Mechanisms of donor-specific tolerance in recipients of haploidentical combined bone marrow/kidney transplantation.

We recently reported long-term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4⁺ CD25⁺ CD127⁻ FOXP3⁺ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥ 18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism.

Efficient characterization of the TCR repertoire in lymph nodes by flow cytometry.

Analysis of the T-cell receptor (TCR) repertoire by flow cytometry proved to be relevant for investigating T-cell diversity and detecting reactive cells in blood samples. We used this approach to characterize non-malignant T-lymphocytes in lymph nodes and give insights into their origin. The TCR repertoire of CD4+ and CD8+ T-cells from 81 lymph nodes was analyzed with a four-color flow cytometer using a wide panel of 25 anti-Vbeta monoclonal antibodies. Flow cytometry proved to be a useful and informative technique. We demonstrated a diversified TCR-Vbeta repertoire, and only low level expansions, in 53% of the samples. They involved nearly all Vbeta families, were more frequent in the CD8+ subset of older patients, but were not related to pathology. No evidence could be demonstrated in favor of stimulation by common antigens. Interestingly, the TCR-Vbeta repertoire proved to be very similar in lymph nodes and blood samples. Our results argue that in the cases studied, lymph node enlargement is mainly due to an increased homing of circulating T-cells. They also provide reference values for expression of 25 TCR-Vbeta in lymph nodes, which could serve as a basis for further applications in diagnosis of T-cell lymphoproliferative disorders.

Resistance to CD95-mediated apoptosis through constitutive c-FLIP expression in a non-Hodgkin's lymphoma B cell line.

CD95 (Fas/Apo-1) is a transmembrane molecule that induces apoptosis and plays a central role in the regulation of the immune response. The present study describes two new B lymphoid cell lines, B593 and BR97, derived from non-Hodgkin's lymphoma, which differ in susceptibility to CD95-mediated apoptosis. While B593 cells are sensitive to CD95mediated apoptosis, BR97 cells are completely resistant. Activation of caspase-8 and caspase-3 proteases plays an important role in the CD95 signalling pathway. CD95 stimulation induced caspase-8 and caspase-3 activation in B593, but not in BR97 cells. However, activation of both caspase-8 and caspase-3 was achieved in BR97 cells treated with staurosporine. Furthermore, protein synthesis inhibition by cycloheximide restored sensitivity to CD95-mediated apoptosis and allowed activation of both caspase-8 and caspase-3 in BR97 cells. These results indicate that, in BR97 cells, both caspases are functional and suggest that CD95-apoptosis resistance may result from the presence of inhibitory factor(s). Constitutive high level expression of the apoptotic inhibitor c-FLIP was observed in the CD95-resistant BR97 cell line compared to B593. Moreover, downregulation of c-FLIP expression level by protein synthesis inhibition strictly correlated with restored sensitivity to CD95-mediated apoptosis in BR97 cells. Furthermore, we demonstrate that c-FLIP is recruited to the CD95 DISC in BR97 cells together with caspase-8 and FADD. The data presented in this study strongly suggests that, in a B-NHL-derived cell line, resistance to CD95-mediated apoptosis results from endogenous high level expression of apoptotic inhibitor c-FLIP.

Accumulation of T-cell clones producing high levels of both granulocyte-macrophage and macrophage colony-stimulating factors (CSF-1) in lymph nodes involved by Hodgkin's disease.

We have previously shown that total T cells derived from lymph nodes (LN) involved by Hodgkin's disease (HD) secrete higher levels of colony-stimulating activity than total T cells present within benign hyperplastic (BH) LN and B-non-Hodgkin's lymphoma (B-NHL) LN, suggesting that T cells with particular properties accumulate in HD LN. To further characterize this T-cell population, we have quantified production of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) production in a total of 98 T-cell clones (TCC) derived from CD25+ activated T cells present in HD LN; TCC derived from CD25+ T cells obtained from B-NHL LN(101 TCC), BH LN(95 TCC), and peripheral blood (PBL; 38 TCC) of healthy donors were used as controls. HD LN were characterized by the presence of an elevated number (44%) of TCC producing particularly high titers of both GM-CSF and M-CSF, whereas only a minority of such TCC was found in control groups (10% in B-NHL, 16% in BH, 8% in PBL). These observations support the hypothesis of a selection of T-cell families with particular properties occurring in contact with Reed-Sternberg (RS) cells. According to the biological properties of GM-CSF and M-CSF, it seems reasonable to suggest the involvement of this particular subset of T cells in the granulomatous process, the peripheral blood polynucleosis, and in the paracrine growth of RS cells.

Stimulation and inhibition of human granulopoiesis in vitro by normal and malignant T4- or T8-lymphocyte subpopulations.

We studied the role of total peripheral blood T-lymphocytes and separated subpopulations of OKT4+ and OKT8+ cells stimulated with concanavalin A in the regulation of human granulopoiesis. Unfractionated total T cells depleted of monocytes are capable of producing colony-stimulating activity (CSA) and colony-forming unit-clone (CFU-C) suppressor activity simultaneously. After positive selection of cell subsets using a fluorescence-activated cell sorter, these two activities were produced by OKT4+ lymphocytes, whereas OKT8+ cells displayed small amounts of CSA and were incapable of releasing suppressor activity. On the other hand, total human thymocytes and subsets defined by monoclonal antibodies Leu2a/Leu3a failed to express any detectable CSA or CFU-C suppressor activity. A total of 14 cases of phenotyped lymphoid malignancies were also studied: the results showed that the production of stimulating and/or inhibiting factors is neither clearly related to a discrete stage of differentiation nor to the OKT4/OKT8 phenotype. Moreover, three monoclonal T leukemias, OKT4+OKT8-, OKT4-OKT8-, and OKT4-OKT8+ have been able in each case to produce simultaneously CSA and CFU-C suppressor activity. Finally, these studies strongly suggest that the activities of T-lymphocytes involved in the regulation of granulopoiesis are not closely linked with OKT4/OKT8 phenotype.

The standard chi 2 test used in limiting dilution assays is insufficient for estimating the goodness-of-fit to the single-hit Poisson model.

Limiting dilution analysis is a common technique that is used in immunology to estimate accurately the frequency of cells possessing a wide variety of functional activities, such as growth, cytotoxicity and production of lymphokines. In the literature, most experiments are fit well by the single-hit Poisson model (SHPM), which assumes that only one cell of one defined cell subset is necessary for a positive response. This is somewhat surprising since other models such as multi-hit or multi-target models that involve the interaction of one or more cells from one or more cell subpopulations for generating or inhibiting a positive response are conceivable. Since the validity of the SHPM is usually investigated by performing a standard chi 2 test, based on the number of observed and expected positive and negative responses, we questioned here the efficiency of this test in comparison with other validity tests for the SHPM, the log likelihood test derived by Cox, and the modified Weibull plot tests, the principles of which are entirely different from that of the standard chi 2 test. We used the following theoretical approach. First, we generated artificial data corresponding to multi-hit and multi-target models. Second, considering that these data were derived from real experiments, we calculated the frequency of the desired cell subset according to the SHPM using the maximum likelihood method. Then, the goodness-of-fit of these data with the SHPM was evaluated. The log likelihood test and the modified Weibull plot tests rejected the SHPM hypothesis, while the standard chi 2 test did not. Thus, the standard chi 2 test is unable to discriminate sensitively between the SHPM and more complicated (non-single-hit) Poisson models. We concluded that the results of limiting dilution studies published thus far must be evaluated with caution. The statistical tests presented here should be routinely applied for each limiting dilution experiment.

Fitting limiting dilution experiments with generalized linear models results in a test of the single-hit Poisson assumption.

Limiting dilution analysis is a common technique that is used in immunology to estimate accurately the frequency of cells possessing a wide variety of functional activities such as growth, cytotoxicity and production of lymphokines. The reliability of the estimated frequency is usually checked by a standard chi-square (x2) test validating the goodness-of-fit to the single-hit Poisson model (SHPM). We present evidence that modelling limiting dilution data according to a generalized linear model offers an alternative to the standard x2 test for detecting departures from the SHPM, with a considerable increase in power compared to the x2 test.

Impaired clonogenic potential of CD25 positive T cells in lymph nodes involved by B cell non-Hodgkin's lymphomas.

In vivo activated T cells (CD25+) present in lymph nodes involved by B-non-Hodgkin's lymphomas (B-NHL) were investigated here for their ability to proliferate in vitro. CD25-/CD25+ T cells were isolated using a rosette method with magnetic beads, then the frequency of proliferating T lymphocyte-precursors (PTL-P) in both populations was assessed by limiting dilution experiments, in the presence of IL2, PHA and allogeneic spleen cells as feeders. In a total of 16 cases studied, growing microcultures were observed in all cases for CD25- T cells (mean value of PTL-P frequency: 1/32; range 1/10 - 1/2899) but in 6 cases only for CD25+ T cells (mean value of PTL-P frequency: 1/441; range 1/119 - 1/3736); the absence of any proliferative cultures in the 10 other cases indicated that the number of PTL-P was inferior to 1/12480. These results suggest that the proliferative potential of CD25+ T cells infiltrating lymph nodes involved by B-NHL is paradoxically decreased.

Valid estimation of IL2 secretion by PHA-stimulated T-cell clones absolutely requires the use of anti-CD25 monoclonal antibody to prevent IL2 consumption.

A major problem encountered for quantification of IL2 production by stimulated T cells is its simultaneous consumption by these activated cells. In the present study, 40 T-cell clones (TCC) derived from normal peripheral blood, hyperplastic lymph nodes (LN) or lymph nodes involved by malignant lymphomas, were studied for their ability to produce IL2. When supernatants were generated in the presence of 20% fetal calf serum (FCS), no IL2 could be detected for 22 of the 40 TCC, whereas very low levels were found for the 18 other TCC (mean value 31 pg/ml; range from 10 pg/ml to 114 pg/ml); in contrast, when conditioned media were produced with reduced amounts of FCS (final concentration, 1%) as well as in the presence of an anti-CD25 monoclonal antibody (final concentration, 50 micrograms/ml), all TCC were found to release IL2, and very high quantities of this lymphokine were measured (mean value: 11,387 pg/ml; range, from 250 pg/ml to 37,000 pg/ml). Consequently, inhibition of IL2 consumption by PHA-stimulated TCC seems to be an absolute requirement for estimating the true capacity of T cells to produce this lymphokine.

Limiting dilution analysis of the frequency of IL2 responsive T cells in lymph nodes involved by B-cell non-Hodgkin's lymphomas.

Total T lymphocytes separated from twelve lymph nodes involved by B-NHL were studied in limiting dilution experiments for their ability to proliferate in the presence of both R-IL2 used at a final concentration of 40 U/ml and irradiated autologous malignant B cells as feeders. The number of proliferating T-lymphocyte precursors (PTL-P) thus estimated was low in each case (mean: 1/4503; range, 1/200 to 1/11013). Once expanded, proliferation of the IL2 responsive T cells in the presence of autologous malignant B cells remained strictly dependent on the addition of exogenous IL2. Control cases consisted of T lymphocytes separated from peripheral blood of six healthy subjects and cultured in the presence of both R-IL2 (40 U/ml) and irradiated autologous total mononuclear cells as feeders; the mean frequency of PTL-P thus obtained (1/173; range, 1/49 to 1/457) was significantly higher than in malignant lymph nodes (p less than 0.01). These findings do not support the hypothesis that, in this series of patients, expansion of malignant B cells may lead to the activation and growth of T cells sensitized against the tumour.

Growth modulation of freshly isolated non-Hodgkin's B-lymphoma cells induced by various cytokines and all-trans-retinoic-acid.

We investigated the potential of ten cytokines (IL2, IL3, IL4, IL6, IL10, IL13, G-CSF, GM-CSF, interferon alpha, interferon gamma) and all-trans-retinoic acid to modulate the spontaneous proliferative response in vitro of purified B-non Hodgkin's lymphoma cells of various histological subtypes. 19 malignant lymph nodes were studied. In each case the growth could be influenced by several of these modulators. Cytokines most often implicated were interferon gamma (14/19 cases, 73.7%), IL4 (13/19 cases, 68.4%), interferon alpha (12/19 cases, 63.1%). IL2 (9/19 cases, 47.3%), IL6, IL10, IL13 and ATRA were less frequently involved (6/19 cases, 31.6%) and hematopoietic growth factors (IL3, GM-CSF, G-CSF) were rarely implicated (2/19 cases, 10.5%). The values of growth stimulation ranged from a 1.1-fold to a 6.1-fold increase, and the values of growth inhibition ranged from 15% to 98%. Each cytokine could be either inhibitory or stimulatory depending on the sample analyzed, and no relationship could be found with the histological subtype. Two notable exceptions were IL2, displaying exclusively a positive effect, and ATRA displaying exclusively a negative effect. Overall, these results may have strong implications for future clinical studies using cytokines in the treatment of lymphomas. Ideally, the pattern of in vitro growth response to cytokines or ATRA should be determined individually before undertaking any cytokine treatment.

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

Myeloma responses and tolerance following combined kidney and nonmyeloablative marrow transplantation: in vivo and in vitro analyses.

Six patients with renal failure due to multiple myeloma (MM) received simultaneous kidney and bone marrow transplantation (BMT) from HLA-identical sibling donors following nonmyeloablative conditioning, including cyclophosphamide (CP), peritransplant antithymocyte globulin and thymic irradiation. Cyclosporine (CyA) was given for approximately 2 months posttransplant, followed by donor leukocyte infusions. All six patients accepted their kidney grafts long-term. Three patients lost detectable chimerism but accepted their kidney grafts off immunosuppression for 1.3 to >7 years. One such patient had strong antidonor cytotoxic T lymphocyte (CTL) responses in association with marrow rejection. Two patients achieved full donor chimerism, but resumed immunosuppression to treat graft-versus-host disease. Only one patient experienced rejection following CyA withdrawal. He responded to immunosuppression, which was later successfully withdrawn. The rejection episode was associated with antidonor Th reactivity. Patients showed CTL unresponsiveness to cultured donor renal tubular epithelial cells. Initially recovering T cells were memory cells and were enriched for CD4+CD25+ cells. Three patients are in sustained complete remissions of MM, despite loss of chimerism in two. Combined kidney/BMT with nonmyeloablative conditioning can achieve renal allograft tolerance and excellent myeloma responses, even in the presence of donor marrow rejection and antidonor alloresponses in vitro.

[Production kinetics of T-lymphocyte activity regulating human granulopoiesis in vitro]. / Cinétique de production des activités lymphocytaires T régulant la granulopoïèse humaine in vitro.

Human T-lymphocytes stimulated with Con A are able to produce both a granulocyte-colony-stimulating activity (G-CSA) and a colony-inhibiting activity. We have studied here the cumulative and daily non-cumulative production of these 2 activities. The release of G-CSA is low on the 1st day of culture, maximal production occurred between the 2nd and the 4th day; from the 6th day on, T-lymphocytes are unable to produce CSA. Colony-inhibiting activity production is usually weak at the beginning of culture, but constantly found on day 5 and on day 7. In conclusion, cumulative culture for 5 days seems optimal for the simultaneous detection of both activities.

[Production of GM-CSF by human T lymphocytes purified and stimulated by Con A]. / Production de GM-CSF par les lymphocytes T humains purifiés et stimulés par la Con A.

Production of granulomacrophage colony stimulating factor (GM-CSF) by human T lymphocytes has not yet been demonstrated due to insufficient monocyte depletion in cell suspensions. We studied GM-CSF production from normal blood T lymphocyte suspensions containing less than 0.1% of monocytes; moreover, human bone marrow samples were also depleted of adherent cells. A colony stimulating activity (CSA) was always found in the 11 healthy subjects studied following Con A stimulation. The colonies were principally of the granulocytic type. These results strongly suggest that normal human T cells have the capacity to produce CSA.

Role of autologous lymph node T cells in membrane expression of mu- or gamma-heavy chain isotype by malignant B NHL cells.

We investigated the possibility that T cells observed in lymph nodes involved by B-non-Hodgkin's lymphomas (B-NHL) may have a direct role in the expression of Mu- or Gamma- heavy chain isotype by autologous malignant B cells. T cells were separated from lymph nodes involved by B-NHL cells expressing either surface IgM (19 cases) or surface IgG (4 cases) and compared to peripheral blood T lymphocytes of healthy subjects (19 cases) for their ability to promote both IgG and IgM secretion in Cowan-activated normal B lymphocytes. The mean values of IgG/IgM ratios obtained under the influence of T cells associated with malignant B cells expressing either surface IgM or surface IgG were not statistically different to that obtained with the help of control T cells (0.60 and 0.62 versus 0.47, respectively). These results do not account for the hypothesis that autologous lymph node T cells may directly affect the expression of the heavy chain isotype by malignant B-NHL cells.

Interest of argyrophilic proteins nucleolar organizer regions (AgNOR) to estimate the reactivity of T cell clones against autologous malignant B-NHL cells.

The silver-stained acidic proteins of interphase nucleolar organizer regions (AgNOR) were studied to assess the reactivity of 11 T cell clones (Tcc) against autologous B-NHL cells. Tcc, derived from tumour-infiltrating T lymphocytes of seven patients, were cultured in the presence of irradiated autologous B-NHL cells with recombinant IL-2. Then the percentage of activated T cells expressing the CD25 antigen and their proliferating rate (measured by [3H]thymidine incorporation) were estimated. Simultaneously, at the end of this culture period, B-NHL cells were eliminated by using a cell-sorter, and the resulting purified T cells were studied for AgNOR expression. Tcc cultured without B-NHL cells served as controls. In eight out of the 11 Tcc, the increase in [3H]thymidine incorporation and CD25 expression paralleled the increase of AgNOR area and number. By contrast, in the three remaining Tcc, we observed a decrease (one Tcc) or an increase (two Tcc) of AgNOR parameters, whereas CD25 expression and/or [3H]thymidine incorporation remained unchanged in comparison to control cultures. We concluded that quantification of AgNOR should be a more sensitive technique than thymidine incorporation and CD25 expression for detecting the activation in vitro of T cells induced by autologous B-NHL cells.

Graphical representation of a generalized linear model-based statistical test estimating the fit of the single-hit Poisson model to limiting dilution assays.

Standardized statistical and graphical methods for analysis of limiting dilution assays are highly desirable to enable investigators to compare and interpret results and conclusions with greater accuracy and precision. According to these requirements, we present in this work a powerful statistical slope test that estimates the fit of the single-hit Poisson model to limiting dilution experiments. This method is readily amenable to a graphical representation. This slope test is obtained by modeling limiting dilution data according to a linear log-log regression model, which is a generalized linear model specially designed for modeling binary data. The result of the statistical slope test can then be graphed to visualize whether the data are compatible or not with the single-hit Poisson model. We demonstrate this statistical test and its graphical representation by using two examples: a real limiting dilution experiment evaluating the growth frequency of IL-2-responsive tumor-infiltrating T cells in a malignant lymph node involved by a B cell non-Hodgkin's lymphoma, and a simulation of a limiting dilution assay corresponding to a theoretical non-single-hit Poisson model, suppressor two-target Poisson model.

Detection, isolation and functional studies of CD25+ T cells in lymph nodes involved by B-cell non-Hodgkin's lymphomas.

We searched for the presence of IL2 receptor (CD25) on T cells as an activation marker in lymph nodes involved by B-cell non-Hodgkin's lymphomas (B-NHL). In 26 malignant lymph nodes studied, the number of CD25+ T cells among total T cells was usually low when assessed by immunofluorescence analysis (mean +/- SD: 6.7% +/- 11.2%), but greatly increased when an immunomagnetic rosette method was used (mean +/- SD: 17.5% +/- 16.6%). In six cases, CD25-/CD25/CD25+ cells were isolated by immunomagnetic separation, with a purity greater than 97% for both populations. Expansion of CD25-/CD25+ T cells was obtained with IL2 and PHA, then conditioned media (CM) were prepared. No IL2 activity was found in CM from both CD25-/CD25+ T cells when tested on CTLL2 cells. BCGF and BCDF mu/gamma activities were assayed on normal B cells stimulated with soluble or insolubilized anti-mu antibodies(BCGF) or with Cowan I (BCDF). Results of production of all these activities were comparable for both populations, and thus do not favour the possibility that CD25+ T cells closely associated with malignant B-NHL cells in lymph nodes may influence their proliferation (BCGF) or expression/secretion of heavy chain isotype (BCDF mu/gamma).

Production of granulopoiesis-stimulating and -inhibiting activities by T cells associated with malignant cells in lymphomas.

The possible role of T lymphocytes in the formation of granulomatous reactions seen in certain malignant lymphoid tumours was investigated by measuring the granulopoietic colony-stimulating activity (CSA) and granulopoietic-inhibiting activity (IA) produced by stimulated T-lymphocytes isolated from peripheral blood, spleen and lymph nodes of patients and normal subjects. Lymph-node T-cells from patients with benign lymphoid hyperplasia, B-cell non-Hodgkin's lymphoma (B-NHL), and non-granulomatous Hodgkin's disease (HD) showed no CSA, but the cells produced IA of 40 +/- 23%, 40 +/- 24% and 50.5 +/- 22.5% respectively. The corresponding cells from patients with HD accompanied by granulomatous reactions produced CSA of 6.85 +/- 6.5 u/microliters and IA of 23.5 +/- 21%. The presence of a granulomatous reaction in malignant lymphoma was correlated with the stimulation of granulopoiesis in vitro by T lymphocytes associated with malignant cells. A correlation was demonstrated between neutrophilic and eosinophilic colonies obtained in vitro under the influence of CSA-producing T cells isolated from malignant lymphomas and the neutrophils and eosinophils present in the granuloma. These results showed that tumour-infiltrating T cells play a role in the presence of granulomatous reactions seen in lymphomas. Peripheral-blood T cells from healthy subjects, and from patients with B-NHL, or with HD unaccompanied by granulocytic reactions produced CSAs of, respectively, 5 +/- 0.5 u/microliter, 4.8 +/- 2.2 u/microliters and 5.3 +/- 0.4 u/microliters, and IAs of 45 +/- 18%. 50 +/- 5.5% and 50.5 +/- 7% respectively. The corresponding values for HD patients with granulocytic reactions were CSA. 17 +/- 15.5 u/microliters, and IA, 9.5 +/- 9%. No correlation was demonstrated between neutrophilic colonies obtained in vitro under the influence of HD blood T cells and neutrophils present in blood. Only one correlation was found: between the percentage of eosinophilic colonies and the number of blood eosinophils. HD blood T cells did not seem to explain completely granulocytic reactions seen in blood.