Compare The Signal Transduction Pathways Of Il-2 And ’Super-il-2-variants’

Interleukin-2(IL-2,also termed T cell growth factor),a type I cytokine produced by activated CD4~+and CD8~+T cells,is a multifunctional cytokine that plays a very important role in lymphocyte homeostasis.IL-2 has been approved by the FDA for the treatment of melanoma and renal cell carcinoma and showed beneficial clinical results.However,due to the short in vivo half-life of IL-2,a high dose of IL-2 is usually required to achieve satisfied efficacy,which often leads to a number of side effects that limits its clinical applications.In the attempt to hurdle the side effects associated with the IL-2 therapy,Levin and colleagues demonstrated that using a IL-2 mutant,named H9,which has a lower binding affinity toward the IL-2 receptor,could decrease IL-2-associated side effects while maintaining its therapeutic effect.In addition,Mitra and colleagues explored novel therapeutic strategy for autoimmune diseases using a variant of the H9 mutant,although the underlying molecular mechanism is largely elusive.To have a mechanic understand of the role of IL-2mutants during autoimmune disease therapy,we set out to compare downstream signaling events initiated by wildtype or mutant IL-2 using biochemical and proteomic methods.To compare the difference of wildtype and mutant IL-2 in promoting signaling complex assembly,we first constructed and purified Flag-tagged IL-2 and its mutants The Flag-tagged wildtype IL-2 is named SF-H2,while the single Flag-tagged H9RETR for SF-H9RETR and the double Flag-tagged H9CRE for DF-H9CRE.Upon confirming the expression of recombinant proteins by Western blotting and mass spectrometry(MS)and in vivo activity by MTT assay,we treated CTLL-2 cells,a cytotoxic T cell line,with the saturating dose(50ng/ml)of above IL-2 variants for 5minutes followed by affinity purification of the IL-2-induced signaling complex using the anti-Flag antibody.Upon tryptic digestion,associated complex components were mapped by mass spectrometry.However,no specific interactors were detected probability due to low affinity purification efficiency of single Flag tag.We also checked downstream signaling pathway activation by probing the AKT/PI3K,the RAS/MAPK and the JAK/STAT5 signal pathways using immunoblotting.We found that the SF-H9RETR was less capable in stimulating these pathways comparing to its wildtype counterpart,likely due to its low affinity towards IL-2R?.These observations are instructive for our subsequent experiments.IL-2 forms intramolecular disulfide bonds between C58 and C105 residues.Correct formation of this disulfide bond is essential for its activity.Therefore,we substituted C125 with serine to eliminating interference from this cysteine residue.In theory,the resulting H9CRE mutant should have increased activity.We have also added double Flag tag to the C-terminus of the protein(termed DF-H9CRE;and its wildtype counterpart is therefore called DF-H2C)to increase affinity purification efficiency.Western blot and mass spectrometry were again used to confirm the expression of these proteins.Both DF-H2C and DF-H9CRE can significantly stimulate the proliferation of CTLL-2 cells as judged by MTT assay.The proliferation curve of DF-H9CRE peaks at 50 ng/mL and is slightly low than that of DF-H2C.Subsequently,we stimulated CTLL-2 with 50 ng/mL DF-H2C and DF-H9CRE,respectively,for 5 min and probed the activation of downstream signaling pathways by immunoblotting.The results showed that extent of signaling intensity induced by DF-H9CRE was slightly lower than that by DF-H2C,which was consistent with results by MTT assays.By IP-MS experiments,we found IL-2Rα,IL-2Rβand JAK1were detectable in the DF-H2C co-IP samples while only IL-2Rβand JAK1 were detectable in the DF-H9CRE samples.Our works compared signaling ability of wildtype IL-2 and its mutants.We found that despite its strong binding to IL-2Rβ,SF-H9RETR could barely activate downstream signaling pathways while DF-H9CRE was much more active,although its activity is still suboptimal comparing to wildtype IL-2.Since both mutants demonstrated enhanced affinity towards IL-2Rβ,they could be used as antagonists in future IL-2 therapies.