Search

Many of the leukocyte cell surface molecules are known by “CD” numbers. In this Appendix, a short introduction describes the history and the use of CD nomenclature and provides a few key references to enable access to the wider literature. This is followed by a table that lists all human molecules with approved CD names, tabulating alternative names, key structural features, cellular expression, major known functions, and usefulness of the molecules or antibodies against them in research or clinical applications. Curr. Protoc. Immunol. 80:A.4A.1-A.4A.73. © 2008 by John Wiley & Sons, Inc. Keywords: CD nomenclature; HLDA; HCDM; leukocyte marker; human leukocyte differentiation; antigens

CD4+ T-cell subsets play a major role in the host response to infection, and a healthy immune system requires a fine balance between reactivity and tolerance. This balance is in part maintained by regulatory T cells (Treg), which promote tolerance, and loss of immune tolerance contributes to autoimmunity. As the T cells which drive immunity are diverse, identifying and understanding how these subsets function requires specific biomarkers. From a human CD4 Tconv/Treg cell genome wide analysis we identified peptidase inhibitor 16 (PI16) as a CD4 subset biomarker and we now show detailed analysis of its distribution, phenotype and links to Treg function in type 1 diabetes. To determine the clinical relevance of Pi16 Treg, we analysed PI16+ Treg cells from type 1 diabetes patient samples. We observed that FOXP3 expression levels declined with disease progression, suggesting loss of functional fitness in these Treg cells in Type 1 diabetes, and in particular the rate of loss of FOXP3 expression was greatest in the PI16+ve Treg. We propose that PI16 has utility as a biomarker of functional human Treg subsets and may be useful for tracking loss of immune function in vivo. The ability to stratify at risk patients so that tailored interventions can be applied would open the door to personalised medicine for Type 1 diabetes.

The Human Leucocyte Differentiation Antigens Workshops (HLDA) have since 1984 provided a forum for the characterization and study of leucocyte surface molecules and antibodies against them. HLDA devised the CD nomenclature, which is sanctioned by IUIS. The HLDA Council reviewed and modified the objectives of HLDA in 2004, and changed the name of the organization to Human Cell Differentiation Molecules (HCDM) to reflect the broader objectives. Workshop studies under the HCDM banner proceeded during 2005 and early 2006, culminating in a meeting in May 2006. At that meeting the Council, acting as Nomenclature Committee, approved a number of new CD designations and changes to some pre-existing CD designations, which are summarized in this report.

The transcription factor FOXP3 is essential for the formation and function of regulatory T cells (Tregs), and Tregs are essential for maintaining immune homeostasis and tolerance. This is demonstrated by a lethal autoimmune defect in mice lacking Foxp3 and in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome patients. However, little is known about the molecular basis of human FOXP3 function or the relationship between direct and indirect targets of FOXP3 in human Tregs. To investigate this, we have performed a comprehensive genome-wide analysis for human FOXP3 target genes from cord blood Tregs using chromatin immunoprecipitation array profiling and expression profiling. We have identified 5579 human FOXP3 target genes and derived a core Treg gene signature conserved across species using mouse chromatin immunoprecipitation data sets. A total of 739 of the 5579 FOXP3 target genes were differentially regulated in Tregs compared with Th cells, thus allowing the identification of a number of pathways and biological functions overrepresented in Tregs. We have identified gene families including cell surface molecules and microRNAs that are differentially expressed in FOXP3+ Tregs. In particular, we have identified a novel role for peptidase inhibitor 16, which is expressed on the cell surface of >80% of resting human CD25+FOXP3+ Tregs, suggesting that in conjunction with CD25 peptidase inhibitor 16 may be a surrogate surface marker for Tregs with potential clinical application.

CD44 is a widely distributed cell surface glycoprotein that participates in a number of cellular adhesion and signal transduction processes. Germinal center B cells express very low levels of CD44, whereas their precursors and differentiation products express much higher levels. In immunofluorescence studies comparing 20 antibodies classified as being against the hematopoietic isoform of CD44, one antibody, AIG3, was unreactive with germinal center B cells, whereas the other antibodies showed low intensity but definite reactivity. Western blotting and sequential immunoprecipitation studies of lysates from density-separated lymphocyte fractions showed two bands that were differentially expressed and reacted differently with AIG3 compared with the other CD44 antibodies. These results suggest that germinal center B cells and non-germinal center B cells express different forms of CD44.Of 21 malignant B-cell populations examined, 5 showed reactivity with a “standard” CD44 reagent and significantly reduced reactivity with AIG3, while one sample showed the opposite pattern and the remainder were positive for both reagents. Of 10 cell lines studied, 5 were differentially stained by AIG3 and a standard CD44 antibody. PCR amplification of reverse transcribed mRNA from sorted human tonsil B-cell subpopulations and Southern blotting showed that B cells express a number of splice isoforms of CD44. These results demonstrate that B cells express multiple forms of CD44; both splice insert isoforms and variants distinguished by AIG3; the form of CD44 expressed depends on B-cell differentiation state.

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century.

There were over 600 antibodies submitted to HLDA8, with many of unknown specificity. Of these, 101 antibodies were selected for a blind panel study that also included 5 negative controls and 27 positive controls of known CD specificity making a total of 133 antibodies in the final panel. Of the 101 unknowns, 31 antibodies were identified during the course of this blind panel study as being specific for known molecules and included some specific for MHC class II antigens, CD45 isoforms and the Dombrock antigen. Several antibody pairs among those in the blind panel were found to have very similar staining patterns and were therefore compared by immunohistochemical and/or Western blot analyses for identity. © 2005 Elsevier B.V. All rights reserved.

Approximately 340 leucocyte plasma membrane proteins have been characterised by the eight Human Leucocyte Differentiation Antigen workshops held between 1982 and 2004, based primarily on their reactivity with monoclonal antibodies. The human genome is predicted to encode approximately 34,000 cDNA transcripts, of which between 15% and 20% are predicted to contain one or more transmembrane helices. We have used SDS-PAGE separation coupled with mass spectrometry-based peptide mass tag identification to identify novel plasma membrane proteins in microsome preparations prepared from mononuclear cells obtained from human peripheral blood. A total of 361 distinct proteins were identified in a single preparation, including 37 known leucocyte plasma membrane proteins, 27 potential novel plasma membrane proteins whose expression on PBMC is poorly characterised, and 51 other proteins for which the subcellular location could not be determined. Expression analysis using cDNA panels indicates that several of these novel plasma membrane proteins are differentially expressed in lymphocyte subsets. These results show that previously unidentified lymphocyte plasma membrane proteins can be identified using this approach.

The 8th International Workshop on Human Leucocyte Differentiation Antigens (chaired by Zola H and managed by Swart B) was run over a 4-year period and culminated in a conference in December 2004. Here we review the achievements of the HLDA Workshops and provide links to information on CD molecules and antibodies against them, including the 93 new CDs assigned in the 8th Workshop. We consider what remains to be achieved (including an estimate of the number of leucocyte surface molecules still to be discovered), and how the field can best move forward.

We have directly compared the use of a CD77 antibody with the binding subunit of Shiga-like toxin 1, Verotoxin 1, and (Stx1B) for delineation on human tonsil cells. We determined that the Stx1B produced a greater intensity of staining than the CD77 antibody, allowing three sub-populations of germinal centre cells to be seen. The populations express high, medium, and low levels of globotriaosylceramide as determined by the binding of the Stx1B reagent. The strong staining patterns of Stx1B suggest that it may be useful in defining germinal center B cell populations.

Toll-like receptors (TLRs) are a family of trans-membrane receptors that play an important role in the innate immune system. Most studies examining the cellular expression of TLRs on immune cells have focussed on neutrophils, monocytes and dendritic cells, but there is little evidence of TLRs being expressed on lymphocytes. Using 3-colour flow cytometry, expression of TLR-1, TLR-2, TLR-3, TLR-4, and TLR-9 on peripheral blood lymphocyte populations was determined. Further examination of TLRs on CD5- and CD5+ CD19+ B cell subsets was performed. The binding of TLR1 and TLR9 antibodies was detected on 15-90% of resting B cells, but not on resting T-cells. The higher expression of TLR1 and TLR9 on CD5+ B cells compared to CD5- B cells may reflect the role of B1 cells in more primitive, less specific antibody responses.

SUMMARY Early diagnosis of neonatal infection has proved problematic due to the inadequacy of currently available laboratory tests. Neonatal sepsis is associated with an increase in plasma-derived cytokine levels, but an increase of a single cytokine cannot identify neonatal sepsis specifically and multiple cytokine levels are required. The time constraints and relatively large volume of plasma required to measure multiple cytokines from newborn infants by conventional enzyme-linked immunosorbent assay (ELISA) techniques is prohibitive. We therefore applied cytometric bead array (CBA) technology for simultaneous measurement of multiple cytokines from a group of 18 term neonates with infection confirmed by culture and a control group. ‘Normal’ ranges were established for each cytokine from 1–7-, 8–14- and 15–21-day-old newborns. There was no significant change in the levels of cytokines from infants in different control age groups, suggesting that basal cytokine levels are unchanged in the first 3 weeks of life. In the patient groups, however, there was a significant difference in several cytokines between the different age groups. Interleukin (IL)-6, IL-10 and IL-12 were increased significantly in the 1–7-day-old patient group compared to either the 8–14 and 15–21 age group, suggesting that infection in utero is associated with increased levels of these cytokines compared to infection acquired following birth. When individual patient cytokine levels were compared to normal control reference ranges, two patients failed to show significant elevation of any cytokine tested. All other patients showed elevated levels of between one and nine cytokines tested (mean of 4·6). There was no correlation between elevated cytokine levels and types of infective organism or patient age. In conclusion, neonatal sepsis is associated with the elevation of multiple plasma cytokines. The use of CBA kits is a rapid, easy, low sample volume and sensitive method to measure multiple plasma cytokines.

Cell separation techniques are important in immunology. Major cell populations can be separated successfully with high purity. However, isolation of cells which are specific for particular antigens is more challenging because of the relatively small numbers of antigen-specific cells, and the lack of independent markers available to determine the purity of the isolated population. In this review, the literature describing three principal techniques used to separate antigen-specific cells has been reviewed. Particular emphasis has been placed on yield and purity; the two most important parameters of any purification method. The most promising isolation methods have used immunomagnetic sorting and multiparametric flow cytometric analysis.

The 7th International Workshop on Human Leucocyte Differentiation Antigens (HLDA7) studied a number of newly characterised molecules relevant to human leucocyte differentiation and function. The HLDA organisation, which devised and continues to maintain the CD nomenclature, is responsible, under the auspices of IUIS and WHO, for the nomenclature of all leucocyte differentiation markers. The 7th Workshop redefined a number of (principally carbohydrate) molecules, and assigned CD names to approximately 80 new molecules. This update lists, in tabular form, the redefined and newly assigned names, together with antibodies, which have been confirmed under Workshop conditions as specific for the new and redefined molecules. The major features of the cellular expression patterns are summarised, and a LocusLink accession number provided to enable the reader to access more detailed information through http://www.ncbi.nlm.nih.gov/LocusLink.

IgG antibody can specifically suppress the antibody response to antigen. This has been explained by the hypothesis that signaling through the B cell antigen receptor is negatively modulated by the co-ligation of immunoglobulin with the receptor for IgG, FcgammaRIIb. We hypothesized that inhibitory signaling through FcgammaRIIb would be counter-productive in germinal center cells undergoing selection by affinity maturation, since these cells are thought to receive a survival/proliferative signal by interacting with antigen displayed on follicular dendritic cells. We have identified and characterized a population of B lymphocytes with low/negative FcgammaRIIb expression that are present in human tonsil. Phenotypically these cells correspond to germinal center B cells and comprise both centroblast and centrocyte populations. In examining expression at the molecular level we determined that these B cells do not express detectable mRNA for FcgammaRIIb. We examined several culture conditions to induce expression of FcgammaRIIb on germinal center cells but could not determine conditions that altered expression. We then examined the functional consequence of cross-linking membrane immunoglobulin and the receptor for IgG on human B lymphocytes. Our results cast some doubt on the value of anti-IgG as a model for antigen-antibody complexes in studying human B cell regulation.

We describe an economical 20 litre bench-top fermenter suitable for production of recombinant antibody fragments in bacterial expression systems. The bacterial culture contained within a polycarbonate carboy is mixed (400–600 rpm) and aerated (1 vessel vol./min) by a high-shear radial flow impeller mounted on a hollow stainless steel shaft, through which pressurised air is pumped. Air is dispersed as fine bubbles into the culture medium by the turbine impeller, without the need for a porous sparger. A stainless steel baffle stabilised by a gliding counterweight increases mixing. The components can easily be disassembled for cleaning and sterilisation. Temperature (range 20–37 °C) and pH (range 7.0–7.5) are controlled manually. Using the apparatus, it proved possible to achieve Escherichia coli cell culture densities equivalent to an optical density at 600 nm (OD600) of 30–32, compared with OD600 4–6 in shake flasks. A yield of 40 mg/litre/day of a recombinant antibody fragment was obtained with the fermenter, which was 15-fold more than the yield of 2.5 mg/litre/day achieved in shake flasks. The fermenter may be particularly suited for research purposes.

Reticulin fibrosis has been recognized in childhood ALL at diagnosis as part of the altered stromal structure in the bone marrow (BM). Increased fibre density is correlated with a higher concentration of leukaemia cells in the BM and lower numbers of blasts in peripheral blood. We hypothesize that these fibres anchor the leukaemia cells within the BM in close proximity to BM stromal cells (BMSC). The BMSC are a rich source of growth factors and cytokines which enhance leukaemia cell growth and provide protection against chemotherapy. Mobilizing the cells by breaking the 'anchoring ropes' could lead to greater exposure to apoptotic signals. Refereed/Peer-reviewed

During the past few years identification and characterization of the surface antigens of B lymphocytes using monoclonal antibodies has continued apace. International Workshops on Leucocyte Differentiation Antigens have categorized many antibodies using the cluster of differentiation (CD) system. Here Heddy Zola reviews the recent advances in antigen identification — including those defined by unclustered antibodies as well as CD antigens — and indicates that the bulk of future work will revolve around attempts to place them within a functional framework.

Monoclonal antibodies (Mab) have defined functional T lymphocyte subsets in mouse and man: Ly-1/OKT4 positive helper T cells, Ly-123/OKT8 positive killer T cells and Ly-23/OKT8 positive suppressor T cells. An equivalent array of useful reagents for B-cell subsets is not yet available-although many laboratories are trying to produce them. In this review Ian Mckenzie and Heddy Zola summarize the Mob which are available and which detect polymorphic or monomorphic determinants on the B-cell surface in both man and mouse.

Alterations in the neuro-immune axis contribute toward viscerosensory nerve sensitivity and symptoms in Irritable Bowel Syndrome (IBS). Inhibitory factors secreted from immune cells inhibit cob-rectal afferents in health, and loss of this inhibition may lead to hypersensitivity and symptoms. We aimed to determine the immune cell type(s) responsible for opioid secretion in humans and whether this is altered in patients with IBS. The beta-endorphin content of specific immune cell lineages in peripheral blood and colonic mucosal biopsies were compared between healthy subjects (HS) and IBS patients. Peripheral blood mononuclear cell (PBMC) supernatants from HS and IBS patients were applied to cob-rectal sensory afferent endings in mice with post-inflammatory chronic visceral hypersensitivity (CVH). beta-Endorphin was identified predominantly in monocyte/macrophages relative to T or B cells in human PBMC and colonic lamina propria. Monocyte derived beta-endorphin levels and colonic macrophage numbers were lower in IBS patients than healthy subjects. PBMC supernatants from healthy subjects had greater inhibitory effects on cob-rectal afferent mechanosensitivity than those from IBS patients. The inhibitory effects of PBMC supernatants were more prominent in CVH mice compared to healthy mice due to an increase in mu-opioid receptor expression in dorsal root ganglia neurons in CVH mice. Monocyte/macrophages are the predominant immune cell type responsible for beta-endorphin secretion in humans. IBS patients have lower monocyte derived beta-endorphin levels than healthy subjects, causing less inhibition of colonic afferent endings. Consequently, altered immune function contributes toward visceral hypersensitivity in IBS. Refereed/Peer-reviewed

It is widely recognized that the newborn immune system has limited functionality. The consequences of immune compromise due to immaturity are seen, for example, in high mortality rates due to infection in historical times, and in poor communities today. It is important to understand neonatal and infant immune compromise in qualitative and quantitative detail, so that immunization campaigns can be delivered effectively – we want to protect the infant as early as possible, yet there is no point immunizing infants before their immune system is able to respond. There is even a risk that early exposure to antigen will prevent an effective immune response later. Immune compromise in the neonate and infant is a physiological state, not a disease, and we should be cautious about “treatment.” It is worth considering the possible evolutionary pressures that have led us to be born into a pathogen-rich environment with immune function that appears to be compromised. Could it be that evolutionary pressures have selected the best balance between different threats, and if we stimulate the immune response excessively at this stage in life there will be adverse consequences to health and reproductive fitness later in life?

Cancer patients may make antibodies against antigens on the surface of their malignant cells due either to the expression of unique antigens or to dysregulated responses to self antigens. Patients with B cell malignancy frequently produce autoantibodies and may therefore be a source of immunoglobulin genes for the production of phage display antibody libraries directed against tumour-associated antigens. Patients with autoimmune disease have circulating antibodies against lymphocyte surface antigens, and may also provide a good starting point for the production of a library of lymphocyte-reactive antibody structures. In this study, plasma and serum samples from patients with B cell malignancy or Sjogren's syndrome and from healthy controls were screened for antibodies against the B cell membrane antigens CD20. While the majority of samples showed very low reactivity, some individuals did show significant and reproducible binding to CD20. To identify a good donor for library construction, it would be advisable to screen donors for antibody against the antigens of interest.

aThe Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, United Kingdom; bCentre d'Immunologie, Parc Scientifique de Luminy, Cedex, France; cFaculte de Medicine de Creteil, Cedex, France; dDivision of Immunity and Infection, MRC Center for Immune Regulation, Department of Rheumatology, University of Birmingham, Edgbaston, Birmingham, United Kingdom; eJohns Hopkins Comprehensive Cancer Center, Baltimore, Maryland, USA; fDepartment of Microbiology, University of Washington, Seattle, Washington, USA; gCLB, Department of Experimental Immunohematology, Amsterdam, The Netherlands; hLaboratory of Molecular Hematology/Division of Molecular Medicine, North Shore University Hospital, Manhasset, New York, USA; iKinderklinik, Medizinische Hochschule Hannover, Hannover, Germany; jMater Medical Research Institute, Aubigny Place, Mater Hospital, Queensland, Australia; kInstitute of Molecular Genetics AS CR, Prague, Czechoslovakia; lInstitut fur Immunologie der Univ. Heidelberg, Heidelberg, Germany, mProfessor of Biochemistry, University of Illinois College of Medicine, Urbana, Illinois, USA; nGerman Cancer Research Center, Heidelberg, Germany; oNational Institute of Health, Bethesda, Maryland, USA; pDirector of Biology Celltech RD qInstitute for Research in Biomedicine, Bellinzona, Switzerland; rUniversity of Adelaide, Department of Pediatrics, Women's and Children's Hospital, South Australia, Australia

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of “blind” antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material. We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp. Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression. The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent.

B cells express an Fc receptor for IgG (FcgammaRII; CD32) which is involved in feedback inhibition of antibody production. Engagement of FcgammaRII during ligation of the antigen receptor provides an inhibitory signal. FcgammaRII exists as several isoforms, with FcgammaRIIb (which carries an immunoreceptor tyrosine-based inhibition motif; ITIM) being predominant form on adult B cells. The inhibitory role of FcgammaRIIb may be unhelpful to the infant, since primary exposure to infectious agents is likely to be in the presence of maternal IgG. We hypothesized that neonatal B cells would be less susceptible to feedback inhibition by antibody, either through the expression of activation-competent FcgammaRII isoforms (FcgammaRIIa and FcgammaRIIc) or through reduced expression of the inhibitory FcgammaRIIb isoforms. Cord and adult B cells were examined for expression of FcgammaRII isoforms using monoclonal antibodies and RT-PCR. In vitro assays were performed to assess susceptibility of cord and adult cells to FcgammaRII-mediated suppression. Although there is no phenotypic difference in FcgammaRII expression (FcgammaRIIb predominating on both adult and cord B cells), FcgammaRIIb is expressed at lower levels on cord cells. This quantitative difference in FcgammaRIIb expression may explain the reduced susceptibility of cord B cells to antibody-mediated inhibition observed in these experiments.

The expression of CD80, CD86, CD28, and CD152 were examined on peripheral blood lymphocytes from adults, neonates (cord blood lymphocytes) and young children (2–20 months of age). There was no difference in the expression of CD80 or CD86 between adult and neonatal B cells, either resting or activated. A higher percentage of resting T cells expressed CD28 in neonates and young children compared to adults. CD28 expression was similar on adult and neonatal T cells activated with PMA and ionomycin. However, CD28 was expressed at greater intensity on a higher percentage of neonatal T cells than adult T cells stimulated with CD3. CD152 expression was lower on neonatal T cells than adult T cells stimulated with PMA and ionomycin and undetectable on neonatal T cells stimulated with CD3. In contrast, intracellular CD152 was equivalent in adult and neonatal T cells stimulated with PMA and ionomycin, suggesting trafficking of CD152 to the cell surface may be differentially regulated in neonatal T cells. Since the T cell response is determined by the balance of signals received from CD28 and CD152, high levels of CD28 expression and lower surface expression of CD152 on neonatal T cells may represent specialisation to promote activation of neonatal T cells.

Umbilical cord blood mononuclear cells isolated by density centrifugation are contaminated by erythrocytes and nucleated erythroid precursors which may exceed 50% of the total cell population, and thus interfere with phenotypic, functional and mRNA analyses. Lysis with hypotonic ammonium chloride can overcome this problem, but interferes with lysosomal function and should be avoided when cell preparations are intended for functional studies. The aim of this study was to develop a technique for removing erythroid cells from cord blood mononuclear cell preparations that would be as effective as ammonium chloride lysis but would not affect cellular function. This paper describes a method using 10F7, a mouse monoclonal antibody against human glycophorin A, and magnetic beads coated with anti-mouse immunoglobulin. The population of cord blood mononuclear cells recovered using this technique was of high purity, good yield and viability, and the cells responded appropriately to stimulation in vitro. To maximise cost-effectiveness, purification with magnetic beads could be performed after two density separations to reduce the quantity of beads required.

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a Ka of 2.3 x 10(-9), compared with 4.2 x 10(-9) for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers.

SUMMARY The antibody response in the young infant is limited in several ways; in particular, responses generally are of low affinity and restricted to IgM. This raises the question whether the affinity maturation process, consisting of somatic mutation of immunoglobulin genes coupled with selection of high-affinity variants, is operative in the neonate. Re-arranged VH6 genes were amplified by polymerase chain reaction (PCR) from cord blood and from peripheral blood of infants. Heteroduplex analysis detected mutation in only 2/18 cord blood samples, while mutations were seen from about 10 days of age onwards. Cloning and sequencing of mutated neonatal VH6 genes showed that mutated sequences contained relatively few mutations (one to three mutations per sequence) compared with published values of about 10 in adult IgM sequences. Selection was not evident in the majority of neonatal samples. Thus mutation can occur in the human neonate, but is minimal and generally not accompanied by selection. The age at which affinity maturation develops effectively is yet to be defined.

The neonatal immune system responds to a restricted range of antigens, producing largely IgM antibody of low affinity. Comparison of the components of the B-cell antigen receptor complex shows significantly elevated membrane levels of IgM in neonatal B cells, compared with adult cells. CD79, which acts as the signal transducer for membrane immunoglobulin, is elevated in parallel with IgM, while IgD is elevated to a lesser degree. CD19, CD21, CD22 and CD81, which are all involved in transmitting activation signals when immunoglobulin is engaged, are not elevated. CD32, which is involved in negative regulation of activation, is present at reduced levels on cord B cells. The elevation of B-cell membrane IgM persists during infancy. Neonatal B cells respond in vitro to interleukin-4 (IL-4) by further elevation of membrane IgM levels. The elevated level of membrane IgM may make neonatal B cells easier to trigger by low concentrations of antigen, but in vitro activation and immunoglobulin modulation experiments did not show significant differences between cord and adult B-cell responses to anti-IgM.

Background T lymphocytes are prevalent in sinus mucosa and are implicated in chronic rhinosinusitis (CRS) pathogenesis. However, the major T-cell subpopulations, helper (CD4+) and cytotoxic (CD8+), have not been adequately examined in CRS. This study was designed to characterize human sinus mucosa and peripheral blood (PB) CD4+ and CD8+ T cells and their level of differentiation in CRS with nasal polyps (NPs), CRS without NPs, and control patients. Methods A prospective study was performed. Percentages of CD4+ and CD8+ T cells and their levels of differentiation were analyzed in sinus mucosa and PB by flow cytometry. Cell populations were defined as naive, central memory, effector memory, and effector T cells using cell surface markers CD45RA, CD62L, and CD27. The influence of coexisting allergy, sinus eosinophilic mucus (EM), and culture results were examined. Results In all patients, sinus mucosa had a lower percentage of CD4+ and a higher percentage of CD8+ T cells compared with PB. However, CRS with NPs (n = 86) had a significantly higher percentage of mucosal CD8+ T cells compared with CRS without NPs (n = 40) in control (n = 13) patients (p < 0.0001). Effector memory T cells were increased in sinuses compared with PB in all patients; however, the percentage of effector memory CD8+ T cells was greatest in CRS with NP mucosa (p = 0.002). Surprisingly coexisting allergy or culture results did not influence the mucosal T-cell phenotype. CRS with NP patients with sinus EM had a significantly higher percentage of mucosal CD8+ T cells. Conclusion Sinus mucosa in CRS with NPs is characterized by a significant enrichment of CD8+ T cells and a relative deficiency of CD4+ T cells. The majority of NP CD8+ T cells had a terminally differentiated, mature, effector memory phenotype, which raises the question, whether these cells are pathogenic or appear as a consequence of inflammation, independent of the presence of allergy or positive microbial culture.

Monoclonal antibodies are protein molecules made in the laboratory from hybridoma cells (stable cell lines derived by fusing antibody-producing cells from immunised animals with cells that confer immortality and high-yield antibody production) or by recombinant deoxyribonucleic acid (DNA) technology. They are originally derived from the immune system and have a number of unique properties that have stimulated their use in medicine, including the ability to bind specifically and with high affinity to almost any molecular structure. They can be made in various expression systems such as bacteria or mammalian cells and modified for enhanced affinity, immune function or half-life in blood. They are made in a homogeneous and reproducible form that allows comparisons across laboratories for diagnosis as well as therapeutics. Current major therapeutic applications of monoclonal antibodies include cancer, chronic inflammatory disease, and infection and they constitute the largest and fastest growing sector of the biological pharmaceutical industry. Key Concepts: Monoclonal antibodies are proteins with high specificity towards targets. Monoclonal antibodies have multiple utilities in therapy as they can recognise specific structures in targets such as bacteria, viruses, cancer cells, etc. Monoclonal antibodies can be produced in large amounts in homogeneous and reproducible form for diagnostic and therapeutic purposes. Monoclonal antibodies have a clear regulatory path for their approval as therapeutics. Monoclonal antibodies can be derived from B-cells from immunised animals, humans with autoimmune diseases and de novo by phage display. Therapeutic monoclonal antibodies can be engineered to be more human-like proteins, to increase their affinity, reduce their immunogenicity and increase their half-life in the circulation, and can be conjugated with toxin for better lytic effect. Monoclonal antibodies can mediate antibody-mediated cytotoxicity by linking the target cells to cytotoxic cells through their binding sites and Fc sites. Keywords: antibody; monoclonal; therapy; immunoconjugate; cancer; transplantation

Diagnosis of disease depends frequently on the identification, quantitation or localization of particular molecules, cells or organisms in tissue or body fluids. Antibodies, because of their exquisite specificity, are particularly useful reagents in this context, and monoclonal antibodies are often superior to polyclonal mixtures of antibodies. Monoclonal antibodies are used widely in the diagnostic laboratory. Keywords: antibody; monoclonal; diagnosis; pathology; immunopathology

The physiological importance of cytokines and other factors which control cell and tissue growth and differentiation is widely appreciated. While physiological studies have included the cellular receptors for these factors, studies on their role in disease have concentrated on the measurement of cytokines themselves or soluble receptor components. This reflects in part the technical difficulty of measuring cell surface expression of receptors, which occur at and are functional at very low concentrations. In this review, the potential va lue of surface-expressed receptors as markers of disease is assessed, and methods are described which allow measurements with standard equipment for flow cytometry and tluorescence microscopy.

Cell surface determinants on typhoid-specific B cells isolated from peripheral blood after oral Ty21 a typhoid vaccination in humans

We have analyzed IgE+ cells in peripheral blood of atopic donors, donors hypersensitive to bee venom, and nonatopic control donors with two- and three-color flow cytometry. Although the percentage of IgE+ cells varied among these groups, the overall phenotypic patterns were similar. Most IgE+ cells do not display typical B-cell markers, such as CD19, CD20, and CD21. A significant proportion of these cells stain for CD38, indicating that they are more differentiated. IgE+ cells express Fc gamma RII and CD45RO, an isoform associated with an advanced level of differentiation. The majority of IgE+ cells do not coexpress other surface immunoglobulin isotypes. In the case of bee venom-hypersensitive donors, we have been able to identify a small population of IgE+ cells with a specificity for phospholipase A2, a major immunogenic component of bee venom. The phospholipase A2+ cells display a phenotype similar to that of the IgE+ cells.

Kohler and Milstein’s1 description of monoclonal antibodies (mAbs) in 1975 revolutionised immunology. With the advent of hybridoma technology, immunologists started to produce an ever increasing number of mAbs directed against leukocyte cell-surface molecules. Inevitably, several mAbs produced by different laboratories and given independent names recognised the same molecule. The First International Human Leucocyte Differentiation Antigen (HLDA) Workshop was organised in the early 1980s, and aimed to identify groups of mAbs reacting with a common human cell-surface antigen and to agree a nomenclature that would facilitate better communication amongst the scientific community.2 The outcomes of the Workshops are the ‘CDs’, an abbreviation for the non-descriptive ‘cluster of differentiation’ number. The CD number is assigned to a group or cluster of mAbs that recognise a molecule expressed on the surface of human leukocytes and other cells relevant to the immune system. The nomenclature has been universally adopted by the scientific community, officially approved by the International Union of Immunological Societies and sanctioned by WHO. Its usage has now evolved and is also commonly used to name the molecules themselves. However, by adding either mAb or molecule/protein it should be made clear whether one means the CDxx mAb or CDxx molecule. Ten HLDA Workshops have been organised to date, with the most recent one held in 2014 in conjunction with the Australasian Society of Immunology in Wollongong, Australia.

Chronic non-healing wounds form a medical need which will expand as the population ages and the obesity epidemic grows. Whilst the complex mechanisms underlying wound repair are not fully understood, remodelling of the actin cytoskeleton plays a critical role. Elevated expression of the actin cytoskeletal protein Flightless I (Flii) is known to impair wound outcomes. To determine if Flii is involved in the impaired healing observed in chronic wounds, its expression in non-healing human wounds from patients with venous leg ulcers was determined and compared to its expression in acute wounds and unwounded skin. Increased expression of Flii was observed in both chronic and acute wounds with wound fluid and plasma also containing secreted Flii protein. Inflammation is a key aspect of wound repair and fluorescence-activated cell sorting (FACS) analysis revealed Flii was located in neutrophils within the blood and that it co-localised with CD16+ neutrophils in chronic wounds. The function of secreted Flii was investigated as both chronic wound fluid and Flii have previously been shown to inhibit fibroblast proliferation. To determine if the inhibitory effect of wound fluid was due in part to the presence of Flii, wound fluids were depleted of Flii using Flii-specific neutralizing antibodies (FnAb). Flii depleted chronic wound fluid no longer inhibited fibroblast proliferation, suggesting that Flii may contribute to the inhibitory effect of chronic wound fluid on fibroblast function. Application of FnAbs to chronic wounds may therefore be a novel approach used to improve the local environment of non-healing wounds and potentially improve healing outcomes.

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal (GI) tract characterized by pain or discomfort from the lower abdominal region, which is associated with altered bowel habit. Despite its prevalence, there is currently a lack of effective treatment options for patients. IBS has long been considered as a neurological condition resulting from alterations in the brain gut axis, but immunological alterations are increasingly reported in IBS patients, consistent with the hypothesis that there is a chronic, but low-grade, immune activation. Mediators released by immune cells act to either dampen or amplify the activity of GI nerves. Release of a number of these mediators correlates with symptoms of IBS, highlighting the importance of interactions between the immune and the nervous systems. Investigation of the role of microbiota in these interactions is in its early stages, but may provide many answers regarding the mechanisms underlying activation of the immune system in IBS. Identifying what the key changes in the GI immune system are in IBS and how these changes modulate viscerosensory nervous function is essential for the development of novel therapies for the underlying disorder.