1. Cloning of recombinant expression vectors that encode Interleukin-15 (IL-15), Interleukin-21 (IL-21) and Luciferase for tumor immunology studies Alberto Del Rosario Harlem Children Society, 3 rd year Memorial Sloan-Kettering Cancer Center Manhattan Center for Science and Mathematics July 13, 2006

2. Experimental Objectives Produce HIV derived retroviral vectors that allow T-cells to express luciferase and IL-15 or IL-21. Produce T-cells that artificially express cytokines in order to learn more about their effects on T-cells in the setting of tumor immunology.

3. Why are cytokines important and what is the role of Luciferase in these experiments? Cytokines are important because they help T-cells to eradicate cancer cells better.Cytokines are important because they help T-cells to eradicate cancer cells better. Cells that express luciferase will produce light when luciferin (the enzyme’s substrate) is injected intravenously. The light can be detected with an infrared camera.Cells that express luciferase will produce light when luciferin (the enzyme’s substrate) is injected intravenously. The light can be detected with an infrared camera. The role of luciferase is to allow us to track down the T- cells in the living body of the mouse.The role of luciferase is to allow us to track down the T- cells in the living body of the mouse.

4. BamHI/NotISpeI/NotIBamHI/SpeI

5. Gel Purification DNA Ligase Gel Purification DNA Ligase BglII 9277 BglII 6126 BglII 3775 BglII 126

6. Results Marker Sample Marker Sample IL-15 lentiviral vector 4,000bp 4,000bp

7. Polymerase Chain Reaction (PCR) Machine that allows us to perform PCR. PCR http://academic.brooklyn.cuny.edu/biology/bio4fv/page/genetic-engin/pcr2484.JPG

8. PCR results The picture shows the band that was desired (that was then cut and the DNA was extracted from the agarose gel), which was at 508 base pairs.The picture shows the band that was desired (that was then cut and the DNA was extracted from the agarose gel), which was at 508 base pairs. This picture represents the cDNA of human IL-21.This picture represents the cDNA of human IL-21. Uncut Cut Uncut Cut Marker Sample Marker Sample

9. BamHI/AvrIIClaI/AvrIIClaI/BamHI

10. Gel Purification DNA Ligase Gel Purification DNA Ligase BamHI 8653 BamHI 7546 BamHI 6762 BamHI 7154 BamHI 5395

11. Marker Sample Marker Sample IL-21 lentiviral vector Results

12. Mentor’s Role After I am done cloning the desired genes, my mentor makes lentiviral vectors.After I am done cloning the desired genes, my mentor makes lentiviral vectors. He will then transduce T-cells with these vectors and inject mice intravenously with the transduced T-cells.He will then transduce T-cells with these vectors and inject mice intravenously with the transduced T-cells. He will check weekly the mice to see if they eradicated the tumors.He will check weekly the mice to see if they eradicated the tumors.

13. References 1. Brentjens, R.J. et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and Interleukin-15. Nature Medicine. 9, 279-286 (2003). 2. Choy, G. et al. Current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research. Mol Imaging. 2. 303- 12 (2003). 3. Klebanoff, C.A. et al. IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T Cells. PNAS. 101. 1969-74 (2004).

14. Acknowledgements Dr. Michel SadelainDr. Michel Sadelain John C. MarkleyJohn C. Markley The Sadelain LabThe Sadelain Lab Dr. Sat BhattacharyaDr. Sat Bhattacharya The Harlem Children SocietyThe Harlem Children Society