Precursors In Adult Bone Marrow Research Articles

Abortive γδTCR rearrangements suggest ILC2s are derived from T-cell precursors

AbstractInnate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.

Murine Bone Marrow Lin−Sca-1+CD45− Very Small Embryonic-Like (VSEL) Cells Are Heterogeneous Population Lacking Oct-4A Expression

Murine very small embryonic-like (VSEL) cells, defined by the Lin−Sca-1+CD45− phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin−Sca+CD45− population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin−Sca-1+CD45− population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit+KDR−, c-Kit−KDR+, and c-Kit−KDR− subpopulations could be distinguished, while c-Kit+KDR+ events were very rare. The c-Kit+KDR− subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit−KDR+ cells. The c-Kit−KDR−FSClow subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin−Sca-1+CD45−FSClow population, even by single-cell qRT-PCR. We also found that the Lin−Sca-1+CD45−c-Kit+ subset did not exhibit hematopoietic potential in a single cell-derived colony in vitro assay, although it comprised the Sca-1+c-Kit+Lin− (SKL) CD34−CD45−CD105+ cells, expressing particular HSC markers. Co-culture of Lin−Sca-1+CD45−FSClow with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45−CD105+ subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin−Sca-1+CD45−FSClow cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin−Sca-1+CD45−c-Kit+KDR− subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.

Distinct requirements for IL-7 in development of TCR gamma delta cells during fetal and adult life.

TCRgammadelta-transgenic IL-7(-/-) mice were generated to determine whether T cells containing productively rearranged TCRgammadelta genes have additional requirements for IL-7 within the thymus or peripheral lymphoid tissues. Differences in developmental requirements for IL-7 by TCRgammadelta cells were noted and were linked to derivation from fetal- vs adult-type precursors in the thymus. Although TCRgammadelta cells are absent from IL-7(-/-) mice, TCRgammadelta cells were restored to the thymus and periphery by expression of TCRgammadelta transgenes. Endogenous TCRgamma chains were expressed by IL-7(+/-) but not IL-7(-/-) TCRgammadelta-transgenic mice, providing direct support for findings that IL-7 is necessary for rearrangement and expression of TCRgamma genes. The number of TCRgammadelta thymocytes was 10-fold reduced in TCRgammadelta-transgenic IL-7(-/-) embryos; however, adult TCRgammadelta-transgenic IL-7(-/-) or IL-7(+/-) mice had similar numbers of fetal thymus-derived TCRgammadelta cells in their skin. Thus, fetal TCRgammadelta cells required IL-7 for TCR rearrangement, but not for proliferation or survival in the periphery. In contrast, the numbers of TCRgammadelta cells in other tissues of TCRgammadelta-transgenic IL-7(-/-) mice were not completely restored. Moreover, coincident with the transition from the first to second wave of T cell precursors maturing in neonatal thymus, thymus cellularity of TCRgammadelta-transgenic IL-7(-/-) mice dropped significantly. These data indicated that in addition to TCRVgamma gene rearrangement, TCRgammadelta cells differentiating from late fetal liver or adult bone marrow precursors have additional requirements for IL-7. BrdU incorporation studies indicated that although IL-7 was not required for TCRgammadelta cell proliferation, it was required to prolong the life span of mature TCRgammadelta cells.

Phenotype of B cells responding to the thymus-independent type-2 antigen polyvinyl pyrrolidinone.

We have determined the origin and cell surface phenotype of B cells producing antibody in response to immunization with the non-self TI-2 antigen polyvinyl pyrrolidinone (PVP). We report that the responding cells are derived from precursors in adult bone marrow and display the phenotype characteristic of B-1 cells. By use of allotype marked chimeric mice, constructed by reconstituting lethally irradiated recipients with adult bone marrow and peritoneal B-1 lymphocytes of recognizably different Ig allotypes, immunized with 1 microgram PVP, we found that although a substantial part of the total IgM produced in these chimeras bore the allotype of the transferred peritoneal B-1 cells, essentially all of the anti-PVP IgM expressed the allotype of the adult bone marrow. Fifteen of 16 hybridomas derived from a normal PVP-immune adult mouse bore N nucleotides at the V-D and D-J junctions of their heavy chain CDR3 regions, indicating their origin from precursors in the adult bone marrow. By use of ELISA spot analysis, we found the cells responding to PVP to be localized in the spleens of normal immunized mice. We then used multiparameter flow cytometric sorting to determine the cell surface phenotype of these cells. We found that the cells producing anti-PVP were greatly enriched in a small subpopulation with the phenotypic characteristics of B-1 cells; they were B220intermediate, CD5low, IgMhigh, IgDlow, CD43+ and CD23-. This subpopulation was also enriched for all cells producing IgM, regardless of specificity (the so-called 'spontaneous' antibody). We conclude that the B-1 phenotype is more likely a marker for a state of differentiation than for a discrete lineage of B cells.

Regulation of VH-gene expression is a lineage-specific developmental marker.

We have previously shown that in IgH congenic mice VH-gene family usage in neonatal spleen B cells and adult Ig-secreting cells is entirely determined by the IgH locus, while in adult resting B cells it is regulated by genetic element(s) located outside the IgH locus. Two observations reported here demonstrate that the differential expression of VH genes is an intrinsic property of the respective cell populations, determined by both the IgH locus and by a cis element(s) operating independently in the same animal. First, the study of F1 hybrids between the IgH congenic B6a and CB.20 strains demonstrates that cis elements control VH-gene family expression. Second, studies in irradiation chimeras showed that the environment in which cell differentiation proceeds is unable to overcome those controls. In chimeras of IgH congenic donors, VH-gene expression in fetal liver-derived splenic B cells and Ig-secreting cells is dictated by the IgH haplotype, while in bone marrow-derived B cells is entirely determined by the cis element(s). These results show a developmental and cell lineage-related restriction in VH-gene expression, and suggest that most adult splenic Ig-secreting cells may originate from precursors originally present in fetal liver, but which are rare among adult bone marrow precursors and CD5+ B cells.

The level of N-region diversity in T cell receptors is not pre-ordained in the stem cell.

The alpha beta T cell repertoires of adults and neonates are distinctly different. For example, T cell receptors (TcR) from adult animals have substantial N-nucleotide addition at their V-D-J junctions while those from neonatal animals do not. This dichotomy reflects a rather abrupt change in expression of the terminal deoxynucleotidyl transferase (TdT) gene in thymocytes on day 4 after birth. We have asked whether this change is due to the differentiation of successive waves of stem cells harboring different potentials for TdT expression, a scenario like the one proposed to explain developmental regulation of gamma delta T cell repertoires. Reconstitution of adult severe combined immunodeficiency mice with either fetal liver or adult bone marrow precursors gave rise to T cells with substantial N-region diversity in their TcR, even at the earliest points of reconstitution. It is most likely, then, that the abrupt change in TdT gene expression in day 4 thymocytes is due to an environmentally induced switch-on.

Origin of CD5+ B cells and natural IgM-secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells.

The bulk of natural IgM secretion is currently attributed to peritoneal CD5+ B cells and their progeny, believed to be independent of adult bone marrow precursors. We have compared the capacity of peritoneal or splenic cells from normal adult mice to generate serum IgM after transfer into allotype-congenic, irradiated and bone marrow-protected mice. Recipients of either cell population produced donor-allotype IgM-secreting cells in the spleen, and had donor-derived serum IgM. In both cases as well, recipient IgM secretion recovered to control levels. Since the spleen cell-derived natural IgM production could result from expansion of CD5+ B cells present in the inoculum, we next investigated the ability of Ig- bone marrow (BM) cells (Ig- BM) to reconstitute natural IgM secretion in irradiated mice. This cell population was most efficient in reconstituting donor-derived IgM secretion. The origin and phenotype (IgM, CD5) of B cells present in spleen and peritoneum of recipient mice were also analyzed. In agreement with the high level of donor IgM-secreting cells, transfers of splenic and Ig- BM cells fully reconstitute donor B cells in spleen and peritoneum and inhibit reconstitution from host origin. In contrast, donor peritoneal cells reconstitute B cells very poorly in spleen and allow for reconstitution by host cells. Furthermore, Ig- BM cells as well as splenic or peritoneal donor cells, all reconstitute CD5+ B cells in the peritoneum of recipient mice. Interestingly, the fraction of IgM+ cells of each allotype that differentiate to IgM secretion varies widely, but normal levels of IgM are established even when the number of donor B cells present in the animal is very limited.

Surface markers, heavy chain sequences and B cell lineages.

A unifying theory of B cell development and lineage commitment is presented. There are two firmly established B lineages: cells which normally arise only from fetal sources and lack N insertions in their rearranged heavy chains; and N-containing cells which arise from adult bone marrow precursors (and perhaps from late fetal sources). Commitment to the expression of CD5 and the capacity for long-life (or self-renewal) are induced as a consequence of sIg cross-linking, typically by a repeating epitope, thymus independent type two antigen. Alternatively, activation resulting from cognate interaction with a helper T cell does not induce CD5 but results in lower expression of J11d. In this case activation occurs in the absence of sIg cross-linking. It is further proposed that differences in the Ig repertoire make it highly likely that fetal/neonatal, but not adult derived B cells will be induced to express CD5. The model offers a plausible explanation for the correlation of CD5 expression and natural autoantibody production by neonatal B cells. Possible sources of pathogenic autoantibody are discussed in the context of this model.

Peritoneal B cells regulate the numbers of allotype-matched pre-B and B cells in bone marrow.

The mechanisms regulating growth and differentiation of B-cell precursors in adult bone marrow (BM) and/or selecting immunocompetent cells for peripheral export are poorly understood. We report here that small numbers of activated peritoneal B cells selectively suppress the numbers of small pre-B (B220+IgM-) and B (B220+IgM+) cells in BM, if transferred into syngeneic adult mice. No significant alterations are detected in other BM cell lineages or in peripheral lymphocytes of recipient mice. Both CD5+ and CD5- peritoneal B cells display this activity, but the same or higher numbers of similarly activated splenic B cells have no effect. Suppression of B-lineage cells is independent of T lymphocytes but requires that both donor and recipient are matched for immunoglobulin allotypes. These findings provide evidence for regulation of BM B-cell production by peripheral B cells, especially when located in the peritoneal cavity, and ascribe regulatory roles to the peritoneal B-cell compartment. They also could contribute to understanding the control of total B-lymphocyte numbers in the organism.