Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. Methods: We developed protocols that minimize the inhibitory effects of 64 Cupyruvaldehyde-bis(N4-methylthiosemicarbazone) ( 64 Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-g-producing CD4 1 T (Th1) cells with 0.7-2.2 MBq of 64 Cu-PTSM and analyzed cell viability, IFN-g production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular 64 Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10 7 64 Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of 64 Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10 7 64 Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. Results: PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, 64 Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered 64 Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of 64 Cu-OVA-Th1 cells in the pulmonary LNs (6.8 6 1.1 percentage injected dose per cubic centimeter [%ID/cm 3 ]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity-diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 6 0.4 %ID/cm 3 ). As expected, 64 Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 6 0.5 %ID/cm 3 ) when compared with phosphate-buffered saline-challenged animals (4.6 6 0.5 %ID/cm 3 ). Conclusion: Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for 64 Cu-PTSM-labeled T cells in clinical trials and novel therapy concepts focusing on T-cell-based immunotherapies of autoimmune diseases or cancer.

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64 Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosisnecrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied 64 Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayedtype hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.PET imaging | mouse T cells | in vivo cell tracking | antibody-based cell labeling | airway DTHR

The metabolites, transporters, and enzymes involved in choline metabolism are regarded as biomarkers for disease progression in a variety of cancers, but their in vivo detection is not ideal. Both magnetic resonance spectroscopy [MRS using chemical shift imaging (CSI) total choline (tCho)] and 11 C-choline positron emission tomography (PET) can probe this pathway, but they have not been compared side by side. In this study, we used the spontaneous murine astrocytoma model SMA560 injected intracranially into syngeneic VM/Dk mice, analyzing animals at various postimplantation time points using dynamic microPET imaging and CSI MRS. We observed an increase in tumor volume and 11 C-choline uptake between days 5 and 18. Similarly, tCho levels decreased at days 5 to 18. We found a negative correlation between the tCho and PET results in the tumor and a positive correlation between the tCho tumor-to-brain ratio and choline uptake in the tumor. PCR results confirmed expected increases in expression levels for most of the transporters and enzymes. Using MRS quantification, a good agreement was found between CSI and 11 C-choline PET data, whereas a negative correlation occurred when CSI was not referenced. Thus, 11 C-choline PET and MRS methods seemed to be complementary in strengths. While advancing tumor proliferation caused an increasing 11 C-choline uptake, gliosis and inflammation potentially accounted for a high peritumoral tCho signal in CSI, as supported by histology and secondary ion mass spectrometry imaging. Our findings provide definitive evidence of the use of MRS, CSI, and PET for imaging tumors in vivo. Cancer Res; 73(5);