Human cord blood progenitors: kinetics, regulation and their use for hemopoietic reconstitution.

The high number of cord blood (CB) hematopoietic progenitors made possible CB transplantation in children with hematologic diseases. We collected and cryopreserved CB from siblings of 11 hematologic pediatric patients for possible transplant. The number of BFU-E+CFU-GEMM and CFU-GM was adequate for engraftment. However the possibility of CB transplantation in adolescents and adults is limited by the amount of CB that can be collected. We studied the possibility of expanding the pool of CB hematopoietic progenitors by preincubation of CB CD 34+ cells with several cytokines. After pre-exposition to rhIL-3, IL-1 beta, rIL-6, GM-CSF and stem cell factor (SCF) and cloning with SCF the number of hematopoietic progenitors increased 14-18 fold compared to basal values.

Genotypic characterization of common acute lymphoblastic leukemia may improve the phenotypic classification.

The reactivity of five anti-B monoclonal antibodies (McAb)-OKB2 (CD24), B4 (CD19), Leu12 (CD19), BA1 (CD24), B1 (CD20)--as well as the presence of cytoplasmic immunoglobulins (CyIg) were assessed in 100 cases of common acute lymphoblastic leukemia (cALL) at presentation (TdT+, J5 [CD10]+, HLA-Dr+). All cases studied revealed one or more B-cell related markers and a hierarchy in their expression was documented: OKB2 was positive in all cases tested (100%), B4 was expressed in 96.4% of cases, Leu12 in 95.8%, BA1 in 94.9%, B1 in 18.3%, and CyIg in 23%. Further evidence of the B-cell origin of cALL was obtained by molecular analyses at the DNA level which demonstrated the presence of an Ig heavy chain gene rearrangement in all 37 cases assessed, while 37.8% showed a light chain gene reorganization. A genomic subclassification of cALL demonstrated that the majority of cases showed an immature molecular configuration with one (8.1%) or both (54.1%) Ig heavy chain alleles rearranged and a germ-line configuration of the light chain genes; 27% revealed a heavy chain gene involvement and one k allele rearranged. Only four cases (10.8%) showed a more mature configuration with both k alleles rearranged or a gamma chain gene involvement. This study confirms that cALL is characterized by the proliferation of immature B-lineage-committed elements and indicates that the leukemic cells are blocked at different levels of B-differentiation which may be recognized with the use of multiple phenotypic or genotypic B-cell-related markers.

Chronic T-cell leukaemias. A variant of T-prolymphocytic leukaemias: morphological, immunological and clinical characterization of 2 cases.

In this study we describe 2 patients who appear to suffer from a morphological, cytochemical and clinico-haematological variant of T-prolymphocytic leukaemia (T-PLL). The cells were smaller than typical prolymphocytes, with a regular nucleus containing a smaller and less prominent nucleolus; the alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP) cytochemical reactions showed a weaker pattern of positivity in this variant compared to T-PLL. No immunological differences were found between the two conditions with regard to membrane expression and functional behavior of the cells. The clinical course and the outcome of the patients appears to be different: aggressive and rapidly fatal in T-PLL; thus far well-controlled in the T-PLL variant. From a molecular point of view, both cases showed a monoclonal rearrangement of the T-cell receptor beta-chain gene.

Heterogeneity of large granular lymphocyte proliferations: morphological, immunological and molecular analysis in seven patients.

The clinical, morphological, immunological and molecular features of seven patients with a stable picture of chronic granular lymphocytosis, observed over a period of up to 4 years, were studied. Mild splenomegaly was detected in one patient, while lymphoadenopathy and hepatomegaly were absent. Surface marker analysis showed in five patients the common membrane phenotype of granular T-cell lymphocytosis (T3+, T4-, T8+, Leu-7+); of the remaining two, one presented an unusual phenotype (T3+, T4+, T8+) and the other showed a marked positivity with the Leu-11 and M1 monoclonal antibodies, but lacked the T3, T4, T8 antigens. Three cases had a low (less than 30%) expression of the T1 antigen. Functional studies showed that the proliferative response to PHA and the NK function were reduced in four of the seven cases. Molecular analysis, performed in six cases, revealed a monoclonal rearrangement of the T-cell receptor beta-chain gene in three, a polyclonal T-cell configuration in two and a germ-line arrangement in the last. All three monoclonal cases showed a depressed NK activity and two a reduced PHA response. The results of this study document the heterogeneity of granular lymphocyte expansions and suggest that the clonal or reactive nature of these often indolent proliferations, suspected on the basis of immunologic functional studies, may be recognized at the DNA level.

Rearrangement of the c-myc oncogene with heavy-chain immunoglobulin enhancer in tumor DNA from an acute lymphoblastic leukemia patient.

The DNA obtained from the leukemia cells of an acute lymphoblastic leukemia (ALL, L3 type) with a pre-B-phenotype and a typical t(8;14) chromosomal translocation showed a rearrangement juxtaposing the c-myc gene and the immunoglobulin (Ig) heavy-chain gene enhancer. This abnormality was only present in the leukemia cells of the patient and correlated with the clinical course of the disease. The breakpoint on chromosome 8 occurred within c-myc intron 1, between 790 and 638 base pairs upstream of c-myc exon 2. This breakpoint position was the nearest to the c-myc exon 2 so far described in Burkitt's type lymphoma-leukemias, and it mapped very near to the location of a major cryptic promoter used by truncated c-myc genes. In spite of what was detected in a human lymphoma cell line (Manca) carrying a similar rearrangement, in this case the amount of c-myc transcript was not increased compared to an Epstein-Barr virus-transformed normal lymphoblastoid cell line obtained from the same patient. This may in part be due to the breakpoint position and to the fact that the efficiency of the major cryptic promoter present within the first intron could have been affected by the translocation event. Finally, as previously suggested by others, the phenotype expressed by the leukemia cells supported the notion that this particular type of rearrangement (linking together the c-myc gene and the Ig heavy-chain gene enhancer element) may be associated with a subgroup of B-ALLs showing an immunologic phenotype relatively more immature than that of classical B-ALL.

Configuration of the immunoglobulin and T cell receptor gene regions in hairy cell leukemia and B-chronic lymphocytic leukemia.

The configuration of the immunoglobulin heavy and light chain genes and of the T cell receptor (TCR) beta region was examined in a series of patients with hairy cell leukemia (HCL) and compared with the findings in B cell chronic lymphocytic leukemia (B-CLL). In all cases of HCL and B-CLL studied a rearrangement of the immunoglobulin heavy chain loci coupled to a reorganization of the light chain genes was documented. However, in all HCL and all but one B-CLL the TCR beta gene region was in a germ-line configuration. These findings confirm that HCL is characterized by the expansion of relatively mature B cell elements with a molecular configuration similar to that of B-CLL and indicate that the reported expression of T cell related markers, particularly in B-CLL, is not coupled to a rearrangement of the TCR beta-chain gene. Analyses of the immunoglobulin gene regions in HCL represent an important diagnostic tool as well as a possible aid toward monitoring the response to treatment.

Rearrangements of immunoglobulin and T cell receptor beta and gamma genes are associated with terminal deoxynucleotidyl transferase expression in acute myeloid leukemia.

The cell origin of the rare terminal deoxynucleotidyl transferase (TdT)-positive acute myeloid leukemias (AML) was investigated at the molecular level, by examining the configuration of the Ig H (Igh) and L (Ig kappa, Ig lambda) chain gene regions, and of the T cell receptor (TCR) beta and T cell rearranging (TRG) gamma loci. In 8 of the 10 TdT+ AML analyzed (classified as myeloid according to morphological and cytochemical criteria, and to the reactivity with one or more antimyeloid mAbs), a rearrangement of the Igh chain gene was found. In TdT- AML, evidence of an Igh gene reorganization was instead observed only in 2 of the 42 patients studied. Furthermore, evidence of TCR-beta and/or TRG-gamma gene rearrangement was observed in four AML, all of which belonged to the Igh-rearranged TdT+ group. In three cases (one TdT+ and two TdT-), the Ig kappa L chain gene was also in a rearranged position. These findings demonstrate a highly significant correlation between TdT expression and DNA rearrangements at the Igh and TCR chain gene regions and support the view that this enzyme plays an important role in the V-(D)-J recombination machinery. Overall, the genomic configuration, i.e., JH gene rearrangement sometimes coupled to a kappa L chain and TCR gene reorganization, similar to that found in non-T-ALL, suggests that in most cases of TdT+ AML, the neoplastic clone, despite the expression of myeloid-related features, is characterized by cells molecularly committed along the B cell lineage.

Molecular and immunological evidence of B-cell commitment in "null" acute lymphoblastic leukaemia.

The DNA configuration of the immunoglobulin (Ig) heavy and light chain genes and the expression of B-cell-related markers were evaluated in 13 cases of non-T, non-B, non-common ("null") acute lymphoblastic leukaemia (ALL). A rearrangement of the Ig heavy-chain gene was found in all cases studied; in 5 of these a structural reorganization of the kappa or lambda light chain gene was also demonstrated. Leukaemic cells from 10 of the 13 cases analysed showed one or more B-cell antigens, the expression of which followed a sequential order of presentation (OKB2, B4, BA-1, B1). The B-cell commitment was confirmed by means of a sensitive immunoperoxidase assay which revealed a weak expression of the common ALL (cALL) antigen in 7/10 cases tested, which were all cALL-negative by conventional immunofluorescence techniques. These findings suggest that in "null" ALL the neoplastic cells show molecular and immunological evidence of B-cell differentiation and that most cases may indeed be characterized by "early" cALL with a very low density expression of the cALL antigen. This was further documented in one case in which the expression of the cALL antigen (and of other B-cell markers) could be induced after exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The presence in a few cases of myeloid features, particularly when the cALL antigen could not be demonstrated by the immunoperoxidase assay, suggests that the leukaemic process may sometimes involve a very early progenitor cell capable of both lymphoid and myeloid phenotypic differentiation. The heterogeneity of "null" ALL documented by this study may help to explain the variable clinical course and prognosis of these patients.

3' c-myc rearrangement in a human leukemic T-cell line.

The human leukemic T-cell line Hut 78, derived from a patient suffering from Sézary syndrome and expressing a mature postthymic membrane phenotype, shows a c-myc rearrangement beginning within 500 base pairs immediately 3' to the c-myc exon 3. Chromosome analysis of the Hut 78 reveals the presence of a hyperdiploid karyotype with a large number of markers and rearrangements, though trisomy is the only cytogenetic anomaly involving chromosome 8. Moreover, as the abnormal c-myc appears to be duplicated, a duplication of the chromosome 8 carrying the abnormal c-myc probably occurred. Unlike four other human leukemic T-cell lines tested, the Hut 78 cells express a high amount of c-myc transcript, suggesting that the 3' c-myc anomaly may cause a deregulation of the expression of this gene.

Relevance of anti-T monoclonal antibodies in the study of children with primary immunodeficiencies.

T-cell phenotypic analysis with anti-T monoclonal antibodies (MoAb) was performed on 37 children with immunologic disorders. Abnormalities of T-cell differentiation and/or of T-cell subset distribution were observed in many patients. In particular two infants with severe combined immunodeficiency showed immunologically incompetent common thymocytes (OKT6+) in the circulation, in one case a proportion of OKT6+ cells was OKT4-, OKT8-. A boy with a selective T-cell defect synthetized normal levels of Ig classes, despite the marked reduction of helper/inducer T-cells (OKT4+). Irregularities of T-cell subsets were also noted in children with Wiskott-Aldrich syndrome and in some patients with selective IgA defect or hypogammaglobulinaemia. In one of these, in whom the agammaglobulinaemia was caused by EBV infection, a persistently reversed OKT4+/OKT8+ ratio together with an excessive suppressor T-cell function were found more than 10 years after the onset of the disease. Such a case supports the hypothesis that a viral infection may cause, in a predisposed host, both the agammaglobulinaemia and an abnormality of the regulatory T-cell subpopulations. Such abnormalities, together with those found in the other children studied, underline the importance of MoAb against different T-cell antigens for a better characterization of primary immunodeficiencies.

Discrepancy between phenotypic and functional features of natural killer T-lymphocytes in B-cell chronic lymphocytic leukaemia.

The phenotypic expression and functional capacity of natural killer (NK) T-lymphocytes (E+, OKT3+) were analysed in a series of untreated patients with B-cell chronic lymphocytic leukaemia (B-CLL). The mean value of NK activity of B-CLL T-lymphocytes, tested against the K562 cell line, was significantly depressed (P less than 0.01) in the 20 cases studied, compared with that of normal T-cells. Incubation with human leucocyte interferon produced an increase (P less than 0.05) in NK activity, although the mean value was still significantly lower (P less than 0.05) than that obtained with normal T-cells. Furthermore, the formation of effector-target conjugates was significantly lower (P less than 0.01) among B-CLL T-cells compared with normal T-lymphocytes. Despite the reduced NK functions observed in the majority of B-CLL patients, the capacity of T-cells to react with the monoclonal antibody (MoAb) Leu-7 (HNK-1 clone), assessed in 60 patients, was significantly higher (P less than 0.001) in B-CLL than in normal blood (mean 24% +/- 10.6 SD v 9% +/- 4.2), irrespective of the clinical stage of the disease. These findings suggest that the reduced cytotoxic ability of B-CLL T-lymphocytes may be due either to an expanded population of immature T-cells which already express a cytotoxic-like phenotype (E+, OKT3+, HNK-1+) but which lack adequate cytotoxic functions, or, alternatively, to an intrinsic defect of the natural effectors present within the T-cell population of B-CLL. The T-cell functional abnormalities documented in this study, together with other defective functions previously described, may be implicated in some of the complications frequently associated with B-CLL, particularly the high incidence of secondary neoplasms. There is growing evidence that natural cytotoxicity may play a major role in the immune surveillance system, both against tumour cells and virus-infected cells (Herberman & Ortaldo, 1981). Natural killer (NK) cells are a morphologically homogeneous population of large granular lymphocytes with azurophilic granules in the cytoplasm (Timonen et al, 1981), which are non-adherent and express receptors for the Fc portion of IgG. About 50% of these cells form rosettes with sheep red blood cells (E-rosettes) (West et al, 1977). A monoclonal antibody (MoAb) which appears to react with practically all human NK cells (HNK-1 clone, Leu-7; Becton Dickinson) has been recently produced (Abo & Balch, 1981).(ABSTRACT TRUNCATED AT 400 WORDS)

Different stages of B cell differentiation in non-T acute lymphoblastic leukemia.

Immunoglobulin heavy chain gene rearrangement was evaluated in 19 cases of acute lymphoblastic leukemia (ALL) and correlated with the immunological phenotypic expression on primary or phorbol diester (12-O-tetradecanoylphorbol-13-acetate [TPA])-induced cells. One case of common ALL (cALL), one case of T-ALL, and one undifferentiated acute leukemia that responded to anti-myeloid drugs after unsuccessful anti-lymphoid induction therapy, had germ line heavy chain genes. Rearranged immunoglobulin genes were instead found in 15 of the 16 cALL cases studied and in a case of non-T, non-B, non-common ("null") ALL, which suggested the B cell origin of the neoplastic cells. All cases bearing a heavy chain gene rearrangement were HLA-DR positive. However, the unique cALL case with a germ line configuration was also HLA-DR positive, which confirmed that both the cALL antigen and HLA-DR antigen were not per se expression of B cell commitment. On the other hand, a complete search for B cell-related markers (BA-1 and B1 monoclonal antibodies, as well as cytoplasmic immunoglobulins [CyIg]) in the cALL cases showed that at least one B cell marker could be detected either on primary or on TPA-induced cells in all cases in which a gene rearrangement had occurred. Incubation with TPA allowed the detection of one B cell marker in a case in which the primary cells were negative, and increased the expression of B cell markers in all but one of the cALLs tested. The only cALL case that was not rearranged expressed no B cell markers either on primary or on TPA-induced cells. The non-T, non-B, non-common ("null") case that was rearranged also showed no phenotypic evidence of B cell markers on primary and induced cells. These findings indicate that: (a) practically all cases of cALL appear to be of B cell origin as shown by gene rearrangement analysis; (b) DNA studies are relevant for a more precise characterization of individual cases of undifferentiated acute leukemia; (c) a complete survey for B cell markers may establish the B cell origin of the cALL blasts, as long as the analysis on primary cells is complemented by differentiation induction assessment; and (d) most cases of non-T ALL appear to be characterized by the expansion of neoplastic cells "frozen" at different levels along the B cell differentiation pathway, the first detectable marker being heavy chain gene rearrangement, followed by BA-1, B1, and CyIg expression.

Immature T lymphocytes in human cord blood identified by monoclonal antibodies: a model for the study of the differentiation pathway of T cells in humans.

The reactivity of human cord blood lymphocytes was assessed against a panel of monoclonal antibodies (MoAb). The mean proportion of OKT3+ cells (pan-T) was significantly lower in cord blood (52 +/- 13.8%; mean +/- SD) compared with that of adult blood (75 +/- 8.9%) and paralleled well with the E-rosette-forming capacity (50 +/- 16.3%). Both the proportions of OKT4+ cells (helper/inducer phenotype) and of OKT8+ cells (suppressor/cytotoxic phenotype) were significantly reduced in cord blood (43 +/- 11.8% vs 50.3 +/- 7.4% and 20 +/- 10.3% vs 25.6 +/- 6.0%, respectively), while the overall OKT4/OKT8 ratio was increased compared with adult blood (2.87 +/- 1.83 vs 2.04 +/- 0.61). Unlike adult blood, in 30 of the 35 samples of cord blood an overlap was observed between the total proportion of OKT4+ and OKT8+ cells (65 +/- 15.2%) and that of OKT3+ cells (52 +/- 14.3%). Although small numbers of cells coexpressing both antigens were occasionally found, double-staining analysis showed that the overlap in cord blood was mostly due to an expanded proportion of OKT3 (Leu-4)-/OKT8 (Leu-2)+ cells. Relevant proportions of OKT6+ (common thymocyte antigen) and OKT10+ (thymocytes, activated T cells, precursor cells) cells were found in cord blood as opposed to adult blood (10.8 +/- 8.6% vs 0.6 +/- 0.6% and 67 +/- 18.0% vs 8 +/- 2.1%, respectively), while terminal deoxynucleotidyl transferase-positive cells were observed only in two samples of cord blood. A small proportion of T cells (E-rosette+) reacted with the MoAb OKIa1 (HLA-DR). Finally, the proportion of cord blood cells recognized by the MoAb Leu-7 (HNK-1 clone) was almost negligible compared with adult blood (2.8 +/- 2.4% vs 15 +/- 7.5%). These data confirm the immaturity and heterogeneity of cord blood lymphocytes and demonstrate the presence at birth of circulating lymphocytes which express a surface phenotype reminiscent of that found in the late stages of intrathymic differentiation and in some human T-cell leukemias. Human cord blood may thus represent a suitable model for the study of the differentiation pathway of normal and pathological T-cells in humans.

Membrane phenotype and functional behaviour of T lymphocytes in multiple myeloma: correlation with clinical stages of the disease.

The distribution of T lymphocyte subsets was assessed using monoclonal antibodies (MoAbs) in 44 untreated patients with multiple myeloma (MM) subdivided according to the clinical stage of the disease. A significant reduction (P less than 0.001) of T lymphocytes was observed only in stage II and III patients. The proportion and absolute number of OKT4 positive cells (helper/inducer phenotype) were significantly reduced in all stages of the disease; this quantitative abnormality was more pronounced in advanced disease. While the proportion of OKT8 positive cells (suppressor/cytotoxic phenotype) was increased above normal in all stages, the absolute number (of OKT8 positive cells) was high only in stage I patients; on the contrary in stage II-III patients the total OKT8 count was reduced compared with normal controls. A significantly reduced OKT4/OKT8 ratio was found in both groups of patients (P less than 0.005). Functional studies, carried out on the unfractionated T cells of patients with MM, demonstrated a consistent helper defect in the ability to induce the differentiation of normal B lymphocytes into antibody producing cells in a pokeweed mitogen driven system. However, the removal of OKT8 positive cells produces a significant increase in helper capacity, suggesting that the reduced helper function of T lymphocytes in toto is probably due to excessive suppressor activity. The possible immunoregulatory role of MM T cell disease is discussed.

Effects of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) on the colony growth of human T lymphocytes.

The T colony promoting activity of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was assessed in a double layer culture assay which is dependent on the simultaneous presence of phytohaemagglutinin (PHA) and a leucocyte rich underlayer. TPA (10(-8) M) incorporated in the overlayer in place of PHA was capable of promoting T cell growth in the form of clusters in all 37 experiments performed and in the form of colonies in more than 50% of the samples tested. However, the T colony promoting activity of TPA alone was markedly less evident and consistent than that of PHA (mean 13 +/- 19.9 s.d. colonies vs 168 +/- 78.6). TPA concentrations of 10(-6) M, 10(-9) M and 10(-10) M were practically ineffective. On the other hand, the number of colonies obtained when both TPA 10(8) M and PHA were incorporated in the overlayer was significantly higher (P less than 0.01) than that observed with PHA alone (mean 250 +/- 108.2 vs 178 +/- 84.5 colonies). When TPA concentrations of 10(-9) M and 10(-10) M were used in addition to PHA, the enhancing effect was less evident, while an inhibition of T colony growth was observed with TPA 10(-6) M + PHA. TPA 10(-8) M was also capable of enhancing T colony growth when incorporated in the leucocyte rich underlayer (222 +/- 98.6 vs 172 +/- 80.9 colonies). In all cultures with TPA the peak of growth was delayed compared with that of control experiments with PHA. These findings demonstrate that TPA, particularly when co-cultured with PHA, is an effective T colony promoting agent. The observation that the number of colonies formed in the presence of TPA plus PHA is higher than the sum of those observed with the two stimulators independently, suggests that their synergistic effect may be mediated via the production of colony stimulating soluble factors.

Lineage relationship of chronic lymphocytic leukemia and hairy cell leukemia: studies with TPA.

The tumor promoting agent TPA (phorbol ester; 1.6 X 10(-8)M) was used to induce the differentiation in vitro of B-chronic lymphocytic leukemia (B-CLL) cells from 14 untreated patients. The uninduced phenotype was SIg+, Mrbc+, RFT-1+, RFA-4-, FMC7-. After 72 h incubation with TPA, B-CLL cells became RFA-4+, FMC7+ and lost the capability of Mrbc rosetting. Large proportions of the "induced" cells also showed morphological and ultrastructural changes, such as undulating membranes and bleblike protusions and became strongly positive for tartrate resistant acid phosphatase (TRAP+) and also contained cytoplasmic immunoglobulins. These features are very similar to the features of hairy cell leukemia (HCL). These observations confirm previous clinical findings that B-CLL and HCL are related disorders of the B lineage. The development of "hairy" features in induced B-CLL and in HCL seems to be a malignancy-associated feature because the Mrbc+ normal B cells (B-CLL-equivalent cells) isolated from tonsil also develop TRAP positivity but no membrane aberrations.

Normal helper T-cell function in hairy-cell leukaemia.

The function of peripheral blood T-lymphocytes was tested in a series of untreated patients with hairy-cell leukaemia (HCL). The in vitro T-colony forming capacity fell within the normal range in 20 of the 22 cases studied. No difference in colony growth was found between splenectomized and non splenectomized patients. The ability to induce differentiation of normal B-lymphocytes into antibody producing plasma cells, in a pokeweed mitogen (PWM) stimulated system, was similar to that of normal T-cells in all 6 cases studied. Both the T-colony growth and the helper capacity were unrelated to the distribution of T-lymphocyte subsets defined by monoclonal antibodies, despite an increase in OKT8+ cells (suppressor/cytotoxic phenotype) and a decrease in OKT4+ cells (helper/inducer phenotype) in 44% of the patients. The significance of these findings is discussed in relation to T-cell abnormalities previously described in other B-cell leukaemias, chiefly chronic lymphocytic leukaemia (B-CLL). The preserved T-cell function in HCL correlates well with the normal humoral immunity found in this disease.

Relevance of monoclonal antibodies in the diagnosis of unusual T-cell acute lymphoblastic leukaemia.

3 cases of adult acute lymphoblastic leukaemia (ALL), the T-cell nature of which was identified only using a panel of monoclonal antibodies (MoAb), are described. All cases were E-rosette negative, surface immunoglobulin (SmIg) negative, common ALL (CALLA) antigen negative, terminal deoxynucleotidyl transferase (TdT) positive, and acid phosphatase positive. The T-cell origin of the blasts was demonstrated by the positivity with RFA-1, a MoAb which detects an antigen of MW 65-69000 present on the membrane of thymocytes and mature T-lymphocytes. In addition, 2 of the 3 cases were positive with OKT6, which recognizes cortical thymocytes. MoAb directed against more mature T lineage cells (OKT3, OKT4, OKT8, OKT11A) were consistently negative (less than or equal to 12%). These findings indicate that the use of a combination of MoAb is important in detecting individual cases of T-ALL, which otherwise might be classified as undifferentiated acute leukaemia or null-ALL. MoAb detecting a T-cell antigenic determinant of MW 65-69000 (e.g. RFA-1, OKT1, Leu1) appear the most specific reagents for T-ALL.

Characterization of T-lymphocyte subsets in hairy-cell leukaemia (HCL) by monoclonal antibodies: comparison with Fc gamma, Fc mu receptors and correlation with disease activity.

T lymphocytes from 22 patients with hairy-cell leukaemia (HCL) were assessed on the basis of their ability to bind the Fc receptors for IgM (T mu) or IgG (T gamma), and by the capacity to react with OKT monoclonal antibodies. T-cell subsets defined by the presence of Fc receptors for IgM or IgG showed an overall increase in the proportion of T gamma cells and a non-significant decrease of T mu cells, regardless of the clinical state of the disease. Results with monoclonal antibodies showed that in patients with HCL in clinical remission T-cell subsets were normally balanced, while in patients with active disease the distribution and absolute number of T-cell subpopulations appeared markedly impaired, with a significant increase of OKT8 positive cells (suppressor/cytotoxic) and a significant reduction of OKT4 positive cells (helper/inducer) compared both with active disease patients and with normal controls. The OKT4+/OKT8+ ratio was also significantly reduced in patients with active disease compared with those in clinical remission and with controls (0.96 v 1.63 and v 1.94, respectively). Our findings confirm the heterogeneity of T-cell subset positivity defined by monoclonal antibodies and by Fc mu and Fc gamma receptors and suggest that in patients with HCL the distribution of OKT4 and OKT8 positive cells is closely correlated to the clinical state of the disease.

[Effect of human plasma on the in vitro formation of T-lymphocyte colonies]. / Effetto del plasma umano sulla formazione in vitro di colonie T-linfocitarie.

T-lymphocyte colonies can be obtained from normal human peripheral blood and bone marrow, using a double layer technique which requires the simultaneous presence of peripheral blood irradiated leucocytes in the underlayer and phytohaemagglutinin in the overlayer (Foa & Catovsky, Clin. Exp. Immunol., 36, 488, 1979). The absence of either of these two factors unables colony formation. In an attempt to improve the plating efficiency 15% autologous plasma was added to the leucocyte rich underlayer, or tested alone. The results demonstrated that both leucocytes and plasma alone show a similar colony promoting activity, giving rise respectively to 160 +/- 80.4 SD and 180 +/- 113.5 SD T-colonies. The combination of both factors produced a significant increase in growth, with an overall mean of 410 +/- 190.5 colonies. Increasing concentration of plasma (1%, 5%, 10%, 15%) gave rise to a dose-dependent increase in colony growth. Furthermore, the simultaneous presence of leucocytes and plasma in the underlayer enabled good colony formation with as little as 0.1 x 105 tes cells in the overlayer. These findings indicate that both human peripheral blood leucocytes and plasma possess T-colony stimulating activity, and that optimal growth is achieved with the combination of these two factors in the underlayer.