Distinct target cells and effects of 1 alpha,25-dihydroxyvitamin D3 and glucocorticoids in the rat thymus gland

Thymocytes are known to possess receptors for glucocorticoids (GC) as well as for alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We have now investigated the distribution of the receptors for GC and 1,25-(OH)2D3 in rat thymocytes and compared the effects of the two steroid hormones on short term primary cultures of these cells. We report that in thymic cells, as in other tissues, 1,25-(OH)2D3 and GC bind specifically to distinct receptor molecules which exhibit sedimentation coefficients of 3.3S and 3.7S, respectively. Furthermore, the thymocytes that express the 1,25-(OH)2D3 receptor belong to a different and distinct subpopulation than the cells that express the glucocorticoid receptor. Specifically, by separating the thymocytes into two subsets by means of agglutination with the lectin peanut agglutinin (PNA), we have determined that the 1,25-(OH)2D3 receptor-positive cells belong to the PNA-negative medullary mature subset, whereas the GC receptor-positive cells belong to the PNA-positive cortical immature subset of thymocytes. Finally, we have compared the effects of the two steroid hormones on primary cultures of each of the two subsets as well as on unseparated thymocytes and found that GC act on PNA-positive cells to induce cell lysis; this leads to an enrichment in 1,25-(OH)2D3 receptor-positive thymocytes, as indicated by an apparent increase (6-fold) in the 1,25-(OH)2D3 binding in the cells surviving at the end of the culture. In contrast, we found that 1,25-(OH)2D3 acts on the PNA-negative cells to decrease the rate of cell lysis. These data indicates that the target cells for GC and 1,25-(OH)2D3 in the thymus are distinct and that these two hormones exert a different regulatory influence on the gland.     

Receptors for peanut agglutinin (Arachus hypogea) in childhood acute lymphoblastic leukemia: possible clinical significance

The presence of lymphocyte receptors for peanut agglutinin in significant numbers (greater than 15%) was identified on leukemic cells from T-cell acute lymphoblastic leukemia (T-ALL) (3/4), B-cell ALL (B- ALL) (2/4), null cell ALL (8/17), and on normal fetal thymic lymphocytes but not on normal human peripheral blood lymphocytes. Peanut agglutinin (PNA) binding was blocked specifically on leukemia lymphoblasts and thymic lymphocytes by the addition of galactose to the medium. When all immunologic subgroups of ALL are combined, preliminary data suggest that of the 13 ALL patients having greater than 15% PNA- positive lymphoblasts, 8 had relapsed, whereas none of the 12 ALL patients with less than 15% PNA-positive cells have recurrent disease at this time. It is likely that analysis of PNA receptors on ALL lymphoblasts may be a useful adjunct to the existing clinical and immunologic prognostic indicators.     

Circulating malignant cells in non-Hodgkin's lymphoma: correlation with binding by peanut agglutinin

A significant number of patients with non-Hodgkin's lymphoma have peripheral blood involvement during the course of their disease. Because the expression of receptor for the lectin peanut agglutinin PNA by normal lymphocytes is associated with noncirculating (stationary phase) cells, we studied the relationship between PNA binding by lymphoma cells and the presence of clonal B cells in the blood of 38 patients with B-cell lymphoma. The binding of PNA by cells in tissues was determined by the immunoperoxidase method and by two-color flow cytometry. Circulating lymphoma cells (clonal B cells) were identified by a sensitive flow-cytometric technique (kappa-lambda analysis) and were also studied for PNA binding in some cases. In all, 16 of 38 (42%) of lymphomas were PNA+, including a spectrum of histologic types. Circulating lymphoma cells were demonstrated in 17 of 22 PNA-lymphomas, whereas only 3 of 16 of PNA+ lymphomas had such circulating cells. Thus, there is a significant association between PNA binding and peripheral blood involvement by lymphoma (P less than .005 by chi- square analysis). In 12 cases, the circulating and tissue lymphoma cells had similar expression of PNA receptor (2 PNA+ and 10 PNA- cases), indicating that modulation of the PNA binding sites did not occur. In three patients who presented with lymphosarcoma cell leukemia, the circulating malignant cells were PNA-. These findings suggest that for both normal and malignant lymphocytes the absence of binding sites for PNA is associated with the capacity of these cells to circulate freely.     

Immunological phenotype of lymphoid cells in regenerating bone marrow of children after treatment for acute lymphoblastic leukemia

Bone marrow samples from 8 children treated for acute lymphoblastic leukemia (ALL) were investigated at cessation of cytostatic treatment and during 18 months thereafter. The course of the percentage of lymphoid cells and characterization of these cells by means of monoclonal antibodies, peanut agglutinin (PNA) binding and S-phase determination are shown. The percentage of lymphocytes rises in the first 1.5 months, followed by a non-significant decline. The percentage of cells in S-phase is higher at 0 months than at 6, 15 and 18 months. The percentage of T-cells does not change significantly. In the first 1.5 months a sudden rise in the percentage of common-ALL-antigen (cALLA)-positive lymphocytes occurs. The number of B-cells rises to a peak at 6 months. PNA positively increases to a maximum at 3 months and is correlated with positivity for markers of the B-cell lineage. The percentages of B-cells, cALLA-positive, and PNA-positive lymphocytes do not change significantly after they reach their maximum values and are still high at 18 months. Our results show that after cessation of chemotherapy for ALL a lymphoid cell regeneration occurs in the bone marrow consisting of cells of the B-cell lineage; many of these are cALLA-positive, but are discernible from their malignant counterparts by PNA-positivity.     

Peanut agglutinin in combination with CD19 monoclonal antibody has potential as a purging agent in myeloma.

Administration of high-dose chemotherapy to patients with myeloma, followed by rescue with autologous bone marrow transplantation (ABMT), sometimes induces complete disease remission but relapse is usual. We have attempted to reduce the risk of relapse by selective in vitro removal of myeloma cells from the autologous graft. A combination of the (gal-galNac)-binding lectin peanut agglutinin (PNA), which binds all plasma cells, and the pan-B monoclonal antibody CD19 was assessed for purging marrow of myeloma cells and their putative precursors using a magnetic bead method. Preliminary experiments performed on peripheral blood mononuclear cells spiked with fluorescent-labeled PNA+ Kirk tumor cells showed that a magnetic bead: target cell ratio of 40:1 resulted in a greater than 3-log reduction in PNA+ cells. This technique was then applied to 17 samples of myeloma bone marrow and to 18 samples of normal bone marrow spiked with PNA+ Kirk cells and CD19+ hairy cell leukemia cells. In each case all detectable plasma cells and CD19+ lymphocytes were effectively removed, and normal hemopoietic progenitor cell recovery was greater than 55%. This purging system deserves further study as a means of reducing relapse rates in myeloma patients treated by a combination of high-dose chemotherapy and ABMT.     

Interleukin 4 is a growth factor for activated thymocytes: possible role in T-cell ontogeny.

We have shown that recombinant or natural interleukin 4 (IL-4) (formerly called B-cell stimulatory factor 1) induces proliferation of activated adult or fetal thymocytes. In the case of adult thymocytes, IL-4 in combination with Con A or phorbol 12-myristate 13-acetate (PMA) stimulated the proliferation of peanut agglutinin (PNA)-negative (-) thymocytes, while PNA-positive (+) thymocytes showed only marginal responses. Further investigation revealed that day 14-17 fetal thymocytes, purified L3T4- LyT2- double-negative adult thymocytes, and single positive L3T4+ LyT2- or L3T4- LyT2+ thymocytes failed to respond to IL-4 or PMA alone but proliferated strongly with both IL-4 and PMA. In contrast, purified double-positive L3T4+ LyT2+ adult thymocytes showed only a marginal proliferative response to these stimuli. Responsiveness of thymic subpopulations to PMA and IL-4 could be inhibited with anti-IL-4 but not with anti-IL-2 monoclonal antibodies, indicating that they were IL-2 independent. Finally, we have observed that supernatants from calcium ionophore and PMA-stimulated adult double-negative L3T4- LyT2- thymocytes induce proliferation of double-negative adult thymocytes. This latter response is inhibited by anti-IL-4 monoclonal antibodies, suggesting that under appropriate stimulation conditions, these immature thymocytes are able to produce IL-4. These observations suggest a role for IL-4 in T-cell ontogeny.     

Insulin receptors on leukemia and lymphoma cells

Tumor cells obtained from leukemia and lymphoma patients were investigated for specific insulin receptors. Using radioactive 125I- labeled insulin, specific insulin binding sites were demonstrated on most acute lymphocytic leukemia (ALL) and acute myelocytic leukemia (AML) cells, including acute promyelocytic leukemia (APL), chronic myelocytic leukemia (CML), and acute monocytic leukemia (AMoL) cells. Insulin receptors were not found on chronic lymphocytic leukemia (CLL) and malignant lymphoma (ML) cells. Specific insulin binding sites were also found on monocytes and thymocytes after treatment with phytohemagglutinin (PHA-P), but not on inactivated tonsil cells, peripheral blood lymphocytes, or thymocytes. There was no inverse correlation between the content of insulin receptors and the basal level of circulating insulin. These data suggest that the insulin receptor may be a new marker of acute leukemia and chronic myelocytic leukemia.     

Glucose transporter 1 expression identifies a population of cycling CD4+ CD8+ human thymocytes with high CXCR4-induced chemotaxis

GLUT1, the major glucose transporter in peripheral T lymphocytes, is induced upon T cell receptor activation. However, the role of GLUT1 during human thymocyte differentiation remains to be evaluated. Our identification of GLUT1 as the human T lymphotrophic virus (HTLV) receptor has enabled us to use tagged HTLV-receptor-binding domain fusion proteins to specifically monitor surface GLUT1 expression. Here, we identify a unique subset of CD4+ CD8+ double-positive (DP) thymocytes, based on their GLUT1 surface expression. Whereas these cells express variable levels of CD8, they express uniformly high levels of CD4. Glucose uptake was 7-fold higher in CD4(hi) DP thymocytes than in CD4(lo) DP thymocytes (P = 0.0002). Further analyses indicated that these GLUT1+ thymocytes are early post-beta-selection, as demonstrated by low levels of T cell receptor (TCR)alphabeta and CD3. This population of immature GLUT1+ DP cells is rapidly cycling and can be further distinguished by specific expression of the transferrin receptor. Importantly, the CXCR4 chemokine receptor is expressed at 15-fold higher levels on GLUT1+ DP thymocytes, as compared with the DP GLUT1- subset, and the former cells show enhanced chemotaxis to the CXCR4 ligand CXCL12. Thus, during human thymopoiesis, GLUT1 is up-regulated after beta-selection, and these immature DP cells constitute a population with distinct metabolic and chemotactic properties.     

Immunologic identification of lymphocyte subsets in experimental murine myocarditis with encephalomyocarditis virus. Different kinetics of lymphocyte subsets between the heart and the peripheral blood, and significance of Thy 1.2+ (pan T) and Lyt 1+, 23+ (immature T) subsets in the development of myocarditis

To clarify the immune mechanism in myocarditis, immunofluorescence techniques with laser flow cytometry were used to examine serial changes in lymphocyte subsets in the heart, spleen, and peripheral blood of DBA/2 and BALB/c mice inoculated with encephalomyocarditis virus (Experiment I). B cells were identified by staining with fluorescein isothiocyanate-labelled rabbit anti-mouse immunoglobulin. T-cell subsets were identified with rat anti-Thy 1.2, and nonpolymorphic Lyt 1 and Lyt 2 monoclonal antibodies plus fluorescein isothiocyanate-labelled anti-mouse immunoglobulin. On days 7 and 14 postinfection, the percentage of Thy 1.2+ (pan T) cells in both strains had decreased in the peripheral blood; B cells showed no significant changes throughout the entire period. On the other hand, Thy 1.2+ (pan T) and Lyt 1+, 23+ (precursor and immature) T cells appeared to occupy the major portion of the myocardium on days 7 and 14 when congestive heart failure developed. To confirm this, serial immunohistologic studies (immunoperoxidase staining) of the hearts of DBA/2 and BALB/c mice with encephalomyocarditis virus-induced myocarditis were performed (Experiment II). In Experiment II, most of the stained cells in the hearts of both strains were Thy 1.2 positive and Lyt 1 and Lyt 2 positive on days 7 and 14. Thus, Experiments I and II demonstrated that lymphocytes at the site of inflammation in acute viral myocarditis carried antigenic markers that differed from those of peripheral lymphocytes and suggested that Thy 1.2+ (pan T) cells, especially the Lyt 1+, 23+ subset (immature T cells and T-cell subset precursors) were involved in the development of myocarditis in these animals.     

Ultrastructural analysis of acute lymphoblastic cells: peanut lectin binding correlates with degree of differentiation of the leukemic cells

Peanut agglutinin (PNA) has been shown to bind selectively to immature cells. Bone marrow cells from some children having acute lymphocytic leukemia (ALL) bind PNA while cells from other ALL patients do not bind, the significance being that the patients whose cells bind PNA have a poorer prognosis than those not binding PNA. In the present study, PNA was conjugated to horseradish peroxidase and the two cell types were compared. Cells binding PNA are immature compared with the non-PNA-binding cells.     

Characterization of B-cell type chronic lymphocytic leukemia cells by surface markers and a monoclonal antibody

This study was carried out to determine the reactivity of the Leu-1 mouse monoclonal antibody with B-chronic lymphocytic leukemia and other B-cell leukemias. This antibody has been previously reported to recognize a surface antigen expressed by almost all human thymocytes and peripheral T cells. It was also detected on surface immunoglobulin-bearing cells of most patients with chronic lymphocytic leukemia but was not detectable in normal B-cells and B-cell lines. In the present series, the neoplastic lymphocytes in 33 cases of B-chronlc lymphocytic leukemia expressed surface immunoglobulin, la antigen, and receptors for Fc, C3, and mouse erythrocytes. All but one case expressed the Leu-1 antigen as detected by indirect immunofluorescence using flow cytometry. In three other cases, the leukemic cells in the peripheral blood reacted with anti-Leu-1, whereas the bone marrow lymphocytes did not. Moreover, quantitative differences in the surface density of Leu-1 were apparent by flow cytometry. The peripheral blood and bone marrow lymphocytes in other B-cell leukemias, including 15 cases of leukemic B-cell lymphomas and three cases of hairy-cell leukemia, failed to stain positively with the anti-Leu-1 antibody. The recognition of the Leu-1 antigen adds to the phenotypic characterization of B-chronic lymphocytic leukemia and may contribute to a better understanding of the pathogenesis of the disease and its expression in different tissues.     

Human T-cell leukemia virus type II transforms normal human lymphocytes.

A unique human retrovirus (human T-cell leukemia virus type II, HTLV-II), isolated from a patient with a T-cell variant of hairy-cell leukemia, has been shown to be distinct from the more common isolates of human T-cell leukemia virus. This virus was tested for its ability to transform normal human peripheral blood lymphocytes. The HTLV-II-infected T-cell line Mo-T was lethally x-irradiated and cocultivated with normal human peripheral blood lymphocytes. The cocultivation of normal cells with Mo-T cells resulted in the transformation of the normal cells as evidenced by the establishment of permanent cell lines. The transformed cells are infected with HTLV-II as shown by immunologic tests and molecular hybridization. The cells are of mature T-cell phenotype and constitutively produce lymphokines. An Epstein-Barr virus-transformed lymphoblast B-cell line established from peripheral blood cells of the patient Mo, designated Mo-B, also was found to be infected with HTLV-II. All HTLV-II-infected cells, including the Mo-B cells, were capable of transforming normal cells of T-cell phenotype by transmission of virus by cocultivation. These results indicate that HTLV-II infects both B and T cells but transforms normal human peripheral blood lymphocytes of T-cell phenotype.     

Flow cytometric detection of CD10 (cALLA) on peripheral blood B lymphocytes of neonates

CD10 (cALLA) was detected on the surface of CD19-positive circulating lymphocytes in the peripheral blood of 32/50 neonates tested. These cells are presumed to represent immature B cells, commonly referred to as haematogones, previously undescribed in peripheral blood. The CD10+/CD19+ cells expressed lower levels of CD22 consistent with these cells being immature B lymphocytes. The presence of CD10+/CD19+ cells in the blood was not significantly correlated with a leucoerythroblastic picture, adjusted gestational age, or the presence in blood smears of medium-to-large lymphocytes with an immature appearance that morphologically resembled classic bone-marrow haematogones.     

Sialyl SSEA-1 antigen as a carbohydrate marker of human natural killer cells and immature lymphoid cells

The distribution of a carbohydrate antigen, the sialyl SSEA-1 (sialyl Lex-i), in human lymphoid cells was investigated by flow cytometry with a specific monoclonal antibody, MoAb FH-6. We concluded that the lymphocytes positive for the sialyl SSEA-1 antigen present in normal peripheral blood (PB) are natural killer (NK) cells since the positive cells had an NK activity toward K562 cells, and most of the sialyl SSEA- 1+ cells were simultaneously positive for Leu-11 (CD-16) and Leu-19. Essentially, no T and B cells, defined by Leu-4 (CD3) and Leu-16 (CD20), were positive for the sialyl SSEA-1 antigen in PB samples taken from healthy donors and patients with disorders unrelated to lymphoid malignancies. Among the malignant lymphoid cells, many sialylated SSEA- 1+ cells were observed in large granular lymphocyte (LGL) leukemia cells and some acute lymphoblastic leukemia (ALL) blasts, but not in CLL cells or malignant lymphoma cells. Sialyl SSEA-1 was also positive in some cultured human lymphoid cell lines. We conclude that expression of the sialyl SSEA-1 antigen is strictly limited to a distinct population of NK cells among the mature lymphocytes in normal PB, but the antigen is present in a wide range of immature lymphoblasts of T- and B-cell lineages as well as the NK-cell lineage. The sialyl SSEA-1 antigen disappears from the surface of immature lymphocytes of T- and B- cell lineages during the course of maturation.     

Monoclonal antibody to human cytotoxic-suppressor T-lymphocytes cross-reacts with canine lymphocytes and inhibits cell-mediated lympholysis of canine cells

Of 19 murine antihuman Tp32 (T8) monoclonal antibodies tested, only antibody T811 showed cross-reactivity with canine lymphoid cells. It recognized an antigen expressed on 19%-27% of peripheral blood mononuclear cells (PBMC), 20%-30% of peripheral Thy 1+ T-lymphocytes, 40% of puppy thymocytes, 35%-45% of alloantigen-activated peripheral lymphocytes, and 100% of PBMC from a dog with acute T-cell leukemia. The antigen was not expressed on peripheral Thy 1- cells, bronchoalveolar cells, or null-type acute leukemia cells. The antibody inhibited cell-mediated lympholysis in the absence of complement, while antibody F3-20-7 directed at the canine Thy 1 antigen did not. It was not possible to precipitate a corresponding cell surface antigen on canine cells using standard 125I radioimmunoprecipitation techniques nor did the antibody cause antigenic modulation. Our data suggest that monoclonal antibody T811 reacts with a functional subset of canine T-lymphocytes similar to that of human cells; however, significant differences may exist between the antigens recognized on these cells in the two species.     

Production of burst-promoting activity by monoclonal antibody defined malignant T lymphocytes from patients with lymphocytic leukemia and lymphoma

We tested conditioned media from 12 patients with T lymphocyte neoplasms and four T cell lines for their ability to stimulate the in vitro growth of erythroid-burst-forming units (BFU-E) from bone marrow mononuclear cells in a methylcellulose culture system. Nine patients suffered from acute lymphocytic leukemia, two from chronic lymphocytic leukemia, and one from non-Hodgkin's lymphoma. The T lymphocytes were characterized by a series of monoclonal antibodies and their stage of development was correlated with their ability to produce burst-promoting activity (BPA). Conditioned media from cells classified as prothymocytes (three cases), common thymocytes (one case), mature thymocytes (three cases), and mature lymphocytes of the helper subtype (two cases) increased BFU-E proliferation four- to 19-fold over control values using normal bone marrow as target cells. Conditioned media from OKT8+ malignant T lymphocytes (three cases) did not enhance BFU-E proliferation. Conditioned media from cells classified as immature T cells stimulated CFU-GM proliferation in only one of seven cases even though they secreted BPA. Conditioned media from three of the four cell lines stimulated by phytohemagglutinin, enhanced BFU-E growth. Our results indicate that malignant cells that have characteristics of immature T cells are able to produce BPA. Studies using techniques to isolate homogeneous populations of normal T cell subsets are required to determine whether normal immature T lymphocytes have the same capability.     

Human immature thymocytes as target cells of the leukemogenic activity of human T-cell leukemia virus type I

The risk of developing adult T-cell leukemia (ATL) associated with neonatal infection by human T-cell leukemia virus type I (HTLV-I) suggests that early events triggered by HTLV-I might be of crucial importance in initiating the multistep lymphoproliferative process leading several decades later to the development of leukemic disease. Thus, infection of thymocytes early in life might be directly correlated with the development of ATL. In the present study, we show that in vitro infection of mature (CD2+CD3+) or immature (CD2+CD3-) thymocytes resulted in the exogenous interleukin (IL)-2-dependent proliferation of HTLV-I-positive thymocytes, most of them displaying a CD2+CD3-CD4+ phenotype and expressing the CD25 molecule, the alpha chain of the IL-2 receptor. Furthermore, the CD80 and CD54 antigens, normally expressed by thymic stromal cells, were detected on these transformed thymocytes, indicating that HTLV-I infection may disturb the cooperation between thymocytes and their thymic environment. These HTLV-I-positive thymocytes were producing significant amounts of IL-6, which was found to be implicated in their proliferation and in the expression of CD25, as demonstrated by blocking experiments using a monoclonal antibody to IL-6. The present study suggests that immature thymocytes may provide an environment favorable to the unfolding of events leading to leukemia.     

Cap formation on lymphocytes from patients with leukemic diseases induced by four different lectins

Summary When ficoll purified peripheral blood lymphocytes were treated with fluorescein conjugated lectins from lentils (LCH), castor beans (RCA) and phaseolus coccineus beans (L- and E-PHA) for 15 min and the percentages of the cap forming cells were examined, the values of leukemic lymphocytes were reduced compared to the values obtained with normal lymphocytes. The reduction was more than half in patients with acute and chronic myelogenous leukemia and immunoblastoma, it was only one quarter in patients with chronic lymphocytic leukemia, Hodgkin's disease and lymphosarcoma. The lowest number of cap forming cells was found in lymphoblasts of established lymphoblastoid cell lines. The four different lectins showed nearly the same capacity in the induction of caps. After successive binding, the different lectins showed cocapping on the lymphocyte surface.     

Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and rhesus monkeys

The expression of receptors for interleukin-6 (IL-6) on human and rhesus monkey peripheral blood and bone marrow (BM) cells was examined by multiparameter flow cytometry after staining with biologically active, biotin-labeled human IL-6 and phycoerythrin-conjugated streptavidin. Consistent with the multiple biologic effects of IL-6 in stimulating immune functions and hematopoiesis, IL-6 receptors were detectable on a wide variety of cell types. In peripheral blood, IL-6 receptors were detectable on monocytes, granulocytes, and on CD4+ T lymphocytes but not on resting, CD19+ B lymphocytes and CD56+ natural killer (NK) cells. CD8+ T lymphocytes also expressed IL-6 receptors but at lower levels than CD4+ cells. The IL-6 receptors on granulocytes were only detectable after staining with high concentrations of biotin- IL-6, suggesting that most IL-6 receptors on these cells represent low- affinity sites. In contrast, IL-6 receptors on both CD4+ and CD8+ T lymphocytes were detectable at biotin-IL-6 concentrations as low as 10 pmol/L, indicating that these cells bind IL-6 with high affinity. IL-6 receptor expression patterns on rhesus monkey and human blood cells were very similar except that receptor levels on granulocytes were lower in humans than in rhesus monkeys. Similar differences in expression levels were observed for IL-6 receptors that were detectable on most granulocyte precursors in the mononuclear fraction of rhesus monkey and human bone marrow. In addition to these relatively mature cell types, IL-6 receptors were detectable on a large fraction of human and rhesus monkey BM blast cells that express the CD34 antigen. The presence of IL-6 receptors on CD34+ BM blast cells, which are the precursor cells of most, if not all, BM-derived blood cells, is consistent with the ability of IL-6, in conjunction with other cytokines, to stimulate immature hemopoietic cells in vitro and to promote blood cell production when administered in vivo.     

Flow Cytometric and Immunohistochemical Evaluation of Ethanol-Induced Changes in Splenic and Thymic Lymphoid Cell Populations

Ethanol-induced alterations in the immune system are thought to play a major role in increasing the susceptibility of alcoholics to infections and tumors. One important change in the immune system is the noted loss of lymphoid cells from the thymus and spleen. To examine these alterations we used a model system where C57Bl/6 mice were pair-fed either a Leiber-DeCarli diet containing 7% (v/v) ethanol or an isocaloric control diet. Mice receiving ETOH for 7 days showed a loss of cells from the spleen and thymus; this loss was even more severe after withdrawal for 1 day. The most profound changes were seen after 2 weeks of ETOH. Spleen and thymus cell numbers were reduced to 36% and 6.2%, respectively compared to control mice. Staining of thymocytes with monoclonal antibodies to lymphocyte surface markers and evaluation with flow cytometry revealed that immature thymocytes (PNA+, CD4+/CD8+) were most reduced. Mature thymocytes (CD4+/CD8- or CD4-/CD8+) were depleted, and the CD4+ to CD8+ ratio was increased. Sections of thymus stained with hematoxylin and eosin or with immunohistochemical methods showed atrophy and lymphoid cell depletion. No cortex was histologically identifiable after 2 weeks of ETOH. The spleen cells most affected by ETOH were the B cells. They were reduced to 8.2 x 10(6) cells/spleen (31.5% of the lymphocytes), as compared to 38.5 x 10(6) cells/spleen (50.3% of the lymphocytes) in the control mice. The spleen was atrophic, but the immunoarchitecture was preserved. Ethanol causes a depletion of lymphocytes from the spleen and thymus with alterations in lymphocyte subpopulations.(ABSTRACT TRUNCATED AT 250 WORDS)