Kerrie J. Sandgren

Vaccine priming is restricted to draining lymph nodes and controlled by adjuvant-mediated antigen uptake

Vaccine responses are initiated in vaccine-draining lymph nodes and depend on the efficiency by which adjuvants influence antigen uptake. Moving beyond mice for vaccine studies Vaccine enhancement by adjuvants has been known for decades, but the mechanistic differences in how specific adjuvants influence the immune response are just beginning to be elucidated. Liang et al. sought a model that closely mimics humans, so they intramuscularly immunized nonhuman primates with a prototypical HIV antigen in combination with various adjuvants. They then inspected the muscles and lymph nodes to characterize antigen-presenting cells and resulting adaptive immune responses. Their findings should provide valuable information on adjuvant selection for vaccine development in humans. The innate immune mechanisms by which adjuvants enhance the potency and protection of vaccine-induced adaptive immunity are largely unknown. We introduce a model to delineate the steps of how adjuvant-driven innate immune activation leads to priming of vaccine responses using rhesus macaques. Fluorescently labeled HIV-1 envelope glycoprotein (Env) was administered together with the conventional aluminum salt (alum) adjuvant. This was compared to Env given with alum with preabsorbed Toll-like receptor 7 (TLR7) ligand (alum-TLR7) or the emulsion MF59 because they show superiority over alum for qualitatively and quantitatively improved vaccine responses. All adjuvants induced rapid and robust immune cell infiltration to the injection site in the muscle. This resulted in substantial uptake of Env by neutrophils, monocytes, and myeloid and plasmacytoid dendritic cells (DCs) and migration exclusively to the vaccine-draining lymph nodes (LNs). Although less proficient than monocytes and DCs, neutrophils were capable of presenting Env to memory CD4+ T cells. MF59 and alum-TLR7 showed more pronounced cell activation and overall higher numbers of Env+ cells compared to alum. This resulted in priming of higher numbers of Env-specific CD4+ T cells in the vaccine-draining LNs, which directly correlated with increased T follicular helper cell differentiation and germinal center formation. Thus, strong innate immune activation promoting efficient vaccine antigen delivery to infiltrating antigen-presenting cells in draining LNs is an important mechanism by which superior adjuvants enhance vaccine responses.

IFN-α produced by human plasmacytoid dendritic cells enhances T cell-dependent naïve B cell differentiation.

The development and quality of a humoral immune response are largely influenced by the environment that supports the activation of naïve B cells. Human PDCs, through their unique capacity to produce high levels of IFN‐α, have been shown earlier to enhance B cell responses stimulated by selected TLR ligands. In this study, we investigated whether PDCs also promote B cell activation induced by Th cell interactions and BCR ligation. Sorted human naive CD19+ CD27– B cells were activated in vitro with anti‐Ig and irradiated CD4+ T cells. Under these conditions, the presence of supernatants from TLR‐stimulated PDCs increased B cell proliferation, the frequency of B cells that differentiated to CD27high CD38high cells, and secretion of IgM. Similar results were observed when the B cells were activated in the presence of purified IFN‐α. In contrast, supernatants from stimulated MDCs did not augment these functions. Also, IFN‐α treatment of B cells up‐regulated the expression of costimulatory molecule CD86 but not CD40, CD80, MHC class II, or CD25. Although direct IFN‐α exposure of T cells suppressed their proliferative capacity, IFN‐α treatment of B cells led to a small increase in their capacity to induce superantigen‐driven activation of autologous CD4+ T cells. In summary, PDCs, via their production of IFN‐α, may render B cells more responsive to T cell contact, which in turn, facilitates B cell proliferation and differentiation to antibody‐producing cells.

Techniques for time-efficient isolation of human skin dendritic cell subsets and assessment of their antigen uptake capacity.

Dendritic cells (DCs) residing in skin are important sentinels for foreign antigens. Methods to facilitate studies of subsets of skin DCs are important to increase the understanding of various pathogens, allergens, topical treatments or vaccine components targeting the skin. In this study, we developed a new DC purification method using a skin graft mesher, clinically used for expansion of skin grafts, to accelerate processing of skin into nets that allowed efficient enzymatic disruption and single cell isolation. The reduction in processing time using the skin graft mesher enabled processing of larger skin samples and also limited the ex vivo handling of the specimens which is associated with maturation of DCs. In addition, a skin explant model to functionally monitor early events of antigen uptake by DC subsets in situ was developed. DCs isolated from epidermis represented a uniform CD1a(+) HLA-DR(+) CD11c(+) Langerin(+) DC-SIGN(-) DC-LAMP(int) DEC-205(int) Langerhans cell (LC) population whereas three subtypes of HLA-DR(+) CD11c(+) DCs were isolated from dermis based on their varying expression of CD1a. Epidermal LCs showed a significantly higher antigen uptake capacity of fluorescently-labelled ovalbumin (OVA) and dextran as compared to any of the dermal DC (dDC) subsets. In contrast, injection of antigen directly into skin explants followed by in situ imaging revealed that the majority of DCs with internalized antigen were localized in the dermis, likely as a consequence of the anatomical site for antigen delivery. These methods offer potency for various applications addressing antigen uptake, microbial DC interactions or other antigenic stimulation targeting the skin and can enhance our knowledge of basic DC biology in human skin.

Human Anti-CD40 Antibody and Poly IC:LC Adjuvant Combination Induces Potent T Cell Responses in the Lung of Nonhuman Primates

Nonlive vaccine platforms that induce potent cellular immune responses in mucosal tissue would have broad application for vaccines against infectious diseases and tumors. Induction of cellular immunity could be optimized by targeted activation of multiple innate and costimulatory signaling pathways, such as CD40 or TLRs. In this study, we evaluated immune activation and elicitation of T cell responses in nonhuman primates after immunization with peptide Ags adjuvanted with an agonistic anti-CD40Ab, with or without the TLR3 ligand poly IC:LC. We found that i.v. administration of the anti-CD40Ab induced rapid and transient innate activation characterized by IL-12 production and upregulated costimulatory and lymph node homing molecules on dendritic cells. Using fluorescently labeled Abs for in vivo tracking, we found that the anti-CD40Ab bound to all leukocytes, except T cells, and disseminated to multiple organs. CD4+ and CD8+ T cell responses were significantly enhanced when the anti-CD40Ab was coadministered with poly IC:LC compared with either adjuvant given alone and were almost exclusively compartmentalized to the lung. Notably, Ag-specific T cells in the bronchoalveolar lavage were sustained at ∼5–10%. These data indicate that systemic administration of anti-CD40Ab may be particularly advantageous for vaccines and/or therapies that require T cell immunity in the lung.

Human Plasmacytoid Dendritic Cells Efficiently Capture HIV-1 Envelope Glycoproteins via CD4 for Antigen Presentation

Advances in HIV-1 vaccine clinical trials and preclinical research indicate that the virus envelope glycoproteins (Env) are likely to be an essential component of a prophylactic vaccine. Efficient Ag uptake and presentation by dendritic cells (DCs) is important for strong CD4+ Th cell responses and the development of effective humoral immune responses. In this study, we examined the capacity of distinct primary human DC subsets to internalize and present recombinant Env to CD4+ T cells. Consistent with their specific receptor expression, skin DCs bound and internalized Env via C-type lectin receptors, whereas blood DC subsets, including CD1c+ myeloid DCs, CD123+ plasmacytoid DCs (PDCs), and CD141+ DCs exhibited a restricted repertoire of C-type lectin receptors and relied on CD4 for uptake of Env. Despite a generally poor capacity for Ag uptake compared with myeloid DCs, the high expression of CD4 on PDCs allowed them to bind and internalize Env very efficiently. CD4-mediated uptake delivered Env to EEA1+ endosomes that progressed to Lamp1+ and MHC class II+ lysosomes where internalized Env was degraded rapidly. Finally, all three blood DC subsets were able to internalize an Env-CMV pp65 fusion protein via CD4 and stimulate pp65-specific CD4+ T cells. Thus, in the in vitro systems described in this paper, CD4-mediated uptake of Env is a functional pathway leading to Ag presentation, and this may therefore be a mechanism used by blood DCs, including PDCs, for generating immune responses to Env-based vaccines.

Plasmacytoid dendritic cells infiltrate the skin in positive tuberculin skin test indurations

Plasmacytoid dendritic cells (pDCs) are rarely present in normal skin but have been shown to infiltrate lesions of infections or autoimmune disorders. Here, we report that several DC subsets including CD123(+) BDCA-2/CD303(+) pDCs accumulate in the dermis in indurations induced by the tuberculin skin test (TST), used to screen immune sensitization by Mycobacterium tuberculosis. Although the purified protein derivate (PPD) used in the TST did not itself induce pDC recruitment or IFN-α production, the positive skin reactions showed high expression of the IFN-α-inducible protein MxA. In contrast, the local immune response to PPD was associated with substantial cell death and high expression of the cationic antimicrobial peptide LL37, which together can provide a means for pDC activation and IFN-α production. In vitro, pDCs showed low uptake of PPD compared with CD11c(+) and BDCA-3/CD141(+) myeloid DC subsets. Furthermore, supernatants from pDCs activated with LL37-DNA complexes reduced the high PPD uptake in myeloid DCs, as well as decreased their capacity to activate T-cell proliferation. Infiltrating pDCs in the TST reaction site may thus have a regulatory effect upon the antigen processing and presentation functions of surrounding potent myeloid DC subsets to limit potentially detrimental and excessive immune stimulation.

Design and evaluation of surface and adjuvant modified PLGA microspheres for uptake by dendritic cells to improve vaccine responses.

Designing strategies for targeting antigens to dendritic cells is a major goal in vaccinology. Here, PLGA (poly lactic-co-glycolic acid) microspheres and with several surface modifications that affect to their uptake by human blood primary dendritic cells and monocytes have been evaluated. Higher uptake was found by all the cell types when cationic microspheres (PLGA modified with polyethylene imine) were used. These cationic particles were in vivo evaluated in mice. In addition, MPLA(1) or poly(I:C)(2) and α-GalCer(3) were also encapsulated to address their adjuvant effect. All the microspheres were able to produce humoral immune responses, albeit they were higher for cationic microspheres. Moreover, surface charge seemed to have a role on biasing the immune response; cationic microspheres induced higher IFN-γ levels, indicative of Th1 activation, while unmodified ones mainly triggered IL4 and IL17A release, showing Th2 activation. Thus, we have shown here the potential and versatility of these MS, which may be tailored to needs.

Dendritic cell recruitment in response to skin antigen tests in HIV-1 infected individuals correlates with the level of T cell infiltration

Objectives:To study whether in-vivo recruitment of dendritic cells in response to antigen administration in the skin is altered during HIV-1 infection. Design:Skin punch biopsies were collected from HIV-1-positive as well as seronegative individuals at 48 h after intradermal injection of inactivated antigens of mumps virus, Candida albicans, or purified protein derivate (PPD) from Mycobacterium tuberculosis. Methods:Cryosections were analyzed by in-situ staining and computerized imaging. Results:Control skin biopsies showed that there was no difference in the number of skin-resident dendritic cells between seronegative and HIV-1-positive individuals. Antigen injection resulted in substantial infiltration of dendritic cells compared to the frequencies found in donor-matched control skin. In HIV-1-positive individuals, CD123+/CD303+ plasmacytoid dendritic cells and CD11c+ myeloid dendritic cells, including the CD141+ cross-presenting subset, were recruited at lower levels compared to healthy controls in response to PPD and mumps but not C. albicans. The level of dendritic cell recruitment correlated with the frequencies of T cells infiltrating the respective antigen sites. Ki67+ cycling T cells at the injection sites were much more frequent in response to each of the antigens in the HIV-1-positive individuals, including those with AIDS, compared to healthy controls. Conclusion:Multiple dendritic cell subsets infiltrate the dermis in response to antigen exposure. There was no obvious depletion or deficiency in mobilization of dendritic cells in response to antigen skin tests during chronic HIV-1 infection. Instead, the levels of antigen-specific memory T cells that accumulate at the antigen site may determine the level of dendritic cell infiltration.

Infiltration of dendritic cells and antigen uptake in the muscle after injection of HIV-1 Env gp120 in adjuvant.

Background Most vaccines are delivered into the muscle although it contains very few potent antigen presenting cells, such as dendritic cells (DCs) that are critical for driving adaptive immune responses. Understanding the early mechanisms that dictate vaccine responses and why some adjuvants like the oil-in-water emulsion MF59 are shown to be more potent than alum is important for the design of new vaccines. Here, we investigated the recruitment of immune cells to the vaccine injection site and uptake of a clinically relevant HIV-1 envelope glycoprotein (Env) and MF59 in a non-human primate (NHP) model.

CTI special feature on innate immune responses and vaccine design

The challenge of developing next generation vaccines against some of the most burdensome diseases—both infectious and inflammatory, will require an injection of basic knowledge as well as scientific innovation. As highlighted in this collection of expert reviews, the innate immune response is a critical stepping-stone between vaccination and the development of effective adaptive immunity, triggering and shaping the ensuing cellular and humoral responses that will ultimately control the disease. An enhanced understanding of how innate immunity develops in different disease states will help inform the design and formulation of new vaccine components. Conversely, an increased understanding of the mechanism of action of novel adjuvants will aid in their rational selection for different diseases and delivery platforms. In this Special Feature of Clinical and Translational Immunology, we present a number of papers that jointly highlight the interplay between innate immune responses and the resulting functional vaccine responses.