Allogeneic and xenogeneic lymphoid reconstitution in a RAG2 -/- IL2RG y /- severe combined immunodeficient pig: A preclinical model for intrauterine hematopoietic transplantation

Mice with severe combined immunodeficiency are commonly used as hosts of human cells. Size, longevity, and physiology, however, limit the extent to which immunodeficient mice can model human systems. To address these limitations, we generated immunodeficient pigs and demonstrate successful engraftment of SLA mismatched allogeneic D42 fetal liver cells, tagged with pH2B-eGFP, and human CD34 hematopoietic stem cells after cell transplantation. Following intrauterine injection at day 42-45 of gestation, fetuses were allowed to gestate to term and analyzed postnatally for the presence of pig (allogeneic) and human (xenogeneic) B cells, -cells and NK cells in peripheral blood and other lymphoid tissues. Engraftment of allogeneic hematopoietic cells was detected based on co-expression of pH2B-eGFP and various markers of differentiation. Analysis of spleen revealed robust generation and engraftment of pH2B-eGFP mature B cells (and IgH recombination) and mature -cells and β recombination), T helper (CD3 CD4 ) and T cytotoxic (CD3 CD8 ) cells. The thymus revealed engraftment of pH2B-eGFP double negative precursors (CD4 CD8 ) as well as double positive (CD4 , CD8 ) precursors and single positive -cells. After intrauterine administration of human CD34 hematopoietic stem cells, analysis of peripheral blood and lymphoid tissues revealed the presence of human -cells (CD3 CD4 and CD3 CD8 ) but no detectable B cells or NK cells. The frequency of human CD45 cells in the circulation decreased rapidly and were undetectable within 2 weeks of age. The frequency of human CD45 cells in the spleen also decreased rapidly, becoming undetectable at 3 weeks. In contrast, human CD45 CD3 -cells comprised >70% of cells in the pig thymus at birth and persisted at the same frequency at 3 weeks. Most human CD3 cells in the pig's thymus expressed CD4 or CD8, but few cells were double positive (CD4 CD8 ). In addition, human CD3 cells in the pig thymus contained human -cell excision circles (TREC), suggesting development. Our data shows that the pig thymus provides a microenvironment conducive to engraftment, survival and development of human -cells and provide evidence that the developing -cell compartment can be populated to a significant extent by human cells in large animals.