chemical-conjugation-purified-dec-205-directed-antibody-with-full

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells <em>In Vitro</em> and <em>In Vivo</em>

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Helmholtz Centre for Infection Research

Otto-von-Guericke University Magdeburg

Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8 memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.

The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.

The survival of peripheral T cells is dependent on their access to peripheral LNs (pLNs) and stimulation by IL-7. In pLNs fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) produce IL-7 suggesting their contribution to the IL-7-dependent survival of T cells. However, IL-7 production is detectable in multiple organs and is not restricted to pLNs. This raises the question whether pLN-derived IL-7 is required for the maintenance of peripheral T cell homeostasis. Here, we show that numbers of naive T cells (T ) remain unaffected in pLNs and spleen of mice lacking Il7 gene activity in pLN FRCs, LECs, or both. In contrast, frequencies of central memory T cells (T ) are reduced in FRC-specific IL-7 KO mice. Thus, steady state IL-7 production by pLN FRCs is critical for the maintenance of T , but not T , indicating that both T cell subsets colonize different ecological niches in vivo.

IL-7 derived from lymph node fibroblastic reticular cells is dispensable for naive T cell homeostasis but crucial for central memory T cell survival.

NKp46 innate lymphoid cells (ILC) modulate tissue homeostasis and anti-microbial immune responses. ILC development and function are regulated by cytokines such as Interleukin (IL)-7 and IL-15. However, the ILC-intrinsic pathways translating cytokine signals into developmental programs are largely unknown. Here we show that the anti-apoptotic molecule cellular FLICE-like inhibitory protein (c-FLIP) is crucial for the generation of IL-7/IL-15-dependent NKp46 ILC1, including conventional natural killer (cNK) cells, and ILC3. Cytokine-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) precedes up-regulation of c-FLIP, which protects developing NKp46 ILC from TNF-induced apoptosis. NKp46 ILC-specific inactivation of c-FLIP leads to the loss of all IL-7/IL-15-dependent NKp46 ILC, thereby inducing early-onset chronic colitis and subsequently microbial dysbiosis; meanwhile, the depletion of cNK, but not NKp46 ILC1/3, aggravates experimental colitis. In summary, our data demonstrate a non-redundant function of c-FLIP for the generation of NKp46 ILC, which protect T/B lymphocyte-sufficient mice from intestinal inflammation.

Laura Knop

Immune Regulation Group

Helmholtz Centre for Infection Research

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo