similar response was observed in WT and Scimp^-/- mice following acute PAM₂CSK₄ treatment.

To elucidate the underlying mechanism, we analyzed the transcriptomic signature of HSCs from the bone marrow of WT and Scimp^-/- mice treated with chronic PAM₂CSK₄ using bulk RNA sequencing. GSEA revealed down-regulation of G2M checkpoint genes and E2F target genes (Cks1b, Mcm3, Mcm5, Prim2, H2az1) in HSCs from Scimp^-/- mice.

Next, we studied whether regulation of TLR2 signaling by SCIMP in HSC is cell intrinsic. We isolated c-Kit enriched bone marrow cells from WT and Scimp^-/- mice, stimulated them with PAM₂CSK₄ (10ug/ml) for one hour and assessed for activation of downstream signaling molecules (phospho- SYK, and phosphor- ERK) involved in TLR2 activation. Flow cytometry analysis revealed that in Scimp^-/- HSCs, phosphorylation of SYK and ERK was decreased by 21.85% and 14.40%, respectively.

Finally, Scimp^-/- HSCs demonstrated a competitive advantage in transplantation experiments of Scimp^-/- vs WT HSCs into lethally irradiated recipient mice (similar to TLR-deficient HSCs). After 24 weeks of secondary transplantation, lethally irradiated mice receiving Scimp^-/- HSCs had a higher HSC percentage (84.77%) in their bone marrow compared to mice receiving WT HSCs. Moreover, Scimp^-/- HSCs treated with chronic TLR agonist PAM₂CSK₄ exhibited a competitive advantage and higher HSC percentage (33.01%) compared to WT HSCs treated with the same agonist_.

Further, we assessed whether overexpressing Scimp increase sensitivity of hematopoietic stem and progenitor cells to stress by serial replating assay. We observed a significantly higher number of colonies in Scimp overexpressing HSPCs compared to empty vector expressing HSPCs by 92% and 10% after 7^th and 14^th day of replating in MethoCult.

Together, these findings suggest that SCIMP is necessary for normal TLR signaling and function in HSCs, highlighting the importance of further investigating its role in stress-induced HSC dysfunction.