T Cell-Mediated Immune Responses to AAV And AAV Vectors

Mechanisms of the Innate Immune Response to AAV

Toll-like receptors (TLRs) are a type of transmembrane receptors that participate in body immunity. Eleven members of the Toll-like receptor family have been discovered in mammals. Among them, the more clearly studied ones are TLR2, TLR4, TLR5 and TLR9. TLRs are like the eyes of innate immunity, monitoring and recognizing various pathogen-associated molecular patterns (PAMPs). Some TLR family members can not only recognize foreign substances such as exogenous pathogens, but also recognize endogenous substances and degradation products. TLR9 is a pattern recognition receptor that recognizes CpGDNA. It can activate NF-κB and AP-1 by activating signal transduction in the monocyte/phagocytic cell system, thereby releasing a large amount of TNF-α, IL-1, IL-6 and IL. 12 and other pro-inflammatory cytokines, causing acute inflammatory response, sepsis and even shock. TLR2 is a Toll-like receptor with the largest recognition range of ligands in the Toll-like family. It can recognize a variety of ligands including lipoproteins, lipopolypeptides, lipoteichoic acid (LTA), arabinomannan (LAM), and zymosan. Activated TLR2 can play an important role in the immune system and trigger specific immune responses. For TLR recognition of ligands, different types of TLRs can be combined to recognize different PAMPs. For example, TLR7 may be combined with TLR9 to mediate CpG activation of immune cells. Among them, TLR4/TLR4 and TLR9/TLR9 are in the form of homodimers; while TLR2/TLR4, TLR2/TLR6 and TLR7/TLR8 are heterodimers, and some dimers have a subunit that has not yet been OK, such as TLR3/TLR, TLR5/TLR.

TLR9 in cells such as plasmacytoid DCs (pDCs) in the blood and Kupffer cells in the liver is the main pattern recognition receptor that recognizes AAV genomic nucleic acids (especially unmethylated CpG DNA). Some researchers have used single-stranded AAV vector stimulated mouse-derived pDC in vitro, and an increase in type I IFN was detected in the culture supernatant. Similar results were obtained using human peripheral mononuclear cells (PBMC). Preclinical studies have also confirmed that activation of the TLR9-MyD88 signaling pathway is one of the causes of immune activation in AAV gene therapy targeting liver and muscle. Single-chain AAV vectors stimulated pDCs from TLR9 knockout mice and did not show an increase in IFN secretion. After treating human pDCs with TLR9 inhibitors, stimulation with single-chain AAV was unable to increase IFN secretion. In addition, studies have shown that the effect of AAV vector on stimulating pDCs to secrete IFN has nothing to do with the AAV vector serotype and the type of genetic element loaded. When single-chain AAV vectors were used to stimulate mouse macrophages, Kupffer cells, and human monocytes, no increase in the secretion of type I IFN was found. The above results indicate that TLR9 is a pathogen pattern recognition receptor in pDC that recognizes AAV vectors. pDC is the most important cell in the body for producing type I IFN, and its efficiency in producing type I IFN is more than a hundred times that of other cells. Researchers have found that type I IFN is an important factor in inducing anti-AAV capsid CD8+ T cell responses in specific immunity. Innate immune cells pDC and cDC work together to activate CD8+ T cell immune responses in specific immune responses, among which TLR9 is pDC is necessary. For cDC, TLR9 is not important. pDC monitors the invasion of AAV vector through TLR9, and cDC is responsible for the presentation of AAV capsid polypeptide antigen. TLR9 knockout mice can restore their CD8+ T cell immune responses against AAV capsids by adoptive infusion of wild-type mouse pDCs. The above studies indicate that the TLR9 innate immune signaling pathway in pDC is an important factor in subsequent adaptive cellular immune CD8+ T activation.