Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system

TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4+ T cell response, while only R848 and Poly I:C induced CD8+ cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans

DC Subsets Regulate Humoral Immune Responses by Supporting the Differentiation of Distinct Tfh Cells

To determine the contribution of skin DC subsets in the regulation of humoral immunity, we used a well-characterized antigen targeting system to limit antigen availability and presentation to certain skin-derived DC subsets. Here we show that delivery of foreign antigen to steady state Langerhans cells (LCs) and cDC1s through the same receptor (Langerin) led to, respectively, robust vs. minimal-to-null humoral immune response. LCs, unlike cDC1s, supported the formation of germinal center T follicular helper cells (GC-Tfh) antigen dose-dependently and then, likely licensed by these T cells, some of the LCs migrated to the B cell area to initiate B cell responses. Furthermore, we found that the cDC1s, probably through their superior T cell activation capacity, prevented the LCs from inducing GC-Tfh cells and humoral immune responses. We further show that targeted delivery of cytokines to DCs can be used to modulate DC-induced humoral immune responses, which has important therapeutic potential. Finally, we show that human LCs, unlike monocyte-derived DCs, can support GC Tfh generation in an in vitro autologous system; and in agreement with mouse data, we provide evidence in NHP studies that targeting LCs without adjuvants is an effective way to induce antibody responses, but does not trigger CD8+ T cell responses. Our findings suggest that the major limitations of some relatively ineffective vaccines currently in use or in development might be that (1) they are not formulated to specifically target a certain subset of DCs and/or (2) the antigen dose is not tailored to maximize the intrinsic/pre-programmed capabilities of the specific DC subset. This new and substantial departure from the status quo is expected to overcome problems that have hindered our ability to generate effective vaccines against some key pathogens

Human thymic stromal lymphopoietin preferentially stimulates myeloid cells

The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R alpha-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Ralpha are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c(+) dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells

Human Dendritic Cells Induce the Differentiation of Interleukin-21-Producing T Follicular Helper-like Cells through Interleukin-12

SummaryT follicular helper (Tfh) cells help development of antibody responses via interleukin-21 (IL-21). Here we show that activated human dendritic cells (DCs) induced naive CD4+ T cells to become IL-21-producing Tfh-like cells through IL-12. CD4+ T cells primed with IL-12 induced B cells to produce immunoglobulins in a fashion dependent on IL-21 and inducible costimulator (ICOS), thus sharing fundamental characteristics with Tfh cells. The induction of Tfh-like cells by activated DCs was inhibited by neutralizing IL-12. IL-12 induced two different IL-21-producers: IL-21+IFN-γ+T-bet+ Th1 cells and IL-21+IFN-γ−T-bet− non-Th1 cells, in a manner dependent on signal transducer and activator of transcription 4 (STAT4). IL-12 also regulated IL-21 secretion by memory CD4+ T cells. Thus, IL-12 produced by activated DCs regulates antibody responses via developing IL-21-producing Tfh-like cells and inducing IL-21 secretion from memory CD4+ T cells. These data suggest that the developmental pathway of Tfh cells differs between mice and humans, which have considerable implications for vaccine development

Skin-Resident Murine Dendritic Cell Subsets Promote Distinct and Opposing Antigen-Specific T Helper Cell Responses

SummarySkin-resident dendritic cells (DCs) are well positioned to encounter cutaneous pathogens and are required for the initiation of adaptive immune responses. There are at least three subsets of skin DC— Langerhans cells (LC), Langerin+ dermal DCs (dDCs), and classic dDCs. Whether these subsets have distinct or redundant function in vivo is poorly understood. Using a Candida albicans skin infection model, we have shown that direct presentation of antigen by LC is necessary and sufficient for the generation of antigen-specific T helper-17 (Th17) cells but not for the generation of cytotoxic lymphocytes (CTLs). In contrast, Langerin+ dDCs are required for the generation of antigen specific CTL and Th1 cells. Langerin+ dDCs also inhibited the ability of LCs and classic DCs to promote Th17 cell responses. This work demonstrates that skin-resident DC subsets promote distinct and opposing antigen-specific responses

Candida albicans morphology and dendritic cell subsets determine T helper cell differentiation

SummaryCandida albicans is a dimorphic fungus responsible for chronic mucocutaneous and systemic infections. Mucocutaneous immunity to C. albicans requires T helper 17 (Th17) cell differentiation that is thought to depend on recognition of filamentous C. albicans. Systemic immunity is considered T cell independent. Using a murine skin infection model, we compared T helper cell responses to yeast and filamentous C. albicans. We found that only yeast induced Th17 cell responses through a mechanism that required Dectin-1-mediated expression of interleukin-6 (IL-6) by Langerhans cells. Filamentous forms induced Th1 without Th17 cell responses due to the absence of Dectin-1 ligation. Notably, Th17 cell responses provided protection against cutaneous infection while Th1 cell responses provided protection against systemic infection. Thus, C. albicans morphology drives distinct T helper cell responses that provide tissue-specific protection. These findings provide insight into compartmentalization of Th cell responses and C. albicans pathogenesis and have critical implications for vaccine strategies

Immunologic Characterization of a Rhesus Macaque H1N1 Challenge Model for Candidate Influenza Virus Vaccine Assessment

Despite the availability of annually formulated vaccines, influenza virus infection remains a worldwide public health burden. Therefore, it is important to develop preclinical challenge models that enable the evaluation of vaccine candidates while elucidating mechanisms of protection. Here, we report that naive rhesus macaques challenged with 2009 pandemic H1N1 (pH1N1) influenza virus do not develop observable clinical symptoms of disease but develop a subclinical biphasic fever on days 1 and 5 to 6 postchallenge. Whole blood microarray analysis further revealed that interferon activity was associated with fever. We then tested whether type I interferon activity in the blood is a correlate of vaccine efficacy. The animals immunized with candidate vaccines carrying hemagglutinin (HA) or nucleoprotein (NP) exhibited significantly reduced interferon activity on days 5 to 6 postchallenge. Supported by cellular and serological data, we conclude that blood interferon activity is a prominent marker that provides a convenient metric of influenza virus vaccine efficacy in the subclinical rhesus macaque model