E. Yuen

The prognostic significance of T cell receptor β gene rearrangements and idiotype-reactive T cells in multiple myeloma

Clonal T cell populations with idiotype specificity are present in the peripheral blood of a proportion of patients with multiple myeloma. We have identified the presence of both T cell sub-populations with a specificity for autologous immunoglobin fragments and T cell receptor β gene rearrangements in peripheral blood samples of patients with myeloma. T cell receptor β gene rearrangements were detected in 38 of 119 patient samples (32%) and were more common in progressive disease (70%), than at diagnosis (25%) or in stable disease (23%). The 38 patients who had T cell receptor β gene rearrangements detected at any time had a better overall survival (median not yet achieved) than the patients who never had rearrangements detected (median 45 months, n = 49;χ 2 = 6.2, P < 0.01). All 12 patients with T cell receptor β gene rearrangements at diagnosis are still alive whereas the median survival for 28 patients with a germline configuration at diagnosis was 40 months (χ 2 = 5.8, P > 0.01). The presence of T cell receptor β gene rearrangements even conferred a survival advantage during progressive disease (median survival 44 months vs 19 months;χ2 = 8.7, P < 0.003). Two colour flow cytometry with biotinylated autologous immunoglobulin fragments demonstrated idiotype-reactive T cells in the peripheral blood of five out of 15 patients all of whom had T cell gene rearrangements. The remaining 10 patients had neither idiotype-reactive T cells nor a detectable T cell receptorβ gene rearrangement in concurrent samples. Thus in patients with myeloma there was a good correlation between the presence of T cell receptor β gene rearrangements and idiotype-reactive T cells. Patients with a rearranged T cell receptor β gene had a significantly better prognosis.

T‐cell expansions in patients with multiple myeloma have a phenotype of cytotoxic T cells

The presence of T‐cell clones in peripheral blood has been previously shown to be associated with a survival advantage in patients with multiple myeloma and suggests that the expanded T‐cell populations may be involved in an anti‐tumour response. We studied the T‐cell receptor (TCR) repertoire of 38 patients with myeloma to identify and characterize the expanded T‐cell populations by flow cytometry. T‐cell expansions were found in 79% of the patients. The expansions occurred randomly among the 21 variable regions of the TCR β chain (Vβ) studied, representing 62% of the V‐β repertoire, and were stable during an 18‐month follow‐up. The phenotype of the expanded V‐β populations was predominantly CD8+, CD57+, CD28− and perforin+, which differed significantly from the other non‐expanded Vβ populations. The expression of the apoptosis markers Fas (CD95) and bcl‐2 were similar between the expanded and non‐expanded Vβ populations. In conclusion, expanded T‐cell populations were frequent in patients with myeloma, they remained unchanged during follow‐up and had phenotypic characteristics of cytotoxic T cells. These data add further support to the concept that the T‐cell expansions may have an immunoregulatory role in myeloma.

The Expression of T Cell Related Costimulatory Molecules in Multiple Myeloma

Presentation of tumour antigen by malignant cells not expressing costimulatory molecules is considered to be a major cause of the failure of the hosts immune response against tumours. This study has determined the expression of the B7 family of costimulatory molecules on malignant plasma cells and the expression of the counter receptor molecules, CD28 and CD152 (CTLA-4), on T cells of patients with multiple myeloma. CD28 expression was present on most CD4 cells but was lower on CD8 cells especially from those patients who also showed evidence of expanded T cell clones (median 40%. z=2.4; p<0.02). CD152 expression was increased in 50% (9/18) of patients with myeloma. CD80 (B7-1) expression was present on the plasma cells of only 1 of 27 samples but CD86 (B7-2) expression within the normal range was present on the plasma cells of 14 of 27 samples. Primitive plasma cells (CD38++ CD45++) had a higher expression of CD86 (median 78%) than mature plasma cells (CD38++ CD45-) (median 19%, z=3.7; p<0.01). Thus patients with expanded T cell clones have a downregulated T cell CD28 expression and lack B7-1 expression on their malignant plasma cells. These results are consistent with the concept that engagement of the T cell receptor by tumour antigen on B7-1 deficient malignant plasma cells would result in T cell anergy rather than productive immunity.

Clonal cytotoxic T cells in myeloma.

Multiple myeloma (MM) is a malignant disease characterized by accumulation of morphologically recognizable plasma cells producing immunoglobulin (Ig) in the bone marrow. The occurrence of clonal T cells in MM, as defined by the presence of rearrangements in the T-cell receptor (TCR)-beta chains detected on Southern blotting, is associated with an improved prognosis. This review aims to describe the various ways in which we have demonstrated the presence of such T cell clones, and to describe the phenotype of these cells. Finally, the specificities of these clinically important CD8+ T cell populations will be discussed in the context of immunotherapy.

An evaluation of serum erythropoietin estimation by a hemagglutination inhibition assay in the differential diagnosis of polycythemia

Summary A commercially available hemagglutination inhibition assay kit for the measurement of erythropoietin (EPO) was evaluated for its usefulness in the differential diagnosis of the polycythemic states. Serum samples were obtained from patients with polycythemia rubra vera (active and controlled), secondary and relative polycythemia and from normal controls. Firstly, we found that the mean EPO level (± SD) of our normal controls (81 ± 69 miu/ml) was higher than the manufacturers quoted normal range (15–59 miu/ml) and that there was a significant spread of values (7‐233 miu/ml). Secondly, within each patient subgroup studied, the spread of data points was so wide that interpretation of individual data points would be impossible. We conclude that this assay kit is of little value for serum EPO estimation and in the differential diagnosis of polycythemia.

Plasma lactoferrin in patients with neutropenia

SummaryThis study examines the role of plasma lactoferrin in the assessment of neutropenia. In particular, we have studied lactoferrin as an inhibitor of granulopoiesis and as an indicator of the size of the total blood granulocyte pool (TBGP). Plasma lactoferrin concentration was determined in a heterogeneous group of 30 patients with neutropenia. Serial plasma lactoferrin levels in a patient with cyclic neutropenia correlated with the cycles of the neutrophil count. Patients with splenomegaly had a grossly elevated lactoferrin: neutrophil ratio. Most chronic idiopathic neutropenia patients had no real clinical problems and a normal plasma lactoferrin level. The results provide further evidence to support the concept that plasma lactoferrin indicates the size of the TBGP and the lactoferrin: neutrophil ratio indicates the degree of granulocyte margination. There was no evidence to suggest that lactoferrin acting as a feedback inhibitor of granulopoiesis caused neutropenia in these patients.

Scanning electron and light microscope correlation of individual human bone marrow cells before and after culture in nutrient agar.

This study was undertaken with the aim of identifying the different cell types found in human bone marrow by examining their surface morphology. In an attempt to obtain a homogeneous cell population, cells were both fractionated by discontinuous albumin density gradient centrifugation (DADGC) and selectively grown in nutrient agar. Both cell preparations underwent the critical point drying technique before examination under both the scanning electron microscope (SEM) and subsequently the light microscope (LM). When the SEM image of individual cells was compared with the corresponding LM image, it was not easy to identify the different cell types, because of the shrinkage and distortion that occurred during their preparation. The shrinkage observed under the SEM amounted to a 45% reduction in mean cell diameter. This shrinkage was confirmed by comparing the SEM and LM images of the same cell. Although shrinkage occurred throughout the dehydration sequence, critical point drying was responsible for a 25% reduction in mean cell diameter. Furthermore, direct observation under LM of fixed cells drying in air from ethanol, revealed visible contraction of the cell and distortion of the cell membrane. We assume that a similar morphological change occurred during critical point drying. We conclude that the shrinkage and distortion, caused by the dehydration process involved in SEM preparation, severely limit the value of a study of surface morphology by SEM in the identification of the different cell types found in human bone marrow.

The use of peripheral blood feeder layers as a source of GM-CSF for human bone marrow cultures

SummaryThe use of peripheral blood feeder layers as a source of stimulus for colony formation by human granulocytic progenitor cells in semi-solid agar cell cultures was examined. Comparison with various conditioned media demonstrated that cultures stimulated by peripheral blood feeder layers produced the greatest number and largest colonies. The cell concentration in the feeder layers was more important than the total cell number. Feeder layer plates containing 2 × 105 cells at a concentration of 1 × 106 cells/ml proved to be just as potent as the conventional feeder layer plates containing 1 × 106 cells/ml in 1 ml. Thus feeder layer plates can be more economical in terms of cell numbers than has previously been reported. By using a known panel of donors for the leukocytes, the number of sub-optimal batches of feeder layers was reduced. Addition of 1 μM adenosine 3′ : 5′-cyclic-monophosphate or 10 μg/ml Li2CO3 to poor feeder layers enhanced their colony-stimulating ability.

The Effect of Cyclic AMP On Human Colony Forming Cell and Colony Stimulating Factor Production

Summary The effect of cyclic AMP (cAMP) and related nucleotides on human colony forming cells (CFC) and those cells producing colony stimulating factor (CSF) was studied in vitro. When added at physiological concentrations (1−2 to 1μM), exogenous cAMP stimulated maximum colony formation in cultures without feeder layers. Related nucleotides stimulated colony formation to a lesser extent and in decreasing order of free energy. All nucleotides inhibited colony formation in concentrations above 1 μM. A velocity sedimentation cell separation technique was used to obtain cell fractions rich in CFC but poor in CSF‐producing cells. Such fractions did not respond to cAMP stimulation. These studies suggest that exogenous cAMP stimulates human bone marrow to form colonies in vitro by increasing the release and/or production of endogenous CSF.

Southern Blotting of IgH Rearrangements in B-Cell Disorders

Southern blotting is a method whereby DNA fragments in the gel are denatured by soaking in an alkali solution, carried out of the gel, and transferred onto a membrane. After drying the membrane, the DNA is fixed irreversibly. The net result is a replica on the membrane of the DNA fragment pattern from the agarose gel. This technique is used to demonstrate B-cell clonality in blood and bone marrow down to the 1% level, though more reliably at the 5% level. The analysis is relatively nonselective and will detect novel rearrangements in relapse that were not seen at diagnosis. Modifications of the technique have been used to determine illegitimate switch recombinations and mutations of oncogenes.