Alistair Noble

Immune regulation: a new role for the CD8+ T cell

During an immune response, peripheral T cells develop into functionally distinct subpopulations that effect cell-mediated immunity and regulate humoral immune responses through the secretion of specific cytokines. Recent data suggest that CD8+ T cells, which have long been regarded simply as cytotoxic cells, play a more active role in the regulation of the immune response. In this article, Mike Kemeny and colleagues suggest that there are functionally distinct subsets of CD8+ T cells that produce different combinations of cytokines and appear to play an important part in determining the pattern of cytokines produced by CD4+ T cells and the isotype of immunoglobulins expressed by B cells.

Cyclic AMP plays a critical role in C3a-receptor mediated regulation of dendritic cells in antigen uptake and T cell stimulation

The biochemical basis for complement acting directly on antigen-presenting cells to enhance their function in T-cell stimulation has been unclear. Here we present evidence that engagement of C3a receptor (C3aR) on the surface of dendritic cells (DCs) leads to alterations in the level of intracellular cyclic adenosine monophosphate (cAMP), a potent negative regulator of inflammatory cytokines. C3aR activation-induced depression of cAMP was associated with enhanced capacity of DCs for antigen uptake and T-cell stimulation. Conversely, C3aR-deficient DCs showed elevation of cAMP and impaired properties for antigen uptake and immune stimulation. Similarities in the phenotype of C3-deficient and C3aR-deficient DCs suggest that local production of C3 with extracellular metabolism to C3a is an important driver of DC alterations in cAMP. The finding of a link between complement and adaptive immune stimulation through cAMP offers new insight into how innate and adaptive immunity combine to generate efficient effector and memory responses.

Th17 Responses in Chronic Allergic Airway Inflammation Abrogate Regulatory T cell-mediated Tolerance and Contribute to Airway Remodeling

The role of T-helper type 17 (Th17) responses in airway remodeling in asthma is currently unknown. We demonstrate that both parenteral and mucosal allergen sensitization, followed by allergen inhalation, leads to Th17-biased lung immune responses. Unlike Th17 cells generated in vitro, lung Th17 cells did not produce tumor necrosis factor-α or interleukin (IL)-22. Eosinophilia predominated in acute inflammation, while neutrophilia and IL-17 increased in chronic disease. Allergen-induced tolerance involved Foxp3-, Helios-, and glycoprotein-A repetitions predominant-expressing regulatory T cells (Treg) and IL-10/interferon-γ priming. This Treg phenotype was altered in inflamed lungs and abrogated by inhalation of IL-17. Using Th17-deficient mice with genetic disruption of gp130 in T cells, we showed that Th17 cells induce airway remodeling independent of the Th2 response. All-trans retinoic acid administration ameliorated Th17-mediated disease and increased Treg activity, while dexamethasone inhibited eosinophilia but not neutrophilia, and enhanced Th17 development in vitro. Targeting the Th17/Treg axis might therefore be therapeutic in neutrophilic and glucocorticoid-refractory asthma.

CD8 T Cells Inhibit IgE Via Dendritic Cell IL-12 Induction That Promotes Th1 T Cell Counter-Regulation

Th1 and Th2 cells are counterinhibitory; their balance determines allergic sensitization. We show here that CD8 T cell subsets break these rules as both T cytotoxic (Tc)1 and Tc2 cells promote Th1 over Th2 immunity. Using IL-12−/−, IFN-γ−/−, and OVA257–264-specific Vα2Vβ5 TCR-transgenic mice, we have identified the key steps involved. OVA-specific IFN-γ−/− CD8 T cells inhibited IgE responses equivalent to wild-type CD8 T cells (up to 98% suppression), indicating that CD8 T cell-derived IFN-γ was not required. However, OVA-specific CD8 T cells could not inhibit IgE in IFN-γ−/− recipients unless reconstituted with naive, wild-type CD4 T cells, suggesting that CD4 T cell-derived IFN-γ did play a role. Transfer of either Tc1 or Tc2 Vα2Vβ5 TCR-transgenic CD8 T cells inhibited IgE and OVA-specific Th2 cells while promoting OVA-specific Th1 cell responses, suggesting a potential role for a type 1 inducing cytokine such as IL-12. CD8 T cells were shown to induce IL-12 in OVA257–264-pulsed dendritic cells (DC) in vitro. Furthermore, CD8 T cells were unable to inhibit IgE responses in IL-12−/− recipients without the addition of naive, wild-type DC, thus demonstrating a pivotal role for IL-12 in this mechanism. These data reveal a mechanism of IgE regulation in which CD8 T cells induce DC IL-12 by an IFN-γ-independent process that subsequently induces Th1 and inhibits Th2 cells. Th1 cell IFN-γ is the final step that inhibits B cell IgE class switching. This demonstrates a novel regulatory network through which CD8 T cells inhibit allergic sensitization.

The Balance of Protein Kinase C and Calcium Signaling Directs T Cell Subset Development

Development of naive T cells into type 1 (Th1, Tc1) or type 2 (Th2, Tc2) effector cells is thought to be under the control of cytokines. In this study, we show that when both IL-12 and IL-4 are present, murine and human T cell differentiation is regulated by the balance of protein kinase C (PKC) and calcium signaling within T cells. Although both biochemical signals were required for T cell activation via the TCR, altering the balance between them redirected type 1 cells to type 2 and vice versa. Stimulation of calcium signaling or inhibition of PKC favored type 1 differentiation, whereas stimulation of PKC or inhibition of calcineurin resulted in type 2 effectors. Altered peptide ligands induced distinct balances of PKC/calcium signaling and altered Tc1/Tc2 development in TCR-transgenic CD8 T cells. The data suggest novel strategies for manipulation of the immune response in vivo.

Effect of Transforming Growth Factor-beta Receptor I Kinase Inhibitor 2,4-Disubstituted Pteridine (SD-208) in Chronic Allergic Airway Inflammation and Remodeling

Transforming growth factor (TGF)-β is a multifunctional regulator of cell growth and differentiation with both pro- and anti-inflammatory properties. We used an inhibitor of TGF-β receptor I (TGF-βRI) kinase, SD-208 (2,4-disubstituted pteridine, a ATP-competitive inhibitor of TGF-βRI kinase), to determine the role of TGF-β in airway allergic inflammation and remodeling. Brown-Norway rats sensitized and repeatedly exposed to ovalbumin (OVA) aerosol challenge were orally administered SD-208 twice daily, before each of six OVA exposures to determine the preventive effects, or only before each of the last three of six OVA exposures to investigate its reversal effects. SD-208 (60 mg/kg) reversed bronchial hyperresponsiveness (BHR) induced by repeated allergen exposure, but it did not prevent it. SD-208 prevented changes in serum total and OVA-specific IgE, but it did not reverse them. SD-208 had both a preventive and reversal effect on airway inflammation as measured by major basic protein-positive eosinophils and CD2+ T-cell counts in mucosal airways, cell proliferation measured by 5-bromo-2′-deoxyuridine expression in airway smooth muscle (ASM) cells and epithelial cells, and goblet cell hyperplasia induced by repeated allergen challenges. There was a significant decrease in intracellular Smad2/3 expression. SD-208 did not significantly decrease the increased ASM thickness induced by allergen exposure. These findings support a proinflammatory and proremodeling role for TGF-β in allergic airway inflammation. Inhibition of TGF-βRI kinase activities by SD-208 may be a useful approach to the reversal of BHR and to the prevention and reversal of inflammatory and remodeling features of chronic asthma.

Blockade of chronic graft-versus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts.

CD4+CD25+ regulatory T cells (Tregs) are well known to suppress immunopathology induced in lymphopenic animals following T cell reconstitution, including acute graft‐versus‐host disease (GVHD) post‐bone marrow transplantation. The regulatory potential of this subset in nonlymphopenic hosts and in chronic, Th2‐mediated GVHD is less clear. We have generated alloantigen‐specific cells from CD4+CD25+ populations stimulated with MHC‐disparate dendritic cells and found them to express a stable Treg forkhead box p3+ phenotype with enhanced suppressive activity mediated by cell contact. When transferred into nonlymphopenic F1 hosts, nonspecific Tregs proliferated as rapidly as CD4+CD25− cells but displayed distinct growth kinetics in vitro. Tregs, expanded in response to alloantigen in vitro, displayed greatly enhanced suppressive activity, which was partially antigen‐specific. They were effective inhibitors of chronic GVHD, blocking donor cell engraftment, splenomegaly, autoantibody production, and glomerulonephritis. CD25+ and CD25− cells were equally susceptible to inhibition by immunosuppressive drugs targeting TCR signaling and rapamycin, but Tregs were resistant to inhibition by dexamethasone. The data indicate that alloantigen‐driven expansion, rather than homeostatic proliferation, is key to the effectiveness of CD4+CD25+ Tregs in GVHD and suggest that cellular therapy with alloantigen‐induced Tregs in combination with glucocorticoid treatment would be effective in prevention of chronic GVHD after immune reconstitution.

Activation-induced cell death of human T-cell subsets is mediated by Fas and granzyme B but is independent of TNF-alpha.

Human primary effector T cells were analyzed for their susceptibility to anti‐CD3‐induced activation‐induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95‐mediated apoptosis, although cell‐surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti‐CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF‐α/TNFR interactions because although type 1 cells secreted more TNF‐α than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z‐AAD‐CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.

Combined triggering of dendritic cell receptors results in synergistic activation and potent cytotoxic immunity.

Elimination of malignant cells and intracellular infections involves collaboration between CTLs and Th1 inflammation. Dendritic cells drive this response via costimulation and cytokines. We have defined key signals required for the exponential expansion of specific CD8+ T cells in vivo in mice. Immunization with two or more TLR agonists, anti-CD40, IFN-γ, and surfactant were sufficient to drive unprecedented levels of CD8 response to peptide or protein Ag and highly polarized Th1 CD4 responses. CD40 signaling was required for CD8 expansion but could be provided by a concomitant CD4 Th response in place of anti-CD40. Triggering of these pathways activated migration and activation of myeloid and plasmacytoid dendritic cells and secretion of IL-12. Cross-presentation can thus be exploited to induce potent cytotoxic responses and long-term memory to peptide/protein Ags. When combined with a tumor-associated peptide from tyrosinase-related protein 2, our combined adjuvant approach effectively halted tumor growth in an in vivo melanoma model and was more effective than anti-CD40 and a single TLR agonist. Antitumor immunity was associated with long-lived effector memory CD8 cells specific for the naturally processed and presented tumor Ag, and tumor protection was partially but not entirely dependent on CD8 T cells. This flexible strategy is more effective than existing adjuvants and provides a technological platform for rapid vaccine development.

Early Th1/Th2 cell polarization in the absence of IL-4 and IL-12: T cell receptor signaling regulates the response to cytokines in CD4 and CD8 T cells.

Differentiation of developing T cells into the type 1 (IFN‐γ‐producing) or type 2 (IL‐4‐producing) subsets is a central theme of immune regulation. The balance of IL‐4 and IL‐12 present during T cell activation has been considered the major influence on type 1 versus type 2 development. Here we show that CD4 T cells can become biased towards type 1 or type 2 phenotypes during their initial activation in the absence of IL‐4 or IL‐12. This type of regulation is dependent on the balance of MAPkinase, protein kinase C, and calcineurin signaling after TCR engagement. Later maturation of Th1 or Th2 effectors is dependent on IL‐12 or IL‐4. However Tc1 CD8 effector development is independent of IL‐12, and Tc2 cell generation requires both appropriate TCR signals and IL‐4 early ineffector development. Using an altered peptide ligand to stimulate TCR transgenic T cells, we show that altered signaling regulates the numbers of CD8 cells capable of developing into Tc2 effectors, and also their responsiveness to IL‐4. Together, the results support a two‐stage model of differentiation in which intermediate cells biased towards the type 1 or type 2 pathways after activation, are subsequently matured in response to IL‐12 or IL‐4, respectively.