George Janossy

Immunologically defined subclasses of acute lymphoblastic leukaemia in children: their relationship to presentation features and prognosis.

Summary. Leukaemic cells from 542 patients under 21 years of age with a diagnosis of acute lymphoblastic leukaemia (ALL) were typed with immunological cell surface markers between June 1975 and December 1979; 379 of these patients entered into the trials up until December 1978 have been followed for more than 1 year. They were divided into four subgroups: common (c) ALL, T (thymic) ALL, ‘null’ (or ‘unclassified’) ALL and a rare lymphoma/leukaemia type B‐ALL. A T‐cell phenotype was found more frequently in boys and was usually but not invariably associated with a high white cell count at presentation. A mediastinal thymic mass was present in 53% of T‐ALL patients but was not observed in any unequivocal non‐T ALL. Clinical prognosis differed substantially between the three major phenotypic classes, remission induction rate and remission duration being lowest in T‐ALL, better in ‘null’ ALL, and highest in cALL (P trend < 0·0001; P=0·0002 for comparison of cALL versus T‐ALL). There was a much higher incidence of CNS involvement in the T‐ALL group than in the cALL group or ‘null’ ALL group and although this was strongly correlated with WBC count it was also significantly associated with T‐ALL independent of WBC count.

Serial CD4 lymphocyte counts and development of AIDS.

Low CD4 lymphocyte counts are associated with increased risk of progression to AIDS in human immunodeficiency virus (HIV) infection. We investigated the extent to which the timing of progression to AIDS can be explained solely in terms of decline of the CD4 lymphocyte count in 111 haemophiliacs followed for up to 11 years since infection with HIV. A median of 10 CD4 lymphocyte counts were made per patient. By applying a simple linear model for the decline in CD4 lymphocyte counts over time, we estimated the date of development of AIDS in 96 patients who had at least 5 determinations. 84% (81 of 96) of patients were correctly classified as to development of AIDS before Jan 1, 1990 (p less than 0.0001), with this model. The results suggest that differences in the time at which patients with HIV will progress to AIDS can largely be explained by differences in rates of decline of CD4 lymphocyte counts.

The synergy between naive and memory T cells during activation

Naive and memory T-cell subsets differ in their ability to synthesize and respond to a variety of cytokines in vitro and each subset can produce cytokines that amplify the response of the other subset. The significance of these interactions to antigen responsiveness has, until now, been unclear. In this article Arne Akbar and colleagues point out that both subsets are activated to the same extent by alloantigen and suggest that synergy may be an important event in initiating potent responses against transplanted allografts.

Intestinal lymphocyte subpopulations in inflammatory bowel disease: an analysis by immunohistological and cell isolation techniques.

Lymphocyte subpopulations in the intestinal mucosa of patients with ulcerative colitis or Crohns disease have been studied using a double marker immunofluorescence technique. Analysis of tissue sections revealed that the majority of intraepithelial lymphocytes (IEL) were T cells (Hle-1+ HuTLA+ UCHT1+). Of these, over 80% were of suppressor-cytotoxic phenotype (OKT8+:83 +/- 10.2%) with a small population of helper type IEL (OKT4+). Only one third of OKT8+ IEL reacted with the T cell antibody, anti-Leu-1. IEL were also Tac-, C3b-receptor- (C3RT05-), and Ig-. Within the lamina propria, OKT4+ T cells predominated (ulcerative colitis 64 +/- 6.0%; Crohns disease 63 +/- 6.0%). Less than half of the smaller OKT8+ population in the lamina propria was Leu-1+. These finding did not differ from those seen in histologically normal tissues from controls, and are similar to those reported in the small intestine. Mononuclear cells were also isolated from the intestinal lamina propria using an enzymatic technique. The majority of lymphocytes obtained were T cells (OKT3+), with populations of OKT4+ and OKT8+ cells. Comparison of the ratio of OKT4+ to OKT8+ lymphocytes determined by immunohistological analysis with that obtained in mucosal isolates, however, suggested that the isolation procedure may deplete OKT8+ cells. These findings indicate that an imbalance of mucosal immunoregulatory T cells, as defined by monoclonal antibodies, does not occur in inflammatory bowel disease. They also emphasize that functional studies of isolated intestinal mucosal cells should be combined with morphological studies of cell populations in situ.

Lymphocyte activation in HIV-1 infection. II: Functional defects of CD28-T cells

Objectives and design:The expression of the accessory molecule CD28 was compared in various populations of T and natural killer (NK) cells from HIV-1-negative and HIV-1-positive individuals and correlated with activation using mitogens in vitro. Methods:Multiparameter flow cytometric analysis using combinations of CD3 CD28 and other markers was performed together with absolute cell counting in peripheral blood. Blast transformation and proliferative responses were also quantitated using the Cytoronabsolute after stimulation with phytohaemagglutinin (PHA) and anti-CD3. CD28− cells were also purified to confirm the observations. Results:In HIV-1-negative individuals >90% of CD3+ T cells were CD28+ and responded to stimulation, while CD3− CD16+ CD57+ NK-like cells were CD28-and failed to respond. In HIV-1-positive individuals the expression of CD28 was greatly reduced and the proportion of CD3+CD28− T cells expanded. CD8 lymphocytosis was caused entirely by the accumulation of CD28− T cells and many of these expressed activation markers human lymphocyte antigen-DR, CD38 and CD45RO on their membrane and molecules such as TIA-1 and perform, associated with cytolytic function, in their cytoplasm. The strong positive correlation (r = 0.66) between the lack of CD28 expression and the poor proliferation from HIV-1-positive individuals was confirmed by demonstrating that only CD28+ cells transformed into lymphoblasts and proliferated. Although the CD28− including CD3+ T cells transiently expressed CD25 (interleukin-2Rct), they did not undergo blastogenesis or activation measured by bromodeoxyuridine uptake and died after 3–4 days in culture. These observations were confirmed in costimulation experiments with anti-CD2 and anti-CD28. Conclusion:In HIV-1 infection activated CD3+CD28− T cells accumulate but are unresponsive to mitogens and anti-CD28. These cells appear to represent terminally differentiated effector cells which fail to respond to further stimuli because of the absence of a CD28 second signal.

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

USE OF ANTI-T-CELL MONOCLONAL ANTIBODY OKT3 TO PREVENT ACUTE GRAFT-VERSUS-HOST DISEASE IN ALLOGENEIC BONE-MARROW TRANSPLANTATION FOR ACUTE LEUKAEMIA

Seventeen patients who received allogeneic bone-marrow transplants from matched or slightly mismatched (in four patients) siblings were observed for at least 60 days or until acute graft-versus-host disease (GvHD) developed. All donor marrows after preliminary manipulation were incubated with 1 mg of the murine monoclonal antibody OKT3 before infusion in an attempt to deplete them of immunocompetent T lymphocytes (opsonisation). In three of the seventeen patients acute GvHD of grade II or greater developed. Two of these patients died, but they had disseminated cytomegalovirus infection as well as GvHD. Eleven patients showed no evidence of acute GvHD, and four had transient limited skin rashes (grade I GvHD). Opsonisation of T lymphocytes has reduced the incidence of severe acute GvHD in this unit from 79% in an earlier group of 14 patients to 18% when added to prophylactic methotrexate.

Laboratory control values for CD4 and CD8 T lymphocytes. Implications for HIV‐1 diagnosis

With the advent of standard flow cytometric methods using two‐colour fluorescence on samples of whole blood, it is possible to establish the ranges of CD3. CD 4 and CDS T lymphocyte subsets in the routine laboratory, and also to assist the definition of HIV‐1‐related deviations from these normal values. In 676 HIV‐1‐seronegative individuals the lymphocyte subset percentages and absolute counts were determined. The samples taken mostly in the morning. The groups included heterosexual controls, people with various clotting disorders but without lymphocyte abnormalities as well as seronegative homosexual men as the appropriate controls for the HIV‐1‐infected groups. The stability of CD4% and CD8% values was demonstrated throughout life, and in children CD4 values < 25% could be regarded as abnormal. The absolute counts of all T cell subsets decreased from birth until the age of 10 years. In adolescents and adults the absolute numbers (mean±s.d.) of lymphocytes, CD3, CD4 and CD8 cells were 1·90±0·55, 1·45±0·46, 0·83±0·29 and 0·56± 0·23 ± 109/l, respectively. In patients with haemophilia A and B the mean values did not differ significantly. In homosexual men higher CD8 levels were seen compared with heterosexual men and 27% had an inverted CD4/CD8 ratio but mostly without CD4 lymphopenia (CD4<0·4 ± 109/l). However, some healthy uninfected people were‘physiologically’ lymphopenic without having inverted CD4/CD8 ratios. When the variations‘within persons’ were studied longitudinally over a 5‐year period, the absolute CD4 counts tended to be fixed at different levels. As a marked contrast, over 60% of asymptomatic HIV‐1+ patients exhibited low CD4 counts <0·4 ± 109/l together with inverted CD4/CD8 ratios. Such combined changes among the heterosexual and HIV‐1‐seronegative homosexual groups were as rare as 1·4% and 3%, respectively. For this reason, when the lymphocyte tests show <0·4 ± 109/l CD4 count and a CD4/CD8 ratio of less than unity, the individuals need to be investigated further for chronicity of this disorder, the signs of viral infections such as HIV‐1 and other causes of immunodeficiency.

Increased numbers of primed activated CD8+CD38+CD45RO+ T cells predict the decline of CD4+ T cells in HIV-1-infected patients.

OBJECTIVE To look for surrogate markers in HIV-1 infection that can predict the decline of CD4+ T cells. METHODS Multiparameter flow cytometric analyses of CD8+ lymphocytes were performed. These cells were investigated for their expression of the activation marker CD38+ within the naive (CD45RA+) and primed (CD45RO+) subsets. Serial CD4 counts were plotted for each patient and the straight line that best fitted was obtained using least squares regression. Differences in rate of decline were tested using analysis of variance, after each patient was weighted by the reciprocal of the variance. RESULTS Baseline levels of percentages of CD8+CD38+ T lymphocytes predict the CD4 decline in HIV-1-infected patients. Within the CD8+ subset, the primed CD8+CD38+CD45RO+ population was responsible for this prediction. Conversely, the naive CD8+CD38+CD45RA+ population was not predictive. Patients who initially showed a percentage of CD8+CD38+ T lymphocytes above the median (> 25%) had a more marked decline in CD4+ T cells when compared to individuals with percentages of CD8+CD38+ T lymphocytes below the median value (79.3 and 21.2 x 10(6)/l mean CD4 cell decline per year, respectively). Similarly, percentages of CD8+CD38+CD45RO+ T cells above the median value (> 7%) were also associated with a more rapid decline (69.4 and 14.2 x 10(6)/l mean CD4+ cell decline per year). These results were statistically significant after adjustment for the baseline CD4 count and beta 2-microglobulin levels. CONCLUSIONS Percentages of activated CD8+ cells expressing CD38+ can predict the rate of decline (slope) of the CD4+ T cells. This resides in the CD45RO+ primed population. An early prediction of the CD4+ slope will allow the clinician to target treatment to those patients that are most likely to benefit.

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells.

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.