Paula M. Chilton

The Vaccine Adjuvant Monophosphoryl Lipid A as a TRIF-Biased Agonist of TLR4

The inflammatory toxicity of lipopolysaccharide (LPS), a component of bacterial cell walls, is driven by the adaptor proteins myeloid differentiation factor 88 (MyD88) and Toll-interleukin 1 receptor domain–containing adapter inducing interferon-β (TRIF), which together mediate signaling by the endotoxin receptor Toll-like receptor 4 (TLR4). Monophosphoryl lipid A (MPLA) is a low-toxicity derivative of LPS with useful immunostimulatory properties, which is nearing regulatory approval for use as a human vaccine adjuvant. We report here that, in mice, the low toxicity of MPLAs adjuvant function is associated with a bias toward TRIF signaling, which we suggest is likely caused by the active suppression, rather than passive loss, of proinflammatory activity of this LPS derivative. This finding may have important implications for the development of future vaccine adjuvants.

Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment

Bone marrow transplantation offers great promise for treating a number of disease states. However, the widespread application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FCs) have been shown to enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. In the present studies, we have identified the main subpopulation of FCs as plasmacytoid precursor dendritic cells (p-preDCs). FCs and p-preDCs share many phenotypic, morphological, and functional features: both produce IFN-α and TNF-α, both are activated by toll-like receptor (TLR)-9 ligand (CpG ODN) stimulation, and both expand and mature after Flt3 ligand (FL) treatment. FL-mobilized FCs, most of which express a preDC phenotype, significantly enhance engraftment of HSCs and induce donor-specific tolerance to skin allografts. However, p-preDCs alone or p-preDCs from the FC population facilitate HSC engraftment less efficiently than total FCs. Moreover, FCs depleted of preDCs completely fail to facilitate HSC engraftment. These results are the first to define a direct functional role for p-preDCs in HSC engraftment, and also suggest that p-preDCs need to be in a certain state of maturation/activation to be fully functional.

The low‐toxicity versions of LPS, MPL® adjuvant and RC529, are efficient adjuvants for CD4+ T cells

Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less‐toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long‐term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon‐γ following a 21‐day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long‐term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low‐inflammation adjuvants, such as MPL and RC529, are capable of enhancing short‐term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long‐term retention of antigen‐specific T cells.

Soluble Cytokine Receptors: Their Roles in Immunoregulation, Disease, and Therapy

Publisher Summary This chapter discusses the mechanisms that regulate cytokine activity and the generation and function of soluble cytokine receptors (sCR) as cytokine antagonists and/or cytokine carriers in vivo and also reviewed, in a more specific manner, information about the different sCR, their potential involvement in diseases, and their use as immunotherapeutic agents. The generation of soluble forms of cytokine receptors appears to be a general mechanism contributing to the regulation of cytokine activity in vivo . The soluble receptor forms are normally generated by the proteolytic cleavage of the extracellular domain of the membrane-bound receptor or by translation from alternatively spliced mRNAs, different from those encoding the membrane-bound receptors. Elucidation of the relationships of the different sCR with the cytokine networks, both under normal conditions and after immunologic stimulation, is necessary in order to fully understand their role in the regulation of the immune system. Such knowledge should allow using sCR more efficiently to modify cytokine activity in vivo for therapeutic purposes. Besides their putative immunoregulatory function, the ability of sCR to specifically alter the biologic activity of their ligand, coupled to a lack of immunogenicity, has generated considerable interest in potential therapeutic applications, particularly in clinical conditions in which cytokines play a prominent pathophysiological role such as inflammatory and autoimmune diseases.

TNF-α Is Critical to Facilitate Hemopoietic Stem Cell Engraftment and Function

The use of tolerogenic cells as an approach to induce tolerance to solid organ allografts is being aggressively pursued. A major limitation to the clinical application of cell-based therapies has been the ability to obtain sufficient numbers and also preserve their tolerogenic state. We previously reported that small numbers of bone marrow-derived CD8+/TCR− graft facilitating cells (FC) significantly enhance hemopoietic stem cell (HSC) engraftment in allogeneic and syngeneic recipients. Although the majority of FC resemble precursor plasmacytoid dendritic cells (p-preDC), p-preDC do not replace FC in facilitating function. In the present studies, we investigated the mechanism of FC function. We show for the first time that FC significantly enhance HSC clonogenicity, increase the proportion of multipotent progenitors, and prevent apoptosis of HSC. These effects require direct cell:cell contact between FC and HSC. Separation of FC from HSC by transwell membranes completely abrogates the FC effect on HSC. p-preDC FC do not replace FC total in these effects on HSC function. FC produce TNF-α, and FC from TNF-α-deficient mice exhibit impaired facilitation in vivo and loss of the in vitro effects on HSC. Neutralizing TNF-α in FC similarly blocks the FC effect. The antiapoptotic effect of FC is associated with up-regulation of Bcl-3 transcripts in HSC and blocking of TNF-α is associated with abrogation of up-regulation of Bcl-3 transcripts. These data demonstrate a critical role for TNF-α in mediating FC function. FC may have a significant impact upon the safe use of chimerism to establish tolerance to transplanted organs and tissue.

Production of Donor T Cells Is Critical for Induction of Donor-Specific Tolerance and Maintenance of Chimerism

Nonmyeloablative conditioning has significantly reduced the morbidity associated with bone marrow transplantation. The donor hemopoietic cell lineage(s) responsible for the induction and maintenance of tolerance in nonmyeloablatively conditioned recipients is not defined. In the present studies we evaluated which hemopoietic stem cell-derived components are critical to the induction of tolerance in a total body irradiation-based model. Recipient B10 mice were pretreated with mAbs and transplanted with allogeneic B10.BR bone marrow after conditioning with 100–300 cGy total body irradiation. The proportion of recipients engrafting increased in a dose-dependent fashion. All chimeric recipients exhibited multilineage donor cell production. However, induction of tolerance correlated strictly with early production of donor T cells. The chimeras without donor T cells rejected donor skin grafts and demonstrated strong antidonor reactivity in vitro, while possessing high levels of donor chimerism. These animals lost chimerism within 8 mo. Differentiation into T cells was aborted at a prethymic stage in recipients that did not produce donor T cells. Moreover, donor Ag-driven clonal deletion of recipient T cells occurred only in chimeras with donor T cells. These results demonstrate that donor T cell production is critical in the induction of transplantation tolerance and the maintenance of durable chimerism. In addition, donor T cell production directly correlates with the deletion of potentially alloreactive cells.

Impaired Bcl3 Up-regulation Leads to Enhanced Lipopolysaccharide-induced Interleukin (IL)-23P19 Gene Expression in IL-10–/– Mice

Genetic and biochemical analyses show that IL-23p19 plays a central role in mediating bacteria-induced colitis in interleukin-10-deficient (IL-10–/–) mice. The molecular mechanisms responsible for the dysregulated innate host response leading to enhanced IL-23 gene expression in IL-10–/– mice are poorly understood. In this study, we investigated the role of Bcl3 in controlling LPS-induced IL-23p19 gene expression in bone marrow-derived dendritic cells (BMDC) isolated from IL-10–/– mice. We report higher IL-23p19 mRNA accumulation and protein secretion in LPS-stimulated BMDC isolated from IL-10–/– compared with WT mice. Lipopolysaccharide (LPS)-induced B cell leukemia 3 (Bcl3) expression was strongly impaired (90% decrease) in IL-10–/– BMDC compared with WT BMDC. Chromatin immunoprecipitation demonstrated enhanced RelA binding to the IL-23p19 promoter in IL-10–/– compared with WT BMDC. Bcl3 overexpression decreased LPS-induced IL-23p19 gene expression in IL-10–/– BMDC, which correlated with enhanced NF-κB p50 binding and decreased RelA binding to the gene promoter. Conversely, Bcl3 knockdown enhanced LPS-induced IL-23p19 gene expression in WT BMDC. Moreover, LPS-induced IL-23p19 gene expression was significantly enhanced in Bcl3–/– BMDC compared with WT BMDC. In conclusion, enhanced LPS-induced IL-23p19 gene expression in IL-10–/– mice is due to impaired Bcl3 expression leading to diminished p50 and enhanced RelA recruitment to the IL-23p19 promoter.

TRIF Is Required for TLR4 Mediated Adjuvant Effects on T Cell Clonal Expansion

Toll like receptor 4 (TLR4) is an important pattern recognition receptor with the ability to drive potent innate immune responses and also to modulate adaptive immune responses needed for long term protection. Activation of TLR4 by its ligands is mediated by engagement of the adapter proteins MyD88 (myeloid differentiation factor 88) and TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta). Previously, we showed that TRIF, but not MyD88, plays an important role in allowing TLR4 agonists to adjuvant early T cell responses. In this study, we investigated the T cell priming events that are regulated specifically by the TRIF signaling branch of TLR4. We found that TRIF deficiency prevented the TLR4 agonist lipid A from enhancing T cell proliferation and survival in an adoptive transfer model of T cell priming. TRIF deficient DC showed defective maturation as evidenced by their failure to upregulate co-stimulatory molecules in response to lipid A stimulation. Importantly, TRIF alone caused CD86 and CD40 upregulation on splenic DC, but both TRIF and MyD88 were required for CD80 upregulation. The impairment of T cell adjuvant effects and defective DC maturation in TRIF lps/lps mice after TLR4 stimulation was mainly due to loss of type I IFN production, indicating that type I interferons are central to TLR4s adjuvant effects. These results are useful for the continued development of TLR4 based vaccine adjuvants that avoid inflammatory risks while retaining beneficial immune response.

CD45 congenic bone marrow transplantation: evidence for T cell-mediated immunity.

CD45 congenic mice have been used to study stem cell engraftment in the absence of alloreactivity. Recently, impaired engraftment was reported in this model and attributed to weak immune reactivity. We have confirmed that there is indeed low‐level reactivity mediated by CD8+ cells and αβ‐TCR+ T cells. B6 (CD45.2) recipients were conditioned with total body irradiation (TBI) and transplanted with increasing doses of B6 (CD45.1) bone marrow cells (BMCs). Although chimerism was present at 1 month in all recipients, durable engraftment did not occur with <150 cGy of TBI, emphasizing the importance of long‐term follow‐up in evaluating nonmyeloablative conditioning approaches. A single dose of cyclophosphamide on day 2 also significantly enhanced engraftment. When B6 TCRβ−/−, TCRδ−/−, or TCRβ−/−/δ−/− (CD45.2) mice were transplanted with CD45.1 bone marrow, significantly enhanced engraftment occurred in recipients lacking αβ‐TCR+ T cells (p < .00005). Similarly, removal of αβ‐TCR+ host T cells in wild‐type recipients resulted in enhanced engraftment. Engraftment was also significantly increased in CD8−/− and CD4−/−/8−/− recipients (p < .0005). Taken together, these results demonstrate that αβ‐TCR+ and CD8+ T cells play a critical role in regulating engraftment of CD45 congenic marrow and suggest that these cells are the main effector cells in low‐level alloreactivity to the CD45 disparity.

Type I interferon signaling contributes to the bias that Toll-like receptor 4 exhibits for signaling mediated by the adaptor protein TRIF

The selective use by Toll-like receptor 4 of an adaptor protein may aid in the development of safer vaccine adjuvants. Toward Better Vaccine Adjuvants The increasing use of vaccines based on components of pathogens rather than on whole pathogens requires the development of vaccine adjuvants to boost immune responses. Toll-like receptor 4 (TLR4) is a pattern-recognition receptor that stimulates immune signaling pathways through two adaptor proteins: MyD88 and TRIF. Whereas MyD88-dependent signaling leads to potentially harmful inflammatory responses, TRIF-dependent signaling leads to immunostimulatory responses. Current adjuvant strategies are based on the idea that the structures of TLR4 agonists determine which adaptor protein the receptor uses. However, Kolb et al. found that TLR4 signaling is inherently biased toward the TRIF-dependent pathway. TRIF-dependent bias of a vaccine adjuvant occurred with concentrations sufficient for TRIF, but limiting for MyD88, signaling. Without type I interferon signaling, TLR4 required higher concentrations of agonist, thereby equivalently stimulating both TRIF- and MyD88-dependent pathways. These data may aid in the development of more effective vaccine adjuvants that enhance the immune response without triggering undesirable inflammatory reactions. Signaling by Toll-like receptor 4 (TLR4) is mediated by either of two adaptor proteins: myeloid differentiation marker 88 (MyD88) or Toll–interleukin-1 (IL-1) receptor (TIR) domain–containing adaptor inducing interferon-β (TRIF). Whereas MyD88-mediated signaling leads to proinflammatory responses, TRIF-mediated signaling leads to less toxic immunostimulatory responses that are beneficial in boosting vaccine responses. The hypothesis that monophosphorylated lipid A structures act as TRIF-biased agonists of TLR4 offered a potential mechanism to explain their clinical value as vaccine adjuvants, but studies of TRIF-biased agonists have been contradictory. In experiments with mouse dendritic cells, we found that irrespective of the agonist used, TLR4 functioned as a TRIF-biased signaling system through a mechanism that depended on the autocrine and paracrine effects of type I interferons. The TLR4 agonist synthetic lipid A induced expression of TRIF-dependent genes at lower concentrations than were necessary to induce the expression of genes that depend on MyD88-mediated signaling. Blockade of type I interferon signaling selectively decreased the potency of lipid A (increased the concentration required) in inducing the expression of TRIF-dependent genes, thereby eliminating adaptor bias. These data may explain how high-potency TLR4 agonists can act as clinically useful vaccine adjuvants by selectively activating TRIF-dependent signaling events required for immunostimulation, without or only weakly activating potentially harmful MyD88-dependent inflammatory responses.