Background Although humanized NSG mice engrafted with hematopoietic stem cells (HSCs), constituting a human immune system, are a robust in vivo platform for analyzing effectiveness of novel immune modulators in cancer, inter and intra-experimental variability in immune subsets and anti-tumor response, resulting from utilization of diverse HSC donors, adds complexity to the data, making analysis challenging. To reduce variability, we acquired a significant amount of HSCs from the same donor and humanized six different NSG strains. Frequency and diversity of human immune subsets were characterized across non-tumor bearing humanized mice strains, and treatment effects of Rituximab on tumor burden and immune profiles in Raji-tumor bearing humanized mice were investigated.
Methods The six different NSG strains (NSG, NSG-IL15, SGM3, SGM3-IL15, NSG-TLR4, and NSG-FLT3) were engrafted with 0084 HSCs. Immune subsets in the periphery were assessed by high-dimensional cytometry. Furthermore, the humanized mice in NSG, NSG-IL15, SGM3 and SGM-IL15 strains were implanted with Raji cells subcutaneously and treated with Rituximab two or three times a week for 3 weeks, whole blood and tumors were collected, and the human immune subsets were profiled by flow cytometry.
Results Although the levels of hCD45 engraftment were comparable across the six different humanized strains, CD3+ T cells were more enriched in SGM3, SGM3-IL15 and NSG-FLT3, while higher frequency of CD56+ NK cell was observed in NSG-IL15, SGM3-IL15 and NSG-FLT3. Furthermore, the human FLT3 transgene expression improved myeloid development in the periphery (NSG-FLT3L ~11.7% vs. NSG ~1%). Interestingly, administration of Rituximab resulted in significant Raji tumor growth inhibition in NSG-IL15, SGM3 and SGM3-IL15 mice, but not in the NSG strain. While CD19+ B cells diminished in all humanized strains, trends in CD3 increases in tumor microenvironment were only observed in strains which demonstrated tumor growth inhibition.
Conclusions Humanization of NSG strains engrafted with HSCs from the same donor not only provided an effective platform to assess the influence of different human transgenes on degree and diversity of human immune subsets, but also allowed for investigating Rituximab response in multiple tumor bearing strains with no donor variability. Interestingly, robust anti-tumor effect upon rituximab treatment was observed in NK rich NSG-IL15 and SGM3-IL15 mice suggesting a possible NK driven antibody-dependent cell-mediated cytotoxicity mechanism contributing to tumor growth inhibition. Additionally, antitumor response also seemed to track with an increase in CD3+ T cell however the mechanism of action of Rituximab mediated by T cells requires further investigations.
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