Memory B-cell diversity: From early generation to tissue residency and reactivation.

Memory B cells (MBCs) have a crucial function in providing an enhanced response to repeated infections. Upon antigen encounter, MBC can either rapidly differentiate to antibody secreting cells or enter germinal centers (GC) to further diversify and affinity mature. Understanding how and when MBC are formed, where they reside and how they select their fate upon reactivation has profound implications for designing strategies to improve targeted, next-generation vaccines. Recent studies have crystallized much of our knowledge on MBC but also reported several surprising discoveries and gaps in our current understanding. Here, we review the latest advancements in the field and highlight current unknowns. In particular, we focus on timing and cues leading to MBC generation before and during the GC reaction, discuss how MBC become resident in mucosal tissues, and finally, provide an overview of factors shaping MBC fate-decision upon reactivation in mucosal and lymphoid tissues.

Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Autoimmunity: Are we asking the right question?

For decades, the main question immunologists have asked about autoimmunity is "what causes a break in self-tolerance?" We have not found good answers to that question, and I believe we are still so ignorant because it's the wrong question. Rather than a break in self-tolerance, I suggest that many autoimmune diseases might be due to defects in normal tissue physiology.

Critical Role of Synovial Tissue-Resident Macrophage and Fibroblast Subsets in the Persistence of Joint Inflammation.

Rheumatoid arthritis (RA) is a chronic prototypic immune-mediated inflammatory disease which is characterized by persistent synovial inflammation, leading to progressive joint destruction. Whilst the introduction of targeted biological drugs has led to a step change in the management of RA, 30-40% of patients do not respond adequately to these treatments, regardless of the mechanism of action of the drug used (ceiling of therapeutic response). In addition, many patients who acheive clinical remission, quickly relapse following the withdrawal of treatment. These observations suggest the existence of additional pathways of disease persistence that remain to be identified and targeted therapeutically. A major barrier for the identification of therapeutic targets and successful clinical translation is the limited understanding of the cellular mechanisms that operate within the synovial microenvironment to sustain joint inflammation. Recent insights into the heterogeneity of tissue resident synovial cells, including macropahges and fibroblasts has revealed distinct subsets of these cells that differentially regulate specific aspects of inflammatory joint pathology, paving the way for targeted interventions to specifically modulate the behaviour of these cells. In this review, we will discuss the phenotypic and functional heterogeneity of tissue resident synovial cells and how this cellular diversity contributes to joint inflammation. We discuss how critical interactions between tissue resident cell types regulate the disease state by establishing critical cellular checkpoints within the synovium designed to suppress inflammation and restore joint homeostasis. We propose that failure of these cellular checkpoints leads to the emergence of imprinted pathogenic fibroblast cell states that drive the persistence of joint inflammation. Finally, we discuss therapeutic strategies that could be employed to specifically target pathogenic subsets of fibroblasts in RA.

MAIT Cells in Barrier Tissues: Lessons from Immediate Neighbors.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells present at considerable frequencies in human blood and barrier tissues, armed with an expanding array of effector functions in response to homeostatic perturbations. Analogous to other barrier immune cells, their phenotype and function is driven by crosstalk with host and dynamic environmental factors, most pertinently the microbiome. Given their distribution, they must function in diverse extracellular milieus. Tissue-specific and adapted functions of barrier immune cells are shaped by transcriptional programs and regulated through a blend of local cellular, inflammatory, physiological, and metabolic mediators unique to each microenvironment. This review compares the phenotype and function of MAIT cells with other barrier immune cells, highlighting potential areas for future exploration. Appreciation of MAIT cell biology within tissues is crucial to understanding their niche in health and disease.

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Tissue-resident lymphocytes: weaponized sentinels at barrier surfaces.

Tissue-resident immune cells stably localize in tissues largely independent of the circulatory system. While initial studies have focused on the recognition of CD8 + tissue-resident memory T (CD8 T RM) cells, it is now clear that numerous cell types such as CD4 + T cells, gd T cells, innate lymphoid cells and mucosal-associated invariant T (MAIT) cells form stable populations in tissues. They are enriched at the barrier surfaces and within non-lymphoid compartments. They provide an extensive immune network capable of sensing local perturbations of the body's homeostasis. This positioning enables immune cells to positively influence immune protection against infection and cancer but paradoxically also augment autoimmunity, allergy and chronic inflammatory diseases. Here, we highlight the recent studies across multiple lymphoid immune cell types that have emerged on this research topic and extend our understanding of this important cellular network. In addition, we highlight the areas that remain gaps in our knowledge of the regulation of these cells and how a deeper understanding may result in new ways to 'target' these cells to influence disease outcome and treatments.

Recent Advances in Psoriasis Research; the Clue to Mysterious Relation to Gut Microbiome.

Psoriasis is a chronic inflammatory cutaneous disease, characterized by activated plasmacytoid dendritic cells, myeloid dendritic cells, Th17 cells, and hyperproliferating keratinocytes. Recent studies revealed skin-resident cells have pivotal roles in developing psoriatic skin lesions. The balance in effector T cells and regulatory T cells is disturbed, leading Foxp3-positive regulatory T cells to produce proinflammatory IL-17. Not only acquired but also innate immunity is important in psoriasis pathogenesis, especially in triggering the disease. Group 3 innate lymphoid cell are considered one of IL-17-producing cells in psoriasis. Short chain fatty acids produced by gut microbiota stabilize expression of Foxp3 in regulatory T cells, thereby stabilizing their function. The composition of gut microbiota influences the systemic inflammatory status, and associations been shown with diabetes mellitus, cardiovascular diseases, psychomotor diseases, and other systemic inflammatory disorders. Psoriasis has been shown to frequently comorbid with diabetes mellitus, cardiovascular diseases, psychomotor disease and obesity, and recent report suggested the similar abnormality in gut microbiota as the above comorbid diseases. However, the precise mechanism and relation between psoriasis pathogenesis and gut microbiota needs further investigation. This review introduces the recent advances in psoriasis research and tries to provide clues to solve the mysterious relation of psoriasis and gut microbiota.

Isolation and Characterization of MAIT Cells from Human Tissue Biopsies.

Human mucosal-associated invariant T (MAIT) cells are unconventional T cells highly enriched in tissues exposed to microbial antigens including the oral, gastrointestinal and genital mucosae, liver, and lung. Here we describe a protocol for isolation and characterization of peripheral blood and tissue-infiltrating MAIT cells by using multicolor flow cytometry. This technology allows the analysis of multiple markers in a single sample at a single-cell level. Study of human samples requires particular care since the sample amount is often limited. We present a protocol optimized for the isolation and characterization of human MAIT cells and the identification of MAIT cell populations detected by simultaneous expression of multiple activation markers and inhibitory receptors.

29-Color Flow Cytometry: Unraveling Human Liver NK Cell Repertoire Diversity.

Recent studies have demonstrated extraordinary diversity in peripheral blood human natural killer (NK) cells and have suggested environmental control of receptor expression patterns on distinct subsets of NK cells. However, tissue localization may influence NK cell differentiation to an even higher extent and less is known about the receptor repertoire of human tissue-resident NK cells. Advances in single-cell technologies have allowed higher resolution studies of these cells. Here, the power of high-dimensional flow cytometry was harnessed to unravel the complexity of NK cell repertoire diversity in liver since recent studies had indicated high heterogeneity within liver NK cells. A 29-color flow cytometry panel allowing simultaneous measurement of surface tissue-residency markers, activating and inhibitory receptors, differentiation markers, chemokine receptors, and transcription factors was established. This panel was applied to lymphocytes across three tissues (liver, peripheral blood, and tonsil) with different distribution of distinct NK cell subsets. Dimensionality reduction of this data ordered events according to their lineage, rather than tissue of origin. Notably, narrowing the scope of the analysis to the NK cell lineage in liver and peripheral blood separated subsets according to tissue, enabling phenotypic characterization of NK cell subpopulations in individual tissues. Such dimensionality reduction, coupled with a clustering algorithm, identified CD49e as the preferred marker for future studies of liver-resident NK cell subsets. We present a robust approach for diversity profiling of tissue-resident NK cells that can be applied in various homeostatic and pathological conditions such as reproduction, infection, and cancer.

Dynamic Metabolic State of Tissue Resident CD8 T Cells.

In the past years, there have been significant advances in the understanding of how environmental conditions alone or in conjunction with pathogen invasion affect the metabolism of T cells, thereby influencing their activation, differentiation, and longevity. Detailed insights of the interlinked processes of activation and metabolism can contribute to major advances in immunotherapies. Naive and memory T cells circulate the body. In a quiescent state with low metabolic demands, they predominantly use oxidative phosphorylation for their energy needs. Recognition of cognate antigen combined with costimulatory signals results in a proliferative burst and effector molecule production, requiring rapid release of energy, achieved via dynamically reprogramming metabolic pathways. After activation, most T cells succumb to activation induced cell death, but few differentiate into memory T cells. Of note, some memory T cells permanently occupy tissues without circulating. These, tissue resident T cells are predominantly CD8 T cells, maintained in a metabolic state distinct from naïve and circulating memory CD8 T cells with elements similar to effector CD8 T cells but without undergoing proliferative burst or secreting immune mediators. They continually interact with tissue cells as part of an immune surveillance network, are well-adapted to the tissues they have made their home and where they may encounter different metabolic environments. In this review, we will discuss recent insights in metabolic characteristics of CD8 T cell biology, with emphasis on tissue resident CD8 T cells at the epithelial barriers.

Hematopoietic stem cell-independent hematopoiesis and the origins of innate-like B lymphocytes.

The current paradigm that a single long-term hematopoietic stem cell can regenerate all components of the mammalian immune system has been challenged by recent findings in mice. These findings show that adult tissue-resident macrophages and innate-like lymphocytes develop early in fetal hematopoiesis from progenitors that emerge prior to, and apparently independently of, conventional long-term hematopoietic stem cells. Here, we discuss these recent findings, which show that an early and distinct wave of hematopoiesis occurs for all major hematopoietic lineages. These data provide evidence that fetal hematopoietic progenitors not derived from the bona fide long-term hematopoietic stem cells give rise to tissue-resident immune cells that persist throughout adulthood. We also discuss recent insights into B lymphocyte development and attempt to synthesize seemingly contradictory recent findings on the origins of innate-like B-1a lymphocytes during fetal hematopoiesis.

A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer.

Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (TRM) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αß and γδ CD8+ intraepithelial T lymphocytes (IELs) and CD4+ T cells. While the development of intestinal γδ CD8+ IELs has been extensively investigated, the origin and function of intestinal CD4+ T cells have not been clarified. We report that CCR9 signals delivered during naïve T cell priming promote the differentiation of a population of α4ß7+ IFN-γ-producing memory CD4+ T cells, which displays a TRM molecular signature, preferentially localizes to the gastrointestinal (GI) tract and associated lymphoid tissue and cannot be mobilized by remote antigenic challenge. We further show that this population shapes the immune microenvironment of GI tissue, thus affecting effector immunity in infection and cancer.

Chasing Mavericks: The quest for defining developmental waves of hematopoiesis.

Hematopoiesis is the process by which mature blood and immune cells are produced from hematopoietic stem and progenitor cells (HSCs and HSPCs). The last several decades of research have shed light on the origin of HSCs, as well as the heterogeneous pools of fetal progenitors that contribute to lifelong hematopoiesis. The overarching concept that hematopoiesis occurs in dynamic, overlapping waves throughout development, with each wave contributing to both continuous and developmentally limited cell types, has been solidified over the years. However, recent advances in our ability to track the production of hematopoietic cells in vivo have challenged several long-held dogmas on the origin and persistence of distinct hematopoietic cell types. In this review, we highlight emerging concepts in hematopoietic development and identify unanswered questions.

Tissue-Specific Roles of NKT Cells in Tumor Immunity.

NKT cells are an unusual population of T cells recognizing lipids presented by CD1d, a non-classical class-I-like molecule, rather than peptides presented by conventional MHC molecules. Type I NKT cells use a semi-invariant T cell receptor and almost all recognize a common prototype lipid, α-galactosylceramide (α-GalCer). Type II NKT cells are any lipid-specific CD1d-restricted T cells that use other receptors and generally don't recognize α-GalCer. They play important regulatory roles in immunity, including tumor immunity. In contrast to type I NKT cells that most have found to promote antitumor immunity, type II NKT cells suppress tumor immunity and the two subsets cross-regulate each other, forming an immunoregulatory axis. They also can promote other regulatory cells including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and can induce MDSCs to secrete TGF-ß, one of the most immunosuppressive cytokines known. In some tumors, both Tregs and type II NKT cells can suppress immunosurveillance, and the balance between these is determined by a type I NKT cell. We have also seen that regulation of tumor immunity can depend on the tissue microenvironment, so the same tumor in the same animal in different tissues may be regulated by different cells, such as type II NKT cells in the lung vs Tregs in the skin. Also, the effector T cells that protect those sites when Tregs are removed do not always act between tissues even in the same animal. Thus, metastases may require different immunotherapy from primary tumors. Newly improved sulfatide-CD1d tetramers are starting to allow better characterization of the elusive type II NKT cells to better understand their function and control it to overcome immunosuppression.

Mycobacterium tuberculosis Invasion of the Human Lung: First Contact.

Early immune responses to Mycobacterium tuberculosis (Mtb) invasion of the human lung play a decisive role in the outcome of infection, leading to either rapid clearance of the pathogen or stable infection. Despite their critical impact on health and disease, these early host-pathogen interactions at the primary site of infection are still poorly understood. In vitro studies cannot fully reflect the complexity of the lung architecture and its impact on host-pathogen interactions, while animal models have their own limitations. In this study, we have investigated the initial responses in human lung tissue explants to Mtb infection, focusing primarily on gene expression patterns in different tissue-resident cell types. As first cell types confronted with pathogens invading the lung, alveolar macrophages, and epithelial cells displayed rapid proinflammatory chemokine and cytokine responses to Mtb infection. Other tissue-resident innate cells like gamma/delta T cells, mucosal associated invariant T cells, and natural killer cells showed partially similar but weaker responses, with a high degree of variability across different donors. Finally, we investigated the responses of tissue-resident innate lymphoid cells to the inflammatory milieu induced by Mtb infection. Our infection model provides a unique approach toward host-pathogen interactions at the natural port of Mtb entry and site of its implantation, i.e., the human lung. Our data provide a first detailed insight into the early responses of different relevant pulmonary cells in the alveolar microenvironment to contact with Mtb. These results can form the basis for the identification of host markers that orchestrate early host defense and provide resistance or susceptibility to stable Mtb infection.

Liver sampling: a vital window into HBV pathogenesis on the path to functional cure.

In order to optimally refine the multiple emerging drug targets for hepatitis B virus (HBV), it is vital to evaluate virological and immunological changes at the site of infection. Traditionally liver biopsy has been the mainstay of HBV disease assessment, but with the emergence of non-invasive markers of liver fibrosis, there has been a move away from tissue sampling. Here we argue that liver biopsy remains an important tool, not only for the clinical assessment of HBV but also for research progress and evaluation of novel agents. The importance of liver sampling has been underscored by recent findings of specialised subsets of tissue-resident immune subsets capable of efficient pathogen surveillance, compartmentalised in the liver and not sampled in the blood. Importantly, the assessment of virological parameters, such as cccDNA quantitation, also requires access to liver tissue. We discuss strategies to maximise information obtained from the site of infection and disease pathology. Fine needle aspirates of the liver may allow longitudinal sampling of the local virus/host landscape. The careful utilisation of liver tissue and aspirates in conjunction with blood will provide critical information in the assessment of new therapeutics for the functional cure of HBV.

Advancement in regional immunity and its clinical implication.

Organs in our body have formed their own unique immune surveillance system that is finely tuned by in situ milieu. Sequestrated tissue-resident immune cells differ from their counterparts in circulation and participate in tissue physiological activities and the maintenance of local homeostasis. Dysregulation of regional immunity leads to organ-specific inflammatory injuries. Here we review the recent developments in the field of tissue-resident immune cells and organ-specific regional immunity, and discuss their clinical implication.

Development and Function of Effector Regulatory T Cells.

Distinguishing self from nonself is a unique feature of the immune system. Although most self-reactive T cells are eliminated in the thymus, a few rogue cells escape the negative selection process and have the potential to mediate autoimmune disease. Over the last decade, there has been a vast improvement in our understanding of the cellular mechanisms that evolved to dampen the deleterious effects of these self-reactive T cells. In particular, T cells expressing the transcription factor FoxP3, known as regulatory T (Treg) cells, play a central role in maintaining immune homeostasis and suppressing autoimmune responses. In addition, Treg cells are endowed with the ability to suppress diverse inflammatory responses both in lymphoid and in nonlymphoid tissues. This requires Treg cells to undergo a peripheral differentiation and specialization program that results in the emergence of effector Treg (eTreg) cells that are characterized by their ability to produce high amounts of immunosuppressive molecules, including IL-10. This chapter discusses the recent advances in our understanding of the mechanisms governing the differentiation, migration, and maintenance of eTreg cells, in particular in nonlymphoid tissues, in health and disease.

Tissue-resident memory T cells: local guards of the thymus.

T-cell surveillance of nonlymphoid tissues has traditionally been ascribed to recirculating memory T cells that continuously patrol the body. Extending this concept, recent evidence suggests that T cells also exist as nonmigratory memory cells that provide local immune protection in a broad range of peripheral tissues, including barrier locations such as skin and mucosa. In this issue of the European Journal of Immunology, Pircher and colleagues [Eur. J. Immunol. 2013. 43: 2295-2304] demonstrate, for the first time, the existence of such permanently tissue-resident CD8(+) memory T (TRM) cells in a primary lymphoid organ, the thymus. TRM cells in this location provide potent local immunity, which may help to preserve thymic integrity and normal T-cell development in the face of infection with thymus-invading pathogens.