In vivo imaging of T cell delivery to tumors after adoptive transfer therapy

Abstract

Adoptive transfer therapy of in vitro-expanded tumor-specific cytolytic T lymphocytes (CTLs) can mediate objective cancer regression in patients. Yet, technical limitations hamper precise monitoring of posttherapy T cell responses. Here we show in a mouse model that fused single photon emission computed tomography and x-ray computed tomography allows quantitative whole-body imaging of (111)In-oxine-labeled CTLs at tumor sites. Assessment of CTL localization is rapid, noninvasive, three-dimensional, and can be repeated for longitudinal analyses. We compared the effects of lymphodepletion before adoptive transfer on CTL recruitment and report that combined treatment increased intratumoral delivery of CTLs and improved antitumor efficacy. Because (111)In-oxine is a Food and Drug Administration-approved clinical agent, and human SPECT-CT systems are available, this approach should be clinically translatable, insofar as it may assess the efficacy of immunization procedures in individual patients and lead to development of more effective therapies.

Fig. 1.

Antigen-specific CTL-mediated antitumor response. (A) Outline of the experimental design. (B) In vitro-stimulated HA-specific CTLs specifically lyse CT44 HA⁺ tumor cells in ⁵¹Cr-release assays. (C) ¹¹¹In-Oxine-labeled HA-specific CTLs selectively accumulate in HA⁺ tumors, as determined by calculating the percentage of ¹¹¹in-injected dose/gram tissue in explanted tumors 96 h after adoptive T cell transfer. (D) Thy1.2⁺ HA-specific CTLs selectively accumulate in HA⁺ tumors implanted in Thy1.1⁺ animals, as determined by immunohistochemical analysis of tumor parenchyma. (E) Administered HA-specific CTLs selectively control HA⁺ CT44 tumor growth; n = 3–10.

Fig. 2.

In vivo SPECT-CT monitoring of CTL distribution after adoptive transfer. (A) Fused SPECT-CT scans (Upper) and 3D virtual rendering (3D VR) images of anesthetized mice obtained 2 h (Left) and 24 h (Right) PI of ¹¹¹In-labeled HA-specific CTLs. Mice received CT44 HA⁺ and CT26 HA⁻ tumor cells in the right and left footpads, respectively, on day 0, and the CTLs on day 7 (the mice did not receive irradiation). The CTLs accumulated in the lung 2-h PI and in the liver and spleen 24 h PI (see also SI Movies 1 and 2). (B) 3D VR view of HA-specific CTL accumulation in HA⁺ and HA⁻ tumors (see also SI Movie 3). (C) CT, SPECT, and SPECT-CT fusion images obtained 2, 24, 48, and 120 h PI show specific accumulation of HA-specific CTLs in HA⁺ tumors. (D) Ratios of SPECT activity to ROI area calculated 2, 24, 48, and 120 h PI indicate specific accumulation of HA-specific CTLs in HA⁺ tumors. (E) Tumor volumes calculated by x-ray CT indicate that administered HA-specific CTLs control HA⁺ CT44 tumor growth. Data shown indicate changes (Δ) in HA⁺ CT44 and HA⁻ CT26 tumor volumes at the indicated time points when compared with tumor volumes at the time of CTL transfer [i.e., day 7 (d7)]; n = 5–10.

Fig. 3.

In vivo SPECT-CT monitoring for comparison of immunotherapeutic strategies. (A) CT44 HA⁺ tumor growth kinetics in mice treated with HA-specific CTLs (administered on day 7) and lymphodepleted (irradiated on day 4), either alone or in combination. Red lines indicate mean values for all mice analyzed; n = 12–30. (B) CT, SPECT, and SPECT-CT fusion images obtained 72 h PI show increased ¹¹¹in-CTL activity in tumors of lymphopenic mice. (C) Quantification of SPECT-CT data reveals increased ¹¹¹in-CTL activity 72 h PI in tumors of lymphopenic mice; n = 3. (D) Flow cytometric analysis of HA-specific CTL accumulation in HA⁺ tumors 72 h PI confirms the SPECT-CT findings; n = 3.