T Cell Immunotherapy Enabled by Imaging-Guided Injections of Lymphoid Organs

Injection of lymphoid organs such as the thymus in mice is a valuable tool at the disposal of developmental biologists and immunologists. In this study we performed for the first time freehand percutaneous ultrasound-guided intrathymic injections in a variety of mouse models to develop novel strategies promoting T cell immunity.

We found that in vitrogenerated T cell precursors can efficiently be delivered via injection into the thymus of a sublethally irradiated allogeneic recipient 3 h after irradiation, and this procedure significantly improved T cell reconstitution. As expected, cell injection into the thymus of non-irradiated syngeneic recipients resulted in low levels of thymic reconstitution, indicating that in the absence of rejection the number of available niches for precursor cells is still a major limiting factor. We next assessed if it was feasible to inject the thymus of mice with significantly decreased thymic volume secondary to either age-related thymic involution or radiation-induced injury (injection 24 h after irradiation). Even though challenging, we were able to perform accurate injections of the target on both settings, highlighting the potential of this approach for the development of protocols to treat aging populations, as well as for radiation countermeasure development.

We next studied the fate of intrathymically injected bone marrow-derived progenitor cell populations including less committed Lin-Sca-1+c-kit+ (LSK) cells and lymphoid-primed multipotent progenitor cells. We found that especially LSK cells had excellent thymus-repopulating capacity and their progeny almost exclusively committed to the T cell lineage. Intrathymic injection of LSK cells into recipients of a T-cell depleted BMT resulted in enhanced T cell reconstitution on day +28.

We finally evaluated the efficacy of 3 known thymopoietic factors (interleukin 7 (IL-7), interleukin 22, keratinocyte growth factor) when injected into the thymus before or after irradiation. Only injection of IL-7, 3 h after irradiation resulted in increased thymic cellularity on day +14. However, a single injection of each of the 3 agents 24 h prior to irradiation resulted in a 2 to 4-fold increase in thymus size by day +28.

In addition to the thymus, lymph nodes provide an even easier, already clinically validated target. Importantly, lymph nodes have been found to support extrathymic T cell development during periods of lymphopenia. When T lineage committed precursor cells were injected into single axillary lymph nodes of irradiated nude mice, stable engraftment was found and 4 weeks after injection, the majority of recovered donor-derived cells were CD8+ T cells with a central memory phenotype.

Our findings indicate that imaging-guided freehand injection of lymphoid organs is feasible, safe, accurate, time saving, and has the potential to open up exciting new avenues for cell, drug, and gene therapy.