New insights into the molecular and cellular requirements of lymphocyte development

The development of functional lymphocytes fending off pathogenic intruders yet tolerating self is dependent on the instructive or permissive action of soluble and/or membrane-bound factors. This is best exemplified by the loss, reduction, or functional impairment of some lymphocyte populations in mouse or man lacking designated cytokines, their receptors or important signal mediators.
The Early Progenitor with Lymphoid and Myeloid potential (or EPLM) has been identified in our laboratory several years ago. This cell population combines two different fate options, namely lymphoid and myeloid, which were long thought being mutually exclusive. However, now we were able to show that the total EPLM population can be further subdivided into subpopulations with differentiation biases. This finding follows the trend that multipotentiality of a given progenitor cell population is often based on heterogeneity amongst the population, rather than single cells having all the fate options still utilizable. EPLM co-express the receptors for Fms-like tyrosine kinase 3 ligand (Flt3L) and interleukin-7 (IL-7) and the Ly6D+ subpopulation of EPLM includes the direct progenitor of committed B-cell precursors. By generating an almost complete set of Flt3L and IL-7 deficient as well as transgenic mice and the respective combinations, we analyzed the involvement of Flt3L and IL-7 in the B-cell commitment process. Strikingly, excess Flt3L, which provides the system with tremendous numbers of functional precursors, rescued the B-cell defect in the absence of IL-7. Even if the anti-apoptotic factor Bcl2 was ectopically expressed, B-cell commitment as well as development were possible in IL-7-/-Bcl2tg mice. These results contradict previous reports describing IL-7 as a non-redundant cytokine. We provide evidence that IL-7 acts in a permissive way mediating survival of Ly6D+CD19- EPLM precursors and proliferation of committed B-cell progenitors. The commitment event itself appears to be independent of the action of IL-7.
Under physiological conditions, hematopoiesis occurs in the specialized microenvironment of the bone marrow. Now we show the successful circumvention of stromal cell dependency in in vitro culture systems for the long-term propagation of mouse progenitor lymphocytes. B-lymphocyte precursors grew in suspension cultures supplemented with Flt3L, IL-7, and stem cell factor (SCF). This result suggests that signals mediated by SCF and Flt3L substitute those delivered by stromal cells, since up to now pro-B cell propagation was only possible in the presence of OP9 stromal cells and IL-7. T-lymphocyte precursors depend on plate-bound Notch1 ligand Delta-like 4 as well as soluble IL-7 and SCF. Such feeder-free proliferation and also differentiation systems have several advantages. Firstly, they provide control over the dose, strength, as well as duration of cytokines and other ligand signaling for instance. Thereby the molecular requirements for lymphocyte development can be further addressed. Subsequently, one could study the molecular and epigenetic changes accompanying the development and differentiation of mouse lymphocytes in vitro. Contaminations originating from stromal cell nucleic acids or proteins can now be completely ruled out. Secondly, in these cultures the progenitor cells remain accessible to manipulations, such as overexpression or shRNA-mediated knock-down of gene transcripts encoding proteins of interest. Since these progenitor lymphocytes maintain in vivo reconstitution capacity and build a functional immune system upon injection into sublethally irradiated recombination deficient hosts, the effect of mutations can be immediately observed in vitro but also in vivo. Thirdly, an efficient in vitro system for the expansion of functional progenitor cells is only likely to become approved for potential therapeutic applications in humans if no co-culture settings have been applied. The next step would be to extend the applicability of this system to human progenitor lymphocytes. This would be an ideal tool to address the still poorly defined requirements for human hematopoiesis and one could potentially use these cells in a therapeutic setting.
The fourth project that is discussed within this thesis addresses the B-cell co-receptor molecule CD19. As a positive regulator of B-cell receptor (BCR) signaling divergent expression of CD19 affects B-cell development. In order to address this subject in further detail we employed the highly effective Nup98-HoxB4 (NH) hematopoietic stem cell (HSC) immortalization system developed by Ruedl et al. We ectopically expressed CD19 or a CD19 mRNA directed shRNA in these immortalized HSCs and reconstituted sublethally irradiated recipient mice. We observed diminished developmental progression from pre/proB cells to immature B cells due to transgene over-expression of CD19. Also reduction of CD19 expression resulted in reduced numbers of mature B cells with a developmental block at the immature to mature B cell transition. These results indicate that aberrant CD19 levels interfere with selection of B cells into the mature B-cell compartment. Tonic signals mediated by the BCR are indispensable for positive selection of B cells whereas a strong signal causes negative selection. CD19 acts as a positive regulator of BCR signaling. Therefore, high expression of CD19 might mimic a strong BCR signal, in this way mediating the developmental block. Low levels of CD19 might interfere with or dampen the tonic BCR signal required for positive selection of non-autoreactive immature B cells. Our results clearly indicate that a certain minimal threshold level of CD19 expression has to be reached but must not be widely exceeded, to mediate normal B-cell development. This highlights the important regulatory role of CD19 in fine-tuning the BCR signal response.
The projects described in this thesis address the molecular requirements for mouse lymphocyte development. Using several mutant and transgenic mice, we could clearly show that commitment to the B-cell lineage is possible without the action of IL-7. Moreover, we successfully developed stromal cell free in vitro culture systems to obtain large numbers of functional lymphocyte progenitors capable of in vivo reconstitution. A second in vitro culture system we successfully implemented is the Nup98-HoxB4 system. Multipotent HSCs can be expanded, manipulated, and the effect of a manipulation can be monitored upon in vivo reconstitution of a fully functional immune system. We found that the selection of mature B cells is critically dependent on the amount of CD19 expressed by B-cell progenitors.