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Congenically marked, CD45 (all hematopoietic cells), mice are surgically joined for ~4 weeks Tracking of chimerism and engraftment of immune cells T cells, B cells, NK cells, Neutrophils, Macrophages, and Dendritic Cells (DCs) Heterochronic Parabiosis: Allowing the Dissection of the Aged Immune System John S. Davies, Heather L. Thompson, Vesna Pulko and Janko Nikolich-Zugich Department of Immunobiology and the Arizona Center on Aging, College of Medicine, University of Arizona, Tucson, AZ 85724 Abstract Parabiosis is the surgical union of two organisms resulting in the development of a single, shared circulatory system. When animals of different ages are conjoined (i.e. heterochronic parabiosis), investigators have shown that blood-borne factors from the younger animal beneficially affect the older animal and recapitulate the youthful phenotype and function in their target tissues. However, the effect of heterochronic parabiosis on the aged immune system remain unexplored. An important question to be answered is whether the cellular defects involved in the aged immune system are due to intrinsic defects or if they can be rescued by extrinsic factors. This experimental technique is ideal to test cellular migration patterns, interrogate the mechanisms driving migration defects that occur with aging, if these defects can be rejuvenated and identify molecules that are targets for intervention. Finally, T cell ontogenesis is known to diminish drastically with age due to thymic involution, but there are also defects in lymphopoiesis that affect early T cell precursors. The heterochronic parabiosis model allows one to test the contributions of the aged thymus, the aged pre-T cells (thymic seeding progenitor cells) or if the aged systemic environment are involved in the defect in thymic output with age. Thus far, the preliminary data demonstrate differential migration patterns of T cells, B cells and dendritic cells. Of interest, pre-T cells from either animal adequately seed the opposing thymus and undergo thymopoiesis and while development in the adult thymus supports normal thymopoesis of adult or old thymocytes, the opposite is not true in the old thymus. This indicates that there are no intrinsic defects in old thymocytes, but rather the old thymic environment and this defect cannot be rejuvenated by heterochronic parabiosis. Total Heterochronic Chimerism is Similar T & B Lymphocyte Bone Marrow Tropism Switch Thymopoiesis Defects Specific to Old Thymus Conclusions B cells are preferentially retained in the bone marrow of the heterochronic partner. CD8 T cells are preferentially retained in the bone marrow of the heterochronic partner. Preliminary evidence indicates that different T cell subsets contribute to the preference to home to the heterochronic partner. Defects in the old thymus were not restored by heterochronic parabiosis. Defects in thymopoiesis is due to the old thymus, not old thymocytes. Preliminary evidence indicates that there is a different developmental block in adult or old thymocytes in the old environment. Acknowledgements Adult EM CD8 T Cells in Adult or Old Bone Marrow Adult Naive CD8 T Cells in Adult or Old Bone Marrow Adult CM CD8 T Cells in Adult or Old Bone Marrow Old EM CD8 T Cells in Adult or Old Bone Marrow Old Naive CD8 T Cells in Adult or Old Bone Marrow Old CM CD8 T Cells in Adult or Old Bone Marrow Heterochronic Chimerism in the Adult Thymus Adult Thymocytes in Adult or Old Thymi Heterochronic Chimerism in the Old Thymus Experimental Design ~4w Separate Mice & Harvest Tissues Assay Cells by Flow Cytometry Anatomical Diagram of Interrogated Tissues Total Chimerism in Adult TissuesTotal Chimerism in Old Tissues Figure 1: Total CD45 Chimerism (A) Total hematopoietic chimerism of adult or old cells measured in adult tissues. (B) Total hematopoietic chimerism of adult or old cells measured in old tissues. The mice below illustrate the color scheme you will see throughout the poster for which cells were observed where. Different T Cell Subsets Contribute to Tropism (A) (B) (A) (B) Figure 2: T & B Cells have a Preference for the Opposing Bone Marrow (A) Measurements of adult or old T cells in the adult bone marrow (left) or the old bone marrow (right). (B) Measurements of adult or old B cells in the adult bone marrow (left) or the old bone marrow (right). T Cell Chimerism in Old Bone Marrow T Cell Chimerism in Adult Bone Marrow B Cell Chimerism in Old Bone Marrow B Cell Chimerism in Adult Bone Marrow Adult Mouse Old Mouse Figure 3: Effector Memory, Central Memory and Naïve CD8 T Cells Differentially Contribute to Bone Marrow Preference (A) Adult effector memory CD8 T cells are preferentially retained in adult bone marrow, while old effector memory CD8 T cells are preferentially retained in old bone marrow. (B) Adult central memory CD8 T cells have no homing preference and redistribute equally across adult and old bone marrow. Old central memory CD8 T cells preferentially home to adult bone marrow. (C) Adult naïve CD8 T cells preferentially home to old bone marrow. Old naïve CD8 T cells have no homing preference and redistribute equally across adult and old bone marrow. These graphs compare adult or old cells across tissues while prior graphs compare adult vs old cells within a given tissue. The comparison here is more suited for comparing homing preferences. (A) (B) (C) (A) (B) (C) Figure 4: Tracking Thymopoeisis of Adult or Old Thymocytes as they Differentiate in Adult or Old Thymic Environments (A) Adult or old thymocytes undergoing thymopoeisis equally in the adult thymic environment. Since there is no difference between adult or old thymocytes in the adult thymic environment there must be no intrinsic defect in old thymocytes. (B) Adult or old thymocytes undergoing thymopoesis differently in the old thymic environment. The old thymic environment induces differential defects in adult or old thymocytes, as they are blocked in the DN4 & DN1 stages, respectively. (C) Adult thymocytes undergoing thymopoesis differently in the adult or old thymic environments. When adult thymocytes are compared across adult and old thymic environments it is clear that the defect in thymopoiesis is imposed on adult thymocytes in the old thymic environment. This work was funded by NIH/NIAID: HSN266200500027C These surgeries could not have been conducted without Vesna Pulko’s help