Bacille Calmette-Guérin (BCG), the live attenuated tuberculosis vaccine, is manufactured under different conditions across the globe generating formulations that may differ in clinical efficacy. Innate immune recognition of live BCG contributes to immunogenicity suggesting that differences in BCG viability may contribute to divergent activity of licensed formulations.

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.

The in vitro Peripheral Tissue Equivalent (PTE) module is a three-dimensional tissue-engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll-like receptor (TLR) agonists, including polyinosine-polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1alpha/beta, 6, 8 and 10 and tumour necrosis factor-alpha. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100-fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up-regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE-derived DC treated with Gardiquimod, a TLR-7 agonist, significantly augmented anti-tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33(+)) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen-presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.

Newborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells ("innate memory"). However, little is known regarding the impact of age on BCG-induced innate responses.

Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization.

Infections are most frequent at the extremes of life, especially among newborns, reflecting age-specific differences in immunity. Monocytes maintain tissue-homeostasis and defence-readiness by escaping circulation in the absence of inflammation to become tissue-resident antigen presenting cells in vivo. Despite equivalent circulating levels, neonates demonstrate lower presence of monocytes inside peripheral tissues as compared to adults. To study the ability of monocytes to undergo autonomous transendothelial extravasation under biologically accurate circumstances we engineered a three-dimensional human vascular-interstitial model including collagen, fibronectin, primary endothelial cells and autologous untreated plasma. This microphysiological tissue construct enabled age-specific autonomous extravasation of monocytes through a confluent human endothelium in the absence of exogenous chemokines and activation. Both CD16- and CD16+ newborn monocytes demonstrated lower adherence and extravasation as compared to adults. In contrast, pre-activated tissue constructs were colonized by newborn monocytes at the same frequency than adult monocytes, suggesting that neonatal monocytes are capable of colonizing inflamed tissues. The presence of autologous plasma neither improved newborn homeostatic extravasation nor shaped age-specific differences in endothelial cytokines that could account for this impairment. Newborn monocytes demonstrated significantly lower surface expression of CD31 and CD11b, and mechanistic experiments using blocking antibodies confirmed a functional role for CD31 and CD54 in neonatal homeostatic extravasation. Our data suggests that newborn monocytes are intrinsically impaired in extravasation through quiescent endothelia, a phenomenon that could contribute to the divergent immune responsiveness to vaccines and susceptibility to infection observed during early life.

Much remains to be learned about the physiologic events that promote monocytes to become lymph-homing dendritic cells (DCs). In a model of transendothelial trafficking, some monocytes become DCs in response to endogenous signals. These DCs migrate across endothelium in the ablumenal-to-lumenal direction (reverse transmigration), reminiscent of the migration into lymphatic vessels. Here we show that the subpopulation of monocytes that expresses CD16 (Fcgamma receptor III) is predisposed to become migratory DCs. The vast majority of cells derived from CD16(+) monocytes reverse transmigrated, and their presence was associated with migratory cells expressing high levels of CD86 and human histocompatibility leukocyte antigen (HLA)-DR, and robust capacity to induce allogeneic T cell proliferation. A minority of CD16(-) monocytes reverse transmigrated, and these cells stimulated T cell proliferation less efficiently. CD16 was not functionally required for reverse transmigration, but promoted cell survival when yeast particles (zymosan) were present as a maturation stimulus in the subendothelial matrix. The cell surface phenotype and migratory characteristics of CD16(+) monocytes were inducible in CD16(-) monocytes by preincubation with TGFbeta1. We propose that CD16(+) monocytes may contribute significantly to precursors for DCs that transiently survey tissues and migrate to lymph nodes via afferent lymphatic vessels.

Current vaccine development disregards human immune ontogeny, relying on animal models to select vaccine candidates targeting human infants, who are at greatest risk of infection worldwide, and receive the largest number of vaccines. To help accelerate and de-risk development of early-life effective immunization, we engineered a human age-specific microphysiologic vascular-interstitial interphase, suitable for pre-clinical modeling of distinct age-targeted immunity in vitro. Our Tissue Constructs (TCs) enable autonomous extravasation of monocytes that undergo rapid self-directed differentiation into migratory Dendritic Cells (DCs) in response to adjuvants and licensed vaccines such as Bacille Calmette-Guérin (BCG) or Hepatitis B virus Vaccine (HBV). TCs contain a confluent human endothelium grown atop a tri-dimensional human extracellular matrix substrate, employ human age-specific monocytes and autologous non heat-treated plasma, and avoid the use of xenogenic materials and exogenous cytokines. Vaccine-pulsed TCs autonomously generated DCs that induced single-antigen recall responses from autologous naïve and memory CD4+ T lymphocytes, matching study participant immune-status, including BCG responses paralleling donor PPD status, BCG-induced adenosine deaminase (ADA) activity paralleling infant cohorts in vivo, and multi-dose HBV antigen-specific responses as demonstrated by lymphoproliferation and TCR sequencing. Overall, our microphysiologic culture method reproduced age- and antigen-specific recall responses to BCG and HBV immunization, closely resembling those observed after a birth immunization of human cohorts in vivo, offering for the first time a new approach to early pre-clinical selection of effective age-targeted vaccine candidates.

More than 98% of the human genome is made up of non-coding DNA, but techniques to ascertain its contribution to human disease have lagged far behind our understanding of protein coding variations. Autism spectrum disorder (ASD) has been mostly associated with coding variations via de novo single nucleotide variants (SNVs), recessive/homozygous SNVs, or de novo copy number variants (CNVs); however, most ASD cases continue to lack a genetic diagnosis. We analyzed 187 consanguineous ASD families for biallelic CNVs. Recessive deletions were significantly enriched in affected individuals relative to their unaffected siblings (17% versus 4%, p < 0.001). Only a small subset of biallelic deletions were predicted to result in coding exon disruption. In contrast, biallelic deletions in individuals with ASD were enriched for overlap with regulatory regions, with 23/28 CNVs disrupting histone peaks in ENCODE (p < 0.009). Overlap with regulatory regions was further demonstrated by comparisons to the 127-epigenome dataset released by the Roadmap Epigenomics project, with enrichment for enhancers found in primary brain tissue and neuronal progenitor cells. Our results suggest a novel noncoding mechanism of ASD, describe a powerful method to identify important noncoding regions in the human genome, and emphasize the potential significance of gene activation and regulation in cognitive and social function.

Adjuvants can enhance vaccine immunogenicity, but their mechanism of action is often incompletely understood, hampering rapid applicability for pandemic vaccines. Herein, we characterized the cellular and molecular activity of adjuvant formulations available for pre-clinical evaluation, including several developed for global open access. We applied four complementary human in vitro platforms to assess individual and combined adjuvants in unformulated, oil-in-water, and liposomal delivery platforms. Liposomal co-formulation of MPLA and QS-21 was most potent in promoting dendritic cell maturation, selective production of Th1-polarizing cytokines, and activation of SARS-CoV-2 Spike-specific CD4+ and CD8+ T cells in a co-culture assay. Select formulations also significantly enhanced Spike antigen-specific humoral immunity in vivo. This study confirms the utility of the cumulative use of human in vitro tools to predict adjuvanticity potential. Thus, human in vitro modeling may advance public health by accelerating the development of affordable and scalable adjuvants for vaccines tailored to vulnerable populations.