Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Johannes L Zakrzewski1, Adam A Kochman1, Sydney X Lu1, Theis H Terwey1, Theo D Kim1, Vanessa M Hubbard1, Stephanie J Muriglan1, David Suh1, Odette M Smith1, Jeremy Grubin1, Neel Patel1, Andrew Chow1, Javier Cabrera-Perez1, Radhika Radhakrishnan1, Adi Diab1, Miguel-Angel Perales1, Gabrielle Rizzuto1, Ewa Menet1, Eric G Pamer1, Glen Heller2, Juan Carlos Zu´n˜iga-Pflu¨cker3, O¨ nder Alpdogan1,4 & Marcel R M van den Brink1,4

Summary:

Allogenic hematopoietic stem cell transplantations (HSCTs) are widely used as a last attempt to treat blood diseases such as lymphoma. Research has shown that the biggest problem after HSCT is poor reconstitution of the T-cell lineage in the host. The authors of the paper propose that if large numbers of T-cell precursors could be generated and transplanted in addition to the bone marrow/HSCs, T-cell reconstitution would be enhanced. In order to produce these T-cell precursors, the researchers co-cultured hematopoietic stem cells (HSCs) with OP9-DL1 mouse stromal cells, which express the ligand that commits HSCs to the T-cell lineage.

First, the researchers had to show that the cells derived from the co-culture were in fact T-cell precursors and that they were at the right stage of differentiation. It is important for the cells to be at the double-negative (CD4^- and CD8^-)stage before they acquire specificity or else the T-cells would start attacking the recipient, causing Graft vs. Host Disease (GVHD). In addition, of the 4 double-negative (DN) stages, only DN2 cells were able to successfully engraft into the recipient thymus.

After determining exactly what types of cells were to be used, the researchers showed that adding OP9-DL1 derived T-cell precursors actually improved the ability of T-cells to reconstitute after transplantation. They used FACS to determine types of cells and number of cells that were generated following the procedure (day 14 and day 28 after injection). They used purified HSCs from bone marrow, which is the current procedure for HSCTs, for control. Not only could these OP9-DL1 derived T-cell precursors engraft into the thymus, but they were also able to migrate to the bone marrow and differentiate into NK cells. Treating recipients with keratinocyte growth factor (KGF) before irradiation was also found to improve thymic reconstitution. The OP9-DL1 derived T-cell precursors were even able to enhance T-cell recovery in thymectomized mice, showing that these cells could also undergo extrathymic T-cell development.

Next, the researchers tested the OP9-DL1 derived T-cells to make sure they had the appropriate response to foreign antigens. They first did a flow cytometric analysis to make sure the TCR repertoire was still intact so that they have the ability to recognize a wide variety of antigens. Then, the researchers exposed allogenic HSCT recipients to antigens in order to see if the appropriate cytokines were activated in response. Lastly, L. monocytogenes was introduced twice in order to compare clearance times of the pathogen during the primary infection versus secondary infection. The time is supposed to decrease because T-cells function in adaptive immunity and accelerates the response time to similar pathogens.

Most importantly, the researchers showed that there is no GVHD associated with the procedure and in fact has Graft vs. Tumor (GVT) effects. This means that not only will the transplanted T-cells not attack the recipient cells, but they will actually attack tumor cells of the recipient (since tumor cells are not technically “self” cells) and delay tumor growth.

Conclusion:

The researchers clearly show that HSCT with OP9-DL1 derived T-cell precursors and purified HSCs is a better method than the current method of just using HSCs. However, a problem they found was that the OP9-DL1 derived T-cell precursors resulted in only a single generation of T-cells. After day 28, OP9-DL1 derived cells were barely detectable. This means that the effects of the transplant may not be long lasting and thus may require more than one HSCT. Also, for the last experiment that tested for GVT, instead of challenging mice with lymphoma after transplantation, they should test on mice that already have lymphoma to see if their method is curative, which is more relevant to clinical situations.