Toll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression also extended to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen-mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.

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Viable Lin(-) CD27(+) c-kit(Hi) Sca-1(Hi) GFP(+) cells recovered from heterozygous RAG1/GFP knockin mice progressed through previously defined stages of B, T, and NK cell lineage differentiation. In contrast to the GFP(-) cohort, there was minimal myeloid or erythroid potential in cells with an active RAG1 locus. Partial overlap with TdT(+) cells suggested that distinctive early lymphocyte characteristics are not synchronously acquired. Rearrangement of Ig genes initiates before typical lymphoid lineage patterns of gene expression are established, and activation of the RAG1 locus transiently occurs in a large fraction of cells destined to become NK cells. These early lymphocyte progenitors (ELP) are distinct from stem cells, previously described prolymphocytes, or progenitors corresponding to other blood cell lineages.

Hematopoietic stem cells (HSC) can be harmed by disease, chemotherapy, radiation and normal aging. We now show that damage also occurs in mice repeatedly treated with very low doses of lipopolysaccharide (LPS). Overall health of the animals was good, and there were relatively minor changes in marrow hematopoietic progenitors. However, HSC were unable to maintain quiescence, and transplantation revealed them to be myeloid skewed. Moreover, HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified monoclonal antibodies that resolve HSC subsets, and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example, minor CD150Hi CD48− populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150Lo/− CD48− HSC and gain of the normally rare subsets, in parallel with diminished transplantation potential would be consistent with age or Tolllike receptor (TLR) related injury. On the other hand, HSC in old mice differed from those in LPS treated animals with respect to VCAM-1 or CD41 expression, and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity.

SummaryA cell adhesion model was previously used to select a series of monoclonal antibodies (mAbs), which were subsequently found to recognize CD44/Pgp-1 . Interest in these reagents increased with the finding that they totally inhibited production of lymphoid or myeloid cells in long-term bone marrow cultures. Further investigation has now revealed that hyaluronate is a potential ligand for CD44 and that hyaluronate recognition accounts for the adhesion between B lineage hybridoma and stromal cells . The hybridoma cells adhered to hyaluronate-coated plastic wells as well as to monolayers of stromal cells. The adhesion in both cases was inhibited by treatment with hyaluronidases, and did not require divalent cations . Addition of exogenous hyaluronate also diminished binding of lymphoid cells to stromal cells. One of several mAbs to Pgp-1/CD44 was particularly effective at blocking these interactions. Since hyaluronate and Pgp-1/CD44 were present on both cell types, experiments were done to determine the cellular location ofinteracting molecules required for the adhesion process . Treatment oflymphoid cells with an anti-Pgp-1/CD44 antibody was more inhibitory than antibody treatment ofthe stromal cells. Conversely, hyaluronidase treatment of stromal cells reduced subsequent binding more than treatment of the lymphoid cells . Adhesive interactions that involve hyaluronate and CD44 could contribute to a number of cell recognition processes, including ones required for normal lympho-hemopoiesis .

SummaryAdhesion molecules are probably required for retention of maturing lymphocyte precursors in bone marrow, where they closely interact with and are dependent on stromal cells . Lymphomyeloid cell lines avidly adhere to cloned stromal cell lines in culture and screening pairs of these resulted in a selection strategy for a new monoclonal antibody to a leukocyte adhesion molecule. Immunoprecipitation analyses and comparison to a previously described antibody showed that it recognizes the a4 chain of the integrin, VLA 4. This antibody totally inhibited lymphopoiesis and retarded myelopoiesis in long-term bone marrow cultures. A similar selection strategy resulted in two additional antibodies which define a single 100-kD species on stromal cells. This stromal cell adhesion molecule is a potential counter-receptor/ligand for VLA4 on murine Lmpho-myeloid cells . Our findings suggest a new role for VLA-4 in lymphoid progenitor-microenvironment interactions. Recognition molecules that function in cell migration and inflammation in peripheral tissues may be important for steady-state lymphopoiesis within bone marrow.

A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.

The subdivision of bone marrow (BM) with surface markers and reporter systems and the use of multiple culture and transplantation assays to assess differentiation potential have led to extraordinary progress in defining stages of B lymphopoiesis between the hematopoietic stem cell and B cell receptor (BCR)-expressing lymphocytes. Despite the lack of standard nomenclature and a series of technical issues that still need to be resolved, there seems to be a general consensus regarding the major route to becoming a B cell. Nevertheless, evidence that additional, minor pathways through which B lineage cells are generated exists, and a new appreciation that lymphoid progenitors are protean and able to alter their differentiation potential during embryogenesis and after birth in response to infections suggests that a full understanding of B cell development and how it is regulated has not yet been attained.