Noninvasive detection of tumor-infiltrating T cells by PET reporter imaging

Abstract

Adoptive transfer of tumor-reactive T cells can successfully reduce tumor burden; however, in rare cases, lethal on-target/off-tumor effects have been reported. A noninvasive method to track engineered cells with high sensitivity and resolution would allow observation of correct cell homing and/or identification of dangerous off-target locations in preclinical and clinical applications. Human deoxycytidine kinase triple mutant (hdCK3mut) is a nonimmunogenic PET reporter that was previously shown to be an effective tool to monitor whole-body hematopoiesis. Here, we engineered a construct in which hdCK3mut is coexpressed with the anti-melanoma T cell receptor F5, introduced this construct into human CD34 cells or PBMCs, and evaluated this approach in multiple immunotherapy models. Expression of hdCK3mut allowed engrafted cells to be visualized within recipient bone marrow, while accumulation of [18F]-L-FMAU in hdCK3mut-expressing T cells permitted detection of intratumoral homing. Animals that received T cells coexpressing hdCK3mut and the anti-melanoma T cell receptor had demonstrably higher signals in HLA-matched tumors compared with those in animals that received cells solely expressing hdCK3mut. Engineered T cells caused cytotoxicity in HLA/antigen-matched tumors and induced IFN-γ production and activation. Moreover, hdCK3mut permitted simultaneous monitoring of engraftment and tumor infiltration, without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations.

Figure 8. Visualization of engrafted stem and progenitor cells expressing the hdCK3mut PET reporter gene.

Engraftment of hdCK3mut-expressing cells could be detected within the bone marrow of (A) F5/hdCK3mut and (B) hdCK3mut recipients. Areas of engraftment are circled in green. (C) Quantification of bone signal plotted as bone signal/muscle signal. “Control bone” signal is from a contralateral region with no visible [¹⁸F]-L-FMAU PET signal. Mean ± SD, *P < 0.05, Student’s t test (n = 4).

Figure 7. Detection of hdCK3mut-engineered tumor-infiltrating lymphocytes by [¹⁸F]-L-FMAU PET reporter imaging.

(A) [¹⁸F]-FDG images from F5/hdCK3mut and hdCK3mut recipient animals. M202 xenografts are circled in red, and M207 xenografts are circled in aqua. (B) Quantification of the tumor/muscle ratio from [¹⁸F]-FDG images (NS by 1-way ANOVA), shown as mean ± SEM (n = 3–7). (C) [¹⁸F]-L-FMAU images from F5/hdCK3mut and hdCK3mut recipient animals. (D) Quantification of the tumor/muscle ratio from [¹⁸F]-L-FMAU images (P = 0.0001, 1-way ANOVA), shown as mean ± SEM (n = 3–11). *P < 0.05, **P < 0.01, ***P < 0.0005, 1-way ANOVA.

Figure 6. Immunohistochemistry of immune infiltrates from xenografts removed from F5/hdCK3mut animals.

(A) Schematic of xenograft location on F5/hdCK3mut animals. (B) H&E histology of M202 (HLA-A*0201⁺) and M207 tumors (HLA-A*0201^–). The white line distinguishes viable tumor from tumor necrosis. (C) Anti-CD3 and (D) anti-CD8 staining of representative sections of M202 or M207 xenografts. Black arrows point to T cells. Scale bar: 50 μM.

Figure 5. hdCK3mut-expressing T cells developed in vivo are capable of cytokine production and activation.

(A) Representative FACS plots of cultures incubated with PMA/ionomycin or (B) untreated. Subgate on CD8 cells showing intracellular IFN-γ by intracellular flow cytometry. IgG isotype was used as a control for background staining. (C) Percentage of CD8 cells that produce IFN-γ, shown as mean ± SD. K562 aAPCs and T cells were cocultured for 72 hours. Representative flow cytometry plots from T cells of F5/hdCK3mut animals cocultured with (D) MART-1–expressing aAPCs or (E) hdCK3mut aAPCs. F5 T cells are shown on the top row, and CD8 T cells are shown on the bottom row. (F) Total activated T cells after 72 hours coculture with aAPCs (CD25⁺CD71⁺) for F5/hdCK3mut or hdCK3mut recipients. NS by 1-way ANOVA, shown as mean + SEM (n = 3). (G) Engineered F5 T cell activation with aAPCs (**P < 0.005), shown as mean + SEM (n = 3).

Figure 4. Engraftment and hematopoiesis of human cells in BLT mice.

Cells were viably isolated from the spleens of BLT animals at the experimental endpoint. Total engraftment and phenotype was analyzed by flow cytometry. Representative engraftment of (A) hdCK3mut and (B) F5/hdCK3mut animals is shown. (C) Average human engraftment is shown as mean ± SD, and (D) distribution of cell phenotypes from all animals is shown as mean + SD. Statistics were analyzed using ANOVA (n = 3). SSC, side scatter.

Figure 3. Establishment of humanized mice harboring F5 TCR against MART-1.

(A) HLA-A*0201 donor tissue was combined to create a human thymus, which was transplanted subrenally as a thymic graft into NSG mice. After 4 months, animals received low-dose full-body irradiation and were transplanted with gene-modified (expressing F5/hdCK3mut or hdCK3mut) hCD34 cells. Two subcutaneous tumors were implanted 12 weeks after HSC transplant: M202 (left side, HLA match) and M207 (right side, HLA mismatch). After 4 weeks, animals were subjected to microPET scans and ex vivo analysis of the engineered cells. (B) Vector diagrams of the F5/hdCK3mut and hdCK3­mut lentivirus used.

Figure 2. Visualization of short-term hdCK3mut-transduced PBMC T cell trafficking into M202 tumors.

(A) PET imaging of adoptive cell transfer with NSG mice with M202 (HLA-A*0201⁺ MART-1⁺) tumors receiving 5 × 10⁶ PBMCs (40% transduced). PBMCs were transferred on day 0 i.v. with 50,000 IU of IL-2 i.p. (B) [¹⁸F]-L-FMAU imaging and quantification was performed on day 1, (C) that for [¹⁸F]-FDG was performed on day 2, and (D) that for [¹⁸F]-L-FMAU was performed on day 8. Quantification is shown as mean ± SD, n = 3. Peripheral blood on day 9 after ACT for total human CD4, CD8, and F5 engineered cells for (E) F5/hdCK3mut and (F) hdCK3mut.

Figure 1. Expression of hdCK3mut does not alter engineered T cell function.

(A) Intracellular flow for IFN-γ production after T cell activation by PMA/ionomycin. (B) IFN-γ production of control untreated PBMCs. (C) Quantification of A (black bars) and B (white bars) IFN-γ production. n = 4 unique PBMC donors, plotted as mean + SD. (D) Representative plots of cytotoxicity (DAPI⁺) of M202 melanoma cells when cocultured with engineered T cells. (E) Quantification of cytotoxicity in tumor cells normalized to cytotoxicity in PBMCs (M202^–HLA⁺MART⁺, M207^–HLA^–MART⁺, M407^–HLA⁺MART^–). n = 2 unique PBMC donors, plotted as mean + SD. **P < 0.001, ANOVA.