A hepatic network of dendritic cells mediates CD4 T cell help outside lymphoid organs.

While CD4+ T cells are a prerequisite for CD8+ T cell-mediated protection against intracellular hepatotropic pathogens, the mechanisms facilitating the transfer of CD4-help to intrahepatic CD8+ T cells are unknown. Here, we developed an experimental system to investigate cognate CD4+ and CD8+ T cell responses to a model-antigen expressed de novo in hepatocytes and reveal that after initial priming, effector CD4+ and CD8+ T cells migrate into portal tracts and peri-central vein regions of the liver where they cluster with type-1 conventional dendritic cells. These dendritic cells are locally licensed by CD4+ T cells and expand the number of CD8+ T cells in situ, resulting in larger effector and memory CD8+ T cell pools. These findings reveal that CD4+ T cells promote intrahepatic immunity by amplifying the CD8+ T cell response via peripheral licensing of hepatic type-1 conventional dendritic cells and identify intrahepatic perivascular compartments specialized in facilitating effector T cell-dendritic cell interactions.

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.

Using Full-Spectrum Flow Cytometry to Phenotype Memory T and NKT Cell Subsets with Optimized Tissue-Specific Preparation Protocols.

Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ. This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full-spectrum flow cytometry. We provide a 21-color antibody panel for identification of different memory subsets, including tissue-resident memory T (TRM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver TRM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Processing mouse liver for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 2: Processing mouse spleen for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 3: Processing mouse lungs for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 4: Processing mouse lymph nodes for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 5: Staining and flow cytometric analysis of samples for memory T and NKT cell subsets Support Protocol: Obtaining cell counts from flow cytometry data.

The liver contains distinct interconnected networks of CX3CR1+ macrophages, XCR1+ type 1 and CD301a+ type 2 conventional dendritic cells embedded within portal tracts.

Portal tracts are key intrahepatic structures where leukocytes accumulate during immune responses. They contain the blood inflow, which includes portal blood from the gut, and lymphatic and biliary outflow of the liver, and as such represent a key interface for potential pathogen entry to the liver. Myeloid cells residing in the interstitium of the portal tract might play an important role in the surveillance or prevention of pathogen dissemination; however, the exact composition and localization of this population has not been explored fully. Our in-depth characterization of portal tract myeloid cells revealed that in addition to T lymphocytes, portal tracts contain a heterogeneous population of MHCIIhigh myeloid cells with potential antigen presenting cell (APC) function. These include a previously unreported subset of CSF1R-dependent CX3CR1+ macrophages that phenotypically and morphologically resemble liver capsular macrophages, as well as the two main dendritic cell subsets (cDC1 and cDC2). These cells are not randomly distributed, but each subset forms interconnected networks intertwined with specific components of the portal tract. The CX3CR1+ cells were preferentially detected along the outer border of the portal tracts, and also in the portal interstitium adjacent to the portal vein, bile duct, lymphatic vessels and hepatic artery. cDC1s abounded along the lymphatic vessels, while cDC2s mostly surrounded the biliary tree. The specific distributions of these discrete subsets predict that they may serve distinct functions in this compartment. Overall, our findings suggest that portal tracts and their embedded cellular networks of myeloid cells form a distinctive lymphoid compartment in the liver that has the potential to orchestrate immune responses in this organ.

6″-Modifed α-GalCer-peptide conjugate vaccine candidates protect against liver-stage malaria.

Self-adjuvanting vaccines consisting of peptide epitopes conjugated to immune adjuvants are a powerful way of generating antigen-specific immune responses. We previously showed that a Plasmodium-derived peptide conjugated to a rearranged form of α-galactosylceramide (α-GalCer) could stimulate liver-resident memory T (TRM) cells that were effective killers of liver-stage Plasmodium berghei ANKA (Pba)-infected cells. To investigate if similar or even superior TRM responses can be induced by modifying the α-GalCer adjuvant, we created new conjugate vaccine cadidates by attaching an immunogenic Plasmodium-derived peptide antigen to 6″-substituted α-GalCer analogues. Vaccine synthesis involved developing an efficient route to α-galactosylphytosphingosine (α-GalPhs), from which the prototypical iNKT cell agonist, α-GalCer, and its 6″-deoxy-6″-thio and -amino analogues were derived. Attaching a cathepsin B-cleavable linker to the 6″-modified α-GalCer created pro-adjuvants bearing a pendant ketone group available for peptide conjugation. Optimized reaction conditions were developed that allow for the efficient conjugation of peptide antigens to the pro-adjuvants via oxime ligation to create new glycolipid-peptide (GLP) conjugate vaccines. A single dose of the vaccine candidates induced acute NKT and Plasmodium-specific CD8+ T cell responses that generated potent hepatic TRM responses in mice. Our findings demonstrate that attaching antigenic peptides to 6″-modifed α-GalCer generates powerful self-adjuvanting conjugate vaccine candidates that could potentially control hepatotropic infections such as liver-stage malaria.

Differential location of NKT and MAIT cells within lymphoid tissue.

Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αß T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.

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.

Adrenergic regulation of the vasculature impairs leukocyte interstitial migration and suppresses immune responses.

The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of ß-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.

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.

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.

CD8+ T Cell Activation Leads to Constitutive Formation of Liver Tissue-Resident Memory T Cells that Seed a Large and Flexible Niche in the Liver.

Liver tissue-resident memory T (Trm) cells migrate throughout the sinusoids and are capable of protecting against malaria sporozoite challenge. To gain an understanding of liver Trm cell development, we examined various conditions for their formation. Although liver Trm cells were found in naive mice, their presence was dictated by antigen specificity and required IL-15. Liver Trm cells also formed after adoptive transfer of in vitro-activated but not naive CD8+ T cells, indicating that activation was essential but that antigen presentation within the liver was not obligatory. These Trm cells patrolled the liver sinusoids with a half-life of 36 days and occupied a large niche that could be added to sequentially without effect on subsequent Trm cell cohorts. Together, our findings indicate that liver Trm cells form as a normal consequence of CD8+ T cell activation during essentially any infection but that inflammatory and antigenic signals preferentially tailor their development.

A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.

The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood-borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver-resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self-renewing. LCM numbers increased after weaning in a microbiota-dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non-overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver.

The pro-fibrotic role of dipeptidyl peptidase 4 in carbon tetrachloride-induced experimental liver injury.

Liver fibrosis is a progressive pathological process involving inflammation and extracellular matrix deposition. Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a cell surface glycoprotein and serine protease. DPP4 binds to fibronectin, can inactivate specific chemokines, incretin hormone and neuropeptides, and influences cell adhesion and migration. Such properties suggest a pro-fibrotic role for this peptidase but this hypothesis needs in vivo examination. Experimental liver injury was induced with carbon tetrachloride (CCl4) in DPP4 gene knockout (gko) mice. DPP4 gko had less liver fibrosis and inflammation and fewer B cell clusters than wild type mice in the fibrosis model. DPP4 inhibitor-treated mice also developed less liver fibrosis. DNA microarray and PCR showed that many immunoglobulin (Ig) genes and some metabolism-associated transcripts were differentially expressed in the gko strain compared with wild type. CCl4-treated DPP4 gko livers had more IgM+ and IgG+ intrahepatic lymphocytes, and fewer CD4+, IgD+ and CD21+ intrahepatic lymphocytes. These data suggest that DPP4 is pro-fibrotic in CCl4-induced liver fibrosis and that the mechanisms of DPP4 pro-fibrotic action include energy metabolism, B cells, NK cells and CD4+ cells.

Protective immunity to liver-stage malaria.

Despite decades of research and recent clinical trials, an efficacious long-lasting preventative vaccine for malaria remains elusive. This parasite infects mammals via mosquito bites, progressing through several stages including the relatively short asymptomatic liver stage followed by the more persistent cyclic blood stage, the latter of which is responsible for all disease symptoms. As the liver acts as a bottleneck to blood-stage infection, it represents a potential site for parasite and disease control. In this review, we discuss immunity to liver-stage malaria. It is hoped that the knowledge gained from animal models of malaria immunity will translate into a more powerful and effective vaccine to reduce this global health problem.

Liver-Resident Memory CD8+ T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection.

In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.

Effector T cell function rather than survival determines extent and duration of hepatitis in mice.

BACKGROUND & AIMS: Acute hepatitis is often mediated by cytotoxic T lymphocytes (CTLs); however, the intrinsic parameters that limit CTL-mediated liver injury are not well understood. METHODS: To investigate whether acute liver damage is limited by molecules that decrease the lifespan or effector function of CTLs, we used a well-characterized transgenic (Tg) mouse model in which acute liver damage develops upon transfer of T cell receptor (TCR) Tg CD8 T cells. Recipient Tg mice received donor TCR Tg T cells deficient for either the pro-apoptotic molecule Bim, which regulates CTL survival, or suppressor of cytokine signaling-1 (SOCS-1), which controls expression of common gamma chain cytokines; the effects of anti-PD-L1 neutralizing antibodies were also assessed. RESULTS: Use of Bim-deficient donor T cells and/or PD-L1 blockade increased the number of intrahepatic T cells without affecting the degree and kinetic of acute hepatitis. In contrast, SOCS-1-deficient T cells induced a heightened, prolonged acute hepatitis caused by their enhanced cytotoxic function and increased expansion. Although they inflicted more severe acute liver damage, SOCS-1-deficient T cells never precipitated chronic hepatitis and became exhausted. CONCLUSIONS: The degree of acute hepatitis is regulated by the function of CD8 T cells, but is not affected by changes in CTL lifespan. Although manipulation of the examined parameters affected acute hepatitis, persistent hepatitis did not ensue, indicating that, in the presence of high intrahepatic antigen load, changes in these factors in isolation were not sufficient to prevent T cell exhaustion and mediate progression to chronic hepatitis.