Key Role of Kupffer Cells in IL-1 Dependent Activation of CD8⁺ T Cell Responses to AAV Transgene Product in Liver

Introduction: Adeno-associated virus (AAV) based viral vectors have emerged as preferred vectors for in vivo gene therapy and are being evaluated in multiple phase I/II and phase III clinical studies. However, immune responses to both the AAV capsid and its encoded transgene still remain a major hurdle. Previously, we demonstrated in a murine model that cellular responses to AAV encoded transgene in the liver are dependent on IL-1α/β mediated activation of IL-1R1-MyD88 signaling. Using flow cytometry and tissue staining we provided evidence that plasmacytoid dendritic cells (pDCs) and Kupffer cells are central players in initiation of IL-1 signaling. Further, we showed that cross-presenting XCR1⁺ dendritic cells (DCs) are critical for antigen presentation to transgene-specific CD8⁺ T cells (Blood 136 Supplement 1: 2-3, 2020). In the present study we tested the impact of Kupffer cells depletion on transgene-specific CD8⁺ T cell response. Since Kupffer cells have been implicated in TLR4-MyD88 mediated secretion of IL-1α in a murine model of acetaminophen induced liver injury (Cell Mol Immunol 15(11): 973, 2018). Therefore, we also explored if innate sensor TLR4 is required for IL-1 mediated CD8⁺ T cell response to AAV encoded transgene in liver microenvironment. Further, we tested if intrinsic IL-1R1-MyD88 signaling in DCs is essential for these cellular responses.

Methods: To study the impact of Kupffer cell depletion on transgene-specific CD8⁺ T cells we intraperitoneally injected wild-type (WT) C57BL6 mice with 200μg of depleting α-mouse F4/80 antibody (BioXcell) one day prior and 2X/week following vector administered for up to 4 weeks. Groups of C57BL6 WT (control), C57BL6 WT (treated with α-mouse F4/80 antibody) and TLR4^-/- mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x10⁹vg) of hepatotropic AAV8 vector encoding ovalbumin (OVA) as the transgene (Mol Ther 25(4): 880, 2017).

To study the role of MyD88 intrinsic signaling in DCs, we generated mice that lacked adaptor protein MyD88 in DCs (DC-MyD88^-/-) using cre-lox system (Blood 129(24): 3184, 2017). Mice were again injected with 1x10⁹vg of AAV8-OVA via IV route.

Four weeks after vector administration PBMCs from each group were analyzed for OVA-specific CD8⁺ T cells using MHC class I tetramer.

Results: As expected, ~62% of WT mice developed OVA-specific CD8⁺ T cell response. Interestingly, only 12% of mice (1 out of 8) treated with α-mouse F4/80 antibody showed CD8⁺ T cells specific to OVA. Whereas similar to WT mice, 50% of TLR4^-/- mice had OVA-specific CD8⁺ T cell. We conclude that Kupffer cells, which show increased surface expression of IL-1R1 within days after AAV vector administration, are essential for the IL-1R-dependent CD8⁺ T cells response to the hepatic transgene product but do not function through a mechanism that requires TLR4. This mechanism therefore shares some aspects of acetaminophen-induced liver injury (critical role of KCs and IL-1) but is distinct with regard to TLR involvement.

IL-1R1 signaling requires the cytosolic MyD88 adapter molecule. We know from prior studies that MHC I antigen presentation of liver-derived AAV encoded transgene product in lymphoid organs is primarily mediated by cross-presenting XCR1⁺ DCs. Thus, we tested the requirement of intrinsic MyD88 signaling in DCs. Our results suggest that intrinsic MyD88 signaling in DCs is not required in lymphoid organs, as the CD8⁺ T cell response in DC-MyD88^-/- mice was comparable to that in WT control mice (these mice lack MyD88 in conventional DCs in lymphoid organs but retain MyD88 in hepatic DCs and in pDCs).

Conclusions: In summary, we established that Kupffer cells play a central role in elicitation of transgene product-specific CD8⁺ T cell responses in hepatic AAV gene transfer (through a TLR4-independent mechanisms). While direct sensing of IL-1 by DCs in the hepatic environment may very well play a role, it is not required in lymphoid organs. Following initial priming of CD8⁺ T cell in the liver, co-stimulation by CD4⁺ T helper cells may replace IL-1 signaling during the subsequent systemic response.

Biswas:Pfizer: Consultancy. Herzog:BioMarin: Consultancy; Regeneron Pharma/Intellia Therapeutics collaboration: Consultancy; Spark Therapeutics: Research Funding; Pfizer: Consultancy.