Noah S. Butler

Therapeutic blockade of PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium infection

Infection of erythrocytes with Plasmodium species induces clinical malaria. Parasite-specific CD4+ T cells correlate with lower parasite burdens and severity of human malaria and are needed to control blood-stage infection in mice. However, the characteristics of CD4+ T cells that determine protection or parasite persistence remain unknown. Here we show that infection of humans with Plasmodium falciparum resulted in higher expression of the inhibitory receptor PD-1 associated with T cell dysfunction. In vivo blockade of the PD-1 ligand PD-L1 and the inhibitory receptor LAG-3 restored CD4+ T cell function, amplified the number of follicular helper T cells and germinal-center B cells and plasmablasts, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice. Thus, chronic malaria drives specific T cell dysfunction, and proper function can be restored by inhibitory therapies to enhance parasite control.

Respiratory Syncytial Virus Up-regulates TLR4 and Sensitizes Airway Epithelial Cells to Endotoxin

Airway epithelial cells are unresponsive to endotoxin (lipopolysaccharide (LPS)) exposure under normal conditions. This study demonstrates that respiratory syncytial virus (RSV) infection results in increased sensitivity to this environmental exposure. Infection with RSV results in increased expression of Toll-like receptor (TLR) 4 mRNA, protein, and increased TLR4 membrane localization. This permits significantly enhanced LPS binding to the epithelial monolayer that is blocked by disruption of the Golgi. The increased TLR4 results in an LPS-induced inflammatory response as demonstrated by increased mitogen-activated protein (MAP) kinase activity, IL-8 production, and tumor necrosis factor α production. RSV infection also allowed for tumor necrosis factor α production subsequent to TLR4 cross-linking with an immobilized antibody. These data suggest that RSV infection sensitizes airway epithelium to a subsequent environmental exposure (LPS) by altered expression and membrane localization of TLR4. The increased interaction between airway epithelial cells and LPS has the potential to profoundly alter airway inflammation.

Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria

Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.

Extreme CD8 t cell requirements for anti-malarial liver-stage immunity following immunization with radiation attenuated sporozoites

Radiation-attenuated Plasmodium sporozoites (RAS) are the only vaccine shown to induce sterilizing protection against malaria in both humans and rodents. Importantly, these “whole-parasite” vaccines are currently under evaluation in human clinical trials. Studies with inbred mice reveal that RAS-induced CD8 T cells targeting liver-stage parasites are critical for protection. However, the paucity of defined T cell epitopes for these parasites has precluded precise understanding of the specific characteristics of RAS-induced protective CD8 T cell responses. Thus, it is not known whether quantitative or qualitative differences in RAS-induced CD8 T cell responses underlie the relative resistance or susceptibility of immune inbred mice to sporozoite challenge. Moreover, whether extraordinarily large CD8 T cell responses are generated and required for protection following RAS immunization, as has been described for CD8 T cell responses following single-antigen subunit vaccination, remains unknown. Here, we used surrogate T cell activation markers to identify and track whole-parasite, RAS-vaccine-induced effector and memory CD8 T cell responses. Our data show that the differential susceptibility of RAS-immune inbred mouse strains to Plasmodium berghei or P. yoelii sporozoite challenge does not result from host- or parasite-specific decreases in the CD8 T cell response. Moreover, the surrogate activation marker approach allowed us for the first time to evaluate CD8 T cell responses and protective immunity following RAS-immunization in outbred hosts. Importantly, we show that compared to a protective subunit vaccine that elicits a CD8 T cell response to a single epitope, diversifying the targeted antigens through whole-parasite RAS immunization only minimally, if at all, reduced the numerical requirements for memory CD8 T cell-mediated protection. Thus, our studies reveal that extremely high frequencies of RAS-induced memory CD8 T cells are required, but may not suffice, for sterilizing anti-Plasmodial immunity. These data provide new insights into protective CD8 T cell responses elicited by RAS-immunization in genetically diverse hosts, information with relevance to developing attenuated whole-parasite vaccines.

Chronic Exposure to Plasmodium falciparum Is Associated with Phenotypic Evidence of B and T Cell Exhaustion

Naturally acquired immunity to malaria develops slowly, requiring several years of repeated exposure to be effective. The cellular and molecular factors underlying this observation are only partially understood. Recent studies suggest that chronic Plasmodium falciparum exposure may induce functional exhaustion of lymphocytes, potentially impeding optimal control of infection. However, it remains unclear whether the “atypical” memory B cells (MBCs) and “exhausted” CD4 T cells described in humans exposed to endemic malaria are driven by P. falciparum per se or by other factors commonly associated with malaria, such as coinfections and malnutrition. To address this critical question we took advantage of a “natural” experiment near Kilifi, Kenya, and compared profiles of B and T cells of children living in a rural community where P. falciparum transmission is ongoing to the profiles of age-matched children living under similar conditions in a nearby community where P. falciparum transmission ceased 5 y prior to this study. We found that continuous exposure to P. falciparum drives the expansion of atypical MBCs. Persistent P. falciparum exposure was associated with an increased frequency of CD4 T cells expressing phenotypic markers of exhaustion, both programmed cell death-1 (PD-1) alone and PD-1 in combination with lymphocyte-activation gene-3 (LAG-3). This expansion of PD-1–expressing and PD-1/LAG-3–coexpressing CD4 T cells was largely confined to CD45RA+ CD4 T cells. The percentage of CD45RA+CD27+ CD4 T cells coexpressing PD-1 and LAG-3 was inversely correlated with frequencies of activated and classical MBCs. Taken together, these results suggest that P. falciparum infection per se drives the expansion of atypical MBCs and phenotypically exhausted CD4 T cells, which has been reported in other endemic areas.

Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA.

Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.

Altered IL-4 mRNA Stability Correlates with Th1 and Th2 Bias and Susceptibility to Hypersensitivity Pneumonitis in Two Inbred Strains of Mice

Previously, we have shown in a model of hypersensitivity pneumonitis that Th1-biased C57BL/6 mice are susceptible and Th2-biased DBA/2 mice are resistant to disease. We also showed that this was explained in part by differential regulation of IL-12 by IL-4. For these reasons, we postulated that C57BL/6 and DBA/2 mice differentially express IL-4. In this study, we show that C57BL/6 immune cells express Th2 but not Th1 cytokines at lower levels than DBA/2 cells. We also found that C57BL/6 splenocytes exhibit decreased mRNA stability of Th2 cytokines, relative to DBA/2 splenocytes. Stability of IL-2 and IFN-γ were similar in the two strains of mice. Differences in Th2 cytokine mRNA stability between C57BL/6 and DBA/2 cells were not due to sequence polymorphism at specific regions of the IL-4/IL-13 locus. Furthermore, expression of Th1- and Th2-specific transcription factors T-bet and GATA-3, as well as the nuclear factor of activated T cells transcription factor, NFATc, was not significantly different between the two mice. Our data suggest that decreased mRNA stability of Th2 cytokines in C57BL/6 splenocytes may underlie the differential susceptibility to hypersensitivity pneumonitis between C57BL/6 and DBA/2 mice. Moreover, our results indicate that regulation of mRNA stability may serve as an important mechanism underlying Th1/Th2 immune polarization.

Differential Effector Pathways Regulate Memory CD8 T Cell Immunity against Plasmodium berghei versus P. yoelii Sporozoites

Malaria results in >1,000,000 deaths per year worldwide. Although no licensed vaccine exists, much effort is currently focused on subunit vaccines that elicit CD8 T cell responses directed against Plasmodium parasite liver stage Ags. Multiple immune-effector molecules play a role in antimicrobial immunity mediated by memory CD8 T cells, including IFN-γ, perforin, TRAIL, Fas ligand, and TNF-α. However, it is not known which pathways are required for memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. In this study, we used a novel immunization strategy to generate memory CD8 T cells in the BALB/c mouse model of P. berghei or P. yoelii sporozoite infection to examine the role of immune-effector molecules in resistance to the liver stage infection. Our studies reveal that endogenous memory CD8 T cell-mediated protection against both parasite species is, in part, dependent on IFN-γ, whereas perforin was only critical in protection against P. yoelii. We further show that neutralization of TNF-α in immunized mice markedly reduces memory CD8 T cell-mediated protection against both parasite species. Thus, our studies identify IFN-γ and TNF-α as important components of the noncytolytic pathways that underlie memory CD8 T cell-mediated immunity against liver stage Plasmodium infection. Our studies also show that the effector pathways that memory CD8 T cells use to eliminate liver stage infection are, in part, Plasmodium species specific.

Whole parasite vaccination approaches for prevention of malaria infection

Malaria is caused by complex protozoan Plasmodium parasites that have foiled efforts to develop a protective vaccine. Despite this, it has been known for more than 40 years that immunization with radiation-attenuated, whole Plasmodium sporozoites confers complete protection against malaria challenge. This model gave the rationale for development of recombinant and vectored subunit vaccination strategies that have, however, not yet matched whole sporozoite protective efficacy. Novel attenuation and immunization approaches for whole sporozoite vaccination and a deeper understanding of cellular and humoral protective immune responses that eliminate pre-erythrocytic stages are paving the way for the development of next-generation vaccination strategies that completely prevent malaria.

Inhibition of Rho Family GTPases Results in Increased TNF-α Production After Lipopolysaccharide Exposure

These studies demonstrate that treatment of macrophages with lovastatin, a cholesterol-lowering drug that blocks farnesylation and geranylgeranylation of target proteins, increases LPS-induced TNF-α production. This is reversed by the addition of mevalonate, which bypasses the lovastatin block. Examination of membrane localization of RhoA, Cdc42, Rac1, and Ras demonstrated decreased membrane localization of the geranylgeranylated Rho family members (RhoA, Cdc42, and Rac1) with no change in the membrane localization of farnesylated Ras. LPS-induced TNF-α production in the presence of the Rho family-specific blocker (toxin B from Clostridium difficile) was significantly enhanced consistent with the lovastatin data. One intracellular signaling pathway that is required for TNF-α production by LPS is the extracellular signal-regulated kinase (ERK). Significantly, we found prolonged ERK activation after LPS stimulation of lovastatin-treated macrophages. When we inhibited ERK, we blocked the lovastatin-induced increase in TNF-α production. As a composite, these studies demonstrate a negative role for one or more Rho family GTPases in LPS-induced TNF-α production.