GIF regulation of CD4 T-dependent immune responses

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DESCRIPTION: (Adapted from applicant's abstract) This proposal is to establish the role of glycosylation-inhibiting factor (GIF), a 13-kDa cytokine in the generation and function of Th effector cells. GIF inhibits IgG1 and IgE antibody formation when administered around the time of immunization with DNP-Ova. GIF secreted from T cells is cysteinylated, i.e., modified by binding of free cysteine to Cys-60 in the sequence. Cys-modified recombinant human GIF(rhGIF) inhibited IgG1 and IgE antibody formation in vivo, whereas unmodified rhGIF did not. The modification is required not only for the bioactivity but also for the capability to bind to receptors on target cells. The receptor for this cytokine is detected on activated T and B cells and fresh NKT cells, whereas it is undetectable on small resting T and B cells, fresh conventional NKT cells, macrophage lines and dendritic cells. GIF is chemically cross-linked to a 50-52 kDa protein on target cells. This cytokine inhibited B cell Ig switch to IgG1 and IgE in vitro. It also inhibited antigen presentation mediated by B cell antigen receptor (BCR), whereas it showed little effect on that independent of BCR. GIF-/- T cells secreted more IL-2 and proliferated more than GIF+/+ T cells following stimulation with anti-CD3 and anti-CD28. GIF -/- T cells differentiated in vitro into Th effectors which secreted more IL-4 than GIF +/+ T cells, whereas the capability to secrete IFNg was similar between GIF -/- and +/+ Th effectors. It is hypothesized that this cytokine regulates the generation and function of Th effector cells by acting on cognate T-B interaction and directly on T cells. The effect of GIF on activation events on T and B cells will be analyzed by using antigen-specific T and B cells derived from transgenic mice that express antigen-specific TCR and BCR, respectively. The effect of GIF on antigen internalization and processing will be determined. Since BCR-mediated signaling regulates antigen presentation, the effect of GIF on signaling events induced by antigen will be investigated. The mechanism by which GIF regulates expansion and differentiation of T cells will be examined in vitro by using GIF -/- and +/+ T cells. To determine the in vivo function of this cytokine, GIF-/- and +/+ mice on BALB/c genetic background will be immunized and challenged with aerosolized Ova and allergic airway inflammation will be analyzed. Finally, in order to define the significance of this cytokine in the immune response, cDNA for GIF receptor will be cloned. For the receptor cloning, monoclonal antibodies (mAb) that specifically block the binding of GIF to target cells will be raised. Receptor protein will be purified by using the mAb and its partial amino acid sequences will be determined. cDNA libraries will be screened based on the amino acid sequences, or by expression cloning of the receptor through direct binding of anti-receptor mAb or 125I-GIF The experimental system proposed here will clarify the role of this cytokine and fill a critical gap in our knowledge regarding cytokine-mediated regulation of allergy.