Clinical utility of molecular and flow cytometric markers in chronic lymphocytic leukaemia.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease. While immunoglobulin variable region heavy chain (IgVH) mutational status remains the 'gold standard' in molecular prognostication, a range of additional markers is increasingly being used in clinical trials. As awareness of trial data increases, requests to determine these prognostic markers for new CLL patients are becoming more prevalent in Australia. AIM: To explore the clinical utility of currently available prognostic markers for CLL in an Australian cohort. METHODS: IgVH mutational status and gene usage was determined and compared with other reported immunophenotypic markers, cytogenetics and clinical outcome as defined by treatment-free survival (TFS), lymphocyte doubling time and clinical stage in a cohort of 65 CLL patients. RESULTS: An unmutated IgVH gene, high expression of CD38, ZAP-70, CD25, CD49d, CD54 or low expression of CD49c was associated with shorter TFS indicating an adverse clinical prognosis in our cohort. High expression of each of CD38, ZAP-70, CD49d and CD54 was significantly associated with an unmutated IgVH gene; however, associations were not absolute. IgVH and CD25 expression retained their significance in multivariate analysis. Concordant CD25(high) /IgVH unmutated CLL patients had the shortest median TFS interval (40 months) in our cohort. CONCLUSIONS: Molecular and immunophenotypic markers remain useful as adjuncts to clinical prognostication; however, as single parameters they are unable to dictate the timing of therapeutic intervention. The combined use of CD25 and IgVH mutational status may be clinically relevant to CLL prognostication while also providing insight into the biological pathways involved in disease progression.

Different temporal expression of immunodominant Ro60/60 kDa-SSA and La/SSB apotopes.

Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH2-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.

Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.

A comparison of Verotoxin B-subunit (Stx1B) and CD77 antibody to define germinal centre populations.

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.

Increased expression of the B-cell-regulatory molecule CD72 in primary Sjögren's syndrome.

To determine whether there is an intrinsic abnormality of B-cell signaling in primary Sjögren's syndrome (pSS), the expression of B-cell coreceptors was determined in patients with primary Sjögren's syndrome and healthy and disease controls. Peripheral blood mononuclear cells were labeled with monoclonal antibodies to CD21, CD22, or CD72, and then the pan B-cell marker CD19. The expression of these coreceptors on the total CD19(+) population was determined. There was a significant increased expression of CD72 on the B cells of pSS patients (MFI, 215 +/- 6) compared to normal controls (MFI, 141 +/- 6). The increased CD72 expression was disease specific for pSS, as it was not observed in systemic lupus erythematosus or rheumatoid arthritis. The effect of B-cell stimulation on coreceptor expression was determined by culturing cells with B-lymphocyte-activating factor (BAFF) and/or pokeweed mitogen (PWM) or without either. Following culture, CD72 expression was decreased in both pSS and normal controls, regardless of the presence of BAFF or PWM. The upregulation of CD72 in pSS might be a compensatory response to increased B-cell receptor stimulation or a primary abnormality leading to uncontrolled B-cell activation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is associated with a naïve B-cell phenotype in human tonsils.

In B cells, signaling through the B-cell antigen receptor (BCR) is negatively modulated by the co-ligation of immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing molecules such as FcgammaRIIB1, B-cell transmembrane protein CD72, paired immunoglobulin-like receptor PIR-B, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Ig-like transcript ILT2, biliary glycoprotein BGP-1 and B-cell co-receptor CD22. The co-expression of multiple Ig-ITIM receptors may provide B cells with different mechanisms of regulating inhibitory pathways at different stages of differentiation. In this study, we have examined the expression of a newly defined Ig-ITIM receptor, PECAM-1 (CD31) on human B-cells. Human tonsillar B cells were purified using negative selection by depleting T cells with a combination of monoclonal antibodies and magnetic bead separation. Following purification, the pattern of PECAM-1 expression was analyzed in B-cell subpopulations using two- and three-colour fluorescence. To complement this work, PECAM-1 localization in the context of distinct areas of human tonsil was defined by immunohistochemical analysis of tonsil sections. Finally to investigate somatic mutation, Ig variable (V) region genes belonging to the nonpolymorphic VH6 family were amplified by polymerase chain reaction (PCR), subcloned and sequenced from sort-purified CD19+ PECAM-1+ and CD19+ PECAM-1- B cells. Our results demonstrate that PECAM-1 is associated with an unstimulated resting B-cell phenotype, localization to the follicular mantle and marginal zones of human tonsil and expression of unmutated Ig V region genes. These studies suggest that PECAM-1 appears on the cell surface at the naive B-cell stage and is lost as B cells differentiate into memory cells, indicating that PECAM-1 is primarily involved in naive or immature B-cell function.

HLA-B27 expression by flow cytometry: an analysis of 7 years quality assurance data.

The Royal College of Pathologists of Australasia Quality Assurance Programs Pty. Ltd. has been monitoring HLA-B27 assignment by flow cytometry for 7 years as part of the Immunology Program. Here we present data that demonstrates a gradual improvement in reports of false positive and negative results. Many participating laboratories demonstrate an ability to assign HLA-B27 status correctly by flow cytometric means. This ability appears to be independent of reagent and methodology. However a small number of laboratories produce consistently unacceptable results that suggest poor quality assurance practice.

Expression of the costimulator molecules, CD40 and CD154, on lymphocytes from neonates and young children.

Differential expression of the costimulator molecules CD40 and CD154 on neonatal lymphocytes may be one explanation for limited T-dependent antibody responses in human neonates. CD40 was expressed at similar levels on resting B cells from adults, young children (2-20 months of age) or cord blood. CD40 expression was higher on cord blood B cells compared to adult B cells after stimulation with PMA and ionomycin, but similar on adult and cord blood B cells activated by CD3-stimulated T cells. In contrast to previous reports, cord blood T cells stimulated with PMA and ionomycin expressed adult levels of CD154 initially, but this expression was more transient on cord blood T cells. When adult and cord blood mononuclear cells were stimulated with CD3 mAb, T cells from some cord blood specimens showed different kinetics of CD154 expression compared with adult T cells. However, some cord blood specimens showed adult patterns of T cell CD154 expression. When mononuclear cells were depleted of B cells and monocytes prior to stimulation with CD3 mAb, the MFI and percentage of T cells expressing CD154 increased, with adult and cord T cells showing similar patterns of expression. These results show some differences in expression of CD40 and CD154 between neonatal and adult lymphocytes, but do not directly account for the relative deficiencies of humoral immunity in neonates.

Expression of the costimulator molecules, CD80, CD86, CD28, and CD152 on lymphocytes from neonates and young children.

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.

Analysis of the cord blood T lymphocyte response to superantigen.

We have characterized the T lymphocyte population of the human neonate in respect of the expression of phenotypic profiles for naive, memory and differentiated populations. We have examined the response of the neonate T cell to the superantigen Staphylococcus enterotoxin B (SEB) and compared the response to T cells from healthy adults. We found that the primary response to SEB is equivalent in neonates and adults but that the secondary response demonstrates hyporesponsiveness in the neonate that is more profound than in adults. This response was associated with increased expression of CD25; the alpha chain of the IL-2 receptor, equivalent to that seen in responding cells from adults. A modest increased expression of CD122 and CD132, the beta and gamma chains of the IL-2 receptor, was also observed. There was no increase in the IL-4 receptor (CD124). The hyporesponsive neonate T cells proliferated in response to exogenous IL-2 but the response was less than none SEB treated cells. The neonate cells did not respond to IL-4. We also examined the expression of MHC class II molecules on SEB stimulated cells and found that both neonate and adult T cells upregulate MHC class II to a similar degree. The difference in the hyporesponsive cells appears to result in part from a lower production of IL-2 and in part from a lower ability of cord cells to respond to IL-2. Since the stimulated cord cells expressed IL-2 receptor at the same levels as similarly treated adult cells; there may be differences in down stream signaling pathways.

Interleukin 2 receptor regulation and IL-2 function in the human infant.

IL-2 receptor is expressed at low levels on adult blood lymphocytes, and at lower levels on cord blood cells. IL-2 receptor alpha and beta chain expression increases gradually from 0-18 months of age. The level of soluble CD25 (IL-2 receptor alpha chain) has been reported to be elevated in cord blood. Quantitative RT-PCR showed that adult cells express 10 times as much CD25 mRNA as cord cells. Cord plasma showed only a marginal ability to strip CD25 from the membrane. To assess the functional consequences of low IL-2 receptor expression, cord and adult cells were activated in vitro. The response was stimulus-dependent, but cord cells upregulated CD25 readily. Cord and adult cells proliferated in an IL-2-dependent assay to a similar extent. Infants suffering acute infection showed marginally higher levels of membrane CD25 expression than infants without overt infection. Thus neonatal and infant lymphocytes express lower levels of IL-2 receptors than adult cells, reflecting lower mRNA concentrations at least for CD25; they are able to up-regulate receptors in response to in vitro stimulation and are able to respond in vitro to IL-2-dependent stimulation; however in vivo there may be a dampening down of the IL-2 system in infancy.

Removal of erythroid cells from umbilical cord blood mononuclear cell preparations using magnetic beads and a monoclonal antibody against glycophorin A.

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 antigen receptor complex on cord B lymphocytes.

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.

Blood group antibodies are made by CD5+ and by CD5- B cells.

The B1 subset of B lymphocytes is associated with the production of low-affinity, polyspecific antibodies. B1 cells are generally recognized by their expression of CD5, and comprise the majority of neonatal B cells. The neonate responds to a restricted range of antigens, and generally makes low-affinity IgM antibody. Published data suggest that antibodies against the blood group antigens A and B are found occasionally in cord blood, and develop rapidly in infants as a result of cross-reactivity with bacterial carbohydrate antigens. This suggests that CD5+ B1 cells may be specialized to make antibodies against such carbohydrate antigens. In this study we evaluated the appearance of antibodies against the blood group (ABO) antigens in human infants, using reagents which specifically distinguish between IgM (made by the infant) and IgG (mainly of maternal origin) and immunofluorescence to detect low levels of antibody. Having established that antibody is always detectable by 8 months of age, and frequently much earlier, we developed a plaque assay to examine the phenotype of cells making antibody against blood group antigens. At 8 months of age, CD5+ and CD5- cells were both capable of making anti-blood group antibody.

The Fas antigen (CD95) on human lymphoid cells: epitope analysis with ten antibodies.

The expression of CD95 antigen was examined on adult and cord blood lymphocytes using a highly sensitive immunofluorescence/flow cytometric procedure. CD95 was expressed by the majority of circulating blood T cells in adults, and by a smaller proportion of CD4+ and CD8+ T cells in cord blood. The majority of circulating B cells did not react with seven CD95 antibodies, but three antibodies did stain B cells. In tonsil sections, CD95 was expressed throughout the tissue but germinal centres showed generally stronger staining than the surrounding follicular mantle and interfollicular areas. This was confirmed by flow cytometry, which showed expression preferentially on B cells with a germinal centre phenotype. Because different antibodies stained different proportions of B cells, CD95 epitopes were examined by inhibition, additive binding and protease susceptibility studies using a panel of ten CD95 antibodies. B cells apparently reacting selectively with CD95 antibodies were sorted and CD95 mRNA was reverse transcribed to cDNA and analyzed, in order to confirm the presence of CD95 in cells which reacted selectively and to explore the possible existence of CD95 isoforms. The major cDNA band was identical in the two populations. Inhibition of N-glycosylation suggested that the epitopes detected differentially could not be accounted for by differential N-glycosylation.

Purification of cord blood lymphocytes.

When cord blood is separated using standard methods based on Ficoll-Hypaque, the mononuclear fraction is contaminated with erythrocytes and also with nucleated cells that do not express the leucocyte marker CD45. The contamination with CD45-negative cells can exceed 50%, and will interfere with phenotypic, mRNA or functional analysis. A large proportion of these cells are erythrocyte precursors. The contaminating cells may be removed by lysis with hypotonic ammonium chloride; when the cells are required for studies which are adversely affected by ammonium chloride (such as antigen processing), high purity can be attained by two rounds of density separation.

Characterization of human leucocytes bearing the IL-3 receptor.

Human leucocytes from peripheral blood and tonsil were examined for the presence of the IL-3 receptor using monoclonal antibodies directed to epitopes of the alpha and beta chains of the receptor. We found that the beta chain, common to IL-3, IL-5, and GM-CSF, was either present at low levels or not detected on the majority of peripheral blood and tonsil B lymphocytes, while the alpha chain showed a distinct but restricted distribution. In peripheral blood the IL-3R alpha chain was limited to a subpopulation of peripheral B lymphocytes and a population of cells which lack lineage-specific markers. Dimly staining cells were identified as B lymphocytes as they coexpressed CD19, CD20, CD22, CD24, and HLA-DR. A brightly staining population lacks T and B lymphocyte, NK specific, and macrophage lineage markers but expresses CD9, CD45RO, CD26, and, in a proportion of cells, CD36 and CD60. This population remains unclassified. In tonsil tissue IL-3R alpha chain expression was strongest on B lymphocytes present in the T cell rich areas of tonsillar tissue. The IL-3R alpha bearing B tonsil cells included cells in both CD23 and IgD positive and negative populations. The phenotype of the IL-3R alpha positive B cells defines them as a population of B lymphocytes distinct from previously characterized cells in the lymphoid architecture. Lymphoblastoid cell lines with a corresponding phenotype were also identified.

Reduced expression of the interleukin-2-receptor gamma chain on cord blood lymphocytes: relationship to functional immaturity of the neonatal immune response.

Mutation of the interleukin-2 (IL-2) receptor gamma chain, which also serves as a component of the receptor complexes for IL-4, 7, 9 and 15, results in severe immune deficiency. We hypothesized that the immunological immaturity of healthy neonates might be associated with low levels of expression of this receptor molecule. Using monoclonal antibody and a highly sensitive immunofluorescence method, we showed that IL-2 receptor gamma chain is expressed at significantly lower levels on cord blood cells compared with adult cells. IL-2-dependent T-cell activation in vitro was reduced in cord blood cells compared with adult cells, but B-cell responses to IL-4 were not obviously impaired. The lower level of expression of the gamma chain and some other cytokine receptor chains may contribute to the immunological immaturity of the newborn, by selectively depressing particular immunological mechanisms.