Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation.

Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 1x PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.

Id2 and Id3 inhibit development of CD34(+) stem cells into predendritic cell (pre-DC)2 but not into pre-DC1. Evidence for a lymphoid origin of pre-DC2.

We found previously that Id3, which inhibits transcriptional activities of many basic helix-loop-helix transcription factors, blocked T and B cell development but stimulated natural killer (NK) cell development. Here we report that ectopic expression of Id3 and another Id protein, Id2, strongly inhibited the development of primitive CD34(+)CD38(-) progenitor cells into CD123(high) dendritic cell (DC)2 precursors. In contrast, development of CD34(+)CD38(-) cells into CD4(+)CD14(+) DC1 precursors and mature DC1 was not affected by ectopic Id2 or Id3 expression. These observations support the notion of a common origin of DC2 precursors, T and B cells. As Id proteins did not block development of NK cells, a model presents itself in which these proteins drive common lymphoid precursors to develop into NK cells by inhibiting their options to develop into T cells, B cells, and pre-DC2.

Effect of cytokines on HIV-induced depletion of thymocytes in vivo.

BACKGROUND: Cytokines play an important role in the differentiation of thymocytes into mature T cells; consequently, certain cytokines could be useful for immune reconstitution after HIV infection without increasing viral load. OBJECTIVE: To investigate whether cytokines affect immune depletion caused by HIV infection with a CXCR4-tropic strain in SCID-hu mice implanted with human fetal thymus and liver (thy/liv) tissue. METHODS: The thy/liv implants were either mock infected or infected with HIV-1 NL4-3, a CXCR4-tropic molecular clone. Interleukin (IL)-2, IL-4, IL-7, interferon-gamma (IFN-gamma) or diluent was administered to the mice during the second and third week postinfection. Viral load and immunophenotype were determined in thymocytes. RESULTS: Thymocyte subset distributions at 3 weeks postinfection were significantly influenced by treatment with certain cytokines. In particular, IL-2 caused the infected mice to retain a thymocyte profile that was more similar to that in mock-infected mice than that in diluent-treated infected mice, in that the percentages of immature CD4+CD8+ and CD5+CD1+ cells were slightly higher and much less variable than in diluent-treated infected mice. The effect of IFN-gamma treatment was similar to IL-2 but did not reach statistical significance. However, after IFN-gamma treatment, normal percentages of mature CD3+CD69+ cells were maintained whereas this population was relatively increased in diluent-treated infected mice. Although treatment with IL-4 and IL-7 delayed depletion of immature thymocytes, these cytokines increased viral load. CONCLUSIONS: Cytokines such as IL-2 and IFN-gamma maintain immature thymocytes without increasing viral load and may be useful as adjuncts to improve immune reconstitution after HIV infection.

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability.

BACKGROUND: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. MATERIALS AND METHODS: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. RESULTS: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. CONCLUSIONS: The results show that cells stained with FITC-labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions.

Primary HIV infection of infants: the effects of somatic growth on lymphocyte and virus dynamics.

Acute HIV infection is characterized by the appearance of high concentrations of virus in the peripheral blood. In adults, this high-level viremia spontaneously abates after several weeks. In contrast, after perinatal infection of infants, blood virus levels remain high for many months, during which the concentration of circulating CD4+ lymphocytes remains well above normal values for adults. Here we suggest an explanation for these differences, based on developmental factors including somatic growth and immunological ontogeny. Flow cytometric analysis revealed that at birth the thymus contains elevated levels of mature T lymphocytes, compared to the thymus after 3 months of age. A mathematical model is proposed incorporating immunological and virological data from longitudinally evaluated infants who acquired infection at the time of birth. This model explains the pattern of high-level viremia in infants as resulting from the replication of HIV within the progressively expanding lymphoid cell mass.

Differential tropism and replication kinetics of human immunodeficiency virus type 1 isolates in thymocytes: coreceptor expression allows viral entry, but productive infection of distinct subsets is determined at the postentry level.

Human thymocytes are readily infected with human immunodeficiency virus type 1 (HIV-1) in vivo and in vitro. In this study, we found that the kinetics of replication and cytopathic effects of two molecular isolates, NL4-3 and JR-CSF, in postnatal thymocytes are best explained by the distribution of chemokine receptors used for viral entry. CXCR4 was expressed at high levels on most thymocytes, whereas CCR5 expression was restricted to only 0.1 to 2% of thymocytes. The difference in the amount of proviral DNA detected after infection of fresh thymocytes with NL4-3 or JR-CSF correlated with the levels of CXCR4 and CCR5 surface expression. Anti-CCR5 blocking studies showed that low levels of CCR5 were necessary and sufficient for JR-CSF entry in thymocytes. Interleukin-2 (IL-2), IL-4, and IL-7, cytokines normally present in the thymus, influenced the expression of CXCR4 and CCR5 on thymocytes and thus increased the infectivity and spread of both NL4-3 and JR-CSF in culture. NL4-3 was produced by both immature and mature thymocytes, whereas JR-CSF production was restricted to the mature CD1(-)/CD69(+) population. Although CXCR4 and CCR5 distribution readily explained viral entry in mature CD69(+) and immature CD69(-) cells, and correlated with proviral DNA distribution, we found that viral production was favored in CD69(+) cells. Therefore, while expression of CD4 and appropriate coreceptors are essential determinants of viral entry, factors related to activation and stage-specific maturation contribute to HIV-1 replication in thymocyte subsets. These results have direct implications for HIV-1 pathogenesis in pediatric patients.

Thymic dendritic cells are primary targets for the oncogenic virus SL3-3.

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70(+) cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70(+) cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70(+) cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70(+) dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.

Apoptosis in cord blood T lymphocytes from infants of human immunodeficiency virus-infected mothers.

Apoptosis continues to be controversial in human immunodeficiency virus (HIV)-induced pathogenesis. To investigate whether apoptosis occurs with HIV exposure with or without subsequent infection, levels of apoptosis were measured in cord blood lymphocytes (CBL) from seven newborns delivered to HIV-infected mothers and seven normal, unexposed newborns. Live cells were costained with antibodies to cell surface markers and the DNA dye 7-amino actinomycin D to immunophenotype apoptotic CBL subsets. Apoptosis was measured in fresh and cultured CBL in the presence and absence of CD3 T-cell receptor stimulation. Compared to the CD4+ CBL from HIV-unexposed newborns, CD4+ CBL from six HIV-exposed, noninfected newborns demonstrated significantly greater apoptosis after overnight culture even in the absence of CD3 stimulation. Compared to HIV-unexposed controls, CD8+ CBL from the six HIV-exposed newborns also demonstrated increased levels of apoptosis after overnight culture without stimulation. The one HIV-infected newborn in this study showed the highest levels of CD4+ and CD8+ apoptotic CBL. The data suggest that levels of apoptosis are increased in infants in association with HIV infection. Furthermore, CD4+ and CD8+ cord blood lymphocytes from HIV-exposed infants behaved differently than T lymphocytes from either normal, unexposed infants or an HIV-infected infant. These results suggest that exposure to HIV or HIV-induced factors increases the levels of apoptosis in CBL.

High viral burden and rapid CD4+ cell depletion in human immunodeficiency virus type 1-infected SCID-hu mice suggest direct viral killing of thymocytes in vivo.

The mechanism of CD4+ cell loss in lymphoid organs is unknown. In this study, human immunodeficiency virus (HIV) infection of human fetal thymus/liver implants in severe combined immunodeficient mice was used to investigate the mechanism of HIV-induced depletion of CD4-bearing cells in vivo. The implants were assessed for depletion of CD4+ thymocytes, apoptosis, and viral burden. We detected two phases of CD4 cell depletion, an initial rapid phase and a more gradual later phase. Compared to mock-infected implants, HIV-infected implants did not demonstrate detectable increases in the levels of apoptosis while severe depletion of CD4-bearing cells was ongoing. During peak loss of CD4+ cells, high viral burden was observed, suggesting that loss of CD4+ cells in this in vivo system is due to direct killing of infected thymocytes. Increased levels of apoptosis were observed during the later phase of thymocyte depletion; however, these apoptotic cells lacked CD4. This finding suggests that a second indirect mechanism may be responsible for the destruction of CD4- CD8+ thymocytes in vivo. Taken together, these results suggest that CD4+ and CD4- cells may die by different mechanism(s).

Mechanism of human immunodeficiency virus type 1 localization in CD4-negative thymocytes: differentiation from a CD4-positive precursor allows productive infection.

Human immunodeficiency virus (HIV) infection of the thymus could have profound effects on development of the immune response, particularly in children. We and others have established that in addition to infecting and depleting CD4-bearing thymocytes, functional HIV proviruses are found in thymocytes lacking surface CD4 expression. Using in vitro thymocyte cultures, we show that neither HIV-mediated down regulation of CD4 nor CD4-independent infection contributes to the localization of HIV in cells lacking the primary virus receptor. Rather, infection of a CD4-positive precursor cell (CD4 positive/CD8 positive) with subsequent differentiation into a mature CD4-negative phenotype results in productively infected CD4-negative cells. This novel mechanism may contribute to pathogenesis by distributing viral sequences into functional subsets of T cells typically refractory to HIV infection and could account for the presence of viral DNA in CD8-positive lymphocytes recently observed in patients.

Galectin-1, an endogenous lectin produced by thymic epithelial cells, induces apoptosis of human thymocytes.

Galectin-1, a beta-galactoside binding protein, is produced by thymic epithelial cells and binds to human thymocytes. We have previously reported that galectin-1 induces the apoptosis of activated T lymphocytes. Because the majority of thymocytes die via apoptosis while still within the thymus, we tested whether galectin-1 could induce the apoptosis of these cells. We now report that in vitro exposure to galectin-1 induced apoptosis of two subsets of CD4(lo) CD8(lo) thymocytes. The phenotypes of susceptible thymocytes were consistent with that of both negatively selected and nonselected cells. Galectin-1-induced apoptosis was enhanced by preexposure of thymocytes to antibody to CD3, suggesting that galectin-1 may be a participant in T-cell- receptor mediated apoptosis. In contrast, pretreatment of thymocytes with dexamethasone had no effect on galectin-1 susceptibility. We noted that 71% of the cells undergoing apoptosis after galectin-1 treatment had a DNA content greater than 2N, indicating that proliferating thymocytes were most sensitive to galectin-1. We propose that galectin-1 plays a role in the apoptosis of both negatively selected and nonselected thymocytes, and that the susceptibility of thymocytes to galectin-1 is regulated, in part, by entry or exit from the cell cycle.

Human thymocyte responsiveness to stem cell factor: synergy with interleukin-2 for the generation of NK/LAK cytotoxicity.

Human recombinant stem cell factor (SCF) increases the viability and cell size of a subset of thymocytes in vitro, but does not independently induce phenotypic changes on thymocytes indicative of T cell differentiation. The SCF-responsive thymocytes have characteristics of large granular cells, that do not express T, B or NK cell-related antigens, and are primarily found in immature thymocyte subsets. These large granular thymocytes do not display cytotoxic activity. However, SCF acts synergistically with IL-2 in the generation of cytotoxic effector cells from thymocyte precursors. Synergy in cytotoxicity is observed to both NK-sensitive and NK-resistant targets. Studies of the SCF receptors on thymocytes show that receptors are expressed on mature 'bright' CD3+ cells, immature 'dim' CD3+ cells as well as CD3- cells. IL-2 increases the frequency of SCF receptor-positive cells in cultured thymocytes, which may explain its synergy with SCF in the generation of NK/LAK cytotoxicity. These data show that SCF enhances the functional development of thymic NK/LAK cells in vitro.

Differential tropism of HIV-1 isolates for distinct thymocyte subsets in vitro.

OBJECTIVE: Understanding the interaction between HIV and developing thymocytes is crucial in determining how HIV infection perturbs the immune system. We determined which thymocyte subsets can harbor and express HIV. DESIGN: HIV expression in mature and immature thymocytes obtained from surgical specimens from non-infected children was determined after in vitro infection with the syncytium-inducing, cytopathic NL4-3 and the non-syncytium-inducing, relatively noncytopathic JR-CSF isolates. METHODS: Intracellular staining for the HIV p24gag antigen was combined with cell surface phenotyping to determine thymocyte subsets expressing HIV. Infection was quantitated by polymerase chain reaction on sorted subsets. RESULTS: NL4-3 replicated faster and to higher titers and caused a more severe decrease of all CD4-bearing thymocytes than did JR-CSF. In addition, both immature CD1+ and mature CD1-thymocytes expressed NL4-3, whereas only mature CD1-cells expressed JR-CSF. The tropism of NL4-3 for these immature cells suggests a mechanism for a more profound impact on T-cell maturation than that seen with JR-CSF. We also found that thymocytes lacking cell surface CD4 (CD4-CD8- and CD4-CD8+ subsets) expressed virus with either isolate late in infection, when viral levels were high. The CD4-CD8- cells expressing HIV were mature CD3bright T-cell receptor (TCR) alpha/beta bright cells. CONCLUSIONS: These results show that NL4-3 can be expressed by thymocytes at immature and mature stages of differentiation and cause severe loss of CD4+ cells. Thus, tropism of a virus for immature cells can affect the capability of the thymus to produce new T lymphocytes leading to a greater impact on development and functions of the immune system. It is proposed that this in vitro model can be used to study pathogenic mechanisms in the thymus.

Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells.

Thymic epithelial cells play a crucial role in the selection of developing thymocytes. Thymocyte-epithelial cell interactions involve a number of adhesion molecules, including members of the integrin and immunoglobulin superfamilies. We found that human thymic epithelial cells synthesize an endogenous lectin, galectin-1, which binds to oligosaccharide ligands on the surface of thymocytes and T lymphoblastoid cells. Binding of T lymphoblastoid cells to thymic epithelial cells was inhibited by antibody to galectin-1 on the epithelial cells, and by two antibodies, T305 and 2B11, that recognize carbohydrate epitopes on the T cell surface glycoproteins CD43 and CD45, respectively. T lymphoblastoid cells and thymocytes bound recombinant galectin-1, as demonstrated by flow cytometric analysis, and lectin binding was completely inhibited in the presence of lactose. The degree of galectin-1 binding to thymocytes correlated with the maturation stage of the cells, as immature thymocytes bound more galectin-1 than did mature thymocytes. Preferential binding of galectin-1 to immature thymocytes may result from regulated expression of preferred oligosaccharide ligands on those cells, since we found that the epitope recognized by the T305 antibody, the core 2 O-glycan structure on CD43, was expressed on cortical, but not medullary cells. The level of expression of the UDP-GlcNAc:Gal beta 1,3GalNAc-R beta 1, 6GlcNAc transferase (core 2 beta 1, 6 GlcNAc transferase, or C2GnT), which creates the core 2 O-glycan structure, correlated with the glycosylation change between cortical and medullary cells. Expression of mRNA encoding the C2GnT was high in subcapsular and cortical thymocytes and low in medullary thymocytes, as demonstrated by in situ hybridization. These results suggest that galectin-1 participates in thymocyte-thymic epithelial cell interactions, and that this interaction may be regulated by expression of relevant oligosaccharide ligands on the thymocyte cell surface.

A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry.

A sensitive method for quantification of cells undergoing apoptosis that permits the simultaneous measurement of dual-color cell surface immunofluorescence is presented. Unfixed cells are stained with 7-amino-actinomycin D (7-AAD) for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). Owing to its spectral characteristics 7-AAD can be combined with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) cell surface staining. After staining, the samples can be treated with paraformaldehyde (PF) solution to eliminate the risk for exposure of laboratory personnel to biohazardous agents and to preserve the cells through fixation for later analysis on the flow cytometer. The value of the method is shown on the measurement of apoptosis in human thymocytes and in human peripheral blood mononuclear cells (PBMC) exposed to various inducers of active cell death. The method is validated by fluorescent activated cell sorting in combination with morphologic examination of the sorted cells. The technique we are presenting is particularly valuable in a clinical setting because it allows rapid multiparameter analysis of apoptosis in combination with cell surface phenotype on biohazardous samples with single laser instrumentation.

The SCID mouse environment causes immunophenotypic changes in human immature T-cell lines.

In order to evaluate whether the SCID mouse can provide the microenvironment for the growth of human immature T-cell leukemias and if in vivo growth alters their phenotype, we examined the behavior of 3 well-characterized T-cell acute lymphoblastic leukemia (T-ALL)-derived T-cell lines which are at different stages of maturation (CEM, SUP-T3 and MOLT-4f) after transfer to non-irradiated SCID mice. All 3 T-cell lines engrafted and proliferated to form tumors in the mice and showed dissemination patterns in the SCID mouse comparable to those of T-ALL in man: i.e., human cells were detectable by flow cytometry or were cultured from mouse bone marrow, spleen, liver or thymus. CEM, which is the most immature T-cell line, readily formed tumors after injection of cells. The more mature T-cell lines, SUP-T3 and MOLT-4f, required a longer time period, even after injection of higher cell numbers. Whereas no changes in the configuration of the rearranged T-cell receptor genes were detected, striking phenotypic changes were observed in all 3 leukemias growing in the SCID mice after injection. SCID-CEM cells showed an increase in the surface expression of CD3 and CD8 and a decrease in the expression of CD1 and CD71 (transferrin-receptor). SCID-MOLT-4f cells showed an increase in CD5 and CD8 expression and a decrease in CD45RA expression. SCID-SUP-T3 cells showed increased expression of CD8 and CD45RA. Apparently, the mouse environment caused changes in cell-surface antigen expression on the T-ALL-derived T-cell lines. Some immunophenotypic changes remained stable during subsequent growth in culture of SUP-T3 cells, suggesting that maturation of the cell line occurred in vivo. The other cell lines, CEM and MOLT-4f after undergoing in vivo-induced changes, reverted to the original immunophenotype, suggesting transitory activation in vivo. These data point out the importance of stromal factors in defining growth and maturation of human leukemic cells in vivo, in SCID mice.

Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry.

A rapid, gentle, and sensitive method for quantification of cells undergoing apoptosis is presented. The method allows the simultaneous determination of dual-color cell surface immunofluorescence. Cells are stained for 7 min with the vital dye Hoechst 33342 (HO342) for identification of live and apoptotic cells. 7-amino-actinomycin D (7-AAD) is added to distinguish cells that have lost membrane integrity from apoptotic and live cells. Due to its spectral properties 7-AAD can be utilized on cells that are dual-surface labelled with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE). The value of the method is demonstrated on human thymocytes, which constitutively undergo programmed cell death and which show an increase in the rate of apoptosis after exposure to the glucocorticoid dexamethasone (DEX). Vital staining with HO342 permits earlier detection of apoptotic changes compared to a staining technique in which cells are treated with a hypotonic citrate solution containing propidium iodide (PI) and the apoptotic cells are represented in a hypodiploid, "sub-G1" peak. The HO342/7-AAD method may be particularly applicable to studies of programmed cell death in cells in which DNA fragmentation is difficult to detect by decreased DNA stainability.

Effects of cytokines on HIV-1 production by thymocytes.

The thymus is essential for normal T cell development and is particularly active during fetal and postnatal life. Here we describe in vitro studies of HIV-infected thymocytes cultured with cytokines normally produced in the thymus. Virus expression was determined by measuring p24 antigen levels in the culture supernatants. Addition of IL-2+IL-4 and IL-4+IL-7 to the HIV-infected cultures of both fetal and postnatal thymocytes resulted in various levels of synergistic expression of p24 antigen. When differences in phenotype between HIV-infected and non-infected (sham-treated) cultures from the same specimen were evaluated, there was a decrease in the percentages and absolute numbers of CD4-bearing cells in HIV-infected thymocytes cultured with IL-2+IL-4. Studies were done to determine if synergy in HIV expression was mediated by activation, proliferation or induction or suppression of other cytokines. We found a higher percentage of activated CD4+CD8+/high cells in thymocytes cultured with IL-2+IL-4 and IL-4+IL-7 than in thymocytes cultured with IL-2+IL-7. Proliferation was higher in thymocytes cultured with cytokine combinations but did not correlate with those conditions showing synergy. IL-4 reduced IFN-gamma production by thymocytes cultured with IL-2 in both HIV-infected and non-infected thymocytes. In addition, exogenous IFN-gamma decreased p24 expression by HIV-infected thymocytes when cultured with IL-4 alone, with IL-2+IL-4 or IL-4+IL-7. These results suggest that suppression of IFN-gamma by IL-4 may combine with cell activation and proliferation to produce synergy of virus expression observed with IL-2+IL-4 and IL-4+IL-7.

Phosphorylation of CD20 in cells from a hairy cell leukemia cell line. Evidence for involvement of calcium/calmodulin-dependent protein kinase II.

Hairy cell leukemia is an uncommon B cell lymphoproliferative disease of unknown etiology. We previously observed that CD20, a membrane protein involved in B cell activation, is hyperphosphorylated on hairy cells and that these cells have unusually high levels of intracellular free Ca2+. Therefore, we used a hairy cell line, HCLL-7876, to study the potential involvement of Ca(2+)-activated protein kinases in CD20 phosphorylation. Addition of the Ca2+ ionophore, ionomycin, increased CD20 phosphorylation both in activated B cells and in cells from the hairy cell line; addition of EGTA to either cell type decreased basal levels of CD20 phosphorylation. Ionomycin treatment of these cells resulted in increased kinase activity of cytosolic extracts toward syntide-2, a synthetic peptide substrate for calcium/calmodulin-dependent kinase II (CaM-KII), with kinetics similar to those of CD20 phosphorylation in the cell line. CD20 isolated from the cell line was a substrate for purified CaM-KII in vitro. Phosphopeptide maps of CD20 from untreated hairy cells or ionomycin-treated HCLL-7876 cells were similar to maps of CD20 that had been phosphorylated in vitro by CaM-KII. These results suggest that the unusually high levels of intracytoplasmic Ca2+ in hairy cells may enhance the phosphorylation of key surface proteins.

Interleukin-7 promotes the generation of phenotypically mature CD45RA positive human thymocytes in vitro.

IL-7 has been shown to support the proliferation of several cell types including human and murine thymocytes. We have investigated the influence of IL-7 on human thymocyte growth and maturation. In a serum-free culture system, recombinant human interleukin-7 (IL-7) (200 U/ml) stimulated nylon wool purified human thymocytes to express a mature phenotype. IL-7 promoted the generation of a population of large thymocytes which expressed high density CD3, CD4 and/or CD8, and high density CD45RA after 10 to 14 days in culture. The induction of CD45RA was not a transient event, since large CD45RA+ cells remained "bright" CD45RA+ up to 12 days after IL-7 was removed from cultures. IL-7 did support thymocyte proliferation. However, the IL-7-induced generation of large CD45RA+ thymocytes was not due to proliferation of a small subset because large CD45RA+ thymocytes also appeared in the presence of proliferation inhibitors. In order to identify IL-7-responsive thymocyte subsets, highly purified CD45RA- subsets at distinct stages of maturation were tested. CD3-, "dim" CD3+ and "bright" CD3+ subsets each responded to IL-7 with de novo expression of CD45RA. These data provide evidence that IL-7 supports phenotypic changes in both mature and immature subsets of human thymocytes, and indicate that IL-7 may play an important role in T cell development in the thymus.