Pathology

Clifford V. Harding, M.D., Ph.D.

Professor, Interim Chair

Mailing Address:
2103 Cornell Rd.
WRB 6522
Cleveland, OH 44106-7288

phone: (216) 368-3611
fax: (216) 368-0494
email: Clifford.Harding@Case.edu

Biography
Dr. Clifford Harding graduated magna cum laude, and with Highest Honors, in Biology from Harvard College in 1979. He completed his M.D. and Ph.D. in Cell Biology at Washington University of St. Louis in 1985. He went on as a Resident and then Chief Resident in Pathology, eventually moving up to an Instructor position in 1989. From 1990 until 1993, Dr. Harding was an Assistant Professor at Washington University as well as Attending Physician at Barnes Hospital of St. Louis. In 1993, he came to Case as an Assistant Professor of Pathology and as Senior Staff Physician at University Hospitals of Cleveland (1993-2003). Presently, Dr. Harding spends his time as a Professor of Pathology (1999-) and Director of the Medical Scientist Training Program (2001-) at Case, along with being on the Adjunct Staff in the Department of Immunology at the Lerner Research Institute of The Cleveland Clinic Foundation.

Research
Antigen processing converts protein antigens to peptide-MHC complexes that can be recognized by T cells. Class I MHC (MHC-I) and class II MHC (MHC-II) molecules are loaded with peptides via two distinct “conventional” processing pathways. MHC-II molecules target to endocytic compartments or phagosomes to bind peptides from exogenous antigens that are cleaved by vacuolar proteases. In contrast, MHC-I molecules are loaded in the endoplasmic reticulum (ER) with peptides that are produced by proteasome cleavage of cytosolic antigens and imported into the ER by TAP. In addition to these “conventional” pathways, we are studying “alternate” pathways with different processing mechanisms.

Much of our effort is now directed to understanding regulation of antigen presenting cell (APC) function in the context of infectious diseases, e.g. tuberculosis and HIV infection. APCs sense pathogens by innate immune receptors, including Toll-like receptors (TLRs), and are regulated by cytokines and interferons that are produced during infection. For example, we are studying recognition of Mycobacterium tuberculosis by TLRs and the dysregulation of type I interferon responses by APCs from HIV-infected patients.

Research areas:

    1. Phagosomal processing of bacteria (e.g., Mycobacterium tuberculosis). Phagosomes are isolated and analyzed by biochemical techniques, T cell assays and flow organellometry (flow cytometry applied to subcellular particles). M. tuberculosis can modify phagosomes and alter antigen processing, contributing to their evasion of immune responses.
    2. Alternate MHC-I antigen processing mechanisms whereby exogenous particulate antigens (e.g. bacteria) are presented by MHC-I molecules, in some cases independent of proteasomes and ER function. These mechanisms may contribute to the generation of T cell responses against tumors as well as infectious organisms.
    3. Modulation of antigen processing and T cell responses by microbial products and adjuvants, including CpG DNA and M. tuberculosis 19 kD lipoprotein. We are determining how these substances modulate antigen processing and immune responses, in some cases by Toll-like receptor signaling.
    4. Recognition of “PAMPs” from Mycobacterium tuberculosis, particularly the recognition of bacterial lipoproteins by TLR2. We are determining the identity of M. tuberculosis “PAMPs” and studying their ability to iinteract with and signal throught TLRs.
    5. Regulation of APC function by type I interferons. We are studying the role of type I interferon in regulation of APCs by CpG DNA and abnormalities in interferon regulation of APCs from HIV- infected patients.



Publications
Harding C, Heuser J & Stahl P. 1983. Receptor mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J. Cell Biol. 97:329 339.

Harding, CV & Unanue ER. 1990. Quantitation of peptide class II MHC complexes generated in antigen presenting cells and necessary for T cell stimulation. Nature 346:574 576.

Harding, CV, Collins, DS, Slot, JW, Geuze, HJ & Unanue ER. 1991. Liposome-encapsulated antigens are processed in lysosomes, recycled and presented to T cells. Cell 64:393 401.

Collins DS, Unanue ER & Harding CV. 1991. Reduction of disulphide bonds in lysosomes is a key step in antigen processing. J Immunol 147:4054 4059.

Harding CV & Geuze HJ. 1992. Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells. J. Cell Biol. 119:531 542.

Pfeifer JD, Wick MJ, Roberts RL, Findlay KF, Normark SJ & Harding CV. 1993. Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature 361:359 362.

Harding CV & Geuze HJ. 1993. Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment. J. Immunol. 151:3988-3998.

Song R & Harding CV. 1996. Roles of proteasomes, TAP and beta2-microglobulin in the processing of bacterial or particulate Ags via an alternate class I MHC processing pathway. J. Immunol. 156: 4182-90.

Griffin J, Chu R & Harding CV. 1997. Early endosomes and a late endocytic compartment generate distinct species of peptide:MHC-II complexes via different mechanisms. J. Immunol. 158: 1523-1532.

Chu R, Targoni OS, Krieg AM, Lehmann PV & Harding CV. 1997. CpG oligodeoxynucleotides provide a danger signal and act as adjuvants that switch on Th1 immunity. J. Exp. Med. 186:1623-1631.

Ramachandra L, Song R & Harding CV. 1999. Phagosomes are fully competent antigen processing organelles that mediate the formation of peptide:class II MHC complexes. J. Immunol. 162:3263-3272.

Chu RS, Askew D, Noss EH, Tobian,A, Krieg AM & Harding CV. 1999. CpG oligodeoxynucleotides down- regulate macrophage class II MHC antigen processing. J. Immunol, 163: 1188-1194.

Johnsen AK, Templeton D, Sy M-S & Harding CV. 1999. Deficiency of TAP in tumor cells allows evasion of immune surveillance and increases tumorigenesis. J Immunol 163: 4224-4231.

Askew D, Chu RS, Krieg AM & Harding CV. 2000. CpG DNA induces maturation of dendritic cells with distinct effects on nascent and recycling MHC-II Ag processing mechanisms. J. Immunol.165: 6889-95.

Noss EH, Pai RK, Sellati TJ, Radolf JD, Belisle J, Golenbock DT, Boom WH & Harding CV. 2001. Toll- like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19 kD lipoprotein of Mycobacterium tuberculosis. J. Immunol., 167: 910-918.

Ramachandra L, Noss EH, Boom WH & Harding CV. 2001. Processing of Mycobacterium tuberculosis antigen 85B involves intra-phagosomal formation of peptide:MHC-II complexes and is inhibited by live bacilli that decrease phagosome maturation. J. Exp. Med., 194:1421-1432.

Sieg, S.F., Harding, C.V. and Lederman, M.M. 2001. HIV-1 infection impairs cell cycle progression of CD4(+) T cells without affecting early activation responses. J. Clin. Invest. 108: 757-764.

Pai RK, Askew D, Boom WH & Harding CV. 2002. Regulation of class II MHC expression in APCs: Roles of types I, III and IV class II transactivator. J. Immunol. 169: 1326-1333.

Canaday DH, Gehring A, Leonard EG, Eilertson B, Schreiber JR, Harding CV* & Boom WH* 2003. T cell hybridomas from HLA-transgenic mice as tools for analysis of human antigen processing. J Immunol. Methods, 281:129-141 (*Shared senior authorship).

Pai RK, Convery M, Hamilton TA, Boom WH & Harding CV. 2003. Inhibition of IFN-γ-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: A potential mechanism for immune evasion. J Immunol 171: 175-184.

Kuchtey J, Pennini M, Pai RK & Harding CV. 2003. CpG DNA induces a class II transactivator- independent increase in class II MHC by stabilizing class II MHC mRNA in B lymphocytes. J Immunol 171: 2320-2325.

Tobian AAR, Canaday DH, Boom WH & Harding CV. 2004. Bacterial heat shock proteins promote CD91-dependent class I MHC cross presentation of chaperoned peptide to CD8+ T cells by cytosolic mechanisms in dendritic cells versus vacuolar mechanisms in macrophages. J. Immunol., 172: 5277-86.

Pai RK, Pennini ME, Tobian AAR, Canaday DH, Boom WH & Harding CV. 2004. Prolonged Toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kDa lipoprotein inhibits interferon-gamma-induced gene regulation in macrophages. Infect Immun 72:6603-6614.

Tobian AAR, Canaday DH & Harding CV. 2004. Bacterial heat shock proteins enhance class II MHC antigen processing and presentation of chaperoned peptides to CD4+ T cells. J Immunol 173: 5130-5137.

Ramachandra L, Smialek JL, Shank SS, Convery M, Boom WH and Harding CV. 2005. Phagosomal processing of Mycobacterium tuberculosis antigen 85B is modulated independently of mycobacterial viability and phagosome maturation. Infect. Immun. 73, 1097-1105.

Jiang, W, Lederman, MM, Salkowitz, JR, Harding, CV & Sieg, SS. 2005. CpG ODN induces monocyte maturation in cells from healthy individuals and HIV-infected patients. J. Virol. 79: 4109-4119.

Kuchtey, J, Chefalo, PJ, Ramachandra, L & Harding, CV. 2005. Enhancement of dendritic cell antigen cross presentation by CpG DNA involves type I IFN and stabilization of class I MHC mRNA. J Immunol 175: 2244-2251.

Rojas, R.E., Thomas, J.J., Gehring, A.J., Hill, P.J., Belisle, J.T., Harding, C.V. and Boom, W.H.* 2006. Phosphatidylinositol mannoside from Mycobacterium tuberculosis binds alpha5-beta1 integrin (VLA-5) on CD4+ T cells and induces adhesion to fibronectin. J. Immunol. 177: 2959-2968. *Shared senior authorship.

Pennini, ME, Pai, RK, Schultz, DC, Boom, WH & Harding, CV. 2006. Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-g-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling. J. Immunol. 176: 4323-4330.

Pecora, ND, Gehring, AJ, Canaday, DH, Boom, WH & Harding, CV. 2006. Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function. J. Immunol. 177:422-429.

Rodriguez, B, Lederman, MM, Jiang, W, Bazdar, DA, Gàrate, K, Harding, CV & Sieg, SF. 2006. Inter- feron-α differentially rescues CD4+ and CD8+ T cells from apoptosis in HIV infection. AIDS 20:1379- 1389.

Gray, R.C., Kuchtey, J. and Harding, C.V. 2007. CpG-B ODNs potently induce low levels of IFNalpha/beta and induce IFNalpha/beta-dependent MHC-I cross presentation in DCs as effectively as CpG-A and CpG-C ODNs. J. Leukocyte Biol. 81:1075-1085.

Pennini, ME, Yang, J, Croniger, CM, Boom, WH and Harding, CV. 2007. C/EBP-beta binds to CIITA promoters and inhibits CIITA expression in response to M. tuberculosis 19-kDa lipoprotein. J. Immunol. 179: 6910– 6918.

Funderberg, N., Lederman, M.M., Feng, Z., Drage, M.G., Jadlowsky, J. Harding, C.V., Weinberg, A. and Sieg, S.F. 2008. Human beta-defensin-3 activates professional antigen-presenting cells via Toll-like receptors 1 and 2. Proc. Natl. Acad. Sci. USA 104: 18631-18635. PMCID:PMC2141828

Askew, D. and Harding, C.V. 2008. Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells. Immunology 123: 447-455. PMCID:PMC2433328

Anis, M.M., Fulton, S.A., Reba, S.M., Liu, Y., Harding, C.V. and Boom, W.H. 2008. Modulation of pulmonary dendritic-cell function during mycobacterial infection. Infect. Immun 76: 671-677. PMCID: PMC2223454

Jiang, W, MM Lederman, RJ Mohner, B Rodriguez, TM Nedrich, CV Harding & SF Sieg. 2008. Impaired naive and memory B cell responsiveness to TLR9 stimulation in HIV-infection. J Virol. 82: 7837- 7845. PMCID:PMC2519583

Pecora, N.D., Fulton, S.A., Reba, S.M., Drage, M.G., Simmons, D.P., Urankar-Nagy, N.J., Boom, W.H. and Harding, C.V. 2009. Mycobacterium bovis BCG decreases MHC-II expression in vivo on murine lung macrophages and dendritic cells during aerosol infection. Cell. Immunol. 254: 94-104. PMCID:PMC2653222

Qu, Y., Ramachandra, L., Mohr, S., Franchi, L., Harding, C.V., Nunez, G. and Dubyak, G.R. 2009. P2X7 receptor-stimulated secreation of MHC-II-containing exosomes requires the ASC/NLRP3 inflammasome but is independent of caspase-1. 2008. J. Immunol. 182: 5052-5062.

Drage, M.G., Pecora, N.D., Hise, A.G., Febbraio, M., Silverstein, R.L., Golenbock, D.T., Boom, W.H. and Harding, C.V. 2009. Differences in expression of TLR2 and its co-receptors determine responses of antigen presenting cells to lipoproteins of Mycobacterium tuberculosis. Cell. Immunol. 258: 29-37.

Hardy, G.A., Sieg, S.F., Rodriguez, B., Jiang, W., Asaad, R., Lederman, M.M. and Harding, C.V. 2009. Desensitization to type I interferon in HIV-1 infection correlates with markers of immune activation and disease progression. Blood, 113: 5497-5505.

Gray, RC, Liu, Y, Hardy, GAD, Kuchtey, J, Abbott, DW, Emancipator, SN and Harding, CV 2009. CpG- B oligodeoxynucleotides inhibit Toll-like receptor-dependent and independent induction of type I IFN in dendritic cells. Submitted.

Gabrilovich, MI, Walrath, J, Harding, CV, Lee, H, Mackay, W, Dixon-Williams, S and Silver, RF. 2009. Hyperresponsiveness to Toll-like receptor 2 ligand 19-kDa lipoprotein of M. tuberculosis (LpqH) in pulmonary sarcoidosis. Submitted.

Sweeney, KA, Dao, DN, Hsu, T, Ramachandra, L, Henao-Tamayo, M, Ordway, D, Jain, P, Chen, B, Chen, M, Kim, J, Harding, CV, Orme, I, Chan, J, Porcelli, SA, and Jacobs, WR 2009. A recombinant Mycobacterium smegmatis that elicits bactericidal immunity against M. tuberculosis. Submitted.

Drage, M.G., Tsai, J., Pecora, N.D., Cheng, T.-Y., Arida, A.R., Rojas, R.E., Moody, D.B., Boom, W.H., Sacchettini, J.C., and Harding, C.V. 2009. Mycobacterium tuberculosis lipoprotein LprG (Rv1411c) is a glycolipid chaperone and delivers triacylated glycolipids to Toll-like receptor 2. Submitted.