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PubMed2002

Homotypic cell-cell adhesion induced by human T cell leukemia virus type 1 tax protein in T cell lines.

Cell-cell adhesion is involved in the processes of cell growth, activation and migration, and inflammation. T cells infected with human T cell leukemia virus type 1 (HTLV-1) exhibit a high degree of homotypic cell-cell adhesion in vitro. In this study, we investigated the involvement of the viral protein Tax in such process. Expression of Tax in an interleukin (IL)-2-dependent mouse T cell line (CTLL-2) increased homotypic cell-cell adhesion; however, less cell adhesion was induced by Tax than that observed in HTLV-1-infected T cell lines. Moreover, Tax induced cell-cell adhesion in a human T cell line, in which the expression of Tax is inducible. Microscopic examination also revealed Tax-induced morphologic changes, including rounding of CTLL-2 cells, increased cell volume, and increased nucleus size. Taken together, our results suggest that Tax induces cell-cell adhesion and morphologic changes in HTLV-1-infected cells. Tax may thus play a role in persistent HTLV-1 infection and the pathogenesis of associated disease.

Animals↗
PubMedDate not supplied

Alkylator resistance in human B lymphoid cell lines: (1). Melphalan accumulation, cytotoxicity, interstrand-DNA-crosslinks, cell cycle analysis, and glutathione content in the melphalan-sensitive B-lymphocytic cell line (WIL2) and in the melphalan-resistant B-CLL cell line (WSU-CLL).

Two human B lymphoid cell lines WIL2 (melphalan sensitive. ***IC50:8.57 +/- 1.08 mM) and WSU-CLL (melphalan resistant, ***IC50:223.18 +/- 6.45 mM) were used as models to study alkylator resistance in human lymphoid cells. Melphalan transport studies demonstrated decreased initial melphalan accumulation in WSU-CLL cells as compared to WIL2 cells. Lineweaver-Burk plots of the rate of initial melphalan uptake showed an approximately 3.5-fold decrease of Vmax in WSU-CLL cells as compared to WIL2 cells. Melphalan transport was inhibited by 2-amino-bicyclo[2,2,1] heptane-2-carboxylic acid(BCH) in both cell lines, indicating that the amino acid transport (System L, which is sodium independent and inhibited by BCH) is functional in these two cell lines. Only a minor degree of inhibition of melphalan transport was noted after sodium depletion (System ASC, which is sodium dependent and unaffected by BCH). Interstrand-DNA-cross-link formation showed a highly significant correlation with in-vitro cytotoxicity in both two cell lines. However, the melphalan concentration at which such interstrand DNA cross-linking occurred differed significantly when WIL2 cells and WSU-CLL cells were compared. The kinetics of interstrand-DNA-cross-link formation and removal following treatment with melphalan also differed significantly, with WSU-CLL cells, showing a much more rapid rate of removal of interstand DNA cross-links as compared to WIL2 cells. Cell cycle analysis showed that melphalan treatment resulted in the progressive arrest of the WSU cells in G1 and G2 phases. But WIL2 cells failed to enter G1 or G2 arrest after melphalan treatment, suggesting an increased rate of DNA repair occurring in melphalan-resistant WSU-CLL cells. There was no significant difference between the two cell lines in regard to either glutathione content or glutathione-S transferase activity. These findings indicate that multiple factors are associated with alkylator resistance in lymphoid cells including alteration of uptake, DNA repair and cell cycle progression. However no evidence for alteration in glutathione content and glutathione-S-transferase activity was found.

Antineoplastic Agents, Alkylating↗
PubMed1993

T cell-dependent differentiation of human B cells into IgM, IgG, IgA, or IgE plasma cells: high rate of antibody production by IgE plasma cells, but limited clonal expansion of IgE precursors.

The development of human functional Ig precursors into plasma cells expressing IgM, IgG, IgA, or IgE was compared. Purified human B cells were stimulated at limiting dilution with irradiated EL4 helper cells, IL-2, and IL-4. B cells proliferated exponentially until Day 8 of culture. Nondividing plasma cells of all isotypes were detectable in ELISPOT assays between Days 8 and 10 and secreted 1.8 +/- 0.7 ng antibody per cell within 24 hr. This indicates that plasma cells of all isotypes, including IgE, bear a comparable potential to secrete antibody. It further shows that Ig switching does not delay the development into IgE plasma cells, despite that switching from IgM to IgE in vitro required 6 days of IL-4 action. The proliferation and Ig production by B cells readily declined after Days 8 and 10, respectively, and could not be prolonged by restimulating B cells with fresh helper cells and lymphokines in secondary cultures. This indicates that B cells have developed into nondividing, high rate Ig-secreting plasma cells within 9 days, and that they do not differentiate any further under the applied conditions. In contrast to IgM, IgG, and IgA committed B cells, IgE switched cells did not undergo clonal expansion, since the numbers of functional IgE precursors corresponded to the maximal numbers of IgE-secreting plasma cells, whereas the numbers of IgM-, IgG-, or IgA-secreting cells exceeded the number of functional precursors 15-fold. The results demonstrate that human B cells of all isotypes, including IgE, have the potential to secrete antibody at a comparably high rate, and that the IL-4-induced switch process does not delay the differentiation into plasma cells.

Animals↗
PubMed2003

Interaction of cutaneous lymphoma cells with reactive T cells and dendritic cells: implications for dendritic cell-based immunotherapy.

BACKGROUND: Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of skin neoplasms that originate from T lymphocytes. An anti-CTCL T-cell immunity has been described but seems to be inefficient to clear CTCL cells. It is not known whether cutaneous dendritic cells (DCs) perpetuate the proliferation of the malignant CTCL cell clone or play a role in the control of this usually slowly progressing disease. OBJECTIVES: To characterize CTCL cell properties in the control of anti-CTCL T cells and to pave the way for a DC-based immunotherapy for CTCL. METHODS: We studied the interaction of a CTCL cell line with DCs and with allogeneic T cells. RESULTS: We found an antigen non-specific capacity of viable but not apoptotic CTCL cells to hamper CD4+ and CD8+ T-cell proliferation in a dose-dependent manner, indicating a suppressive potential of CTCL cells. Both viable and apoptotic CTCL cells were phagocytosed by immature DCs but only apoptotic CTCL cells induced an upregulation of DC maturation markers to a degree which enabled classification of these DCs as semimature. CTCL cells did not respond with proliferation when encountering allogeneic, mature DCs either loaded with CTCL cell material or unloaded, indicating a role for DCs in the induction of anti-CTCL T-cell immunity rather than in perturbation of clonal proliferation. For the loading of DCs with CTCL material lysate seems to be optimal as apoptotic cells were not phagocytosed extensively and necrotic CTCL material induced a partial cellular toxicity in DCs. DCs loaded with CTCL material were cryopreservable without significant loss of DC viability, surface marker expression or allostimulatory activity. CONCLUSIONS: Together, these data argue in favour for a DC-based immunotherapy for CTCL patients and provide an experimental protocol for preparing CTCL cell-loaded DCs.

Apoptosis↗
PubMed2007

Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells.

OBJECTIVE: Human embryonic stem cells (hESCs) have been shown to generate CD34(+) primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73(+) multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34(+) hematopoietic cells and CD73(+) mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73(+) cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34(+) cells are generated. MATERIALS AND METHODS: We cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34(+) and CD73(+) cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34(+) cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73(+) cells generated using in vitro differentiation assays. RESULTS: We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34(+) hematopoietic and CD73(+) MSC generation follows a similar pattern. We sorted the CD34(+) cells and showed that they can generate hematopoietic progenitor colonies. Starting with cocultured cells on day 8, and through an enrichment procedure, we also could generate a pure population of MSCs. These hESC-derived MSCs had typical morphological and cell surface marker characteristics of adult bone marrow-derived MSCs, and could be differentiated toward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs. CONCLUSIONS: OP9 cells when cocultured with hESCs support simultaneous generation of CD34(+) primitive hematopoietic cells and CD73(+) MSCs from hESCs.

5'-Nucleotidase↗
PubMed1995

Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-to-cell interactions and colony-stimulating factors.

Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353-fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).

Animals↗
PubMed1980

Role of accessory cells in B cell activation. III. Cellular analysis of primary immune response deficits in CBA/N mice: presence of an accessory cell-B cell interaction defect.

The effect of the X-linked CBA/N genetic defect on the ability of mice to generate primary responses to thymic-dependent and thymic-independent antigens was assessed by comparing the ability of abnormal (CBA/N x DBA/2)F1 male mice and normal (DBA/2 x CBA/N)F1 male mice to generate 2,4,6-trinitrophenyl (TNP)-specific plaque-forming cell responses to TNP-keyhole limpet hemocyanin (KLH), TNP-conjugated Ficoll (TNP-Ficoll), TNP-Brucella abortus (BA), and TNP-lipopolysaccharide (LPS). The reciprocal F1 combinations used in this study differ genetically only in the origin of their X chromosome, but differ immunologically in that (CBA/N x DBA/2)F1 male mice express all the CBA/N immune abnormalities, whereas (DBA/2 x CBA/N)F1 male mice are immunologically normal. Analysis of thymic-dependent responses to TNP-KLH revealed that abnormal F1 mice were capable of generating primary responses in vivo to high doses of TNP-KLH, but failed to generate responses to suboptimal doses of TNP-KLH that were still immunogenic for normal F1 mice. Furthermore, under limiting in vitro micro-culture conditions, the abnormal F1 mice failed to generate primary thymic-dependent responses to any dose of TNP-KLH, even though under the identical conditions normal F1 mice consistently responded to a wide antigen dose range. The cellular basis of the failure of abnormal F1 mice to respond in vitro to TNP-KLH was investigated by assaying the ability of purified populations of accessory cells, T cells, and B cells from these mice to function in responses to TNP-KLH. The results of these experiments demonstrated that helper T cells and antigen-presenting accessory cells from abnormal F1 mice were competent and functioned as well as the equivalent cell populations from normal F1 mice. Instead, the failure of CBA/N mice to generate primary in vitro responses to TNP-KLH was solely the result of a defect in their B cell population such that B cells from these mice failed to be triggered by competent helper T cells and/or competent accessory cells. Similarly, the failure of abnormal F1 mice to respond either in vivo or in vitro to TNP-Ficoll was not the result of defective accessory cell presentation of TNP-Ficoll, but was the result of the failure of B cells from these mice to be activated by competent TNP-Ficoll-presenting accessory cells. In contrast to the failure of B cells from abnormal F1 mice to be activated in vitro in response to either TNP-KLH or TNP-Ficoll, B cells from abnormal F1 mice were triggered to respond to TNP-BA and TNP-LPS, antigens that did not require accessory cell presentation. The specific failure of B cells fron abnormal F1 mice to be activated in responses that required antigen-presentation by accessory cells suggested the possibility that the X-linked CBA/N genetic defect resulted in B cell populations that might be deficient in their ability to interact with antigen-presenting accessory cells...

Animals↗
PubMed1981

Role of the major histocompatibility complex in T cell activation of B cell subpopulations. Major histocompatibility complex-restricted and -unrestricted B cell responses are mediated by distinct B cell subpopulations.

The present study has evaluated the identity of the B cell subpopulations participating in T dependent antibody responses that differ in their requirements for major histocompatibility complex-restricted T cell recognition. In vitro responses of keyhole limpet hemocyanin (KLH)-primed T cells and trinitrophenyl (TNP)-primed B cells were studied to both low and high concentrations of the antigen TNP-KLH. It was first demonstrated that for responses to low concentrations of TNP-KLH, (A x B)F(1) {arrow} parent(A) chimeric helper T cells were restricted in their ability to recognize parent(A) but not parent(B) H-2 determinants expressed by both B cells and antigen-presenting cells (APC). In contrast, at higher antigen concentrations, helper T cells were not restricted in their interaction with B cells. It was then determined whether these observed differences in T cell recognition resulted from the activation of distinct B cell subpopulations with different activation requirements. At low concentrations of TNP-KLH it was demonstrated that Lyb-5(-) B cells were activated, and that it was thus the activation of the Lyb-5(-) subpopulation that required T cell recognition of B cell H-2 under these conditions. In contrast, responses to high concentration of antigen required the participation of Lyb-5(+) B cells, and these Lyb-5(+) B cells were activated by a pathway that required H-2- restricted T cell interaction with APC, but not with B cells. The findings presented here have demonstrated that Lyb-5(-) and Lyb-5(+) B cells constitute B cell subpopulations that differ significantly in their activation requirements for T cell-dependent antibody responses to TNP-KLH. In so doing, these findings have established that the function of genetic restrictions in immune response regulation is critically dependent upon the activation pathways employed by functionally distinct subpopulations of B, as well as T, lymphocytes.

Animals↗
PubMed1994

T cell-independent and T cell-dependent B cell activation increases IFN-gamma R expression and renders B cells sensitive to IFN-gamma-mediated inhibition.

We have studied the relationship between B cell activation and the ability of IFN-gamma to inhibit B cell differentiation. The LPS activation of conventional and CD5+ B cells resulted in increased IFN-gamma R expression and increased the ability of IFN-gamma to inhibit LPS-induced B cell differentiation correlated with increased IFN-gamma R expression. We detected increased B cell IFN-gamma R expression 12 h after activation, and maximal IFN-gamma R expression was observed at 24 h. Activation of B cells by F(ab2)' anti-IgM induced a similar increase in IFN-gamma R expression. In autoimmune New Zealand Black mice, both conventional and CD5+ B cells showed a pattern of IFN-gamma R expression similar to that seen in DBA/2 mice, and both populations of B cells were sensitive to inhibition by IFN-gamma. To examine the role of IFN-gamma in the regulation of T cell-dependent B cell responses, we activated B cells with the CDC35 T cell line (which is specific for rabbit IgG). When rabbit anti-mouse Ig-treated B cells were activated by CDC35 T cells, we found that B cells exhibited increased IFN-gamma R expression by 48 h; we also found that IFN-gamma inhibited CDC35-mediated IgM secretion to a degree similar to IFN-gamma inhibition of T cell-independent B cell differentiation. Additionally, IFN-gamma inhibited CDC35-stimulated B cells even in the presence of exogenous IL-4 and IL-5. This study establishes the importance of IFN-gamma as a regulator of both T cell-independent and T cell-dependent B cell differentiation.

Animals↗
PubMed1987

Immunohistochemical study of epidermal Langerhans cells and dermal dendritic cells in benign and malignant skin lesions characterized by a dermal lymphoid infiltrate consisting either of B-cells or T-cells.

Skin biopsies from 43 patients with a rather dense dermal lymphoid infiltrate of either inflammatory or neoplastic nature have been investigated. We studied the number, distribution and immunophenotype of epidermal Langerhans cells and dermal dendritic cells. As previously reported, differences in epidermal Langerhans cell and dermal dendritic cell numbers between skin biopsies with a B-cell infiltrate and skin biopsies with a T-cell infiltrate were found, dendritic cells being more numerous in the latter. The main finding of this study was an uneven distribution of epidermal Langerhans cells and dermal dendritic cells in skin biopsies with a T-cell infiltrate: in skin lesions with an inflammatory lymphoid infiltrate, small clusters of epidermal and dermal dendritic cells admixed with T-lymphocytes (predominantly T-helper/inducer cells) and small blood vessels were present at areas of exocytosis. In skin lesions with a neoplastic lymphoid infiltrate larger, more loosely arranged aggregates of dendritic cells and T-cells were seen. These cell aggregations composed of activated (inflammatory or neoplastic) T-cells and dendritic cells may represent the cutaneous homologue of the secondary T-nodule in the lymph node. Both types of cell aggregates may correspond to the dendritic cell-T cell clusters observed in in vitro induced immune responses.

B-Lymphocytes↗
PubMed1989

The role of dendritic cells as stimulators of minor lymphocyte-stimulating locus-specific T cell responses in the mouse. I. Differential capacity of dendritic cells to stimulate minor lymphocyte-stimulating locus-reactive T cell hybridomas and the primary anti-minor lymphocyte-stimulating locus mixed lymphocyte reaction.

The response of T cells to minor lymphocyte-stimulating locus (Mls) determinants remains poorly understood with respect to the antigenic determinants responsible for T cell stimulation and the types of APC capable of stimulating the response. In this report, we demonstrate that highly purified dendritic cells (DC) as well as B cells have the capacity to stimulate Mls-specific responses. Unseparated spleen cells, purified DC, resting B cells, and activated B cells were compared for their capacity to stimulate several Mls-reactive T cell hybridomas. Whereas the entire panel of Mls-reactive T cell hybridomas was stimulated strongly by unseparated spleen cells and activated B cells, the hybridomas responded only weakly to purified DC or resting B cells. Activation of resting B cells with either B cell stimulatory factor-1 (1 day pre-treatment) or LPS/dextran (2 or 3 day pre-treatment) greatly augmented their Mls-stimulatory capacity. In contrast, the Mls-stimulatory capacity of DC was not augmented by a 1-day pre-treatment with either B cell stimulatory factor-1 or supernatant from the DC-induced primary anti-Mls-MLR. In the primary anti-Mls-MLR, both purified DC and LPS/dextran-stimulated B blasts were found to elicit vigorous T cell proliferative responses. Much weaker responses were elicited by unseparated spleen cells. The stimulation of the primary anti-Mls-MLR by purified DC was further confirmed by producing Mls-specific T cell clones which were preferentially stimulated by DC. Autologous (Mlsb) DC were found to markedly enhance the primary anti-Mls-MLR response to small numbers of Mlsa B blasts. Thus, DC possess other "accessory cell" properties that augment the primary anti-Mls-MLR despite the predicted low level of Mls determinant expression on DC based on the results obtained with Mls-reactive hybridomas. Possible accessory cell properties of DC relevant to this phenomenon are discussed.

Animals↗
PubMed1984

Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines.

A glycoprotein complex of 210,000 and 130,000 m.w., found on mitogen or alloantigen-stimulated human T cells and not on other hematopoietic cells, has been defined by a monoclonal antibody (Mab). The components of this complex are a subset of a larger family of proteins (210,000, 165,000 and 130,000 m.w.) defined by a second Mab. In a panel of hematopoietic cell lines and cell types, only activated T cells (including the cell line HUT-102) express the 210,000/130,000 complex and these cells also express the IL 2 receptor, a characteristic marker for activated T cells. The 210,000/130,000 m.w. complex (reactive with the Mab TS2/7) is present on all long-term activated T cells, including both the OKT4 and OKT8 subsets. The 210,000 m.w. subunit is expressed only on activated T cells. Other lymphoid cells express either the 130,000 m.w. subunit alone (unactivated lymphocytes, thymocytes, HUT-78) or the 130,000 subunit together with a 165,000 subunit (MOLT-4, HSB, and other leukemic T cell lines). The 210,000/130,000 m.w., 165,000/130,000 m.w. and 130,000 m.w. complexes are antigenically related in that all share reactivity with the Mab A- 1A5 . Among non-lymphoid hematopoietic cells and cell lines, none express the 210,000 m.w. chain; adherent cells (monocytes) and myeloid cell lines each express single proteins of 130,000 to 155,000 m.w. Granulocytes and red blood cells are negative and platelets express multiple bands (165,000 and 140,000 m.w.). Immunoperoxidase staining of tissue sections showed that a broad range of tissues and cell types had material cross-reactive with the lymphoid 130,000 m.w. protein. However, only a discrete subset of those tissues and cells including blood vessel walls, connective tissue, smooth muscle, kidney mesangial cells, and some non-cellular matrix tissue, had material cross-reactive with the 210,000 m.w. protein on activated T lymphocytes.

Animals↗
PubMed1997

Class I-deficient hemopoietic cells and nonhemopoietic cells dominantly induce unresponsiveness of natural killer cells to class I-deficient bone marrow cell grafts.

NK cells in normal mice reject bone marrow transplants from class I-deficient mice. In contrast, class I-deficient mice do not reject autologous cells, suggesting that NK cell tolerance is acquired. We employed fetal liver irradiation chimeras to assess two potential mechanisms that could account for the tolerance of NK cells in class I-deficient mice to class I-deficient cells: 1) a positive model, in which recognition of class I+ cells molecules by NK cells is necessary to induce functional NK cell maturation; and 2) a negative model, in which interactions of NK cells with class I-deficient cells induce tolerance. In class I+ chimeras reconstituted with mixtures of class I+ and class I-deficient fetal liver cells, the rejection of class I-deficient bone marrow cell grafts was significantly impaired, supporting the negative model. We further addressed whether nonhemopoietic cells are also able to induce NK cell tolerance. Class I- mice reconstituted with class I+ fetal liver cells were tolerant of class I-deficient cells, favoring this idea. Furthermore, class I-deficient mice reconstituted with a mixture of class I-deficient and class I+ fetal liver cells were more tolerant to class I-deficient cells than were class I+ mice reconstituted with the same fetal liver cell mixture. These results suggest that maximal tolerance induction requires the presence of class I-deficient nonhemopoietic cells.

Animals↗
PubMed1986

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells.

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2Kk. In the absence of exogenous IL-2 the clones require the presence of Ia+, Thy-1- accessory cells and of Thy-1+, Lyt-1+2- cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A alpha A beta and/or E alpha E beta had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2+ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E alpha E beta when B10.A(4R) spleen cells, which do not express E alpha E beta, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R] F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2Kd). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A alpha A beta and E alpha E beta molecules with syngeneic accessory cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34+ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures.

Porcine microvascular endothelial cells (PMVECs) plus cytokines support a rapid proliferation and expansion of human CD34+CD38- cells that are capable of multilineage engraftment within the bone marrow of a secondary host. CD34+CD38- cells contain the self-renewing, long-term culture-initiating cells (LTC-IC) that are ideal targets for retroviral gene transfer experiments. Previous experiments attempting retroviral infection of CD34+CD38- cells have failed partly because these cells do not enter cell cycle in response to cytokine combinations. In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34+ cells and the CD34+CD38- subset before and after ex vivo expansion on PMVECs. Purified human CD34+ cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF) + Flt-3 ligand. The total CD34+ population and the CD34+CD38- subset increased 8.4- and 67-fold, respectively, with absolute increases in the number of colony-forming unit-granulocyte macrophage (CFU-GM) (28.2-fold), CFU-Mix (8.7 fold), and burst-forming unit-erythroid (BFU-E) (4.0-fold) progenitor cells. After 7 days of coculture with PMVECs, 44% of the CD34+CD38+ subset were found to be in G1, and 51% were in G2/S/M phase of the cell cycle. More remarkably, 53% of the CD34+CD38- subset were in G1, and 17% were in G2/S/M phase after 7 days of PMVEC coculture. In contrast, only 22% of the CD34+CD38- subset remaining after 7 days of stroma-free culture were in G1, and 6% were in G2/S/M phase. Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expression of adhesion molecules CD11a (LFA-1), CD11b, CD15s (sialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34+ population was maintained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, and CD62L (L selectin) increased after ex vivo expansion. In contrast, CD34+ cells expanded on stroma-free cultures showed lower and more variable expression of CD62L and CD15s. These findings demonstrate that the primitive CD34+CD38- subset of marrow progenitor cells can be induced to enter cell cycle and can be significantly expanded ex vivo on a hematopoietic supportive microenvironment (PMVECs) while preserving the expression of cell adhesion molecules that may be important in stem cell homing and engraftment.

ADP-ribosyl Cyclase↗
PubMed1999

Monocytoid B cells are distinct from splenic marginal zone cells and commonly derive from unmutated naive B cells and less frequently from postgerminal center B cells by polyclonal transformation.

Monocytoid B cells represent a morphologically conspicuous B-cell population that constantly occurs in Toxoplasma gondii-induced Piringer's lymphadenopathy. Although widely believed to be closely related to splenic marginal zone B cells, neither this relationship, nor the B-cell differentiation stage of monocytoid B cells, nor their cellular precursors have been established. We have therefore examined monocytoid B cells for their expression of B-cell differentiation markers and the Ig isotypes at the RNA and protein level as well as for rearranged Ig heavy chain (H) genes and somatic mutations within the variable (V) region. The results obtained were compared with the corresponding features of other B-cell populations. The monocytoid B cells displayed immunophenotypical differences to all other B-cell populations. IgM and IgD expression was absent from most monocytoid B cells at the RNA and protein levels. Unrelated (polyclonal) Ig rearrangements were found in 85 of the 95 cells studied. Seventy-four percent of the rearranged VH genes were devoid of somatic mutations, whereas the remaining 26% carried a low number of somatic mutations. The majority of these showed no significant signs of antigen selection. This finding in conjunction with the predominantly unrelated Ig gene rearrangements indicates that most monocytoid B cells arise not by clonal proliferation but by transformation of polyclonal B cells. The B cells undergoing a monocytoid B-cell transformation are in the majority (74%) naive B cells, and only a minority are (26%) non-antigen-selected postgerminal center B cells. Thus, our data show that monocytoid B cells represent a distinct B-cell subpopulation.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1984

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity.

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.

Animals↗
PubMed1996

"Harpoon" model for cell-cell adhesion and recognition of target cells by the natural killer cells.

The mechanism of recognition by natural killer (NK) cells is still unknown. A dynamic model is formulated describing recognition of NK-sensitive target cells (TCs) by NK cells or NK-like cells. This model does not assume the presence of the specific NK-receptor(s) on the membrane of NK cells and corresponding specific ligands on the NK-sensitive TCs. We suggest: (1) the expression of various kinds of "non-NK receptors" and corresponding ligands (counter-receptors) on the plasma membrane of the same NK cell and, possibly, of TCs (e.g. LFA-1 and ICAM-1-ICAM3, CD2 and LFA-3; receptors for TNF and corresponding ligand etc.0; (2) the presence of multiple disorders in the organization of "extracellular matrix-surface membrane-submembrane cytoskeleton" assembly of the NK-sensitive TCs; (3) non-specific primary linking of NK cell with TCs, which induces a transfer of vesicles or membrane fragments from the NK surface to the target cell surface (and perhaps vice versa). These processes may also permit the transfer of many types of receptor and counter-receptor molecules from the surface of one conjugated cell to another by vesicles or membrane fragments. After transferral through the intercellular cleft, the free receptors and counter-receptors will be localized on both cell surfaces at the contact region between conjugated cells. By this model the NK cell can "harpoon" the TC and enhance the binding forces between cells up to the critical level and then switch on killing mechanisms for the TC. By means of this "harpoon" model of cell recognition, it seems possible to explain the nature of the wide polymorphism of TCs which are sensitive to the effect of NK and NK-like cells. A mathematical model of the NK cell cytotoxic reaction is described. The model describes many nonlinear peculiarities of the cytotoxic process and predicts some new phenomena. We suggest new approaches of manipulation of cell membranes which can transform NK-resistant target cells in NK sensitive cells and vice versa.

Animals↗