In vitro production of granulocyte-macrophage colony-stimulating factor in aplastic anemia: possible mechanisms of action of antithymocyte globulin

Various abnormalities of lymphokine production have been described in patients with aplastic anemia. To determine if abnormal production of hematopoietic growth factors could contribute to the process of aplastic anemia we studied the in vitro production of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) by phytohemagglutinin (PHA)- and antithymocyte globulin (ATG)-stimulated peripheral blood lymphocytes from 29 patients with aplastic anemia and 15 normal controls. GM-CSF production in response to 1% PHA was seen in nearly all samples (43 of 44) and similar amounts of GM-CSF were produced by patients with aplastic anemia and normal controls. Production of GM-CSF by ATG-stimulated lymphocytes was seen in 7 of 23 patients with aplastic anemia (30%); two of these patients also demonstrated low-level spontaneous production of GM-CSF. Production of GM-CSF in response to ATG was also seen in 2 of 11 normal controls (18%) and barely detectable spontaneous production of GM-CSF was seen in both. Biologically active IL-3 could also be detected in PHA- or ATG-stimulated peripheral blood mononuclear cells in several patients and normal controls. Our results indicate that lymphocytes from patients with aplastic anemia can be stimulated in vitro to produce normal quantities of GM-CSF, suggesting that impaired potential for production of T-cell derived hematopoietic growth factors is unlikely to account for the marrow hypoplasia seen. In several patients overproduction of GM-CSF was observed, consistent with the notion that some patients with aplastic anemia may have circulating activated T cells. We also demonstrate that ATG can stimulate the production of growth factors such as IL-3 and GM-CSF, supporting the role for ATG in stimulating hematopoiesis.     

Decreased interleukin-1 production in aplastic anemia

PURPOSE: Interleukin-1 (IL-1), a monocyte factor, plays a central role in the regulation of the immune response; recent data have suggested that IL-1 is the same molecule as hemopoietin-1, a growth factor acting on the multipotent hematopoietic stem cell. IL-1 affects hematopoiesis (1) in vitro, by inducing the release of colony-stimulating factors and regulating early hematopoietic progenitor cells, and (2) in vivo, by stimulating stem cell recovery in irradiated or chemotherapy-treated mice. Several lines of evidence suggest that aplastic anemia may be mediated by cells of the immune system. We address the issue of abnormal IL-1 production in severe aplastic anemia and attempt to correlate normalization of the levels with response to anti-thymocyte globulin (ATG) therapy. PATIENTS AND METHODS: We studied IL-1 production by monocytes from 21 patients with aplastic anemia using a bioassay for IL-1 activity. Fifteen patients were evaluated before ATG therapy. Eight patients were studied before and three months after ATG. In addition, five patients were evaluated only after ATG treatment. One patient did not respond to ATG but did respond to intravenous acyclovir, and was studied before and after acyclovir therapy. Twenty patients with other hematologic disorders requiring transfusions and 30 normal healthy volunteers were also assessed. RESULTS: IL-1 production was markedly decreased in 75 percent of patients with aplastic anemia when compared with that in normal control subjects (p less than 0.005). Hematologic recovery correlated with normalization of IL-1 production in all but two cases (p less than 0.04). CONCLUSION: These observations represent the first evidence of monocyte dysfunction and deficient hematopoietic growth factor production in aplastic anemia. Decreased IL-1 production may have a pathologic role in some cases of aplastic anemia.     

In vitro immunoglobulin production, proliferation, and cell markers before and after antithymocyte globulin therapy in patients with aplastic anemia

Peripheral blood lymphocytes from 16 aplastic anemia patients were studied for in vitro biosynthesis of immunoglobulins (Ig), proliferative responses, and cell markers before and after antithymocyte globulin (ATG) treatment in an attempt to identify immune functions that would be useful in predicting responses to ATG therapy. Six of the 16 aplastic anemia patients were complete responders to ATG therapy, two were partial responders, and eight failed to respond to ATG therapy. The proportion of E+, CD4, CD8, and surface Ig-positive cells did not correlate with in vitro lymphocyte functions nor clinical responses before or after ATG therapy. Lymphocyte proliferative responses to phytohemagglutinin, tetanus toxoid, alloantigens, or pokeweed mitogen were generally present before and after ATG therapy. When pokeweed mitogen, herpes simplex type I virus, and tetanus toxoid were used as probes to elicit in vitro Ig production using a hemolytic plaque assay, some patients had 1) B cells that failed to produce Ig, 2) T cells that failed to provide helper activity, and 3) T cells that exhibited excessive suppressor activity in the various antibody production systems. These measures of immune function, however, did not correlate with clinical responses to ATG therapy.     

Antithymocyte globulin reacts with many normal human cell types

Antithymocyte globulin (ATG) is frequently effective therapy for aplastic anemia. Its mechanism of action is often assumed to be upon a lymphocyte inhibitor of hematopoiesis. However, specificity for T lymphocytes would not be anticipated from consideration of the method of preparing ATG. In fact, using flow microfluorometry and fluorescence immunohistochemistry, we have found that ATG binds to virtually all circulating lymphocytes, granulocytes, and platelets, as well as to bone marrow cells. Extensive absorption of ATG with either granulocytes or lymphocytes does not eliminate reactivity with the opposite cells, indicating that ATG recognizes some distinct antigens on each cell type. Treatment of cells with ATG blocks the binding of monoclonal antibodies directed against either lymphocyte differentiation or histocompatibility antigens. ATG also binds to visceral tissues, including thymus and testis cell membranes and the nuclear and cytoplasmic components of tonsil, kidney, liver, breast, lung, and intestine. In vitro cytotoxicity of ATG was demonstrated for both T and non- T lymphocytes and platelets. Despite its name, ATG is not specific for a particular cell type, and it would be premature to conclude that its effect is mediated through a specific lymphocyte population.     

Persistent Remission after Immunosuppressive Therapy of Hairy Cell Leukemia Mimicking Aplastic Anemia: Two Case Reports

Some patients with hairy cell leukemia (HCL) manifest pancytopenia and bone marrow hypoplasia without an apparent increase in atypical cells, so their disease resembles severe aplastic anemia at onset. We treated 2 HCL patients, who were initially diagnosed with aplastic anemia, with antithymocyte globulin (ATG) in combination with cyclosporine or antilymphocyte globulin (ALG). Both patients obtained partial remission in response to the immunosuppressive therapy and did not need transfusion treatment for more than 3 years. Sustained improvement of hematopoiesis in such B-cell malignancies after ATG/ ALG therapy suggests that the mechanisms underlying successful immunosuppressive therapy for aplastic anemia may involve B-cell suppression, inhibiting hematopoietic stem cells.     

Effectiveness of antilymphocyte and antithymocyte globulins for patients with severe aplastic anemia and pure red cell aplasia--analysis of immunologic parameters of peripheral lymphocytes concerning ALG and ATG therapy

Four patients with severe aplastic anemia and one patient with pure red cell aplasia (PRCA) were treated with antilymphocyte and antithymocyte globulins. One patient in aplastic anemia who achieved good response by ALG administration had a possible diagnosis of myelodysplastic syndrome. ATG was administered to only one case of aplastic anemia and ALG was administered to the remainder. In the result, three out of 4 patients with aplastic anemia and one patient with PRCA achieved good response without severe side effects. Three patients with aplastic anemia had high CD4/CD8 ratio in their peripheral lymphocytes. This ratio normalized after ALG therapy in effective cases, but not in ineffective case. Natural killer activity elevated after ALG therapy in two effective cases of aplastic anemia and PRCA, but not in one ineffective case of aplastic anemia.     

Release of interleukin-1 and interleukin-6 from human monocytes by antithymocyte globulin: requirement for de novo synthesis.

Antithymocyte globulin (ATG) is an effective treatment in patients with severe aplastic anemia (SAA). Its mechanism of action remains unclear, although it has been assumed to be immunosuppressive. However, ATG has also been shown by several laboratories to be immunostimulatory. Recently, interleukin-1 (IL-1) production has been found to be decreased in lipopolysaccharide-stimulated peripheral blood monocytes obtained from SAA patients. We have investigated the ability of ATG to function as an immunostimulatory agent via the production of IL-1 and IL-6 by normal human monocytes in vitro. Supernatants from ATG-stimulated monocytes were assayed for biologically active and immunoreactive IL-1 and IL-6. We have found that ATG, via its F(ab')2 fragment is a powerful inducer of IL-1 and IL-6 production. Furthermore, ATG induction of both cytokines from normal monocytes required de novo synthesis, as determined by 35S-methionine incorporation. Because these two cytokines synergize with other cytokines at both the stem cell and progenitor levels, these stimulatory properties of ATG may be relevant to the treatment of SAA. This would favor the hypothesis of a bimodal mechanism for ATG as an inducer of hematopoietic growth factors and as an immunosuppressive agent.     

Analysis of natural killer cells in patients with aplastic anemia

We have analyzed natural killer (NK) cells in 43 patients with severe aplastic anemia, using cytotoxicity assays and microfluorometry with monoclonal antibodies, prior to and after treatment with antithymocyte globulin (ATG). Before treatment, natural killer cell activity (NKa) in both peripheral blood and bone marrow was markedly decreased in 76% of patients as compared with normal controls. Although we have measured low NKa in patients receiving large numbers of blood transfusions (means = 150 U of RBCs), six aplastic patients had low NKa in the absence of transfusions, and the average number of transfusions in the total population was low (means = 24). Purification of larger granular lymphocytes (LGLs) from peripheral blood of aplastic anemia patients failed to recover significant NKa. Most of these large granular lymphocytes showed few azurophilic granules. NKa was appropriately enhanced in these patients samples by exposure of mononuclear cells to either interleukin 2 (IL-2) or interferon (IFN). Analysis of peripheral blood phenotypic markers showed that cells bearing Leu 7 antigen were in the normal range in aplastic anemia (means = 12% +/- 2%; normal = 16% +/- 2%), but there was a deficiency of Leu 11+ cells (means = 8% +/- 2%; normal = 15% +/- 2%). The number of Leu 11+ cells was well correlated with NKa. In 13 of 22 patients treated with ATG, NKa returned to the normal range, and recovery of NKa was correlated to hematopoietic recovery. Our results suggest that deficient NKa is an intrinsic feature of aplastic anemia, and that the circulating cells in this disease are of the pre-NK cell stage.     

Antithymocyte globulin stimulates human hematopoietic progenitor cells.

Antithymocyte globulin (ATG), a horse antihuman thymus antiserum highly effective in the majority of patients with aplastic anemia, was studied for its in vitro effects on hematopoietic progenitor cells. Marrow cells isolated by an immunoadherence technique with the HPCA-1 (human progenitor cell antigen) monoclonal antibody after removal of contaminating T cells and macrophages formed erythroid colonies in methyl cellulose media in the presence of ATG at concentrations of 25-50 micrograms/ml. ATG also stimulated continuous production of hemoglobin-containing erythroid colonies beyond 35 days of culture when it was added to the culture weekly. ATG also had an indirect effect on myeloid (granulocytic and macrophagic) colony growth in vitro. At a concentration of 10 micrograms/ml, ATG stimulated late but not early myeloid progenitor cells to form mature colonies. This effect required the participation of lymphocytes containing the Leu-11 antigen and macrophages or supernatant fluids made from these two types of cells that had been preincubated with ATG for 3 hr and then cultured for 5 additional days. The supernatant fluids produced in such a manner showed characteristics similar to granulocyte colony-stimulating factor and had an activity peak that eluted at a volume corresponding to a 20,000 Da molecular mass protein by high-resolution liquid chromatography.     

Lymphocyte phenotype and lymphokines following anti-thymocyte globulin therapy in patients with aplastic anaemia

Twenty-two patients with adult onset aplastic anaemia were analysed before and after therapy with anti-thymocyte globulin (ATG). Lymphocyte phenotype, lymphokine levels or production, and haematopoietic progenitor cell number were measured 3 months after therapy; clinical response was determined 1 year post-therapy. By flow cytometry there was a significant reduction in both the proportion and absolute number of peripheral blood lymphocytes expressing activation antigen Tac (IL-2 receptor) and in the proportion of HLA-DR+ lymphocytes. For T cells bearing HLA-DR, there were proportional decreases in both activated helper and suppressor cells. There was no statistically significant difference pre-ATG to post-ATG in the absolute numbers of total, helper and suppressor lymphocytes. In all 10 haematologic responders the number of Tac bearing lymphocytes after ATG therapy was in the normal range, but half of 12 non-responding patients continued to have abnormally elevated numbers of Tac+ T cells. The proportion of Tac+ cells were not related to transfusion history. Gamma-interferon levels in serum by radioimmunoassay were elevated in almost half the aplastic patients; post-ATG, gamma-interferon was detectable in only three patients. Haematologic response to ATG therapy was associated with increased numbers of haematopoietic progenitors post-treatment, but pre-treatment values were not predictive of a response. These results are consistent with a pathogenic role for activated T-cells and their lymphokine products and suggest that the target of ATG therapy may be a Tac+ lymphocyte.     

Immunosuppressive therapy with antithymocyte globulin and cyclosporine for prolonged marrow failure after hemophagocytic syndrome

We describe a patient with typical hemophagocytic syndrome (HPS) in whom pancytopenia was refractory to steroid pulse therapy. He was successfully treated with immunosuppressive therapy using antithymocyte globulin (ATG) and cyclosporine (CyA), which is known to be effective for aplastic anemia (AA). Activation of histiocytes occurs in HPS as a response to several cytokines produced by activated T lymphocytes, while apoptosis of hematopoietic stem cells in AA is caused by T lymphocyte-derived cytokines. The response of this patient indicated that both diseases may have some similar immune-mediated conditions involving the activation of T lymphocytes and that intensive immunosuppressive therapy with ATG and CyA might be a useful strategy for steroid-resistant HPS.     

Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo.

We have investigated interferon as a mediator of hematopoietic suppression in bone marrow failure. Interferon production by stimulated peripheral blood mononuclear cells from patients with aplastic anemia was significantly higher than that observed in controls; spontaneous interferon production by these cells was also high for more than half of aplastic anemia patients. Circulating interferon, not detectable in normal individuals, was detected in 10 of 24 patients. Interferon is a potent inhibitor of hematopoietic cell proliferation and, therefore, may be the mediator of suppression in many in vitro models employing patients' cells and sera. The possible pathogenic importance of interferon in aplastic anemia was suggested by an increase in hematopoietic colony formation in vitro after exposure of bone marrow cells to antiinterferon antisera (277 +/- 71% increase for patients compared to 1.6 +/- 1.6% for normal individuals). Interferon levels in the bone marrow sera of aplastic anemia patients were high (mean = 203 international units (IU)/ml, n = 8), even in comparison to circulating levels in the same patients. Normal bone marrow sera also contained measurable interferon but at lower levels (41 IU/ml, n = 16), indicating that interferon may be a normal bone marrow product. High concentration of bone marrow interferon, possibly due to abnormal immunologic activity or a reaction to virus infection of the bone marrow, may mediate hematopoietic suppression in aplastic anemia patients.     

Regulation of erythropoietin and burst-promoting activity production in patients with aplastic anemia and iron deficiency anemia

To clarify the control mechanism of production of erythropoietic growth factors in anemic states, we compared erythropoietin (Epo) and burst-promoting activity (BPA) in patients with aplastic anemia and iron deficiency anemia, using in vitro erythroid progenitor assays. Although serum levels of Epo activity increased in the presence of anemia, the rise was more marked in patients with aplastic anemia. BPA was high only in the sera of aplastic anemia patients. Serum levels of BPA of patients with aplastic anemia negatively correlated with hemoglobin concentrations, while those of patients with iron deficiency anemia did not correlate. In 2 patients with aplastic anemia who responded well to androgen therapy, serum levels of Epo activity and BPA decreased after the hemopoiesis had recovered. These results suggest that serum levels of BPA do not rise in response to anemia only. The elevated BPA levels in sera in cases of aplastic anemia are probably related to a reduction in the number of hemopoietic stem cells. Moreover, we observed that BPA in bone-marrow-conditioned medium (BMCM) from patients with severe aplastic anemia increased more than in the BMCM from patients with severe iron deficiency anemia. Therefore, our findings suggest that the enhanced BPA production depends on a decrease in hemopoietic precursors rather than the anemic state.     

Interferon-gamma constitutively expressed in the stromal microenvironment of human marrow cultures mediates potent hematopoietic inhibition

Clinical and laboratory studies have suggested involvement of interferon-gamma (IFN-gamma) in the pathophysiology of aplastic anemia. T cells from aplastic anemia (AA) patients secrete IFN-gamma in vitro, activated cytotoxic lymphocytes infiltrate aplastic bone marrow (BM), and IFN-gamma mRNA, not detected in normal BM, is present in BM from most AA patients. Many patients respond to immunosuppressive therapy with antithymocyte globulin and cyclosporine. Using long-term BM cultures (LTBMC) as a tissue culture model of hematopoiesis, we show that IFN-gamma is a potent inhibitor in the long-term culture- initiating cell (LTC-IC) assay, the best in vitro surrogate test for human hematopoietic stem cells, as well as of the output of committed progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]). In LTBMC, continuous addition of relatively high IFN-gamma concentrations (1,000 U/mL weekly or 200 U/mL every 2 days) was required for inhibition of secondary colony formation, a measure of LTC-IC number and clonogenicity. To mimick local production of IFN-gamma, human stromal cells were engineered by retroviral-mediated gene transfer to express a transduced IFN-gamma gene. IFN-gamma secreted by stromal cells was far more potent than exogenous IFN-gamma in its effects in the LTC-IC assay. For purified CD34+ cells culture in the presence of IFN-gamma stroma dramatically reduced secondary colony numbers as well as production of CFU-GM and BFU-E. Supernatants from these cultures contained only about 20 U/mL of IFN-gamma; this quantity of cytokine, when added to LTBMC, had little effect on hematopoiesis. The mechanism of hematopoietic suppression was related to the inhibition of cell cycle progression and induction of apoptosis of CD34+ cells. There was no apparent effect of local low-level IFN-gamma production on stromal cell function, as reflected in cell morphology, cell surface phenotype, or expression of hematopoietic growth factor genes. LTBMC with genetically altered stromal cells offers an in vitro model of immune suppression of hematopoiesis in AA and may be helpful in testing certain therapeutic modalities. We infer from our data that local production of low levels of inhibitory cytokine is sufficient to markedly inhibit hematopoiesis and to destroy stem cells and more mature progenitor cells.     

Hairy cell leukemia responsive to anti-thymocyte globulin used as immunosuppressive therapy for aplastic anemia

Hairy cell leukemia (HCL) is occasionally misdiagnosed as aplastic anemia when only a few leukemic cells are present in the circulation. Here, we describe a patient with HCL who initially presented with pancytopenia and received a diagnosis of aplastic anemia. The patient was treated with immunosuppressive therapy including cyclosporine A and anti-thymocyte globulin (ATG). No blood cell transfusion was required for approximately 3 years after ATG therapy. She was referred to our hospital because of an abdominal mass and requiring periodic blood transfusions. A bone marrow biopsy at this time revealed proliferation of lymphocytes with a fried egg appearance and an increase in reticulin fibers that are typical findings of HCL. It is notable that our patient with a presumably long history of HCL and an increase in marrow reticulin fibers showed good recovery of hematopoiesis after cladribine therapy. Some HCL patients may receive an initial diagnosis of aplastic anemia and may show a good response to ATG masking the underlying HCL.     

Lymphoproliferative disease of granular T lymphocytes presenting as aplastic anemia

Lymphoproliferative disease of granular T lymphocyte (T-LDGL), also known as T-cell large granular lymphocyte leukemia, is a clonal disorder of cytotoxic T lymphocytes that is clinically manifested as chronic neutropenia and anemia. Association with autoimmune disorders is common. In 9 patients, T-LDGL is reported as presenting as aplastic anemia. The clinical characteristics were similar to acquired aplastic anemia. Morphologic evidence of increased granular lymphocytes in the peripheral blood and an excess of CD3(+)/CD8(+)/CD57(+) cells in the bone marrow were found in most cases. Cyclophosphamide was ineffective, but noncytotoxic immunosuppressive agents generally produced a good response. After a median follow-up of 49 months, 5 patients had died from the disease or related complications. Median survival was 40 months. Aplastic anemia can be a presenting manifestation of T-LDGL, and T-LDGL should be considered in the differential diagnosis of acquired aplastic anemia.     

Circulating Suppressor Cells in Aplastic Anemia

In vitro proliferation and differentiation of hematopoietic stem cells was studied in a patient with aplastic anemia. Prior to therapy his peripheral blood contained a very low number of myeloid progenitors but a normal number of cells forming lymphoid colonies. Moreover, peripheral blood lymphocytes of this patient were able to suppress in vitro formation of myeloid colonies but not lymphoid colonies. This suppression effect was found to be sensitive to prednisolone and antithymocytic globulin. Following treatment with prednisolone, during which an apparent hematological recovery was observed, the level of lymphoid progenitors fell, but myeloid committed cells returned to normal and hematopoietic suppression was no longer detectable. These results indicate that cells suppressing hematopoiesis may circulate in the peripheral blood of some patients with aplastic anemia and the detection and testing of suceptibility of these cells to immunosuppressive drugs may be helpful in monitoring treatment and prognosis.     

Differentiation of normal marrow and HL60 cells induced by antithymocyte globulin.

Antithymocyte globulin (ATG) therapy is an important treatment alternative for patients with acquired aplastic anemia. The mechanism by which it exerts its effects on hematopoiesis is unknown. In this report, we describe the ability of horse ATG to induce growth and differentiation of normal bone marrow. A single cell suspension of normal human bone marrow was cultured in methylcellulose medium and examined for the growth and maturation after incubation with ATG (10 micrograms/ml). After 3-4 days of culture, spherical colonies containing mature myeloid elements were found in cultures containing ATG but not in cultures containing medium or preimmunization horse IgG. The addition of 10% colony-stimulating factor increased growth by 40%. The number of spherical colonies is not dependent on the presence of macrophages or T lymphocytes. This property of ATG may be relevant to the mechanism behind the hematologic recovery in some patients with acquired aplastic anemia. We also describe the ability of ATG to induce terminal differentiation in the HL60 leukemic cell line. ATG binds to HL60 cells and at concentrations between 10 and 100 micrograms/ml, 50% of the cells become mature granulocytes, acquire the ability to reduce nitroblue tetrazolium, and lose their proliferative capacity in the clonogenic assay. These new observations of ATG-induced differentiation of normal marrow myeloid elements and terminal differentiation of the HL60 cell line point to different avenues for future search of differentiation-inducing agents.     

Hematopoietic growth factors in the pathogenesis and for the treatment of aplastic anemia

The production and release of hematopoietic growth factors from bone marrow stromas established in vitro from patients with aplastic anemia is normal or increased. Addition of hematopoietic growth factors to aplastic anemia bone marrow cells results in only modest increases in colony growth, with the exception of granulocyte colony-stimulating factor (G-CSF), which corrects their impaired cloning efficiency to normal. Most clinical data on the use of hematopoietic growth factors in aplastic anemia have derived from uncontrolled and small single-arm studies or case reports. Sustained trilineage hematologic responses have not been observed when hematopoietic growth factors have been used alone or in combination. Serious side effects have been reported for most of the hematopoietic growth factors in patients with aplastic anemia, with the exception of G-CSF. There is a major concern that they may further increase the risk of clonal disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Hematopoietic growth factors should not be used alone in newly diagnosed patients as specific treatment for aplastic anemia, and their use in combination with immunosuppressive therapy should be confined to multicenter, prospective randomized studies.     

Involvement of Fas and TNF pathways in the induction of apoptosis of T cells by antithymocyte globulin

Antithymocyte globulin (ATG) is the treatment of choice for those aplastic anemia patients who are not suitable for bone marrow transplantation (BMT). ATG is also used for the treatment of rejections in organ transplantation and as a conditioning regimen in BMT. Despite the proven efficacy of ATG in these areas, its mechanism of action is not known. Profound T-cell lymphopenia observed in vivo with ATG treatment is supposed to contribute to its therapeutic effect. We have previously shown that apoptosis is one of the mechanisms responsible for ATG-induced lymphopenia. Our next objective was to investigate the effect of ATG on modulation of Fas and TNF pathways, the two main pathways of T-cell apoptosis. Maximum surface expression of Fas on T cells was observed after 24 h at an ATG dose of 100 microg/ml; at this dose 88% of cells expressed Fas as compared to 26% of untreated cells. Surface expression of FasL was found to peak after 24 h at an ATG dose of 1000 microg/ml when 34% of cells were positive for FasL as compared to 1.5% of untreated T cells. Tumor necrosis factor (TNF)-alpha production was found to be maximum after 6 h at 1000 microg/ml dose (20%) as measured by intracellular cytokine staining of T cells. TNF-alpha production was also measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of lymphocytes treated with ATG for 6 h. A dose-dependent increase in TNF-alpha production was found in these supernatants with a plateau being achieved at an ATG dose of 1000 micro g/ml. We conclude that ATG-induced apoptosis in T cells involves both Fas and TNF pathways and TNF-alpha is produced much earlier than Fas and FasL expression.