Insight into the dynamics, localization and magnitude of antigen-specific immune responses by [¹⁸F]FLT PET imaging

Abstract

In order for cellular therapeutics to succeed, comprehensive monitoring of the transplanted cells in vivo is required i.e., their localization, functionality and numbers in a longitudinal manner. Recently, dendritic cell based vaccines have been monitored by their effect on lymphocyte activation using [¹⁸F]FLT PET in cancer patients.

Keywords: :

• cell therapy
• cell tracking
• [¹⁸F]FLT-PET
• immunomonitoring
• dendritic cell-based vaccines

The field of vaccination has expanded over the last few years to include the development of therapeutic vaccines against infections and cancer.¹ One technique to induce antigen-specific immune responses in cancer patients is by exploiting autologous dendritic cells (DCs) that are "educated" ex vivo i.e., DCs that are appropriately activated and loaded with tumor antigens.¹ DCs are the most potent antigen presenting cells of the immune system and play a central role in the induction and maintenance of antigen-specific immunity. These cells capture and process antigen and migrate to the lymph nodes (LNs) where they present the antigen to the adaptive arm of the immune system, inducing antigen-specific T and B cell responses.

The development of appropriate metrics is necessary to optimize these novel cellular therapies, in terms of the types of cells, dosage, activation status and route of delivery. Furthermore, this monitoring needs to be noninvasive and capable of obtaining longitudinal data. For these reasons, in vivo imaging is gaining in popularity. Monitoring of therapeutic cells post-delivery requires a multi-pronged approach, where the cell number, localization and functionality (or viability) need to be measured in order to fully understand the fate of the transplanted therapeutic cells. The utility of imaging for this purpose has already been demonstrated, for example in determining the accuracy of delivery.² While the localization and numbers of transplanted cells in vivo is well-established, using scintigraphy or MRI,³ the measurement of cell functionality in vivo has proven more difficult. However, such measurements are crucial to determine whether the therapy should be continued, modified or stopped completely in an individual patient. [¹⁸F]-labeled 3'-fluoro-3'-deoxy-L-thymidine ([¹⁸F]FLT) has been used to monitor cell proliferation, primarily in tumors.⁴ We adapted it to the measurement of lymphocyte proliferation instead.⁵ FLT is a thymidine analog and accumulates in dividing cells, although it is not incorporated in to DNA. We compared this tracer to the commonly used [¹⁸F]-labeled fluoro-2-deoxy-2-D-glucose (FDG), a glucose analog which accumulates in metabolically active cells.

Melanoma patients were treated with a therapeutic DC vaccine. DCs were generated ex vivo, activated and loaded with melanoma-associated antigens and keyhole limpet hemocyanin (KLH) as an immunogenic control antigen. Vaccines were targeted directly to LNs under ultrasound guidance, and contralateral LNs were injected with saline or DCs not loaded with antigen to serve as negative control.

We first demonstrated that [¹⁸F]FLT PET signals co-localized with vaccinated antigen-loaded DCs, labeled ex vivo with [¹¹¹In]oxine and superparamagnetic iron oxide (SPIO). Scintigraphy was performed immediately following PET/CT scanning and showed profound [¹⁸F]FLT uptake even when only 4.5 x 10⁵ antigen-loaded DCs were present. This was confirmed by immunohistochemical staining of the LNs, showing that the SPIO-labeled DC which have dispersed into the T cell areas and induced activation of CD4⁺ and CD8⁺ T cells.

We identified the optimal time window of [18F]FLT imaging, a significant increase in the [¹⁸F]FLT signal was observed shortly after the very first vaccination. Although de novo immune responses are readily visualized, vaccinated LNs remained positive up to 3 weeks after the last vaccination. The clearest signals [¹⁸F]FLT were observed, exclusively in vaccinated LNs, from day 3 to 6 post-vaccination.

We observed a further increase in [¹⁸F]FLT accumulation (p < 0.05) in LNs that received three subsequent intranodal vaccinations, but not in control LNs. This indicates that the observed increase in [¹⁸F]FLT signal upon vaccination cannot be attributed to the effect of tissue damage by intranodal injection or to the presence of dendritic cells alone, but requires the presence of antigen to be recognized by lymphocytes.

Lastly, the level of [¹⁸F]FLT uptake in the LN was compared with the levels of antigen-specific T cells and B cell antibody responses to the highly immunogenic KLH in peripheral blood. We observed a significant correlation between [¹⁸F]FLT accumulation and the level of circulating KLH-specific IgG antibodies as well as KLH-specific proliferation of T cells, underlining our hypothesis that [¹⁸F]FLT imaging is a sensitive technique to follow the development of immune responses in vivo. Note that PET can be used to detect lymphocyte proliferation even when KLH is not used as a marker antigen.

In conclusion, we have demonstrated that [¹⁸F]FLT PET can be used to directly monitor antigen-specific immune responses in vivo shortly after vaccination. Here, we applied PET/CT imaging in a therapeutic anti-cancer vaccine setting to discriminate potentially responding from non-responding patients as a first step toward personalized medicine. In combination with other cell tracking techniques (Fig. 1), multimodal imaging offers a powerful technique for the comprehensive monitoring of therapeutic cells post-transfer to the patient.⁶

Figure 1. Strategy to monitor the functionality of transplanted therapeutic cells in vivo by using imaging in patients. In our study, dendritic cell (DC) vaccines were cultured from patient blood and loaded with tumor antigen ex vivo before transfer to the patient. A control antigen, KLH, was added as a readily identifiable marker. Imaging labels, such as iron oxide for MRI or radiolabels for scintigraphy can also be added at this stage. Once transferred to the patient, the DCs activate antigen-specific lymphocytes in lymph nodes, resulting in their activation and proliferation. We showed that this antigen-specific proliferation of lymphocytes can be detected using [¹⁸F]FLT PET in melanoma patients,⁵ and validated this using conventional blood tests on the control antigen KLH and histology on biopsy material. Thus, the functionality of the therapeutic cells can be monitored in vivo. We have shown previously that the localization and numbers of transferred cells can be monitored using MRI and scintigraphy.² In the figure, detected cells are encircled in green. This powerful multimodal imaging approach allows for comprehensive monitoring of therapeutic cells, and can readily be adapted to different cell types.⁶