Systemic inflammatory response syndrome triggered by blood-borne pathogens induces prolonged dendritic cell paralysis and immunosuppression.

Blood-borne pathogens can cause systemic inflammatory response syndrome (SIRS) followed by protracted, potentially lethal immunosuppression. The mechanisms responsible for impaired immunity post-SIRS remain unclear. We show that SIRS triggered by pathogen mimics or malaria infection leads to functional paralysis of conventional dendritic cells (cDCs). Paralysis affects several generations of cDCs and impairs immunity for 3-4 weeks. Paralyzed cDCs display distinct transcriptomic and phenotypic signatures and show impaired capacity to capture and present antigens in vivo. They also display altered cytokine production patterns upon stimulation. The paralysis program is not initiated in the bone marrow but during final cDC differentiation in peripheral tissues under the influence of local secondary signals that persist after resolution of SIRS. Vaccination with monoclonal antibodies that target cDC receptors or blockade of transforming growth factor ß partially overcomes paralysis and immunosuppression. This work provides insights into the mechanisms of paralysis and describes strategies to restore immunocompetence post-SIRS.

mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Complexing CpG adjuvants with cationic liposomes enhances vaccine-induced formation of liver TRM cells.

Tissue resident memory T cells (TRM cells) can provide effective tissue surveillance and can respond rapidly to infection. Vaccination strategies aimed at generating TRM cells have shown promise against a range of pathogens. We have previously shown that the choice of adjuvant critically influences CD8+ TRM cell formation in the liver. However, the range of adjuvants tested was limited. Here, we assessed the ability of a broad range of adjuvants stimulating membrane (TLR4), endosomal (TLR3, TLR7 and TLR9) and cytosolic (cGAS, RIG-I) pathogen recognition receptors for their capacity to induce CD8+ TRM formation in a subunit vaccination model. We show that CpG oligodeoxynucleotides (ODN) remain the most efficient inducers of liver TRM cells among all adjuvants tested. Moreover, their combination with the cationic liposome DOTAP further enhances the potency, particularly of the class B ODN CpG 1668 and the human TLR9 ligand CpG 2006 (CpG 7909). This study informs the design of efficient liver TRM-based vaccines for their potential translation.

Type I interferon production elicits differential CD4+ T-cell responses in mice infected with Plasmodium berghei ANKA and P. chabaudi.

Plasmodium parasites that infect humans are highly polymorphic, and induce various infections ranging from an asymptomatic state to life-threatening diseases. However, how the differences between the parasites affect host immune responses during blood-stage infection remains largely unknown. We investigated the CD4+ T-cell immune responses in mice infected with P. berghei ANKA (PbA) or P. chabaudi chabaudi AS (Pcc) using PbT-II cells, which recognize a common epitope of these parasites. In the acute phase of infection, CD4+ T-cell responses in PbA-infected mice showed a lower involvement of Th1 cells and a lower proportion of Ly6Clo effector CD4+ T cells than those in Pcc-infected mice. Transcriptome analysis of PbT-II cells indicated that type I interferon (IFN)-regulated genes were expressed at higher levels in both Th1- and Tfh-type PbT-II cells from PbA-infected mice than those from Pcc-infected mice. Moreover, IFN-α levels were considerably higher in PbA-infected mice than in Pcc-infected mice. Inhibition of type I IFN signaling increased PbT-II and partially reversed the Th1 over Tfh bias of the PbT-II cells in both PbA- and Pcc-infected mice. In the memory phase, PbT-II cells in PbA-primed mice maintained higher numbers and exhibited a better recall response to the antigen. However, recall responses were not significantly different between the infection groups after re-challenge with PbA, suggesting the effect of the inflammatory environment by the infection. These observations suggest that the differences in Plasmodium-specific CD4+ T-cell responses between PbA- and Pcc-infected mice were associated with the difference in type I IFN production during the early phase of the infection.

The Batman and Robin of liver-stage immunity to malaria.

Malaria parasites replicate within the liver shortly after infection. This stage can be controlled by CD8 T cells, but which subsets undertake this function is unclear. Lefebvre et al. now elegantly show that effector memory T (TEM) cells are avid participants, working as a dynamic duo with liver tissue-resident memory T (TRM) cells to combat infection.

Elucidating the Motif for CpG Oligonucleotide Binding to the Dendritic Cell Receptor DEC-205 Leads to Improved Adjuvants for Liver-Resident Memory.

DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.

Hemozoin-mediated inflammasome activation limits long-lived anti-malarial immunity.

During acute malaria, most individuals mount robust inflammatory responses that limit parasite burden. However, long-lived sterilizing anti-malarial memory responses are not efficiently induced, even following repeated Plasmodium exposures. Using multiple Plasmodium species, genetically modified parasites, and combinations of host genetic and pharmacologic approaches, we find that the deposition of the malarial pigment hemozoin directly limits the abundance and capacity of conventional type 1 dendritic cells to prime helper T cell responses. Hemozoin-induced dendritic cell dysfunction results in aberrant Plasmodium-specific CD4 T follicular helper cell differentiation, which constrains memory B cell and long-lived plasma cell formation. Mechanistically, we identify that dendritic cell-intrinsic NLRP3 inflammasome activation reduces conventional type 1 dendritic cell abundance, phagocytosis, and T cell priming functions in vivo. These data identify biological consequences of hemozoin deposition during malaria and highlight the capacity of the malarial pigment to program immune evasion during the earliest events following an initial Plasmodium exposure.

CD8+ and CD4+ T Cells Infiltrate into the Brain during Plasmodium berghei ANKA Infection and Form Long-Term Resident Memory.

In the Plasmodium berghei ANKA mouse model of malaria, accumulation of CD8+ T cells and infected RBCs in the brain promotes the development of experimental cerebral malaria (ECM). In this study, we used malaria-specific transgenic CD4+ and CD8+ T cells to track evolution of T cell immunity during the acute and memory phases of P. berghei ANKA infection. Using a combination of techniques, including intravital multiphoton and confocal microscopy and flow cytometric analysis, we showed that, shortly before onset of ECM, both CD4+ and CD8+ T cell populations exit the spleen and begin infiltrating the brain blood vessels. Although dominated by CD8+ T cells, a proportion of both T cell subsets enter the brain parenchyma, where they are largely associated with blood vessels. Intravital imaging shows these cells moving freely within the brain parenchyma. Near the onset of ECM, leakage of RBCs into areas of the brain can be seen, implicating severe damage. If mice are cured before ECM onset, brain infiltration by T cells still occurs, but ECM is prevented, allowing development of long-term resident memory T cell populations within the brain. This study shows that infiltration of malaria-specific T cells into the brain parenchyma is associated with cerebral immunopathology and the formation of brain-resident memory T cells. The consequences of these resident memory populations is unclear but raises concerns about pathology upon secondary infection.

CD49d marks Th1 and Tfh-like antigen-specific CD4+ T cells during Plasmodium chabaudi infection.

Upon activation, specific CD4+ T cells up-regulate the expression of CD11a and CD49d, surrogate markers of pathogen-specific CD4+ T cells. However, using T-cell receptor transgenic mice specific for a Plasmodium antigen, termed PbT-II, we found that activated CD4+ T cells develop not only to CD11ahiCD49dhi cells, but also to CD11ahiCD49dlo cells during acute Plasmodium infection. CD49dhi PbT-II cells, localized in the red pulp of spleens, expressed transcription factor T-bet and produced IFN-γ, indicating that they were type 1 helper T (Th1)-type cells. In contrast, CD49dlo PbT-II cells resided in the white pulp/marginal zones and were a heterogeneous population, with approximately half of them expressing CXCR5 and a third expressing Bcl-6, a master regulator of follicular helper T (Tfh) cells. In adoptive transfer experiments, both CD49dhi and CD49dlo PbT-II cells differentiated into CD49dhi Th1-type cells after stimulation with antigen-pulsed dendritic cells, while CD49dhi and CD49dlo phenotypes were generally maintained in mice infected with Plasmodium chabaudi. These results suggest that CD49d is expressed on Th1-type Plasmodium-specific CD4+ T cells, which are localized in the red pulp of the spleen, and can be used as a marker of antigen-specific Th1 CD4+ T cells, rather than that of all pathogen-specific CD4+ T cells.

Extrafollicular CD4 T cell-derived IL-10 functions rapidly and transiently to support anti-Plasmodium humoral immunity.

Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti-Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria.

Harnessing liver-resident memory T cells for protection against malaria.

INTRODUCTION: Tissue-resident memory T cells (TRM cells) are powerful mediators of protracted adaptive immunity to infection in peripheral organs. Harnessing TRM cells through vaccination hence promises unprecedented potential for protection against infection. A paramount example of this is malaria, a major infectious disease for which immunity through traditional vaccination strategies remains challenging. Liver TRM cells appear to be highly protective against malaria, and recent developments in our knowledge of the biology of these cells have defined promising, novel strategies for their induction. AREAS COVERED: Here, we describe the path that led to the discovery of TRM cells and discuss the importance of liver TRM cells in immunity against Plasmodium spp. infection; we summarize current knowledge on TRM cell biology and discuss the current state and potential of TRM-based vaccination against malaria. EXPERT OPINION: TRM based vaccination has emerged as a promising means to achieve efficient protection against malaria. Recent advances provide a solid basis for continuing the development of this area of research. Deeper understanding of the mechanisms that mediate TRM formation and maintenance and identification of immunogenic and protective target epitopes suitable for human vaccination remain the main challenges for translation of these discoveries.

Development of Plasmodium-specific liver-resident memory CD8+ T cells after heat-killed sporozoite immunization in mice.

Malaria remains a major cause of mortality in the world and an efficient vaccine is the best chance of reducing the disease burden. Vaccination strategies for the liver stage of disease that utilise injection of live radiation-attenuated sporozoites (RAS) confer sterile immunity, which is mediated by CD8+ memory T cells, with liver-resident memory T cells (TRM ) being particularly important. We have previously described a TCR transgenic mouse, termed PbT-I, where all CD8+ T cells recognize a specific peptide from Plasmodium. PbT-I form liver TRM cells upon RAS injection and are capable of protecting mice against challenge infection. Here, we utilize this transgenic system to examine whether nonliving sporozoites, killed by heat treatment (HKS), could trigger the development of Plasmodium-specific liver TRM cells. We found that HKS vaccination induced the formation of memory CD8+ T cells in the spleen and liver, and importantly, liver TRM cells were fewer in number than that induced by RAS. Crucially, we showed the number of TRM cells was significantly higher when HKS were combined with the glycolipid α-galactosylceramide as an adjuvant. In the future, this work could lead to development of an antimalaria vaccination strategy that does not require live sporozoites, providing greater utility.

Plasmodium berghei Hsp90 contains a natural immunogenic I-Ab-restricted antigen common to rodent and human Plasmodium species.

Thorough understanding of the role of CD4 T cells in immunity can be greatly assisted by the study of responses to defined specificities. This requires knowledge of Plasmodium-derived immunogenic epitopes, of which only a few have been identified, especially for the mouse C57BL/6 background. We recently developed a TCR transgenic mouse line, termed PbT-II, that produces CD4+ T cells specific for an MHC class II (I-Ab)-restricted Plasmodium epitope and is responsive to both sporozoites and blood-stage P. berghei. Here, we identify a peptide within the P. berghei heat shock protein 90 as the cognate epitope recognised by PbT-II cells. We show that C57BL/6 mice infected with P. berghei blood-stage induce an endogenous CD4 T cell response specific for this epitope, indicating cells of similar specificity to PbT-II cells are present in the naïve repertoire. Adoptive transfer of in vitro activated TH1-, or particularly TH2-polarised PbT-II cells improved control of P. berghei parasitemia in C57BL/6 mice and drastically reduced the onset of experimental cerebral malaria. Our results identify a versatile, potentially protective MHC-II restricted epitope useful for exploration of CD4 T cell-mediated immunity and vaccination strategies against malaria.

Resident Memory T Cells and Their Role within the Liver.

Immunological memory is fundamental to maintain immunity against re-invading pathogens. It is the basis for prolonged protection induced by vaccines and can be mediated by humoral or cellular responses-the latter largely mediated by T cells. Memory T cells belong to different subsets with specialized functions and distributions within the body. They can be broadly separated into circulating memory cells, which pace the entire body through the lymphatics and blood, and tissue-resident memory T (TRM) cells, which are constrained to peripheral tissues. Retained in the tissues where they form, TRM cells provide a frontline defense against reinfection. Here, we review this population of cells with specific attention to the liver, where TRM cells have been found to protect against infections, in particular those by Plasmodium species that cause malaria.

Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination.

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination.

Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against rodent malaria.

Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver TRM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.

A Natural Peptide Antigen within the Plasmodium Ribosomal Protein RPL6 Confers Liver TRM Cell-Mediated Immunity against Malaria in Mice.

Liver-resident memory CD8+ T (TRM) cells remain in and constantly patrol the liver to elicit rapid immunity upon antigen encounter and can mediate efficient protection against liver-stage Plasmodium infection. This finding has prompted the development of immunization strategies where T cells are activated in the spleen and then trapped in the liver to form TRM cells. Here, we identify PbRPL6120-127, a H2-Kb-restricted epitope from the putative 60S ribosomal protein L6 (RPL6) of Plasmodium berghei ANKA, as an optimal antigen for endogenous liver TRM cell generation and protection against malaria. A single dose vaccination targeting RPL6 provided effective and prolonged sterilizing immunity against high dose sporozoite challenges. Expressed throughout the parasite life cycle, across Plasmodium species, and highly conserved, RPL6 exhibits strong translation potential as a vaccine candidate. This is further advocated by the identification of a broadly conserved, immunogenic HLA-A∗02:01-restricted epitope in P. falciparum RPL6.

Microbiota-Derived Short-Chain Fatty Acids Promote the Memory Potential of Antigen-Activated CD8+ T Cells.

Interactions with the microbiota influence many aspects of immunity, including immune cell development, differentiation, and function. Here, we examined the impact of the microbiota on CD8+ T cell memory. Antigen-activated CD8+ T cells transferred into germ-free mice failed to transition into long-lived memory cells and had transcriptional impairments in core genes associated with oxidative metabolism. The microbiota-derived short-chain fatty acid (SCFA) butyrate promoted cellular metabolism, enhanced memory potential of activated CD8+ T cells, and SCFAs were required for optimal recall responses upon antigen re-encounter. Mechanistic experiments revealed that butyrate uncoupled the tricarboxylic acid cycle from glycolytic input in CD8+ T cells, which allowed preferential fueling of oxidative phosphorylation through sustained glutamine utilization and fatty acid catabolism. Our findings reveal a role for the microbiota in promoting CD8+ T cell long-term survival as memory cells and suggest that microbial metabolites guide the metabolic rewiring of activated CD8+ T cells to enable this transition.