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Mark Pezzano Ph.D.

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Mark Pezzano, Ph.D.

Associate Professor Department of Biology

Deputy Director RCMI Center for the Study of the Cellular and Molecular Basis of Development

Department of Biology
138th Street and Convent Ave

New York , NY 10031
Email:
Office Phone: (212) 650-8559

Education:

  1. BS Biology 1985 William Paterson University
  2. Ph.D. Cell and Molecular Biology 1993 The City University of New York

Biography:


Mark Pezzano is a cellular immunologist studying thymic epithelial stem cells and their contribution to the development and maintenance of thymic microenvironments. His research is directed at understanding how the immune system learns to distinguish self from non-self; this basic problem is fundamental to adaptive immune responses, while defects lead to development of autoimmune disease. His work has clinical applications in counteracting age-associated thymic involution, as well as enhancing bone marrow transplants used for cancer therapy. 
 
Research Projects:
The thymus represents the key organ where the development and selection of T cells occurs. This process is critical to the function of the adaptive immune response.   The thymus is composed of a mixture of endoderm derived epithelial cells, mesenchyme derived fibroblasts and haematopoietic derived macrophages, and dendritic cells, which interact to provide a series of complex microenvironments responsible for regulating the attraction, migration, expansion, survival and differentiation of developing T cells.  Most of these functions are mediated through the actions of as yet poorly defined subsets of thymic epithelial cells.  Research in my lab is focused on defining the function of specific thymic epithelial subsets in thymocyte selection, as well as understanding the cross talk mechanisms, which regulate thymic organogenesis.   TECs have recently been shown to express a wide range of tissue specific antigens (TSAs) that are critical to ensuring a self-tolerant mature T cell repertoire.  Understanding the complex interactions that occur between developing thymocytes and TECs, as well as the crosstalk mechanisms that regulate TEC expansion and differentiation will be critical for intervention in autoimmune disease, thymic recovery after cancer therapy and in regulating immune function in the aging population.  We will continue our productive collaboration with Jerry Guyden studying the function of thymic nurse cells in T cell development and selection (Martinez, M.*, Samms, M.*, Hendrix, T.*, Adeosun, O.*, Pezzano, M., and Guyden, J.C. (2007) The Organization of the Thymic Nurse Cell Multi-Cellular complex within the Thymus of HY-TCR Transgenic Mice Demonstrates Their Association with MHC Restriction Journal of Experimental Biology and Medicine 232 (6):780-8 (Feature Article for June).  In addition we have two NIH funded research projects in the lab for which we are currently recruiting graduate students.


Crosstalk Control of Thymic Epithelial Development

The thymic epithelial cells (TECs) are responsible for regulating the development of immature thymocytes into functional self-tolerant mature T cells.  A common endoderm derived TEC progenitor was recently shown to give rise to both cortical and medullary TEC subsets, however, the signaling pathways and cell-to-cell interactions that regulate this process are poorly defined.  TEC development and organization is dependent on crosstalk signals received from developing thymocytes as they migrate through the epithelial network.  The goal of this study will be to define both the nature and function of the SP thymocyte/TEC interactions that regulate development of the thymic medulla, the thymic microenvironment that is critically responsible for central tolerance.  The medullary reconstitution induced by adoptive transfer of mature T cells into mice with blocks in T cell development, will be used to test the contribution of specific T cell/TEC interactions.  We propose to: 1) use both fetal thymic organ cultures and medullary reconstitution assays in a newly developed Wnt signaling reporter mouse strain, to access the contribution of Wnt signaling in mTEC development and determine if soluble Wnt inhibitory molecules secreted by SP thymocytes inhibit the canonical Wnt signaling pathway in TECs, thereby providing a molecular switch that is necessary for mTEC development from precursor cells; 2) use MHC class I/ MHC class II double KO mice to determine if the expansion and differentiation of the thymic medulla is dependent on TCR/MHC interactions and identify the specific contributions of CD4 SP, CD8 SP and Tregs, in this process Fully understanding the signaling pathways and cellular interactions which contribute to TEC development and organization will be critical to designing rational therapeutic strategies to counteract age associated thymic involution and thymic architecture defects associated with autoimmunity.  These strategies will also significantly impact the effectiveness of bone marrow transplantation for cancer treatment by counteracting the severe premature thymic degeneration associated with preparative cytoablative treatments including chemotherapy and radiation, as well as post transplant GVHD.
 
Progress to date:  Development of thymic epithelial microenvironments is critical to the development and selection of thymocytes.  Developing thymocytes also provide crosstalk signals that are required for proper organization and differentiation of thymic epithelial cells (TECs), however, the nature of this crosstalk is not well defined. Since Wnts and Wnt inhibitors are soluble factors secreted by both TECs and thymocytes we propose that they may contribute to the crosstalk between developing thymocytes and TECs. We further examined TEC development and organization in mice with sequential blocks in T cell development including SCID, Rag-/- and CaKO both before and after reconstitution with intra-peritoneal-injected WT thymocytes.  Rag-/- and CaKO mice with blocks at DN3 and DP respectively, show an abundance of immature TEC types including K5K8DP and MTS10 UEA1 DP cells.  SCID mice with an earlier block in T cell development at DN2 show an abundance of MTS10 SP cells throughout the thymus with almost no UEA1+ mTECs. D10 TCR transgenic mice with a limited TCR repertoire display an intermediate phenotype complete with patchy partially expanded medullary areas. Subsequent to reconstitution with WT thymocytes, the medulla of both SCID and CaKO 4-week-old mice dramatically expands in response to SP thymocytes homing to the thymus and entering the medulla. Histology of the expanded medullary areas reveals increased numbers of UEA1MTS10DP mTECs in intimate association with the injected SP thymocytes. When identical experiments were performed in MHC deficient mice, no medullary reconstitution occurred, suggesting that MHC/TCR interactions are required for mTEC development. Analysis of Kremen1 KO mice, with increased canonical Wnt signaling, identified severe disruptions in thymic architecture and an abundance of immature K5K8DP TECs scattered throughout the thymus and MTS10UEA1DP mTECs.  Preliminary analysis of E14.5 FTOCs cultured with Wnt3a shows the TECs maintain a more immature phenotype complete with a disorganized structure and abundant K5K8DP cells and limited MTS10UEA1DP mTECS.  In contrast, treatment of FTOCs with the soluble Wnt inhibitor Dkk1 resulted in a remarkably organized architecture with defined cortical and medullary areas and the development of mature UEA1SP and MTS10 SP mTECs. Together these results suggest that the medulla of mice deficient in mature SP thymocytes is maintained at an immature state. The entry of SP thymocytes, through crosstalk signals, then leads to expansion of this immature MTS10 UEA1 DP subset followed by differentiation of mature UEA1 SP and MTS10 SP subsets. Regulation of canonical Wnt signaling, possibly through Wnt inhibitors secreted by SP thymocytes, appears to be critical for proper TEC development. Further, these data demonstrate that even the adult thymus contains TEC progenitors capable of responding to crosstalk signals.  These results are the subject of a published manuscript (Osada, M., Ito, E., Vazquez-Cintron, E*., Venkatesh, T., Friedel, R.H., and Pezzano, M.  (2006) The Wnt Signaling Antagonist Kremen1 is Required for the Development of Thymic Architecture Clinical and Developmental Immunology 13 (2-4) 299-319.) as well as a manuscript in preparation (Osada, M., Fermin, H., Sissa, A.*, Sant’Angelo, D.B. and Pezzano, M. (2008) MHC/TCR interactions are required for thymic medullary epithelial development.) and have provided the preliminary data for our NCI funded collaborative pilot study to examine crosstalk (NIH/NCI U56 CA96299-05) as well as our recently funded NIH/NIAID grant application (1SC1AI081527-01) to continue these studies.  We have also acquired K14 WNT7a transgenic mice from Sarah Millar at U. Penn. that over-express WNT7a specifically in K14/K5 expressing TECs. In addition she provided us DKK1 transgenic mice that express the Wnt signaling inhibitor DKK1 under the control of a tetracycline response element. These mice were bred to a K5TET-on strain (generously provided by Adam Glick at Penn State).  We are currently beginning to feed the resulting K5TETon Dkk1double transgenic mice with doxycycline food to activate expression of DKK1 in K5+TECs.  These experiments will allow us to examine the effect of altered Wnt signaling on TEC development and maintenance in vivo to complement our in vitro FTOC results. In preliminary histological analysis the K14 Wnt7a mice show a similar phenotype to or Wnt3a treated FTOCs including an immature TEC phenotype and a peripheral distribution of MTS15+ thymic fibroblasts.  Our medullary reconstitution assay results and were presented at the International Workshop on T cell Development in Kerkrade The Netherlands, were we won the BD outstanding poster award at the meeting and a description of our work was published both the European and US version of Hotlines.

Characterization of Thymic Epithelial Progenitors and their Capacity to Reconstitute Thymic Function
(collaborative project with Derek B. Sant' Angelo at memorial Sloan Kettering Cancer Center and funded through NCI as part of the CCNY/MSKCC Collaborative Cancer Center)

Hematopoietic stem cell transplantation (HSCT) can cure many forms of blood-derived cancers.   Unfortunately, particularly in adult patients, HSCT is associated with a period of immune incompetence due to a loss of capacity to generate functional T cells. The structural and functional changes in thymic epithelial cells (TECs), induced by HSCT, directly impact the capacity of the thymus to facilitate T cell development. Clinical treatment strategies that enhance T cell reconstitution could significantly improve the survival of HSCT recipients through reduced incidence of fatal infectious complications and enhanced T-cell mediated tumor activity.  The goal of this study will be to identify thymic epithelial stem cells/progenitors and test the capacity of grafting of these populations to speed the recovery of thymic function after HSCT.
      Aim 1 of this project will be to purify and characterize thymic epithelial progenitor cells.  Due to their slow turnover rate, stem cells in a variety of tissues have been identified by their capacity to retain labeled nucleotides, so called “label retaining cells” (LRCs).  A novel transgenic model, which utilizes a histone2B-GFP fusion protein (H2B-GFP), under control of a tetracycline response element driven by a K5 promoter, was used by Elaine Fuch’s group at Rockefeller to identify epithelial stem cells in the hair follicle bulge [1].   This model is unique because it allows sorting of viable stem cells, in the absence of defining cell surface markers.  Since putative thymic epithelial progenitors also express K5, we have obtained this transgenic model and will apply the method to identify thymic epithelial stem/progenitor populations.  Further separation of putative stem cell populations will be performed using previously characterized epithelial stem cell surface characteristics. Sorted subsets will be assayed for progenitor potential based on their capacity to reform a functional thymus after transfer under the kidney capsule of nude mice. 
     Aim 2 will assess the ex vivo growth potential of thymic epithelial progenitors and their subsequent capacity to enhance immune reconstitution following hematopoietic stem cell transplantation (HSCT).  Optimal culture conditions for expansion and maintenance of sorted TEC progenitors will be assessed.  Maintenance of a stem cell phenotype in expanded TEC progenitors will then be assayed by testing their capacity to reconstitute a full thymus after grafting under the nude mice kidney capsule.  The capacity of similarly prepared stem cell populations to enhance thymic recovery and reconstitution of immune function in mice following HSCT will be tested using both intrathymic injection and kidney capsule grafting of synegeneic TEC stem cells, followed by quantitative and functional T cell analysis.

This work has resulted in 8 presentations and 5 published abstracts.

Recent Publications

1.    Webb, O.*, Kelly, F.*, Benitez, J.*, Li, J., Parker, M.*, Martinez, M.*, Samms, M.*, Blake, A.*, Pezzano, M. and Guyden, J.C.  (2004) The Identification of Thymic Nurse Cells In Vivo and the Role of Cytoskeletal Proteins in Thymocyte Internalization Cellular Immunology 228(2):119-29.
2.    Osada, M., Ito, E., Vazquez-Cintron, E*., Venkatesh, T., Friedel, R.H., and Pezzano, M.  (2006) The Wnt Signaling Antagonist Kremen1 is Required for the Development of Thymic Architecture Clinical and Developmental Immunology 13 (2-4) 299-319.
3.    Martinez, M.*, Samms, M.*, Hendrix, T.*, Adeosun, O.*, Pezzano, M., and Guyden, J.C. (2007) The Organization of the Thymic Nurse Cell Multi-Cellular complex within the Thymus of HY-TCR Transgenic Mice Demonstrates Their Association with MHC Restriction Journal of Experimental Biology and Medicine 232 (6):780-8 (Feature Article for June)
4.    Kovolovski, D., Pezzano, M., Ortiz, B. and Sant ‘Angelo, D.B. (2010) A Novel TCR Transgenic Model Reveals that Negative Selection Involves an Immediate, Bim-Dependent Pathway and a Delayed, Bim-Independent Pathway PLoS One 13;5(1):e8675. PMID: 20072628. PMCID: PMC2800196.
 
5.    Pezzano, M., Kovolovski, D., Osada, M., and Sant ‘Angelo, D.B. (2010) Growth of the Thymic Medulla requires Negative Selection. Manuscript in Preparation for JI.
6.     Osada M, Jardine L, Misir R, Andl T, Millar SE and Mark Pezzano (2010) DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration. PLoS ONE 5(2): e9062. PMID: 20161711 PMCID: PMC2817005

Presentations
1.    Mark Pezzano, Juncheng Li, Marcia Martinez*, Michael Samms*, and Masako Osada.  Presentation of Peripheral Antigens to Developing Thymocytes by PECs.  Oral Platform Presentation RCMI International Symposium on Health Disparities Baltimore Maryland Dec. 2004 A# G34.
2.    Vasquez-Cintron, E.*, Osada, M, Ito, E.*, Venkatesh, T., Friedel, R.H. and Pezzano, M. Kremen-1 Knockout Mice Exhibit Altered Thymic Architecture Nov 3, 2005 ABRCMS Meeting Atlanta Georgia.  (Outstanding Poster Award)
3.    Pezzano, M. The Wnt Signaling Antagonist Kremen1 is required for Development of Thymic Architecture-Oral Presentation Memorial Sloan Kettering-Sloan Kettering Institute Immunology Seminar Series, Feb. 27th 2006
4.    Pezzano, M. The Wnt Signaling Antagonist Kremen1 is required for Development of Thymic Architecture-Oral Presentation Biology Department and MARC program University of Texas El Paso, March 10th 2006.
5.    Vasquez, E.*, Osada, M,  Ito, E.*, Venkatesh, T., Friedel, R.H. and Pezzano, M. Kremen-1 Knockout Mice Exhibit Altered Thymic Architecture. Collegiate Science and Technology Entry Program (CSTEP) 14th Annual Journeys Beyond Excellence Statewide Student Conference. The Sagamore on Lake George, NY, April 21-23. (Second Place in the Outstanding Poster Awards)
6.    Osada, M, Ito, E., Vasquez, E.*, Venkatesh, T., Friedel, R.H. and Pezzano, M.  Kremen-1 Knockout Mice Exhibit Altered Thymic Architecture AAI Meeting May 12-16, 2006 Boston MA (Abstract J. Immunol. C67 p.2).
7.    Osada, M, Ito, E., Vasquez, E.*, Venkatesh, T., Friedel, R.H. and Pezzano, M.  Kremen-1 Knockout Mice Exhibit Altered Thymic Architecture (Platform Presentation) RCMI International Symposium on Health Disparities San Juan PR Dec.15 2006.
8.    The Microenvironment Niche Provided by Thymic Nurse Cells Facilitates the Attraction of Thymocytes and Macrophages.  Oluwaseun, O Adeosun*, Jerry Guyden, Tonya Hendrix*, Marcia Martinez*, Michael Samms* and Mark Pezzano ABRCMS 2006 06-A-1451 (Outstanding Poster Award).
9.    Characterization of Kremen-1 Functional Domains. Logan Jardine*, Masako Osada and Mark Pezzano.  CCAAP Poster Presentation CCNY Nov. 2006
10.    Use of Medullary Reconstitution to Analyze  mTEC Development. Osada, M., Fermin, H.* Sant’Angelo, D.B. and Pezzano, M. (2007) THYMUS- Workshop on T cell Development 2007 Rolduc Netherlands May 19-22 ) (BD-Outstanding Poster Award) Abstract P81.
11.    Crosstalk control of thymic epithelial development.  M. Pezzano, M. Osada, H. Fermin and D.B. Sant’Angelo (2007) Guest Lecture Immunology and Microbial Pathogenesis Program-Scientific Retreat. Mohonk Mountain House Conference Center, New Paltz, New York.
12.    The Use of Medullary Reconstitution to Analyze mTEC Development Agnes Sisa, Masako Osada, Hector Fermin, Derek B. Sant’Angelo and Mark Pezzano Einsteins in the City II International Student Research Conference Oct 30-31st 2007.
13.    Characterization of Kremen 1 Function.  Logan Jardin, Masako Osada, Hector Fermin, and Mark Pezzano Einsteins in the City II International Student Research Conference Oct 30-31st 2007.
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14.   Use of Medullary Reconstitution to Analyze mTEC Development Masako Osada1, Agnes Sisa, Hector Fermin1, Derek. B. Sant’Angelo2 and Mark Pezzano1 International Symposium on Health Disparities Honolulu Hawaii Dec 1-4 2008.

15.   DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration Masako Osada, Logan Jardine, Ruth Misir, Thomas Andl, Sarah E. Millar and Mark Pezzano  AAI meeting  Oral Platform Presentation May 7th-11th Baltimore Maryland 2010  A# 36.32.

16.  Characterization of Thymic Epithelial Stem Cells and their Capacity to Reconstitute Thymic Function Masako Osada, Logan Jardine, Ruth Misir, Derek B. Sant’ Angelo and Mark Pezzano MSKCC/CCNY Cancer Research Symposium Cancer Heath Disparities: A Translational Approach April 23 2010.

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