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CMV infection alters NK cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the FcRγ-chain (gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced Ab-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives enhanced ADCC and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. We ablated genes that encode molecules in the ADCC pathway, such as FcRγ, CD3ζ, SYK, SHP-1, ZAP70, and the transcription factor PLZF, and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ-chain caused a modest increase in TNF-α production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced cytotoxicity, cytokine production, and target cell conjugation, whereas ZAP70 kinase ablation diminished function. Ablating the phosphatase SHP-1 enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. We found the lack of SYK expression could improve target cell conjugation through enhanced CD2 expression or limit SHP-1–mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production.

There is no cure for HIV infection, and lifelong antiretroviral therapy (ART) is required. N-803 is an IL-15 superagonist comprised of an N72D mutant IL-15 molecule attached to its alpha receptor and a human IgG1 fragment designed to increase IL-15 activity. Preclinical studies with both HIV and SIV suggest that the drug has potential to reduce virus reservoirs by activating virus from latency and enhancing effector function. We conducted a phase 1 study of N-803 ( NCT02191098 ) in people living with HIV, the primary objective of which was to assess the safety and tolerability of the drug, with an exploratory objective of assessing the impact on peripheral virus reservoirs. ART-suppressed individuals were enrolled into a dose-escalation study of N-803 in four different cohorts (0.3, 1.0, 3.0 and 6.0 mcg kg

The replacement of chloroquine with artemisinin-based combination therapies (ACTs) for over a decade has had varying impacts on the ability of malaria parasite to sustain its chloroquine resistance prowess in different malaria-endemic regions. We evaluated the frequency of Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations in an endemic area of southwest Nigeria 17 years after replacement of chloroquine with ACTs for malaria treatment. Genomic DNA was isolated from dried blood spot samples obtained from 129 patients (aged 1-35 years) with microscopically confirmed P. falciparum infection. PfCRT fragments covering codons 72-76, CVMNK (wildtype) and A220 were amplified and sequenced. Two mutant PfCRT haplotypes on residues 72-76 (CVIET and CVINT) were identified with a prevalence of 18.6% and 2.3%, respectively. Interestingly, the CVINT haplotype was identified for the first time in this region. A220S changes were found in 16.3% of samples occurring concurrently with the CVIET haplotype, while a Q271E mutation occurred in a wildtype isolate. The reduced prevalence of the PfCRT mutant alleles in this study may suggest a gradual disappearance of chloroquine-resistant malaria parasites following reduced drug pressure. It may also be an indicator of the ability of malaria parasites to develop resistance gradually against the current first-line regimen.

Metastatic castration-resistant prostate cancer (mCRPC) has been largely resistant to immunotherapy. Natural killer (NK) cells are cytotoxic lymphocytes that detect and kill transformed cells without prior sensitization, and their infiltration into prostate tumors corresponds with an increased overall survival among patients with mCRPC. We sought to harness this knowledge to develop an approach to NK-cell based immunotherapy for mCRPC. We engineered an NK cell line (NK-92MI) to express CD64, the sole human high-affinity IgG Fcγ receptor (FcγR1), and bound these cells with antibodies to provide interchangeable tumor-targeting elements. NK-92MICD64 cells were evaluated for cell-activation mechanisms and antibody-dependent cell-mediated cytotoxicity (ADCC). A combination of mAbs was used to target the prostate tumor antigen tumor-associated calcium signal transducer 2 (TROP2) and the cancer-associated fibroblast marker fibroblast activation protein alpha (FAP). We found that CD64, which is normally expressed by myeloid cells and associates with the adaptor molecule FcRγ, can be expressed by NK-92MI cells and mediate ADCC through an association with CD3ζ. Cytotoxicity from the combination approach was two-fold higher compared to treatment with NK-92MICD64 cells and either mAb alone, and seven-fold higher than NK-92MICD64 cells alone at an effector–target cell ratio of 20:1. The cytotoxic effect was lost when using isotype control antibodies, indicating a selective targeting mechanism. The combination approach demonstrated efficacy in vivo as well and significantly reduced tumor growth compared with the saline control. This combination therapy presents a potential approach for treating mCRPC and could improve immunotherapy response.

Vaccines and mAbs offer promising strategies to treat substance use disorders (SUDs) and prevent overdose. Despite vaccines and mAbs against SUDs demonstrating proof of efficacy, selectivity, and safety in animal models, it is unknown whether the mechanism of action of these immunotherapeutics relies exclusively on the formation of Ab/drug complexes, or also involves Ab-mediated effector functions. Hence, this study tested whether the efficacy of active and passive immunization against drugs of abuse requires phagocytosis, the intact Fc portion of the anti-drug Ab, FcγRs, or the neonatal FcR (FcRn). The efficacy of a lead vaccine against oxycodone was not diminished in mice after depletion of macrophages or granulocytes. Anti-oxycodone F(ab′)2 fragments resulted in lower serum levels of F(ab′)2 compared with intact mAbs, and F(ab′)2s were not as effective as the parent mAbs in reducing distribution of oxycodone to the brain. The efficacy of vaccines and mAbs against oxycodone was preserved in either FcγIII or FcγI–IV ablated mice, suggesting that FcγRs are not required for Ab efficacy. Finally, both active and passive immunization against oxycodone in FcRn−/− mice yielded reduced efficacy compared with wild-type control mice. These data identified a role for FcRn, but not for phagocytosis or Fc-dependent effector functions, in mediating the efficacy of vaccines and mAbs against SUD. This study supports rational design of vaccines and mAbs engineered for maximal neutralization activity and optimal FcRn binding.

Cytomegalovirus (CMV) infection alters natural killer (NK) cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the Fc receptor γ chain (Gene:FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (FcγRIIIA) signaling apparatus, which includes FcRγ, CD3ζ, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ chain caused a modest increase in TNFα production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3ζ chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.

OBJECTIVES/GOALS: The innate immune responses to Multisystem Inflammatory Syndrome in Children (MIS-C) are not fully known. Using samples from MIS-C, we will assess the cellular responses and develop a novel Tri-Specific Killer Engager (TRiKE) that engages innate immune cells to improve those responses. METHODS/STUDY POPULATION: We collected blood samples from 60 pediatric patients from which we isolated plasma and peripheral blood mononuclear cells. We received blood samples from 13 MIS-C, 32 severe acute COVID, 5 COVID-19 asymptomatic, and 15 COVID-19 negative patients. Using plasma, we then performed ELISAs to determine IgG antibody levels against SARS-CoV-2 and plaque reduction neutralization tests to determine neutralizing antibody functions. We isolated DNA to look at Fc receptor genetics. We also utilized utilize flow cytometry assays determine the phagocytosis and killing abilities of the innate cells from these patients. This data will be correlated with clinical outcomes. Additionally, we have developed a novel SARS-CoV-2 TRiKE which directs natural killer (NK) cell killing specifically to of COVID-19 infected cells. RESULTS/ANTICIPATED RESULTS: MIS-C patients had higher IgG antibody titers against SARS-CoV-2 compared to children with symptomatic or asymptomatic COVID. MIS-C patients also neutralized SARS-CoV-2 more effectively than children with acute symptomatic or asymptomatic COVID-19. We found natural killer cells and monocytes are dysfunctional in MIS-C patients and do not kill SARS-CoV-2 infected cells as well. Specifically, NK cells do not kill COVID-19 infected cells as well. To combat this, we have successfully generated and are now testing a Tri-Specific Killer engager (TRiKE) which binds one ends to NK cells, one end to the Spike protein on COVID-19 infected cells and contains IL-15 to improve NK cell function. We anticipate that we can improve NK cell killing of COVID-19 infected cells with this TRiKE. DISCUSSION/SIGNIFICANCE: We found that MIS-C patients have antibodies that can neutralize SARS-CoV-2 but that that innate immune cells that engage antibodies are dysfunctional. We are have successfully developed and are targeting this response with a TRiKE to improve innate immune cell functional; this may serve as an adjunctive therapeutic if proven successful.

Chimeric antigen receptor (CAR)-T cells show great promise in treating cancers and viral infections. However, most protocols developed to expand T cells require relatively long periods of time in culture, potentially leading to progression toward populations of terminally differentiated effector memory cells. Here, we describe in detail a 9-day protocol for CAR gene transduction and expansion of primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Cells produced and expanded with this method show high levels of viability, high levels of co-expression of two transduced genes, retention of the central memory phenotype, and sufficient quantity for immunotherapeutic infusion of 1–2 × 108 cells/kg in a 10 kg rhesus macaque. This 9-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches to decrease culture time and increase maintenance of central memory populations., Graphical Abstract

How antibodies naturally acquired during Plasmodium falciparum infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. P. falciparum–infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of P. falciparum–infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to P. falciparum antigens is therefore warranted in the design of malaria vaccines.

The magnitude of SARS-CoV-2–specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Background Chronic active Epstein-Barr virus (CAEBV) presents with high levels of viral genomes in blood and tissue infiltration with Epstein-Barr virus (EBV)–positive lymphocytes. The pathogenesis of CAEBV is poorly understood. Methods We evaluated 2 patients with natural killer (NK) cell CAEBV and studied their NK cell phenotype and signaling pathways in cells. Results Both patients had increased numbers of NK cells, EBV predominantly in NK cells, and immature NK cells in the blood. Both patients had increased phosphorylation of Akt, S6, and STAT1 in NK cells, and increased total STAT1. Treatment of 1 patient with sirolimus reduced phosphorylation of S6 in T and B cells, but not in NK cells and did not reduce levels of NK cells or EBV DNA in the blood. Treatment of both patients’ cells with JAK inhibitors in vitro reduced phosphorylated STAT1 to normal. Patients with T- or B-cell CAEBV had increased phosphorylation of Akt and S6 in NK cells, but no increase in total STAT1. Conclusions The increase in phosphorylated Akt, S6, and STAT1, as well as immature NK cells describe a new phenotype for NK cell CAEBV. The reduction of STAT1 phosphorylation in their NK cells with JAK inhibitors suggests a novel approach to therapy.

Natural killer (NK) cells are important innate effector cells that are well described in their ability to kill virally-infected cells and tumors. However, there is increasing appreciation for the role of NK cells in the control of other pathogens, including intracellular parasites such as Plasmodium, the cause of malaria. NK cells may be beneficial during the early phase of Plasmodium infection—prior to the activation and expansion of antigen-specific T cells—through cooperation with myeloid cells to produce inflammatory cytokines like IFNγ. Recent work has defined how Plasmodium can activate NK cells to respond with natural cytotoxicity, and inhibit the growth of parasites via antibody-dependent cellular cytotoxicity mechanisms (ADCC). A specialized subset of adaptive NK cells that are negative for the Fc receptor γ chain have enhanced ADCC function and correlate with protection from malaria. Additionally, production of the regulatory cytokine IL-10 by NK cells prevents overt pathology and death during experimental cerebral malaria. Now that conditional NK cell mouse models have been developed, previous studies need to be reevaluated in the context of what is now known about other immune populations with similarity to NK cells (i.e., NKT cells and type I innate lymphoid cells). This brief review summarizes recent findings which support the potentially beneficial roles of NK cells during Plasmodium infection in mice and humans. Also highlighted are how the actions of NK cells can be explored using new experimental strategies, and the potential to harness NK cell function in vaccination regimens.

Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment, upregulation of T helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses, and p53 activation associated with control of malarial fever and coordinated with Pf-specific immunoglobulin G (IgG) and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever.

Plasmodium falciparum–infected individuals in a malaria-endemic region have an abundance of adaptive NK cells that correlates with resistance to malaria. Adaptive NK cells dominate antibody-dependent responses to P. falciparum–infected red blood cells and may contribute to acquired immunity to malaria., How antibodies naturally acquired during Plasmodium falciparum infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. P. falciparum–infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of P. falciparum–infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to P. falciparum antigens is therefore warranted in the design of malaria vaccines.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action., eLife digest Malaria is a deadly disease caused by a parasite transmitted by mosquitoes. The parasite infects red blood cells, causing fever with flu-like symptoms. In some people, particularly pregnant women and children, the disease may be very serious and even lead to death. An effective malaria vaccine is urgently needed because malaria parasites are developing resistance to current drugs. People living in areas where malaria is common develop specific proteins called antibodies that protect them from malaria. Learning more about how the antibodies achieve this, could help to develop better vaccines. Scientists already know some antibodies bind to the malaria parasites and prevent them from entering red blood cells. Some vaccines have been based on these antibodies. Other antibodies bind to infected cells flagging them for destruction by cells of the immune system. Immune cells called natural killer cells can eliminate viruses or cancer cells this way, but it was not clear if they could also eliminate malaria parasite-infected red blood cells. Now, Arora et al. show that natural killer cells can selectively destroy malaria-infected red blood cells flagged with antibodies from people who live in areas where malaria is common. In laboratory experiments, natural killer cells from US volunteers, who were never exposed to malaria, did not kill normal or malaria-infected red blood cells. Adding antibodies collected from malaria-resistant volunteers from Africa allowed these natural killer cells from unexposed people to selectively seek out and destroy malaria-infected cells and leave uninfected red blood cells intact. Arora et al. also found that the antibodies from the malaria-resistant volunteers bound to parasite proteins on the surface of infected blood cells. The experiments suggest that vaccines designed to stimulate the production of antibodies to malaria proteins that are displayed on infected red blood cells, could destroy the parasite in infected people and help prevent disease and save lives.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

Summary Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA-infected red blood cells develop a fatal CM-like disease caused by CD8 + T cell-mediated pathology. We found that treatment with interleukin-15 complex (IL-15C) prevented ECM, whereas IL-2C treatment had no effect. IL-15C-expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL-15C treatment also decreased CD8 + T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL-15C induced NK cells to express IL-10, which was required for IL-15C-mediated protection against ECM. Finally, we show that ALT-803, a modified human IL-15C, mediates similar induction of IL-10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine-stimulated NK cells in the prevention of a pathogenic immune response.

Malaria is a mosquito-borne disease caused by parasites of the obligate intracellular Apicomplexa phylum the most deadly of which, Plasmodium falciparum, prevails in Africa. Malaria imposes a huge health burden on the world's most vulnerable populations, claiming the lives of nearly one million children and pregnant women each year. Although there is keen interest in eradicating malaria, we do not yet have the necessary tools to meet this challenge, including an effective malaria vaccine and adequate vector control strategies. Here we review what is known about the mechanisms at play in immune resistance to malaria in both the human and mosquito hosts at each step in the parasite's complex life cycle with a view toward developing the tools that will contribute to the prevention of disease and death and, ultimately, to the goal of malaria eradication. In so doing, we hope to inspire immunologists to participate in defeating this devastating disease.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature., Author Summary Cerebral malaria (CM) is a severe and potentially fatal complication of malaria in humans that results in swelling and bleeding within the brain. The mechanisms that cause this fatal disease in humans are not completely understood. We studied an animal model known as experimental cerebral malaria to learn more about the factors that drive this disease process. Using a technique referred to as intravital microscopy, we captured movies of immune cells operating in the living brain as the disease developed. At the peak of disease, we observed evidence of immune cells interacting with and aggregating along blood vessels throughout the brain. These interactions were directly associated vascular leakage. This caused the brain to swell, which gave rise to an unsustainable pressure that ultimately killed neurons responsible for heart and lung function. The fatal swelling was induced by immune cells (referred to as T cells) interacting with bits of parasite presented by blood vessels in the brain. Removal of this parasite presentation protected the mice from fatal disease. We also evaluated a straightforward therapy that involved intravenous administration of antibodies that interfered with T cell sticking to blood vessels. Our movies revealed that this therapeutic approach rapidly displaced T cells from the blood vessels in the brain and prevented fatal disease.

Epstein-Barr virus (EBV) infection is usually asymptomatic and latency occurs in B cells. We studied three patients with NK cell chronic active EBV (CAEBV) disease and EBV hepatitis. All patients had an increased number of NK cells, and high levels of EBV in the blood, with EBV predominantly in NK cells; two patients who were tested had an NK cell receptor repertoire consistent with immature cells. All three patients had increased phosphorylation of Akt and ribosomal protein S6 in NK cells and a marked increase in STAT1; two of the patients had increased phosphorylation of STAT1 in their T cells, NK cells, and monocytes. Treatment of one of these patients with sirolimus, an mTOR inhibitor, reduced phosphorylation of S6 in the patient’s T and B cells, but not in her NK cells and did not reduce the level of NK cells or EBV DNA in the peripheral blood. The increased STAT1 activity observed in two of the patients was reduced to normal when the cells were treated with JAK inhibitors in vitro. Unlike other patients with STAT1 gain-of-function (GOF) mutations, no mutations were identified in STAT1 or STAT1 regulatory proteins. Patients with T or B cell CAEBV also had increased phosphorylation of Akt and S6, but not STAT1. In summary, the increase in Akt and S6 phosphorylation, and the increase in STAT1 protein, as well as immature NK cells in our NK cell patients describe a novel phenotype in NK cell CAEBV disease.

Plasmodium falciparum malaria is a deadly infectious disease in which Abs play a critical role in naturally acquired immunity. However, the specificity and nature of Abs elicited in response to malaria are only partially understood. Autoreactivity and polyreactivity are common features of Ab responses in several infections and were suggested to contribute to effective pathogen-specific Ab responses. In this article, we report on the regulation of B cells expressing the inherently autoreactive VH4-34 H chain (identified by the 9G4 mAb) and 9G4+ plasma IgG in adults and children living in a P. falciparum malaria-endemic area in West Africa. The frequency of 9G4+ peripheral blood CD19+ B cells was similar in United States adults and African adults and children; however, more 9G4+ B cells appeared in classical and atypical memory B cell compartments in African children and adults compared with United States adults. The levels of 9G4+ IgG increased following acute febrile malaria but did not increase with age as humoral immunity is acquired or correlate with protection from acute disease. This was the case, even though a portion of 9G4+ B cells acquired phenotypes of atypical and classical memory B cells and 9G4+ IgG contained equivalent numbers of somatic hypermutations compared with all other VHs, a characteristic of secondary Ab repertoire diversification in response to Ag stimulation. Determining the origin and function of 9G4+ B cells and 9G4+ IgG in malaria may contribute to a better understanding of the varied roles of autoreactivity in infectious diseases.

Several recent studies reported that Krüppel-like factor (KLF)2 controls trafficking, development, and function of B cells. Conditional B cell KLF2 knockout mice have increased numbers of marginal zone B cells and decreased numbers of B1 phenoytpe cells. However, it was unclear whether KLF2 is required for B1 B cell development, survival, or phenotypic maintenance. We show that B1 phenotype B cells are present in neonatal mice with B cell-specific KLF2 deficiency, suggesting that B1 differentiation can occur even in the absence of KLF2. Furthermore, by use of an inducible knockout strategy, we show that deletion of KLF2 in mature B1 cells causes loss of phenotypic markers associated with B1 cell identity, but it has a minimal effect on short-term cell survival. Taken together, our findings suggest that KLF2 is necessary for the maintenance of B1 cell identity rather than differentiation or survival of the population.

Plasmodium falciparum (P.f) infection is a major cause of morbidity and mortality world-wide where symptoms and death occur as a result of the blood stage of the P.f. life cycle. To date, there is no effective blood stage malarial vaccine. Natural Killer (NK) cells are key players in the control of hematopoietic cancers and viral infections, however their role in blood stage malaria is unknown. We undertook a comprehensive analysis of NK cell phenotype and function in a cohort of subjects from a malaria clinical study in Mali, Africa. Using an unbiased analysis of different NK cell subsets, we found three major findings: 1. Fc receptor gamma chain (−) or PLZF (−) NK cells correlate with reduced parasitemia 2. Fc receptor gamma chain (−) NK cells correlate with protection from malaria symptoms 3. Fc receptor gamma chain (−) NK cells are enhanced for antibody dependent cellular cytotoxicity (ADCC). Antibodies are known to be protective in malaria, however the mechanism of this protection is still being elucidated. We hypothesized Fc receptor gamma chain (−) NK cells may be clearing opsonized, malaria-infected RBCs in vivo via ADCC. We show that Fc receptor gamma chain (−) NK cells specifically release lytic granules in response to opsonized, infected RBCs. This work indicates the host may increase the number of Fc receptor gamma chain (−) NK cells to aide in clearance of infected RBCs through ADCC. Because the antigens on the surface of infected RBCs are different than the invading parasite, these results could improve the identification of effective targets for blood stage vaccines.

The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood-brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8(+) effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood-brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8(+) T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children.

Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host’s immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism., IMPORTANCE Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection is cerebral malaria with a fatality rate of 15 to 20%, despite treatment with antimalarial drugs. Cerebral malaria takes a second toll on African children, leaving survivors at high risk of debilitating neurological defects. At present, we have no effective adjunctive therapies for cerebral malaria, and developing such therapies would have a large impact on saving young lives in Africa. Here we report results that open a new avenue for the development of highly selective adjunctive therapies for cerebral malaria by targeting pathways that regulate host and parasite metabolism.

Periodontal disease is one of the most prevalent chronic inflammatory diseases. There is a genetic component to susceptibility and resistance to this disease. Using a mouse model, we investigated the progression of alveolar bone loss by gene expression profiling of susceptible and resistant mouse strains (BALB/cByJ and A/J, respectively). We employed a novel and sensitive quantitative real-time PCR method to compare basal RNA transcription of a 48-gene set in the gingiva and the spleen and the subsequent changes in gene expression due to Porphyromonas gingivalis oral infection. Basal expression of interleukin-1 beta ( Il1b ) and tumor necrosis factor alpha ( Tnf ) mRNA was higher in the gingiva of the susceptible BALB/cByJ mice than in the gingiva of resistant A/J mice. Gingival Il1b gene expression increased further and Stat6 gene expression was turned on after P. gingivalis infection in BALB/cByJ mice but not in A/J mice. The basal expression of interleukin-15 ( Il15 ) in the gingiva and the basal expression of p-selectin ( Selp ) in the spleen were higher in the resistant A/J mice than in the susceptible BALB/cByJ mice. In the resistant A/J mice the expression of no genes detectably changed in the gingiva after infection. These results suggest a molecular phenotype in which discrete sets of differentially expressed genes are associated with genetically determined susceptibility ( Il1b , Tnf , and Stat6 ) or resistance ( Il15 and Selp ) to alveolar bone loss, providing insight into the genetic etiology of this complex disease.

Plasmodium falciparum (P.f) infection is a major cause of morbidity and mortality world-wide where symptoms and death occur as a result of the blood stage of the P.f. life cycle. To date, there is no effective blood stage malarial vaccine. Natural Killer (NK) cells are key players in the control of hematopoietic cancers and viral infections, however their role in blood stage malaria is unknown. We undertook a comprehensive analysis of NK cell phenotype and function in a cohort of subjects from a malaria clinical study in Mali, Africa. Using an unbiased analysis of different NK cell subsets, we found that ‘adaptive’ NK cells (lacking FcR gamma chain) increased with age and correlated with reduced malaria risk. Adaptive NK cells had enhanced antibody dependent cellular cytotoxicity (ADCC) function relative to other NK cell subsets. Antibodies are known to be protective in malaria, however the mechanism of this protection is still being elucidated. We hypothesized the protective mechanism of adaptive NK cells may be adaptive NK cells pairing with malaria antibodies to clear infected RBCs via ADCC. In a parallel study, we have shown that NK cells from healthy US donors can inhibit the growth of Plasmodium falciparum 3D7 strain in vitro in the presence of IgG from malaria-exposed adults. Adaptive NK cells from Mali also have enhanced ADCC to infected RBCs. This work indicates that protective antibodies may pair with the strong ADCC activity of adaptive NK cells to protect human subjects from malaria symptoms. These findings thus open a new avenue for vaccine development exploring antibody-dependent cell mediated killing of malaria parasites.

Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection, responsible for a large fraction of the nearly 500,000 malaria-related deaths annually. In the experimental mouse model of CM (ECM), mice are inoculated with red blood cells infected with Plasmodium berghei ANKA (PbA) parasites and develop a CM-like disease within 6–10 days post-infection (dpi) resulting from CD8 T cell-mediated damage to the CNS. We found that treatment of C57Bl/6 mice with interleukin (IL)-15 complexes (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) prevented the development of PbA-induced ECM. IL-15C treatment stimulates natural killer (NK) and CD8 T cells. Interestingly, adoptive transfer of IL-15C-stimulated NK cells, but not CD8 T cells, prevented ECM. Similar complexes formed with IL-2 (IL-2C; IL-2 bound to the anti-IL-2 S4B6 antibody) also induce robust expansion and activation of NK cells, but fail to protect against ECM. Rescue from ECM was not associated with reduced parasitemia at 6 dpi. Instead, IL-15C treatment resulted in reduced CD8 T cell activation in the brain at 6 dpi and reduced blood brain barrier breakdown. Moreover, we found that in vivo IL-15C, but not IL-2C, treatment of mice induces IL-10 expression in NK cells, and that IL-10 is required for IL-15C-mediated protection from ECM. These data indicate that NK cells – which are typically involved in promoting inflammatory responses – can also restrain damaging immune responses, and that differences between the activation of NK cells by IL-2C and IL-15C regulates their capacity to control the inflammatory response to blood stage malaria infection.

In areas of high Plasmodium transmission, humans develop clinical immunity only after years of recurrent exposure. This naturally acquired immunity depends primarily on antibodies specific for parasite antigens. The underlying basis of this protective response remains unclear. In particular, the contribution of antibody-dependent cellular cytotoxicity (ADCC) to malaria immunity remains largely unexplored. Peripheral blood natural killer (NK) cells exhibit potent ADCC through FcγRIII (CD16) binding to antibody-coated target cells. We have developed sensitive and quantitative in vitro assays to evaluate outcome of infected red blood cells (RBC) incubation with NK cells. Without specific antibodies, NK cell-mediated natural cytotoxicity was undetectable towards both uninfected and infected RBCs. Addition of serum antibodies raised against human RBCs in immunized rabbits, however, produced NK cell-dependent lysis of both uninfected and infected RBCs. Rabbit serum antibodies specific for PfEMP1, an immunodominant P. falciparum variant antigen at the surface of infected RBCs, yielded selective lysis of infected RBCs. Plasma from malaria-immune individuals also triggered selective NK-mediated ADCC of infected RBCs. Finally, incubation of infected RBCs with NK cells in the presence of plasma from malaria-immune individuals inhibited P. falciparum growth, as determined by reinvasion of fresh, uninfected RBCs. Therefore, primary human NK cells can limit growth of bloodstream P. falciparum through specific ADCC-mediated lysis of infected RBCs.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. Pathological studies of CM victims reveal vascular hemorrhaging and edema within the brain, yet the underlying mechanisms that lead to this fatal condition are unknown. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the factors that drive the pathogenesis of CM. We found that ECM is associated with a large influx of immune cells into the central nervous system (CNS) at the peak of infection. By depleting individual innate and adaptive immune cell populations, we discovered that CD8 T cells alone were capable of mediating the fatal pathology associated with ECM, including breakdown of the blood brain barrier, edema, and extensive neuronal death in the brainstem. Using two-photon microscopy, we observed parasite-specific CD8 T cells slowly crawling and arresting along the CNS vasculature in vivo during ECM. This behavior was facilitated by the IFN-γ-dependent upregulation of antigen presentation and adhesion molecules on CNS endothelial cells (ECs). Antibody therapy against the adhesion molecules VLA-4 and LFA-1 disrupted CD8 T cell interactions with the CNS vasculature and prevented disease. Importantly, infected mice rendered genetically unable to present antigen on ECs had reduced CD8 T cell-brain EC interactions and were resistant to ECM. These results demonstrate that parasite-specific CD8 T cells interact with brain vascular ECs in an antigen-dependent manner to induce fatal vascular and neuronal pathology during ECM.

The Krüppel-like factor family of transcription factors plays an important role in differentiation, function, and homeostasis of many cell types. While their role in lymphocytes is still being determined, it is clear that these factors influence processes as varied as lymphocyte quiescence, trafficking, differentiation, and function. This review will present an overview of how these factors operate and coordinate with each other in lymphocyte regulation.

The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to decreased expression of the trafficking molecules CD62L and β7-integrin, yet expression of sphingosine-1 phosphate receptor 1 (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly, Klf2 deletion led to a drastic reduction in the B1 B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells.

The transcription factor Kruppel-like factor 2 (KLF2) was proposed to regulate genes involved in cell cycle entry and T cell trafficking; however, the physiological role of its expression in postactivated T cells is not well defined. Previous studies suggested that the cytokines IL-2 and IL-15 differentially regulate KLF2 re-expression in postactivation T cells and that these cytokines also influence effector versus memory T cell differentiation. Using conditional and inducible KLF2-knockout model systems, we tested the specific role of KLF2 expression in activated CD8+ T cells cultured with these cytokines. KLF2 was required for effective transcription of sphingosine-1-phosphate receptor-1 (S1P1) and CD62L in postactivation T cells. However, although different cytokines dramatically altered the expression of cell-cycle–related genes, endogenous KLF2 had a minimal impact. Correspondingly, KLF2-deficient T cells showed dysregulated trafficking but not altered proliferative characteristics following in vivo responses to Ag. Thus, our data help to define KLF2-dependent and -independent aspects of activatedCD8+ T cell differentiation and argue against a physiological role in cell cycle regulation.

New techniques are being applied to identify all the genes involved in mammalian gonad development and differentiation. As this list of genes increases, understanding the potential interactions between these genes will become increasingly difficult. We used a real time reverse transcription PCR (real time RTPCR) protocol to examine and compare the relative expression levels of 55 genes in individual mouse fetal gonads. Real time PCR analysis demonstrated that except for Sry, no differences in relative gene expression were detectable between XX and XY gonad/mesonephroi complexes at embryonic day (E)11.5. Following Sry peak expression at E11.5, a number of genes were expressed at significantly higher relative levels in E12-14 XY than XX gonads. Of six genes expressed at higher levels in E12.5-14 XX than XY gonads, three, Bmp2, Emx2, and Fgfr2, had not been reported previously. Our results caution that differential localization patterns observed with whole mount in situ hybridization techniques may not accurately reflect changes in transcript levels. We conclude that real time PCR is an efficient and powerful tool for studying multiple gene expression patterns during gonad development and differentiation, and can provide insight into gene interactions.