The present study demonstrates that lentiviral vectors can be used to express cytokine genes in HIS mice following a single intravenous injection. This approach enabled hIL-7 to be expressed at consistent levels long-term, and improved human T lymphocyte numbers in the HIS model. Increases in T cell numbers occurred in all compartments analyzed, consistent with IL-7 functioning as a mediator of homeostatic proliferation. Additionally, the direct effects induced by IL-7 were entirely confined to the human graft, because the common γ chain required for IL-7R signaling is deficient in the host mouse strain.
Our approach resulted in hIL-7 serum concentrations higher than those found in healthy human adults, which ranges from 0.27 to 8.7 pg/ml (average 2.2 pg/ml), yet within the range of some HIV-positive lymphopenic individuals (which can be up to 70 pg/ml)
. Our experiments found that the impact of hIL-7 on human T cells was reduced in animals expressing lower concentrations of cytokine (10–20 pg/ml vs. 100 pg/ml hIL-7). This suggests that the HIS mouse model may require super-physiological levels of certain cytokines to induce meaningful changes in cellular compartments. The reasons for this are presently unclear, but it may reflect insufficient niche specific expression of hIL-7, which may be higher than systemic levels. Therefore, our expression platform might not be producing enough hIL-7 to reach such locations in sufficient quantities until we boost the overall systemic levels to super-physiological concentrations. Alternatively, other human cytokines not present in the HIS model might sensitize target cells to lower amounts of IL-7, but this also remains to be determined. It is also plausible that insufficient niche specific hIL-7 levels are the reason why a recent study did not observe differences in HIS mouse peripheral T cell levels following direct injection of recombinant hIL-7
. In fact, an earlier approach in which an Fc-IL-7 fusion protein was delivered via weekly injections led to increased T cell: B cell ratios in the spleens of humanized NOD-scid γc-/- mice
. This approach stabilized hIL-7 serum levels, resulting in a biological impact similar to that observed following constant hIL-7 production by our expression system in humanized Rag2-/-γc-/- mice.
We observed a direct correlation between vector dose and cytokine expression indicating that lentiviral based cytokine delivery can be easily adjusted to deliver a desired dose of cytokine. Transgene expression was also highest in the liver, bone marrow and spleen, consistent with an earlier study that used VSVG-pseudotyped lentivirus to deliver human factor IX to non-chimeric SCID mice
. This infection pattern exhibits significant overlap with the tissues and organs engrafted by the human immune cells such as spleen and bone marrow. Liver tissue was also efficiently targeted, mimicking one of the natural sites of IL-7 production in vivo. Given this overlap in infection and human cell engraftment patterns, it may also be possible to deliver molecules in addition to cytokines, such as transmembrane receptors, that require direct cell-to-cell contact to mediate their effects. Furthermore, because the technology to target lentiviral vectors to specific cell types in vivo is making substantial progress
, delivering molecules to specific tissues in vivo may soon be feasible in the HIS model.
Recently, clinical trials have shown that hIL-7 can increase T cell numbers in people, indicating a promising therapeutic role for this cytokine
. Two of these studies examined HIV infected patients undergoing HAART therapy, resulting in low viral loads in these individuals and making it hard to assess the impact IL-7 on HIV replication. Our HIS mouse experiments suggest that IL-7 can improve T cell levels even in the absence of antiretroviral drugs, and do so without boosting HIV titers. This finding highlights the utility of HIS mice as models for human-specific disease.
IL-7 has been shown to function as an adjuvant in mice
. However, despite increased T cell numbers and lymphoid follicle size in the spleen, hIL-7 expressing HIS mice had only limited immune function similar to controls. Ovalbumin immunization resulted in modest antigen specific IgM responses and virtually no IgG production in both control as well as hIL-7 expressing HIS mice (Figure S4a
). Additionally, JR-CSF infected hIL-7 or control mice did not develop any appreciable antibody responses to virus antigens as determined by western blotting (Figure S4b
), further supporting our assessment that IL-7 expression alone was insufficient to restore normal immune function. Despite its inability to improve antigen specific immune responses, hIL-7 is likely an important piece of the puzzle given that T cells play a central role in immune responses to antigens. Consistent with this, we observed increased total serum IgM levels in hIL-7 expressing mice, suggesting that improved T-cell survival by hIL-7 resulted in increased B-cell output. Beyond immune function, IL-7 did not improve the mouse to mouse, or donor to donor, variability observed in the HIS model, suggesting that additional cytokines known to act on HSCs are likely necessary to impact overall human cell engraftment. Importantly, the modular nature of our system will permit the delivery of multiple cytokines at one time, and therefore future studies will investigate the effect of various cytokine combinations with the goal of enhancing human immune cell development and function in the HIS model.