Immortalized multipotential mesenchymal cells and the hematopoietic microenvironment.

In an attempt to analyze the cellular and molecular basis of the capacity of bone marrow stromal cells to support hematopoiesis in culture, we developed a series of murine stromal cell lines from a single long-term bone marrow culture (BMC). The cytokines produced by these cells were analyzed using immunohistochemical techniques, ribonuclease protection assays (RPA) and RT-PCR. We examined the capacity of these cloned cell lines to replace primary bone marrow-derived stromal cells in long-term bone marrow cultures (LT-BMC) and sought correlations between the capacity to support hematopoiesis in culture with the production of known cytokines. These immortalized lines replicate many of the functions of the hematopoietic microenvironment. They express cytokines known to play a role in hematopoiesis. All of the lines constitutively express mRNA for PBSF (SDF-1), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), FLT-3, thrombopoietin (TPO), interleukin 7 (IL-7), leukemia inhibitory factor (LIF), tumor necrosis factor-beta (TNF-beta), and interferon-gamma (IFN-gamma). Most lines also express granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF. They vary in their expression of IL-6, tumor growth factor-beta1 (TGF-beta1), TGF-beta2, and TNF-alpha. Growing these lines in the presence of cytokines that influence hematopoiesis alters the levels of cytokine message. The most striking effects were produced by TNF-alpha. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as osteoblast-specific factor-2 (OSF-2) and bone morphogenetic protein-1 (BMP-1). They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Several of the lines can maintain hematopoiesis in culture, as measured by the continuous production of myeloid colony-forming cells (CFU-c), for months. This capacity to support hematopoiesis does not correlate with any pattern of cytokine expression. Several of these lines also support the growth of human hematopoietic cells, and human CFU-c can be detected in the cultures in which CD34(+) bone marrow cells (BMC) are cultured on murine stromal cells. No correlation between the production of any of the known cytokines and the ability to support murine hematopoiesis was detected. In addition, there was no correlation between the capacity to support murine hematopoiesis and the capacity to maintain human HSC. Despite repeated cloning, the lines remain heterogeneous and are capable of producing cells with the properties of fibroblasts, osteoblasts, adipocytes, and myoblasts. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as OSF-2 and BMP-1. They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including NGF and BDNF.

Identification of four human cDNAs that are differentially expressed by early hematopoietic progenitors.

The molecular processes that maintain the stem cell pool are largely unknown. Using polymerase chain reaction-driven subtraction, we examined genes that are differentially expressed by early hematopoietic progenitors. We expected that identifying genes that are uniquely expressed by the earliest precursors would provide insight into the mechanism(s) through which stem cell number is maintained and differentiation is regulated. Using CD34(+)CD38(-) cells as starting material, we identified four mRNAs, expressed by these cells, that are either absent or present in reduced amounts in more mature CD34(+)CD38(+) cells. One of these cDNAs (C40) encodes a known member of the subfamily of protein phosphatases (CL100) that exhibits dual substrate specificity for phosphotyrosine- and phosphoserine/threonine-containing substrates and specifically inactivates MAP kinases. This phosphatase has been shown to play a role in regulating the differentiation of several cell types. The second cDNA (C23) is identical to LR11 (gp250), a member of the low-density lipoprotein receptor family. LR11 is unusual in that, in addition to 11 ligand-binding repeats, it contains a series of fibronectin type III repeats near its carboxyl terminal end that are similar to those found in cytokine receptors. It is highly expressed in developing brain, but hematopoietic expression has not been reported. The 178-bp fragment that we originally cloned is part of a 4,145-bp 3' untranslated region (UTR) that had not been previously sequenced and is among the largest human 3' UTRs ever reported. The other isolates (C21 and C12) do not correspond to known protein sequences. They are homologous to EST sequences from a fetal brain library. C21 encodes a previously unknown gene that is a member of the WD-40 family. An open reading frame encoding a 515 amino acid protein has been identified. Four mRNAs, differentially expressed by CD34(+)CD38(-) human bone marrow cells, have been identified. Although this population is highly enriched for early hematopoietic progenitors, none of these genes encodes a message whose expression is limited to the hematopoietic system. They all are expressed in a variety of tissues, suggesting that they are involved in processes that are fundamental to the development of many cell types. All of these cDNAs possess atypically long 3' UTRs, and one of them is among the longest ever described. Their differential expression by immature hematopoietic cells, in contrast to more mature cells, suggest that long 3' UTRs may be characteristic of genes that play a regulatory role during development.

Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6).

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively gamma-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be gamma-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells.

Expression of CD41 and c-mpl does not indicate commitment to the megakaryocyte lineage during haemopoietic development.

Haemopoietic progenitors with the phenotype expected of early megakaryocyte precursors (CD34+ CD41+) were isolated from normal human bone marrow or induced in culture from CD34+ CD41- bone marrow cells by treatment with thrombopoietin (TPO) or IL-3. We found that although this population included the majority of cells that can form CFU-MK in culture, it also contained both erythroid and myeloid progenitors. The clonogenic potential of the CD34+ CD41+-induced cells was greater than that of isolated CD34+ CD41+ cells in that the isolated cells only formed CFU-MK and BFU-e, whereas the induced cells formed myeloid colonies as well. Glycophorin was found on isolated CD34+ CD41+ cells, not on induced cells. Its presence distinguished between MK and erythroid progenitors. Separation of a CD34+ CD41+ glycophorin A+ population resulted in the isolation of a highly purified population of BFU-e. A major portion of the cells that expressed CD34+ CD41+, in either cohort, were of the erythroid lineage. True MK progenitors were present in the CD34+ population in greater proportion than in whole marrow and were further enriched amongst CD34+ populations that expressed CD41. The presence of the thrombopoietin (TPO) receptor, c-mpl, did not correlate with inducibility of the gpIIbIIIa complex since essentially all CD34+ progenitors, including the earliest identifiable human haemopoietic progenitors (CD34+ CD38- cells), expressed c-mpl mRNA detectable by PCR regardless of their ultimate fate. Thus neither the expression of CD41 nor the expression of c-mpl was predictive of commitment to the MK lineage.

Complementary and antagonistic effects of IL-3 in the early development of human megakaryocytes in culture.

The effect of IL-3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41+ MK from pre-MK, the number of cells produced was greatly augmented by the addition of IL-3 and SCF. IL-3 was also able to support the growth of MK colonies in semi-solid media (CFU-MK). The CD41+ cells that developed in suspension cultures containing IL-3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL-3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb-IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL-3. The inhibitory effects of IL-3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL-3 did not stimulate MK growth. The results obtained in these cultures suggest that IL-3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL-3 results in the appearance of cells that do not mature normally.

Growth of human hematopoietic cells in immunodeficient mice conditioned with cyclophosphamide and busulfan.

Human hematopoietic cells survive and proliferate for at least 10 weeks in severe combined immunodeficient mice prepared with the cytotoxic drugs busulfan and cyclophosphamide. The human cells growing in the mice can be detected by in situ hybridization using a probe detecting human repetitive DNA or by staining the cells with antihuman antibodies (anti-CD45 and anti-HLA I). Busulfan/cyclophosphamide-treated mice were injected with a wide range of cell doses, ranging from 5 to 50 million unfractionated bone marrow cells and 2 to 40 million low density bone marrow cells. Animals were killed at 1, 3, 5, 7 and 10 weeks after transplantation. Human cells were found in many animals and could be detected as early as one week after transplantation. The peak of repopulation was at two to five weeks, but in some animals human cells could be detected for as long as 10 weeks. Many of the human cells expressed high levels of glycophorin, but mature human erythrocytes were not found. The human cells were not uniformly distributed throughout the marrow. They grew in small clusters in the subepiphyseal region. The extent of human hematopoietic repopulation in the mouse was extremely variable. At no time and at no dose was repopulation achieved in all of the animals. Treatment with human growth factors is not necessary for the survival of the human hematopoietic cells but, in their absence, normal hematopoiesis does not occur.

The development of human megakaryocytes: III. Development of mature megakaryocytes from highly purified committed progenitors in synthetic culture media and inhibition of thrombopoietin-induced polyploidization by interleukin-3.

Megakaryocyte (MK) progenitors, CD34+CD41+ cells, were isolated from human bone marrow with a purity greater than 98% and a viability of 95%, using affinity techniques with magnetic beads followed by fluorescence-activated cell sorting. These cells were incubated in synthetic media containing the cytokines thrombopoietin (TPO), interleukin-3 (IL-3), stem cell factor (SCF), and IL-6, obviating the confounding effects of serum growth factors or cytokine secretions of non-MK cells on MK maturation. MK number, MK colony-forming units (CFU-MK), and MK ploidy and phenotype were examined during 7 days in culture. TPO in serum-free cultures without any other exogenously added cytokine supported MK growth and maturation. SCF synergized with TPO to augment MK production and maturation and could partially replace it under some conditions. Both TPO and IL-3 alone increased MK number (12- and 5-fold, respectively) and CFU-MK (approximately 15-fold each). SCF alone had no effect on MK proliferation in the absence of TPO, but increased both MK number and CFU-MK by 1.5- to 2.0-fold in the presence of TPO. When combined with IL-3, SCF increased both MK number and CFU-MK by 15- to 20-fold in the absence of TPO. In the presence of TPO, the combination of IL-3 and SCF produced only modest increases (1.5- to 2.0-fold) in both MK number and CFU-MK. The proportion of polyploid MK increased greater than fivefold in the presence of TPO. SCF had little effect on MK ploidy in the presence of TPO, but enhanced ploidy twofold to threefold in the absence of TPO. IL-3 alone never increased the level of polyploidization. Rather, it consistently inhibited TPO- and SCF-induced polyploidization of MK. This inhibition was observed in cultures with or without SCF or IL-6. Although IL-3 also supported the proliferation of CD41+ cells and CFU-MK production, the cells that developed under the influence of IL-3 were phenotypically unusual (CD41dim, CD42dim) and of relatively low ploidy. Mature MK were not produced. When added with TPO, IL-3 suppressed polyploidization. Therefore, TPO stimulates MK growth and maturation, whereas IL-3 stimulates growth without maturation and may serve to conserve the immature MK compartment.

The development of human megakaryocytes. II. CD4 expression occurs during haemopoietic differentiation and is an early step in megakaryocyte maturation.

CD4 expression is not limited to T cells and monocytes. In both mouse and man the antigen has been detected on some early haemopoietic progenitors and we have shown that some mature megakaryocytes (MK) express CD4, the surface molecule that serves as the high-affinity receptor for human immunodeficiency virus type-1 (HIV-1). Using a serum-free culture system in which sorted CD34+ haemopoietic progenitors are cultured with thrombopoietin (TPO), IL-3, IL-6 and SCF, we now show that CD4 expression is induced in virtually all developing haemopoietic cells. This phenomenon was particularly striking in the MK lineage, where CD4 expression began whilst the cells were still CD34+ but after they expressed CD41 (GPIIb/IIIa). CD4 expression and endomitotic polyploidization occur at the same time in MK development. In culture, maximum CD4 expression occurred 4-6 d after CD41 expression and lasted for a few days. Expression of CD4 declined gradually thereafter and most MK were CD4- by the end of the culture period. The amount of CD4 on the surface of some MK, as measured by intensity of fluorescence staining, exceeded that of normal monocytes and approached the brightness of T cells. Appearance of the surface antigen correlated with the presence of mRNA for CD4, as measured by RT-PCR.

Evaluation of presence and functional activity of potentially self-reactive T cells in aged mice.

Autoimmunity is known to increase in aging. A possible factor could be an alteration in the T cell repertoire with advancing age. Antibodies to the variable region of the beta chain of the TCR activate T cells and can serve as probes for analysis of the T cell repertoire. We have used V beta 3 and V beta 17a antibodies to determine the presence and functionality of normally deleted T cells bearing potentially self-reactive TCR in peripheral lymphoid tissue and blood from aged (SJL/J x BALB/c) F1, LAF1 and BALB/c mice. Although an occasional 20- to 24-month-old mouse exhibited V beta 3+ or V beta 17a+ T cells in their lymph nodes or peripheral blood lymphocytes (PBL) slightly above the range for normal young mice of these I-E+ strains, there was no striking 'escape' from the normal thymic deletion process. However, responsiveness to anti-V beta 3 and anti-V beta 17a was slightly higher in aged, and particularly in aged thymectomized (TX), than in young mice. This was in contrast to proliferative responses to stimulation with antibody to the normally expressed V beta 8, which were lower in the lymph nodes from aged than from young mice. The PBL of some 30- to 36-month-old mice were also examined. Enhanced numbers of 'forbidden' V beta bearing T cells were seen more frequently at this age. In spite of the age-related decrease in overall CD4/CD8 T cell ratios in all organs, the mice with relatively high V beta 17a + T cells exhibited proportionally more CD4+ cells in that V beta population. We conclude that the 'forbidden' T cells that respond to anti-V beta stimulation in the 20- to 24-month-old mice are most likely to extra-thymic origin, since they were more readily detectable in aged TX mice. Potentially self-reactive Cd4 (and CD8) single-positive T cells were detectable in PBL only in very aged (30-36 months old) euthymic mice.

Effects of fibroblast growth factor-4 (k-FGF) on long-term cultures of human bone marrow cells.

Fibroblast growth factor-4 (FGF-4), a highly mitogenic protein encoded by the k-fgf/hst oncogene, stimulates the growth of a variety of cells of mesenchymal and neuroectodermal origin. Addition of FGF-4 to human long-term bone marrow cultures increased both the cell density of the stromal layer and the number of hematopoietic colony forming cells in the cultures in a dose-dependent manner. Hematopoiesis in the stromal layer persisted for up to 8 months. Erythropoiesis was maintained for up to 4 weeks, but granulocytes were the predominant nonadherent cell type. Cultures treated with FGF had increased numbers of monocytes compared with control cultures and some CD14+, CD45+ monocytes could still be detected after 8 months of continuous culture. The addition of the growth factor increased the rate of growth of the stromal layer and appeared to delay its senescence. Subcultures made in the presence of FGF-4 had up to 10-fold increases in plating efficiency and grew as relatively uniform monolayers. These subcultures retained the capacity to support hematopoiesis for several months, while untreated subcultures, made without FGF-4, grew erratically and generally lost the capacity to support hematopoiesis within 4 to 6 weeks. The improved growth after subculture greatly enhanced the reliability of limit-dilution assays of multipotential hematopoietic stem cells that use stromal cell monolayers. The primary effect of FGF-4 appeared to be on the stromal cells of the long-term bone marrow cultures, but a direct effect on hematopoietic progenitors could not be ruled out.

Development of human megakaryocytes: I. Hematopoietic progenitors (CD34+ bone marrow cells) are enriched with megakaryocytes expressing CD4.

CD34 is expressed by essentially all human hematopoietic progenitors including cells of the megakaryocyte (MK) lineage. We have previously reported CD4 expression by some human MK (Blood 81:2,664, 1993). To study the role of maturation on CD4 expression by MK, we examined CD34+ bone marrow cells for their expression of CD41 (GPIIb-GPIIIa) and CD4 with specific monoclonal antibody (MoAb)-fluorochrome conjugates and for DNA polyploidization with propidium iodide or 7-aminoactinomycin D (7-AAD). Surprisingly, MK were at least 20-fold more common in the CD34+ progenitor pool (approximately 10%) than in the more mature CD34+ population (approximately 0.5%) of low density bone marrow cells. CD4 expression correlated with markers of immaturity in that CD4 was enriched among CD34+ cells, and the proportion of CD4+ MK declined with increasing ploidy. Almost all CD34+ polyploid ( > or = 8N) cells were CD4+. Despite these correlations with immaturity, CD34+CD4+ MK precursors were unable to produce MK colony-forming units (CFU-MK) when cultured under conditions that supported the growth of CFU-MK from CD34+CD4- MK lineage cells. MK became polyploid before the loss of either CD34 or CD4 expression. The presence of CD4 on these cells correlates with the onset of endomitotic reduplication and is associated with the loss of the ability of these cells to undergo normal mitotic division. The role of CD4 on immature MK as a differentiation antigen and/or receptor for the human immunodeficiency virus (HIV)-1 virus remains to be determined.

Flow cytometric determination of apoptosis in heterogeneous cell populations.

We have shown that apoptosis and surface antigen expression can be detected simultaneously using multicolor flow cytometry. Apoptosis was measured with an in situ assay that makes use of the ability of the enzyme terminal deoxyribonucleotidyl transferase to catalyze the addition of biotinylated nucleotides to the free 3'-OH groups produced during endonucleolytic cleavage of DNA. The incorporation of the biotinylated nucleotides was quantified using fluorochrome-coupled streptavidin. Immunofluorescence was used to identify the phenotype of apoptotic cells using three color flow cytometry. In model systems, apoptosis was detected in human PBMNC treated with the DNA topoisomerase I inhibitor CAM and in murine thymocytes treated with DEX. The simultaneous application of this method for detecting apoptosis and immunofluorescence offers several advantages. (1) Apoptotic and necrotic cells can be distinguished. (2) The phenotype of the cells undergoing apoptosis can be determined in heterogeneous systems. Thus, we could show that T cells are relatively resistant to CAM-induced apoptosis compared to other peripheral blood mononuclear cells and that both double negative and single positive thymocytes are resistant to steroid-induced apoptosis. (3) The flow cytometric TdT assay to detect apoptosis-associated DNA degradation method is extremely sensitive compared with gel electrophoresis. As few as 2-5 x 10(3) cells give an adequate signal and it is possible to detect DNA strand breaks even when only a small proportion of the cells are undergoing apoptosis. Neither the sensitivity nor specificity of this method can be matched by any electrophoretic method of detecting apoptosis.

Early thymic regeneration after irradiation.

Whole body irradiation produces profound thymic atrophy. After sublethal irradiation, regeneration begins promptly and the earliest regeneration is from radioresistant intrathymic precursors. The progeny of these precursors expand rapidly and restore thymic cellularity to near normal within 2 weeks. We have used monoclonal antibodies specific for a variety of differentiation markers of the T lineage to analyze the early events in thymic regeneration. A three-color flow microfluorometric analysis revealed that the majority of the cells found early in the regenerative process have the phenotype of mature T cells. These include CD4-/CD8-; CD3hi as well as CD4+/CD8-; CD3hi and Cd4-/CD8+; CD3hi. The proportion of cells with mature phenotypes declines rapidly between day 6 and day 12. Not all of the early appearing cells have mature phenotypes. Among the early cells that do not express CD3 are both CD4 and CD8 single positive cells that express HSA and resemble the intrathymic precursors found in other systems. In these mice CD4 single positive predominate. There are other cells that are HSA positive but express low levels of CD4 and very low levels of Thy-1. These appear to include the earliest members of the T-lineage. In addition to relatively mature conventional T cells and early progenitors, the early developing population includes cells that express markers of the T-cell lineage including the T-cell receptor but do not express Thy-1. These Thy-1 negative T cells comprise a significant number of the earliest cells found after regeneration.

Human megakaryocytes have a CD4 molecule capable of binding human immunodeficiency virus-1.

Most human megakaryocytes (MGKs) express the CD4 antigen on their surface. Approximately 25% have a CD4 receptor density comparable to that of CD4+ T cells (Basch et al, Proc Natl Acad Sci USA 87:8085, 1990). In these studies, we show: (1) the presence of mRNA for CD4 in human MGKs; (2) the binding of human immunodeficiency virus-1 (HIV-1) to human MGKs; (3) the inhibition of binding by anti-CD4 (Leu3a) antibody or rCD4; (4) the infection of a human MGK line, CHRF-288 with HIV-1; and (5) inhibition of infection with anti-CD4. Human MGKs have mRNA for CD4 as shown by in situ hybridization with an RNA probe synthesized from a 3-kb cDNA sequence of plasmid pSP65.T4.8 containing the full-length CD4 sequence. MGKs (23% +/- 17%) bound HIV-1, as determined by anti-gp120 and anti-CD41 staining. Binding to human MGKs could be inhibited 55% to 75% with anti-CD4 or rCD4, respectively. Infection of a CD4+ MGK line (CHRF-288) could be accomplished with HIV-1, as determined by proviral DNA polymerase chain reaction and p24 production. Preincubation with anti-CD4 inhibited apparent proviral DNA infection by 100% and p24 production by 65% to 70%. Thus, human MGKs have a CD4 receptor capable of binding HIV-1. Using this receptor, HIV-1 can infect cells representative of the MGK lineage.

B-cell subsets and platelet counts in HIV-1 seropositive subjects.

A subset of B lymphocytes positive for the CD5 antigen have been implicated in several autoimmune disorders. To investigate their role in human immunodeficiency virus type 1 (HIV-1) infection, we studied peripheral-blood B and T lymphocytes from HIV-1-positive patients with (n = 13) and without (n = 18) thrombocytopenia, 8 patients with classic autoimmune thrombocytopenia, and 16 healthy controls. The proportion of CD5-positive B cells was significantly higher in the HIV-1-positive thrombocytopenic patients than in the healthy controls, as a result of both higher numbers of CD5-positive B cells and lower numbers of CD5-negative B cells. Platelet count was positively correlated with CD5-negative B-cell count (r = 0.6, p less than 0.001) and negatively correlated with proportion of B cells that were CD5 positive (r = -0.5, p less than 0.01) among the HIV-1-positive patients. The high concentrations of IgM-containing immune complexes in HIV-1-positive patients with autoimmune disorders may be due to changes in the CD5-positive B-cell subset.

T560: an (H-2b x H-2a) F1 hybrid, phosphorylcholine (PC)-binding, murine B cell lymphoma that bears receptors for IgA and IgG, presents antigen and secretes IL-4.

We describe T560, a tissue culture-adapted B lymphoma derived from the gut-associated lymphoid tissue (GALT) of a (B10 x B10.H-2a H-4b)F1 hybrid mouse. This lymphoma is interesting and useful not only because it bears an unusual IgA receptor, fully described elsewhere, but also because it is potentially capable of presenting antigen to T cells restricted by the MHC of either parent. Here we document that T560 cells are IgG2a kappa +, Ia+, B220+, J11d.2+, CD3-, CD4-, CD5-, Mac 1-, Mac 2-, non-specific esterase-. They bind bromelain-treated mouse RBC (BrMRBC) in a PC chloride-inhibitable manner but do not bind SRBC, ox RBC (ORBC) or TNP-ORBC. Two lines, T560.1 and T560.2, and several clones are available. T560.1 and its clones contain low numbers of IgA rosette-forming cells (RFC), intermediate numbers of IgG2a RFC and moderately high numbers of IgG2b RFC; T560.2 and its clones contain moderately high numbers of IgA RFC and low numbers of both IgG2a and IgG2b RFC. Both lines stimulate both B10 and B10.A cells in mixed lymphocyte reactions (MLR) and present keyhole limpet hemocyanin (KLH) to KLH-reactive T cells. T560.2 populations are, however, more efficient possibly because they have somewhat higher proportions of brightly fluorescent Ia+ cells and secrete larger quantities of lymphokine than T560.1 cells. They present PC-conjugated KLH (PC-KLH) approximately 20 times more efficiently than unconjugated KLH, suggesting that their PC binding receptors function in antigen uptake. They constitutively produce IL-1, IL-4 and IL-6, but not IL-2, IL-5 or TGF beta. Neither their IgA nor their IgG receptor expression is affected by IL-4 or by IFNs-alpha, -beta, or -gamma. In their ability to bind BrMRBC and secrete IL-4, they resemble the CH12 lymphoma but differ from it in that they are of F1 hybrid origin, are CD5-, bear IgG2a rather than IgM, do not bind sheep erythrocytes and have a receptor for IgA not present on CH12.

Separation of self-renewing hematopoietic progenitors on the basis of CD45 (T-200) antigen expression.

Using monoclonal antibodies recognizing the shared determinants of the CD45 (T-200) antigen we have been able to distinguish spleen colony-forming units (CFU-s) whose progeny are self-renewing from other colony formers on the basis of their quantitative expression of this antigen(s). We have also been able to identify a population of cells that is capable of producing 12-day colonies but has only a limited capacity to produce 8-day colonies. CFU-s8 are found primarily in the dim T-200 population, whereas CFU-s12 were found in both the bright and dim population, but the cells within the colonies produced by these two populations differ in their capacity for self-renewal as CFU-s. Only the colonies dissected after 12 days from the spleens of mice receiving T-200-bright bone marrow cells contained significant numbers of cells that were capable of forming colonies after retransplantation. We calculate the frequency of these cells in total bone marrow to be approximately 1 in 5000.

Thymic stromal cells in culture. 2. Binding of normal thymocytes to a cloned thymic stromal cell line.

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3-/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23,gp45 complex expressed on murine stromal cells.