Carolyn R. Casella

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.

Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant

Abstract.The development of non-infectious subunit vaccines greatly increases the safety of prophylactic immunization, but also reinforces the need for a new generation of immunostimulatory adjuvants. Because adverse effects are a paramount concern in prophylactic immunization, few new adjuvants have received approval for use anywhere in the developed world. The vaccine adjuvant monophosphoryl lipid A is a detoxified form of the endotoxin lipopolysaccharide, and is among the first of a new generation of Toll-like receptor agonists likely to be used as vaccine adjuvants on a mass scale in human populations. Much remains to be learned about this compound’s mechanism of action, but recent developments have made clear that it is unlikely to be simply a weak version of lipopolysaccharide. Instead, monophosphoryl lipid A’s structure seems to have fortuitously retained several functions needed for stimulation of adaptive immune responses, while shedding those associated with pro-inflammatory side effects.

Selective Activation of the p38 MAPK Pathway by Synthetic Monophosphoryl Lipid A

TLR4 stimulation by lipopolysaccharide can cause both MAL/MyD88- and TRAM/TRIF (Toll IL-1 receptor domain-containing adaptor-inducing IFNβ)-dependent signaling events. Monophosphoryl lipid A (MPLA), a low toxicity derivative of endotoxic lipopolysaccharide, enhances antibody responses, T cell expansion, and recall responses against antigens without causing excessive inflammatory side effects. Previously, we proposed that TRIF-biased activation of TLR4 by MPLA is responsible for its reduced toxicity while retaining potent adjuvant effects. However, some TRIF-associated genes, such as MCP-1, are only weakly expressed, and some MyD88-associated inflammatory and anti-inflammatory cytokines, such as tumor necrosis factor α and interleukin-10, are strongly activated after MPLA stimulation despite weak NF-κB but strong IRF3 activation. We now report that synthetic derivatives of MPLA retained TRIF bias as compared with synthetic diphosphoryl lipid A, indicating a change in a single phosphoryl group is sufficient for TRIF-biased TLR4 stimulation. We extend our previous observations by showing that sMLA induces strong p38 MAPK but weak JNK activation, resulting in high IP-10 (interferon-inducible protein 10), tumor necrosis factor α, and interleukin-10 but low MCP-1 transcript levels. Results of this study identify a novel biochemical mechanism for regulation of sMLA-induced gene expression.

Mechanisms of lymphocyte killing by HIV.

One of the remaining mysteries of HIV infection is what causes the destruction of the CD4+ T cells. Several reports in the past year have linked viral load to disease progression, strengthening the conclusion that the virus is responsible for CD4+ T‐cell depletion. We discuss several possible mechanisms of T‐cell death, including the killing of uninfected CD4+ T cells. We also discuss the possibility that the virus protects the cell it infects at least until viral replication can be completed.

MyD88-Dependent SHIP1 Regulates Proinflammatory Signaling Pathways in Dendritic Cells after Monophosphoryl Lipid A Stimulation of TLR4

We previously showed that monophosphoryl lipid A (MLA) activates TLR4 in dendritic cells (DCs) in a Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF)–biased manner: MLA produced from Salmonella minnesota Re595 induced signaling events and expression of gene products that were primarily TRIF dependent, whereas MyD88-dependent signaling was impaired. Moreover, when tested in TRIF-intact/MyD88-deficient DCs, synthetic MLA of the Escherichia coli chemotype (sMLA) showed the same activity as its diphosphoryl, inflammatory counterpart (synthetic diphosphoryl lipid A), indicating that TRIF-mediated signaling is fully induced by sMLA. Unexpectedly, we found that the transcript level of one proinflammatory cytokine was increased in sMLA-treated cells by MyD88 deficiency to the higher level induced by synthetic diphosphoryl lipid A, which suggested MyD88 may paradoxically help restrain proinflammatory signaling by TRIF-biased sMLA. In this article, we demonstrate that sMLA induces MyD88 recruitment to TLR4 and activates the anti-inflammatory lipid phosphatase SHIP1 in an MyD88-dependent manner. At the same time, MyD88-dependent signaling activity at the level of IL-1R–associated kinase 1 is markedly reduced. Increased SHIP1 activity is associated with reductions in sMLA-induced IκB kinase α/β and IFN regulatory factor 3 activation and with restrained expression of their downstream targets, endothelin-1 and IFN-β, respectively. Results of this study identify a pattern that is desirable in the context of vaccine adjuvant design: TRIF-biased sMLA can stimulate partial MyD88 activity, with MyD88-dependent SHIP1 helping to reduce proinflammatory signaling in DCs.

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.

Unrestrained Glycogen Synthase Kinase-3β Activity Leads to Activated T Cell Death and Can Be Inhibited by Natural Adjuvant

Activated T cell death (ATCD) after peak clonal expansion is required for effective homeostasis of the immune system. Using a mouse model of T cell clonal expansion and contraction, we found that regulation of the proapoptotic kinase glycogen synthase kinase (GSK)-3β plays a decisive role in determining the extent to which T cells are eliminated after activation. Involvement of GSK-3β in ATCD was tested by measuring T cell survival after GSK-3β inhibition, either ex vivo with chemical and pharmacological inhibitors or in vivo by retroviral expression of a dominant-negative form of GSK-3. We also measured amounts of inactivating phosphorylation of GSK-3β (Ser9) in T cells primed in the presence or absence of LPS. Our results show that GSK-3β activity is required for ATCD and that its inhibition promoted T cell survival. Adjuvant treatment in vivo maintained GSK-3β (Ser9) phosphorylation in activated T cells, whereas with adjuvant-free stimulation it peaked and then decayed as the cells became susceptible to ATCD. We conclude that the duration of GSK-3β inactivation determines activated T cell survival and that natural adjuvant stimulation decreases the severity of clonal contraction in part by keeping a critical proapoptotic regulatory factor, GSK-3β, inactivated.

Inefficient TLR4/MD-2 heterotetramerization by monophosphoryl lipid A.

Synthetic forms of E. coli monophosphoryl lipid A (sMLA) weakly activate the MyD88 (myeloid differentiation primary response protein) branch of the bifurcated TLR4 (Toll-like receptor 4) signaling pathway, in contrast to diphosphoryl lipid A (sDLA), which is a strong activator of both branches of TLR4. sMLA’s weak MyD88 signaling activity is apparent downstream of TLR4/MyD88 signaling as we show that sMLA, unlike sDLA, is unable to efficiently recruit the TNF receptor-associated factor 6 (TRAF6) to the Interleukin-1 receptor-associated kinase 1 (IRAK1). This reduced recruitment of TRAF6 explains MLA’s lower MAPK (Mitogen Activated Protein Kinase) and NF-κB activity. As further tests of sMLA’s ability to activate TLR4/Myeloid differentiation factor 2 (MD-2), we used the antibody MTS510 as an indicator for TLR4/MD-2 heterotetramer formation. Staining patterns with this antibody indicated that sMLA does not effectively drive heterotetramerization of TLR4/MD-2 when compared to sDLA. However, a F126A mutant of MD-2, which allows lipid A binding but interferes with TLR4/MD-2 heterotetramerization, revealed that while sMLA is unable to efficiently form TLR4/MD-2 heterotetramers, it still needs heterotetramer formation for the full extent of signaling it is able to achieve. Monophosphoryl lipid A’s weak ability to form TLR4/MD-2 heterotetramers was not restricted to synthetic E. coli type because cells exposed to a biological preparation of S. minnesota monophosphoryl lipid A (MPLA) also showed reduced TLR4/MD-2 heterotetramer formation. The low potency with which sMLA and MPLA drive heterotetramerization of TLR4/MD-2 contributes to their weak MyD88 signaling activities.

A Short Domain within Bcl-3 Is Responsible for Its Lymphocyte Survival Activity

Abstract: The NFκB factor Bcl‐3 influences the survival of T cells when they are activated to take part in immune responses. Because treatment of mice with adjuvant results in the increased expression of Bcl‐3 in T cells, where it has survival‐promoting effects, Bcl‐3 may be an important, limiting factor that is supplied to T cells only when they are contributing to an appropriate immune response to infection, and not when spuriously activated by self‐antigens. Although Bcl‐3 is a member of the NFκB/Rel/IκB family of transcription factors, the means by which it promotes T cell survival is not obvious because Bcl‐3 is unique in having an ankyrin repeat domain, like inhibitory IκB proteins, while also possessing domains capable of transcriptional activation, like Rel proteins. In order to understand the basis for the survival activity of Bcl‐3, deletion mutants were engineered and tested in a retroviral gene transfer sytem. We report that most of Bcl‐3 can be deleted without diminishing its ability to prolong the survival of activated T and B cells, and find that its lymphocyte survival domain maps to the vicinity of its first and second ankryin repeats. This information sets the stage for experiments in which a focused search can be made for mediators of Bcl‐3 survival effects.

Peptide-stimulated DO11.10 T cells divide well but accumulate poorly in the absence of TLR agonist treatment

Immunological adjuvants increase the clonal burst size of antigen‐specific T cell populations by mechanisms that remain incompletely understood. Using the DO11.10 adoptive transfer system to study peptide‐stimulated T cell responses, we found that TLR agonist treatment increased the extent of cellular division undergone by responding T cells, but not by enough to explain the net increases in T cell yield that were achieved. Two novel analyses involving CFSE dye dilution analysis were used to characterize the shortfall, both of which were consistent with the idea that DO11.10 T cells are frequently lost during proliferation unless TLR agonists are present. T cell loss during clonal expansion was correlated with decreased levels of Bcl‐2, but TLR agonists did not appear to afford protection by restoring levels of Bcl‐2 or of cell surface IL‐7Rα chain expression. TLR‐mediated protection also failed to correlate with increased expression of Bcl‐x or decreased expression of pro‐apoptotic Bim. Our findings suggest that DO11.10 T cells stimulated by antigenic peptide in vivo divide well, but fail to accumulate efficiently unless TLR agonists are present.