Tailoring activation of MSC immunomodulation via genetic priming

Introduction: Mesenchymal Stem/Stromal Cells (MSCs) have been studied in over 1200 clinical trials as of 2020. Two of the most studied MSC disease indications include Osteoarthritis and Coronary Artery Disease, which affect 32.5M² patients and 20.1M³ patients respectively in the US as of 2020. MSC are attractive candidates for cell therapy in these and many other settings due to their ability to produce a large spectrum of different immunomodulatory and restorative effector molecules. Yet, clinical responses to MSC therapy have largely been underwhelming. Poor cell potency is among the core reasons for these deficiencies. It is widely known that MSCs do not express their many anti-inflammatory molecules unless they are activated by an inflammatory stimulus such as IFNγ, TNFα, LPS, or others⁶. These stimuli activate different gene sets depending on the intracellular pathway through which these signals are transduced. Some of these pathways can lead to the promotion rather than suppression of immune cell activation. With IFNγ activation, MSCs express beneficial genes such as IDO1, but also detrimental genes such as MHC class II molecules that promote their clearance by CD4 T cells and molecules that activate macrophages. Biochemical stimulation can also increase expression of inflammatory cell death genes and decrease growth rates. Herein, we describe the concept of ‘genetic activation’ or ‘genetic priming’ whereby a synthetic transgene for a transcription factor (TF-A) is used to controllably activate MSCs’ production of many advantageous molecules while avoiding production of unwanted molecules.


Materials and Methods: hTERT-MSCs were stably transduced with transcription factor transgenes using lentiviral constructs. The activation of MSCs at the transcriptional, translational, and functional levels were analyzed and compared for native unmodified control, TF-A, and IFNγ-activated (50ng/mL for 24h) MSCs via a battery of assays that included RT-qPCR, bulk RNA sequencing, immunocytochemistry, western blotting, and flow cytometry. Functional analyses with immune cells focused on multiparameter immunophenotyping that characterized the suppression of T cell proliferation and activation. This was done at a 1:1 cell ratio in a secretion-mediated context that utilized anti-CD3 and anti-CD28 as T cell-specific stimuli as well as in a contact-mediated context that did not use any other stimulus (MSCs activated with IFNγ for 72h at 50ng/mL). Contact-mediated MSC immunogenicity was analyzed via flow cytometry by assessing surface expression of HLA-DR, one of several MHC Class II molecules. To assay macrophage activation, control, TF-A, and IFNγ-activated hTERT-MSCs were cocultured in transwells with macrophages derived from CD14+ monocytes, which were isolated from freshly thawed PBMCs. MSCs and macrophages were cocultured at a 1:10 MSC to macrophage ratio with LPS, and a TNFα ELISA was done on the cocultured media.


Results, Conclusions, and Discussions:
Results and Discussion: RNA sequencing revealed biochemical and genetic priming both activated many of the same genes responsible for suppressing T cell proliferation (Fig1b) and activation (Fig 1c). However, only IFNγ upregulated genes responsible for T cell activation and macrophage activation (Fig. 1c). Priming with IFNγ or TF-A-transgenes both significantly attenuated PBMC proliferation in response to a T cell-specific stimulus and CD4 T cell activation in the form of TNFα expression (Fig 1d) as predicted by RNAseq. RNA sequencing also revealed that IFNγ activates many proinflammatory MHC class II genes while TF-A avoids much of this expression, and staining confirmed this for one representative MHC class II molecule: HLA-DR (Fig. 1e). MHC class II molecules were likely involved when hTERT-MSCs and PBMCs were directly cocultured. Immunophenotyping confirmed this behavior (Fig. 1f). CD4 T cells and not other immune cell types were activated and expressing significantly more TNFα in response to IFNγ-activated hTERT-MSCs compared to TF-A hTERT-MSCs (Fig. 1f), which would be expected if MHC class II genes were at play.


Conclusion: TF-A engineered MSCs recapitulated IFNγ-responsive activation of therapeutically beneficial genes that suppress T cell proliferation and activation in response to inflammation. TF-A also avoided the activation of deleterious genes that can promote MSC clearance and macrophage inflammation. This work therefore highlights the utility of transcription factor expression engineering for controlling activated MSC phenotypes and optimizing MSC properties for therapy.


References (Optional):
1. Levy, O. et al. Shattering barriers toward clinically meaningful MSC therapies. Sci. Adv. 6, eaba6884–eaba6884 (2020).
2. Osteoarthritis (OA) | Arthritis | CDC. https://www.cdc.gov/arthritis