mRNA incorporated in lipid nanoparticles (LNPs) became a new class of vaccine modality for induction of immunity against COVID-19 and ushered in a new era in vaccine development. Here, we report a novel, easy-to-execute, and cost effective engineered extracellular vesicles (EVs)-based combined mRNA and protein vaccine platform (EVX-M+P vaccine) and explore its utility in proof-of-concept immunity studies in the settings of cancer and infectious disease. As a first example, we engineered EVs, natural nanoparticle carriers shed by all cells, to contain ovalbumin mRNA and protein (EVOvaM+P vaccine) to serve as cancer vaccine against ovalbumin-expressing melanoma tumors. EVOvaM+P administration to mice with established melanoma tumors resulted in tumor regression associated with effective humoral and adaptive immune responses. As a second example, we generated engineered EVs that contain Spike (S) mRNA and protein to serve as a combined mRNA and protein vaccine (EVSpikeM+P vaccine) against SARS-CoV-2 infection. EVSpikeM+P vaccine administration in mice and baboons elicited robust production of neutralizing IgG antibodies against RBD (receptor binding domain) of S protein and S protein specific T cell responses. Our proof-of-concept study describes a new platform with an ability for rapid development of combination mRNA and protein vaccines employing EVs for deployment against cancer and other diseases.
Copyright © 2024. Published by Elsevier B.V.

Dendritic cells (DCs) populate nearly all tissues and represent the central orchestrators of immunity. Here, we present a protocol for the mild but efficient preparation of single-cell suspensions from multiple murine tissues and the downstream analysis of the DC network via high-parameter flow cytometry. Additionally, we provide evaluation strategies that facilitate the stringent separation of the DC family from other myeloid cells, particularly macrophages and monocytes, and include an in-depth assessment of DC-intrinsic heterogeneity. For complete details on the use and execution of this protocol, please refer to Amon et al.1.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Breast milk is a complex mixture of nutrients and bioactives that promote infant development and decrease the incidence of chronic inflammatory disease. We investigated the role of one milk-derived bioactive, Immunoglobulin A (IgA) on the developing small intestinal microbiota and immune system. We demonstrate that early in life, milk-derived IgA suppressed colonization of the small intestine by Enterobacteriaceae and regulated the maturation of the small intestinal epithelium and the development of intestinal IL-17-producing CD4 + T cells. Enterobacteriaceae - specific CD4 + T cells, induced in the first weeks of life in the absence of milk-derived IgA, persisted in the intestine as memory T cells that can contribute to inflammatory disease later in life. Our study suggests that milk-derived IgA shapes mucosal immunity by regulating the neonatal microbiota thus preventing the development of long-lived intestinal microbiota-specific T cells.

Oncogenic KRASG12D (KRAS∗) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC) and is a known repressor of tumor immunity. Conditional elimination of KRAS∗ in genetic mouse models of PDAC leads to the reactivation of FAS, CD8+ T cell-mediated apoptosis, and complete eradication of tumors. KRAS∗ elimination recruits activated CD4+ and CD8+ T cells and promotes the activation of antigen-presenting cells. Mechanistically, KRAS∗-mediated immune evasion involves the epigenetic regulation of Fas death receptor in cancer cells, via methylation of its promoter region. Furthermore, analysis of human RNA sequencing identifies that high KRAS expression in PDAC tumors shows a lower proportion of CD8+ T cells and demonstrates shorter survival compared with tumors with low KRAS expression. This study highlights the role of CD8+ T cells in the eradication of PDAC following KRAS∗ elimination and provides a rationale for the combination of KRAS∗ targeting with immunotherapy to control PDAC.
Copyright © 2023 Elsevier Inc. All rights reserved.

Low-dose radiation therapy (LDRT) can suppress intractable inflammation, such as that in rheumatoid arthritis, and is used for treating more than 10,000 rheumatoid arthritis patients annually in Europe. Several recent clinical trials have reported that LDRT can effectively reduce the severity of coronavirus disease (COVID-19) and other cases of viral pneumonia. However, the therapeutic mechanism of LDRT remains unelucidated. Therefore, in the current study, we aimed to investigate the molecular mechanism underlying immunological alterations in influenza pneumonia after LDRT. Mice were irradiated to the whole lung 1 day post-infection. The changes in levels of inflammatory mediators (cytokines and chemokines) and immune cell populations in the bronchoalveolar lavage (BALF), lungs, and serum were examined. LDRT-treated mice displayed markedly increased survival rates and reduced lung edema and airway and vascular inflammation in the lung; however, the viral titers in the lungs were unaffected. Levels of primary inflammatory cytokines were reduced after LDRT, and transforming growth factor-β (TGF-β) levels increased significantly on day 1 following LDRT. Levels of chemokines increased from day 3 following LDRT. Additionally, M2 macrophage polarization or recruitment was increased following LDRT. We found that LDRT-induced TGF-β reduced the levels of cytokines and polarized M2 cells and blocked immune cell infiltration, including neutrophils, in BALF. LDRT-induced early TGF-β production was shown to be a key regulator involved in broad-spectrum anti-inflammatory activity in virus-infected lungs. Therefore, LDRT or TGF-β may be an alternative therapy for viral pneumonia.
Copyright © 2023 Song, Chen, Park, Han, Ji, Byun, Kwon, Kim, Ahn and Seo.