Suppressor of cytokine signaling (SOCS) proteins are induced by IL-7 and the interaction of SOCS proteins with the IL-7 receptor alpha (CD127) may play a role in regulating CD127 expression in human CD8 T cells Feras Al-Ghazawi, Parmvir Parmar, Elliott Faller and Paul MacPherson University of Ottawa and The Ottawa Hospital Research Institute

Antigen-specific CTL response Homeostatic response CD8 T cell Apoptosis + IL-7 Survival Proliferation Apoptosis Antigen-specific CTL response Memory Infected/cancer cell MHC I TCR Ag Cytotoxic molecules + IL-7 Clonal Expansion Clonal Expansion CTL response CTL response Memory Memory

IL-7 Receptor Heterodimer composed of a unique α-chain (CD127) and a common γ-chain (CD132) that is shared with the receptors for IL-2, -4, -9, -15 and -21. IL-7Rα (CD127) is a 75 kDa type-I transmembrane protein with an extracellular domain and a cytoplasmic tail. IL-7Rα is majorly expressed on T cells and their precursors. http://en.wikipedia.org

IL-7 signaling is dysregulated in HIV infection HIV infected individuals have increased plasma concentrations of IL-7 averaging 18 pg/ml compared to the much lower average 2.2 pg/ml found in the sera of HIV-negative controls. Surface expression of CD127 is down regulated on CD8 T-cells in HIV infected patients and is partially restored in patients receiving HAART. IL-7 is currently being examined in clinical trials to increase T cell numbers in lymphopenic patients due to BMT, chemotherapy and HIV infection. Therefore, it is important to understand how does IL-7 regulate the expression of its own receptor CD127.

IL-7 Down Regulates Total CD127 Protein IL-7, 10 ng/ml CD127 Hsp70 M 3 6 12 24 Hours N=6 IL-7=10 ng/ml 5

IL-7-mediated CD127 Down regulation is Dependent on 26S Proteasome β actin M IL-7 MG MG+IL-7 N=8 IL-7=10 ng/ml MG132=5 µM t=6hrs 6

Suppressor of cytokine signalling (SOCS) proteins Cytokine-inducible negative regulators of cytokine signaling. Eight members (SOCS1-7 and CIS). Common SH2 domain (interacts with phospho. machinery), C-term SOCS box (recruits E3 unbiquitin ligases), N-term. of varying lengths. Akhtar LN, Benveniste EN.J Virol. 2011 Mar;85(5):1912-21

SOCS1-7 and CIS transcripts are expressed in human CD8 T cells Basal levels – unstimulated CD8 T cells N=5

IL-7 induces the expression of CIS/SOCS1-2 mRNA transcripts in CD8 T cells

IL-7 mediated induction of SOCS mRNA is JAK-dependent IL-7=10 ng/ml JAKi=1.5 µM PI3Ki=50 µM t=3hrs

IL-7 mediated induction of SOCS mRNA is STAT5-dependent IL-7=10 ng/ml STAT5i=0.5 mM t=3hrs

IL-7 mediated induction of SOCS mRNA does not depend on new protein synthesis IL-7=10 ng/ml CHX=100 µM t=1hr

IL-7 induces the expression of CIS and SOCS1-2 proteins M 30’ 1h 2h 3h 6h 12h 24h 48h CIS β actin SOCS1 β actin SOCS2 β actin N=4 IL-7=10 ng/ml 13

CIS, SOCS1 and 2 proteins physically bind to CD127 in CD8 T cells Media - IP: Goat Isotype Media - IP: Goat anti-CD127 (M) IL-7 3h - IP: Goat anti-CD127 (IL-7) N=5 IL-7=10 ng/ml

IL-7 induces co-localization of CIS and SOCS2 with CD127 and with the early endosome marker EEA1 B CID127 co-localization with: EEA1 co-localization with: Media Media CIS CIS IL-7 IL-7 Media Media SOCS2 SOCS2 IL-7 IL-7 Media Media SOCS1 SOCS1 IL-7 IL-7

LAMP1 co-localization with: CIS and SOCS2 co-localize with CD127 and with markers of the proteasome but not with markers of the lysosome upon IL-7 stimulation C D LAMP1 co-localization with: MCP20 co-localization with: Media Media CIS CIS IL-7 IL-7 Media Media SOCS2 SOCS2 IL-7 IL-7 Media Media SOCS1 SOCS1 IL-7 IL-7

The degradation of CD127 and SOCS proteins are dependent on E3 ubiquitin ligases CIS SOCS2 β actin M IL-7 SMER3 SMER3+IL7 N=3 IL-7=10 ng/ml SMER3=30M

Contribution of SOCS1, SOCS2 and CIS proteins in the IL-7 mediated down regulation of CD127 as measured by siRNA-mediated knock-down assays A B CIS β actin M siRNAαCIS SOCS1 β actin M siRNAαSOCS1 SOCS2 β actin M siRNAαSOCS2 N=4 IL-7=10 ng/ml

Acknowledgements Blood donors

A B C pY449-CD127 CD127 CD127 β actin Media Jaki Jaki+IL-7 IL-7 Media