Christine Deppong

Cutting Edge: B and T Lymphocyte Attenuator and Programmed Death Receptor-1 Inhibitory Receptors Are Required for Termination of Acute Allergic Airway Inflammation

T cell activation is regulated by coordinate interaction of the T cell Ag receptor and costimulatory signals. Although there is considerable insight into processes that regulate the initiation of inflammation, less is known about the signals that terminate immune responses. We have examined the role of the inhibitory receptors programmed death receptor-1 and B and T lymphocyte attenuator in the regulation of allergic airway inflammation. Our results demonstrate that there is a temporally regulated expression of both the receptors and their ligands during the course of allergic airway inflammation. Following a single inhaled challenge, sensitized wild-type mice exhibit peak inflammation on day 3, which resolves by day 10. In contrast, mice deficient in the expression of programmed death receptor-1 or B and T lymphocyte attenuator have persistent inflammation out to 15 days following challenge. Thus, these receptors are critical determinants of the duration of allergic airway inflammation.

Negligible Role of Antibodies and C5 in Pregnancy Loss Associated Exclusively with C3-Dependent Mechanisms through Complement Alternative Pathway

Factors involved in pregnancy failure due to abnormal fetomaternal tolerance are poorly understood. Here we describe distinct defects in placenta formation and subsequent pregnancy loss solely dependent on the activation of the complement alternative pathway and the effector mechanisms provided by the maternal C3. Surprisingly, this effect is independent of other complement activation pathways and of the effector mechanisms provided by other complement components. These findings provide significant insight into the role of the innate immune system in human pregnancy failure, a frequent clinical outcome.

A Role for the Cr2 Gene in Modifying Autoantibody Production in Systemic Lupus Erythematosus

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibody production against nuclear Ags. Recent studies suggest that the Cr2 gene, which encodes for complement receptor (CR)1 and CR2, is important in disease susceptibility. Because the precise disease phenotype related to this gene, in isolation or in relation to other genetic loci, is not known, we analyzed C57BL/6 mice with a targeted mutation in Cr2 (C57BL/6.Cr2−/−) with or without a concomitant mutation in Fas (C57BL/6.lpr Cr2−/−). The Cr2null mutation in a C57BL/6.lpr background markedly increases the serum concentrations of IgG1 and IgG2b and the levels of antinuclear and anti-dsDNA Abs as compared with C57BL/6.lpr controls. There is also a trend for higher concentrations of IgG2a and IgG3. In contrast, isolated deficiencies in either these CRs or Fas have a limited effect in the production of anti-dsDNA Abs. Moreover, the Cr2null mutation does not affect other disease manifestations. These findings demonstrate that abnormalities in CR1 and CR2 may be linked to the production of autoantibodies by modifying the effect of other systemic lupus erythematosus susceptibility genes. Phenotypic expression of other disease manifestations need additional Cr2-independent genetic factors.

Targeted knock-in mice expressing mutations of CD28 reveal an essential pathway for costimulation.

ABSTRACT Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-XL, or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase Cθ, and glycogen synthase kinase 3β, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.

CD28 Facilitates the Generation of Foxp3− Cytokine Responsive Regulatory T Cell Precursors

The T cell costimulatory molecule CD28 plays an important role in the thymic generation of Foxp3+ regulatory T cells (Tregs) essential for the maintenance of self-tolerance. In this study, we show that a cell-intrinsic signal from CD28 is involved in the generation of cytokine-responsive Foxp3− precursors using studies of mixed bone marrow chimeras as well as TCR-specific generation of Foxp3+ cells using intrathymic transfer of TCR-transgenic thymocytes expressing a natural Treg TCR. Contrary to a previous report, the analysis of CD28 mutant knockin mice revealed that this cell-intrinsic signal is only partially dependent on the Lck-binding PYAP motif. Surprisingly, even though the absence of CD28 resulted in a 6-fold decrease in thymic Tregs, the TCR repertoires of CD28-deficient and sufficient cells were largely overlapping. Thus, these data suggest that CD28 does not operate by markedly enlarging the repertoire of TCRs available for Treg development, but rather by improving the efficiency of Treg development of thymocytes expressing natural Treg TCRs.

A dose-dependent requirement for the proline motif of CD28 in cellular and humoral immunity revealed by a targeted knockin mutant

Activation of naive T cells requires the integration of signals through the antigen receptor and CD28. Although there is agreement on the importance of CD28, there remains controversy on the mechanism by which CD28 regulates T cell function. We have generated a gene-targeted knockin mouse expressing a mutation in the C-terminal proline-rich region of the cytoplasmic tail of CD28. Our analysis conclusively showed that this motif is essential for CD28-dependent regulation of interleukin 2 secretion and proliferation. In vivo analysis revealed that mutation of this motif-dissociated CD28-dependent regulation of cellular and humoral responses in an allergic airway inflammation model. Furthermore, we find an important gene dosage effect on the phenotype of the mutation and provide a mechanistic explanation for the conflicting data on the significance of this motif in CD28 function.

CTLA4Ig Inhibits Effector T Cells through Regulatory T Cells and TGF-β

The CD28 costimulatory receptor is a critical regulator of T cell function, making it an attractive therapeutic target for the treatment of immune-mediated diseases. CTLA4Ig, now approved for use in humans, prevents naive T cell activation by binding to B7 proteins and blocking engagement of CD28. However, CTLA4Ig suppresses inflammation even if administered when disease is established, suggesting alternative mechanisms. We identified a novel, CD28-independent mechanism by which CTLA4Ig inhibits activated T cells. We show that in vitro, CTLA4Ig synergizes with NO from bone marrow–derived macrophages to inhibit T cell proliferation. Depletion of regulatory T cells (Tregs) or interference with TGF-β signaling abrogated the inhibitory effect of CTLA4Ig. Parallel in vivo experiments using an allergic airway inflammation model demonstrated that this novel mechanism required both macrophages and regulatory T cells. Furthermore, CTLA4Ig was ineffective in SMAD3-deficient mice, supporting a requirement for TGF-β signaling. Thus, in addition to preventing naive T cells from being fully activated, CTLA4Ig can turn off already activated effector T cells by an NO/regulatory T cell/TGF-β–dependent pathway. This mechanism is similar to cell-extrinsic effects of endogenous CTLA4 and may be particularly important in the ability of CTLA4Ig to treat chronic inflammatory disease.

B and T lymphocyte attenuator regulates T cell survival in the lung.

The initiation, intensity, and duration of T cell-directed inflammatory responses are dependent upon the coordination of both activating and inhibitory signals mediated by specific receptors on the T lymphocyte. The recently described receptor, B and T lymphocyte attenuator (BTLA), has been demonstrated to have an important role in limiting the duration of inflammation in a murine model of allergic asthma. In this study, we have examined the role of BTLA on the proliferation, recruitment, and survival of T cells in response to inhaled allergen. We find that there is decreased cell death in T cells from BTLA-deficient mice, whereas proliferation and recruitment to the lungs are unchanged. Thus, the regulation of cell death through BTLA signaling is a key determinant of the inflammatory response in the lung.

A Randomized Controlled Trial to Evaluate Inhibition of T-Cell Costimulation in Allergen-induced Airway Inflammation

RATIONALE T lymphocytes are important in the pathogenesis of allergic asthma. Costimulation through CD28 is critical for optimal activation of T cells, and inhibition of this pathway with CTLA4Ig has been shown to be effective in preventing airway inflammation and hyperresponsiveness in animal models of asthma. Abatacept, a humanized version of CTLA4Ig, has been approved for treatment of rheumatoid arthritis, providing the opportunity to test whether inhibition of costimulation is an effective strategy to treat people with asthma. OBJECTIVES To determine if 3 months of treatment with abatacept reduced allergen-induced airway inflammation in people with mild atopic asthma. METHODS Randomized, placebo-controlled, double-blinded study. Bronchoscopically directed segmental allergen challenge was performed on 24 subjects followed by bronchoalveolar lavage 48 hours later. Subjects were randomized 1:1 to receive abatacept or placebo, followed by a second allergen challenge protocol after 3 months of study drug. MEASUREMENTS AND MAIN RESULTS There was no significant reduction in allergen-induced eosinophilic inflammation in the abatacept-treated group compared with placebo (17.71% ± 17.25% vs. 46.39% ± 29.21%; P = 0.26). In addition, we did not detect an effect of abatacept on FEV1, provocative concentration of methacholine sufficient to induce a 20% decline in FEV1, or asthma symptoms. Subjects treated with abatacept had an increased percentage of naive and a corresponding decrease in memory CD4(+) T cells in the blood compared with placebo. CONCLUSIONS Inhibition of CD28-mediated costimulation with abatacept does not seem to alter the inflammatory response to segmental allergen challenge or clinical measures of asthma symptoms in people with mild atopic asthma. Clinical trial registered with ClinicalTrials.gov (NCT 00784459).

CTLA4-Ig inhibits allergic airway inflammation by a novel CD28-independent, nitric oxide synthase-dependent mechanism

The T‐cell response to antigen depends on coordinate signaling between costimulatory and inhibitory receptors. The altered function of either may underlie the pathophysiology of autoimmune and/or chronic inflammatory diseases and manipulation of these pathways is an important emerging area of therapeutics. We report here that the immunosuppressant drug CTLA4‐Ig inhibits the effector phase of allergic airway inflammation through a CD28‐independent, nitric oxide synthase (NOS)‐dependent mechanism. Using mice deficient in both B‐ and T‐lymphocyte attenuator (BTLA) and CD28, we demonstrate that simultaneous deficiency of an inhibitory receptor can rescue the in vivo but not the in vitro CD28‐deficient phenotype. Furthermore, we demonstrate that inflammation in CD28/BTLA‐double‐deficient mice is suppressed by CTLA4‐Ig. This suppression is reversed by treatment with the NOS inhibitor, N6‐methyl‐L‐arginine acetate (L‐NMMA). In addition, CTLA4‐Ig is ineffective at inhibiting inflammation in NOS2‐deficient mice when given at the effector phase. Thus, CD28 and BTLA coordinately regulate the in vivo response to inhaled allergen, and CTLA4‐Ig binding to B7‐proteins inhibits the effector phase of inflammation by a CD28‐independent, NOS‐dependent mechanism.