In HIV infection a large frequency of HIV specific CD8+ and CD4+ T lymphocytes are dysfunctional and coincidentally show an increased expression of the negative regulator of T cell activation PD-1, PD1 ligation by PD-L1 and PD-L2 leads to the inhibition of most T cell functions including proliferation, cytokine production and survival. However, the molecular mechanisms downstream of PD-1 remain unknown. The major objective of this project is to define the molecular mechanisms that render T cells dysfunctional following PD-1 ligation and to identify strategies that will allow the rescue of such dysfunction in HIV specific CD8 cells. We will test the hypothesis that PD1 interferes with upstream TCR signaling pathways in order to affect all T cell functions. We have shown that PD-1 ligation leads to the phosphorylation of SHP-1 and SHP-2 phosphatases. Moreover PD1 crosslinking leads to the inhibition of p56Lck activation through the phosphorylation of the tyrosine Y505 by the inhibitory kinase Csk.
In Specific Aim 1 we will determine the proximal signaling defect in PD-1 hi exhausted cells from HIV infected patients and assess whether Csk is recruited to the immunological synapse by PD1 ligation or whether it is released from Cbp/PAG by the SHP- 2 phosphatase. We will determine by quantitative immunoprecipitation if the inhibitory kinase Csk and the in phosphatases SHP-1 and SHP-2 are recruited to the TCR complex. Fluorescence (Forster) resonance energy transfer (FRET) experiments will be carried out to identify the partners involved in the negative regulation of p56Lck activity. The recruitment of these negative regulators will be assessed in cells showing varying degrees of dysfunction . We will use siRNAs specific for Csk, SHP-1, SHP-2 and Fyn-T and activated forms of Lck to rescue T cell function in T cells at the extremes of this gradient of dysfunction.
In Aim 2, live cell imaging using total internal reflection fluorescence microscopy (TIRFM) will be used to visualize, (in collaboration with Core C) the dynamics of the immunological synapse formation upon PD-1 crosslinking in HIV specific CD8+ T cells showing varying degrees of dysfunction. . We will assess whether inhibition of the kinase Csk and the phosphatases SHP-112 will restore the integrity of the synapse as observed in efficiently activated T cells. Results emanating from this project may lead to the identification of novel therapeutic targets that can allow immune reconstitution during HIV infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program Projects (P01)
Project #
5P01AI080192-05
Application #
8380114
Study Section
Special Emphasis Panel (ZAI1-PRJ-A)
Project Start
Project End
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
5
Fiscal Year
2012
Total Cost
$267,411
Indirect Cost
$47,177
Name
Emory University
Department
Type
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Kamphorst, Alice O; Araki, Koichi; Ahmed, Rafi (2015) Beyond adjuvants: immunomodulation strategies to enhance T cell immunity. Vaccine 33 Suppl 2:B21-8
Chetty, Shivan; Govender, Pamla; Zupkosky, Jennifer et al. (2015) Co-infection with Mycobacterium tuberculosis impairs HIV-Specific CD8+ and CD4+ T cell functionality. PLoS One 10:e0118654
Porichis, Filippos; Hart, Meghan G; Griesbeck, Morgane et al. (2014) High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat Commun 5:5641
Penaloza-MacMaster, Pablo; Kamphorst, Alice O; Wieland, Andreas et al. (2014) Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection. J Exp Med 211:1905-18
Xiao, Yanping; Yu, Sanhong; Zhu, Baogong et al. (2014) RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance. J Exp Med 211:943-59
Morou, Antigoni; Palmer, Brent E; Kaufmann, Daniel E (2014) Distinctive features of CD4+ T cell dysfunction in chronic viral infections. Curr Opin HIV AIDS 9:446-51
Lu, Peiyuan; Youngblood, Benjamin A; Austin, James W et al. (2014) Blimp-1 represses CD8 T cell expression of PD-1 using a feed-forward transcriptional circuit during acute viral infection. J Exp Med 211:515-27
Porichis, Filippos; Hart, Meghan G; Zupkosky, Jennifer et al. (2014) Differential impact of PD-1 and/or interleukin-10 blockade on HIV-1-specific CD4 T cell and antigen-presenting cell functions. J Virol 88:2508-18
Chetty, Shivan; Porichis, Filippos; Govender, Pamla et al. (2014) Tuberculosis distorts the inhibitory impact of interleukin-10 in HIV infection. AIDS 28:2671-6
Austin, James W; Lu, Peiyuan; Majumder, Parimal et al. (2014) STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells. J Immunol 192:4876-86

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