CD Maps-Dynamic Profiling of CD1-CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases

Differential effects of Cytomegalovirus carriage on the immune phenotype of middle-aged males and females

The elderly population is more susceptible to infections as a result of an altered immune response, commonly referred to as immunosenescence. Cytomegalovirus (CMV)-infection associated changes in blood lymphocytes are known to impact this process, but the interaction with gender remains unclear. Therefore, we analysed the effects and interaction of gender and CMV on the absolute numbers of a comprehensive set of naive and memory T- and B-cell subsets in people between 50 and 65 years of age. Enumeration and characterisation of lymphocyte subsets by flow cytometry was performed on fresh whole blood samples from 255 middle-aged persons. CMV-IgG serostatus was determined by ELISA. Gender was a major factor affecting immune cell numbers. CMV infection was mainly associated with an expansion of late-differentiated T-cell subsets. CMV+ males carried lower numbers of total CD4+, CD4+ central memory (CM) and follicular helper T-cells than females and CMV-males. Moreover, CMV+ males had significantly lower numbers of regulatory T (Treg)-cells and memory B-cells than CMV+ females. We here demonstrate an interaction between the effects of CMV infection and gender on T-and B-cells in middle-aged individuals. These differential effects on adaptive immunity between males and females may have implications for vaccination strategies at middle-age

B-Cell Dysregulation in Crohn's Disease Is Partially Restored with Infliximab Therapy

<div><p>Background</p><p>B-cell depletion can improve a variety of chronic inflammatory diseases, but does not appear beneficial for patients with Crohn’s disease.</p><p>Objective</p><p>To elucidate the involvement of B cells in Crohn’s disease, we here performed an ‘in depth’ analysis of intestinal and blood B-cells in this chronic inflammatory disease.</p><p>Methods</p><p>Patients with Crohn’s disease were recruited to study B-cell infiltrates in intestinal biopsies (n = 5), serum immunoglobulin levels and the phenotype and molecular characteristics of blood B-cell subsets (n = 21). The effects of infliximab treatment were studied in 9 patients.</p><p>Results</p><p>Granulomatous tissue showed infiltrates of B lymphocytes rather than Ig-secreting plasma cells. Circulating transitional B cells and CD21^low B cells were elevated. IgM memory B cells were reduced and natural effector cells showed decreased replication histories and somatic hypermutation (SHM) levels. In contrast, IgG and IgA memory B cells were normally present and their Ig gene transcripts carried increased SHM levels. The numbers of transitional and natural effector cells were normal in patients who responded clinically well to infliximab.</p><p>Conclusions</p><p>B cells in patients with Crohn’s disease showed signs of chronic stimulation with localization to granulomatous tissue and increased molecular maturation of IgA and IgG. Therapy with TNFα-blockers restored the defect in IgM memory B-cell generation and normalized transitional B-cell levels, making these subsets candidate markers for treatment monitoring. Together, these results suggest a chronic, aberrant B-cell response in patients with Crohn’s disease, which could be targeted with new therapeutics that specifically regulate B-cell function.</p></div

Composition of the blood B-cell compartment in patients with Crohn’s disease.

<p><b>A.</b> Schematic overview of peripheral B-cell subsets. <b>B.</b> Average numbers of blood B cell subsets of 21 patients affected with Crohn’s disease (black bars) and 28 healthy controls (light grey bars). <b>C.</b> Distribution of IgM, IgA and IgG within CD21^low in patients and controls <b>D.</b> Total CD21^low B cells in relation to disease duration. Statistical analyses were performed with the Mann-Whitney test or Spearman correlation; *, P<0.05; **, P<0.01.</p

Clinical and basic immunological characteristics of patients with Crohn’s disease.

<p>Clinical and basic immunological characteristics of patients with Crohn’s disease.</p

IgA and IgG subclass analysis.

<p><b>A,</b> Schematic representation of the constant region of the human IGH locus. <b>B,</b> Distribution of IgA and IgG subclass use in switched transcripts of healthy controls and patients with Crohn’s disease. Total numbers of analyzed sequences are indicated in the middle of the plots. χ² Test was performed to analyze differences in distributions. <b>C,</b> Combined <i>IGHV</i> mutation frequencies in IgA and IgG transcripts in patients and controls (total numbers of sequences indicated between brackets). Grey dots represent unique sequences; red lines represent median values. Statistical analysis was performed with the Mann-Whitney test; *, P<0.05; **, P<0.01; ***, P<0.001.</p

Replication history and SHM levels in <i>IGHV</i> genes of natural effector B cells.

<p><b>A,</b> Replication history of naïve and natural effector B cells as assessed using the KREC assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160103#pone.0160103.ref025" target="_blank">25</a>]. <b>B,</b> <i>IGHV</i> mutation frequencies in rearranged <i>IGH</i> genes of natural effector B cells in patients and controls (total numbers of sequences indicated between brackets). Grey dots represent unique sequences; red lines represent median values. <b>C</b>, Selection for replacement mutation in IGHV-CDR (red line) and IGHV-FR regions (blue lines) as determined with the BASELINe program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160103#pone.0160103.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160103#pone.0160103.ref029" target="_blank">29</a>]. Solid lines represent patients; dashed lines represent healthy controls. Selection Strengths >0 indicate positive selection. <b>D,</b> IGH-CDR3 size distributions. All individual sizes are indicated as grey dots, red lines representing median values. The dashed line represents median values for centroblasts and centrocytes. Sorted cells were analyzed from patients 15, 16, 18 and 19. Controls were published previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160103#pone.0160103.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160103#pone.0160103.ref031" target="_blank">31</a>]. Statistical analysis was performed with the Mann-Whitney test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.</p