ICOS-Expressing Lymphocytes Promote Resolution of CD8-Mediated Lung Injury in a Mouse Model of Lung Rejection

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8+ T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8+ T cell–mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG-/- mice, we found that CD4+ T cells and ICOS+/+ T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4+Foxp3 + ICOS+ T cells were enriched in the lungs of animals surviving lung injury and ICOS+/+ Tregs promoted survival in animals that received ICOS-/- T cells. Direct comparison of ICOS-/- Tregs to ICOS+/+ Tregs found defects in vitro but no differences in the ability of ICOS-/- Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function

Inducible Costimulator Expression Regulates the Magnitude of Th2-Mediated Airway Inflammation by Regulating the Number of Th2 Cells

Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5' promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology.We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/- mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/- mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/- mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/- is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4.These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation

Protective Effector Memory CD4 T Cells Depend on ICOS for Survival

Memory CD4 T cells play a vital role in protection against re-infection by pathogens as diverse as helminthes or influenza viruses. Inducible costimulator (ICOS) is highly expressed on memory CD4 T cells and has been shown to augment proliferation and survival of activated CD4 T cells. However, the role of ICOS costimulation on the development and maintenance of memory CD4 T cells remains controversial. Herein, we describe a significant defect in the number of effector memory (EM) phenotype cells in ICOS−/− and ICOSL−/− mice that becomes progressively more dramatic as the mice age. This decrease was not due to a defect in the homeostatic proliferation of EM phenotype CD4 T cells in ICOS−/− or ICOSL−/− mice. To determine whether ICOS regulated the development or survival of EM CD4 T cells, we utilized an adoptive transfer model. We found no defect in development of EM CD4 T cells, but long-term survival of ICOS−/− EM CD4 T cells was significantly compromised compared to wild-type cells. The defect in survival was specific to EM cells as the central memory (CM) ICOS−/− CD4 T cells persisted as well as wild type cells. To determine the physiological consequences of a specific defect in EM CD4 T cells, wild-type and ICOS−/− mice were infected with influenza virus. ICOS−/− mice developed significantly fewer influenza-specific EM CD4 T cells and were more susceptible to re-infection than wild-type mice. Collectively, our findings demonstrate a role for ICOS costimulation in the maintenance of EM but not CM CD4 T cells

Inducible Costimulator Expression Regulates the Magnitude of Th2-Mediated Airway Inflammation by Regulating the Number of Th2 Cells

Background: Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5′ promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology.Methodology/Principal Findings: We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/− mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/− mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/− mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/− is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4.Conclusion: These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.</p

Protective Effector Memory CD4 T Cells Depend on ICOS for Survival

Memory CD4 T cells play a vital role in protection against re-infection by pathogens as diverse as helminthes or influenza viruses. Inducible costimulator (ICOS) is highly expressed on memory CD4 T cells and has been shown to augment proliferation and survival of activated CD4 T cells. However, the role of ICOS costimulation on the development and maintenance of memory CD4 T cells remains controversial. Herein, we describe a significant defect in the number of effector memory (EM) phenotype cells in ICOS−/− and ICOSL−/− mice that becomes progressively more dramatic as the mice age. This decrease was not due to a defect in the homeostatic proliferation of EM phenotype CD4 T cells in ICOS−/− or ICOSL−/− mice. To determine whether ICOS regulated the development or survival of EM CD4 T cells, we utilized an adoptive transfer model. We found no defect in development of EM CD4 T cells, but long-term survival of ICOS−/− EM CD4 T cells was significantly compromised compared to wild-type cells. The defect in survival was specific to EM cells as the central memory (CM) ICOS−/− CD4 T cells persisted as well as wild type cells. To determine the physiological consequences of a specific defect in EM CD4 T cells, wild-type and ICOS−/− mice were infected with influenza virus. ICOS−/− mice developed significantly fewer influenza-specific EM CD4 T cells and were more susceptible to re-infection than wild-type mice. Collectively, our findings demonstrate a role for ICOS costimulation in the maintenance of EM but not CM CD4 T cells.</p

Decreased Th2 response in B7RP-1^+/− mice.

<p>Splenocytes from Wild-type and B7RP-1^+/− mice were removed and single-cell suspensions were made. The cells were stained with B7RP-1 and either CD19 or CD11c. Black = WT Grey = B7RP-1^+/−. (B) B6. B7RP-1^+/+ (black bars), B6. B7RP-1^+/− (grey bars), and B6. B7RP-1^−/− (white bars) mice were sensitized and challenged as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007525#s2" target="_blank">Materials and Methods</a>. Total cells in BAL fluid were counted and percent CCR3⁺ eosinophils, CD4⁺ T cells and CD8⁺ T cells were analyzed via flow cytometry and used to calculate total cell numbers and normalized to the average of the wild-type value. ICOS expression on BAL CD4⁺ T cells isolated from the mediastinal lymph nodes of mice is shown. Serum IgE levels were analyzed via ELISA.</p

ICOS^+/− T cells have decreased ICOS expression but equal activation status.

<p>ICOS^+/+ (black lines) and ICOS^+/− (grey line) T cells were isolated and stimulated with anti-CD3 and anti-CD28 antibodies in media alone or in the presence of IL-4 or anti-IL-4 antibodies. After 48 hours the cells were removed and expression of the indicated surface markers were analyzed via flow cytometry. (A) ICOS expression on CD4⁺ T cells stimulated under the indicated conditions. (B) Expression of the indicated cell-surface markers after stimulation for 48 hours in media alone cultures.</p

Reduced number of Th2 cells in the airways of ICOS^+/− mice.

<p>(A) ICOS^+/+.4get (black bars), ICOS^+/−.4get (grey bars) mice were activated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007525#s2" target="_blank">Materials and Methods</a>. Total cells in BAL fluid were counted and percent CCR3⁺ eosinophils, total CD4⁺ T cells, CD4⁺GFP⁺ and CD4⁺GFP⁻ T cells were analyzed via flow cytometry and used to calculate total cell numbers. (B) The percent of CD4⁺GFP⁺ cells from the indicated organs are shown. The results are from separate experiments that have been combined (ICOS^+/+ n = 7, ICOS^+/− n = 9). (C) CD4⁺GFP⁻ and CD4⁺GFP⁺ cells were sorted from the mediastinal lymph nodes and lungs of ICOS^+/+.4get and ICOS^+/−.4get mice. The cells were equalized and stimulated with anti-CD3 and anti-CD28 antibodies for 48 hours and the IL-4 production was analyzed via ELISPOT.</p