Poster CC18 - Comparative effects of Proteins and Exosomes Derived from CD4+ T-cell Subsets on Satellite Cell Activity in vitro.

Introduction: Severe and persistent inflammation is a major impediment to muscle recovery post-volumetric muscle loss (VML). Helper CD4+ T-cells and their subsets (Th1, Th2, Th17, and Treg) are known to be recruited at the skeletal muscle injury site to produce an immune response and play an important role in providing microenvironmental cues to satellite cells for effective muscle regeneration. CD4+ T-cell subtypes mediate their function through secreted products such as trophic factors and/or exosomes. For instance, Th1 produces IFN-γ and TNF-α to promote inflammation and satellite cell (SC) proliferation, while the release of interleukin (IL)-4 and IL-13 by Th2 and amphiregulin by Treg suppresses inflammation and promotes the differentiation of SC. Th17 produces IL-17 A, which promotes angiogenesis and myogenesis. However, the exact contribution of CD4+ T cell derived products remains poorly understood in context of muscle repair. The goal of this study was to determine the role of CD4+ T-cell derived secreted products in supporting SC activity in vitro. We hypothesize that trophic factors and/or exosomes derived from Th2 and Treg will support SC differentiation and fusion, while those derived from Th1 and Th17 subsets will enhance SC proliferation.

Materials and

Methods: - CD4+ T-cells were isolated from spleens of male Lewis rat and cultured under standard conditions in T-cell culture media containing specific cytokines and antibodies to polarize them into Th1, Th2, Th17, and Treg. Unpolarized CD4+ T-cells (Th0) served as controls. T-cell culture media was collected from each subset on days 5 and 7 of culture and was used for exosome isolation using Exo-Quick TC (System Biosciences). The exosome depleted culture media containing proteins and growth factors secreted by T-cell subsets will be called conditioned media (CM). Exosomes were characterized using Exo-Check Array and Nanoparticle Tracking Analysis (NTA). For the in vitro experiment, SCs were isolated from the hindlimb muscles of rats and were seeded in two separate 48 well plates at a density of 100,000 cells/well (n=4/group). In the first plate, SCs were incubated with growth media containing PBS (control group) or 25 µg/ml exosomes derived from Th0, Th1, Th2, Th17 or Treg for 72 hours. In the second plate, SCs were incubated with growth media containing PBS or CM diluted by a factor 2 in growth media. PBS or CM were replenished every 24 hours for 3 days. Post 3 days, brightfield images were taken at 20X magnification to observe myotube formation. Supernatants were collected and saved at -20 degree for ELISAs. The cell layer was washed twice with 1X sterile PBS and CyQuant Cell Proliferation Assay Kit (Invitrogen) was used to perform a fluorescence-based assay to quantify the DNA concentration in live SCs, indicative of proliferation.

Results, Conclusions, and Discussions: Expression of IFN-γ by Th1, IL-4 and IL-13 by Th2, IL-17 A and IL-22 by Th17 and FoxP3 by Treg was confirmed. Th0 exosomes showed characteristic surface marker expression (CD81, TSG101) and were in characteristic size range of 30-200 nm (Fig 1). SCs differentiated into myotubes in response to T-cell exosomes and CM from all subsets (Fig 2), quantification is ongoing.
While inflammatory cytokines like IL-6 and TNF-α activate SCs and promote their proliferation, anti-inflammatory growth factors like VEGF and IGF-1 support SC proliferation and differentiation. The release of these factors by SCs in response CD4+ T-cell derived secreted products was quantified (Fig 3). Th17 exosomes and CM increased IL-6 production from SCs. Th2 exosomes but not CM enhanced the production of IL-6 by SCs. TNF-α, VEGF and IGF-1 production was similar in response to T-cell exosomes. Th1, Th2, and Th17 CM elicited more TNF-α production from SCs compared to Th0 and Treg CM. VEGF production was increased in response to Th17 and Treg CM compared to other subsets. IGF-1 production was significantly higher in response to Th0 CM relative to other T-cell subsets.
CyQuant Assay showed that exosomes from all T-cell subsets promoted proliferation of SCs. However, Th0 and Th2 CM enhanced SC proliferation more than Th1, Th17 and Treg CM (Fig 4).
Conclusion- Our results would indicate that Th2 can stimulate pro-inflammatory cytokine production while Th17 can elicit both pro- and anti-inflammatory trophic factor release from SCs in vitro. Taken together, these results were unexpected and highlighted the complex interplay between immune cell subsets and muscle resident stem cells. Furthermore, these results indicate different biological effects of helper T-cell exosomes and CM on SCs, possibly attributable to difference in their biological cargo.

Discussion: Elucidation of the biological effects and underlying mechanism of T-cell subset secreted products in context of immunomodulation and regeneration can help utilize their therapeutic potential. Our results imply differences in the effects of T-cell subset derived trophic factors and exosomes on SC activity in vitro. Future studies are needed to compare in vivo biological activity of T-cell secreted products in a rat VML injury model.

Acknowledgements (Optional): This work was supported by the NIGMS 1R15GM129731-02 awarded to KG.