Adult Human Hematopoietic Cells Differentiate into Mature T Cells Via a CD3-4+8- Intermediate within the Mouse Thymic Microenvironment; a New Model System for the Study of Human Thymocyte Development.
Authors: Kraft Daniel L. , Czechowicz Agnieszka , Weissman Irving L. 
Published: April 2020

Normal human T lymphocyte differentiation occurs within the thymus. We previously determined that human thymocyte precursors develop via a novel CD3-4+8- intermediate population, utilizing SCID-hu thymic grafts (Kraft, Weissman &Waller, JEM). We have recently worked to develop a more convenient and robust model of human hematopoiesis utilizing RAG2/Common gamma chain double knock out mice (RAG2 DKO) transplanted with hematopoietic stem and progenitor cells from adult human donors.

Methods: Human peripheral blood derived CD34+ cells were obtained to &gt;90% purity by magnetic bead positive selection following apheresis from healthy GCSF mobilized donors. 200,000-800,000 human CD34+ cells were injected intrahepatically into 0–2 day old RAG2/CG DKO pups following 2Gy x 2 of irradiation. At serial time points following intrahepatic transplantation, human CD45+ chimerism and donor derived phenotype was measured within the bone marrow, peripheral blood, spleen, liver, lymph node and thymus by flow cytometry. Thymuses were further analyzed utilizing antibodies to human CD3, CD4 and CD8. Results: Robust human thymopoeisis was observed in CD34+ transplanted mice. The degree of human engraftment increased with the number of CD34+ cells transplanted. Overall &gt;70% of recipient mice successfully engrafted with &gt;10% human CD45+ expression in analyzed tissues, with a mean of 63% of cells within the thymus being human derived. At earlier time points (4–6 weeks post transplant) the thymus of recipient mice were found to contain high fractions of immature CD45+ CD3-4-8- cells (making up 31–43% of human cells within the thymus) and the CD3-4+8- intermediate (22–30%) and CD4+8+ double positive (34–71%) populations with very rare mature CD3+4+8- or CD3+4-8+ T-cells (figure A). At later time points the fraction of immature CD3-4-8- and CD3-4+8- populations declined and increasing fractions of mature CD3+4+8- and CD3+4-8+ populations were identified (Figure B), in distributions similar to those found in normal human thymus.

Conclusions: Human T-cells can differentiate within the mouse thymus derived from adult human CD34+ cells, and development appears to progress normally within a murine thymic microenvironment. The early developement of a CD3-4+8- intermediate suggests that T-cell development occurs via this population, unlike the CD3-4-8+ intermediate found in murine thymopoesis, suggesting that the pathway of human T-cell differentiation is intrinisic to the human thymocytes, and is independant of whether the thymic stroma is human (as found in SCID-hu) or murine (in Rag2 DKO). This robust model system enabling study of human thymopoesis utilizing hematopoietic stem cells from normal and diseased adults human donors may provide significant advantages for the study of human intrathymic T-cell differentiation and function in-vivo.

Human thymocyte profile in the RAG2 DKO thymus A) 4 and B) 8 weeks following intrahepatic transplantation of human CD34+ cells. Right panels: CD3 profile of CD4+8− population reveals a CD3−4+8− population Human thymocyte profile in the RAG2 DKO thymus A) 4 and B) 8 weeks following intrahepatic transplantation of human CD34+ cells. Right panels: CD3 profile of CD4+8− population reveals a CD3−4+8− population