Thymocyte Suspensions Research Articles

Circulating CD3+/T cell receptor V gamma 3+ fetal murine thymocytes home to the skin and give rise to proliferating dendritic epidermal T cells.

The presence of CD3/TCR V gamma 3 moieties on both dendritic epidermal T cells (DETC) and fetal murine thymocytes has led to the concept that fetal thymocytes expressing this particular TCR phenotype are the actual DETC precursors. To test this assumption, we injected i.v. thymocyte suspensions prepared from day 16 and day 19 fetal mice as well as from adult animals, into syngeneic and Thy-1-disparate nude mice, the epidermis of which contains only Thy-1+/CD3- lymphocytes. Phenotypic analysis of the recipient epidermis by in situ immunolabeling revealed that injection of day 16 and day 19 fetal, but not of adult, thymocytes resulted in the appearance of distinct clusters of DETC as judged by their dendritic morphology and uniform expression of CD3/TCR V gamma 3 receptors. The presence of CD3+/TCR V gamma 3+ cells in the fetal, but not in the adult, thymocyte population(s) together with the failure to detect DETC after transfer of Thy-1+/CD3- fetal thymocytes strongly suggest that CD3+/TCR V gamma 3+ thymocytes are the DETC precursors. Kinetic studies of the DETC population from 2 to 12 wk after cell transfer revealed a substantial increase in the cell density within the DETC clusters that was not accompanied by an increase in the number of clusters. Thus, it appears that newly arriving DETC undergo proliferative activity in situ. Collectively, our results show that, under the experimental conditions chosen, CD3+/TCR V gamma 3+ fetal thymocytes are actual DETC precursors. Although it is not clear whether these experimental conditions are representative of the in vivo situation, they may serve as a useful model for studying the mechanisms underlying the homing properties of different lymphocyte subsets.

T-Cell Membrane CD45RA (2H4) and CD45RO (UCHL1) Determinants: II, Aberrant HLA-ABC Expression by CD45RA and CD45RO Cell Subpopulations of Mature CD4(+) T-Cell Leukaemias.

Six thymocyte suspensions, 10 normal blood CD4(+) CD8(-) lymphocyte-enriched fractions and leukaemic cells from 24 patients with CD4(+) mature T-cell lymphoid malignancy (five Sezary Syndrome, six adult T-cell leukaemia-lymphoma and 13 cases of T-cell prolymphocytic leukaemia) were examined in this study for the expression of membrane HLA-ABC by CD45RA (2H4) and CD45RO (UCHL1) subpopulations. These analyses showed that the main increase in HLA-ABC expression by normal CD4(+) CD8(-) blood lymphocytes (mean 490 to 760 FITC units) paralleled the loss of membrane 2H4 whilst the acquisition of UCHL1 was not associated with any significant change in HLA-ABC staining intensity. The sequence of 2H4 differentiation by normal thymocytes, based on the observed increasing levels of HLA-ABC staining intensity appeared to be (a) CD1a (+) 2H4(-) UCHL1(+) (25 HLA-ABC fluorescent units), (b)CD1a(-)2H4(int)UCHL1(+) (134 units), and (c) CD 1a(-) 2H4 (+) UCHL1 (-) (197 units). Quantitative estimates of membrane HLA-ABC expression by leukaemic T-cells revealed marked heterogeneity between individual cases irrespective of diagnostic subgroup. Based on the lower observed limits for normal CD4(+) 2H4(+) (318 units) and CD4 (+) 2H4(-) (478 units) fractions, 14% and 38% respectively of the leukaemic 2H4(+) and 2H4(-) components examined showed reduced HLA-ABC expression. Two cases showed very low membrane HLA-ABC levels that were within the range observed for normal CD1a(-) thymocytes. In contrast, HLA-ABC staining intensities exceeding that of corresponding normal CD4(+) 2H4(+) (710 units) and CD4(+) 2H4(-) (1286 units) subpopulations were seen in a high proportion (65%) of leukaemic 2H4 (+) components, with only 14% of 2H4(-) fractions showing raised levels and, in two cases, these staining intensities exceeded three times the normal observed limits. In addition to the quantitative differences in HLA-ABC expression, a remarkably consistent (81% of evaluable cases) feature of the leukaemic T-cells was that the 2H4(-)UCHL1(+) subpopulation in CD4(+) malignancies had a lower HLA-ABC level than the 2H4(+)UCHL1 subpopulation. This was in marked contrast to normal post-thymic T-cells where increasing HLA-ABC expression was seen with increasing UCHL1 (or decreasing 2H4) staining. These results suggest that leukaemic T-cells have an aberrant intra-thymic and post-thymic sequence of 2H4/UCHL1 expression which has become 'uncoupled' from CD1a/HLA-ABC expression.

Analysis of clones derived from human CD7+CD4-CD8-CD3- thymocytes

The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR gamma delta cells, 12% of clones were TCR alpha beta cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR alpha beta, TCR gamma delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR gamma, delta, and beta gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR alpha beta and TCR gamma delta lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR alpha beta and TCR gamma delta lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR gamma delta and NK cells.

Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex.

Development of tolerance to self Ag occurs during a negative cell selection process in the thymus. This selection process is thought to involve interactions between Ag-specific thymocyte receptors and self Ag presented by the MHC proteins on accessory cells, resulting in deletion of potentially harmful self-reactive precursors. However, the mechanisms underlying this clonal deletion have not been identified. In confirmation of previous findings (C. A. Smith, G. T. Williams, R. Kingston, E. J. Jenkins, and J. J. T. Owen, 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181), we have found that an anti-CD3 antibody stimulated DNA fragmentation, characteristic of a suicide mechanism known as apoptosis or programmed cell death (PCD), in suspensions of human thymocytes. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin. Although the magnitude and duration of the Ca2+ increase were similar to those observed in response to Con A, the mitogen did not stimulate DNA fragmentation or cell death. Phorbol ester prevented Ca2+-dependent DNA fragmentation and cell killing in response to anti-CD3 or other agents that stimulated PCD, suggesting that activation of protein kinase C abrogated cell suicide. Disappearance of CD4+CD8+ immature thymocytes was generally observed in response to all agents that stimulated PCD, whereas mature PBL were insensitive to stimulation of PCD. Our results suggest that antibody-mediated stimulation of immature thymocytes via the TCR complex results in Ca2+-dependent, endonuclease-mediated cell killing, depending on the activation status of protein kinase C.

Evidence that activation of 2-deoxy-D-glucose transport in rat thymocyte suspensions results from enhanced coupling between transport and hexokinase activity.

1. Suspensions of rat thymocytes accumulate free 2-deoxy-D-glucose (2-dGlc) within the cytosol to a concentration approx. 25-fold above the external concentration. This active accumulation was enhanced by 40 nM-phorbol 12-myristate 13-acetate (phorbol). 2. The Km for zero-trans uptake in control cells was 2.3 +/- 0.14 mM and Vmax. was 0.41 +/- 0.08 mumol/min per 10(10) cells (n = 6). In cells treated with phorbol (40 nM) the Km for zero-trans uptake was 1.2 +/- 0.13 mM and Vmax. 0.46 +/- 0.03 mumol/min per 10(10) cells (n = 6). The Km was decreased significantly by phorbol (P less than 0.01). 3. Phorbol-dependent activation of thymocytes delayed exit of free 2-dGlc into sugar-free solution and prevented exchange exit. Activation had no effect on 3-O-methyl D-glucoside (3-OMG) exit. 4. Coupling of 2-dGlc transport to hexokinase activity was determined by observing the effects of various concentrations of unlabelled cytosolic 2-dGlc on influx of labelled 2-dGlc into the hexose phosphate pool. In control cells this coupling was 0.81 +/- 0.02 and in phorbol-activated cells it was 0.92 +/- 0.01 (P less than 0.01). 5. The high-affinity inhibitor of hexokinase, mannoheptulose, inhibited uptake of 2-dGlc in both control and phorbol-treated cells. These data are consistent with a model for activation of sugar transport in which hexokinase activity is integrated with the sugar transporter at the endofacial surface. The results suggest that phorbol increases the degree of coupling transport with hexokinase activity, thereby leading to an increase in the rate of uptake of 2-dGlc, a decrease in exit of free 2-dGlc from the cytosol and an increase in free 2-dGlc accumulation.

2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) kills glucocorticoid-sensitive thymocytes invivo

The effect of in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on glucocorticoid- and calcium ionophore-induced DNA fragmentation in rat thymocytes was investigated. TCDD dose-dependently abolished DNA fragmentation in response to both agents after 7 days of exposure. Analysis of the time dependence of the effect revealed that after 1 or 2 days TCDD potentiated DNA fragmentation in untreated and glucocorticoid-treated thymocyte suspensions relative to controls. The DNA fragmentation in untreated thymocyte suspensions from TCDD-treated rats was completely prevented by inhibitors that block glucocorticoid-induced thymocyte suicide. Our results suggest that TCDD-induced thymic atrophy is due to Ca2+-dependent endonuclease activation.

Thymic stroma is required for the development of human T cell lineages in vitro

Development of the T cell lineage is characterized by the homing of hematopoietic precursors to thymus, followed by their acquisition of receptors for antigen. T cell receptors are alpha beta or gamma delta heterodimers associated with CD3 (TCR-CD3). Very early T cell precursors in humans have been characterized as CD7+ 45+ cells which lack the T cell differentiation antigens CD1, CD2, CD3, CD4, and CD8. A phenotypically equivalent early thymocyte population also occurs in postnatal life, and we have previously shown that interleukin 2 (IL2) promotes the development in vitro of both the alpha beta and the gamma delta T cells from these early thymocytes. Here we have analyzed the requirements of the induction of the IL2 pathway in early thymocytes, and their developmental potential. We show that: (i) thymic stromal cells, which are present in thymocyte suspensions, are necessary to induce the IL2 pathway and the development of alpha beta or gamma delta T cell lineages from early thymocytes in vitro; and (ii) when removed from the in vivo environment, early thymocytes can develop in vitro into TCR-CD3- cells of the natural killer (NK) lineage. We conclude that CD7+ 45+, CD1-2-3-4-8- early thymocytes are multipotential progenitors that, at least, have the capacity to develop into alpha beta or gamma delta T cell and NK lineages. The analysis of the mechanisms of generation and selection of human T and NK cell diversity, not feasible in bone marrow cultures, is now possible.

Alterations in thymocyte subpopulations in Down's syndrome (trisomy 21)

To correlate the histologically observed thymic abnormalities with the cellular immunodeficiency found in Down's syndrome (DS), thymus fragments and thymocyte suspensions from 14 noninstitutionalized DS subjects were studied. Histologic examination and immunohistologic studies using an anticluster of differentiation (CD) 1 monoclonal antibody showed a contracted cortex due to cortical thymocyte depletion. When DS unselected thymocytes were phenotyped, a significant reduction of CD3-, CD1-, CD4-, and CD8-positive cells was found as compared to controls. To evaluate if the deficient expression of these markers was due to the reduction of thymocyte subsets identifiable on the basis of their physical properties, we separated DS unselected thymocytes into 10 fractions by continuous Percoll density gradient centrifugation. DS thymuses were almost completely devoid of high density thymocytes. Since in normal thymus, these cells correspond to small CD1+, CD4+, CD8+, and 50% CD3+ cortical thymocytes, their absence may explain the unrestricted reduction of markers on DS unfractionated thymocytes. Furthermore DS thymuses appeared to be enriched in CD1+ first fraction (Fr1) low density thymocytes of the Percoll gradient. Fr1 CD1+ cells constitute the main spontaneously proliferating pool in normal human thymus. When the spontaneous proliferating activity of DS Fr1 was compared to that of the control, a significant reduction was observed. This reduction associated with the absence of high density thymocytes, with the reduction of cells expressing α- and β-chains of the T cell receptor and in conclusion with the lymphocyte depletion, suggests that in DS thymuses there is a deficient expansion of immature T cells resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool able to differentiate into functionally mature T cells.

Clonal analysis of CD4-CD8- human thymocytes expressing a T cell receptor gamma/delta chain. Direct evidence for the de novo expression of CD8 surface antigen and of cytolytic activity against tumor targets.

CD4-CD8- human thymocytes were obtained by treating total thymocyte suspensions with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. The resulting cell populations contained virtually no CD4+, CD8+ or WT31+ cells and 17-65% CD3+ cells. In addition, analysis of cell reactivity with delta-TCS-1 mAb (specific for the C gamma 2-encoded, nondisulfide-linked form of TcR gamma/delta), revealed the presence of a variable proportion of delta-TCS-1+ cells (the % of delta-TCS-1+ cells were lower than the percentage of CD3+ cells). Upon culture in recombinant interleukin 2 (IL2, in the presence of irradiated mononuclear cells), CD4-CD8- thymocytes underwent extensive proliferation. In addition, a progressive increase of CD8+ cells (but not of CD4+ or WT31+ cells) could be detected. Cells also progressively acquired cytolytic activity against K-562 or fresh melanoma cells. Fresh CD4-CD8- thymocytes were cloned under limiting dilution conditions. The cloning efficiencies were relatively high (1/3 cells); in addition, virtually all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31-delta-TCS-1+ surface phenotype. About half of the clones analyzed were CD8+, whereas none expressed CD4 antigens. We conclude that (a) only delta-TCS-1-reactive, TcR gamma/delta+ cells can be isolated from CD4-CD8- thymocytes cultured in IL2, and (b) the expression of CD8 antigen and of cytolytic activity reflects a true in vitro phenotypic change of CD8-, noncytolytic precursors (and not the preferential growth of few contaminating cells).

2,3,7,8-Tetrachlorodibenzo- p -Dioxin Kills Immature Thymocytes by Ca 2+ -Mediated Endonuclease Activation

Suspensions of thymocytes from young rats were incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which resulted in a sustained increase in cytosolic free Ca2+ concentration followed by DNA fragmentation and loss of cell viability. Both the Ca2+ increase and DNA fragmentation were prevented in cells treated with the inhibitor of protein synthesis, cycloheximide, and DNA fragmentation and cell killing were not detected when cells were incubated in a "Ca2+-free" medium or pretreated with high concentrations of the calcium probe, quin-2 tetraacetoxymethyl ester. These results indicate that TCDD can kill immature thymocytes by initiating a suicide process similar to that previously described for glucocorticoid hormones.

Dissociation of the res and immune components in the transient splenic response of embryos and neonatal chicks to immunization

Immunization with mouse erythrocytes (MRBC) elicited transient splenic enlargement as well as PFC formation in immunocompetent 7, 10, and 14 day chicks. An early enlargement within two days was followed by a decline and a second enlargement by day six. Only the early enlargement was observed when MRBC or Percoll was administered to neonatal chicks and late embryos. No significant splenomegaly was observed following cell transfer of bone marrow, spleen, leucocyte, thymocyte or bursal cell suspensions from B haplotype-compatible donors. During the adoptive immune response in embryo hosts, a minor early peak was observed that was followed by an elevated or progressive increase in spleen size. The data suggest that the early splenic response reflects primarily RES-vascular activity and the later enlargement is the consequence of immune proliferation.

Thymus and autoimmunity. Transplantation of the thymus from cyclosporin A-treated mice causes organ-specific autoimmune disease in athymic nude mice.

Organ-specific autoimmune diseases such as gastritis, oophoritis, thyroiditis, or insulitis developed in athymic nu/nu mice after engraftment of the thymus from euthymic nu/+ mice treated with cyclosporin A (CsA), a potent immuno-suppressant. The development of autoimmune disease in the nu/nu mice was prevented by inoculation of thymocyte suspensions prepared from normal nu/+ mice, but not by thymocyte suspensions from CsA-treated nu/+ mice. Cotransplantation of normal nu/+ mouse thymus with CsA-treated thymus also suppressed the development of autoimmune disease. Inoculation of spleen cell suspensions prepared from normal adult nu/+ mice prevented autoimmune disease, but inoculation of those from newborn nu/+ mice did not. Thus, CsA appears to interfere selectively with the thymic production of certain suppressor T cells controlling self-reactive (autoimmune) T cells, allowing the latter to expand and cause autoimmune disease.

Production of a Monoclonal Anti-Hamster Thymocyte Antibody with Mitogenic Properties

A monoclonal antibody (MAb), designated H3, with specificity for hamster lymphocytes, was produced by somatic cell hybridization of myeloma Sp 2/0 and spleen cells of Balb/c mice immunized with suspensions of viable hamster thymocytes. The H3 MAb (IgG 3) reacted specifically with hamster thymocyte surface membranes (immunofluorescent assay). The antibody recognized a protein of an approximate molecular weight of 44,000 Daltons in immunoblots of hamster thymocyte extracts. The soluble H3 MAb presented potent mitogenic properties as indicated by the DNA synthesis induced in in vitro hamster lymphocyte cultures.

Interleukin-2-Induced proliferation of CD4-CD8- human thymocytes.In vitro expression of CD3 and CD8 antigens and cytolytic activity

Human thymocytes lacking both CD4 and CD8 differentiation antigens were prepared by treating total thymocyte suspensions with a mixture of anti-CD4 and anti-CD8 monoclonal antibodies and complement. The resulting populations contained less than 2% CD4+, CD8+ or WT31+ cells and variable percentages (less than 20%) of CD3+ cells. These cell populations were cultured in recombinant IL-2 in the presence of peripheral blood mononuclear cells as feeder cells. Cells underwent extensive proliferation accompanied by a progressive increase of CD3+ and CD8+ cells. On the other hand, appearance of neither WT31+, alpha/beta-positive T cell receptor (TCR), nor CD4+ cells could be observed in several independent experiments. Functional analyses revealed the appearance and the progressive increase of cytolytic activity against the natural killer (NK)-sensitive K562 cells as well as the NK-resistant fresh melanoma cells. Experiments of T cell cloning indicated that both the expression of CD8 and CD3 antigens and the appearance of cytolytic activity were consequent to cell maturation occurring at the level of CD4-CD8- non-cytolytic cell precursors. In these experiments, more than 30% of cells underwent clonal expansion and all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31- surface phenotype. The expression of CD8 was variable, whereas no CD4+ clones could be obtained. Cells expressing such surface phenotype are known to belong to the TCR gamma-positive T lymphocyte subset lacking the typical alpha/beta TCR and thus appear to be the only T cell type capable of in vitro proliferation and maturation under easily reproducible culture conditions.

Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis).

Thymocytes are highly radiosensitive and show 'interphase death' within a few hours after low doses of irradiation. However, the mechanisms responsible for this type of death remain ill-defined. Separation of the dead thymocyte fraction from irradiated thymocyte suspensions by centrifugation on Percoll gradients provided homogeneous populations of dead cells suitable for detailed study. Using this method, radiation-induced interphase death of thymocytes was found to involve a sharp but transient increase in buoyant density, concomitant with the appearance of distinctive morphologic changes which included disappearance of microvilli and blistering of the cell surface. The chromatin in the dead cells had a molecular weight sufficiently low to resist sedimentation, and consisted of short oligonucleosome chains. We were unable to detect populations of cells intermediate between the dead and normal in the above characteristics. Interphase death thus involves a discrete, abrupt transition from the normal state and is not merely the consequence of progressive and degenerative changes. Furthermore, immediate cessation of development of interphase death by cycloheximide suggested a possible involvement of protein synthesis on this transition step.

Differential effects of tri- n-butylin chloride on macromolecular synthesis and ATP levels of rat thymocyte subpopulations obtained by centrifugal elutriation

Using centrifugal elutration, rat thymocytes were separated into three fractions by size. Fraction 1 (F1) consisted of a large population (>88% of all cells) of small, nonproliferating thymocytes. Fraction 2 (F2; 7% of all cells) was enriched in medium-sized thymocytes and showed an increased macromolecular synthesis. In the small fraction 3 (F3; <5% of all cells), large thymocytes, monocytes, granulocytes and cells in mitosis were concentrated. F3 demonstrated the highest proliferative activity. Fractions were characterized by size, cell morphology, DNA, RNA and protein synthesis and steady state ATP levels. The effects of the inhibitor of oxidative phosphorylaiton, tri- n-butyltin chloride (TBTC) on ATP levels and the incorporation of DNA, RNA and protein precursors were investigated for each thymocyte fraction. Although ATP levels increased with increasing thymocyte volume, TBTC reduced ATP levels in each subfraction proportionally. The incorporation of thymidine and leucine was markedly reduced in all fractions by TBTC, but not to the same extend. Dependent on the TBTC concentration, the rapidly proliferating cells of F2 and F3 were less affected compared to the noncycling cells of F1. The incorporation of uridine in the unfractionated cells and in F2 and F3 was also decreased by TBTC. However, at concentrations between 0.1 and 1 μM, TBTB stimulated uridine incorporation in the small thymocytes of F1. Therefore, fractionation of thymocyte suspensions was necessary to defect differential effects of TBTC on subpopulations. Centrifugal elutriation was found to be a useful toll with which to obtain subfractions of isolated rat thymocytes.

Activation of mouse lymphocytes inhibits induction of rapid cell death by x-irradiation.

A distinctive property of the resting lymphocyte is its ability to die rapidly in interphase after x-irradiation. Suspensions of thymocytes and peripheral lymphocytes from BALB/c mice were irradiated with doses ranging from 10 to 10,000 rad (0.1 to 100 Grays), and their viability was measured by eosin dye exclusion at intervals through 3 days of culture. After an initial latent period of a few hours, viability declined exponentially in a dose-dependent fashion. Doses as low as 20 rad caused some lymphocytes to die rapidly. After 1000 rad, 90% of the cells became nonviable in 15 to 20 hr and 99% in 25 to 35 hr. Peripheral lymphocytes showed a somewhat earlier loss of viability than did thymocytes, and were killed especially rapidly by 10,000 rad. Enriched T cells and B cells were killed by irradiation at equal rates, and medullary thymocytes were killed at the same rate as the whole thymocyte population. In contrast with resting cells, T and B lymphocytes activated by mitogens were not subject to such rapid induction of cell death. Irradiation with 1000 rad reduced the viability of activated cells by only 50% at a time when less than 1% of nonstimulated lymphocytes remained alive. Similarly, cloned lines of antigen-specific helper and cytotoxic T cells showed only a delayed and slow loss of viability after receiving 1000 rad. The state of activation can therefore be a significant determinant of the immunologic consequences of irradiation.

Estrogen receptors and estrogen sensitivity of fetal thymocytes are restricted to blast lymphoid cells.

We have observed [3H]estradiol binding sites in the cytosol of lymphoid cell suspensions obtained from the fetal thymus of guinea pig. Scatchard analysis [dissociation constant (Kd), 0.5 +/- 0.02 (SE) nM], binding specificity, and diethylaminoethyl chromatography of these [3H]estradiol binding sites are similar to those described for estrogen receptors. Estrogen receptor levels in the thymic lymphoid cell population have been studied after fractionating cells by Percoll discontinuous density gradients into large and low density cells (alpha-cells) and small and high density cells (beta-cells). Estrogen receptor levels are higher in alpha-cells [1002 +/- 200 (SE) sites per cell] than beta-cells [61 +/- 6 (SE) sites per cell]. Electron microscopy shows that the ultrastructural characteristics of 95% alpha-cells are consistent with their lymphoblastoid nature, whereas 90% of the beta-cells represented here can be categorized as typical small lymphocytes. To study whether cellular estrogen receptor expression was related to blastogenesis, fetal thymocyte suspensions were cultured throughout 48 h in the presence of the mitogen Concanavalin A. A significant increase in estrogen receptor levels was observed in thymic lymphoid cells cultured in the presence of the mitogen [1500 +/- 193 (SE) sites per cell] with respect to cells cultured in the absence of the mitogen [400 +/- 54 (SE) sites per cell]. One-day and 3-day in vivo estrogen treatments decrease significantly the [3H]thymidine uptake [by 40 +/- 1% (SE) and 66 +/- 5% (SE), respectively], the mitotic index [by 82 +/- 8% (SE) and 96 +/- 2% (SE), respectively] and the frequency of lymphoblastoid cells [by 15 +/- 4% (SE) and 50 +/- 2% (SE), respectively] in fetal thymocyte suspensions. A 29 +/- 0.7% (SE), 31 +/- 0.8% (SE), and 35 +/- 2% (SE) decrease of [3H]thymidine incorporation in the DNA of alpha-cells cultured throughout 24 h, 48 h, and 72 h, respectively, in the presence of estradiol (10 nM) was observed with respect to untreated alpha-cells (P less than 0.01), while no effect of estradiol was observed on [3H]thymidine uptake by beta-cells. The mitotic index of cultured alpha-cells was significantly decreased [by 67 +/- 5% (SE)] 48 h after the addition of estradiol to the culture medium. It is concluded that: 1) estrogen receptors and estrogen sensitivity are restricted to lymphoblastoid thymic cells, and 2) estrogen receptor levels are increased after lymphoid blast transformation.

IMMUNOHISTOCHEMICAL DEMONSTRATION OF ADA AND ADCP ON THE CELL SURFACE OF HUMAN T-LYMPHOID CELLS: 54

ADA plays an important role in the development of the immune system and the enzyme is probably associated with T-lymphocyte differentiation. ADA has been shown to exist in different molecular weight forms. In man the major forms are the red cell ADA or ADA-S and the tissue specific ADA or ADA-L. ADA-S can be converted to ADA-L by complexing with ADCP. Intact human peripheral T lymphocytes and human thymocytes were first incubated with rabbit anti-ADA and anti-ADCP antisera, followed by incubation with FITC-labeled swine anti-rabbit IgG. Since only cell surface ADA and ADCP would be exposed to the antisera, fluorescence indicates ADA and ADCP expression on the outer cell membrane. Labeled cells were analyzed by fluorescence microscopy and FACS analysis. ADCP is present at the cell membrane of about 50% of the T lymphocytes; 10-20% of the cells contain membrane-bound ADA. When these cells were incubated with a lysate of human erythrocytes (as source of ADA-S) about 50% of the cells were found to display ADA at the membrane. Human thymocyte suspensions contained 20-30% membrane-ADA positive cells and 10-20% membrane-ADCP positive cells. The physiological significance of ADA and ADCP expression at the extracellular surface of human T-lymphoid cells will be resolved.

Mitochondrial membrane potential in lymphocytes as monitored by fluorescent cation diS-C3-(5).

A lipophilic fluorescent cation diS-C3-(5) and rotenone suppress the oxygen consumption rate of thymocytes in similar concentrations. Seventy percent inhibition corresponds to an inhibitor:cytochrome a molar ratio of about 1:1. Addition of uncouplers decreases the inhibition of respiration by diS-C3-(5) (but not rotenone). FCCP in similar concentrations increases O2 consumption in the absence of diS-C3-(5) and the diS-C3-(5) fluorescence intensity in the presence of TMPD in thymocyte suspensions. In most thymocyte preparations, oligomycin (0.05-0.1 microgram/mL) increases the fluorescence of diS-C3-(5) and further addition of TMPD (50-100 microM) decreases the fluorescence. Addition of NaCN (400 microM) after oligomycin leads to a fluorescence increase that is hardly affected by subsequent addition of 0.2 microM FCCP. Nigericin (10-50 nM) decreases the diS-C3-(5) fluorescence. The data indicate that the diS-C3-(5) fluorescence associated with mitochondrial transmembrane potential (delta psi m) may be an essential part of the diS-C3-(5) fluorescence in lymphocyte suspensions. The changes of the diS-C3-(5) fluorescence intensity in the presence of TMPD after FCCP addition reflect delta psi m.