David A. Christian

Polymersome Carriers: from Self-Assembly to siRNA and Protein Therapeutics

Polymersomes are polymer-based vesicular shells that form upon hydration of amphiphilic block copolymers. These high molecular weight amphiphiles impart physicochemical properties that allow polymersomes to stably encapsulate or integrate a broad range of active molecules. This robustness together with recently described mechanisms for controlled breakdown of degradable polymersomes as well as escape from endolysosomes suggests that polymersomes might be usefully viewed as having structure/property/function relationships somewhere between lipid vesicles and viral capsids. Here we summarize the assembly and development of controlled release polymersomes to encapsulate therapeutics ranging from small molecule anti-cancer drugs to siRNA and therapeutic proteins.

Spotted vesicles, striped micelles and Janus assemblies induced by ligand binding

Selective binding of multivalent ligands within a mixture of polyvalent amphiphiles provides, in principle, a mechanism to drive domain formation in self-assemblies. Divalent cations are shown here to crossbridge polyanionic amphiphiles that thereby demix from neutral amphiphiles and form spots or rafts within vesicles as well as stripes within cylindrical micelles. Calcium and copper crossbridged domains of synthetic block copolymers or natural lipid (PIP2, phosphatidylinositol-4,5-bisphosphate) possess tunable sizes, shapes, and/or spacings that can last for years. Lateral segregation in these ‘responsive Janus assemblies’ couples weakly to curvature and proves restricted within phase diagrams to narrow regimes of pH and cation concentration that are centered near the characteristic binding constants for polyacid interactions. Remixing at high pH is surprising, but a theory for Strong Lateral Segregation (SLS) shows that counterion entropy dominates electrostatic crossbridges, thus illustrating the insights gained into ligand induced pattern formation within self-assemblies.

Generalized Levy walks and the role of chemokines in migration of effector CD8+ T cells

Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8+ T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8+ T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8+ T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets.

Immune response and immunopathology during toxoplasmosis

Toxoplasma gondii is a protozoan parasite of medical and veterinary significance that is able to infect any warm-blooded vertebrate host. In addition to its importance to public health, several inherent features of the biology of T. gondii have made it an important model organism to study host–pathogen interactions. One factor is the genetic tractability of the parasite, which allows studies on the microbial factors that affect virulence and allows the development of tools that facilitate immune studies. Additionally, mice are natural hosts for T. gondii, and the availability of numerous reagents to study the murine immune system makes this an ideal experimental system to understand the functions of cytokines and effector mechanisms involved in immunity to intracellular microorganisms. In this article, we will review current knowledge of the innate and adaptive immune responses required for resistance to toxoplasmosis, the events that lead to the development of immunopathology, and the natural regulatory mechanisms that limit excessive inflammation during this infection.

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.

Calcium-dependent lateral organization in phosphatidylinositol (4,5) bisphosphate (PIP2)- and cholesterol-containing monolayers

Biological membrane function, in part, depends upon the local regulation of lipid composition. The spatial heterogeneity of membrane lipids has been extensively explored in the context of cholesterol and phospholipid acyl-chain-dependent domain formation, but the effects of lipid head groups and soluble factors in lateral lipid organization are less clear. In this contribution, the effects of divalent calcium ions on domain formation in monolayers containing phosphatidylinositol 4,5-bisphosphate (PIP2), a polyanionic, multifunctional lipid of the cytosolic leaflet of the plasma bilayer, are reported. In binary monolayers of PIP2 mixed with zwitterionic lipids, calcium induced a rapid, PIP2-dependent surface pressure drop, with the concomitant formation of laterally segregated, PIP2-rich domains. The effect was dependent upon head-group multivalency, because lowered pH suppressed the surface-pressure effect and domain formation. In accordance with previous observations, inclusion of cholesterol in lipid mixtures induced coexistence of two liquid phases. Phase separation strongly segregated PIP2 to the cholesterol-poor phase, suggesting a role for cholesterol-dependent lipid demixing in regulating PIP2 localization and local concentration. Similar to binary mixtures, subphase calcium induced contraction of ternary cholesterol-containing monolayers; however, in these mixtures, calcium induced an unexpected, PIP2- and multivalency-dependent decrease in the miscibility phase transition surface pressure, resulting in rapid dissolution of the domains. This result emphasizes the likely critical role of subphase factors and lipid head-group specificity in the formation and stability of cholesterol-dependent domains in cellular plasma membranes.

Toxoplasma Co-opts Host Cells It Does Not Invade

Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.

Contractile Forces Sustain and Polarize Hematopoiesis from Stem and Progenitor Cells

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Divalent Cation-Dependent Formation of Electrostatic PIP2 Clusters in Lipid Monolayers

Polyphosphoinositides are among the most highly charged molecules in the cell membrane, and the most common polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate (PIP(2)), is involved in many mechanical and biochemical processes in the cell membrane. Divalent cations such as calcium can cause clustering of the polyanionic PIP(2), but the origin and strength of the effective attractions leading to clustering has been unclear. In addition, the question of whether the ion-mediated attractions could be strong enough to alter the mechanical properties of the membrane, to our knowledge, has not been addressed. We study phase separation in mixed monolayers of neutral and highly negatively charged lipids, induced by the addition of divalent positively charged counterions, both experimentally and numerically. We find good agreement between experiments on mixtures of PIP(2) and 1-stearoyl-2-oleoyl phosphatidylcholine and simulations of a simplified model in which only the essential electrostatic interactions are retained. In addition, we find numerically that under certain conditions the effective attractions can rigidify the resulting clusters. Our results support an interpretation of PIP(2) clustering as governed primarily by electrostatic interactions. At physiological pH, the simulations suggest that the effective attractions are strong enough to give nearly pure clusters of PIP(2) even at small overall concentrations of PIP(2).

Parasite Fate and Involvement of Infected Cells in the Induction of CD4+ and CD8+ T Cell Responses to Toxoplasma gondii

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses.