Public Health Research Institute Center
New Jersey Medical School - Rutgers, The State University of New Jersey
225 Warren Street
Newark, New Jersey 07103
Phone: (973) 854-3350
This research program was historically focused on biology and function of MHC class I proteins in antigen presentation during infection and in tissue transplantation. Recently, our interest in the network of receptor-mediated responses provided for a renewed focus on immunology of tuberculosis. The program has four areas of interest discussed below.
Single T cell immunity functional signatures in tuberculosis. Stimulation of T cells with specific functions (effector, memory or regulatory) through Ag:T cell receptor (TCR) interactions initiates a signaling cascade that alters gene expression in responding cells. These changes can be detected by mRNA enumeration using single molecule fluorescence in situ hybridization (smFISH) and flow cytometry as a read-out. The FISH-Flow platform can identify key markers of T cell activation defined as such due to their characteristic expression in functional signatures of T cells associated with evolution of an infection and disease. Tuberculosis (TB) is a complex disease with remarkable plasticity. The progression of infection is generally moving from preclinical to active disease or to latent TB. Reactivation of latent TB to a preclinical stage and then active disease is also common due to the changes in the immune system status (cancer, HIV co-infection). Since functions of many cell populations are defined by expression of specific markers including pro-inflammatory or suppressive cytokines, distinct proportions of T cell subsets expressing different cytokines can serve as biomarkers of TB infection stages. The latter goal certainly implies identification of novel key markers of T cell activation and/or function in addition to canonic IL-2, IFNγ and TNFα.
Our recent NIH-funded studies involve participation of a multidisciplinary team encompassing four laboratories at PHRI. In this program, we combine expertise in T cell immunology (Bushkin), mycobacterial Ag (Gennaro), signaling and gene expression (Pine), and smFISH (Tyagi). We design and optimize ex vivo T cell assays utilizing peripheral blood cells obtained from human donors and stimulated with selected mixtures of mycobacterial Ags. The next step entails unraveling the complexity of T cell functional signatures comprised by RNA targets identified by downstream “walking” the pathways involved in receptor-ligand interactions. Each of these goals involves the design and preparation of a new class of probes with multiple fluorophores for detection of RNA in situ. The methodology explored here applies beyond the field of TB to other infectious and non-infectious pathologies and to the characterization of various cell populations at the single-cell level.
Th1 immunity in tuberculosis. Recent data suggest that Th1-type cytotoxic T lymphocyte (CTL) responses against novel M. tb targets identified in our lab, alanine dehydrogenase (Rv2780) and glutamine synthetase (Rv2220), can be detected only in non-vaccinated, tuberculin skin test-positive asymptomatic individuals but not in active tuberculosis patients, while responses to superoxide dismutase (Rv3846) were present in both groups. This finding supports the notion that in humans the profile of Ag-specific responses associated with active disease differs from that observed during latent infection. Complex interactions between the bacterial pathogen and host immune system underline this dynamics. For example, selection of initial CTL targets and possible 're-targeting' in the course of infection may be related to certain profound changes in the program of mycobacterial gene expression during infection. Additionally, CTL responses are likely to be affected by the Ag load and presentation pathways. It is possible that in latent infections, when the pathogen Ag levels are quite low, soluble MHC class I can serve as enrichment mechanism for the pools of specific high-affinity peptides providing for CTL stimulation and/or memory maintenance via cross-presentation pathways (see below).
Mechanisms of release of soluble MHC class I. In mammals, MHC class I molecules are expressed on the surface of nearly all nucleated cells as integral membrane proteins and also as soluble proteins in many body fluids. The mechanisms of release of soluble MHC class I involve alternate splicing of transcripts and shedding of integral proteins. Most of previous studies focused on shedding at the cell surface, and thus the question of potential shedding in intracellular compartments remained much less investigated. Our latest data using siRNA-mediated knock-out demonstrate that the two closely related surface proteases, ADAM10 and ADAM17, critically contribute to MHC class I processing. The release is augmented by stimulation of monocytic THP-1 cell line and primary monocytes and macrophages with immunomodulating cytokines TNFα, IFNγ, GM-CSF and IL-10. The cytokine-mediated regulation of MHC class I release involves calcium- and calmodulin-dependent mechanisms. Analysis of subcellular fractions for the presence of cleavage products (i.e., soluble ectodomains of MHC class I) revealed that lysosomal compartments contained ectodomains derived from internalized intact MHC class I molecules. Thus, MHC class I undergo cleavage by ADAM10 and ADAM17 in response to cytokines and this processing occurs predominantly in lysosomal compartments.
Soluble MHC class I in alloantigen presentation. Accumulated evidence obtained, in part, in transplantation studies suggests an important role for soluble MHC class I in Ag presentation. The MHC class I processing pathways mediated by ADAM10 and ADAM17 generate ectodomains as final products. These soluble proteins can be captured by DC or other APC and further processed to generate linear peptides for presentation on MHC class II or present intact alloAg on their cell surface inducing T cell-dependent Ab responses. Additionally, since MHC class I ectodomains retain stably bound peptides, these soluble complexes captured by APC could be utilized for intracellular re-loading of matching MHC class I with peptides, as previously hypothesized. The latter process may serve as an enrichment mechanism for rarely or otherwise inefficiently presented peptides and thus allowing for successful cross-priming of naïve T cells during infections. This may contribute to the robust capacity of DC to capture soluble antigens and process them for cross-presentation on MHC class I.
Arrigucci R, Bushkin Y, Radford F, Lakehal K, Vir P, Pine R, Martin D, Sugarman J, Zhao Y, Yap GS, Lardizabal AA, Tyagi S, Gennaro ML (2017) FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Protoc 12: 1245-1260. PMI: 28518171
Radford F, Tyagi S, Gennaro ML, Pine R, Bushkin Y (2016) Flow cytometric characterization of antigen-specific T cells based on RNA and its advantages in detecting infections and immunological disorders. Critical Reviews in Immunology 5: 359-378. PMI:
Vir P, Arrigucci R, Lakehal K, Davidow AL, Pine R, Tyagi S, Bushkin Y, Lardizabal A, Gennaro ML (2015) Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status. PLoS One 10: e0144904. PMI: 26658491
Bushkin Y, Radford F, Pine R, Lardizabal A, Mangura BT, Gennaro ML, Tyagi S (2015) Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry. J Immunol 194: 836-841. PMI: 25505292
Pine R, Bushkin Y, Gennaro ML (2013) Immunological biomarkers for tuberculosis: potential for a combinatorial approach. In McFadden J, Beste D, and Kierzek A (eds.), Systems Biology of Tuberculosis. Springer, pp. 193-219.
Zhao J, Guo Y, Yan Z, Zhang J, Bushkin Y, Liang P (2011) Soluble MHC I and soluble MIC molecules: potential therapeutic targets for cancer. Int Rev Immunol 30: 35-43. PMI: 21235324
Mizrahi S, Markel G, Porgador A, Bushkin Y, Mandelboim O (2007) CD100 on NK cells enhance IFNgamma secretion and killing of target cells expressing CD72. PLoS One 2: e818. PMI: 17786190
Dong Y, Demaria S, Sun X, Santori FR, Jesdale BM, De Groot AS, Rom WN, Bushkin Y (2004) HLA-A2-restricted CD8+-cytotoxic-T-cell responses to novel epitopes in Mycobacterium tuberculosis superoxide dismutase, alanine dehydrogenase, and glutamine synthetase. Infect Immun 72: 2412-2415. PMI: 15039371
Dong Y, Lieskovska J, Kedrin D, Porcelli S, Mandelboim O, Bushkin Y (2003) Soluble nonclassical HLA generated by the metalloproteinase pathway. Hum Immunol 64: 802-810. PMI: 12878359
Haynes LD, Bushkin Y, Love RB, Burlingham WJ (2002) Interferon-gamma drives the metalloproteinase-dependent cleavage of HLA class I soluble forms from primary human bronchial epithelial cells. Hum Immunol 63: 893-901. PMI: 12368042
Mandelboim O, Lieberman N, Lev M, Paul L, Arnon TI, Bushkin Y, Davis DM, Strominger JL, Yewdell JW, Porgador A (2001) Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature 409: 1055-1060. PMI: 11234016
Latvian State University, Riga, Latvia, and Institute of Cytology, St. Petersburg, Russia, M.S., 1971 (Cytogenetics); Weizmann Institute of Science, Rehovot, Israel, Ph.D., 1978 (Cell Biol. and Genetics); Sloan-Kettering Institute for Cancer Research, New York, NY, Post Doc., 1977-1981 (Immunogenetics).
Sloan-Kettering Institute for Cancer Research, New York, NY, Research Associate, 1981-1986; New York University Medical Center, Research Associate Professor, New York, NY, Department of Pathology, 1989-present; PHRI, Associate, Department of Retroviral Biology, 1986-1988, Assistant Member, Head of the Laboratory of Molecular Immunology, 1988-1996, Associate Member, Head of the Laboratory of Molecular Immunology, 1996-present, University of Medicine and Dentistry, New Jersey Medical School, Adjunct Professor (Dept. of Medicine) 2006-present.