Cell composition and expansion strategy can reduce the beneficial effect of AKT-inhibition on functionality of CD8+ T cells.

AKT-inhibition is a promising approach to improve T cell therapies; however, its effect on CD4+ T cells is insufficiently explored. Previously, we and others showed that AKT-inhibition during ex vivo CD8+ T cell expansion facilitates the generation of polyfunctional T cells with stem cell memory-like traits. However, most therapeutic T cell products are generated from lymphocytes, containing CD4+ T cells that can affect CD8+ T cells dependent on the Th-subset. Here, we investigated the effect of AKT-inhibition on CD4+ T cells, during separate as well as total T cell expansions. Interestingly, ex vivo AKT-inhibition preserved the early memory phenotype of CD4+ T cells based on higher CD62L, CXCR4 and CCR7 expression. However, in the presence of AKT-inhibition, Th-differentiation was skewed toward more Th2-associated at the expense of Th1-associated cells. Importantly, the favorable effect of AKT-inhibition on the functionality of CD8+ T cells drastically diminished in the presence of CD4+ T cells. Moreover, also the expansion method influenced the effect of AKT-inhibition on CD8+ T cells. These findings indicate that the effect of AKT-inhibition on CD8+ T cells is dependent on cell composition and expansion strategy, where presence of CD4+ T cells as well as polyclonal stimulation impede the favorable effect of AKT-inhibition.

A cis-regulatory element regulates ERAP2 expression through autoimmune disease risk SNPs.

Single-nucleotide polymorphisms (SNPs) near the ERAP2 gene are associated with various autoimmune conditions, as well as protection against lethal infections. Due to high linkage disequilibrium, numerous trait-associated SNPs are correlated with ERAP2 expression; however, their functional mechanisms remain unidentified. We show by reciprocal allelic replacement that ERAP2 expression is directly controlled by the splice region variant rs2248374. However, disease-associated variants in the downstream LNPEP gene promoter are independently associated with ERAP2 expression. Allele-specific conformation capture assays revealed long-range chromatin contacts between the gene promoters of LNPEP and ERAP2 and showed that interactions were stronger in patients carrying the alleles that increase susceptibility to autoimmune diseases. Replacing the SNPs in the LNPEP promoter by reference sequences lowered ERAP2 expression. These findings show that multiple SNPs act in concert to regulate ERAP2 expression and that disease-associated variants can convert a gene promoter region into a potent enhancer of a distal gene.

Vaccination with a bacterial peptide conjugated to SARS-CoV-2 receptor-binding domain accelerates immunity and protects against COVID-19.

Poor immunogenicity of critical epitopes can hamper vaccine efficacy. To boost immune recognition of non- or low-immunogenic antigens, we developed a vaccine platform based on the conjugation of a target protein to a chimeric designer peptide (CDP) of bacterial origin. Here, we exploited this immune Boost (iBoost) technology to enhance the immune response against the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. Despite its fundamental role during viral infection, RBD is only moderately immunogenic. Immunization studies in mice showed that the conjugation of CDP to RBD induced superior immune responses compared to RBD alone. CDP-RBD elicited cross-reactive antibodies against the variants of concern Delta and Omicron. Furthermore, hamsters vaccinated with CDP-RBD developed potent neutralizing antibody responses and were fully protected from lung lesion formation upon challenge with SARS-CoV-2. In sum, we show that the iBoost conjugate vaccine technology provides a valuable tool for both quantitatively and qualitatively enhancing anti-viral immunity.

Endothelial RhoB and RhoC are dispensable for leukocyte diapedesis and for maintaining vascular integrity during diapedesis.

Active remodeling of the actin cytoskeleton in endothelial cells is necessary for allowing leukocytes to cross the barrier during the process of transendothelial migration (TEM). Involvement of RhoGTPases to regulate actin organization is inevitable, and we recently reported on the local function of RhoA in limiting vascular leakage during leukocyte TEM. As a follow-up we investigated here the possible involvement of two other closely-related GTPases; RhoB and RhoC, in regulating leukocyte TEM and vascular barrier maintenance. Physiological flow experiments showed no substantial involvement of either endothelial RhoB or RhoC in neutrophil adhesion and transmigration efficiency. Besides neutrophil TEM, we did not observe a role for endothelial RhoB or RhoC in limiting vascular leakage in both inflammatory conditions and during TEM. In conclusion, endothelial RhoB and RhoC are both dispensable for regulating leukocyte diapedesis and for maintaining vascular barrier function under inflammatory conditions and during leukocyte diapedesis.

Stiffness-Induced Endothelial DLC-1 Expression Forces Leukocyte Spreading through Stabilization of the ICAM-1 Adhesome.

Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration.