Heterogeneity of B-cell growth factor receptor reactivity in healthy donors and in patients with chronic lymphatic leukemia: relationship to B-cell-derived lymphokines

Twenty-five long-term B-cell lines were studied for B-BCGF activity. The cell lines were cultured in the presence or absence of the new tumor promoter teleocidin, and control and teleocidin-treated derived supernatants were cocultured with purified B cells obtained from healthy donors and patients with B chronic lymphatic leukemia (B-CLL), in the presence of anti-mu. In attempt to delineate the role of other B-cell lymphokines in promoting proliferation of activated B cells, the supernatants were also studied for interleukin 1 (IL-1), interleukin 2 (IL-2), and interferon gamma (IFN-gamma). The effect of B-cell-derived lymphokines on the proliferation of activated B cells obtained from the 14 donors was heterogeneous, and three types of response were observed: In four healthy donors there was induction of B-cell proliferation by B-cell lymphokines derived from both control cells and teleocidin-treated cells. In cells obtained from the other five healthy donors there was induction of B-cell proliferation by B-cell lymphokines derived from teleocidin-treated cells. In the five B-CLL patients, B-cell proliferative response to B-cell lymphokines derived from both control cells and teleocidin-activated cells was absent. Comparison of B-BCGF reactivity to T-BCGF reactivity demonstrated that B-CLL B lymphocytes did not respond to either B-BCGF or T-BCGF, whereas normal B cells responded to T-BCGF and may proliferate upon stimulation with B-cell-derived IL-2 and/or B-BCGF. These results suggest heterogeneity of B-BCGF receptor reactivity in B lymphocytes derived from healthy donors, and lack of both B-BCGF and T-BCGF receptor reactivities in B lymphocytes derived from B-CLL patients; B-cell-derived lymphokines influence normal B-cell response but not leukemic B cells; B-BCGF optimal effect is in large part due to other B-cell lymphokines, especially B-cell-derived IL-2; the possible existence of various B-BCGFs.     

A novel role for accessory cells in T cell-dependent B cell differentiation

The monocyte requirement for pokeweed mitogen-induced T cell-dependent B cell activation was reexamined. We report a dichotomy in the requirement for accessory cells in B cell proliferation and differentiation. Adherent cell-depleted human peripheral blood mononuclear cells which contained only 5% monocytes generated sufficient T cell help for optimal B cell proliferation. However, the presence of 10 to 20% monocytes were required during the last 5 days of culture for stimulated B cells to become IgG-secreting cells. Similar numbers of monocytes were also needed for anti-CD3-induced B cell differentiation. Moreover, monocytes alone added to previously activated B cells could support B cell differentiation in the absence of T cells. To determine the role of cytokines in this system, we demonstrated that supernatants of adherent cell-depleted PBMC contained decreased IL-6 activity in comparison with unseparated PBMC, but not IL-1, IL-2, or BCGF. Recombinant IL-6, however, added back either alone or with other cytokines could not replace the effects of intact monocytes on B cell differentiation. Physical interaction between the accessory cells and the responder cells was also required. As a minimum, paraformaldehyde-fixed monocytes, IL-6, and IL-1 were needed to reconstitute maximal IgG secretion. These studies suggest that accessory cells capable of producing IL-1 and IL-6 can have direct effects on the terminal differentiation of stimulated B cells.     

Antigen presentation by human B cells: T cell proliferation induced by Epstein Barr virus B lymphoblastoid cells

T cell proliferation in response to antigen requires the presence of an antigen-presenting accessory cell. The accessory cell most commonly associated with antigen presentation has been the macrophage (Mo). This study demonstrates that B cells in the form of Epstein Barr virus-transformed B lymphoblasts (EBV-B) are able to present tetanus toxoid (TT) to human T cells and induce proliferation of these cells. T cells freshly isolated from peripheral blood were shown to undergo blast transformation and proliferation in response to TT and irradiated EBV-B cells. Furthermore, TT-reactive T cell blasts were shown to proliferate in the presence of EBV-B cells and TT, but not with other antigens. There was a progressive increase to a plateau in T blast proliferation with increasing numbers of EBV-B cells added to the culture. The concentration of TT required for optimal antigen presentation was similar for EBV-B cells and for Mo. TT-specific cloned T cells also proliferated in response to TT and EBV-B cells and could be continuously grown in culture with TT, interleukin 2-containing supernatant, and EBV-B cells in place of autologous Mo. EBV-B cells pulsed with TT could also act as antigen-presenting cells. The proliferative response of T cell clones to TT in the presence of EBV-B cells was inhibited by antiserum to human p29,34 glycoprotein but not by anti-beta 2-microglobulin. This inhibition was shown to result from interaction with Ia-like determinants on EBV-B cells. These results indicate that B lymphoblastoid cells in man are able, like Mo, to present antigen to T cells in the context of Ia-like determinants.     

Autoreactive T cells in MRL/Mpr-lpr/lpr mice. Characterization of the lymphokines produced and analysis of antigen-presenting cells required

Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene.     

Development of a serum-free medium which supports the long-term growth of human and nonhuman primate lymphoid cells

The ability to grow lymphoid cells in serum-free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, β-mercaptoethanol, L-α-phosphatidylcholine, and transferrin supported the long-term proliferation of a gibbon ape lymphoma T-cell line, MLA144. These cells continue to produce Interleukin 2 (IL-2, T-cell growth factor) constitutively in the serum-free medium. IL-2-dependent human T cells initiated and maintained in culture in serum-free medium containing IL-2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL-2-dependent marmoset T-cell line, OH-1, also proliferated on the serum-free medium when supplemented with IL-2. Several established primate B-cell lines which do not require IL-2 for growth were able to proliferate in the serum-free medium. These B-cell lines included B95-8, an Epstein-Barr virus (EBV)-transformed marmoset cell line, HuCo/R-H, a human cord B-lymphocyte line transformed with EBV, and Namalwa, an EBV-positive B-cell line established from a Burkitt's lymphoma. B95-8 cells grown on serum-free medium showed high levels of EBV antigen-positive cells after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA).     

The role of IL 1 in the antigen-specific activation of murine class II-restricted T lymphocyte clones

The role of IL 1 in the antigen-specific activation of class II-restricted T lymphocytes was examined by using a model system consisting of cloned WEHI 5 B lymphoma accessory cells and class II-restricted, soluble antigen- or alloantigen-reactive T cell clones. The addition of exogenous recombinant IL 1 to the T cell cultures resulted in a significant enhancement of the antigen-specific T cell proliferation response, but at best, only small increases in IL 2 release. Goat IgG anti-IL 1 antibodies were added to the T cell cultures to assess their effect on T cell activation. The IL 1 enhancement of the T cell proliferation response was inhibited by the anti-IL 1 antibodies in a dose-dependent manner. In contrast, only modest levels (10 to 25%) of proliferation inhibition were observed in T cell cultures containing either WEHI 5 or splenocyte accessory cells but no exogenous IL 1. When the anti-IL 1 antibodies were added to primary mixed lymphocyte cultures stimulated by WEHI 5 cells in the absence of exogenous IL 1, no significant inhibition of proliferation was observed. A small but statistically significant proliferation inhibition was observed when anti-IL 1 antibodies were added to mixed lymphocyte reaction cultures stimulated by splenocytes. Two-color cytofluorometric analysis of the effects of IL 1 on antigen-activated T cell clones demonstrated that under suboptimal stimulation conditions, IL 1 stimulated a small but significant increase in the number of T cells bearing IL 2 receptors. In the presence of optimal numbers of WEHI 5 accessory cells, IL 1 enhanced T cell proliferation in the absence of a detectable increase in the number of T cells bearing IL 2 receptors, the number of IL 2 receptors per T cell, or the levels of IL 2 released. Finally, exogenous IL 1 can be added as late as 18 to 24 hr after culture initiation without significantly reducing its ability to enhance the T cell proliferation response. These data indicate that IL 1 has pleiotropic effects on murine T lymphocytes and can function to enhance T cell activation at multiple points during the activation sequence.     

Proliferative response of Tax1-transduced primary human T cells to anti-CD3 antibody stimulation by an interleukin-2-independent pathway.

The growth properties of human T-cell leukemia virus Tax1-transduced primary human T cells derived from peripheral blood lymphocytes were compared with those of the same subset of T cells transduced with a control vector. Tax1-transduced T cells exhibited slightly elevated responsiveness to externally added interleukin-2 (IL-2) and a markedly higher proliferative response to stimulation with anti-CD3 antibody. The proliferation after anti-CD3 antibody stimulation was mainly via an IL-2-independent pathway. Therefore, some other mechanism than the previously proposed IL-2 autocrine model seems to be involved in the process of deregulation of T-cell proliferation by Tax1. Moreover, Tax1-transduced T cells have continued to proliferate in medium containing IL-2 long after control T cells ceased to grow, and so they are considered to be immortalized.     

Preferential proliferation of T cell receptor V gamma 3-positive cells in IL-2-stimulated fetal thymocytes

Thymocyte cell suspensions, prepared from mice at different ages, were cultured in vitro with human rIL-2. This stimulation resulted in a cell population that contained almost 50% TCR-gamma delta-positive cells if thymocytes were taken from fetal day 17 until just after birth. Analysis of the variable (V gamma) region used by the TCR-gamma delta cells revealed that 90% of them expressed TCR-V gamma 3, and less than 5% expressed TCR-V gamma 2. Cells positive for TCR-alpha beta were barely detectable. If fetal day 18 organ cultured thymus lobes, instead of a cell suspension, were stimulated with IL-2, no rise in the number of TCR-V gamma 3+ or TCR-delta+ cells was observed, whereas a partial outgrowth of TCR-alpha beta+ cells occurred. From day 1 after birth, the number of TCR-gamma delta cells recovered from an IL-2-stimulated thymocyte cell suspension dropped to reach a plateau of 15% of the total cell number, whereas TCR-V gamma 3+ cells became undetectable in older animals. TCR-alpha beta+ cells, on the other hand, quickly rose in cell number after birth. Kinetic analysis showed that the preferential outgrowth of TCR-V gamma 3+ cells in IL-2-stimulated fetal day 18 thymocyte cell suspensions was present from the onset of the culture; a significant proliferation of CD4 or CD8 single positive TCR-alpha beta cells was never observed. This lack of proliferation of TCR-alpha beta cells was not due to inhibition by the activated TCR-V gamma 3+ cells. Throughout the IL-2 culture, one-fourth of the TCR-V gamma 3+ thymocytes was positive for CD8. Analysis of the DNA content and the IL-2 receptor (IL-2R) p55 expression showed that during the first days of culture the TCR-V gamma 3+ cells had a much higher proliferation rate than the TCR-V gamma 3- cells, although TCR-V gamma 3+ IL2R p55+ cells could not be detected. From day 3 to 4 of culture, the proliferation rate of TCR-V gamma 3+ cells equaled that of the rest of the cells and less than 20% of the TCR-V gamma 3+ cells expressed the IL-2R p55. The biologic significance of our findings is discussed.     

Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells

A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.     

Molecular analysis of the dissociation between IL-2 production and proliferation in a response of a T cell clone to the antigen presented by B cells

Using B cells as APC, antigen specific responses of two murine T cell clones, 34-7F and 35-8H, were analyzed. 34-7F cells produced IL-2 but failed to proliferate, whereas 35-8H cells both produced IL-2 and proliferate. The antigenic stimulation increased intracellular free Ca2+ concentration in both clones, but enhanced inositol phospholipid metabolism only in 35-8H cells. The treatment of 34-7F cells with PMA, an activator of protein kinase C, synergized with the antigenic stimulation to induce the proliferation of the T cells. Thus, the failure of 34-7F cells to proliferate in the Ag response appears to result from the absence of an increase in inositol phospholipid metabolism. The absence is likely due to the defect in B cells as APC, inasmuch as the antigenic stimulation of 34-7F cells with whole spleen cells induced increases in inositol phospholipid metabolism and proliferation. The PMA treatment synergized with the Ag on B cells to enhance IL-2R expression, which was not inhibited by the addition of nifedipine, a calcium channel blocker. The agent inhibited the IL-2 production. Taken together, the results in the present experiments suggest the association of IL-2 production with increases in intracellular free Ca2+ concentration but not in inositol phospholipid metabolism, and that of IL-2R expression with increases in the metabolism but not in intracellular free Ca2+ concentration.     

Isolated peripheral T cells from GvHR recipients exhibit defective IL-2R expression, IL-2 production, and proliferation in response to activation stimuli

Graft-vs-host reactions (GvHR) following the injection of class I/II MHC disparate parental cells into unirradiated F1 recipient mice result in the development of marked immune dysfunction. Following negative selection using adherence and antibody and complement depletion, highly purified T cells were examined to determine their ability to undergo activation. Three weeks after GvHR initiation, unstimulated splenic T cells from GvHR mice displayed normal CD3 and IL-2R expression but elevated expression of class I MHC and Ly-6A/E antigens. Despite culture with normal F1 accessory cells, both CD4+ and CD8+ GvHR T cells exhibited low levels of proliferation to both Con A and anti-CD3 mAb. Although following exposure for 12 h to either of these stimuli, GvHR T cells expressed normal levels of IL-2R, expression was greatly decreased vs normal T cells between 24 and 48 h. In addition, at no timepoint was detectable IL-2 produced by GvHR T cells. Importantly, mixing experiments did not demonstrate detectable suppressive activity in the purified GvHR T cell subsets. GvHR T cells were also tested for their ability to respond to stimuli in the absence of any accessory cell population. These cells again did not proliferate to levels equivalent to normal T cells. Incubation with PMA and either cytokines (Con A supernatant, rIL-7) or anti-CD3 mAb resulted in only low levels of proliferation in GvHR T cells. Notably, at high ionomycin concentrations together with PMA, GvHR T cells did proliferate to equivalent levels as normal cells. However, with decreasing concentrations of ionophore, these cells failed to proliferate as well as normal cells. In total, these findings demonstrate that GvHR T cells are phenotypically and functionally distinct from normal T cells. The results suggest that GvHR T cells themselves may contribute to the well-characterized immune depression occurring in recipients undergoing GvHR.     

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity,tolerance, and suppression

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN- and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN- and IL-10, and down-regulation of TNF- production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.     

Protein kinase C is required for responses to T cell receptor ligands but not to interleukin-2 in T cells

We have tested the role of protein kinase C in mRNA expression and T cell proliferation mediated through the T cell receptor and through the interleukin-2 (IL-2) receptor. Chronic treatment of a mouse T cell clone with phorbol esters caused a complete loss of protein kinase C activity and a concomitant loss of proliferation to T cell receptor ligands (antigen, lectins, antireceptor antibodies). In contrast, kinase C-depleted T cells retained the ability to proliferate to IL-2. Loss of the T cell receptor response was not due to decreased cell surface expression of receptor or impairment of early receptor function (phosphatidylinositol turnover, calcium mobilization). Kinase C-depleted T cells showed no induction of mRNAs for activation-associated genes on exposure to the T cell receptor ligand Concanavalin A; expression of a subset of the same mRNAs in response to IL-2 was unaffected. We conclude that kinase C is required for mRNA expression and subsequent proliferation mediated through the T cell receptor pathway but is not involved in mRNA expression and proliferation in response to IL-2.     

Cyclosporin A inhibits initiation but not progression of human T cell proliferation triggered by phorbol esters and calcium ionophores

Cyclosporin A (CsA) is a potent inhibitor of T lymphocyte proliferation induced by Ag and mitogens. In an attempt to further delineate the mechanism of action of CsA, we have examined its effects on T cell proliferation induced by the combination of the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin. T cells were rendered competent as the result of a 30-min initial incubation with both drugs, after which the drugs were washed out. Competence is defined as the ability to subsequently proliferate in response to exogenously added IL-2 or PDB in the second phase of the culture, but not to synthesize IL-2 or proliferate without these additions. Addition of CsA (1 microgram/ml) to the cells in the initial, competence-inducing 30-min incubation with PDB/ionomycin abrogated their subsequent response to IL-2 or PDB. In contrast, addition of CsA to cells after they had been treated for 30 min with PDB/ionomycin and then washed did not affect their responses to subsequent addition of either IL-2 or PDB. Treatment with CsA during induction of competence prevented the expression of the 55-kDa IL-2R gene during competence induction and inhibited IL-2 gene expression and IL-2 production in response to PDB in the second phase. These results indicate that the effects of CsA are limited to the initiation (competence induction) period of T cell activation, that CsA apparently affects expression of more than one gene, and in competent cells, CsA does not affect their ability to progress to DNA synthesis.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

T cell activation induced by anti-CD3 antibodies requires prolonged stimulation of protein kinase C

Accessory cell-depleted T cells required the presence of a protein kinase C (PKC) stimulating phorbol ester, such as phorbol 12,13-dibutyrate (PDB), to be activated by soluble antibodies to the CD3 molecular complex. To determine the duration of PDB costimulation necessary to induce a proliferative response, highly purified T cells were pulsed with anti-CD3, incubated with PDB for limited periods of time, and then washed and recultured in the absence of PDB. T cells stimulated with anti-CD3 and PDB for 2 hr were unable to proliferate unless IL-2 or PDB was added to the second culture. With more prolonged exposure to PDB (4-18 hr), anti-CD3-pulsed cells exhibited an increased capacity to proliferate in the absence of additional PDB. Proliferation could be augmented by exogenous IL-2, but remained submaximal. Optimal DNA synthetic responses required the presence of PDB throughout the entire culture. Despite this, costimulation with anti-CD3 and PDB induced a significant number of cells to express IL-2 receptors and enter the cell cycle after 18 hr of costimulation with PDB. Moreover, T cells costimulated by anti-CD3 and PDB produced IL-2 within 4 hr. However, T cells that were stimulated with anti-CD3 and PDB for 4 hr, washed, and recultured rapidly lost the ability to continue to produce IL-2, which reflected a decrease in the content of mRNA encoding IL-2. This loss of IL-2 production was prevented by reculturing the cells with PDB. These studies therefore indicate that after initial T cell activation by anti-CD3, continued stimulation of PKC is necessary for ongoing IL-2 production. These results suggest a model of T cell activation in which sustained stimulation of PKC after cell cycle entry is required to maintain growth factor production and continued proliferation.