Inhibition of macrophage-induced antigen-specific T-cell proliferation by interferon-gamma

The effects of IFN-gamma on macrophage (M phi)-mediated antigen-specific T-cell proliferation was investigated. A well-defined assay system using purified resident populations of antigen-pulsed peritoneal M phi and immune T cells was used to measure M phi-induced antigen-specific T-cell proliferation. Antibody affinity purified or recombinant IFN-gamma inhibited M phi-induced T-cell proliferation when KLH-pulsed M phi from mice given IFN-gamma prior to KLH were cultured with KLH immune T cells from normal mice. Monoclonal rat anti-IFN-gamma antibody neutralized the inhibitory effect of IFN-gamma. This inhibition of T-cell proliferation occurred despite the fact that these M phi appeared to be activated by IFN-gamma treatment as measured by increased tumoricidal activity. The mechanism for the inhibition was unrelated to class II (Ia) expression, IL-1 secretion, and prostaglandin secretion. These results demonstrate the complex and sensitive role IFN-gamma has in regulating the immune response.     

Inhibition of macrophage-induced antigen-specific T lymphocyte proliferation by poly I:C: strain and antigen independence

We have previously demonstrated that IFN-alpha/beta, poly I:C (an inducer of IFN-alpha/beta), and IFN-gamma can inhibit the ability of KLH-pulsed peritoneal macrophages to induce proliferation of syngeneic, KLH immune T lymphocytes in CBA/J mice. In this study, we show that this IFN-induced immunosuppression is not restricted to CBA/J (H-2k) mice but is also seen in BALB/cJ (H-2d) mice. A similar inhibition of proliferation is observed with the KLH-specific T cell hybridoma BDK, 100, which requires KLH-pulsed macrophages for optimum proliferation and IL-2 production. The immunosuppression produced by IFN was also independent of the antigen employed. Inhibition of T lymphocyte proliferation was observed when casein, instead of KLH, was used to immunize T cells and to pulse peritoneal macrophages in vivo. Utilizing KLH and casein, the antigen specificity of the inhibition was demonstrated. Therefore, the inhibition by the IFN-inducer poly I:C of macrophage-induced, antigen-specific T cell proliferation is not limited by H-2 type of the mice or to one antigen.     

The proliferative response of rat T cells to calcium ionophores increases with age

Splenocytes and T cells from both old and young rats proliferate to A23187 and ionomycin, and this response increases 3- to 10-fold in aged animals. Augmented responsiveness to ionomycin occurs in the absence of any defect in Con A-induced proliferation of T lymphocytes of aged rats and is dependent upon the addition of thiol compounds to the tissue culture medium. Augmented proliferative responses to ionomycin precede the significant but much smaller decline (30 to 40%) in Con A-induced proliferative responsiveness of splenocytes, which is evident only when rats reach 24 months of age. Heightened proliferation to calcium ionophores is not caused by a greater ability of T lymphocytes from aged rats to increase [Ca2+]i in response to ionomycin. The increased proliferative response of lymphocytes from aged rats to ionomycin occurs in the absence of detectable amounts of secreted IL 2 or IL 4. The ionophore response is a much more sensitive biomarker of age than the decline in Con A-induced proliferative responses of lymphocytes and identifies an activity of T lymphocytes that increases rather than decreases during the aging process.     

The effect of bone marrow depletion on prostaglandin E-producing suppressor macrophages in mouse spleen

The i.p. injection of Corynebacterium parvum (CP) into CBA/J mice effected increases in macrophage colony-forming cells (M-CFC) when spleen cells were cultured with L cell culture filtrate as a source of colony-stimulating factor. Significant increases in phagocytic macrophages (M phi) with Fc receptors for IgG2a and IgG2b immune complexes were additionally noted among the spleen cells in these mice. These M phi effectively inhibited Con A-induced lymphocyte proliferation, probably reflecting a 10-fold increase above normal controls in prostaglandin E to 47 ng/3 X 10(6) spleen cells/ml. To determine whether the suppressor M phi are immediate derivatives of splenic M-CFC, we tried to induce suppressor M phi by the injection of CP into mice depleted of bone marrow M-CFC by the earlier administration of the bone-seeking isotope, 89Sr. This procedure reduced M-CFC in the bone marrow to less than 1% of normal for more than 30 days. Monocytes in the blood fell to 5% of normal by day 10 and were 30% on day 30. Levels of resident peritoneal M phi showed relatively little change in this period. By contrast, splenic M-CFC increased to 20-fold higher than the "cold" 88Sr controls. CP-induced suppressor M phi activity, however, was sharply reduced in 89Sr marrow-depleted mice on day 10, despite the striking increase in M-CFC. There was a threefold increase in the number of phagocytic M phi binding IgG2a immune complexes, with no significant increase in IgG2b binding M phi. The kinetics of recovery of suppressor M phi activity showed that on days 20, 30, and 50 after 89Sr injection the activities reached 20%, 30%, and 70% of the "cold" control, respectively, and correlated with the recovery of significant levels of M-CFC in the bone marrow. Taken together, these observations suggest that splenic M-CFC are not an immediate source of PGE-suppressor M phi in vivo. It appears more likely that the CP-inducible suppressor M phi, in particular, originate from radiosensitive bone marrow cells or require for differentiation a microenvironment provided by bone marrow cells. The data also suggest that the expression of the Fc gamma 2b receptor and of suppressor activity by CP-induced splenic M phi are related phenomena.     

Induction of Apoptosis and Tyrosine Phosphorylation of Cellular Proteins in T Cells and Non-T Cells by Stimulation with Concanavalin A

A high concentration (30 μg/ml or more) of Con A caused the death of not only thymocytes but also splenic cells of BALB/c mice, whereas a moderate concentration (3 μg/ml) of Con A induced proliferation of these cells. A high concentration of Con A also induced the death of splenic cells of athymic BALB/c-nu/nu mice and the bone marrow cells of BALB/c mice which mainly consist of non-T cells. However, any concentration (1-30 μg/ml) of Con A failed to induce the proliferation of these cells. Specific binding of tetrameric Con A to mannose-containing receptors was required for the induction of cell death. DNA fragmentation was observed by both laser flow cytometry and electrophoresis in Con A-stimulated T cells and non-T cells. This indicated that the mechanism of induction of apoptosis with Con A is not necessarily TCR-dependent. Con A induced tyrosine phosphorylation of a number of proteins in various types of cells. Interestingly, phosphorylation of the 40 kDa protein developed only in the thymocytes and spleen cells that contain T cells, whereas phosphorylation of the 80 and 120 kDa proteins appeared in both T cells and non-T cells. These results suggested that the Con A-induced apoptosis of T cells and non-T cells involves different but possibly mutually related protein tyrosine phosphorylation-linked signals.     

Characteristics of immunosuppressive macrophages induced in spleen cells by Mycobacterium avium complex infections in mice

The profile of generation and characteristics of splenic macrophages (M phi s) which suppress the concanavalin A (Con A) mitogenic response of splenic T cells (designated as 'immunosuppressive M phi s') in host CBA/JN mice during the course of Mycobacterium avium complex (MAC) infection were investigated. In MAC-infected mice, reductions in some cellular functions of host splenic T cells, such as the Con A mitogenic response and mixed leucocyte reaction, were seen around 2 weeks after challenge of organisms, and this was accompanied by appearance of immunosuppressive M phi s in spleen cells. In this case, increase in immunosuppressive M phi activity was seen in terms of both activity per spleen and activity per individual M phi. In this phase of the infection, MAC-induced splenic M phi s showed a markedly increased ability to produce reactive oxygen radicals in response to phorbol myristate acetate. Thus, the expression of suppressor activity of MAC-induced M phi s seems to be closely linked to their activated state. A large proportion of the immunosuppressive M phi s exhibited suppressor activity dependent on prostaglandins and membrane functions related to microfilaments. It was also found that the generation of IL-2-reactive T cell populations in response to Con A was markedly inhibited by MAC-induced splenic M phi s, whereas they caused no significant reduction in the IL-2-producing ability of normal spleen cells.     

Immunodeficiency of aging: restorative effects of phorbol ester combined with calcium ionophore

Current models for lectin-induced T cell proliferation suggest that activation of protein kinase C (PK-C) and elevation of cytoplasmic Ca2+ may both play important roles in the earliest phases of signal transduction. To learn more about the relative inability of T cells from old mice to proliferate in response to mitogenic stimuli, we attempted to stimulate T cells by the synergistic effects of a PK-C activator, phorbol myristate acetate (PMA), and the calcium ionophore ionomycin. T cells from young mice respond as well to optimal combinations of these agents as they do to the strong polyclonal activator Con A, but T cells from old mice respond much better to PMA plus ionomycin than they do to Con A. This result suggests that an inability to transduce the signal supplied by extracellular ligands into the intracellular signals represented by Ca2+ and PK-C activators may underlie the age-associated loss of T cell reactivity. We also found evidence for a second defect in old T cells related to their response to elevated intracellular Ca2+: old T cells, compared with young, required higher levels of ionomycin for maximal proliferation.     

Apolipoprotein A-II suppressed concanavalin A-induced hepatitis via the inhibition of CD4 T cell function

Con A-induced hepatitis has been used as a model of human autoimmune or viral hepatitis. During the process of identifying immunologically bioactive proteins in human plasma, we found that apolipoprotein A-II (ApoA-II), the second major apolipoprotein of high-density lipoprotein, inhibited the production of IFN-γ by Con A-stimulated mouse and human CD4 T cells. Con A-induced hepatitis was attenuated by the administration of ApoA-II. The beneficial effect of ApoA-II was associated with reduced leukocyte infiltration and decreased production of T cell-related cytokines and chemokines in the liver. ApoA-II inhibited the Con A-induced activation of ERK-MAPK and nuclear translocation of NFAT in CD4 T cells. Interestingly, exacerbated hepatitis was observed in ApoA-II-deficient mice, indicating that ApoA-II plays a suppressive role in Con A-induced hepatitis under physiological conditions. Moreover, the administration of ApoA-II after the onset of Con A-induced hepatitis was sufficient to suppress disease. Thus, the therapeutic effect of ApoA-II could be useful for patients with CD4 T cell-related autoimmune and viral hepatitis.     

Diminished calcium influx in lectin-stimulated T cells from old mice

T lymphocytes from aged donors function poorly, but the biochemical basis for the defect remains uncertain. We tested the hypothesis that T cells from old mice had a diminished ability to transmit extracellular signals into the cytoplasm, by measuring intracellular free calcium concentrations (Cai) in T cells stimulated by the polyclonal activator concanavalin A (Con A). Using the second-generation fluorochrome indo-1 as a reporter of Cai, we found that the Con A-induced elevation of Cai levels is reduced both in rate and extent in old T cells, as compared to T cells from young mice. Flow cytometric analysis showed that this age-sensitive change represents a decline, with age, in the number of T cells that can respond to Con A by increasing their Cai above resting baseline levels (100-120 nM). These results thus show that defects in activation are manifested by T cells from old donors within the first 5 minutes of the activation process, and suggest that aging may lead to alterations either in the surface molecules that receive extracellular signals, or in the sequence of coupled events by which these extracellular signals bring about alterations in the intracellular ionic milieu.     

The effect of aging on the induction of humoral and cellular immunity and tolerance in two long-lived mouse strains.

Aging is a complex process that adversely affects most if not all components of the immune system. In this report, two long-lived mouse strains have been compared in ability to generate both antigen-specific immunity and tolerance. Although CBA/CaJ mice produced high levels of antibody following injection of aqueous preparations of aggregated human gamma-globulin (AHGG), C57BL/6 mice made only meager antibody responses to such preparations. Age dramatically affects the humoral anti-HGG response to aqueous AHGG in both strains, but the meager response of young C57BL/6 mice was at insignificant levels in aged C57BL/6 mice. Conversely, both mouse strains generated good responses following injection of HGG in complete Freund's adjuvant at both the T and B cell level as evidenced by in vitro antigen-specific T cell proliferation and anti-HGG antibody production. Aged mice of both strains showed a marked decrease in the production of serum anti-HGG antibody in comparison to young mice. Although the antigen-specific T cell proliferative response was significantly decreased in aged CBA/CaJ mice, such proliferation was not affected in aged mice of the C57BL/6 strain. Removal of CD8+ cells from lymph node T cells of either young or aged C57BL/6 mice did not increase the antigen-specific proliferative response, suggesting that loss of CD8+ suppressors during the aging process is not responsible for the high level of antigen-specific T cell proliferation in aged C57BL/6 mice. Tolerance to HGG was readily induced in both young and aged C57BL/6 and CBA/CaJ mice although aged mice demonstrate a modest resistance to tolerance induction when compared to their young counterparts. This resistance was observed in both antibody production and antigen-specific T cell proliferation.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Polyclonal activation of xid B cells by auto-Ia-reactive T cell clones

The mechanism of T cell-dependent activation of xid B cells into Ig-producing cells was studied by employing H-2-restricted, antigen-specific T cell clones. Helper factors (B cell stimulatory factors, BSF) released from KLH-specific T cell lines could induce polyclonal Ig production in B cells from (CBA/N X BALB/c)F1 (NBF1) female mice but not from CBA/N or NBF1 male mice. Direct addition of helper T cell lines induced Ig production in xid B cells from CBA/N or NBF1 male mice. A T cell clone, MK6, which was derived from NBF1 male mice and specific against Iad determinant, could activate NBF1 male but not CBA/N B cells. Another clone, CK4, derived from CBA/N mice and having specificity against KLH plus I-Ak determinant could activate both CBA/N and NBF1 male B cells into IgM- and IgG-producing cells in the absence of KLH, and monoclonal anti-I-Ak antibody specifically blocked such activation. These results suggest that xid B cells are able to be activated by the signal provided by the recognition of Ia molecules on B cells by auto-Ia-reactive T cells. Xid B cells from CBA/N mice that had been co-cultured with a T cell line specific against I-Ak determinant for 24 hr became reactive to BSF and capable of differentiating into Ig-producing cells in the presence of BSF. The results showed that even xid B cells could be responsive to BSF if they were in a certain activation stage.     

Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-beta-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted [3H]thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of AC contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by planning, even when these cells were cultured at high density. When PMA was added to T cell cultures supported by optimal numbers of M phi, catalase-reversible suppression of responses was noted. Even in cultures containing catalase, PMA failed to enhance responsiveness above that supported by optimal numbers of M phi. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. PMA could not support responses even in cultures supplemented with interleukin 1-containing M phi supernatants or purified interleukin 2 alone or in combination. Similar results were found in high-density cultures of T cells depleted of Ia-bearing cells. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.     

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.     

The effect of aging on the induction of experimental autoimmune thyroiditis

The effect of age on the ability to elicit the various immune functions comprising experimental autoimmune thyroiditis in mice has been examined. Compared with young mice (2 to 3 mo), CBA/CaJ and A/J aged mice (20 to 30 mo) show a drastic reduction in their ability to develop circulating antibody after injection of mouse thyroglobulin (MTg) or mouse thyroid extract (MTE) in complete Freund's adjuvant (CFA). Delayed-type hypersensitivity responses were also depressed, as well as the ability of aged lymph node cells to proliferate in vitro to antigen and the ability of aged splenic T cells to function as helper cells for in vitro antibody production. However, after injection of these thyroid antigens in CFA, aged mice developed thyroid lesions either comparable to or only slightly less intense than those observed in young mice. The disparity between the levels of immune responses and thyroid lesions observed in aged mice can be explained by the greater susceptibility of aged thyroids to tissue damage, since transfer of identical numbers of Con A-activated MTE-primed young splenocytes to young and aged recipients results in a more severe thyroiditis in the aged recipients. Priming mice to MTE in CFA at 9 mo of age, at which time mice are responsive to MTE, did not enhance either T or B cell responsiveness to injection of MTE in CFA at 24 mo of age. Lymphocytes from MTE-injected aged mice also failed to transfer thyroiditis to young recipients after in vitro activation of the lymphocytes with Con A.     

The effect of hemopoietic microenvironment on splenic suppressor macrophages in congenitally anemic mice of genotype Sl/Sld

Mechanisms underlying mononuclear phagocyte specialization are being probed by studying suppressor macrophages (M phi) as a reference population in mouse models with impaired blood monocyte formation. Splenic suppressor M phi, defined by PGE-mediated inhibition of Con A-induced T lymphocyte proliferation are induced by the i.p. administration of Corynebacterium parvum (CP). Mice severely depleted of bone marrow and blood monocytes by treatment with 89Sr fail to show this suppressor M phi response to CP, although M phi-forming stem cells, assessed as splenic M-CFC in vitro, are increased 20-fold. These observations suggest that radiosensitive bone marrow stem cells are necessary for the generation of both suppressor M phi and monocytes and that one such stem cell may be common to both types of mononuclear phagocytes. This notion was explored further by employing congenitally anemic mice of the genotype S1/S1d in which the hemopoietic microenvironment is genetically defective and thus unable to support the proliferation, differentiation, and function of stem cells. The congenital defect was found to be additionally expressed in the S1/S1d mouse by a monocytopenia of less than 10% of the values in normal congenic littermate controls and by the failure of splenic M-CFC to increase in response to CP. PGE-producing suppressor M phi expressing Fc gamma 2b receptors, however, were induced by CP in S1/S1d mice with no significant diminution of suppressor activity. These data establish the fact that significant impairment of the formation of monocytes is part of the overall hemopoietic defect in S1/S1d mice. PGE-producing suppressor M phi, however, were inducible at normal functional levels in the presence of a profound monocytopenia, and therefore appear to be independent of the mechanisms that regulate blood monocyte formation. Ablation of the bone marrow with 89Sr resulted in failure of CP to induce suppressor M phi in the spleens of the S1/S1d mice as in the littermate controls. Other observations in the present study, when taken with data from the 89Sr model, show the additional independence of these suppressor M phi from splenic M-CFC. In aggregate, these findings delineate three functionally definable populations of mononuclear phagocytes that appear to be independently regulated.     

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)     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Dissociation between macrophage tumoricidal capacity and suppressive activity: analysis with macrophage-defective mouse strains

Macrophages (M phi diameter) from three mouse strains with genetically distinct M phi diameter deficits (C3H/HeJ, A/J, and P/J) were unable to develop high cytolytic and cytotoxic activity against tumor cells in vitro when exposed to agents (MAF and IFN-beta) that strongly increased the tumoricidal capacity of M phi diameter from nondefective C3H/HeN mice. Nevertheless, the tumoricidal deficits of M phi diameter from the defective strains did not affect their suppressive capacity on Con A-induced lymphoproliferation, nor their ability to react to IFN-beta by decreasing suppressive activity. In fact, natural suppressive activity and IFN-beta-induced changes in the suppression of M phi diameter from C3H/HeJ, A/J, and P/J mice were highly comparable to those of C3H/HeN M phi diameter, thus stressing the dissociation between the mechanisms governing M phi diameter suppression and M phi diameter tumoricidal activity. Analysis of the modulation by MAF and IFN-beta of M phi diameter ability to release the oxygen metabolites O2- and H2O2, molecules possibly involved in the effector mechanism of both M phi diameter cytotoxicity and suppression, revealed a close correlation with the patterns of suppressive activity in both nondefective and defective strains. In contrast, no correlation between the production of oxygen-reactive species and M phi diameter tumoricidal activity was observed. The ability of MAF- and IFN-beta-treated M phi diameter to produce PGE, a molecule of major importance in M phi diameter-mediated suppression and possibly involved also in the regulation of M phi diameter tumoricidal activity, again paralleled M phi diameter suppressive capacity. Thus, the mechanisms controlling M phi diameter antitumor activity appeared to be clearly distinct from those involved in M phi diameter suppression.     

Simultaneous suppression of allogeneic cytolytic activity and stimulation of lectin-dependent cytolytic activity by con A.

It is well known that when mouse lymphocytes are cultured with irradiated allogeneic stimulator lymphocytes, T cells capable of mediating specific cytolysis are generated. We noted that when the stimulator cells were pretreated with concanavalin A (Con A), the generation of T cell-mediated specific lysis (TSL) is largely abolished, but large amounts of T cell-mediated lectin-dependent lytic (LDL) activity are nevertheless produced. Data are presented indicating that generation of TSL is inhibited by suppressor cells which are generated by the Con A in the culture.One possible source of the LDL would be a specific clonal response to Con A-altered H-2 molecules. Evidence concerning this was equivocal since the amount of lectin required tor expression of the LDL is suffcient to cause non-specific T cell-mediated lysis of any target cell type, making inoperative the conventional tests for H-2 restricted recognition.Use of dead cell fragments for stimulation in MLC has previously been reported to produce LDL without TSL, but Con A-induced premature death of the stimulator cells was ruled out as a source of the LDL in our cultures.Another possible source of LDL would be suppressor-induced blockade of killer cell differentiation at a hypothetical stage expressing LDL but not TSL. However, delayed addition of Con A induced suppressor cells to M LC's never allowed generation of LDL when TSL was suppressed. Therefore, such a blocked differentiation mechanism did not contribute significantly to the LDL produced.The LDL activity results largely from Con A-induced polyclonal activation of cytolytic progenitor cells. It is shown that generation of LDL by Con A is able to be suppressed by Con A-induced suppressor cells added at the initiation of culture. However, in cultures in which Con A is simultaneously generating LDL and suppressor activities, the LDL is generated rapidly (in 2 days) and apparently passes the suppressable stage before the suppressor cells become active.