Colin Stephen Scott

Decreased T helper cells in the myelodysplastic syndromes

Summary. The myelodysplastic syndromes (MDS) are disorders in which the abnormalities are thought to be confined to cells of the myeloid series. However, examination of peripheral blood from 56 patients with MDS showed that the majority had low lymphocyte counts. In a detailed study of 25 patients, using OKT3, OKT4 and OKT8 monoclonal antisera together with SRBC rosettes, it was shown that there is a consistent and significant reduction in peripheral blood T lymphocytes. This decrease was primarily confined to the OKT4‐defined helper subpopulation. There is a consequent relative increase in the OKT8‐defined subset but in absolute numbers however, suppressor cells are reduced compared to normal particularly in those patients with lymphopenia. No correlations between T cell abnormalities and the different morphological groups of MDS were found. The possible implications of the reversed helper/suppressor ratio in the pathogenesis of MDS are discussed.

Transient and persistent expansions of large granular lymphocytes (LGL) and NK‐associated (NKa) cells: the Yorkshire Leukaemia Group study

Summary. A survey of 870 different adult blood samples (primarily from patients with non‐haematological disorders) found that 269 (31%) had increased proportions (>25%) and/or absolute numbers (>1.0 × 109/l) of morphologically‐defined large granular lymphocytes (LGL), and/or pheno‐typically‐defined NK‐associated (NKa) cells. Of these, 112 were re‐analysed at least 6 months after initial presentation and were classified as‘persistent’(92/112) or ‘transient’(20/112) according to whether or not the original abnormality was still present. Lymphocyte counts in most patients with persistent abnormalities were within normal limits (18/92) or slightly increased (68/92), with only six having a lymphocytosis exceeding 10.0 × 109/l. With the exception of five persistent LGL expansions in which the granular lymphocytes did not express NKa determinants (designated LGL+NKa‐), the remaining 87 cases could be phenotypically grouped according to their primary abnormality as CD8+NKa+ (n = 33), CD4+ NKa+ (n=14), CD8dim+NKa+ (n=7) or CD8−NKa+ (n=33). TCR genotypic studies in 58 patients showed that the 16 patients with rearranged TCR components were restricted to the CD8+NKa+ group and that, in most of these, the CD8+ fraction showed abnormal relative CD16/CD56 expression. Persistent neutropenia (n=15) also appeared to be associated with primary abnormalities of CD8+NKa+ cells (12/15). with 10 of these additionally showing rearranged TCR genes. In contrast, persistently increased CD8dim+NKa+ and CD8–NKa+ components did not appear to phenotypically differ from their corresponding ‘counterparts’in normal bloods or in patients with transient LGL/NKa+ abnormalities. This survey has therefore established that persistent LGL/NKa+ abnormalities are considerably more common than suggested in published work, that a high proportion of patients with expanded CD8+NKa+ components, with quite diverse clinical histories, show evidence of clonal lymphoid populations, and that the clonal nature of such disorders appears to be associated with abnormal NKa phenotypic patterns.

Diagnostic and prognostic factors in acute monocytic leukaemia: an analysis of 51 cases

Summary. Diagnostic features (cytochemistry, immunophenotyping and serum biochemistry) were examined in 51 cases of acute monocytic leukaemia (AMoL). Peroxidase, Sudan black B and alpha naphthyl acetate esterase (ANAE) cytochemical reactions were unrelated to morphological (FAB groups M5a and M5b) or immunological subtype. ANAE cytochemistry, however, indicated that AMoL cases could be subdivided into those with typical (M‐type) reactions and those with insignificant staining or monocytic ANAE isoenzymes (defined by IEF). All cases were phenotypically CD13/CD33 positive and, with one exception, had > 30% HLA‐DR positive cells. Membrane CD14 expression was insignificant or variable in 33% of M5a cases in contrast to 23/24 M5b cases which showed high proportions of CD14‐staining cells with at least two monoclonal antibodies. Serum lysozyme, LDH and beta‐2 microglobulin (β2m) were increased in 88%, 68% and 81% of cases respectively but, with the exception of statistically higher lysozyme levels in CD14+ cases, were unrelated to the morphological, cytochemical or immunological diagnostic subgroups. Clinical and diagnostic features were also examined as possible prognostic indicators. The morphological, cytochemical and immunological subgroups of AMoL were not found to be of prognostic relevance but age (P=0.004), renal failure (P=0.005) and serum β2m levels (P=0.002) were related to patient survival. Moreover, renal failure and serum β2m remained significant (P=0.012 respectively) when age was taken into account and were shown to be independent prognostic variables.

A distinct large granular lymphocyte (LGL)/NK-associated (NKa) abnormality characterized by membrane CD4 and CD8 coexpression

Summary. In a study of 870 individual patients with either lymphocytosis (excluding known lymphoproliferative disease), increased proportions of blood lymphocytes with granular morphology (LGL), or neutropenia, 14 cases were found with abnormally increased CD3+CD4+CD8+ components. Eleven of these were further investigated and 10 shown in follow‐up studies to be persistent in nature. Morphological assessments revealed increased LGL in 9/11 cases, and in seven of these > 50% lymphocytes had discernable cytoplasmic granulation. Immunophenotypic studies indicated that CD8 expression by CD4+ lymphocytes in these patients was of low density (CD8dim+), and that both the CD4+CD8− and CD4+CD8dim+ fractions in each patient was characterized by a CD11b+ CD16−CD56+CD57+ composite NK‐associated (NKa) phenotype (in contrast to normal CD4+ CD8− blood lymphocytes and CD4+CD8+ thymocytes which were consistently CD11b−CD16−CD56−CD57−). TCR genotypic studies revealed rearranged components (β plus 7. or β alone) in 5/11 cases, but there were no obvious relationships between TCR configuration (including rearranged band densities) and immunophenotypes, absolute lymphocyte or neutrophil numbers, the proportions of blood LGL, or the proportions of CD4+ cells coexpressing CD8. The occurrence of identical NKa phenotypic profiles in both germline and rearranged TCR cases does, however, suggest the possiblity of an evolutionary process from a non‐clonal expansion to a clonal state. Serum studies, including soluble CD4. CD8 and IL2‐R concentrations and autoantibody investigations, of representative germline and rearranged TCR cases failed to indicate any consistent abnormalities, but there was some suggestion for the existence of a chronic reactive process in some of the patients with germline TCR. These findings suggest that expanded LGL/NKa+ components with phenotypic evidence of CD4/CD8 coexpression should be regarded as a distinct diagnostic category and that persistent CD4+CD8+ abnormalities with germline TCR should be monitored for possible clonal transition.

Human NK cells in health and disease: clinical, functional, phenotypic and DNA genotypic characteristics.

Natural killer (NK) cells are the subject of great current interest because of their possible (in vivo) role in tumour cell surveillance and killing, and because of the potential application of cytokine-modulated NK cells in cancer immunotherapy. In addition, clonal proliferations of NK-associated (NKa) cell populations represent a high proportion of chronic (non-B) lymphoid malignancies and abnormal (both clonal and non-clonal) NKa components are being increasingly reported in association with diverse clinical pictures such as autoimmune disease. This communication extensively reviews what is presently known regarding normal and leukaemic NKa phenotypic diversity, the mechanisms of NK-mediated cytolysis, the role of NK cells in malignancy, and the diagnostic and cellular aspects of malignant NKa proliferations.

Immunophenotypic Dissection of Normal Peripheral Blood NK Associated (NKa) Subpopulations by Flow Cytometry: Morphological Features and Relationships Between Membrane NKa (CD11b, CD 16, CD56 and CD57) arid T-cell (CD2, CD3, TCR, CD5, CD7, CD8 and CD38) Associated Determinant Expression.

Using single and multiple colour flow cytometry (FACSCAN), this study has examined the expression of NK-associated (NKa) CD11b, CD16, CD56 and CD57 membrane antigens by normal lymphocyte subpopulations. In addition to determining the normal proportions and absolute numbers of NKa(+) cells, this investigation also related the expression of these four NKa markers to the presence of; (a) morphologically-defined large granular lymphocytes, (LGL); and (b) membrane CD2, CD3, CD8 and TCR chain expression. This was achieved by preparing highly enriched normal blood CD4-CD8(+)/CD4 CD8(+) and CD4(-)CD8(-) lymphocyte fractions (n = 6) by immunomagnetic depletion of CD4(+)/CD14(+)/CD19(+) or CD4(+)/CD8(+)/CD14(+)/CD19(+) components respectively. Morphological assessment of these fractions showed that the sequential depletion procedures resulted in the enrichment of LGL, from a mean of 9% for the whole lymphocyte (pre-depletion) fraction, to 35% following removal of CD4(+), CD14(+) and CD19(+) cells and to 80%) following the additional removal of CD8(+) cells. FACS analysis revealed the presence of three CD8 subgroups (CD8(-), CD8(dim+) and CD8(+)), defined by differences in membrane staining intensity, and subsequent three-colour (CD2/CD3/CD8) studies indicated that the main composite CD2/CD3 phenotypes for these CD8 groups were (relative frequencies in parenthesis); (a) CD8(+), CD2(+)CD3(+) (94%); (b) CD8(dim+), CD2(+) CD3(+) (31%) and CD2(+)CD3(-) (52%); and (c) CDS(-), CD2(+)CD3(+) (20%), CD2(+)CD3(-) (58%) and CD2(-)CD3(-) (22%). Analysis of paired NKa marker expression (CD11b and CD56; CD11b and CD57; CD16 and CD57), when correlated with CD3 and CD8, showed that (a) the predominant NKa phenotype of CD3(+)CD4(-)CD8(+) cells was CD11b(-)CD16(-)CD56(-)CD57(-); (b) CD3(+)CD4(-)CD8(dim+) and CD3(+)CD4(-)CD8(-) subpopulations showed CD11b(-)CD16(-)CD56(-)CD57(±) and CD11b(±)CD16(-)CD56(-)CD57(-) composite phenotypes respectively (where - and ± denote <20% and 21-60%0 NKa(+) positive cells); and (c) the highest proportions of NKa(+) cells were associated with CD3- subpopulations, with composite phenotypes for CD3-CD4-CD8(dim+) and CD3(-)CD4(-)CD8(-) components being CD11b(-)CD16(±)CD56(+)CD57(±) and CD11b(+)CDl6(+)CD56(+)CD57(±) (where + denotes >60% NKa(+) cells). Expression of NKa determinants by normal CD4(-) lymphoid subpopulations was thus shown to increase in the order CD3(+)CD8(+) > CD3(+) CD8(dim+) or CD3(+)CD8(-) > CD3(-)CD8(dim+) > CD3(-)CD8(-). Studies of TCR chain expression by NKa(+) (CD16/CD56) and NKa(-) subgroups of the CD4(-)CD8(-) fraction additionally showed that the presence of membrane TCRαδ (mean 18%) or TCRγδ (mean 35%) chains were primarily associated with the NKa(-) component and that virtually all (93%) NKa(+) cells were TCRαβ-TCRγδ. Examination of CD4(-)CD8(-)NKa(+) cells for the expression of other T-cell associated markers (CD5, CD7 and CD38) also indicated that this NKa(+) LGL-rich fraction could be further subdivided into CD2(+)CD3-CD5(-)CD7(+)CD38(+) and CD2-CD3(-)CD5(-)-CD7(+)CD38(+) subpopulations.

Flow cytometric analysis of membrane CD11b, CD11c and CD14 expression in acute myeloid leukaemia: relationships with monocytic subtypes and the concept of relative antigen expression

Abstract: Blast cells from 26 cases of acute myeloid leukaemia (AML) were examined, by single and “two‐colour” flow cytometry, for relationships between membrane CD11b (monoclonal antibody OKM1), CD11c (KB90) and CD14 (Leu‐M3). Increased expression of all three determinants was associated with myelomonocytic leukaemias, with their relative diagnostic value in discriminating monocytic (M4 and M5) from non‐monocytic (M1, M2 and M3) subtypes being CD14 > CD11c > CD11b. However, the results also indicated, because of the heterogenous expression of CD11c in particular, and to a lesser extent CD11b, that the patterns or histograms of fluorescent staining were potentially more informative than an empirical subdivision of blasts into positive and negative sub‐populations. In addition, analysis of phenotypic correlations by simultaneous two‐colour fluorescence showed that the expression of CD11b and CD11c determinants by leukaemic myeloid blasts was highly correlated, in contrast to the expression of CD14 and CD11c which were relatively independent. Consequently, CD11c + myeloid blasts almost always co‐expressed CD11b whereas CD14 + cases of AML often comprised CD14 + CD11c + and CD14 + CD11c‐ subpopulations. It is concluded from these observations that CD11c immunophenotyping is a useful supplementary investigation, particularly in CD14‐ cases of myelomonocytic leukaemia. However, it is also apparent that the presence of membrane CD11c per se is not lineage‐specific and that the level of expression is perhaps a more discriminatory factor.

Abnormal CD45R expression in patients with common variable immunodeficiency and X-linked agammaglobulinaemia

This study has investigated the patterns of membrane 2H4 (CD45RA) andUCHL1 (CD45RO) expression by CD4+ and CD8 + lymphocyte subpopulations in 10 adults and seven children with common variable immunodeficiency (CVI) and X‐linked hypogammaglobulinaemia (XLA). Of the 10 adults (CVI, n= 8; XLA, n= 2), only one with a diagnosis of CVI showed normal CD45R expression. For the remaining seven adult CVI patients the abnormal CD45R profiles were primarily associated with CD4+ lymphocytes in three and CD8+ lymphocytes in four. Specific increases in CD45RA‐CD45RO+ fractions were found in four CVI patients and in all of these there was a concomitant reduction in circulating CD19+ B‐cell numbers. Two of the CVI cases were identical twin sisters and both had the same CD45R abnormality. The two adults with XLA also showed abnormal CD45R expression (increased CD45RA+CD45RO∼ components) but of particular note, in view of the fact that these were brothers, one was associated with the CD4+ subpopulation and the other with the CD8+ fraction. Similar analyses for the paediatric group revealed, in distinct contrast to the adult patients, no significant abnormalities of CD45R expression suggesting that these defects may not become apparent until a later age. Further investigations of in vitro lymphocyte proliferation (PHA and PWM) in the eight adults with CVI showed a significant correlation between abnormal responses and disordered CD45R expression. It is proposed that these abnormal patterns of CD45R expression by lymphocyte subpopulations in CVI and XLA may be of fundamental importance with respect to the pathogenesis of these particular immunodeficiencies.

Lineage-Specific Monocytic Esterase, a Distinct Marker for Leukemias of Monocytic Origin: Cytochemical, Isoenzymatic and Biochemical Features

Cytochemical staining of non-specific esterases is commonly used in the classification of acute leukemias. In the biochemical nomenclature these enzymes represent the carboxylesterases (EC 3.1.1.1) and are mostly visualized by a substrate-diazonium dye reaction using alpha-naphthyl acetate which led to the term alpha-naphthyl acetate esterases (ANAE). Separation of cell extracts by polyacrylamide gel electrophoresis (PAGE) or isoelectric focusing (IEF) have demonstrated that the carboxylic esterase (system) comprises a number of distinct isoenzymes. Included among these is a distinct isoenzyme (group), defined by IEF with acidic isoelectric points (pI) of approximately 6.0, that is characterized by its strong staining intensity and inhibition by sodium fluoride (NaF). Due to its selective expression in normal and malignant monocytes and its unique plasmalemmal location as an ectoenzyme on monocytes, this isoenzyme (the monocyte esterase) appears to be specific for cells of monocytic origin. While a number of studies have analyzed the biochemical features of the monocyte esterase, little is known about its physiological function. Examining purified normal cell populations and fresh or cultured leukemia cells, it was found that the expression of the monocyte esterase is, indeed, restricted to cells of monocytoid/macrophage differentiation. Expression of this protein can be induced in-vitro by physiological or pharmacological agents in responsive cell populations which can be triggered to differentiate along the monocyte/macrophage cell axis. Compared with the level of enzymatic activity in normal monocytes, there seems to be an overexpression of monocyte esterase in some leukemic cells. Molecular cloning of the gene encoding this protein, an emphasis on studies of function and further detailed characterization of the biochemistry are future avenues of research.

Diagnostic differentiation of chronic B‐cell malignancies using monoclonal antibody L161 (CD1c)

The expression of membrane CD1c, as defined by monoclonal antibody L161, was examined on malignant lymphoid cells from 191 cases of chronic lymphoproliferative disease and on eight ‘normal’ enriched tonsil B‐cell extracts. Of 79 cases of chronic lymphocytic leukaemia (CLL) studied, 77 showed low (<20% positive cells) CD1c expression whereas 63/71 (89%) cases of B‐PLL, HCL and B‐NHL showed increased CD1c+ (but not CD1a or CD1b) components. In contrast, malignancies corresponding to terminal stages of B‐cell differentiation (immunocytoma and myeloma) generally showed low CD1c expression as did lymphoid cells from 10 cases of post‐thymic malignancy. Although there was some correlation between the expression of membrane CD1c and immunoglobulin (SIg) light chain densities (P<0.001), it is relevant in diagnostic terms that seven cases of B‐NHL, with low SIg staining intensities more typically associated with CLL were CD1c+. CD1c expression was not, however, correlated with the presence of CD23 or FMC7 determinants but did show a similar pattern of expression to that previously reported for beta‐2 microglobulin. Determination of cellular CD1c by APAAP immunocyto‐chemistry confirmed the presence of higher antigen densities in malignant B‐cells at intermediate/late stages of differentiation and this interpretation was further supported by the finding that the majority of phenotypically mature tonsil B‐cells were also CD1c+. The determination of CD1c expression by malignant B‐cells may therefore be of particular value in the diagnostic differentiation of chronic lymphoproliferative disorders.