Modulation of Fibroblastic Reticular Cell Immunomodulatory Phenotype via CRISPR Activation and Interference Platform for Applications in Type 1 Diabetes

Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of insulin-producing pancreatic β cells. Existing autoimmunity treatments largely target immune cells indiscriminately and systemically leading to generalized immunosuppression. An antigen-specific immunomodulatory platform that targets β cell autoreactive T cells could increase safety and efficacy of T1D treatment. Lymph node (LN) derived fibroblastic reticular cells (FRCs) are non-professional antigen-presenting cells that can mediate antigen-specific T cells by inducing T cell anergy, deletion, and regulatory T cell differentiation. Treating FRCs with interferon-γ (IFNγ) enhances their antigen-specific immunomodulatory function, but this effect is transient and broadly affects FRC phenotype. Instead, the non-canonical CRISPR activation and interference (CRISPRa/i) system can be used to transiently or persistently modulate expression of specific genes of interest (GOI) at the epigenetic level while avoiding genomic sequence alterations. The targeted effects of CRISPRa/i can simultaneously and independently overexpress (dCas9-VPR) or repress (dCas9-KRAB) select GOI in a multiplex manner, and is used here to enhance FRC immunomodulatory phenotype to facilitate robust T cell-tolerizing effects. Proposed key GOI of FRC inducible tolerance include the class II major histocompatibility complex transactivator (Ciita), programmed death ligand 1 (PD-L1 /Cd274) and transforming growth factor beta 2 (Tgfb2).

Materials and

Methods: FRCs were isolated from skin-draining LNs (sdLN) of 12 week-old female non-obese diabetic (NOD) mice and expanded in culture. FRCs were then transduced with lentiviral constructs encoding the targetable transcriptional activator dCas9-VPR and the targetable transcriptional repressor dCas9-KRAB, both of which incorporate a puromycin resistance gene in the same open reading frame as the dCas9 effector molecules. Transduced cells were selected on the puromycin resistance gene via puromycin challenge (1ug/mL). Post selection, dCas9 effector protein expression was confirmed via intracellular flow cytometry staining using an anti-Cas9 antibody. Guide RNA (gRNA) sequences were designed via CRISPick to target the core promoter regions of select GOIs (Ciita, CD274 , and Tgfb2) by picking sequences -300 to 0 base pairs (bp) from the predicted transcription start site. Guide RNA sequences were synthesized by IDT as single guide RNAs (sgRNA) containing the 20bp targeting sequence along with the 80bp tracrRNA sequence. Transduced FRCs were transfected via lipofection with sgRNA molecules targeting the various GOI with IFNγ treated FRCs functioning as a positive control for gene upregulation. Dose titration of sgRNA was carried out with a single GOI and analyzed via flow cytometry for the GOI protein product. Multiplex sgRNA studies were also performed where multiple GOI were targeted. Time course studies were also conducted at 24hr, 48hr, 72hr, and 96hr timepoints.

Results, Conclusions, and Discussions: Intracellular Cas9 staining and analysis by flow cytometry of dCas9-VPR and dCas9-KRAB cell lines indicate that FRCs can be transduced with and express the prerequisite transgenes. Single GOI targeting of CD274 in dCas9-VPR cells yielded a distinct positive signal in CD274 in a dose-dependent manner with diminishing differential response at higher doses, suggesting a plateaued response at higher sgRNA concentrations. In dCas9-VPR FRCs, multiplex transfection targeting multiple GOIs (CD274 and Ciita) in addition to single target transfections increased expression of the respective protein product. These data indicate that the cellular response is specific to the GOI sgRNA target with no detectable off target effects in those proteins measured. An additional preliminary finding is that co-transfection with CD274 and Ciita sgRNAs in dCas9-VPR cells yields an increased upregulation in CD274 median fluorescence intensity compared to CD274 sgRNA alone, which suggests a synergistic response that merits further study. Longitudinal studies of multiplex sgRNA treated samples show that the maximal effect of sgRNA transfection on GOI activation and repression occurs at 96 hours post-transfection. Longer timepoints will yield further information on whether this trend of increased effect size continues and when the effects of sgRNA transfection begin to diminish. Taken together, these data support the use of the CRISPRa/i system for phenotype modulation of FRCs, allowing for tight control of specific GOI expression, individually or in combination, and the elucidation of the key FRC molecular patterns that facilitate FRC-mediated antigen-specific T cell tolerance. Further work will validate and refine this system with respect to transfection efficiency, sgRNA design, duration of effect, and pathway interactions between the targeted GOIs. Ultimately, co-culture studies between T1D relevant antigen specific T cells and CRISPRa/i modulated FRCs will be used to assess the differences in tolerizing capabilities between different FRC phenotypes. Applications of this platform in addition to gaining an insight into different cell-cell interactions include the ability to harness an enhanced FRC tolerogenic phenotype to induce tolerance in those autoreactive T cells from peripheral circulation that are the major contributors to the pathogenesis of T1D.

Acknowledgements (Optional): Greatest thanks to Kristen Brennand for providing both lenti-EF1a-dCas9-VPR-Puro and lenti-EF1a-dCas9-KRAB-Puro gene constructs