CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology.

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.

Immune derived opioidergic inhibition of viscerosensory afferents is decreased in Irritable Bowel Syndrome patients.

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 colo-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 ß-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 colo-rectal sensory afferent endings in mice with post-inflammatory chronic visceral hypersensitivity (CVH). ß-Endorphin was identified predominantly in monocyte/macrophages relative to T or B cells in human PBMC and colonic lamina propria. Monocyte derived ß-endorphin levels and colonic macrophage numbers were lower in IBS patients than healthy subjects. PBMC supernatants from healthy subjects had greater inhibitory effects on colo-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 µ-opioid receptor expression in dorsal root ganglia neurons in CVH mice. Monocyte/macrophages are the predominant immune cell type responsible for ß-endorphin secretion in humans. IBS patients have lower monocyte derived ß-endorphin levels than healthy subjects, causing less inhibition of colonic afferent endings. Consequently, altered immune function contributes toward visceral hypersensitivity in IBS.

Accumulation of effector memory CD8+ T cells in nasal polyps.

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.

Elevated serum cytokine levels using cytometric bead arrays predict culture-positive infections in childhood oncology patients with febrile neutropenia.

Neutropenic patients with bacteraemia need prolonged intravenous antibiotic treatment. Using cytometric bead array technology, we show in children with febrile neutropenia that bacteraemia is associated with an elevation of at least 1 of 3 plasma cytokines plus C-reactive protein. The combination of interleukin (IL)-8, IL-6, IL-10, and C-reactive protein values above operator-defined cutoff levels identified 15 of 16 episodes of bacteraemia, making this a potentially useful technique in identifying high-risk patients who should not be discharged early from hospital. Furthermore, low risk of bacteraemia may be predicted by a combination of below threshold cytokines and negative clinical examination.

Reticulin fibres anchor leukaemic blasts in the marrow of patients with acute lymphoblastic leukaemia.

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.

ProNGF mediates death of Natural Killer cells through activation of the p75NTR-sortilin complex.

The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage resistant proNGF123 killed NK cells in the presence of IL-12 after 20h and without IL-12 in serum free conditions at 48h. This was reduced by blocking sortilin with neurotensin. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.

Isolation of whole mononuclear cells from peripheral blood and cord blood.

Peripheral blood is the primary source of lymphoid cells for investigation of the human immune system. Its use is facilitated by Ficoll-Hypaque density gradient centrifugation-a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. Thus, cells are distributed in the solution in layers based on the differences in their density/size. Additional purification methods can be employed as the mononuclear cell sample can be purified from monocytes by adherence or by exposure to L-leucine methyl ester; these methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, which can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues.

Development of CD4+CD25+FoxP3+ regulatory T cells from cord blood hematopoietic progenitor cells.

Adult stem cells are capable of generating all of the cells of the hematopoietic system, and this process is orchestrated in part by the interactions between these cells and the stroma. T cell progenitors emerge from the stem cell compartment and migrate to the thymus, where their terminal differentiation and maturation occur, and it is during this phase that selection shapes the immune repertoire. Notch ligands, including Delta-like 1 (DL1), play a critical role in this lymphoid differentiation. To mimic this in vitro, stroma-expressing DL1 have been used to generate CD4(+)CD8(+) double-positive and single-positive T cells from hematopoietic stem/progenitor cells. This system provides a robust tool to investigate thymopoiesis; however, its capacity to generate regulatory T cells (Tregs) has yet to be reported. Natural Tregs (nTregs) develop in the thymus and help maintain immune homeostasis and have potential clinical use as a cell therapy for modulation of autoimmune disease or for transplant tolerization. Here, we describe for the first time the development of a population of CD4(+)CD25(+) CD127(lo)FoxP3(+) cells that emerge in coculture of cord blood (CB) CD34(+) progenitors on OP9-DL1 stroma. These hematopoietic progenitor-derived CD4(+)CD25(+) Tregs have comparable suppressor function with CB nTregs in vitro. The addition of IL-2 to the coculture enhanced the expansion and survival of this population significantly. This manipulable culture system, therefore, generates functional Tregs and provides a system to elucidate the mechanism of Treg development.

1D09C3, an mAb specific for MHC-II.

GPC Biotech AG is developing 1D09C3, an anti-MHC class II (HLA-DR) fully human IgG4 antibody isolated by MorphoSys AG (from its HuCAL library of human antibodies), for the potential treatment of hematological malignancies. In December 2006, positive safety data from two phase I clinical trials were reported. Final phase I data were expected in mid-2007; however, no additional data have been released at the time of publication.

Multiple Myeloma Includes Phenotypically Defined Subsets of Clonotypic CD20+ B Cells that Persist During Treatment with Rituximab.

Potential progenitor B cell compartments in multiple myeloma (MM) are clinically important. MM B cells and some circulating MM plasma cells express CD20, predicting their clearance by treatment with anti-CD20. Here we describe two types of clonotypic CD20+ B cell in peripheral blood of myeloma patients, identified by their expression of CD19 and CD20 epitopes, their expression of CD45RA and their light scatter properties. Thus, the circulating component of the MM clone includes at least two distinct CD19+ CD20+ B cell compartments, as well as CD138+ CD20+ plasma cells. To determine whether either or both B cell subsets and the CD20+ plasma cell subset were depleted by anti-CD20 therapy, they were evaluated before, during and after treatment of patients with rituximab (anti-CD20), followed by quantifying B cell subsets over a 5 month period during and after treatment. Overall, all three types of circulating B lineage cells persist despite treatment with rituximab. The inability of rituximab to prolong survival in MM may result from this failure to deplete CD20+ B and plasma cells in MM.

Isolation, propagation and characterization of cord blood derived CD4+ CD25+ regulatory T cells.

Regulatory T cells (Treg) have recently come to the fore in studies of immune regulation, particularly in autoimmune disease and cancer. While there appear to be several distinct subsets of T cells with regulatory function, a population described as natural Treg and characterized by expression of the transcription factor FOXP3 has attracted particular interest. These cells can be enriched using the surface markers CD4 and CD25, and cord blood is a convenient source of CD25+ Treg. We present detailed protocols for the enrichment of Treg from cord blood using CD25 and a magnetic bead procedure, yielding populations >80% positive for CD25 and 50-65% FOXP3 positive. This enrichment can be followed by a second magnetic bead or a flow sorting step, yielding >95% CD25 and >65% FOXP3 positive populations. Protocols are presented for propagation of these cells in culture (yielding >80% FOXP3 positive cells) and for their phenotypic and functional characterization.

Medical applications of leukocyte surface molecules--the CD molecules.

Leukocytes are the cells of the immune system and are centrally involved in defense against infection, in autoimmune disease, allergy, inflammation, and in organ graft rejection. Lymphomas and leukemias are malignancies of leukocytes, and the immune system is almost certainly involved in most other cancers. Each leukocyte expresses a selection of cell surface glycoproteins and glycolipids which mediate its interaction with antigen, with other components of the immune system, and with other tissues. It is therefore not surprising that the leukocyte surface molecules (CD molecules) have provided targets for diagnosis and therapy. Among the "celebrities" are CD20, a target for lymphoma therapeutic antibodies which earns $2 billion annually (and makes a significant difference to lymphoma patients), and CD4, the molecule used by the human immunodeficiency virus (HIV) as an entry portal into cells of the immune system. This short review provides a background to the CD molecules and antibodies against them, and summarizes research, diagnostic, and therapeutic applications of antibodies against these molecules.

Expression of toll-like receptors on B lymphocytes.

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.

Cross-reactivity of anti-human chemokine receptor and anti-TNF family antibodies with common marmoset (Callithrix jacchus) leukocytes.

The common marmoset (Callithrix jacchus) is a New World primate species frequently employed for immunological models of human disease. We used flow cytometry to screen a panel of new anti-human antibodies from the HLDA8 workshop to establish cross-reactivity with marmoset peripheral blood mononuclear cells. Seventy-seven antibodies were screened of which nine antibodies showed binding. Cross-reactivity of anti-human monoclonal antibodies with CC and CXC chemokine receptors CCR3, CCR6, CCR7, and CCR8 was demonstrated on untreated marmoset mononuclear cells. Stimulation of marmoset mononuclear cells with ConA and/or PMA-ionomycin resulted in an up-regulated expression of CXCR1, CXCR3, and CXCR4. The expression of TNF-family related molecules TACI and APRIL on marmoset mononuclear cells was also identified. These studies extend the range of cross-reactive antibodies to now include anti-chemokine and anti-TNF family antibodies for this important pre-clinical model species and should provide useful tools for investigation of immunological processes in marmoset monkey models.

Characterization of colonic and mesenteric lymph node dendritic cell subpopulations in a murine adoptive transfer model of inflammatory bowel disease.

Ulcerative colitis and Crohn's disease, collectively termed inflammatory bowel diseases (IBD), are chronic inflammatory diseases of the intestine that afflict more than 4 million people worldwide. Intestinal inflammation is characterized by an abnormal mucosal immune response to normally harmless antigens in the gut flora. In Crohn's disease, the pathogenic mucosal immune response is a typical T helper (TH1) type cell response, whereas ulcerative colitis is predominantly associated with a TH2 response. We are interested in the role of dendritic cells in early immunologic events leading to T cell activation and chronic intestinal inflammation. Using a murine adoptive transfer model of IBD, we found an accumulation of dendritic cells in colon and mesenteric lymph nodes during the early stage of IBD before the appearance of epithelial lesions and tissue degradation. In situ immunostaining and flow-cytometric analysis revealed that approximately 50% of colonic dendritic cells were CD11b B220 myeloid dendritic cells and 50% expressed the CD11b B220 plasmacytoid phenotype. In corresponding mesenteric lymph nodes, approximately 16% were plasmacytoid dendritic cells. Colonic myeloid dendritic cells were shown to express the co-stimulatory molecule CD40. Both, colonic myeloid and plasmacytoid dendritic cells released interferon-alpha in situ and stimulated T cell proliferation ex vivo. Our results show that dendritic cells can mature in the intestine without migrating to mesenteric lymph nodes. Mature intestinal dendritic cells may form a nucleation site for a local T cell response and play an important role in the pathogenesis of IBD.

Tolerization as a tool for generating novel monoclonal antibodies.

Standard hybridoma production involves the fusion of spleen cells from an immunized mouse with a non-secretory murine myeloma cell line. While this technology has provided numerous reagents that are highly valuable, demand is now increasing for monoclonal antibodies which can distinguish between closely related antigens. Induction of tolerance towards common antigens enables the recovery of high-specificity reagents that have previously proved elusive. This review details a number of strategies using either complex protein mixtures or purified proteins as tolerogens and subsequent immunization with a closely related immunogen.

FcgammaRIIb expression on human germinal center B lymphocytes.

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.