Comparison between Pathogenic and Nonpathogenic SIV Infections and Focus on Mucosal Tissue Compartment Reveal a Critical Role for the Adenosine Pathway in the Control of SIV-Related Immune Activation and Inflammation Tianyu He, Egidio Brocca-Cofano, Delbert G. Gillespie, Cuiling Xu, Jan Kristoff, Samantha Ross, Dongzhu Ma, George Haret-Richter, Charles R. Rinaldo, Cristian Apetrei, Edwin K. Jackson, Bernard J.C. Macatangay, Ivona Pandrea

Background Despite ART, mortality rate is still high in HIV-1 infected patients, due to end-organ diseases caused by persistent immune activation/inflammation. CD39/CD73/Adenosine pathway has been reported as one of the mechanisms used by Tregs to suppress immune activation/inflammation. CD26 + Its role in HIV/SIV infection?

NHP models of SIVagm infection AFRICAN GREEN MONKEY Natural host of SIVagm Maintenance of mucosal barrier Lack of microbial translocation Lack of chronic IA/INFL Lack of comorbidities Lack of disease progression PIGTAILED MACAQUE Non natural host of SIVagm Altered mucosal barrier Microbial translocation Chronic IA/INFL SIV comorbidities (CVD) Progression to AIDS

Experimental Strategy African green monkeys (AGMs): no progression to AIDS, no immune activation and inflammation (IA/INFL) Pigtail macaques (PTMs): progression to AIDS, substantial IA/INFL. Non-Progressive : AGMs (n=5) Non-Progressive : AGMs (n=5) Progressive: PTMs (n=5) Progressive: PTMs (n=5) Infect with SIVagm ADO/INO level in tissues was measured with mass spectrometry CD39/CD73 and CD26 expression is assessed by flow cytometry on T cells from blood, lymph nodes (LNs) and intestine (INT) Correlate with immune activation and proliferation markers

Mucosal Site Presents Substantial Coexpression of CD39 and CD73 on Tregs CD39 CD73 PBMC LN INT AGMs PTMs

Mucosal Site Presents Substantial Coexpression of CD39 and CD73 on Tregs  Almost no coexpression of CD39 and CD73 on Tregs in the blood, in both AGMs and PTMs.  Prior to infection, AGMs extensively coexpress CD39 and CD73 on Tregs in the intestine.  Such coexpression is not seen in PTMs prior to infection.

Mucosal Site Presents Substantial Coexpression of CD39 and CD73 on Tregs  After infection, the coexpression of CD39 and CD73 on Tregs increased significantly in PTMs in the INT.  Suggest possible increase in ADO production during progressive infection.

Adenosine Levels in the Tissues  ADO is intrinsically higher in AGMs in the INT and further increases in acute infection.  No significant change of ADO level was observed in lymph node.

CD26 Expression Increase Dramatically after Infection in PTMs in Intestine  In the intestine, CD26 expression increases dramatically after infection in PTMs.  In AGMs, CD26 expression remains around pre- infection level throughout the infection.

Inosine Levels in the Tissues  INO increased significantly at chronic infection stage in PTMs.  No significant change of INO level was observed in lymph node.

CD26 Expression on CD4 + T cells Correlates with Inosine Level  CD26 expression on CD4 + T cells correlates with INO level, and inversely correlates with ADO level in the gut.

ADO/INO Levels Correlates with Immune Activation/Proliferation Markers  Adenosine level in the gut inversely correlates with T cell proliferation.  Inosine level directly correlates with both CD4 + and CD8 + T cell proliferation and activation.

Exogenous ADO Suppress IFN-γ Production ADENOSINE CONTROL CD4 CD8 IFN-γ PBMCs CD3 CD28 stimulation + ADO - ADO cytokine production

Exogenous ADO Suppress Cytokine Production

Summary  Increased levels of ADO is sustained in the intestine of AGMs during SIVagm infection.  In PTMs, although CD39 and CD73 increased after infection, ADO production appears to be counteracted by a massive increase of CD26 which occurs very early after infection.  ADO plays a role in suppressing IA/INFL.  The adenosine pathway is significantly involved in the control of IA/INFL in the nonprogressive non-human primate model.  Changes of ADO-associated markers predominately occur in the gut, suggesting that future study about this pathway in the context of HIV/SIV infection should focus on the mucosal site.

Acknowledgement Pandrea/Apetrei lab Egidio Brocca-Cofano Jan Kristoff George Haret-Richter Ross Samantha Cuiling Xu Jennifer Stock Cristian Apetrei Ivona Pandrea Edwin Jackson lab Delbert G. Gillespie Edwin K. Jackson Bernard J.C. Macatangay MD Charles R. Rinaldo, Jr. PhD