Molecular imaging of lymphoid organs and immune activation by positron emission tomography with a new [18F]-labeled 2'-deoxycytidine analog

Abstract

Monitoring immune function with molecular imaging could have a considerable impact on the diagnosis and treatment evaluation of immunological disorders and therapeutic immune responses. Positron emission tomography (PET) is a molecular imaging modality with applications in cancer and other diseases. PET studies of immune function have been limited by a lack of specialized probes. We identified [(18)F]FAC (1-(2'-deoxy-2'-[(18)F]fluoroarabinofuranosyl) cytosine) by differential screening as a new PET probe for the deoxyribonucleotide salvage pathway. [(18)F]FAC enabled visualization of lymphoid organs and was sensitive to localized immune activation in a mouse model of antitumor immunity. [(18)F]FAC microPET also detected early changes in lymphoid mass in systemic autoimmunity and allowed evaluation of immunosuppressive therapy. These data support the use of [(18)F]FAC PET for immune monitoring and suggest a wide range of clinical applications in immune disorders and in certain types of cancer.

Figure 1. Identification of fluorinated deoxycytidine analogs retained in activated vs. naïve T cells

(a) Primary CD8⁺ T cells were stimulated ex vivo for 72 hrs and then were incubated for 1 hr with [³H]-labeled NAs; following successive washes, intracellular radioactivity was measured by scintillation counting. (b) FAC is a dFdC analog amenable to ¹⁸F labeling. (c) Similar retention of [³H]dFdC and [³H]FAC by activated mouse CD8+ T cells; the retention of [³H]FAC in naïve CD8+ T cells was 18+/−4.5 fmoles/10⁵ cells (d) Increased uptake of [³H]FAC in NIH3T3 fibroblasts engineered to overexpress nucleoside kinases (dCK, TK1) and the nucleoside transporter SLC29A1. [³H]FLT was used as a positive control for TK1 expressing cells. (e) [³H]FAC and the parental nucleoside 2′ deoxycytidine ([³H]dCyd, used as a positive control) are incorporated in the DNA of proliferating CD8+ T cells as a function of time (see text for details). * P values of <0.05. Results are representative of two independent experiments.

Figure 2. [¹⁸F]FAC has better selectivity for lymphoid organs compared with other PET probes for nucleoside metabolism and glycolysis

(a) [¹⁸F]FAC DWBA shown along with the corresponding tissue sections. (b,c) C57/BL6 mice were scanned by microPET/CT using [¹⁸F]FAC, [¹⁸F]FLT, [¹⁸F]D-FMAU and [¹⁸F]FDG. Mice were imaged 60 min after i.v. injection of probes. The orientation of saggital, coronal and transverse sections is depicted in the 3D microCT image in panel b. Images are 1 mm thick sagittal, coronal and transverse slices. Percent ID/g, percent injected dose per gram of tissue; B, Bone; BL, Bladder; BR, Brain; GB, Gall Bladder; GI, Gastrointestinal tract; H, heart; K, Kidney; L, Liver; LU, Lung; SP, Spleen; Thy, thymus; BM, bone marrow; ST, stomach. (d) [¹⁸F]FAC retention/cell number in thymocytes and splenocytes. (d) Proportion of [¹⁸F]FAC retention/cell lineage per lymphoid organ (see text for details).

Figure 3. Increased [¹⁸F]FAC retention in spleen and lymph nodes at the peak of the primary anti-tumor immune response

Images are 1 mm coronal sections from microPET/CT scans using [¹⁸F]FAC (Day -1 and Day 15, panel a), [¹⁸F]FDG (Day 13, panel d) and [¹⁸F]FLT (Day 14, panel e). B, Bone; BL, Bladder; GI, Gastrointestinal tract; H, heart; SP, Spleen; TU, tumor; Thy, thymus; LN, lymph node. (b) Quantification of [¹⁸F]FAC retention in spleen and lymph nodes on Day -1 and Day 15; Number of mice =3. (c) Increased in vivo accumulation of [¹⁸F]FAC in effector CD8+ T cells vs. naïve CD8+ T cells. Mice were challenged with the MoMSV onco-retrovirus and 14 days later were injected with 1 mCi [¹⁸F]FAC. Following 1 hr in vivo uptake, mice were sacrificed to isolate splenocytes which were fractionated by flow cytometry into naïve CD8⁺ T cells (CD44^LOW/CD62L^HIGH) and effector CD8⁺ T cells (CD44^HIGH/CD62L^LOW). Radioactivity accumulated by these cells was measured using a well counter. Results are representative of two independent experiments.

Figure 4. [¹⁸F]FAC microPET/CT allows visualization of increased lymphoid mass in systemic autoimmunity and can be used to monitor immunosuppressive therapeutic interventions

Images are 60 minutes after i.v. injection of [¹⁸F]FAC and show three 1 mm thick coronal slices from (a) wild-type (C57BL/6J) and B6.MRL-Fas^lpr/J (b) before and (c) after treatment with DEX. [¹⁸F]FAC positive LNs were scored blindly. Thy, thymus; LN, lymph nodes; BM, bone-marrow. Results are representative of two independent experiments.