Functional characterization of Con A-responsive Lyt2-positive mouse small intestinal intraepithelial lymphocytes.

The surface phenotypes of resting vs Con A-stimulated intraepithelial lymphocytes (IEL) from mouse small intestine were directly determined by immunofluorescence with double labelling. Both Thy 1.2+, Lyt 2+ and Thy 1.2+ Lyt 2- IEL underwent blastogenesis and expressed interleukin-2 (IL-2) receptors. The Lyt 2+ subsets of IEL (which represent 80% of the total cells) were isolated by panning and shown to proliferate in response to Con A and IL-2, although the frequency of responsive precursors was dramatically lower than that seen in the splenic Lyt 2+ T-cell population (1 in 500 vs 1 in 8, respectively). Con A-stimulated Lyt 2+ IEL produced lymphokines supporting the growth of the interleukin-3 (IL-3)-dependent cell line DA-1, and of the FDC-P2 cell line that proliferates in response to both IL-3 and GM-CSF. The results therefore support the possibility that Lyt 2+ IEL act as inducers of local cell-mediated immune reactions by producing haematopoietic lymphokines.     

During recovery from cytomegalovirus infection T-lymphocyte subsets become selectively responsive to activation and have depressed interleukin 2 (IL2) secretion and IL2 receptor expression

The ability to induce lymphocyte activation with Concanavalin A (Con A) was suppressed in spleen cell cultures derived from BALB/c mice acutely infected with murine cytomegalovirus (MCMV) when compared to cultures derived from control, uninfected mice. This immunosuppression was observed as a reduced incorporation of 3H-thymidine in the lymphocyte proliferative response to Con A, was highly correlated with reduced secretion of interleukin 2 (IL2), and could not be augmented by addition of exogenous IL2. The degree of suppression of both the proliferative response and IL2 secretion was directly dependent on the infecting virus dose and on the time post-infection. At weekly intervals during infection, fluorescein-labeled monoclonal antibodies to various T-lymphocyte surface markers were used to stain spleen cells from control or MCMV-infected mice before or after Con A activation. Analysis of stained spleen cells by flow cytometry revealed several unusual responses to activation signals in lymphocytes derived from mice with acute MCMV-induced immunosuppression. At 4 days post-infection T-lymphocytes of each subset [Thy 1.2 (pan T), L3T4 (T-helper/inducer), Lyt 2 (T-cytotoxic/suppressor) and Lyt 1 (subset of Thy 1.2)] were each present in the spleen but each was in reduced percentage of the total spleen T cell population compared to control (52% to 75% of controls). At this time post-infection these cells were non-responsive to Con A activation as shown by inability to induce 3H-thymidine uptake, IL2 secretion or IL2 responsiveness and an inability to demonstrate an IL2 receptor-positive (IL2R+) population. By 11 days post-infection all tested subsets of T-lymphocytes (Thy 1.2+, L3T4+, Lyt 2+ and Lyt 1+) were in normal control range in fresh spleen. However, only Thy 1.2+L3T4+ (T-helper/inducer) cells were responsive to Con A activation. 3H-thymidine uptake and IL2 secretion were at levels of about 60% of the control but, surprisingly, cells were unresponsive to addition of exogenous IL2 and few, if any, of these cells were found to express IL2 receptors. By 18 days post-infection, when 3H-thymidine uptake could be induced at control level, Thy 1.2+, L3T4+, and Lyt 2+ (T-cytotoxic/suppressor) cells were each responsive to Con A activation at levels comparable to control but Lyt 1+ and IL2 R+ cells were still substantially suppressed (ca. 35% to 40% of control value). Detection of Lyt 1+ subset and IL2 R+ cells after Con A activation did not near control levels until very late in the recovery process (day 25).(ABSTRACT TRUNCATED AT 400 WORDS)     

B cells as accessory cells in a Con A response of a T cell clone

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.     

Production of lymphokines and interferon by immune cells involved in recovery of mice from herpes simplex virus type 2 hepatitis

Adoptive transfer of spleen cells from mice 4 days after infection with herpes simplex virus type 2 (HSV-2) reduced the virus titer in the liver of recipient mice infected 24 h before transfer. Macrophage chemotactic factor (CF) and macrophage migration inhibition factor (MIF) were produced by day 3 of infection in spleen cell cultures stimulated with HSV-2, but not with control antigen, i.e. 1 day before the cells are active in adoptive transfer. Interferon was produced in cultures established throughout the infection but not in normal spleen cells. From days 1 to 5 of infection interferon was produced irrespective of in vitro restimulation, although the highest amounts were always produced after stimulation with the specific antigen. Spleen cells from mice infected for 6 days produced interferon only when stimulated with HSV-2. The cells from 6-day-immune mice active in adoptive transfer and CF and MIF production were found to be Thy 1+, Ig- and Lyt2-. Both Thy 1+ and plastic adherent cells were necessary for interferon production, whereas Ig+ and Lyt2+ cells did not produce interferon. The interferon was acid stable and neutralized by antiserum against alpha/beta-interferon and thus has the characteristics of alpha-interferon. The data indicate that a delayed type hypersensitivity reaction with lymphokine-induced macrophage recruitment into infectious foci may be a central feature of the recovery process in HSV-2-induced hepatitis. A possible role of interferon produced by the accumulated cells needs further investigation.     

Production of Interferon-β by Murine T-Cell Lines Induced by 10-Carboxymethyl-9-Acridanone

Besides the established T-cell property of producing gamma interferon (IFN-gamma), murine T cells additionally possess the ability to produce IFN-alpha and IFN-beta when appropriate inducers such as 10-carboxymethyl-9-acridanone (CMA) or Newcastle disease virus (NDV) are used. Interleukin 2 (IL-2)-dependent murine T-cell lines, but not purified resting splenic T cells, responded to CMA and NDV with production of IFN-alpha, beta. The IFN production by these T cells was not restricted to a special subset, since T cells expressing the Lyt 1+2- and the Lyt 1-2+ phenotype responded to these inducers with IFN production. After prolonged passaging of the T-cell lines in IL-2-containing medium, the ability to respond to CMA with production of antiviral activity was sustained longer than the ability for concanavalin A-induced IFN-gamma production. Whereas the NDV-induced T-cell supernates contained both IFN-alpha and IFN-beta, the induction with CMA resulted exclusively in the synthesis of IFN-beta by the T-cell lines.     

T-cell control of IgA production. I. Distribution, activation conditions and culture of isotype-specific regulatory helper cells.

The capacity of T cells from different sites to augment IgA production by LPS-stimulated B cells has been investigated. Peyer's patch T lymphocytes activated with Con A induced up to a 20-fold increase in IgA production. The effect was isotype-specific, in that no consistent effect on IgG and a diminution of IgM synthesis were observed. Less activity was recorded in spleen and mesenteric lymph node T cells. Optimal activation of the Thy 1+ Lyt 1+2- helper cells required the addition of splenic adherent cells and the elimination of Thy 1+ Lyt 2+ suppressor cells prior to activation. T lymphocytes maintained regulatory activity for several months after expansion in medium supplemented with IL-2 and are now being cloned. We conclude that IgA production is under control of T cells sited preferentially, but not exclusively, in gut-associated lymphoid tissue, and that these T cells can augment IgA production by B lymphocytes from sites with low commitment to production of this isotype.     

Phorbol ester replacement of helper cell and interleukin 2 requirements in gamma interferon production.

Immune interferon (IFN-gamma) production by mitogen-stimulated C57BL/6 mouse spleen cells required both Lyt 1+,2- and Lyt 1-,2+ lymphocytes. The Lyt 1-,2+ cells were the IFN-gamma producers, whereas the Lyt 1+,2- cells were the helpers. Interleukin 2 mediated the Lyt 1+,2- cell help. Phorbol myristic acetate (10 ng/ml) completely replaced Lyt 1+,2- helper cell or interleukin 2 requirements in IFN-gamma production. The phorbol ester exerted its helper effects by direct interaction with IFN-gamma-producing Lyt 1-,2+ cells and not through stimulation of production of interleukin 2 by Lyt 1+, 2- cells. Suppressor cell effects in IFN-gamma production were also completely abrogated by phorbol myristic acetate. The data suggest a mechanism for phorbol myristic acetate regulation of IFN-gamma production at the cellular level.     

Flip-flop in the Lyt 2 phenotype of T cells from radiation chimaeras between Thy 1 congenic donor and recipient mice.

We present further evidence that T helper cells change their surface phenotype from Lyt 2- to Lyt 2+, after secondary adoptive transfer. Chimaeras were established with Lyt 2- spleen cells from normal, or carrier-primed A-Thy 1a donors and irradiated, A-Thy 1b recipients. Despite efficient depletion of Lyt 2+ cells from the initial donor, in the fluorescence-activated cell sorter, the majority of chimaeric T cells expressed Lyt 2 and were resistant to monoclonal Thy 1.2 alloantibody and complement treatment. In functional studies, the T helper activity of chimaeras, reconstituted with carrier-primed spleen cells, was present in both Lyt 2+ and Lyt 2- subsets.     

Positive selection of T-cell subsets. I. Proliferative responses of Lyt 2 separated thymocytes and splenic T cells.

Flow microfluorometry analysis of peanut lectin non-agglutinable (PNA-) thymocytes (ThC) reveals the existence of 30%-50% Lyt 1,2,3+ and 50%-70% Lyt 1+,2,3- subpopulations. Using positive selection on anti-immunoglobulin-coated (Mage) plates, we selected PNA- Lyt 2+ and PNA- Lyt 2- ThC as well as their peripheral counterparts in the spleen. These populations were tested in parallel for their ability to respond to concanavalin A (Con A) and phytohaemagglutinin (PHA), to respond to allogeneic stimulation in the mixed lymphocyte reaction (MLR); ThC subpopulations were also tested for their ability to provide synergy with lymph node cells (LNC) in the MLR. It was found that (a) Lyt 2- cells of both thymic and splenic origin responded to all doses of Con A or PHA; (b) PNA- Lyt 2+ ThC were unresponsive to Con A or PHA, whereas splenic Lyt 2+ T cells responded to low doses of mitogens; and (c) PNA- ThC of both Lyt phenotypes responded in a MLR and provided synergy with LNC in the MLR. These data support the notion that Lyt 2+ cells of either PNA- or PNA+ subpopulations must undergo post-thymic maturation before becoming responsive to low doses of T-cell mitogens.     

Interleukin 4 and Interferon Gamma Production in Restimulated CD4+ and CD8+ Cells Indicates Memory Type Responsiveness

Interleukin 4 (IL-4) and interferon gamma (IFN-gamma) production was analysed in murine spleen cells during primary and secondary mitogen stimulation in vitro. The kinetics, frequency and phenotype of single lymphokine-producing cells were studied by combining intracytoplasmatic immunofluorescence and surface staining. Both IL-4 and IFN-gamma was produced by CD4+ as well as CD8+ cells, however 75-80% of IL-4 producers were CD4+ and 90% of IFN-gamma+ cells were CD8+. In primary stimulations, concanavalin A (Con A) activation or anti-CD3 antibody together with phorbol 12-myristate 13-acetate (PMA) induced different patterns of lymphokine production. Approximately the same frequency of IFN-gamma+ cells was induced by both stimulation procedures but the kinetics was different with a peak at 30 h using Con A and at 52 h using anti-CD3 and PMA. IL-4 production peaked at 52 h, but the frequency of IL-4+ cells was 8-10 times higher after stimulation by anti-CD3 and PMA than after Con A stimulation. During restimulation of the mitogen activated cells, lymphokines were rapidly produced; both IL-4 and IFN-gamma production peaked at 8-11 h. Only a small increase in the frequency of IL-4+ cells was seen, at most two to three times. No evidence for a major shift of lymphokines produced between primary and secondary stimulations could be found. Instead, the pattern of lymphokine production induced by the primary stimulus was dominant also in secondary cultures irrespective of stimulation condition.     

The growth of concanavalin-A activated, Lyt selected subsets in IL-2 containing supernatants

The growth properties of Con A activated, Lyt selected splenic T lymphocytes were examined by limiting dilution analysis and clonally by single cell picking. Under the conditions used, a comparable frequency of Lyt 1+ and Lyt 2+ cells grew after Con A activation in the presence of Con A rat spleen supernatant. At a clonal level, however, the growth of these subsets differed qualitatively and quantitatively. While Lyt 2+ cells obtained clone sizes of several hundred cells, Lyt 1+ clone sizes were usually less than 100 cells, and many clones aborted their growth after a few days. Morphologically, the Lyt 1+ cell was smaller and usually showed fewer pseudopodial protusions as compared to the Lyt 2+ cell.     

Selective effects of vasoactive intestinal peptide on the mitogenic response of murine T cells.

Murine lymphocytes have been shown previously to possess high-affinity specific receptors for the neuropeptide vasoactive intestinal peptide (VIP). This study examines the cellular basis for modulation of concanavalin A (Con A)-induced T-cell responses by this neuropeptide. VIP was most effective as an inhibitor when added at the initiation of the mitogen response. The loss of potency when VIP was added later in the response was accompanied by a decrease in the affinity of stimulated cells for the neuropeptide. The inhibitory influence of VIP was reversible if the neuropeptide was removed from stimulated cell cultures up to 6 hr after the initiation of stimulation. In contrast, VIP-mediated inhibition was fully developed once the stimulated cells had been exposed to the neuropeptide for 24 hr. The presence of VIP led to a decreased production of interleukin-2 (IL-2) by the stimulated lymphocytes, but did not affect the expression of Con A-induced suppressor cell activity by cultured lymphocytes. Studies of the effect of selective, complement-mediated killing of cells with Thy 1, L3T4 and Lyt 2 monoclonal antibodies showed that the majority of the VIP bound by the lymphocytes was accounted for by binding to L3T4+, Lyt 2- T cells. It was concluded that VIP exerts its influence over Con A-stimulated proliferation by selective regulation of T-cell subsets.     

Establishment and characterization of a permanent T-cell line producing an antigen non-specific suppressor factor.

A permanent cell line designated SL4c has been established from a primary culture of murine BALB/c spleen cells regularly stimulated with large doses of irradiated allogeneic cells plus exogenous interleukin-2(IL-2). After 8 months of cultivation, the cells of the SL4c line proliferate spontaneously and do not respond with an increase in proliferation to alloantigenic stimulation. The cells have the Lyt 1.2+, Lyt 2.2-, L3T4a+, Thy 1.2+ phenotype and exert a strong suppressive effect upon stimulation with freshly explanted cells. The SL4c line produces a suppressor factor (SF4c), which inhibits the mitogen-induced proliferation of normal lymphoid cells but does not suppress the proliferation of fibroblasts and sarcoma cells. The suppression is antigen non-specific, is not limited by H-2 restriction nor by interspecies barrier, and is not due to cytotoxic effect. However, the suppression is only detectable if the SF4c is added to the stimulated cells during the early stages of mitogen-induced proliferation. A tentative characterization of the relative molecular weight (MW) of the suppressor molecule based upon fractionation of SF4c supernatant on a Sepharose 6B column shows that the inhibitory activity is confined to the high MW fractions (300,000-350,000). Translation material obtained from Xenopus laevis oocytes, which were injected with RNA preparations isolated from SL4c cells, also shows the suppressive effect.     

Role of interferons in natural killer cell generation from primitive bone marrow precursors

We studied the possible role of interferons (IFNs) on the interleukin-2 (IL-2)-dependent development of mouse natural killer (NK) cells from undifferentiated bone marrow (BM) precursors. Results indicate that alpha-IFN is able to synergize with IL-2 in the induction of lytic effectors. The stimulating effect of alpha-IFN is dose-dependent and is also obtained when BM cells are pretreated shortly before culturing. The precursor cells are resistant to treatment with 5-fluorouracil (FU, 150 mg/kg i.v.). Treatments which have been shown to eliminate more differentiated cells but spare less differentiated BM precursors, are asialoGM1-, Thy1+, Lyt1- and Lyt2-. Effector cells generated by culturing with IL-2 and alpha-IFN are typical NK cells in that they lyse only NK-susceptible targets. These are Thy1+, asialoGM1+, Lyt5+, Lyt1- and Lyt2-. These data suggest that alpha-IFN may represent a maturational signal for the generation of NK cells from undifferentiated BM precursors.     

Characterization studies of suppressor cells in murine bone marrow chimaeras.

When BALB/c bone marrow cells were transferred to lethally X-irradiated C3H/He mice either intrasplenically (i.s.) or intravenously (i.v.), suppressor cells were detected by means of MLR coculture assays in the spleen of i.s. and i.v. chimaeras. Some but not all of the suppressor cells expressed a Thy 1.2 antigen, indicating that suppressor cells either sensitive or insensitive to anti-Thy 1.2 antibody plus complement treatment were generated in the spleen of i.s. and i.v. chimaeras. According to the examination of Lyt alloantigen expression on suppressor T cells, Lyt 1+2-, Lyt 1-2+, and Lyt 1+2+ suppressor T cells appeared to be present. The culture supernatants from several T-cell clones showed the suppressor activity against alloreactive MLR. Cell surface markers of these clones were composed of Lyt 1+2-, Lyt 1+2+ and Lyt 1-2+. In addition, since Carrageenan treatment abrogated the suppressor activity of plastic dish adherent cells, we conclude that some of them were composed mainly of macrophages.     

Production of high titers of interferon-gamma by prestimulated murine spleen cells

Concanavalin A (Con A)-induced interferon-gamma (IFN-γ) production by murine spleen cells prestimulated with Con A for different periods of time has been studied. Highest titers of antiviral activity were obtained by restimulation of cells that had been prestimulated with Con A for 3 days. These cells produced up to 30 times more IFN than freshly isolated spleen cells stimulated under the same conditions. In an effort to explain this rise in the capacity to produce IFN-γ, the possibility that it was due to the inactivation of a suppressor cell was excluded. The addition of freshly isolated spleen cells at different concentrations to prestimulated cells did not affect subsequent Con A-induced IFN-γ production. Separation of freshly isolated or prestimulated spleen cells by velocity sedimentation at unit gravity showed that in the latter case most of the IFN-γ was produced by a population of large-sized cells not present in the former population. It was concluded from these experiments that prestimulation of spleen cells with Con A gives rise to a population of large-sized cells that produce, upon restimulation with the same mitogen, much higher titers of IFN-γ than those obtained upon primary stimulation of small resting lymphocytes.     

Modification of suppressor/cytotoxic and helper subsets in lentil lectin-pretreated mice

The effect of lentil lectin (LCA) on various lymphocyte subpopulations in the spleen, lymph nodes and thymus of CBA mice was studied. The most marked effect after five 1-mg LCA doses was observed in the spleen. LCA caused a drop in both the total and relative number of cells carrying all the markers examined, i.e., Thy 1, L3/T4a, Lyt 2, and sIg, so that large numbers of lymphocytes did not express any of them. A dramatic decrease in the Thy 1-positive cell number is mainly due to depletion of the L3/T4a (helper) T cell subpopulation, whereas the Lyt 2+ (suppressor/cytotoxic) cells are less affected. The decrease in the Thy 1+, Lyt 1+ and L3/T4a+ cell numbers in the thymus and lymph nodes is smaller, the percentage of Lyt 2+ cells is even increased after LCA treatment. Our results indicate that two mechanisms play a role in the induction of allotransplantation tolerance by LCA: 'depletion' of helper T cells and a relative increase in the regulatory (suppressor) cell ratio, which may support allograft survival.