Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice

Ceramide generated by lysosomal acid sphingomyelinase (aSMase) has been proposed to contribute to CD28 co-stimulatory signaling pathways. We used an aSMase-deficient mouse line (asmase-/-) to elucidate the role of the aSMase in splenocytes stimulated with either a combination of anti-CD3 and anti-CD28 antibodies, the lectin concanavalin A (Con A) or the superantigen staphylococcal enterotoxin B. All stimuli were shown to induce IL-2 expression, Con A additionally triggered the expression of high-affinity IL-2 receptor. However, in asmase-/- mice secretion of IL-2 was significantly reduced, whereas the intracellular IL-2 levels were elevated. Proliferation of anti-CD3/anti-CD28 or Con A-stimulated aSMase-deficient splenocytes was reduced up to 50% after 72 h in comparison to wild-type cells. We conclude that ceramide generated by aSMase is not involved in CD28 signal transduction, but rather a perturbation of the secretory system is responsible for the impaired proliferation of aSMase-deficient splenocytes.     

Tumor-specific activation of mitogen-activated protein kinase in human colorectal and gastric carcinoma tissues

In the present study we investigated the effect of anti-CD3 stimulation on IL-3-induced histamine, IL-6, and IL-4 synthesis by murine hematopoietic precursor cells. These activities were strikingly decreased in splenocytes from mice that had received a single intravenous injection of 10 microg of anti-CD3 monoclonal antibody (mAb) 24 hours previously. A similar inhibition occurred after 24-hour in vitro stimulation of normal spleen cells with 1 microg/mL of anti-CD3 mAb. In both situations the inhibitory effect depended on T cell activation in that treatment with F(ab')2 fragments of anti-CD3 did not diminish secretion of histamine and cytokines. Cross-linking of Fas antigen on spleen cells mimicked the action of anti-CD3, provided that interferon (IFN)-gamma was present during the incubation period. Substantial amounts of this cytokine were detected in spleen cell supernatants, which were able to replace recombinant IFN-gamma during Fas receptor cross-linking. This effect was entirely mediated by IFN-gamma, as assessed by its neutralization in the presence of anti-IFN-gamma mAbs. In contrast to splenocytes, bone marrow cells responded normally to IL-3 after in vivo or in vitro stimulation with anti-CD3. They were also not affected by combined treatment with anti-Fas mAb and IFN-gamma. Together, our data support the notion that the decrease in IL-3-induced histamine and IL-6 production by splenocytes pretreated with anti-CD3 is mediated, at least in part, by Fas/FasL interactions, suggesting that the activity of extramedullary myeloid precursor cells can be modulated by molecules involved in apoptosis.     

T cell stimulus-induced crosstalk between lymphocytes and liver macrophages results in augmented cytokine release

Polyclonal T cell stimulation in humans leads to a cytokine burst syndrome that may result in organ failure or lethality. Mechanisms of such cytokine-dependent morbidity can be studied in mice challenged with the T cell mitogen concanavalin A (Con A). In this model tumor necrosis factor (TNF)-dependent toxicity is characterized by a relatively selective liver failure. We examined here whether a crosstalk between liver macrophages and lymphocytes may be the underlying cause for the overshooting TNF response. Lymphocytes from lymph nodes, thymus, or the spleen were cocultured with Kupffer cells and stimulated with the polyclonal T cell stimuli Con A, anti-CD3 mAb, or staphylococcal enterotoxin B. We observed a rapid and synergistically augmented release of TNF, and also of IL-1, IL-2, IL-4, IL-6, and IFN-gamma, compared to stimulation of the individual cell types alone. This dramatically upregulated cytokine response did not require direct cell contact, but was mediated by a soluble factor. In order to find out whether TNF upregulation would require additional cell types in the liver, we used cocultures of T cells and a macrophage cell line and confirmed our previous results. In this model system an increase in TNF mRNA was observed in macrophages, but not in T cells. We conclude that the T cell-macrophage crosstalk following polyclonal T cell stimulation may be responsible for an overshooting TNF release from macrophages. This mechanism finally may lead to organ damage such as liver injury upon Con A injection into mice.     

Glucose-modified low density lipoprotein enhances human monocyte chemotaxis

In response to stimulation with immobilized anti-CD3 antibody, splenocytes from C57BL/6 and BALB/c mice principally produced INF-gamma and IL-4, respectively. However, both splenocytes equally proliferated in response to ConA. We compared the changes after inoculation with BCG (1 mg/mouse) in their capacity to produce IL-4 or IFN-gamma in response to anti-CD3 antibody and to proliferate in response to ConA. Splenocytes from C57BL/6 and BALB/c mice, that had been inoculated with BCG 4 weeks before, produced IFN-gamma with diminished IL-4 production in response to anti-CD3 antibody. Furthermore these splenocytes became anergic to ConA stimulation and died due to cell apoptosis in stead of proliferation. However, we observed the strain difference at 12 weeks after BCG-infection. BCG-primed C57BL/6 splenocytes, that continuously produced IFN-gamma in response to anti-CD3 antibody, failed to proliferate in response to ConA. In contrast, BCG-primed BALB/c splenocytes, that increased IL-4 production but decreased IFN-gamma production when stimulated with anti-CD3 antibody, could proliferate well in response to ConA. Since the splenocytes of BALB/c mice became ConA responsive along with their shifting from Th1 dominant immune response at 4 weeks to Th2 dominant immune response at 12 weeks after BCG-inoculation, IL-4 was assumed to play a crucial role in activation of anergic T cells. Therefore, we stimulated splenocytes from both strains of mice infected with BCG 4 weeks before with ConA in the presence or absence of IL-4. Splenocytes from BCG-infected BALB/c mice showed marked proliferation, while those from BCG-infected C57BL/6 mice failed. We found that IL-4 protected against ConA-induced cell apoptosis in BALB/c splenocytes but not C57BL/6 splenocytes.     

Human interleukin-10 can directly inhibit T-cell growth

Human interleukin-10 (IL-10) inhibits T-cell proliferation and cytokine production in the presence of monocytes. In this study, we have investigated whether IL-10 can directly inhibit T cells. Highly purified peripheral blood T cells containing less than 0.1% CD14+ cells and unresponsive to phytohemagglutinin (PHA), were growth-inhibited by IL-10 when stimulated with immobilized OKT3 monoclonal antibody (MoAb; 55.4% inhibition). This effect was neutralized by the murine MoAb 19F1 directed against human IL-10. In addition, IL-10 inhibited by 52.5% the proliferation of a human tetanus toxoid-specific T-cell clone (TM11) induced by immobilized OKT3 MoAb in the absence of antigen-presenting function. T-cell growth inhibition by IL-10 did not reflect a cytokine-induced change in the kinetics of T-cell response to immobilized OKT3 MoAb, and was observed over a wide range of cell and OKT3 MoAb concentrations. Addition of 1% to 5% monocytes to highly purified peripheral blood T cells resulted in the emergence of proliferation to PHA and to soluble OKT3 MoAb, but did not significantly affect levels of growth inhibition by IL-10 in the presence of immobilized OKT3 MoAb. Similarly, addition of 10% monocytes to the TM11 T-cell clone resulted in the emergence of proliferation in response to tetanus toxoid, but did not significantly influence growth inhibition by IL-10 in the presence of immobilized OKT3 MoAb. When stimulated with immobilized OKT3 MoAb in the absence of accessory cells, T cells secreted IL-2. Secretion of IL-2 under these conditions was inhibited by IL-10 (51.5% inhibition). Thus, IL-10 can directly inhibit growth and IL-2 production in T cells triggered by immobilized OKT3 MoAb in the absence of monocytes.     

Adoptive transfer of anti-CD3-activated CD4+ T cells plus cyclophosphamide and liposome-encapsulated interleukin-2 cure murine MC-38 and 3LL tumors and establish tumor-specific immunity

The infusion of anti-CD3-activated murine T cells plus interleukin-2 (IL-2) exerts antitumor effects against several tumors in murine immunotherapy models. This study compares the therapeutic efficacy of anti-CD3-activated CD4+ or CD8+ T-cell subsets, when given with cyclophosphamide (Cy) and liposome-encapsulated IL-2 (L-IL2) in a murine model. C57BL/6 mice bearing subcutaneous (S.C.) MC-38 colon adenocarcinoma, 3LL Lewis lung carcinoma, or 38C13 lymphoma for 7 to 14 days were pretreated with low-dose intraperitoneal (I.P.) Cy before intravenous (I.V.) injection of anti-CD3-activated T cells or T-cell subsets. Cell administration was followed by I.P. administration of L-IL2 for 5 days. Mice receiving activated CD4+ T cells showed significantly reduced tumor growth or complete remissions with prolonged disease-free survival in MC-38, 3LL, and 38C13. The timing of Cy doses in relation to adoptive transfer was critical in obtaining the optimal antitumor effect by CD4+ cells. Injecting Cy 4 days before the infusion of CD4+ cells greatly enhanced the antitumor effect of the CD4+ cells and improved survival of the mice compared with other Cy regimens. C57BL/6 mice cured of MC-38 after treatment with CD4+ T cells developed tumor-type immunologic memory as demonstrated by their ability to reject rechallenges with MC-38, but not 3LL. Similarly, mice cured of 3LL tumors rejected rechallenges of 3LL, but not MC-38. The immunologic memory could be transferred with an I.V. injection of splenocytes from mice cured of MC-38 or 3LL. No cytotoxic T-lymphocyte activity was detected in T cells or T-cell subsets from mice cured of MC-38 or 3LL. Increased IL-2 and interferon-gamma (IFN-gamma) production was observed from CD4+ subsets in cured animals when stimulated in vitro with the original tumor, but not with an unrelated syngeneic tumor. These results suggest that tumor-specific immunity can be achieved in vivo with anti-CD3-stimulated CD4+ T cells in this cellular therapy model.     

Autologous bone marrow support and bone disease in metastatic breast cancer

Production of IgM, IgG and IgA was induced from human blood B lymphocytes by culturing with a CD40 MoAb and IL-2 for 9 days. Replacement of IL-2 by IL-10 markedly enhanced production of all three isotypes. High levels of immunoglobulin production also occurred when activated irradiated autologous T cells replaced the CD40 MoAb, and when IL-10 replaced IL-2 in these cultures a spectacular increase in IgG production occurred. The effectiveness of the T cell stimulus depended on the mode of purification of the T cells and the nature of the stimulant used to activate them. Differences in the kinetics and level of expression of CD40L on the various T cell preparations were observed, but did not account for variations in immunoglobulin-inducing efficiency. Immunoglobulin production from sIgD+ and sIgD- B cells was investigated. IgG and IgA were found in sIgD+ cultures, indicating that some isotype switching had occurred, but the major part of the IgG and IgA secreted was from cells already committed to these isotypes. Anti-IgD or anti-IgM MoAbs enhanced the proliferation of B cells induced by anti-CD40 antibody, but immunoglobulin production was not enhanced. Factors affecting the balance of proliferation and differentiation are discussed.     

T cell activation modulates retrovirus-mediated gene expression

Important considerations for T lymphocyte-based gene therapy include efficient gene delivery and expression in primary, human T cells. In this study, retrovirus-mediated gene transfer and the fate of proviral gene expression were evaluated in human T cells activated using (1) immobilized anti-CD3 monoclonal antibody (MAb) plus interleukin 2, or (2) cis costimulation using beads carrying coimmobilized anti-CD3 and anti-CD28 MAbs. By cross-linking the CD3 and CD28 receptors, these MAbs mimic in vivo signaling events, leading to cytokine production and proliferation. A modified human interleukin 1beta (IL-1beta) cDNA inserted into the MFG retroviral vector served as an indicator gene. Retroviral transduction frequencies were similar for T lymphocytes activated by the respective methods. However, early after MAb stimulation and virus exposure, proviral gene expression was greater at the RNA and protein levels in optimized anti-CD3/anti-CD28 bead-activated T cells, corresponding with augmented endogenous cytokine responses and mitogenesis. Proviral gene expression was not regulated by extrinsic cell factors present in activated T cell supernatants. Regardless of the MAb stimulation method, proviral IL-1beta expression declined in later T cell cultures concomitant with a decrease in cellular cytokines. Restimulation by either method reinduced both T cell activity and vector expression. Our finding that proviral gene regulation is downmodulated in the absence of T cell signaling events has implications for clinical strategies using retrovirus-modified T cells.     

Cytokine production by lymph node cells from mice infested with Ixodes ricinus ticks and the effect of tick salivary gland extracts on IL-2 production

In BALB/c mice repeatedly infested with nymphal Ixodes ricinus ticks, lymphocytes from axillary and brachial lymph nodes which drain the tick attachment site produced significant levels of IL-2, TNF-alpha and GM-CSF when stimulated in vitro with Con A or anti-CD3 antibodies. Cytokine production by cells from lymph nodes of the opposite flank was equivalent to that of cells from uninfested mice. Nine days after the first infestation and IL-2, GM-CSF were produced primarily by the CD4+ T cells, while some other cell types contributed also to the TNF-alpha production. In mice repeatedly infested, a gradual increase of lymph node cell production of IL-2 was observed. The IL-2 levels regularly increased from the first to the third infestation compared to TNF-alpha levels which gradually decreased. The in vitro production of GM-CSF was not affected by successive infestations. Spleen lymphocytes from naive mice produced higher levels of IL-2 than lymphocytes from axillary and brachial lymph nodes. Both tick salivary gland extracts and D-mannose inhibited IL-2 production by these lymphocytes.     

A T cell activation antigen, Ly6C, induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8+ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8+ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C+ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G0/G1 to S+G2/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4+ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4+ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.     

Injection of complementary DNA encoding interleukin-12 inhibits tumor establishment at a distant site in a murine renal carcinoma model

Interleukin (IL-12) protein has been shown to elicit diverse immunological responses and potent antitumor activity. We demonstrate here that intradermal injection of IL-12 cDNA induces systemic biological effects characteristic of the cytokine in vivo. Intradermal injection of IL-12 cDNA resulted in local expression of IL-12 mRNA, which correlated with a 10-fold increase in natural killer activity and a 3-4-fold increase in anti-CD3-induced IFN-gamma production in cultured splenocytes. Furthermore, when challenged with Renca tumor cells at a distant site, the day of tumor emergence was significantly delayed, and tumor growth was reduced in mice that received IL-12 cDNA, compared to mice given injections of plasmid vector alone. A number of the mice receiving IL-12 cDNA injections remained tumor free months after tumor challenge. In contrast to mice receiving recombinant IL-12 protein, no splenomegaly was detected when natural killer activity was significantly induced in mice receiving injections of IL-12 cDNA. Because purified plasmid DNA is more economical to prepare and has a longer shelf-life than recombinant proteins, and intradermal administration of cDNA encoding IL-12 did not cause splenomegaly, our findings suggest that the in vivo injection of cDNA encoding IL-12 may be a less toxic and more cost-effective alternative to IL-12 protein therapy in some clinical or experimental therapeutic applications.     

MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.     

Costimulation through CD86 is involved in airway antigen-presenting cell and T cell responses to allergen in atopic asthmatics

Atopic allergic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Previous reports have suggested an important role for costimulation through the CD28/CTLA4-CD80/CD86 pathway in allergen activation of T cells in animal models of inhaled allergen challenge. However, human allergen-specific lines and clones were reported to be costimulation independent. We therefore examined CD80 and CD86 dependence of allergen-induced T cell proliferation and cytokine production in peripheral blood and bronchoalveolar lavage from atopic asthmatic subjects and controls. Both allergen-induced proliferation and IL-5 production from PBMC were inhibited by CTLA4-Ig fusion protein and anti-CD86, but not anti-CD80 mAbs. When allergen-specific CD4+ T cell lines from peripheral blood were examined, proliferation and cytokine production were found to be independent of CD80 or CD86 costimulation. However, when cells obtained directly from the airways were examined, allergen-induced proliferation of bronchoalveolar lavage T cells from atopic asthmatic subjects was inhibited by anti-CD86 but not anti-CD80. In addition, bronchoalveolar lavage-adherent cells from asthmatic, but not control subjects showed APC activity to autologous T cells. This was also inhibited by anti-CD86 but not anti-CD80. Thus allergen-induced T cell activation and IL-5 production in the airway in asthmatic subjects is susceptible to blockade by agents interfering with costimulation via CD86, and this may hold therapeutic potential in asthma.     

Augmentation of naive, Th1 and Th2 effector CD4 responses by IL-6, IL-1 and TNF

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-gamma, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-gamma and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.     

Identification of the immunodominant T cell epitope of p38, a major egg antigen, and characterization of the epitope-specific Th responsiveness during murine schistosomiasis mansoni

A recently cloned major Schistosoma mansoni egg Ag p38 induced and elicited strong Th1-type responsiveness in mice of H-2k haplotype. Now, we have identified the immunodominant T cell epitope of p38 and analyzed the dynamics of epitope-specific Th responsiveness during murine schistosomiasis mansoni. Overlapping recombinant and synthetic peptides that encompassed the full-length 354 amino acid of p38 were tested for proliferation and cytokine production in peptide- or p38-sensitized mice. The immunodominant T cell epitope of p38 that elicited pulmonary granuloma formation was localized within peptide P4 (amino acids 235-249). The P4-specific cytokine response of splenocytes that had been sensitized s.c. with p38, P4 or soluble egg Ags in IFA, or i.p. with 3000 eggs was predominantly as the Th1 type, with strong IL-2 and IFN-gamma, but trace amounts of IL-4 and IL-5 secretion. At 6.5 wk of infection, splenocytes and mesenteric lymph node cells responded to p38/P4 peptides with predominantly Th1-type responsiveness. This response did not switch to a Th2-type pattern from 8 wk onwards; rather, it underwent down-modulation. Moreover, the hepatic granuloma lymphocytes at 6.5 wk responded to p38/P4 predominantly with Th1-type cytokine production, indicating that they participate in early granuloma formation. From 8 wk onwards an immune deviation to the p38-specific response was observed that was manifested by rising IgG1, IgE, and IgG2a Ab production as opposed to declining Th1- and Th2-type cytokine secretion.     

Antigen-specific T-cell activations distinguished by in vivo anti-CD4 antibody treatment

This study identifies activation characteristics of PPD-responsive T-cells that emerge after treatment with anti-CD4 monoclonal antibody (Mab). PPD-stimulated T-cell proliferations, OX40 phenotype and protein tyrosine phosphorylations involving p56lck (pp56lck) were compared to Con A stimulations using T-cells isolated from spleen and draining lymph node of CFA/PPD-immunized rats either untreated or treated in vivo with anti-CD4 Mab. Splenocytes stimulated by concanavalin A (Con A) showed correlated increases in proliferation, levels of pp56lck, and OX40 expression; these parameters were not correlated in splenocytes after PPD-stimulations. T-cells isolated from lymph nodes draining the site of CFA/PPD immunization proliferated in response to stimulation by either PPD or Con A, but only PPD-responsive cells showed correlation to the OX40 activation phenotype and increased levels of pp56lck. CD4+ T-cells isolated from either tissue compartment after anti-CD4 Mab treatments showed higher background and PPD-stimulated proliferations, and expressed lower levels of OX40. In contrast, anti-CD4 Mab treatments reduced (60%) and abolished Con A-stimulated proliferations of splenocytes and lymph node T-cells, respectively. The effects of anti-CD4 Mab treatment on pp56lck levels correlated only to the changes observed for Con A stimulations of splenocytes. These results demonstrate that PPD antigen-specific T-cell populations recovered from different tissue compartments were resistant to in vivo anti-CD4 Mab treatments and did not show the activation changes characteristics of CD4+ T-cells after Con A stimulation.