Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy

Abstract

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rg -/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.

Adjuvant effects of a sequence-engineered mRNA vaccine: translational profiling demonstrates similar human and murine innate response

“…Because a mouse cannot be considered a “small human” the development of better methods for the study and analysis of human-based immune system models has been identified as an area of critical need in vaccinology. The need for human-based methods has begun to be filled with the continued development in vitro assays [7, 8] and humanized mice (HM) that harbor a human immune system [9, 10], both of which show promise but neither of which are currently widely used. Human-based in vitro models may not always replicate the entire immunomodulatory activity of an adjuvant or vaccine and are thus a logical complement to in vivo studies [1].…”

Immunotherapies and Metastatic Cancers: Understanding Utility and Predictivity of Human Immune Cell Engrafted Mice in Preclinical Drug Development

“…Furthermore, T cells educated in donor-matched thymus implants and liver implants provide important cytokines for immune cell maturation, which results in improved antigen presentation and immune responses in the model. The BLT-model can be extended by a secondary transfer of bone marrow and thymus implants from BLT mice to super-immunodeficient mice to create so-called propagated BLT mice [71]. In this model, the quantity of immune cells is comparable to the "original" BLT mice and less fetal samples are needed, making this method an easier and less material consuming approach in generating BLT mice.…”

Targeting monoamine oxidase A-regulated TAM polarization for cancer immunotherapy

“…The Retro/ESO-TCR vector was constructed by inserting into the parental pMSGV vector a synthetic gene encoding an HLA-A2-restricted, NY-ESO-1 tumour antigen-speci c human CD8 TCR (clone 3A1) 98 . Vsv-gpseudotyped Retro/ESO-TCR retroviruses were generated by transfecting HEK 293T cells following a standard calcium precipitation protocol and an ultracentrifugation concentration protocol 102 ; the viruses were then used to transduce PG13 cells to generate a stable retroviral packaging cell line producing GALV-pseudotyped Retro/ESO-TCR retroviruses (denoted as the PG13-ESO-TCR cell line). For virus production, the PG13-ESO-TCR cells were seeded at a density of 0.8 x 10 6 cells per ml in D10 medium, and cultured in a 15-cm dish (30 ml per dish) for 2 days; virus supernatants were then harvested and stored at -80 °C for future use.…”