Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Engagement of CD14 on human monocytes terminates T cell proliferation by delivering a negative signal to T cells.

We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti-CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative signal to T cells, rather than from disruption of a costimulatory monocyte-derived signal, because incubation of monocytes with anti-CD14 mAb also inhibited monocyte-independent T cell proliferation induced by PMA and ionophore. These results, together, point to a role of CD14 in the monocyte-dependent regulation of T cell proliferation.     

CD11b+Ly-6C(hi) suppressive monocytes in experimental autoimmune encephalomyelitis

Innate immune cells may regulate adaptive immunity by balancing different lineages of T cells and providing negative costimulation. In addition, CD11b(+)Gr-1(+) myeloid-derived suppressor cells have been described in tumor, parasite infection, and severe trauma models. In this study, we observe that splenic CD11b(+) cells markedly increase after experimental autoimmune encephalomyelitis (EAE) immunization, and they suppress T cell proliferation in vitro. Although >80% of CD11b(+) cells express varying levels of Gr-1, only a small population of CD11b(+)Ly-6C(high) inflammatory monocytes (IMC) can efficiently suppress T cell proliferation and induce T cell apoptosis through the production of NO. IFN-gamma produced by activated T cells is essential to induce IMC suppressive function. EAE immunization increases the frequencies of IMC in the bone marrow, spleen, and blood, but not in the lymph nodes. At the peak of EAE, IMC represent approximately 30% of inflammatory cells in the CNS. IMC express F4/80 and CD93 but not CD31, suggesting that they are immature monocytes. Furthermore, IMC have the plasticity to up-regulate NO synthase 2 or arginase 1 expression upon different cytokine treatments. These findings indicate that CD11b(+)Ly-6C(high) IMC induced during EAE priming are powerful suppressors of activated T cells. Further understanding of suppressive monocytes in autoimmune disease models may have important clinical implications for human autoimmune diseases.     

Platelet factor 4 inhibits proliferation and cytokine release of activated human T cells

Platelet factor 4 (PF-4), a platelet-derived CXC chemokine, has been shown to induce the differentiation of monocytes into a subset of macrophages that lack the expression of HLA-DR Ag. This suggests a potential role for PF-4 in the modulation of monocyte-dependent T cell activation. Using an Ag-specific stimulation model in which T cells were cocultured with monocytes in the presence of recall Ags, we could show that under these conditions PF-4-treatment caused a strong decrease of T cell proliferation as well as of IFN-gamma release. However, inhibition of T cell functions such as proliferation, IL-2 release, and IL-2 mRNA production did also occur when isolated T cells were activated in the absence of monocytes with immobilized Abs directed against CD3 in combination with cross-linked anti-CD28 Abs. The effect could be reversed when low concentrations of exogenous IL-2 instead of anti-CD28 were used as a costimulus in combination with anti-CD3 Abs. Further evidence for direct modulation of T cell function by PF-4 was obtained by the detection of specific binding sites for the chemokine on the surface of these cells. Taken together, our results show that specific binding of PF-4, resulting in the down-regulation of the IL-2-release correlates with the inhibition of functions in activated T cells.     

Analysis of the role of leukocyte function-associated antigen-1 in activation of human influenza virus-specific T cell clones

The role of leukocyte function-associated Ag-1 (LFA-1) in intercellular adhesion is well documented. Previously, we demonstrated that the LFA-1 molecule (CD11a/CD18) can also regulate the induction of proliferation of peripheral blood T cells. In these studies, we observed opposite effects of antibodies against CD11a (LFA-1-alpha-chain) or CD18 (LFA-1-beta-chain). Here, we determined the effects of anti-CD11a and anti-CD18 mAb on proliferation of cloned influenza virus-specific T cells. Anti-CD18 mAb had similar inhibiting effects on the proliferative response of T cell clones induced by immobilized anti-CD3 mAb as it had on the response of peripheral blood T cells. In contrast to its costimulatory effect on resting peripheral blood T cells, anti-CD11a mAb did not increase the proliferation of cloned T cells. Similar differences in effects of anti-CD11a and anti-CD18 mAb were observed when proliferation of the T cell clones was induced by immobilized anti-TCR mAb. When proliferation was induced by influenza virus presented by monocytes as APC, both anti-CD11a and anti-CD18 mAb inhibited T cell proliferation. However, when EBV-transformed B cells were used as APC, neither anti-CD11a nor anti-CD18 mAb inhibited proliferation. These results demonstrate that the effects of antibodies against CD11a (LFA-1-alpha) or CD18 (LFA-1-beta) on T cell proliferation depend on 1) the stage of activation of the T cells, 2) the activation stimulus and its requirement for intercellular adhesion involving LFA-1, and 3) the type of cell used to present Ag.     

Engagement of CD28 modulates CXC chemokine receptor 4 surface expression in both resting and CD3-stimulated CD4+ T cells

Optimal CD4+ T cell activation requires the cooperation of multiple signaling pathways coupled to the TCR-CD3 complex and to the CD28 costimulatory molecule. In this study, we have investigated the expression of surface CXC chemokine receptor 4 (CXCR4) in enriched populations of CD4+ T PBL, stimulated with anti-CD3 and anti-CD28 mAbs, immobilized on plastic. Anti-CD3 alone induced a progressive down-regulation of surface CXCR4, accompanied by a significant decline in the entry of the HXB2 T cell line-tropic (X4-tropic) HIV-1 clone in CD4+ T cells. Of note, this effect was strictly dependent on the presence in culture of CD14+ monocytes. On the other hand, anti-CD28 alone induced a small but reproducible increase in the expression of surface CXCR4 as well as in the entry of HXB2 HIV-1 clone in resting CD4+ T cells. When the two mAbs were used in combination, anti-CD28 potently synergized with anti-CD3 in inducing the expression of CD69 activation marker and stimulating the proliferation of CD4+ T cells. On the other hand, anti-CD28 counteracted the CXCR4 down-modulation induced by anti-CD3. The latter effect was particularly evident when anti-CD28 was associated to suboptimal concentrations of anti-CD3. Because CXCR4 is the major coreceptor for the highly cytopathic X4-tropic HIV-1 strains, which preferentially replicate in proliferating CD4+ T cells, the ability of anti-CD28 to up-regulate the surface expression of CXCR4 in both resting and activated CD4+ T cells provides one relevant mechanism for the progression of HIV-1 disease.     

Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3

The capacity of the monoclonal antibodies (Mab) 64.1 and OKT3 directed at CD3 molecules to induce T4 cell proliferation and interleukin 2 (IL 2) production was examined. Each was tested in soluble form or was immobilized by adhering it to the wells of plastic microtiter wells. Soluble anti-CD3 did not induce proliferation of accessory cell (AC)-depleted T4 cells. In contrast, immobilized anti-CD3 induced T4 cell IL 2 production and proliferation in the complete absence of AC. When T4 cells were stimulated with high density immobilized anti-CD3, responses did not require AC, IL 2, or Mab directed at the Tp44 molecule (9.3). In contrast, responses stimulated by lower densities of immobilized anti-CD3 were enhanced by IL 2, AC, and 9.3, and with even lower densities of immobilized anti-CD3 proliferation, required these additional signals. A variety of other immobilized Mab directed at T cell surface proteins including class I major histocompatibility complex encoded gene products, CD2, CD5, 4F2, and Tp44, did not induce proliferation even in the presence of IL 2. Anti-CD4 Mab (66.1) inhibited immobilized anti-CD3-stimulated T4 cell responses, with a greater degree of inhibition noted when lower densities of immobilized anti-CD3 were used to stimulate T4 cells. The data demonstrate that stimulation of T4 cells by anti-CD3 is completely AC independent when the antibody is immobilized onto a surface. Furthermore, the results indicate that maximal stimulation requires multiple interactions with anti-CD3 without internalization of the CD3 molecule. The observation that additional signals are required to support T4 cell proliferation when the density of immobilized anti-CD3 is diminished suggests that these are necessary only when insufficient interactions with the CD3 molecule have occurred to transmit a maximal activation signal to the cell. Finally, the results indicate that anti-CD4 provides a direct inhibitory signal to the T4 cell, the effect of which is inversely proportional to the intensity of the activation signal.     

Activated peripheral T lymphocytes undergo apoptosis when cultured with monocytes activated by HLA class II ligation

We treated PBMC with anti-MHC class II mAb known to inhibit T lymphocyte proliferation. Adherent cells from mAb-treated PBMC showed increased metabolic activity by the MTS assay that was not due to cell proliferation. PBMC cultured with solid-phase anti-class II mAb in chamber inserts inhibited, across a membrane, the proliferation of PBMC cultured with soluble anti-CD3 mAb. PBMC treated with both soluble mAb underwent apoptosis as shown by nucleosomal DNA fragmentation. The monocytes formed multinucleated giant cells as shown by fluorescent microscopy, and contained apoptotic bodies as shown by the TUNEL method and by electron microscopy. The apoptotic cells were identified as T cells by double-staining with anti-CD4/CD8-PE and annexin-V-FITC. Thus, MHC class II ligation stimulates monocytes to increase their metabolic activity, induce apoptosis of activated T lymphocytes, and phagocytize the apoptotic cells. TCR-mediated ligation of MHC class II may play a role in the downregulation of immune responses.     

Role of IL-2 in the generation of CD4+ suppressors of human B cell responsiveness

The capacity of human T4 cells stimulated by immobilized monoclonal antibodies to the CD3 molecular complex (64.1 and OKT3) to induce and regulate B cell responsiveness was examined. T4 cells stimulated by low concentrations of immobilized 64.1 (3.0 ng/well) and all concentrations of immobilized OKT3 supported B cell proliferation and differentiation. High concentrations of immobilized 64.1 (200 ng/well) failed to stimulate help but rather induced suppression by T4 cells. Suppression was prevented by treating the T4 cells with mitomycin C. Suppression could not be accounted for by deprivation of IL-2. In contrast, induction of suppressor T4 cell activity was closely related to the amount of IL-2 produced by anti-CD3 stimulated T4 cells. Moreover, IL 2 appeared to facilitate the generation of suppressor T4 cell activity. Suppressor cell activity could be generated from unseparated T4 cells as well as from highly purified T4 cell subsets, including Leu 8-, CD45R+, and CD45R- T4 cells, after stimulation with immobilized 64.1. A primary action of suppressor T4 cells appeared to be the direct inhibition of B cell function, as evidenced by the finding that immobilized anti-CD3 activated T4 cells directly suppressed B cell responses stimulated by Staphylococcus aureus and IL-2. Anti-CD3 activated T4 cells did not inhibit initial B cell activation, but suppressed the capacity of the activated B cells to differentiate into ISC. The suppressive influence of anti-CD3 activated T4 cells was reversible as evidenced by the finding that removal of the activated T4 cells from the culture permitted B cell differentiation to proceed. Moreover, anti-CD3-activated T4 cells were able to stimulate initial B cell activation that became apparent when the T cells and B cells were separated. Inhibition of B cell responsiveness by 64.1-activated T4 cells was the result of a block at the G1-S interphase of the cell cycle. The data indicate that anti-CD3-stimulated T4 cells directly and reversibly suppress human B cell function. Moreover, IL 2 appears to play an important role in the differentiation of functionally effective suppressor cells from activated T4 cells.     

Cytochalasins enhance the proliferation of CD4 cells through the CD3-Ti antigen receptor complex or the CD2 molecule through an effect on early events of activation.

Cytochalasins are known to inhibit or enhance the proliferation of T cells induced by mitogens in a concentration-dependent fashion. To clarify the mechanism by which cytochalasins enhance T cell proliferation, we examined which activation pathways and events in signal transduction were affected by cytochalasins. We also examined subsets of CD4 cells for a preferential response to cytochalasins. Cytochalasins enhanced the proliferation of CD4 cells induced by optimal doses of anti-CD3 antibody or suboptimal doses of anti-CD2 antibodies. Cytochalasins, at low concentrations, enhanced the rise in intracellular Ca2+ and production of IP3 in CD4 cells activated by anti-CD2 or CD3 antibodies. Cytochalasins also enhanced the modulation of CD3 induced by anti-CD3 antibody. These results suggest that cytochalasins enhance the proliferation of CD4 cells by affecting early events in signal transduction after activation through the CD3-Ti Ag-receptor complex or CD2 molecule. At the doses used, cytochalasins appear to interact with cytochalasin-binding sites in the cell membrane. Cytochalasins predominantly enhanced CD3-mediated proliferation in the CD29-subset of CD4 cells.     

Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation

The CD44 inhibitor Lutheran [In(Lu)]-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule. We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC. CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody. CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid. CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies. CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells. In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms. First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies. Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation. Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.     

Monocyte activation by circulating fibronectin fragments in HIV-1-infected patients

To identify signals that can alter leukocyte function in patients receiving highly active antiretroviral therapy (HAART), we analyzed single blood samples from 74 HIV-1-infected patients and additional blood was collected at 90-day intervals from 51 HIV-1-infected patients over a 516 +/- 172 (mean +/- SD) day interval. Despite the absence of circulating immune complexes and normalization of phagocytic function, compared with controls, the fraction of patients' monocytes expressing CD49e and CD62L was decreased and expression of CD11b and CD86 increased. Plasma from 63% of patients but none from normal controls contained 110-120 kDa fibronectin fragments (FNf). Presence of FNf did not reflect poor adherence to therapy. Addition of FNf to normal donor blood in vitro replicated changes in monocyte CD49e, CD62L, CD11b, and CD86 seen in vivo. FNf also induced monocytes to release a serine proteinase, nominally identified as proteinase-3, that hydrolyzed cell surface CD49e. alpha(1)-Antitrypsin blocked FNf-induced shedding of CD49e in a dose-dependent manner. Plasma with a normal frequency of CD49e(+) monocytes contained antiproteases that partially blocked FNf-induced monocyte CD49e shedding, whereas plasma from patients with a low frequency of CD49e(+) monocytes did not block this effect of FNf. Electrophoretic analyses of plasma from the latter group of patients suggested that a significant fraction of their alpha(1)-antitrypsin was tied up in high molecular mass complexes. These results suggest that monocyte behavior in HIV-1-infected patients may be influenced by FNf and the ratio of protease and antiproteases in the cells' microenvironment.     

Human T cell activation induced by a monoclonal mouse IgG3 anti-CD3 antibody (RIV9) requires binding of the Fc part of the antibody to the monocytic 72-kDa high-affinity Fc receptor (FcRI)

The mitogenic activity of anti-CD3 mouse monoclonal antibodies (mAb) in cultures of human peripheral blood mononuclear cells (PBMC) depends on the ability of the mAb to interact with CD3 molecules on the T cells, and with Fc receptors (FcR) on monocytes. Two types of FcR with distinct specificity for murine (m) IgG subclasses are involved: a 72-kDa receptor (FcRI) binds mIgG2a and a 40-kDa receptor (FcRII) binds mIgG1. In this study we examined the mitogenic activity of mIgG3 anti-CD3 mAb RIV9. In cultures of human PBMC, the mAb induced T cell proliferation and interleukin 2 production. We found that subjects, unresponsive to mIgG2a anti-CD3 (e.g., OKT3), were also RIV9 nonresponders. In contrast, nonresponders to mIgG1 anti-CD3 (e.g., anti-Leu4) had a normal response to RIV9. Our results therefore suggested that anti-CD3 mAb of the mIgG2a and mIgG3 subclass bind to the same monocytic FcR. Human monomeric IgG, which has been shown to bind to FcRI only, blocked T cell proliferation induced by mIgG2a and mIgG3 anti-CD3, but had no effect on T cell proliferation induced by mIgG1 anti-CD3. In contrast, a mAb (IV.3) to FcRII, which blocks ligand binding of the receptor, blocked the mitogenic activity of mIgG1 anti-CD3 antibodies, but had no effect on T cell proliferation induced by mIgG3 anti-CD3 or by mIgG2a anti-CD3. Binding of RIV9 to FcR of responder monocytes could be demonstrated in immunofluorescence. Monocytes from the RIV9 nonresponder subjects however were unable to bind the Fc portion of this antibody. The binding of fluorescein (FITC)-conjugated mIgG3 or FITC-conjugated mIgG2a to responder monocytes could be inhibited by human monomeric IgG and by mIgG2a and mIgG3, but not by the mAb to FcRII. The results demonstrate that mIgG3 binds to FcRI on human monocytes and that this binding is needed for the mitogenic activity of mIgG3 anti-CD3.     

Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules.

The capacity of purified fibronectin to costimulate human T cell DNA synthesis was examined. Low concentrations of immobilized fibronectin, but not soluble fibronectin, augmented anti-CD3-induced proliferation of highly purified human T cells. In the absence of anti-CD3 stimulation, immobilized fibronectin did not induce T cell proliferation alone or in the presence of IL-2 or phorbol dibutyrate. Although fibronectin is present in high concentrations in the serum, immobilized fibronectin was able to costimulate T cell proliferation when cells were cultured in serum-containing medium. Immobilized collagen type I did not enhance anti-CD3 stimulated T cell responses, whereas gelatin (denatured collagen) and laminin were able to enhance anti-CD3 stimulated T cell responses modestly. The effects of gelatin, however, appeared to be indirect, because it could not enhance responses in medium devoid of fibronectin. Immobilized fibronectin enhanced anti-CD3 induced proliferation of both CD45RA dim and CD45RA bright subsets within both the CD4+ and CD8+ subpopulations of T cells, although cells with the CD45RA dim phenotype were costimulated by lower concentrations of immobilized fibronectin. Enhancement of anti-CD3 induced proliferation by immobilized fibronectin was completely inhibited by a mAb to CD29, the integrin beta 1-chain (4B4) and not by a variety of other mAb. In contrast to its effects on proliferation, 4B4 only partially blocked T cell binding to anti-CD3 and fibronectin-coated macrowells. These findings suggested that the interaction between fibronectin and its receptor transduced a signal to the T cell and did not merely stabilize the interaction between anti-CD3 and the CD3 complex. Further experiments confirmed this observation. Thus fibronectin could enhance anti-CD3 responses when it was immobilized to a separate surface. The augmentation of anti-CD3 stimulated proliferation induced by immobilized fibronectin was also inhibited partially by mAb to either VLA-4 or VLA-5 and completely by a combination of the two mAb. The mAb to VLA-4 not only blocked the capacity of immobilized fibronectin to enhance anti-CD3-induced T cell proliferation but also directly costimulated T cell responses. Thus, at least two fibronectin receptors are involved in fibronectin-mediated costimulation of T cell proliferation. These studies indicate that signals are transduced through the fibronectin receptors, VLA-4 and VLA-5, that augment T cell responses and therefore implicate the extracellular matrix protein fibronectin as an important influence regulating T cell responsiveness in vivo.     

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.     

Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.     

Cutting edge: depletion of CD4+CD25+ regulatory T cells is necessary,but not sufficient,for induction of organ-specific autoimmune disease

Thymectomy of BALB/c mice on day 3 of life results in the development of autoimmune gastritis (AIG) due to the absence of CD4(+)CD25(+) regulatory T cells. However, depletion of CD4(+)CD25(+) T cells by treatment with anti-CD25 rarely resulted in AIG. Depletion was efficient, as transfer of splenocytes from depleted mice induced AIG in nu/nu mice. One explanation for this result is that CD4(+)CD25(-) T cells upon transfer to nude recipients undergo lymphopenia-induced proliferation, providing a signal for T cell activation. Cotransfer of CD25(+) T cells did not inhibit initial proliferation but did suppress AIG. Surprisingly, immunization with the AIG target Ag, H/K ATPase, in IFA failed to induce disease in normal animals but induced severe AIG in CD25-depleted mice. These results demonstrate that second signals (nonspecific proliferation, TCR activation, or inflammation) are needed for induction of autoimmunity in the absence of CD25(+) regulatory T cells.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.