An ES cell–derived immune system

In this issue of Blood, Chan and colleagues test the limits of the current technology for enabling hematopoietic reconstitution from embryonic stem (ES) cells.

Although the advantages of ES-derived cellular therapies are compelling, the difficulties of turning theory into practice are considerable. Chief among these for the hematopoietic system is that ES cells differentiated in vitro follow an early embryonic program of differentiation, generating hematopoietic stem cells (HSCs) akin to those of the precirculation yolk sac rather than the bone marrow. As the early embryo has no need of lymphocytes, and indeed has neither bone nor marrow, these so-called primitive HSCs are considerably different from the definitive (adult) variety. In particular, they lack the ability to differentiate into T lymphocytes efficiently, and also lack the ability for long-term engraftment in irradiated adult recipients.

Hematopoietic engraftment of these progenitors can be enabled by overexpression of the transcription factor HoxB4.¹  Whether this is due to a reprogramming of developmental potential, or is simply a consequence of enhanced self-renewal, is currently unknown. However, it is clear from numerous studies that myeloid cells predominate in the peripheral blood of these recipients, and lymphoid engraftment, although detectable, is minimal.^2-4  The question Chan and colleagues ask is: do these few lymphocytes form a true immune system, or are they merely impotent bystanders, expressing the appropriate markers but being too few in number or intrinsically unable to coordinate an immune response? In their experimental system, ES-derived, HoxB4-expressing HSCs marked with GFP to facilitate tracking were transplanted into Rag2^−/− γ_C^−/− mice, which lack functional lymphocytes. An immune reaction was then provoked by infection with lymphocytic choriomeningitis virus (LCMV) or vaccination with TNP-conjugated carrier antigens.

In contrast to previous studies, which have monitored lymphocytes in unimmunized mice, infection or immunization elicited a remarkable increase in donor-derived T cells in the peripheral blood. Furthermore, T cells purified from the spleens of LCMV-infected mice could be induced to express IFN-γ by short-term exposure to the LCMV-specific peptide, NP396-404, demonstrating their epitope-specific activation potential (see figure). The same spleens also contained ES-derived (GFP⁺) APCs, which, when purified and pulsed with the LCMV peptide antigen, were able to activate T cells from previously infected wild-type mice. It is important to note that the immune reactions elicited in mice that had received transplants, although clearly evident, were much weaker than the same reactions in wild-type mice (compare both the frequency and intensity of IFN-γ expression in ES-derived versus wild-type splenocytes in the figure). In addition to the T-cell responses provoked by LCMV, transplant-recipient mice were also able to generate TNP-specific antibodies after immunization with either TNP-KLH or TNP-LPS. Again, although clearly evident, these immune responses were attenuated compared with the responses seen in wild-type mice.

This work clearly demonstrates the potential ES cells exhibit for immune reconstitution. However, it also makes clear the limitations of using HoxB4 to enable engraftment, and therefore represents a benchmark against which novel methods should be measured.