1. 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

2. 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

3. 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.

4. 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.

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

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

7. 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 Mar;85(5):

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

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

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

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

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

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

15. 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

16. IL-7 induces co-localization of CIS and SOCS2 with CD127 and with the early endosome marker EEA1B
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

17. 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

18. 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

19. 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

21. Acknowledgements
Blood donors

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