In Vivo Tracking of Th1 Cells by PET Reveals Quantitative and Temporal Distribution and Specific Homing in Lymphatic Tissue

Abstract

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.

64 Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

“…2C) (7,10,17). This high trapping stability enables the use of less activity combined with a short exposure time, thereby preventing cell damage while still yielding quantitative, high-quality, low-noise PET images.…”

“…The slow accumulation dynamics of the mAbs into the cell might explain the less-severe damage to the cOVA-TCRtg-TH1 cells compared with [ 64 Cu]PTSM-labeling, in which 64 Cu accumulates rapidly in the cell nucleus (10). The delayed accumulation of the radioactive 64 Cu in the T cells might also explain the reduced induction of DNA damage, annexin expression, and apoptosis/necrosis compared with [ 64 Cu]PTSM-labeling.…”

“…They were then adoptively transferred into the experimental animals. For some comparative studies, cOVA-TCRtg-TH1 cells were labeled with 0.7 MBq [ 64 Cu]PTSM for 3 h, as described previously (10).…”

“…One frequently used cell-labeling compound for PET is [ 64 Cu]Pyruvaldehyde bis(N 4 -methylthiosemicarbazone) ([ 64 Cu]PTSM), which has been applied to mouse lymphocytes, primate stem cells, and human dendritic cells (7)(8)(9). We recently established a [ 64 Cu]PTSMlabeling protocol that minimizes the harmful effects on T-cell functions (10). Nevertheless, intracellular [ 64 Cu]PTSM labeling continues to impair viability and functionality as well as to induce apoptosis/necrosis and DNA damage in TH1 cells.…”

Immuno-PET Imaging of Engineered Human T Cells in Tumors

“…However, most T-cell imaging approaches using PET are far from clinical application and none has been yet clinically approved. This is mainly caused by speciesspecific tool development in non-humans, the often applied clinically non-significant ex vivo labeling procedures providing only short-term information after adoptive T-cell transfer and the negative impact of the labeling on T-cell function (6)(7)(8)(9)(10). Promising novel approaches include the usage of reporter genes or antibody derivatives for imaging.…”