Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens.

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.

Illuminating T cell-dendritic cell interactions in vivo by FlAsHing antigens.

Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.

Contributions of Diet and Age to Ulcerative Dermatitis in Female C57BL/6J Mice.

C57BL/6J (B6) mice are commonly affected by ulcerative dermatitis (UD), a disease of unknown etiology with poor response to treatment. To study the possible role of diet in UD, we compared skin changes in B6 female mice fed a high-fat diet with those of mice fed a control diet. In addition, skin samples from mice with no, mild, moderate, and severe clinical signs of UD were examined by light and transmission electron microscopy (TEM). Mice fed a high-fat diet for 2 mo had more skin mast cell degranulation than did mice fed the control diet for the same period. Regardless of diet, older mice had more skin mast cells and more of these cells were degranulating as compared with younger mice. Microscopic changes in very early lesions were characterized by an increase in dermal mast cells and degranulation with focal areas of epidermal hyperplasia with or without hyperkeratosis. As the condition progressed, a mixed but predominantly neutrophilic inflammatory cell infiltrate appeared in the dermis, with or without epidermal erosion and scab formation. TEM showed that dermal mast cell membranes had disrupted and released of large number of electron dense granules, whereas degranulated mast cells were filled with isolated and coalescing empty spaces due to fusion of granule membranes. Ulceration appeared to occur very quickly, probably as result of intense scratching due to the pruritogenic properties of the histamine released from mast cell granules. This study showed a direct correlation between dietary fat and skin mast cell degranulation in female B6 mice. In addition, the number of skin mast cells and degranulation rates was higher in older mice. Treatments directed at preventing mast cell degranulation may result in better outcomes when applied early in UD cases. As noted previously in studies using caloric restriction, lower fat content in rodent diets may help prevent UD.

Cerebral Malaria Is Regulated by Host-Mediated Changes in Plasmodium Gene Expression.

Cerebral malaria (CM), the deadliest complication of Plasmodium infection, is a complex and unpredictable disease. However, our understanding of the host and parasite factors that cause CM is limited. Using a mouse model of CM, experimental CM (ECM), we performed a three-way comparison between ECM-susceptible C57BL/6 mice infected with ECM-causing Plasmodium ANKA parasites [ANKA(C57BL/6)], ECM-resistant BALB/c mice infected with Plasmodium ANKA [ANKA(BALB/c)], and C57BL/6 mice infected with Plasmodium NK65 that does not cause ECM [NK65(C57BL/6)]. All ANKA(C57BL/6) mice developed CM. In contrast, in ANKA(BALB/c) and NK65(C57BL/6), infections do not result in CM and proceed similarly in terms of parasite growth, disease course, and host immune response. However, parasite gene expression in ANKA(BALB/c) was remarkably different than that in ANKA(C57BL/6) but similar to the gene expression in NK65(C57BL/6). Thus, Plasmodium ANKA has an ECM-specific gene expression profile that is activated only in susceptible hosts, providing evidence that the host has a critical influence on the outcome of infection. IMPORTANCE Hundreds of thousands of lives are lost each year due to the brain damage caused by malaria disease. The overwhelming majority of these deaths occur in young children living in sub-Saharan Africa. Thus far, there are no vaccines against this deadly disease, and we still do not know why fatal brain damage occurs in some children while others have milder, self-limiting disease progression. Our research provides an important clue to this problem. Here, we showed that the genetic background of the host has an important role in determining the course and the outcome of the disease. Our research also identified parasite molecules that can potentially be targeted in vaccination and therapy approaches.

Analysis of Mitochondrial Performance in Lymphocytes Using Fluorescent Lifetime Imaging Microscopy.

During lymphocyte maturation and differentiation, cells undergo a series of proliferative stages interrupted with stages of low activity. The rapid proliferation stages are marked by changes in metabolic outputs-adapting to energy demands by either hindering or utilizing metabolic pathways. As such, it is necessary to view these changes in real time; however, current strategies for metabolomics are time consuming and very rarely provide a holistic profile of the cellular metabolism while also characterizing mitochondrial metabolism. Here, we devised a fluorescence lifetime imaging microscopy (FLIM) strategy to image mitochondrial metabolic profiles in lymphocytes as they go through changes in metabolic activity. Our method provides not only a comprehensive view of cellular metabolism but also narrow in mitochondrial contributions while also efficiently excluding non-viable cells with and without the use of a viability dye. Our novel imaging strategy offers a reliable tool to study changes in mitochondrial metabolism.

The Metabolic Clock Model of B Cell Activation and Differentiation.

Recent advancements in the field of B cell immunometabolism have provided mechanistic insights to B cell activation and fate determination. Here, in this short article, I will explain the main principles of our novel metabolic clock model and how it may reshape our perspective on longstanding immunological questions related to pathologies arising from out of context B cell activation.

High-Resolution Multicolor Imaging of Mitochondria in Lymphocytes.

The identification of cellular changes that accompany immune activation has been a long-standing interest for immunologists. Among these, alterations in the metabolic states of these cells have gained particular attention in the last decade due to the emergence of the field of immunometabolism. A thorough investigation of these metabolic changes can only be achieved with an in-depth visualization of mitochondrial organization; however, current strategies for mitochondrial imaging have been optimized in model cells with a high cytoplasm-to-nucleus ratio and thus are not readily adaptable for many immune cells. Here, we devised a multicolor high-resolution microscopy strategy to image mitochondrial morphology in lymphocytes at both their resting and activated states. Our method allowed us to stain both the mitochondrial surface (by targeting TOM-20) and the mitochondrial matrix (through the use of Mitotracker dyes) while efficiently excluding nonviable cells. Our novel imaging strategy offers a powerful tool to study changes in mitochondrial morphology and complements any research focusing on lymphocyte metabolism.

Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.

A single-nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor alters the development of host immunity.

The acquisition of malaria immunity is both remarkably slow and unpredictable. At present, we know little about the malaria parasite genes that influence the host's ability to mount a protective immune response. Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasite Plasmodium berghei (Pb) NK65 allowed infected mice to mount a T helper cell 1 (TH1)-type immune response that controlled subsequent infections. As compared to PbNK65S, PbNK65F parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. PbNK65F infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell-specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies. Thus, the Pb ApiAP2 transcription factor functions as a critical parasite virulence factor in malaria infections.

B cell memory: building two walls of protection against pathogens.

Surviving a single infection often results in lifelong immunity to the infecting pathogen. Such protection is mediated, in large part, by two main B cell memory 'walls' - namely, long-lived plasma cells and memory B cells. The cellular and molecular processes that drive the production of long-lived plasma cells and memory B cells are subjects of intensive research and have important implications for global health. Indeed, although nearly all vaccines in use today depend on their ability to induce B cell memory, we have not yet succeeded in developing vaccines for some of the world's most deadly diseases, including AIDS and malaria. Here, we describe the two-phase process by which antigen drives the generation of long-lived plasma cells and memory B cells and highlight the challenges for successful vaccine development in each phase.

B cell signaling in context.

Over the past several decades, B cell antigen receptor (BCR)-induced signaling pathways have been described in extraordinary molecular detail, mainly from studies of B cell responses to antigens in vitro. BCR signaling has been shown to govern the initiation of transcriptional programs associated with B cell activation and fate decisions, as well as the BCR-dependent processing of antigen and presentation of antigen to T cells. However, although the potential of the BCR to orchestrate B cell behavior was known, there was no clear appreciation of the context in which B cells signal in secondary lymphoid organs in vivo or how that context influences signaling. In this Review, we describe the current view of the cellular consequences of BCR signaling and advances in the understanding of B cell signaling in context in vivo.

Regulatory T cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells.

Regulatory T cells (Treg cells) can activate multiple suppressive mechanisms in vitro after activation via the T cell antigen receptor, resulting in antigen-independent suppression. However, it remains unclear whether similar pathways operate in vivo. Here we found that antigen-specific Treg cells activated by dendritic cells (DCs) pulsed with two antigens suppressed conventional naive T cells (Tnaive cells) specific for both cognate antigens and non-cognate antigens in vitro but suppressed only Tnaive cells specific for cognate antigen in vivo. Antigen-specific Treg cells formed strong interactions with DCs, resulting in selective inhibition of the binding of Tnaive cells to cognate antigen yet allowing bystander Tnaive cell access. Strong binding resulted in the removal of the complex of cognate peptide and major histocompatibility complex class II (pMHCII) from the DC surface, reducing the capacity of DCs to present antigen. The enhanced binding of Treg cells to DCs, coupled with their capacity to deplete pMHCII, represents a novel pathway for Treg cell-mediated suppression and may be a mechanism by which Treg cells maintain immune homeostasis.

From zero to sixty and back to zero again: the metabolic life of B cells.

Throughout their lifetimes B cells shift metabolic gears to move rapidly from quiescent states to full out proliferative expansion and back again. Here we discuss recent findings that shed light on how B cells rapidly shift gears to metabolically fuel expansion and then just as rapidly down shift during phases of receptor rearrangements to ensure genome stability. We also discuss the link between metabolic activity and fate decisions in B cells.

Increased Mitochondrial Biogenesis and Reactive Oxygen Species Production Accompany Prolonged CD4+ T Cell Activation.

Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for 4 d following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.

Second signals rescue B cells from activation-induced mitochondrial dysfunction and death.

B cells are activated by two temporally distinct signals, the first provided by the binding of antigen to the B cell antigen receptor (BCR), and the second provided by helper T cells. Here we found that B cells responded to antigen by rapidly increasing their metabolic activity, including both oxidative phosphorylation and glycolysis. In the absence of a second signal, B cells progressively lost mitochondrial function and glycolytic capacity, which led to apoptosis. Mitochondrial dysfunction was a result of the gradual accumulation of intracellular calcium through calcium response-activated calcium channels that, for approximately 9 h after the binding of B cell antigens, was preventable by either helper T cells or signaling via the receptor TLR9. Thus, BCR signaling seems to activate a metabolic program that imposes a limited time frame during which B cells either receive a second signal and survive or are eliminated.

Toll-like receptor 9 antagonizes antibody affinity maturation.

Key events in T cell-dependent antibody responses, including affinity maturation, are dependent on the B cell's presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells' capture and presentation of antigen.

T cell-dependent antigen adjuvanted with DOTAP-CpG-B but not DOTAP-CpG-A induces robust germinal center responses and high affinity antibodies in mice.

The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. Here we compared the outcome of immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with the toll-like receptor 9 (TLR9) ligands, CpG-A or CpG-B, alone or conjugated with the cationic lipid carrier, DOTAP. We provide evidence that only NP-CGG adjuvanted with DOTAP-CpG-B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP-specific antibodies. The effectiveness of DOTAP-CpG-B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP-CpG-B but not DOTAP-CpG-A is an effective adjuvant for T cell-dependent protein antigen-based vaccines.

B Cells Produce Type 1 IFNs in Response to the TLR9 Agonist CpG-A Conjugated to Cationic Lipids.

B cells express the innate receptor, TLR9, which signals in response to unmethylated CpG sequences in microbial DNA. Of the two major classes of CpG-containing oligonucleotides, CpG-A appears restricted to inducing type 1 IFN in innate immune cells and CpG-B to activating B cells to proliferate and produce Abs and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. In this study we show that both CpG-B and CpG-A activated B cells in vitro to proliferate, secrete Abs and IL-6, and that neither CpG-B nor CpG-A alone induced type 1 IFN production. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B, induced a type 1 IFN response in B cells in vitro and in vivo. We provide evidence that differences in the function of CpG-A and CpG-B may be related to their intracellular trafficking in B cells. These findings fill an important gap in our understanding of the B cell response to CpGs, with implications for the use of CpG-A and CpG-B as immunomodulators.

Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies.

Ex-vivo differentiation of regulatory T cells (Tregs) from naïve CD4+ T-cells has been widely used in immunological research. Isolation of a highly pure naïve T cell population is the key factor that determines the efficiency of subsequent Treg differentiation. Currently, this step relies mostly on FACS sorting, which is often costly, time consuming, and inconvenient. Alternatively, magnetic separation of T-cells can be performed; yet, available protocols fail to reach sort level purity and consequently result in low Treg differentiation efficiency. Here, we present the results of a comprehensive side-by-side comparison of various magnetic separation strategies and FACS sorting in multiple levels. Additionally, we propose a novel optimized custom made magnetic separation protocol, which not only yields sort level purity and Treg differentiation but also lowers the reagent costs up to 75% compared to the commercially available purification kits. The highly pure naïve CD4+ T-cell population obtained by this versatile method can also be used for differentiation of other T-cell subsets; therefore this protocol may have broad applications in T-cell research.

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.