Multi-spectral fluorescence imaging of adoptive immune cell therapy using a cell membrane probe

Objectives The overall objective of this study is to non-invasively assess in-vivo targeting and retention of adoptively transferred T-lymphocytes at the tumor site.

Methods 4T1 specific T-lymphocytes obtained from draining lymph nodes of 4T1 sensitized BALB/C mice were pulsed in-vitro with Bryostatin/ Ionomycin for 18 hours, and then divided into two populations that were grown in either Interleukin-2 (IL2) or a combination of interleukins 7 & 15 (IL7/15) for 10 to 13 days. T-lymphocytes were then directly labeled with a lipophilic NIR fluorescent probe (Xenolight DiR). In experiment one, recipient mice having a 4T1 tumor were injected with labeled cells. In experiment two, 4T1 tumor cells were implanted 1-week post-injection of labeled T-lymphocytes. Multi-spectral fluorescence imaging was done at various time points up to 24 days. The viability of lymphocytes and their functions were assessed by ATP based cell viability assay, flowcytometry and interferon- gamma (IFNγ) ELISA.

Results IL7/15 is superior to IL2 for ex vivo expansion, but the in-vivo imaging data showed higher tumor retention of labeled T-lymphocytes for IL2 than IL7/15 and the signal persisted at the tumor site for weeks with a peak on day 6 post-injection of IL2 grown cells. In experiment two, IL2 grown cells moved out of lymphoid compartments to the site of 4T1 inoculation within two hours and peaked on day 3. Good visualization of labeled lymphocytes in tumor, liver, spleen, gut, lymph nodes, and in bone/ bone marrow was noted ex vivo. Flowcytometry, cell viability assay and IFNγ ELISA showed that, the proliferation, viability and function of stained 4T1 specific T-lymphocytes was not affected.

Conclusions Direct labeling of 4T1 specific T-lymphocytes by a fluorescent dye yielded relatively stable labeling and provided in vivo data on trafficking of these cells over an extended period of time