Most HIV-infected people ultimately succumb to chronic infection if not adherent to combination antiretroviral therapy (cART). Some, however, known as elite controllers (EC), demonstrate superior virus control maintaining virtually undetectable viral loads even in the absence of treatment. While it is well known that HIV- specific cytotoxic lymphocytes (CTL) are critical for viral control and that HLA haplotype is associated with viral progression status, the mechanisms underlying EC virus control remain unknown. Recently, we showed that HIV-specific CTL from EC form better quality immunological synapses (IS) with HIV-infected target cells, with more rapid biogenesis of perforin and more effective lytic granule loading. We also found that engagement of inhibitory receptor programmed death-1 (PD-1, a marker for CTL exhaustion) on HIV-specific CTL specifically induces phosphorylation of the adaptor protein Chicken Tumor Virus #10 Regulator of Kinase (Crk). We hypothesize that chronic HIV leads to functional impairment of the CTL IS via upregulation of inhibitory receptors like PD-1 and that the resulting downstream phosphorylation of Crk, in turn, prevents CTL activation by disrupting the structure and signaling of the CTL-target cell synapse. Increased inhibitory receptor expression is one of the earliest markers of CTL exhaustion in chronic HIV. EC patients express lower levels of PD-1 than do their chronic progressor (CP) counterparts.
In Aim 1 we will compare synapse structure and function in exhausted (CP) vs. non-exhausted (EC) HIV-specific CTL. HIV-specific CTL will be stimulated by a reductionist lipid bilayer system or by HIV-infected target cells in the presence o absence of PD-1's primary ligand PD-L1. IS will be visualized using super-resolution stimulated emission depletion microscopy and total internal reflection fluorescence (TIRF) microscopy. There are a number of important questions about IS that can only be addressed through super-resolution imaging (e.g. distribution of F-actin and lytic granules at IS). The proposed high-resolution, single cell assays will allow us to determine the specific impact of inhibitory recepto signaling on IS structure and function and will yield critical insights into how these relate to vial control (EC vs. CP) and haplotype. We hypothesize that upregulation of inhibitory receptor in exhausted cells results in increased pCrk, blocking critical Crk-dependent activation signals like Vav-1.
Aim 2 will dissect the dynamics of signal integration at the IS of exhausted HIV-specific CTL, relating Crk phosphorylation and localization of key regulators, as well as cytotoxicity and viral inhibition, to inhibitory receptor expression level, haplotype, and viral progression status. Signaling will be tracked at a single-cell, synapse level using fluorescently tagged signal molecules and super-resolution STED microscopy. While other groups are attempting to boost T cell responses by targeting individual inhibitory receptors (e.g. PD-1 and CTLA-4), the proposed experiments will be the first to address a downstream, central signaling hub. It is our long-term goal to use the knowledge to augment T cell responses in HIV and other chronic viral infections.

Public Health Relevance

Cytotoxic lymphocytes or 'killer cells' play a critical role in human host defense against viruses and cancer. Here, cutting-edge imaging techniques will be used to define how Crk (an adaptor protein) and its phosphorylation act as a molecular switch to turn killer cell function on or off at the level of single cell synapse. These techniques and the data they yield will be used to identify specific molecules that can restore killer cell function i malignancy and HIV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI124769-02
Application #
9310372
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Lawrence, Diane M
Project Start
2016-07-05
Project End
2019-06-30
Budget Start
2017-07-01
Budget End
2019-06-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Methodist Hospital Research Institute
Department
Type
DUNS #
185641052
City
Houston
State
TX
Country
United States
Zip Code
77030
Xiong, Wei; Chen, Yuhui; Kang, Xi et al. (2018) Immunological Synapse Predicts Effectiveness of Chimeric Antigen Receptor Cells. Mol Ther 26:963-975
Liu, Dongfang; Tian, Shuo; Zhang, Kai et al. (2018) Erratum to: Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV. Protein Cell 9:902
Nabekura, Tsukasa; Chen, Zhiying; Schroeder, Casey et al. (2018) Crk Adaptor Proteins Regulate NK Cell Expansion and Differentiation during Mouse Cytomegalovirus Infection. J Immunol 200:3420-3428
Liu, Dongfang; Tian, Shuo; Zhang, Kai et al. (2017) Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV. Protein Cell 8:861-877
Bertolet, Grant; Liu, Dongfang (2016) The Planar Lipid Bilayer System Serves as a Reductionist Approach for Studying NK Cell Immunological Synapses and Their Functions. Methods Mol Biol 1441:151-65
Jang, Joon Hee; Huang, Yu; Zheng, Peilin et al. (2015) Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics. J Immunol 195:1320-30
Pone, Egest J; Lam, Tonika; Lou, Zheng et al. (2015) B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses. J Immunol 194:3065-78
Watkin, Levi B; Jessen, Birthe; Wiszniewski, Wojciech et al. (2015) COPA mutations impair ER-Golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis. Nat Genet 47:654-60
Zheng, Peilin; Noroski, Lenora M; Hanson, Imelda C et al. (2015) Molecular mechanisms of functional natural killer deficiency in patients with partial DiGeorge syndrome. J Allergy Clin Immunol 135:1293-302
Zheng, Peilin; Bertolet, Grant; Chen, Yuhui et al. (2015) Super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer. J Vis Exp :

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