Gut-associated lymphoid tissues (GALT) are the key antigen sampling and adaptive immune inductive sites within the intestinal wall. Human GALT includes the multi-follicular Peyer's patches of the ...ileum, the vermiform appendix, and the numerous isolated lymphoid follicles (ILF) which are distributed along the length of the intestine. Our current understanding of GALT diversity and function derives primarily from studies in mice, and the relevance of many of these findings to human GALT remains unclear. Here we review our current understanding of human GALT diversity, structure, and composition as well as their potential for regulating intestinal immune responses during homeostasis and inflammatory bowel disease (IBD). Finally, we outline some key remaining questions regarding human GALT, the answers to which will advance our understanding of intestinal immune responses and provide potential opportunities to improve the treatment of intestinal diseases.
Sepsis remains a major cause of mortality in critical care, for which specific treatments are lacking. The dysregulated response to infection seen in sepsis includes features of lymphocyte ...dysfunction and exhaustion, suggesting that immune-stimulatory therapy may improve outcomes in certain patient groups. Monoclonal antibodies targeting checkpoint molecules, such as programmed-death 1 protein (PD-1) and its ligand PD-L1, have shown success in stimulating the immune response in patients with cancer, and are being considered for future sepsis trials. The aims of this pilot study were to compare lymphocyte subset expression of PD-1 and its ligands between patients with sepsis and controls; to characterize serum levels of PD-1 and PD-L1 in patients with sepsis and controls, and determine if serum concentrations correlated with cell surface expression.
Expression levels of PD-1, PD-L1 and PD-L2 on four lymphocyte subsets (CD27 + CD19+ B cells, CD27-CD19+ B cells, CD27 + CD4+ T cells and CD27-CD4+ T cells) were compared between 22 patients with sepsis (including 11 survivors and 11 non-survivors) and 11 healthy controls using flow cytometry. Levels of soluble PD-1 and PD-L1 were also compared using commercially available ELISA kits.
Expression of PD-1 and PD-L1 was higher on all lymphocyte subsets in patients with sepsis compared to controls (p < 0.05). PD-L2 expression on CD27+ B cells was also higher in patients with sepsis (p = 0.0317). There was differential expression of PD-1 by CD27 status, with expression being higher in the B and T cell subsets associated with memory status (CD27+ and CD27-, respectively; p < 0.001). Higher PD-1 and PD-L1 expression was not associated with mortality or with a higher risk of nosocomial infection. There were no differences in levels of soluble PD-1 or PD-L1 between patients with sepsis and controls.
Higher expression of PD-1 by memory subpopulations of B cells and CD4+ T cells, with normal soluble PD-1 and PD-L1 in patients with sepsis, are novel findings. This information may be useful to enrich sepsis populations for trials of PD-1/PD-L1 blockade.
Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune ...diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.
Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Igamma-Cmu circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.
Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell-depleting therapies.
Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is ...controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27
CD45RB
population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.
Sepsis represents a dysregulated host response to infection, the extent of which determines the severity of organ dysfunction and subsequent outcome. All trialled immunomodulatory strategies to date ...have resulted in either outright failure or inconsistent degrees of success. Intravenous immunoglobulin (IVIg) therapy falls into the latter category with opinion still divided as to its utility. This article provides a narrative review of the biological rationale for using IVIg in sepsis. A literature search was conducted using the PubMed database (1966 to February 2011). The strategy included the following text terms and combinations of these: IVIg, intravenous immune globulin, intravenous immunoglobulin, immunoglobulin, immunoglobulin therapy, pentaglobin, sepsis, inflammation, immune modulation, apoptosis. Preclinical and extrapolated clinical data of IVIg therapy in sepsis suggests improved bacterial clearance, inhibitory effects upon upstream mediators of the host response (for example, the nuclear factor kappa B (NF-κB) transcription factor), scavenging of downstream inflammatory mediators (for example, cytokines), direct anti-inflammatory effects mediated via Fcγ receptors, and a potential ability to attenuate lymphocyte apoptosis and thus sepsis-related immunosuppression. Characterizing the trajectory of change in immunoglobulin levels during sepsis, understanding mechanisms contributing to these changes, and undertaking IVIg dose-finding studies should be performed prior to further large-scale interventional trials to enhance the likelihood of a successful outcome.
...an important role of the microbiota and their associated antigens is to maintain GALT in an activated, yet balanced, state.2 Here, we discuss some ways in which the chronically activated GALT, ...that are comprised mostly of B cells, can impact intestinal and systemic inflammatory diseases. ...GALT are stimulated by microbiota throughout life. A study that used deep multiplexed methods to investigate the immune drivers associated with ulcerative colitis severity observed that a major parameter associated with the disease is IgG.6 Biases towards IgG-producing plasma cells in inflammatory bowel disease (IBD), which also include IgG subclass biases, have been acknowledged for decades.2 Cutting-edge methods to study cells and tissues now give us the ability to identify the important detail among the many facts we already know. ...Breg cells belong to a network of events driven by the microbiota in a butyrate-dependent way that modulates local and systemic immune responses.
Multiplexed imaging technologies enable the study of biological tissues at single-cell resolution while preserving spatial information. Currently, high-dimension imaging data analysis is ...technology-specific and requires multiple tools, restricting analytical scalability and result reproducibility. Here we present SIMPLI (Single-cell Identification from MultiPLexed Images), a flexible and technology-agnostic software that unifies all steps of multiplexed imaging data analysis. After raw image processing, SIMPLI performs a spatially resolved, single-cell analysis of the tissue slide as well as cell-independent quantifications of marker expression to investigate features undetectable at the cell level. SIMPLI is highly customisable and can run on desktop computers as well as high-performance computing environments, enabling workflow parallelisation for large datasets. SIMPLI produces multiple tabular and graphical outputs at each step of the analysis. Its containerised implementation and minimum configuration requirements make SIMPLI a portable and reproducible solution for multiplexed imaging data analysis. Software is available at "SIMPLI https://github.com/ciccalab/SIMPLI ".
Intestinal homeostasis is maintained by the response of gut-associated lymphoid tissue to bacteria transported across the follicle associated epithelium into the subepithelial dome. The initial ...response to antigens and how bacteria are handled is incompletely understood. By iterative application of spatial transcriptomics and multiplexed single-cell technologies, we identify that the double negative 2 subset of B cells, previously associated with autoimmune diseases, is present in the subepithelial dome in health. We show that in this location double negative 2 B cells interact with dendritic cells co-expressing the lupus autoantigens DNASE1L3 and C1q and microbicides. We observe that in humans, but not in mice, dendritic cells expressing DNASE1L3 are associated with sampled bacteria but not DNA derived from apoptotic cells. We propose that fundamental features of autoimmune diseases are microbiota-associated, interacting components of normal intestinal immunity.
Synchronous colorectal cancers (syCRCs) are physically separated tumours that develop simultaneously. To understand how the genetic and environmental background influences the development of multiple ...tumours, here we conduct a comparative analysis of 20 syCRCs from 10 patients. We show that syCRCs have independent genetic origins, acquire dissimilar somatic alterations, and have different clone composition. This inter- and intratumour heterogeneity must be considered in the selection of therapy and in the monitoring of resistance. SyCRC patients show a higher occurrence of inherited damaging mutations in immune-related genes compared to patients with solitary colorectal cancer and to healthy individuals from the 1,000 Genomes Project. Moreover, they have a different composition of immune cell populations in tumour and normal mucosa, and transcriptional differences in immune-related biological processes. This suggests an environmental field effect that promotes multiple tumours likely in the background of inflammation.
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