Jastaranpreet Singh, Génève Awong and Juan Carlos Zúñiga-Pflücker

Department of Immunology, University of Toronto, and Sunnybrook Research Instute, Toronto, Canada.

Introducon Methods

+/- OPG-Fc § Hematopoietic stem cell transplantation (HSCT) is ProT Cells, 15K associated with prolonged and profound T-cell 10 days

lymphopenia, often making patients susceptible to CD38 potential morbidity/mortality. CD34+CD38-/lo HSCs CD34+CD7++ ProTs CD34 § Aging, and HSCT conditioning regiments induce defects in thymic niches for progenitors, and these ProT Cells E15 NSG Fetal Thymus Hanging Drop, 28h FTOC, 5 days defects are concomitant with a decrease in production § Capable of maturation § In vivo engraftment Proof of concept for in vivo experimentation. of thymus-seeding progenitors by the host. (ProT2 > ProT1) T+Cells+ T Cells Do proT cells with or without OPG-Fc induce thymic architectural changes in the same way? NK+Cells+ B+Cells+

Lymphoid+ Results Myeloid+ Macrophages+ Progenitor+ In vitro-derived ProT cells restore thymic OPG-Fc can block RANKL on in vitro-derived Eosinophils+ Basophils+ architecture in host NSG mice proT cells CD34+CD7+-gated proT cells 0.015 PTPRC 8 Ccl25 A B ** Non-injected 6 Neutrophils+ 0.010 ProT-injected 4

0.005 Mul)potent++ 2 Hematopoie)c+ Myeloid+ Stem+Cell+ Relative-actin β to 0.000 Relative-actin β to 0 Stem+Cell+ Erythroid+ RBCs+ 0.025 2.0 Progenitor+ Platelets+ Ccl19 Ccl21 0.020 Max %of 1.5 * 0.015 1.0 Figure 1 | Reconstitution of immune lineages from bone marrow-derived hematopoietic 0.010 0.5 stem cells. 0.005

Relative-actin β to 0.000 Relative-actin β to 0.0 Anti-Fc

0.025 Selp 15 Krt5 Figure 7 | OPG-Fc-mediated blocking of RANK-RANKL interactions between thymic 0.020 epithelial cells and proT cells. (A) Mechanism of OPG-Fc-mediated blocking and (B) 10 * 0.015 flow cytometric analysis of day 10 in vitro-derived CD34+CD7+-gated proT cells 0.010 5 showing OPG-Fc staining (shaded) and IgG control (open). Thymus' 0.005

Relative-actin β to 0.000 Relative-actin β to 0 0.008 PTPRC No Cells 15 0.006 ProT Cells Cytokera6n'8'(cortex)' H2-Ab1 Tnfrsf11a 0.006 ProT Cells + OPG-Fc 10 0.004 ProT$ PreT$ 0.004

0.002 CCL25 5 0.002 Relative-actin β to

3 Relative-actin β to Relative-actin β to 0.000 0 0.000 5 DP$ Figure 4 | Gene expression analyses from thymi obtained from in vitro-derived 0.06 Ccl19 Ccl21 2 Thymus$ CCL21 Cor6co?medullary'Junc6on' proT-injected mice. QPCR analysis for the expression of human PTPRC (CD45), 4 Seeding$ and mouse Ccl25, Ccl19, Ccl21, Selp (P-selectin), Krt5 (Cytokeratin-5), H2-Ab1 0.04 3 CCL19 CD4$ (MHC Class II), and Tnfrsf11a (RANK) from mouse thymus extracts of NOD/SCID/ 2 null γc mice injected with proT cells or control non-injected mice. Transcript levels 0.02 1 MTEC CD8$ for all genes were normalized to mouse β-actin. Asterisks represent statistical CD4$ CD8$ Relative-actin β to Relative-actin β to HSC$ significance as determined by t-tests 0 1 0.00 Periphery' (* p<0.05 ; ** p<0.005). Bone'Marrow' Cytokera6n'5'(medulla)' 6 Ccl25 2.5 Krt8 Non-injected NSG Thymus 2.0 Wild-type Adult Thymus 4 1.5

1.0 Figure 2 | T-cell development and journey through the thymus. 2 0.5 Relative-actin β to Relative-actin β to 0 0.0 Caveats in thymic reconstitution: Figure 8 | Gene expression analyses from day 5 NOD/SCID/γcnull fetal thymic organ cultures. QPCR analysis for the expression of human PTPRC (CD45), and mouse Ccl25, Ccl19, Ccl21, and Krt8 (Cytokeratin-8) for lobes containing no cells 1 HSCs are unable to directly home to the (n=8), proT cells (n=7) or proT cells with OPG-Fc (n=7). Transcript levels for all thymus. genes were normalized to mouse β-actin. ProT-injected NSG Thymus ProT-injected NSG Thymus Generang gene-modified RANKL-deficient 2 The thymus requires continuous seeding by bone marrow-derived thymus-seeding proT cells using CRISPR/Cas9 progenitors. IC TM EC +1TG 3 An intact thymic microenvironment is A Membrane-Bound RANKL E1D E2 E3 E4 E5/3’ UTR needed, and this is rendered defective by A+1TG conditioning regiments. Secreted RANKL 1A 1B E1C E2 E3 E4 E5/3’ UTR

Figure 5 | Immunohistological analysis of the thymus. Sections of thymus from an § In vivo modeling of RANKL-deficient adult wild-type mouse; control NOD/SCID/γcnull non-injected mouse; and NOD/ SCID/γcnul in vitro-derived proT-injected mouse, 6 weeks after intrahepatic injection proT cells. into neonates. Thymus tissues were stained with anti-Cytokeratin 8 (green; cortical), Experimental Queson anti-Cytokeratin 5 (blue; medullary), and anti-human CD45 (red). § In vitro-derived human progenitor T-cells (proT; ProT cells express RANKL at higher levels Conclusions CD34+CD7++) represent a source of thymus-seeding than HSCs § ProT cells transferred together with HSCs enhance progenitors that enhance HSC-derived engraftment in HSC-derived engraftment in the thymus in vivo after the thymus in vivo after HSCT. HSCT.

§ Injection of proT cells into NSG mice results in % of Max %of Max %of restoration of thymus architecture.

RANKL RANKL § ProT cells express RANKL, which suggests a Figure 6 | Flow cytometric analysis of RANK ligand (RANKL) expression on (A) potential mechanism for proT cell-induced purified CD34+CD38- HSCs (open) and day 11 in vitro-derived CD34+CD7+-gated proT cells (shaded); (B) proT2 cells (dark), proT1 cells (light), and unstained (open). reorganization of the thymic architecture.

Figure 3 | Analysis of early and late engraftment and differentiation of HSCs and proT2 cells in the thymus of immunodeficient mice. Flow cytometric analysis of CD45 in the thymus at 3 weeks and 12 weeks post-injection of neonatal mice receiving HSCs, HSCs with proT2 cells, or proT2 cells. Acknowledgements The Zúñiga-Pflücker Laboratory Flow Cytometry & Cell Sorting: How are proT cells able to enhance human Dr. Mahmood Mohtashami Gisele Knowles, Génève Awong & Courtney McIntosh hematopoiec stem cell-derived T-lymphopoiesis? Dr. Maria Malm Cord Blood: Dr. Elaine Herer M25 Antibody: Dr. Charles Surh