CD8 T cells protect the host against intracellular pathogens and some cancerous cells by direct killing of these deranged cells (cytotoxicity) and by altering the tissue microenvironment. To mount such a response, CD8 T cell undergo priming by dendritic cells (0-36 hrs), followed by “clonal expansion” (36 hr-4 days) to increase their numbers, and the then development into Cytotoxic effector T Lymphocytes (CTL) (>4d) that can kill deranged cells. A subset of CD4 positive T cells will also take on a cytotoxic phenotype in the context of viral infections and autoimmune diseases. The CD8 and CD4 CTLs store cytotoxic proteins perforin and granzyme B in secretory vesicles. CTL release the granules upon re-activation through their T cell receptors (TCR). We have recently discovered that in CD8 CTL, cytotoxic proteins are released in ~100 nm SupraMolecular Attack Particles (SMAPs)- in which many copies of the cytotoxic proteins along with chemokines and cytokines are packaged in a glycoprotein shell (Balint et al, submitted). It remains to be investigated whether CD4 CTL produce similar particles. CD8+ T cells (but not CD4+ T cells) make large amounts of granzyme B during priming (Biorxiv- 456392), but it is unclear how these Pre-Mitotic CD8 CTL (PMC) package and regulate release of granzyme B. The goal of the project is to determine if CD4 CTL and PMC make SMAPs, how PMC regulate immune responses and to better understand the role of SMAPs in infectious and autoimmune disease.
The project will make use of mouse models, human cells from healthy individuals and human cells from rheumatoid synovium. Mouse models will include Listeria infection, a classic way to generate CD8 CTL responses and the main system in which we have studied PMC. We will use human peripheral blood from individuals who are cytomegalovirus positive as a source of human CD8 and CD4 CTL. Rheumatoid Arthritis (RA) synovium is known to contain CD8 and CD4 CTL. The main method to evaluate SMAPs from murine and human sources will be proteomics (with Target Discovery Institute Proteomics Facility), super-resolution microscopy methods (stochastic optical reconstruction microscopy), immunofluorescence analysis of tissues, and functional assays investigating killing and other activities of SMAPs including chemotaxis and cytokine function assays.
The hypothesis is that SMAPs possess innate targeting machinery in the glycoprotein shell that facilitates target cell killing. Further, SMAPs can integrate into extracellular matrices in sites of infection and inflammation, where they continue to recruit immune cells, modify cell behaviour and kill targets. Even if CD4 CTL and PMC don’t make SMAPs, which would be a publishable negative result, there will be important work to do to understand the role of conventional CD8 CTL in RA and whether SMAPs made in this context have normal composition and function. We anticipate that SMAPs will present a means to improve specificity, but also may play a role in pathogenic mechanism due to their potential persistence/accumulation in sites of chronic inflammation.
Mouse models of infection and inflammatory disease. Human antigen specific T cells (CMV). Advanced microscopy. Design of proteomics experiments and immunological assays. Flow cytometry.
Beal AM, Anikeeva N, Varma R, Cameron TO, Vasiliver-Shamis G, Norris PJ, Dustin ML, Sykulev Y. Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain. Immunity. 2009;31(4):632-42.
Dustin ML, Long EO. Cytotoxic immunological synapses. Immunol Rev. 2010;235(1):24-34.
Fuller MJ, Callendret B, Zhu B, Freeman GJ, Hasselschwert DL, Satterfield W, Sharpe AH, Dustin LB, Rice CM, Grakoui A, Ahmed R, Walker CM. Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1). Proc Natl Acad Sci U S A. 2013;110(37):15001-6.
Balint S, Lopes FB, Davis DM. A nanoscale reorganization of the IL-15 receptor is triggered by NKG2D in a ligand-dependent manner. Sci Signal. 2018;11(525). Epub 2018/04/11. doi: 10.1126/scisignal.aal3606.
Dandamudi DB, Blair DA, Arifa R, Lafaille JJ, Dustin ML, Mayya V. Multifaceted influence of pre-mitotic cytotoxicity of primed CD8 T cells on immunity and infection. bioRxiv. 2018:456392. doi: 10.1101/456392.
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