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Conference Abstract: Tracer for CD8 T-cell tumor infiltration

Development of a CD8 tracer for in vivo evaluation of CD8 T cell tumor infiltration during immunotherapy (Conference Abstract)

René Raavé, Milou Boswinkel, Gerwin Sandker, Peter Wierstra, Erik H. J. G. Aarntzen, Sandra Heskamp

EMIM 2019, Glasgow: #858. EMIM 2019 Abstracts.



Immunotherapy is considered a hallmark in cancer treatment by its profound and durable clinical responses in patients with various types of cancer. However, only a subgroup of patients responds to immunotherapy and methods for accurate early response monitoring are lacking. Noninvasive quantitative imaging of CD8+ cytotoxic T cells can provide dynamic and spatial information of anti-tumor response. In the present study we characterized an 111In-labeled anti-mouse CD8 antibody for imaging of tumor infiltrating CD8+ cytotoxic T cells in vitro and in vivo in mice bearing murine CT26 colon tumors.


An anti-mouse CD8 antibody (clone: YTS 169.4) was randomly conjugated with a 30 times molar excess of NCS-DTPA and radiolabeled with 111In. Using CD8+ TK1 mice lymphoma cells, the immunoreactivity, IC50, internalization and affinity characteristics were determined. CT26 tumor bearing BALB/c mice (10-12 weeks old) were intravenously injected with 8.5 µg (8.5 MBq) [111In]In-DTPA-anti-CD8. One group received an excess of non-radiolabeled CD8 antibody (250 µg). SPECT/CT imaging was performed and organs were collected to quantify tracer uptake at 4h, 24h, 48h and 72h after injection. Autoradiography and immunohistochemistry were performed on paraffin embedded tissue sections of tumor, spleen, lymph nodes and duodenum.


In vitro assays demonstrated that the immunoreactive fraction was 44%, IC50 was 1.77 nM, Kd was 3.83 nM, and 6.5% internalization of the total membrane bound activity after 4.5 h of incubation. CD8+ T cell containing organs (lymph nodes, spleen and duodenum) were clearly visible on SPECT scans of mice injected with [111In]In-DTPA-CD8-antibody at all time points. Mice that received an excess of non-radiolabeled CD8-antibody showed most uptake in the spleen. Low to moderate tumor uptake was visible in all groups. Ex vivo biodistribution data confirmed results from SPECT imaging. In the lymph nodes, spleen, duodenum and tumor, an uptake of 38.6 ± 12.3% ID/g (±SD), 87.1 ± 18.0% ID/g, 31.7 ± 16.9% ID/g, and 12.9 ±2.9% ID/g at 24h after injection, respectively. The tumor-to-blood ratio increased from 0.48 at 4h after injection to 2.23 at 72h after injection. Autoradiography and immunohistochemistry confirmed these findings.


The CD8 antibody showed specific uptake in CD8+ T cell containing tissues in vivo, but uptake in the tumor was limited because of presence low number of CD8+ T cells. In the future, this tracer has potential for in vivo evaluation of CD8+ T cell infiltration in tumors and lymphoid tissues before and during immunotherapy.