Human anergic CD4+ T cells can act as suppressor cells by affecting autologous dendritic cell conditioning and survival.

T cell suppression exerted by regulatory T cells represents a well-established phenomenon, but the mechanisms involved are still a matter of debate. Recent data suggest that anergic T cells can suppress responder T cell activation by inhibiting Ag presentation by dendritic cells (DC). In this study, we focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and analyzed the fate of DC and responder T cells. To address this issue, we have cocultured human alloreactive or Ag-specific CD4+ T cell clones, rendered anergic by incubation with immobilized anti-CD3 Ab, with autologous DC and responder T cells. We show that anergic T cells affect either Ag-presenting functions or survival of DC, depending whether immature or mature DC are used as APC. Indeed, MHC and costimulatory molecule expression on immature DC activated by responder T cells is inhibited, while apoptotic programs are induced in mature DC and in turn in responder T cells. Ligation of CD95 by CD95L expressed on anergic T cells in the absence of CD40-CD40L (CD154) interaction are critical parameters in eliciting apoptosis in both DC and responder T cells. In conclusion, these findings indicate that the defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cells could be the primary event in determining T cell suppression and support the role of CD40 signaling in regulating both conditioning and survival of DC.     

Dendritic cell secretion of IL-15 is induced by recombinant huCD40LT and augments the stimulation of antigen-specific cytolytic T cells

Dendritic cells (DC) are professional antigen-presenting cells which stimulate strong proliferative and cytolytic T cell responses. Stimulation of CD40 on dendritic cells by its ligands and anti-CD40 antibodies induces maturation and enhances DC stimulatory ability. In order to understand the mechanism by which ligand:CD40 interactions augment DC function, we assessed the role of T cell stimulatory cytokines IL-12 and IL-15 in the function of DC stimulated with soluble trimeric CD40L, a recombinant fusion protein incorporating three covalently linked extracellular CD40L domains (huCD40LT). Peripheral blood derived DC treated with huCD40LT and/or IFN-gamma were used to stimulate T cell responses in vitro to specific antigens. DC treated with huCD40LT or IFN-gamma/huCD40LT stimulated enhanced T cell proliferation to CASTA, a soluble protein from C. albicans, induced T cells with augmented antigen-specific lysis, and increased the yield of antigen-specific IFN-gamma-producing T cells. IL-15 production by DC was enhanced in cultures treated with huCD40LT and correlated with expansion of antigen-specific cytolytic T cells. Addition of a neutralizing anti-IL-15 monoclonal antibody inhibited the expansion of viral and tumor antigen-specific T cells stimulated by IFN-gamma and huCD40LT-treated DC. In contrast, this enhanced stimulatory ability of DC did not appear to depend on synthesis of IL-12 since huCD40LT treatment stimulated the generation of antigen-specific cytokine producing and cytolytic T cells without increased IL-12 production. Addition of anti-IL-12 monoclonal antibody did not inhibit expansion of these cells. These data suggest that production of IL-15 but not IL-12 is an important factor in the enhanced immunostimulatory ability of huCD40LT-treated DC.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

Type 1 IFN maintains the survival of anergic CD4+ T cells

Anergic T cells have immunoregulatory activity and can survive for extended periods in vivo. It is unclear how anergic T cells escape from deletion, because both anergy and apoptosis can occur after TCR ligation. Stimulation of human CD4+ T cell clones reactive to influenza hemagglutinin peptides can occur in the absence of APCs when MHC class II-expressing, activated T cells present peptide to each other. This T:T peptide presentation can induce CD95-mediated apoptosis, while the cells that do not die are anergic. We found that the death after peptide or anti-CD3 treatment of a panel of CD4+ T cell clones is blocked by IFN-beta secreted by fibroblasts and also by IFN-alpha. This increases cell recovery after stimulation, which is not due to T cell proliferation. This mechanism for apoptosis inhibition rapidly stops protein kinase C-delta translocation from the cytoplasm to the nucleus, which is an early event in the death process. A central observation was that CD4+ T cells that are rescued from apoptosis after T:T presentation of peptide by IFN-alphabeta remain profoundly anergic to rechallenge with Ag-pulsed APCs. However, anergized cells retain the ability to respond to IL-2, showing that they are nonresponsive but functional. The prevention of peptide-induced apoptosis in activated T cells by IFN-alphabeta is a novel mechanism that may enable the survival and maintenance of anergic T cell populations after TCR engagement. This has important implications for the persistence of anergic T cells with the potential for immunoregulatory function in vivo.     

CD8+ T cells can be primed in vitro to produce IL-4.

IL-4 production by T lymphocytes from naive mice in response to stimulation by plate-bound anti-CD3 is concentrated among CD4+ T cells. In vitro stimulation of lymph node T cells with anti-CD3 plus IL-2 and IL-4 strikingly increases the frequency of cells that produce IL-4 in response to subsequent stimulation with anti-CD3 plus IL-2. Separation of these primed cell populations into CD4+ and CD8+ T cell by cell sorting reveals that the frequency of IL-4-producing cells in both population is similar. Verification that CD8+ T cells produce IL-4 is provided by the capacity of anti-IL-4 mAb to inhibit the response of the indicator cell line to the growth factor produced by the primed cells and by detection of IL-4 by an IL-4-specific ELISA. The in vitro "priming" of CD8+ T cells to produce IL-4 is not dependent on the presence of CD4+ T cells because highly purified CD8+ T cells can be stimulated to develop into cells capable of producing IL-4 by culture with plate-bound anti-CD3 plus IL-2 and IL-4.     

Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production

Alcohol consumption inhibits accessory cell function and Ag-specific T cell responses. Myeloid dendritic cells (DCs) coordinate innate immune responses and T cell activation. In this report, we found that in vivo moderate alcohol intake (0.8 g/kg of body weight) in normal volunteers inhibited DC allostimulatory capacity. Furthermore, in vitro alcohol treatment during DC differentiation significantly reduced allostimulatory activity in a MLR using naive CD4(+) T cells, and inhibited tetanus toxoid Ag presentation by DCs. Alcohol-treated DCs showed reduced IL-12, increased IL-10 production, and a decrease in expression of the costimulatory molecules CD80 and CD86. Addition of exogenous IL-12 and IL-2, but not neutralization of IL-10, during MLR ameliorated the reduced allostimulatory capacity of alcohol-treated DCs. Naive CD4(+) T cells primed with alcohol-treated DCs showed decreased IFN-gamma production that was restored by exogenous IL-12, indicating inhibition of Th1 responses. Furthermore, CD4(+) T cells primed with alcohol-treated DCs were hyporesponsive to subsequent stimulation with the same donor-derived normal DCs, suggesting the ability of alcohol-treated DCs to induce T cell anergy. LPS-induced maturation of alcohol-treated immature DCs partially restored the reduced allostimulatory activity, whereas alcohol given only during DC maturation failed to inhibit DC functions, suggesting that alcohol primarily impairs DC differentiation rather than maturation. NFkappaB activation, a marker of DC maturation was not affected by alcohol. Taken together, alcohol both in vitro and in vivo can impair generation of Th1 immune responses via inhibition of DC differentiation and accessory cell function through mechanisms that involve decreased IL-12 induction.     

CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice

Transfer of CD45RB(high) CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin alpha(4)beta(7). Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.     

Manipulation of CD98 resolves type 1 diabetes in nonobese diabetic mice

The interplay of CD4(+) and CD8(+) T cells targeting autoantigens is responsible for the progression of a number of autoimmune diseases, including type 1 diabetes mellitus (T1D). Understanding the molecular mechanisms that regulate T cell activation is crucial for designing effective therapies for autoimmune diseases. We probed a panel of Abs with T cell-modulating activity and identified a mAb specific for the H chain of CD98 (CD98hc) that was able to suppress T cell proliferation. The anti-CD98hc mAb also inhibited Ag-specific proliferation and the acquisition of effector function by CD4(+) and CD8(+) T cells in vitro and in vivo. Injection of the anti-CD98hc mAb completely prevented the onset of cyclophosphamide-induced diabetes in NOD mice. Treatment of diabetic NOD mice with anti-CD98hc reversed the diabetic state to normal levels, coincident with decreased proliferation of CD4(+) T cells. Furthermore, treatment of diabetic NOD mice with CD98hc small interfering RNA resolved T1D. These data indicate that strategies targeting CD98hc might have clinical application for treating T1D and other T cell-mediated autoimmune diseases.     

Foxp3+CD25+ T regulatory cells stimulate IFN-gamma-independent CD152-mediated activation of tryptophan catabolism that provides dendritic cells with immune regulatory activity in mice unresponsive to staphylococcal enterotoxin B

Mice made unresponsive by repeated injection of staphylococcal enterotoxin B (SEB) contained SEB-specific CD25(+)CD4(+)TCRBV8(+) T cells that were able to transfer their state of unresponsiveness to primary-stimulated T cells. About one-half of these cells stably up-regulated the expression of CD152. We undertook the present study to determine whether CD152(high) cells seen in this system were T regulatory cells responsible for suppression or whether they represented SEB-activated CD4(+) T effector cells. Our results show that, among SEB-specific TCRBV8(+) T cells isolated from unresponsive mice, all CD152(high)CD25(+)CD4(+) T cells expressed Foxp3, the NF required for differentiation and function of natural T regulatory cells. Moreover, suppression by CD25(+)CD4(+)TCRBV8(+) T cells was fully inhibited by anti-CD152 Abs. Following stimulation by soluble CD152-Ig, dendritic cells (DC) isolated from unresponsive mice strongly increased the expression and the function of indoleamine-2,3-dioxygenase (IDO), the enzyme responsible for the catabolism of tryptophan. This capacity to activate IDO was independent of IFN-gamma production by DC because CD152-Ig stimulation of DC isolated from SEB-treated IFN-gamma-deficient animals activated IDO expression and function. Finally, adding 1-methyl-tryptophan, an inhibitor of tryptophan catabolism, increased substantially the capacity of DC from unresponsive animals to stimulate primary T cell response toward SEB. Thus, we conclude that IFN-gamma-independent CD152-mediated activation of tryptophan catabolism by Foxp3(+)CD25(+) T regulatory cells provides DC with immune regulatory activity in mice unresponsive to SEB.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

Blockade of CD200 in the presence or absence of antibody effector function: implications for anti-CD200 therapy

CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.     

Lipopolysaccharide modulation of dendritic cells is insufficient to mature dendritic cells to generate CTLs from naive polyclonal CD8+ T cells in vitro, whereas CD40 ligation is essential.

Many cytotoxic CD8+ T cell responses are dependent on the interactions between CD40 ligand on the helper CD4+ T cell and CD40 on the APC. Although CD40 triggering of dendritic cells (DC) has been shown to mature the DC by increasing the level of expression of costimulatory molecules and inducing IL-12 secretion, the precise mechanisms by which CD40-CD40 ligand interactions allow DC to drive CTL responses remain unknown. We have used an in vitro model in which naive polyclonal CD8+ T cells can be activated by bone marrow-derived DC to investigate factor(s) that are responsible for this CD40-dependent generation of CTLs. DC modulated with agonistic anti-CD40 mAb (aCD40) are able to generate Ag-specific CTL responses while DC modulated with the microbial stimulus LPS alone do not. We compared the Ag-presenting capacity, levels of costimulatory molecules, and release of cytokines and chemokines of DC modulated with aCD40 to that of DC modulated by LPS. None of the factors assayed account for the unique capacity of anti-CD40-matured DC to drive CTL but this model provides a simplified system for further investigation. Although we attempted to use an LPS-free system for these studies, we are unable to rule out the possibility that very low levels of endotoxin (<20 pg/ml) may synergize with CD40 ligation in the generation of CTLs.     

Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells

The terminal fate of dendritic cells (DC) remains relatively uncertain. In this study, we tested the hypothesis that DC undergo apoptosis after Ag-specific interaction with T cells. When splenic DC isolated from BALB/c mice were cocultured with HDK-1 T cells (a keyhole limpet hemocyanin (KLH)-specific CD4+ Th1 clone) in the presence of KLH, they showed conspicuous cell death as measured by propidium iodide (PI) uptake and chromatin condensation, whereas they remained relatively intact when incubated with either T cells or KLH alone. Likewise, the long term DC line XS52, which was established from BALB/c mouse epidermis, also died rapidly (within 2 h), and they exhibited characteristic DNA laddering when cocultured with HDK-1 T cells in the presence of KLH. RT-PCR and FACS analyses revealed the expression of CD95 (Fas) by XS52 DC and of CD95 ligand (CD95L) (Fas ligand) by activated HDK-1 T cells, suggesting a functional role for these molecules. In fact, anti-CD95L mAb inhibited partially (50%) T cell-mediated XS52 cell death, and coupling of surface CD95 with anti-CD95 mAb triggered significant XS52 cell death, but only in the presence of cycloheximide. Thus, ligation of CD95 (on DC) with CD95L (on T cells) is one, but not the only, mechanism by which T cells induce DC death. Finally, DC isolated from the CD95-deficient mice were found to be significantly more efficient than DC from control mice in their capacity to induce delayed type hypersensitivity responses in vivo. We propose that T cell-induced DC apoptosis serves as a unique down-regulatory mechanism that prevents the interminable activation of T cells by Ag-bearing DC.     

Enhancement of T helper2 response in the absence of interleukin (IL-)6; an inhibition of IL-4-mediated T helper2 cell differentiation by IL-6

Functional roles of interleukin (IL-)6 in T cell response were investigated. Mice deficient in IL-6 and wild mice were immunized with antigens (myelin oligodendrocyte glycoprotein or methylated BSA) and production of IL-4 and interferon (IFN)-gamma by regional lymph nodes was measured. IL-6 deficiency led to an enhancement of IL-4 and an inhibition of IFN-gamma production. Moreover, polyclonal stimulation of spleen T cells from unimmunized IL-6-deficient mice with anti-CD3 plus anti-CD28 antibodies (Abs) demonstrated an enhancement of T helper (Th)(2)responses. The presence of IL-6, however, augmented IL-4 production but it inhibited IFN-gamma expression by spleen T cells in response to polyclonal stimulation and by antigen-primed spleen T cells in response to re-challenge with the antigen. In contrast, the induction of spleen CD4-positive T cells into Th(2)cells in vitro by the anti-CD3 plus IL-4 was completely suppressed by exogenously added IL-6, whereas Th(1)differentiation of T cells by the anti-CD3 plus IL-12 was not inhibited by the presence of IL-6. Thus, these results indicate that IL-6 physiologically could modulate qualitative T cell response and suggest that it augments Th(1)responses partly through its inhibitory capability of IL-4-induced Th(2)differentiation of naive T cells.     

On the mechanisms of T cell silencing by IL-10 DNA: direct and indirect inhibition of T cell functions

Previously we reported that mucosal IL-10 DNA administration resulted in long-term suppression of virus-induced inflammatory responses by silencing Th1-type CD4+ T cell functions. However, the mechanism by which IL-10 silences the activity of CD4+ T cells was not clear. The present report has shown that mucosal IL-10 DNA administration led to the reduction of reactivity of T cells following TCR stimulation. IL-10 DNA also downregulated APC functions to stimulate T cells but the effect was temporary. Bystander suppression, including that of IL-10 producing regulatory cells, appeared not to be directly involved in the inhibition of T cell reactivity because both anti-IL-10 and anti-IL-10R could not block the suppression of T cell functions. This silenced state could be maintained following adoptive transfer to untreated animals. The nature of the silencing appears to be a reversible anergic state since Ag stimulation in the presence of exogenous IL-2 restored T cell reactivity. Furthermore, IL-10-induced silenced T cells could be induced in vitro by culturing the T cells with rIL-10 in the presence or the absence of antigen stimulation. This state persisted in the absence of rIL-10 and persisted for at least 3 days. A more notable effect, however, was observed when the T cells were incubated with IL-10 in the presence of APC and Ag. These results indicate that IL-10 induced a long-term silenced state in T cells by direct and indirect inhibition of T cell functions.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

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.