Inducible Costimulatory Molecule Research Articles

Immunologic impact of anti-CTLA-4 therapy

3019 Background: Blockade of the T-cell inhibitory molecule known as cytotoxic T lymphocyte antigen-4 (CTLA-4) results in antitumor responses. To date, trials have been conducted with over 4,000 patients with various malignancies in the metastatic disease setting, which allow for correlation of therapy with clinical outcome but do not provide analyses of relevant biomarkers in the systemic circulation that reflect changes within the tumor microenvironment. Approximately 10% of treated patients respond to therapy. Why some patients respond while others do not remains unknown. The identification of intermediate biomarkers are essential for us to understand whether anti-CTLA-4 antibody has “hit its target” to affect human immune responses and whether these identified immune responses can serve as predictive markers of clinical outcomes. Methods: We conducted a presurgical clinical trial with anti-CTLA-4 antibody. Patients with localized bladder cancer (N=10) were given two doses of antibody prior to undergoing surgery. Immunological analyses were conducted on patients’ tissues and blood samples. Results: Expression of the inducible costimulator (ICOS) molecule was increased on CD4 T cells within tumor tissues and systemic circulation. ICOS-expressing T-cells have not previously been reported to have a role in anti-tumor responses. We showed that CD4+ICOShi T cells produced IFNγ and could recognize the NY-ESO-1 tumor antigen. Pre- and post-therapy CD4 T cells were analyzed by reverse-phase protein array and microRNA array, which led to the identification of signaling pathways and regulatory mechanisms that play a role in enhanced T-cell function. Furthermore, murine models confirmed our initial findings and implied a role for ICOS-expressing T-cells in antitumor responses. Finally, we extended our findings to the metastatic disease setting of melanoma patients (N=14) and our preliminary data indicate an improved survival for anti-CTLA-4 treated patients who have a sustained increase in ICOS-expressing CD4 T cells. Conclusions: Our presurgical clinical trial allowed for the correlation of data from tumor tissues with data from peripheral blood, thus identifying ICOS-expressing T-cells as a relevant biomarker that can be used to monitor patients who receive anti-CTLA-4 therapy. [Table: see text]

Flt3-L Increases CD4+CD25+Foxp3+ICOS+Cells in the Lungs of Cockroach-Sensitized and -Challenged Mice

We previously reported in an ovalbumin-induced model of allergic asthma that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11c(high)CD8 alpha(high)CD11b(low) dendritic cells in the lung. In this study, we investigated the effect of Flt3-L in a clinically relevant aeroallergen-induced asthma on the phenotypic expression of lung T cells. Balb/c mice were sensitized and challenged with cockroach antigen (CRA), and AHR to methacholine was established. These mice received three intraperitoneal injections of anti-CD25 antibody (PC61; 250 microg) and Flt3-L (3 microg) daily for 10 days. Cytokines and Ig levels in the serum were measured and differential bronchoalveolar lavage fluid (BALF) cell counts were examined. Flt3-L reversed AHR to methacholine to the control level. Flt3-L significantly decreased levels of BALF IL-5, IFN-gamma, eosinophilia and substantially increased IL-10 and the number of CD4(+)CD25(+) Forkhead winged helix transcription factor box P3 (Foxp3(+)) IL-10(+) T cells in the lung. Administration of PC61 antibody blocked the effect of Flt3-L and substantially increased AHR, eosinophilia, and BALF IL-5 and IFN-gamma levels, and decreased BALF IL-10 levels and the number of CD4(+)CD25(+)Foxp3(+)IL-10(+) T cells. Flt3-L significantly decreased CD62-L, but increased inducible costimulatory molecule and Foxp3 mRNA expression in the CD4(+)CD25(+) T cells isolated from lungs of Flt3-L-treated, CRA-sensitized mice compared to CRA-sensitized mice without Flt3-L treatment and PBS control group. Flt3-L significantly inhibited the effect of CRA sensitization and challenge to increase GATA3 expression in lung CD4(+)CD25(+) T cells. Collectively, these data suggest that the therapeutic effect of Flt3-L is mediated by increased density of naturally occurring CD4(+)CD25(+)Foxp3(+)IL-10(+)ICOS(+) T-regulatory cells in the lung. Flt3-L could be a therapeutic strategy for the management and prevention of allergic asthma.

Naturally Occurring and Inducible T-Regulatory Cells Modulating Immune Response in Allergic Asthma

T-regulatory cells (Tregs) are potent immunomodulators in allergic asthma. We evaluated the functional effects of Tregs by adoptively transferring naturally occurring CD4(+)CD25(+) Tregs (NTregs) and CD4(+)CD25(-) inducible Tregs (iTregs) from lung and spleens of green fluorescent protein (GFP)-transgenic Balb/c mice into cockroach-sensitized and -challenged mice. GFP-labeled NTregs and iTregs were adoptively transferred into cockroach-sensitized and -challenged mice. Airway hyperresponsiveness (AHR) to methacholine was examined using a single-chamber, whole-body plethysmograph and invasive tracheostomy. Adoptive transfer of either NTregs or iTregs from lung or spleen reversed airway inflammation and AHR to methacholine, and the effect lasted for at least 4 weeks. GFP-labeled iTregs up-regulated CD25 and forkhead-winged transcriptional factor box protein 3 and migrated to lymph node and lung. Lung CD4(+)CD25(+) T cells isolated from each group of recipient mice were inducible costimulatory molecule-high and programmed death (PD)-1-positive; however, higher expression of PD-1 was found in the spleen iTregs (S25(-)) and lung iTregs (L25(-)) groups. Higher levels of transforming growth factor-beta and IL-10 mRNA transcripts and bronchoalveolar lavage fluid IL-10 and INF-gamma levels were observed in lung CD4(+)CD25(+) cells from the L25(-) and S25(-) cell-recipient mice than from lung NTregs (L25(+)) and spleen NTregs (S25(+)) cell-recipient mice. Adoptive transfer of either cell type significantly reduced bronchoalveolar lavage fluid IL-4, IL-5, and IL-13 levels. Tregs reverse AHR and airway inflammation; however iTregs that differentiated into IL-10-producing CD4(+) type 1 cells in the lung exert their suppressive activity likely by higher levels of transforming growth factor-beta, IL-10, IFN-gamma, and elevated levels of PD-1 compared with NTregs. Hence, PD-1 may be a conduit for reversing AHR by Tregs and a plausible target for treating asthma.

ICOS‐gene variants are not associated with atopic disease susceptibility in European children

The inducible co-stimulatory molecule, ICOS, is an important regulator of T cell differentiation and effector function. Previously, it was reported that two variants in the ICOS promotor region, g.1-1413G>A and g.1-693G>A, were associated with sensitization to airborne allergens, elevated serum IgE levels and Th2 cytokine production in a Hutterite population. The aim of this study was to evaluate these two and four other selected ICOS variants for association with atopic phenotypes in two large European prospective pediatric cohorts. We investigated subjects from the German Multicenter Allergy Study (MAS), which followed over 800 children with atopic family history from birth until 13 yr of age, and from the Early Treatment of the Allergic Child Study (ETAC), which collected DNA and clinical data of over 330 children with atopic dermatitis during their first 2 yr of life. We genotyped DNA from these children by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. We could not confirm the previously reported association of g.1-1413G>A and g.1-693G>A with atopic phenotypes in our pediatric cohorts. Also four other ICOS variants at putative binding sites for transcription factors showed no association with atopic dermatitis, asthma, allergic sensitization and allergic rhinitis. Our data suggest that these ICOS variants do not play a major role in the development of atopy in European children.

ICOS Deficiency Results in Exacerbated IL-17 Mediated Experimental Autoimmune Encephalomyelitis

Inducible costimulatory molecule (ICOS) is important for the effector function of T cells, especially for Th2 and T cell dependent B cell responses. However, it has been shown that ICOS is required for the differentiation of Th17 cells. Since IL-17 has been identified as a major cytokine involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the enhanced severity of EAE in ICOS-deficient mice (ICOS(-/-)) mice is unexpected. To better understand the role of ICOS and of IL-17 in EAE, we induced EAE in ICOS(-/-) by immunization with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)) in complete Freund's adjuvant. As previously reported, we found that ICOS(-/-) mice developed more severe EAE. Upon restimulation with MOG(35-55,) splenocytes from ICOS(-/-) mice with EAE produced higher amounts of IL-17 and ICOS(-/-) mice had a higher expression of IL-17, IL-6, and TGF-beta mRNA in the spinal cords at the onset of the disease. Finally, the blockade of IL-17 strongly inhibited disease even in ICOS(-/-) mice, showing that IL-17 is playing a major role in the pathogenesis of EAE both in WT and ICOS(-/-) mice. In conclusion, MOG immunization induces MOG-specific Th17 cells also in ICOS(-/-) mice, and a higher expression of IL-17 and of Th17-driving cytokines IL-6 and TGF-beta in the central nervous system at the onset of EAE that correlates with their more severe disease.

Endogenous 4-1BB Ligand Plays a Critical Role in Protection from Influenza-Induced Disease

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells

The inducible costimulatory molecule ICOS has been suggested to be important in the development of interleukin 17 (IL-17)-producing helper T cells (T(H)-17 cells) and of follicular helper T cells (T(FH) cells). Here we show that ICOS-deficient mice had no defect in T(H)-17 differentiation but had fewer T(H)-17 cells after IL-23 stimulation and fewer T(FH) cells. We also show that T(FH) cells produced IL-17 and that T(FH) cells in ICOS-deficient mice were defective in IL-17 production. Both T(H)-17 and T(FH) cells had higher expression of the transcription factor c-Maf. Genetic loss of c-Maf resulted in a defect in IL-21 production and fewer T(H)-17 and T(FH) cells. Thus our data suggest that ICOS-induced c-Maf regulates IL-21 production that in turn regulates the expansion of T(H)-17 and T(FH) cells.

Costimulatory Blockade Enhances Engraftment and Prevents Anti-Donor Antibody Generation in Nonmyeloablative Conditioned Prediabetic NOD Mice

Chimerism induces tolerance to organ and tissue grafts. Although the toxicity of conditioning has been significantly reduced with nonmyeloablative approaches, it would be beneficial to further reduce the intensity of conditioning in organ transplant recipients. Studies in normal mice and autoimmune NOD mice have revealed that the primary role for conditioning is to control host-versus-graft alloreactivity, allowing for immune-based conditioning to establish chimerism. Recent insights into the mechanism of interaction and crossregulation between innate and adaptive immune responses have led to the development of new approaches for immune-based conditioning. We previously demonstrated that preconditioning of diabetes-prone NOD mice with anti-CD8 monoclonal antibody (mAb) combined with anti-CD154 mAb allowed chimerism to be established with 550 cGy total body irradiation (TBI). Here we investigated whether combined costimulatory blockade can improve the nonmyeloablative regimen and enhance bone marrow chimerism in autoimmune recipients. Prediabetic NOD (H-2d) mice were treated with anti-CD8 antibody (day -3). After 500 cGy TBI (day 0), recipient NOD mice were transplanted with 30 × 106 B10.BR (H-2k) bone marrow cells (day 0) and received anti-CD154, anti-OX40L, and anti- inducible co-stimulatory molecule (ICOS) mAbs intraperitoneally (IP) (day 0, 1, 2, 3). Chimerism and multilineage analysis were quantitated by flow cytometry. Chimeric animals produced multilineage donor chimerism. While 18 prediabetic NOD mice demonstrated an average PB chimerism of ~37% at 1 month and 61% engraftment, 15 of 18 mice lost PB chimerism by 6 months. NOD mice were then transplanted with B10.BR donor skin grafts 1 month post BM transplant. Flow cytometry cross-match assays were performed to detect anti-donor antibody (Ab) in the sera collected 1 month post skin grafting. 44% of chimeric NOD mice (n=9) accepted their skin grafts for over 90 days, while 100% of non-chimeric animals (n=4) rejected their grafts within 60 days. NOD mice transplanted with B10.BR skin grafts alone rejected their grafts within 2 weeks and generated significantly higher levels of donor-specific Abs (mean fluorescence intensity: 251.2 ± 61.5, P<0.0001, n=6) compared to chimeric NOD (4.5 ± 0.6, P<0.0005, n=17) or non-chimeric NOD (12.6 ± 2.4, P<0.0005, n=10). Both chimeric NOD and non-chimeric NOD mice transplanted with 3rd party B6 skin grafts rejected their grafts within 30 days. These results suggest that rejection of skin grafts in chimeric and nonchimeric animals is not associated with anti-donor antibody generation in preconditioned animals. Studies are currently underway to determine if impaired generation of effector/memory T cells (CD44high/CD62Llow/−) is responsible for enhanced engraftment and to determine the potential mechanisms that promote tolerance in prediabetic NOD mice after loss of PB chimerism.

Adenovirus‐mediated ICOSIg gene transfer alleviates cardiac remodeling in experimental autoimmune myocarditis

To explore the therapeutic effects of adenovirus vector mediated transfer of the ICOSIg gene on immuno-inflammation-mediated cardiac remodeling in an experimental autoimmune myocarditis (EAM) model, pAdeno-ICOSIg was constructed and transfected into HEK 293 cells to produce the ICOSIg adenovirus. Ad-CMV-GFP was used as a control. EAM was induced in Lewis rats by injection of porcine cardiac myosin. The immunized rats were divided into two groups. The inducible co-stimulatory molecule (ICOS) group received the adenovirus containing ICOSIg on day 14; the green fluorescent protein (GFP) group received the adenovirus containing GFP as the control adenovirus and 15 normal rats (Control group) consisted of the normal controls that were not immunized. On day 28, all rats were euthanized after echocardiography and histopathologically examined for cardiac fibrosis. Western blotting was performed to detect ICOS, ICOS ligand (ICOSL), matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and real-time RT-PCR was performed to detect B7-1, B7-2 and interleukin (IL)-17 expression. ELISPOT was applied to detect Th1 and Th2 cytokine production. Collagen concentration and collagen cross-linking were determined as markers of cardiac fibrosis. It was found that blockade with ICOSIg exerted antifibrotic effects on cardiac remodeling in EAM. On day 28, cardiac function and inflammatory myocardial fibrosis improved significantly in the ICOS group compared to the GFP group. The expression of ICOS, the ICOSL, B7-1 and IL-17 was statistically significantly lower in the ICOS and Control groups compared to the GFP group. ICOSIg significantly augmented Th2 cytokine production and diminished Th1 and Th17 cytokine production. This blockade of the ICOS co-stimulatory pathway with ICOSIg alleviated autoimmune inflammation-mediated cardiac remodeling and improved cardiac function. Regulation of the Th1/Th2/Th17 balance may be one of the underlying mechanisms responsible for this effect.

Expression of inducible co-stimulatory molecule in cornea of rats

目的 探讨可诱导的共刺激分子(ICOS)在大鼠角膜移植排斥反应中角膜上的表达和意义.方法 采用逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹分析检测大鼠角膜移植后角膜中ICOS mRNA和蛋白的表达;免疫组织化学方法 检测ICOS蛋白在角膜中的表达部位.结果 ICOS在正常的角膜中无表达;同种异体角膜移植组术后角膜中的ICOS mRNA转录及蛋白的表达从第1 d起即呈明显上升趋势,第7 d达高峰;免疫组织化学显示同种异体角膜移植术后ICOS阳性表达见于角膜基质层浸润的淋巴细胞.结论 同种异体角膜移植术后ICOS在角膜中表达较对照组明显升高,可能在同种异体角膜术后的免疫排斥反应中起重要作用。

Importance of CD80/CD86–CD28 interactions in the recognition of target cells by CD8+CD122+ regulatory T cells

CD8+CD122+ regulatory T cells are a newly identified, naturally occurring type of regulatory T cell that produce interleukin-10 (IL-10) and effectively suppress interferon-gamma (IFN-gamma) production from both CD8+ and CD4+ target cells. Molecular mechanisms responsible for the recognition of target cells by CD8+CD122+ regulatory T cells were investigated in this study by using an in vitro culture system that reconstitutes the regulatory action of these cells. CD8+CD122( regulatory T cells did not produce IL-10 and did not suppress the IFN-gamma production of allogeneic target T cells when they were stimulated by immobilized anti-CD3 antibody alone, but they clearly produced IL-10 and suppressed the IFN-gamma production of target cells when stimulated by anti-CD3 plus anti-CD28-coated beads. IFN-gamma production by major histocompatibility complex-class I-deficient T cells was also suppressed by CD8+CD122+ regulatory T cells stimulated with anti-CD3 plus anti-CD28 antibody but was not suppressed by cells stimulated by anti-CD3 alone. Experiments examining the blockade of cell surface molecules expressed on either the regulatory cells or the target cells by adding specific neutralizing antibodies in the culture indicated that CD80, CD86, and CD28 molecules were involved in the regulatory action, but cytotoxic T lymphocyte antigen-4, inducible costimulatory molecule (ICOS) and programmed death-1 (PD-1) molecules were not. Finally, CD8+CD122+ cells isolated from CD28-knockout (CD28-/-) mice showed no regulatory activity. These results indicate that CD8+CD122(+) regulatory T cells recognize target T cells via the interaction of CD80/CD86-CD28 molecules to become active regulatory cells that produce suppressive factors such as IL-10.

Analysis of genetic defects in patients with the common variable immunodeficiency phenotype in a single Taiwanese tertiary care hospital

Seven known genetic defects, including Bruton tyrosine kinase (Btk), CD4OL, and signaling lymphocyte activation molecule-associated protein (SAP) (all X-linked) and inducible costimulator molecule (ICOS), transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI), B-cell-activating factor of the tumor necrosis family receptor (BAFFR), and CD19 (all autosomal recessive), were found in patients with the phenotype of common variable immunodeficiency (CVID). To investigate these 7 candidate protein expressions and candidate gene sequences for comprehensive analysis of known genetic defects in patients with CVID. These 7 candidate protein expressions were evaluated by flow cytometry or Western blot, and candidate genes were evaluated by direct sequencing. Of 9 CVID patients from a single Taiwanese tertiary care hospital, we identified 2 cousins with decreased Btk expression who had a mutated (Asp521Val) kinase domain of Btk (1694A>T in exon 15) and 1 patient with decreased CD40L expression who had a mutated (Thr254Met) extracellular domain of CD40L (782T>C in exon 5). This comprehensive approach revealed that, in Taiwan, in some patients mild forms of X-linked agammaglobulinemia and hyper-IgM syndrome caused the CVID phenotype. No mutations of SAP, ICOS, TACI, BAFFR, and CD19 were identified in this study, although selection bias among the small study population and genetic variation may exist.

The Influence of the Regulatory T Lymphocytes on Atherosclerosis

Atherosclerosis is complex inflammatory disease of the arterial wall, in which T lymphocytes play a significant role.1 Since the recognition that T lymphocytes are present in human atherosclerotic plaque nearly 2 decades ago,2 research has focused on the functional importance of these cells in the atherosclerotic process. The majority of T lymphocytes in atherosclerotic lesions are CD4+ T-helper cells with a phenotype characteristic of the proinflammatory T-helper 1 (Th1) subset. These cells recognize specifically antigens that are produced in relative abundance in hypercholesterolemic individuals or in plaques including oxidatively modified LDL (Ox-LDL) and HSP60/65. The T cells are activated when macrophage or dendritic cells present these antigens to the T cells in plaques or lymphoid tissues. The Th1 cells produce inflammatory cytokines IFN-γ, tumor necrosis factor (TNF)-α, and membrane CD40-ligand, which amplify the immune response through activation of macrophages, vascular smooth muscle cells, and endothelial cells.1 See page 2691 Many mechanisms have evolved to maintain immunologic self-tolerance and to limit responses to foreign antigens. One of these mechanisms involves regulatory T cells (Treg) that actively suppress responses of effector T cells. The best- characterized Treg are the natural CD4+CD25+ Treg that mature in the thymus and comprise 5% to 10% of peripheral CD4+ T cells.3 Other surface markers expressed by Treg include CTLA-4 and GITR. FoxP3, a forkhead family transcription factor, is a lineage specification factor for Treg and plays a crucial role in their suppressive function.4 Natural Treg are generated during thymic development, but Treg are also induced in peripheral tissues during immune responses. Treg express antigen receptors typical of effector T cells and are presumably activated by peptide antigens presented by APCs. They also require interleukin (IL)-2 for development and survival. Once activated, Treg may suppress other T …

ICOS/B7RP-1 Interference in Mouse Kidney Transplantation

Activated T cells play a key role in allograft rejection. T cell activation requires signaling via the T cell receptor as well as costimulatory signals. Inducible costimulatory molecule (ICOS), with its ligand B7RP-1, is a recently discovered costimulatory molecule of the CD28 family. The role of this signaling pathway during the early phases of kidney allograft rejection is not clear so far. Kidneys were orthotopically transplanted from BALB/c to C57BL/6 mice. Animals were assigned to five experimental groups: blocking anti-ICOS monoclonal antibody, ICOS fusion protein, anti-B7RP1 monoclonal antibody, B7RP-1 fusion protein, and control immunoglobulin G. Survival was significantly reduced in animals treated with ICOS monoclonal antibody (mAb) and B7RP-1 Fc as compared with controls. These animals had also a lower number of apoptotic graft infiltrating T cells, whereas the expression of intracellular interferon-gamma in CD3CD4 T cells was increased. Animals treated with ICOS Fc and B7RP-1 mAb had similar survival and numbers of apoptotic T cells as compared with controls. In summary, the blockade of ICOS with ICOS mAb or B7RP-1 Fc reduced the amount of apoptosis of infiltrating lymphocytes and resulted in continuous inflammatory processes with progressive tissue damage and graft failure.

Translational Mini-Review Series on Immunodeficiency:&amp;#x2028;Molecular defects in common variable immunodeficiency

Common variable immunodeficiency (CVID) is a primary immunodeficiency that typically affects adults and is characterized by abnormalities of quantative and qualitative humoral function that are heterogeneous in their immunological profile and clinical manifestations. The recent identification of four monogenic defects that result in the CVID phenotype also demonstrates that the genetic basis of CVID is highly variable. Mutations in the genes encoding the tumour necrosis factor (TNF) superfamily receptors transmembrane activator and calcium-modulating ligand interactor (TACI) and B cell activation factor of the TNF family receptor (BAFF-R), CD19 and the co-stimulatory molecule inducible co-stimulator molecule (ICOS) all lead to CVID and illustrate the complex interplay required to co-ordinate an effective humoral immune response. The molecular mechanisms leading to the immune defect are still not understood clearly and particularly in the case of TACI, where a number of heterozygous mutations have been found in affected individuals, the molecular pathogenesis of disease requires further elucidation. Together these defects account for perhaps 10-15% of all cases of CVID and it is highly likely that further genetic defects will be identified.

Differential CD28 and Inducible Costimulatory Molecule Signaling Requirements for Protective CD4+T-Cell-Mediated Immunity against Genital TractChlamydia trachomatisInfection

Th1 cells and gamma interferon (IFN-gamma) production play critical roles in protective immunity against genital tract infections by Chlamydia trachomatis. Here we show that inducible costimulatory molecule (ICOS)(-/-) mice develop greatly augmented host resistance against chlamydial infection. Protection following a primary infection was characterized by strong Th1 immunity with enhanced CD4(+) T-cell-mediated IFN-gamma production in the genital tract and high expression of T-bet in the draining para-aortic lymph node. This Th1 dominance was associated with low expression of interleukin 10 (IL-10) mRNA in the uteruses of protected ICOS(-/-) mice. By contrast, CD28(-/-) mice were severely impaired in their adaptive immune response, demonstrating a lack of CD4(+) T cells and IFN-gamma in the genital tract, with a substantial delay in bacterial elimination compared to that seen in wild-type (WT) mice. Upon reinfection, WT mice exhibited a transient local infection with evidence of regulatory T-cell (Treg)/Foxp3 mRNA and a more balanced Th1 and Th2 response in the genital tract than ICOS(-/-) mice, whereas 90% of the latter mice developed sterile immunity, poor expression of local Treg/Foxp3 mRNA, and macroscopic signs of enhanced local immunopathology. Therefore, different requirements for CD28 signaling and ICOS signaling clearly apply to host protection against a genital tract infection by C. trachomatis. Whereas, CD28 signaling is critical, ICOS appears to be dispensable and can have a dampening effect on Th1 development by driving Th2 immunity and anti-inflammation through IL-10 production and promotion of the Foxp3(+) Treg populations in the genital tract. Both the CD28-deficient and the ICOS-deficient mice demonstrated poor specific antibody production, supporting the fact that antibodies are not needed for protection against genital tract chlamydial infections.

ICOS and B7 costimulatory molecule expression identifies activated cellular subsets in rheumatoid arthritis

To better define important cell subsets expressing activation markers in rheumatoid arthritis (RA), we compared selective lymphocyte and monocyte B7H1, B7H2, B7RP.1, B7RP.2, and inducible costimulatory molecule (ICOS) expression from normal peripheral blood (NL PB), RA PB, and RA synovial fluid (SF) by multicolor flow cytometry and immunohistochemistry. RA SF memory lymphocytes expressed B7RP.1 and B7RP.2, suggesting that T-cells may function as antigen presenting cells (APCs) in RA joints. We found similar results for ICOS expression. RA SF CD14+ monocytes also expressed B7RP.1 (an ICOS ligand) and the homologous ligand B7RP.2, identifying monocytes as potential mediators of antigen processing and lymphocyte activation in RA. Furthermore, we found an increased population of RA SF CD14+ monocytes expressing B7H1 and B7H2. [The FACS analysis was supported by immunohistochemistry, showing intense lymphocyte and APC (macrophages with dendritic morphology) ICOS staining in RA synovial tissue (ST). Overall, these results define elevated populations of memoryT-lymphocytes expressing proinflammatory B7 molecules in RA SF that either stimulate T cells through ICOS (via ICOS ligands B7RP.1 and B7RP.2), or down-regulate RA ST T-lymphocytes through B7H1 and B7H2.] Therefore, in the same joint, there may exist positive and negative influences on the inflammatory response, and perhaps, the negative signals dominate as joint inflammation resolves.

Enhanced Osteoclastogenesis in 4-1BB—Deficient Mice Caused by Reduced Interleukin-10

Enhanced osteoclastogenesis was observed in bone marrow-derived macrophage cells from 4-1BB-deficient mice than in those from wildtype mice. 4-1BB and 4-1BB ligand interaction may play a role at a certain stage of osteoclast formation through increased level of IL-10, a negative regulator of osteoclastogenesis. 4-1BB is an inducible T-cell costimulatory molecule and a member of the TNF receptor family. The expression pattern of 4-1BB and 4-1BB ligand (4-1BBL) has suggested that 4-1BB plays a role not only in various responses related to innate immunity but also in bone metabolism. Osteoclast formation was evaluated in bone marrow-derived macrophage cells (BMMs) from wildtype and 4-1BB-deficient (4-1BB-/-) mice. Expression of interleukin-10 (IL-10) during osteoclast formation was analyzed at the mRNA and protein levels. Expression of IL-10 was higher in RANKL-stimulated wildtype BMMs than 4-1BB-/- BMMs. When 4-1BBL was stimulated with 4-1BB-Fc fusion protein, the expression of IL-10 in BMMs increased. Neutralization of IL-10 was not as effective in preventing inhibition by IL-10 of osteoclast differentiation in 4-1BB-/- BMMs as in wildtype BMMs. When IL-10 was added to the culture medium, osteoclast formation was inhibited more efficiently in the 4-1BB-/- BMMs than in the wildtype BMMs. Interaction of 4-1BB and 4-1BBL stimulates IL-10 production through 4-1BBL signaling. 4-1BBL plays a role at a certain stage of osteoclast formation, and IL-10 may mediate this effect. The elevated level of osteoclastogenesis in 4-1BB-/- BMMs may thus be caused, in part, by a lower level of IL-10.

Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory Molecule

T-cell-mediated immunity contributes to the pathogenesis of atherosclerosis, but little is known about how these responses are regulated. We explored the influence of the inducible costimulatory molecule (ICOS) on atherosclerosis and associated immune responses. Bone marrow chimeras were generated by transplanting ICOS-deficient or wild-type bone marrow into irradiated atherosclerosis-prone, LDR receptor-deficient mice, and the chimeric mice were fed a high-cholesterol diet for 8 weeks. Compared with controls, mice transplanted with ICOS-deficient marrow had a 43% increase in the atherosclerotic burden, and importantly, their lesions had a 3-fold increase in CD4+ T cells, as well as increased macrophage, smooth muscle cell, and collagen content. CD4+ T cells from ICOS-deficient chimeras proliferated more and secreted more interferon-gamma and tumor necrosis factor-alpha than T cells from control mice, which suggests a lack of regulation. FoxP3+ regulatory T cells (Treg) were found to constitutively express high ICOS levels, which suggests a role for ICOS in Treg function. ICOS-deficient mice had decreased numbers of FoxP3+ Treg and impaired in vitro Treg suppressive function compared with control mice. ICOS has a key role in regulation of atherosclerosis, through its effect on regulatory T-cell responses.

Differential Effects of Costimulatory Pathway Modulation on Corneal Allograft Survival

T lymphocytes have a central role in allograft rejection. On engagement of the T cell receptor by antigenic peptide-major histocompatibility complex (MHC) complex, a second "costimulatory" signal is critical to full T-cell activation or downregulation. In this study, the effect on corneal allograft survival of modulation of the costimulatory molecules programmed death-1 (PD-1) and inducible costimulatory (ICOS) molecule was examined. These molecules are known to modulate, respectively, negative or positive T-cell activation signals. A dimeric PD-L1 immunoglobulin (Ig) fusion protein was generated to stimulate the inhibitory receptor PD-1, and a monoclonal antibody was used to block ICOS. The effect of PD-1 engagement and ICOS blockade on lymphocyte activation by in vitro T-cell proliferation and the effect on orthotopic corneal allograft survival in BALB/c mice were determined. Both reagents demonstrated T-cell inhibition in vitro. PD-L1.Ig treatment of BALB/c mice prolonged fully MHC-mismatched C3H donor corneal allograft survival, with a median survival time (MST) of 21 days. This was significantly prolonged compared to isotype control protein-treated recipients (MST 13 days, P < 0.003). Allograft survival in BALB/c recipients treated with anti-ICOS antibody showed no prolongation of survival compared with the isotype control antibody (MST, 12 days in both groups). Augmented ligation of the PD-1 negative costimulatory molecule significantly prolongs corneal allograft survival. However, in contrast to findings in other allograft models, signaling through the positive costimulatory molecule ICOS appears to be less important in allogeneic rejection of cornea.