Darrell J. Irvine

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel–Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.

Direct observation of ligand recognition by T cells.

The activation of T cells through interaction of their T-cell receptors with antigenic peptide bound to major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a crucial step in adaptive immunity. Here we use three-dimensional fluorescence microscopy to visualize individual peptide–I-Ek class II MHC complexes labelled with the phycobiliprotein phycoerythrin in an effort to characterize T-cell sensitivity and the requirements for forming an immunological synapse in single cells. We show that T cells expressing the CD4 antigen respond with transient calcium signalling to even a single agonist peptide–MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present. This sensitivity is highly dependant on CD4, because blocking this molecule with antibodies renders T cells unable to detect less than about 30 ligands.

Therapeutic cell engineering with surface-conjugated synthetic nanoparticles

A major limitation of cell therapies is the rapid decline in viability and function of the transplanted cells. Here we describe a strategy to enhance cell therapy via the conjugation of adjuvant drug–loaded nanoparticles to the surfaces of therapeutic cells. With this method of providing sustained pseudoautocrine stimulation to donor cells, we elicited marked enhancements in tumor elimination in a model of adoptive T cell therapy for cancer. We also increased the in vivo repopulation rate of hematopoietic stem cell grafts with very low doses of adjuvant drugs that were ineffective when given systemically. This approach is a simple and generalizable strategy to augment cytoreagents while minimizing the systemic side effects of adjuvant drugs. In addition, these results suggest therapeutic cells are promising vectors for actively targeted drug delivery.

Structure-based programming of lymph-node targeting in molecular vaccines

In cancer patients, visual identification of sentinel lymph nodes (LNs) is achieved by the injection of dyes that bind avidly to endogenous albumin, targeting these compounds to LNs, where they are efficiently filtered by resident phagocytes. Here we translate this ‘albumin hitchhiking’ approach to molecular vaccines, through the synthesis of amphiphiles (amph-vaccines) comprising an antigen or adjuvant cargo linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain. Administration of structurally optimized CpG-DNA/peptide amph-vaccines in mice resulted in marked increases in LN accumulation and decreased systemic dissemination relative to their parent compounds, leading to 30-fold increases in T-cell priming and enhanced anti-tumour efficacy while greatly reducing systemic toxicity. Amph-vaccines provide a simple, broadly applicable strategy to simultaneously increase the potency and safety of subunit vaccines.

Engineering Nano- and Microparticles to Tune Immunity

The immune system can be a cure or cause of disease, fulfilling a protective role in attacking cancer or pathogenic microbes but also causing tissue destruction in autoimmune disorders. Thus, therapies aimed to amplify or suppress immune reactions are of great interest. However, the complex regulation of the immune system, coupled with the potential systemic side effects associated with traditional systemic drug therapies, has presented a major hurdle for the development of successful immunotherapies. Recent progress in the design of synthetic micro- and nano-particles that can target drugs, deliver imaging agents, or stimulate immune cells directly through their physical and chemical properties is leading to new approaches to deliver vaccines, promote immune responses against tumors, and suppress autoimmunity. In addition, novel strategies, such as the use of particle-laden immune cells as living targeting agents for drugs, are providing exciting new approaches for immunotherapy. This progress report describes recent advances in the design of micro- and nano-particles for immunotherapies and diagnostics.

CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse

How T cells respond with extraordinary sensitivity to minute amounts of agonist peptide and major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells bearing large numbers of endogenous pMHC molecules is not understood. Here we present evidence that CD4 affects the responsiveness of T helper cells by controlling spatial localization of the tyrosine kinase Lck in the synapse. This finding, as well as further in silico and in vitro experiments, led us to develop a molecular model in which endogenous and agonist pMHC molecules act cooperatively to amplify T cell receptor signaling. At the same time, activation due to endogenous pMHC molecules alone is inhibited. A key feature is that the binding of agonist pMHC molecules to the T cell receptor results in CD4-mediated spatial localization of Lck, which in turn enables endogenous pMHC molecules to trigger many T cell receptors. We also discuss broader implications for T cell biology, including thymic selection, diversity of the repertoire of self pMHC molecules and serial triggering.

Synthetic Nanoparticles for Vaccines and Immunotherapy

National Institutes of Health (U.S.) (Grants AI111860, CA174795, CA172164, AI091693, and AI095109)

A role for the immunological synapse in lineage commitment of CD4 lymphocytes

Activation of the naive T-helper lymphocyte (Thp) directs it down one of two major developmental pathways called Th1 and Th2. Signals transmitted by T cell, co-stimulatory and cytokine receptors control Thp lineage commitment but the mechanism by which these signals are integrated remains a mystery. The interferon-γ (IFNGR) and interleukin 4 (IL-4R) cytokine receptors, in particular, direct the earliest stages of T-helper commitment. Here we report that on engagement of the T-cell receptor (TCR) on Thp cells, rapid co-polarization of IFNGR with the TCR occurs within the developing immunological synapse. Thp cells from the intrinsically Th1-like C57BL/6 mouse strain have significantly more receptor co-polarization than Th2-prone BALB/c Thp cells. Remarkably, in the presence of IL-4, a cytokine required for Th2 differentiation, IFNGR co-polarization with TCR is prevented. This inhibition depends on Stat6, the transcription factor downstream of IL-4R that is required for Th2 differentiation. This cytokine receptor crossregulation provides an explanation for the effect of IL-4 in inhibiting Th1 differentiation. These observations suggest a scenario in which physical co-polarization of critical receptors directs the fate of the naive Thp, and offer a novel function for the immunological synapse in directing cell differentiation. They further suggest a new mechanism of membrane-bound signalling control by the physical disruption of large receptor-rich domains on signalling through a functionally antagonistic receptor.

Particulate vaccines: on the quest for optimal delivery and immune response.

Subunit vaccines offer a safer alternative to traditional organism-based vaccines, but their immunogenicity is impaired. This hurdle might be overcome by the use of micro- and nanodelivery systems carrying the antigen(s). This review discusses the rationale for the use of particulate vaccines and provides an overview of antigen-delivery vehicles currently under investigation. It further highlights the cellular uptake, antigen processing and the presentation by antigen-presenting cells because these processes are partially governed by particle characteristics and eventually determine the immunological outcome. Finally, we address the attractive concept of concomitant delivery of antigens and immunopotentiators. The condensed knowledge could be an asset for rationally designing antigen-delivery vehicles to obtain safe and efficacious vaccines.

Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction.

For subunit vaccines, adjuvants play a key role in shaping immunological memory. Nanoparticle (NP) delivery systems for antigens and/or molecular danger signals are promising adjuvants capable of promoting both cellular and humoral immune responses, but in most cases the mechanisms of action of these materials are poorly understood. Here, we studied the immune response elicited by NPs composed of multilamellar “stapled” lipid vesicles carrying a recombinant Plasmodium vivax circumsporozoite antigen, VMP001, both entrapped in the aqueous core and anchored to the lipid bilayer surfaces. Immunization with these particles and monophosphoryl lipid A (MPLA), a US Food and Drug Administration–approved immunostimulatory agonist for Toll-like receptor-4, promoted high-titer, high-avidity antibody responses against VMP001, lasting more than 1 y in mice at 10-fold lower doses than conventional adjuvants. Compared to soluble VMP001 mixed with MPLA, VMP001-NPs promoted broader humoral responses, targeting multiple epitopes of the protein and a more balanced Th1/Th2 cytokine profile from antigen-specific T cells. To begin to understand the underlying mechanisms, we examined components of the B-cell response and found that NPs promoted robust germinal center (GC) formation at low doses of antigen where no GC induction occurred with soluble protein immunization, and that GCs nucleated near depots of NPs accumulating in the draining lymph nodes over time. In parallel, NP vaccination enhanced the expansion of antigen-specific follicular helper T cells (Tfh), compared to vaccinations with soluble VMP001 or alum. Thus, NP vaccines may be a promising strategy to enhance the durability, breadth, and potency of humoral immunity by enhancing key elements of the B-cell response.