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

A detailed phenotypic analysis of immune cell populations in the bronchoalveolar lavage fluid of atopic asthmatics after segmental allergen challenge

BACKGROUND: Atopic asthma is characterized by intermittent exacerbations triggered by exposure to allergen. Exacerbations are characterized by an acute inflammatory reaction in the airways, with recruitment of both innate and adaptive immune cells. These cell populations as well as soluble factors are critical for initiating and controlling the inflammatory processes in allergic asthma. Detailed data on the numbers and types of cells recruited following allergen challenge is lacking. In this paper we present an extensive phenotypic analysis of the inflammatory cell infiltrate present in the bronchoalveolar lavage (BAL) fluid following bronchoscopically directed allergen challenge in mild atopic asthmatics. METHODS: A re-analysis of pooled data obtained prior to intervention in our randomized, placebo controlled, double blinded study (costimulation inhibition in asthma trial [CIA]) was performed. Twenty-four subjects underwent bronchoscopically directed segmental allergen challenge followed by BAL collection 48 hours later. The BAL fluid was analyzed by multi-color flow cytometry for immune cell populations and multi-plex ELISA for cytokine detection. RESULTS: Allergen instillation induced pro-inflammatory cytokines (IL-6) and immune modulating cytokines (IL-2, IFN-γ, and IL-10) along with an increase in lymphocytes and suppressor cells (Tregs and MDSC). Interestingly, membrane expression of CD30 was identified on lymphocytes, especially Tregs, but not eosinophils. Soluble CD30 was also detected in the BAL fluid after allergen challenge in adult atopic asthmatics. CONCLUSIONS: After segmental allergen challenge of adult atopic asthmatics, cell types associated with a pro-inflammatory as well as an anti-inflammatory response are detected within the BAL fluid of the lung

Targeted Knock-In Mice Expressing Mutations of CD28 Reveal an Essential Pathway for Costimulation▿ †

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