Abstract
Murine transplantation studies are the gold standard to assess functional potential of normal and malignant hematopoietic stem cells (HSCs). As an easily identifiable marker, the green fluorescent protein (GFP) has revolutionized these studies, proving to be an invaluable tool in identifying the cell type of interest via flow cytometry or microscopy, but it has its limitations. Work done by a group in Seattle (Grzelak, C. A. et. al., Cancer Cell, 2023) shows that GFP+ 4T1 tumor cells are rejected when injected into immunocompetent mice, preventing the generation of tumor and dissemination to metastasis. Here, we show the presence of high levels of enhanced GFP (eGFP) affects HSC self-renewal potentially by acting as a neoantigen, flagging these GFP-expressing cells for T cell-mediated clearance.
In numerous competitive transplantation studies where equal ratios of whole bone marrow (WBM) cells from wild-type (WT) mice (CD45.2) and WT mice expressing eGFP under the CAG promoter (CD45.1/2) were injected into lethally irradiated CD45.1 recipient mice, GFP+ HSC numbers were significantly decreased compared to WT. Based on these findings, we hypothesized two possible mechanisms that could lead to this phenotype - the first was that GFP acts as a neoantigen, flagging these GFP+ stem cells for removal by T cells. Alternately, we hypothesized that the presence of the foreign reporter protein was hindering stem cell engraftment and self-renewal. To test this hypothesis, we sorted 100 HSCs from either WT/WT:GFP+ CD45.1/2 mice and transplanted it with 0.3 million support bone marrow from a CD45.2 WT mouse or from a Rag1-/- mouse lacking mature T and B cells. While we observed low peripheral blood engraftment of CD45.1/2 GFP+ cells in the mice containing WT support, there was a significant increase in engraftment in the group containing Rag1-/- support, implying that the absence of mature T cells allowed these GFP+ cells to engraft and expand as normal. No differences were observed between the groups that received WT HSCs.
Our data suggests a crucial role for T cells in the elimination of GFP+ stem cells from the blood and bone marrow. While there were no overall differences in T cell numbers between the groups, we observed a significant increase in activated CD8a+ CD25+ T cells in the groups expressing GFP in both blood and splenocytes. Based on the engraftment data seen from the peripheral blood, we anticipated that the GFP+ HSCs in the Rag1-/- group would have a significant increase in numbers due to the absence of T cells. Interestingly, we observed no change in HSC numbers between the groups. This could imply that while the lack of T cells enables engraftment in the blood, the prolonged expression of GFP on the stem cells leads to impaired self-renewal through another mechanism possibly via disruption of proteostasis (Chua, B. A. et. al., Cell Stem Cell, 2023), which might explain the low numbers in the bone marrow at terminal takedown. This needs further experimentation and analysis.Overall, our data suggests that the presence of fluorescent reporter proteins like GFP, while useful in tracking cell populations and easy identification of target cells, affects stem cell self-renewal and engraftment. We show that these cells identify the foreign fluorescent protein and present them to T cells for rapid elimination, leading to reduced engraftment. While the use of fluorescent markers is nearly unavoidable in transplantation studies, this study demonstrates the need for caution while using these markers in studies focused around understanding stem cell self-renewal and fitness, and the use of alternate markers for identification is encouraged.
