Junctional Adhesion Molecule 2 Intensifies T Lymphopoiesis of Hematopoietic Stem Cells By Facilitating Notch/Delta Signaling
Authors: Radulovic Visnja , Van Der Garde Mark , Sigurdsson Valgardur , Zriwil Alya , Soboleva Svetlana , Sitnicka Quinn Ewa , Kaneko Shin , Miharada Kenichi 
Published: April 2021

Abstract
Phenotypically described hematopoietic stem cells (HSCs) represent a functionally heterogeneous pool of primitive cells with conceivable potential to replenish and maintain the whole hematopoietic system. The diverse lineage potential of HSCs is supposed to play a significant role in the response to different kinds of hematopoietic stress. Since subcategorization of HSCs biased towards specific lineage(s) highly relies on the retrospective information, e.g. transplantation assay, exploring additional markers will allow us to understand further molecular mechanisms of HSC regulation such as activation and lineage choice but also the degree of correlation between them.
Here, we show that the cell surface protein Junctional adhesion molecule 2 (Jam2) serves as an amplifier of the Notch/Delta signal thereby representing the higher T cell potential of HSCs. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0%), while other Jam family member, Jam1 (F11r), was expressed on all HSCs and Jam3 was not detected. Transplantation assay using 30 Jam2+ or Jam2-HSCs revealed that Jam2+HSCs reconstituted lethally irradiated mice more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3% in peripheral blood, respectively). Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100%. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell potential of Jam2+HSCs is independent of the cell cycle progression.
Jam2 has been reported to mediate the Notch signaling through an interaction with Jam1 (Kobayashi et al., Nature, 2014). In addition, Jam2+HSCs express Notch1 at a higher level than Jam2-HSCs (23.6 ±6.7 and 9.05 ±5.8%, respectively). We therefore analyzed the functional role of Jam2 in the Notch/Delta-oriented T cell production using a competitive feeder-free T cell culture system. At a low concentration of DLL1, that is insufficient to promote T cell production by itself, Jam2+HSCs effectively produced T cell lineages only in the presence of recombinant Jam1 protein, but not Jam2 or Jam3. In contrast, Jam2+HSCs did not require Jam1 protein with a higher concentration of DLL1. These differences were not observed with Jam2-HSCs, indicating that Jam2/Jam1 interaction amplifies Notch signal transduction and is crucial for the subsequent T cell specification of Jam2+HSCs.
To elucidate the molecular signature of Jam2+HSCs, gene expression profiling was performed using a microarray analysis. Gene set enrichment analysis (GSEA) observed that Jam2+HSCs were significantly enriched for common lymphoid progenitor (CLP) and early T cell gene expression. Of note, Jam2+HSCs were also enriched for E2F target genes, G2M checkpoint genes and glycolysis related genes, which potentially explains the reason why Jam2+HSCs display a bivalent phenotype: being more dormant compared to Jam2-HSCs at the steady state but at the same time having the capacity to reconstitute more actively upon engraftment.
Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoietic environment affects the proportion of Jam2+HSCs. In vivo T cell depletion resulted in significantly higher frequency of Jam2+HSCs but not upon other stress inducers, such as 5-FU treatment, suggesting that the increase in Jam2+HSC pool was specifically due to the T cell deficiency. These findings indicate that the lack of T cells, which also means a requirement for immediate T cell replenishment, leads to an increase of Jam2+HSC fraction.
Our findings suggest that Jam2 is the key protein that controls T lymphopoiesis by enhancing the Notch/Delta signal transduction via interaction with Jam1. It also means that the lineage balance particularly towards T lymphopoiesis might be regulated at a higher stage of hematopoietic hierarchy than currently understood. Thus, Jam2 is a new marker representing the T lymphocyte potential of HSCs, as the frequency of Jam2+HSCs sensitively reflects the state of the T cell environment.

Disclosures
No relevant conflicts of interest to declare.