Leonardo Aguila, Ph.D. Assistant Professor Email: aguila@nso1.uchc.edu

Two major areas of interest are the focus of our laboratory:

1.Development and function of NK cell compartment
NK cells are one of the main effector arms of innate immunity. Their ability to produce cytokines that affect multiple hematopoietic compartments makes them attractive candidates to modulate the outcome of adaptive immune responses. As cytotoxic cells, they can efficiently participate in the clearance of intracellular pathogens and tumors. Their implementation as vectors in adoptive immunotherapy protocols has been promising. The understanding of the development, heterogeneity and function of this compartment is important if we would like to design protocols aimed to modulate or alter NK cell functions. In our laboratory we are studying the heterogeneity of NK cells from a developmental as well as from a functional prospective. The major goals for this project are:

A. To isolate and characterize NK cell committed progenitors from adult bone marrow.
Using in vitro progenitor assays, and populations of cells fractionated by flow cytometry, we can identify cells enriched in NK cells progenitor activity, these cells are being tested in clonal assays by their ability to exclusively generate NK cells in vitro as well as in vivo. Ultimately, these progenitors will be used to study the kinetics of appearance of surface markers and the development of effector functions, to define pathways leading to the generation of the heterogeneity observed in the NK cell compartment.

B. To define the phenotypic and functional heterogeneity of the NK cell compartment.
Using flow cytometry techniques we are analyzing the heterogeneity of the murine NK cell compartment by the expression of various hematopoietic markers. Sorted cells are evaluated by their cytotoxic potential and by their lymphokine production. With these studies we expect to define if a functional commitment occurs within the NK cell compartment. In addition, we have generated novel monoclonal antibodies that specifically react with murine NK cells. These antibodies are being used to deplete defined populations of NK cells in vivo in order to evaluate the role of sub populations of NK cells in processes as: rejection of allogeneic bone marrow transplants, control of tumor metastases, and induction and maintenance of allergic asthma.

C. To establish methods to grow and propagate clonal cell lines of murine NK cells.
The availability of cloned populations of NK cells will allow the dissection NK cell functions and will provide tools to evaluate the recognition and functional diversity of the compartment. Also, these clones could serve as vectors for immunotherapy protocols. We are studying the NK cell compartment in strains of mice deficient in programmed cell death mechanisms, principally p53 deficient mice and mice that over express bcl-2 in their NK cell compartment. We have being able to propagate NK cells in long term cultures under limiting activation, and we are in the process of cloning individual entities from those cultures. If cloning is possible, and distinct clones are obtained, they will be used in adoptive transfer protocols for the treatment of tumors, and to test their ability to alter the course of induction and/ or progression of autoimmunities in a murine model of allergic asthma.

2.Factors affecting engraftment of hematopoietic progenitors during development and after bone marrow transplantation.

Hematopoiesis is the process by which pluripotent Hematopoietic Stem Cells (HSCs) generate and replenish hematopoietic progenitors and all mature blood cells. During development, hematopoiesis is established in different anatomical locations. In adult mammals the bone marrow is the principal site of hematopoiesis. In myeloablated individuals the success on recovering the hematopoietic compartment, after bone marrow transplantation, is dependent on the ability of donor HSCs to engraft in the host marrow. Thus, defined homing properties have been proposed for HSCs. Advances have made possible to identify and isolate pure populations of HSCs, and to study the interactions between progenitors and mature hematopoietic components. As a result, bone marrow transplantation has become the chosen therapy for immunodeficiency disorders and hematopoietic malignancies. However, many aspects of the engraftment process are not fully understood, in both syngeneic and allogeneic settings.

We have found that NK cells, one of the main mediators of bone marrow rejection, do not use cytotoxicity as the main effector function to eliminate allogeneic hematopoietic progenitors. We are now analyzing other NK cell effector functions as well as other cell components that could account for the ability of NK cell compartment to mediate the rejection phenomenon.

Hematopoiesis occurs in the context of a bone marrow microenvironment that includes hematopoietic progenitors, bone marrow stroma cells, and cells involved in bone remodeling. The contribution of bone components to the hematopoietic process has been difficult to define due to the lack of good models that would allow the study of their interactions with hematopoietic progenitors. We are analyzing the dynamics of hematopoiesis in mouse models with alterations in bone remodeling processes. We found that in a transgenic mouse in which osteoblasts can be eliminated on demand, the ability to mount an efficient hematopoiesis is severely compromised in the bone marrow. Alternatively, active hematopoiesis is established in spleen and liver. We are now defining the role of the interactions between osteoblasts and hematopoietic stem cells using this model on transplantation studies.

The understanding of barriers to engraftment and the definition of bone marrow microenvironments will allow us to understand better the biology of hematopoiesis, and will help to design better protocols for bone marrow transplantation.

Selected publications

1. Makki A, Weidt G, Blachere N, LeFrancois L, and Srivastava PK. Abrogation of tumore protection by immunization against a dominant tumor antigen. Cancer Immunity, Vol. 2, pg. 4, 2002.

2. Aguila, H.L., Hershberger, R.J., and Weissman, I.L.. "Transgenic mice carrying the Diphtheria toxin A chain gene under the control of the Granzyme A promoter: Expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells". Proc. Natl. Acad Sci. USA. 92: 10192-10196. 1995.

3. Aguila, H.L., and Weissman, I.L.. " Hematopoietic stem cells are not direct targets of natural killer cells". Blood. 87: 1225-1231. 1996.

4. Aguila, H.L., Akashi, K., Domen, J., Gandy, K.L., Lagasse, E., Mebius, R.E., Morrison, S.J., Shizuru, J., Strober, S., Uchida, N., Wright, D.E., and Weissman, I.L.. "From stem cells to lymphocytes: Biology and transplantation". Immunol. Rev. 157: 13-40. 1997.

5. Gandy, K.L., Domen, J., Aguila, H.L., and Weissman, I.L.. "CD8+TCR- cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers". Immunity. 11:579-590. 1999.

6. Kim, S-J, Iizuka, K., Aguila, H.L., Weissman, I.L., and Yokoyama, W. "In vivo Natural Killer Cell Activities revealed by NK Cell-Deficient Mice". Proc. Natl. Acad Sci. USA. 97:2731-2736. 2000

7. Visnjic, D., Kalajzic, I., Gronowicz, G., Aguila, H.L., Clark, S.H., Lichtler, A.C., and Rowe, D.W. "Conditional ablation of the osteoblast lineage in Col2.3DTK transgenic mice". J Bone Miner Res 16: 2222-2231. 2001

8. Cooper, M.A., J.E. Bush, T.A. Fehniger, J.B. VanDeusen, R.E. Waite, Y. Liu, H.L. Aguila, and M.A. Caliguri. "In Vivo evidence for a Dependence on Interleukin-15 for Natural Killer Cell Survival". Blood. 100:3633-3638. 2002.

9. Katavic, V., Grcevic, D., Lee, S.K., Kalinowski, J., Jastrzebski, S., Dougall, W., Anderson, D., Puddington, L., Aguila, H.L., and J.A. Lorenzo. "The surface antigen CD45R identifies a population of estrogen regulated murine marrow cells that contain osteoclast precursors". Bone 32:581-590. 2003.

10. Visnjic, D., Kalajzic, Z., Rowe, D.W., Katavic, V., Lorenzo, J., and H.L. Aguila. "Hematopoiesis is severely altered in mice with an induced osteoblast deficiency". Blood. 103:3258-3264. 2004.

11. Schluns, K.S., Nowak, E.C., Cabrera-Hernandez, A., Puddington, L., Lefrançois, L., and H.L. Aguila. "Distinct Cell Types Control Lymphoid Subset Development Via IL-15 and IL-15Ra Expression". Proc. Natl. Acad Sci. USA. 101:5616-5621. 2004.