The finding that VCAM-1+ stroma express 4–1BBL, CCL19, CXCL12, and IL-7 and that adoptively transferred CD8+ memory T cells are often found in

proximity to VCAM-1+CD45− cells in the BM demonstrates the plausibility of the VCAM-1+ stromal cell as Erlotinib in vitro the radioresistant cell that provides 4–1BBL to memory CD8+ T cells in the BM. These data support a model in which a radioresistant VCAM-1+ stromal cell attracts the VLA-4+ CD8+ memory T cells via CCL19, where they can receive 4–1BB-4–1BBL induced survival signals. As the VCAM-1-positive stromal population is very abundant in the BM, there may be heterogeneity in the VCAM-1+ stroma with respect to 4–1BBL, cytokines, and chemokines that contribute to CD8+ T-cell memory maintenance. Further analysis will be required to definitively identify the 4–1BBL-expressing radioresistant cell that contributes to CD8+ T-cell memory. C57BL/6 WT mice were obtained from Charles River Laboratories (St. Constant, QC, Canada).

4–1BB−/− mice [47] extensively backcrossed to the C57BL/6 (n = 10) background were bred in our facility. These mice were previously provided to us by Dr. Byoung S. Kwon (National Cancer Center, Ilsan, Korea). 4–1BBL-deficient (4–1BBL−/−) mice were originally obtained under a materials transfer agreement from Immunex (Amgen, Thousand Oaks, CA, USA) and further backcrossed to the C57BL/6 background in our facility (total n = 9). OT-I

and CD45.1 congenic mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and crossed to PS-341 solubility dmso generate CD45.1+/+ or CD45.1+/− OT-I mice. TCRα−/– mice were kindly provided by Dr. Cynthia Guidos (Hospital for Sick Children, Toronto). FoxP3gfp knock-in mice on the C57BL/6 background were kindly provided by Dr. Mohamed Oukka (Harvard Medical School) [48]. ACTB-DsRed transgenic mice expressing DsRed protein under control of the β-actin promoter and backcrossed to B6 mice for five generations (B6.Cg-Tg (ACTB-DsRed*MST) 1Nagy/J) were obtained from the Jackson laboratories and crossed with OT-I mice to obtain OT-I ACTB-DsRed mice (OT-I-DsRed). Mice were maintained under specific pathogen-free conditions in sterile microisolators at the University of Toronto. All mouse experiments were approved of by the University of Toronto animal care committee in accordance with the regulations of the Canadian Council on animal care (University of Toronto approved protocol #20007828). CD8+ T cells with a central memory phenotype were generated by culture with Ag followed by IL-15 using a variation of a previous protocol [7, 29]. In brief, OT-I splenocytes were stimulated with 0.1 μg/mL SIINFEKL peptide and 1 μg/mL of LPS for 1 day, and then the nonadherent cells were rested for 2 days in fresh media (RPMI-1640 with 10% heat-inactivated FCS, 0.03% L-glutamine, antibiotics, and 2-mercaptoethanol).