Some mechanisms of lymphoid cell differentiation

Lymphoid cells differentiation is a well coordinated and highly integrated process, which is defined by the interaction of genes and cell signaling pathways of the maturing cell with the microenvironment factors. In the course of such interaction in the maturing cell, new ways of signal transduction appear, and on the basis of such new ways new gene networks are formed, which start to play rapidly, even during the course of the present or following cell specialization stage, the key role in the further cell fate.     

Apoptosis and neutrophils in the regulation of Ph-positive myeloid cell proliferation and differentiation ex vivo

Ex vivo proliferation and differentiation of Philadelphia chromosome-positive (Ph⁺) human myeloid cells (Ph+ cells) from chronic myeloid leukemia (CML) proceed via alternation stages of cell proliferation and neutrophil maturation. To regulate them, apoptosis is alternately blocked or induced with the help of neutrophils and expression of bcr/abl, bax, and bcl2. The regulation of apoptosis in main types of Ph⁺ cells depends on the alternation of (1) Ph⁺ cell proliferation and (2) neutrophil maturation and may follow two pathways. One consists in alternating blockages and inductions of apoptosis with initial maturation and subsequent proliferation under alternation stages as (2)-(1)-(2) and has not been described as yet. Neutrophil accumulation blocks apoptosis. As neutrophils are depleted, apoptosis is induced again. Its block accelerates proliferation with a new accumulation of neutrophils, which is followed by regular neutrophil death and a new induction of apoptosis. The way optimizes the proliferation efficiency (P/D index) with a regular alternation of maturation and proliferation, allowing the cycle of proliferation and differentiation to be completed. In another way, the alternation starts with proliferation as (1)-(2)-(1) at a lower neutrophil content) and leads to resistant decrease of the maximal apoptosis level by a factor of 3–8 as compared with (2)-(1)-(2) alternation. A stable block of apoptosis is observed in cells with prolonged stages of proliferation and maturation, leading to an accumulation of blasts and myelocytes with elevated bcr/abl expression and expression of bcl2 > bax. A stable block of apoptosis is associated with CML progression and in Ph⁺ cell lines. Cells follow the first pathway of the apoptotic regulation in chronic-phase CML. Ex vivo cultivation of Ph⁺ cells from individual CML patients was assumed to provide for a more exact diagnosis of the CML phase and optimizing the treatment.     

Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha.

The human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product was sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow lambda gt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5' and 3' untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon-inducible genes: 204 and interferon regulatory factor 2. In addition, a 12-base sequence matching the interferon-stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5' untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon alpha. The MNDA mRNA was not induced by interferon alpha in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell-specific response to interferon.     

Idiotypic regulation of B cell differentiation

We study the equilibrium properties of idiotypically interacting B cell clones in the case where only the differentiation of B cells is affected by idiotypic interactions. Furthermore, we assume that clones may recognize and be stimulated by self antigen in the same fashion as by antiantibodies. For idiotypically interacting pairs of non-autoreactive clones we observe three qualitatively different dynamical regimes. In the first regime, at small antibody production an antibody-free fixed point, the virgin state, is the only attractor of the system. For intermediate antibody production, a symmetric activated state replaces the virgin state as the only attractor of the system. For large antibody production, finally, the symmetric activated state gives way to two asymmetric activated states where one clone suppresses the other clone. If one or both clones in the pair are autoreactive there is no virgin state. However, we still observe the switch from an almost symmetric activated state to two asymmetric activated states. The two asymmetric activated states at high antibody production have profoundly different implications for a self antigen which is recognized by one of the clones of the pair. In the attractor characterized by high autoantibody concentration the self antigen is attacked vigorously by the immune system while in the opposite steady state the tiny amount of autoantibody hardly affects the self antigen. Accordingly, we call the first state the autoimmune state and the second the tolerant state. In the tolerant state the autoreactive clone is down-regulated by its anti-idiotype providing an efficient mechanism to prevent an autoimmune reaction. However, the antibody production required to achieve this anti-idiotypic control of autoantibodies is rather large.     

Epigenetic regulation of germ cell differentiation

Germ cells and somatic cells have the identical genome. However, unlike the mortal fate of somatic cells, germ cells have the unique ability to differentiate into gametes that retain totipotency and produce an entire organism upon fertilization. The processes by which germ cells differentiate into gametes, and those by which gametes become embryos, involve dramatic cellular differentiation accompanied by drastic changes in gene expression, which are tightly regulated by genetic circuitries as well as epigenetic mechanisms. Epigenetic regulation refers to heritable changes in gene expression that are not due to changes in primary DNA sequence. The past decade has witnessed an ever-increasing understanding of epigenetic regulation in many different cell types/tissues during embryonic development and adult homeostasis. In this review, we focus on recent discoveries of epigenetic regulation of germ cell differentiation in various metazoan model organisms, including worms, flies, and mammals.     

Autocrine regulation of mammary cell differentiation

Summary Milk secretion and mammary function in dairy animals are regulated by local mechanisms sensitive to the frequency or efficiency of milking. Acute local control of milk secretion occurs through autocrine feedback inhibition by a milk protein. Sustained changes in milking frequency and milk secretion are associated with longer-term adaptations in the degree of differentiation and, ultimately, the number of mammary epithelial cells. Differentiation of cultured mammary cells is suppressed by a milk fraction containing the inhibitor, suggesting that intra-mammary regulation of differentiation in vivo is elicited by the same autocrine regulator subsequent to its acute effect on milk secretion. The autocrine factor may affect mammary cell differentiation by modulating the number of cell surface hormone receptors for prolactin, thereby changing their sensitivity to circulating hormones.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia.

Multiple members of the A, B, and C clusters of Hox genes are expressed in hematopoietic cells. Several of these Hox genes have been found to display distinctive expression patterns, with genes located at the 3' side of the clusters being expressed at their highest levels in the most primitive subpopulation of human CD34+ bone marrow cells and genes located at the 5' end having a broader range of expression, with downregulation at later stages of hematopoietic differentiation. To explore if these patterns reflect different functional activities, we have retrovirally engineered the overexpression of a 5'-located gene, HOXA10, in murine bone marrow cells and demonstrate effects strikingly different from those induced by overexpression of a 3'-located gene, HOXB4. In contrast to HOXB4, which causes selective expansion of primitive hematopoietic cells without altering their differentiation, overexpression of HOXA10 profoundly perturbed myeloid and B-lymphoid differentiation. The bone marrow of mice reconstituted with HOXA10-transduced bone marrow cells contained in high frequency a unique progenitor cell with megakaryocytic colony-forming ability and was virtually devoid of unilineage macrophage and pre-B-lymphoid progenitor cells derived from the transduced cells. Moreover, and again in contrast to HOXB4, a significant proportion of HOXA10 mice developed a transplantable acute myeloid leukemia with a latency of 19 to 50 weeks. These results thus add to recognition of Hox genes as important regulators of hematopoiesis and provide important new evidence of Hox gene-specific functions that may correlate with their normal expression pattern.     

Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs

Naive T cell circulation is restricted to secondary lymphoid organs. Effector and memory T cells, in contrast, acquire the ability to migrate to nonlymphoid tissues. In this study we examined whether nonlymphoid tissues contribute to the differentiation of effector T cells to memory cells and the long-term maintenance of memory T cells. We found that CD4, but not CD8, effector T cell differentiation to memory cells is impaired in adoptive hosts that lack secondary lymphoid organs. In contrast, established CD4 and CD8 memory T cells underwent basal homeostatic proliferation in the liver, lungs, and bone marrow, were maintained long-term, and functioned in the absence of secondary lymphoid organs. CD8 memory T cells found in nonlymphoid tissues expressed both central and effector memory phenotypes, whereas CD4 memory T cells displayed predominantly an effector memory phenotype. These findings indicate that secondary lymphoid organs are not necessary for the maintenance and function of memory T cell populations, whereas the optimal differentiation of CD4 effectors to memory T cells is dependent on these organs. The ability of memory T cells to persist and respond to foreign Ag independently of secondary lymphoid tissues supports the existence of nonlymphoid memory T cell pools that provide essential immune surveillance in the periphery.     

Growth factor-dependent differentiation along the myeloid and lymphoid lineages in an immature acute T lymphocytic leukemia

Bone marrow cells from a child with an immature (CD2+, CD5+, CD7+) acute T lymphocytic leukemia (T-ALL) were cultured in the presence and absence of human rIL-2, IL-3, or granulocyte-macrophage (GM)-CSF. Cells cultured without growth factors failed to divide and those initiated in the presence of IL-2 or GM-CSF underwent maturation and terminal T lymphoid or myelomonocytic differentiation, respectively. In contrast, a permanent growth factor-dependent cell line, designated TALL-103/3, was established upon culture in IL-3. The TALL-103/3 cells gradually lost the T cell-specific markers and acquired a myeloid phenotype (CD15+, CD33+). Switching of the IL-3-dependent cells at an early passage to medium containing only human rIL-2 resulted in the establishment of a subline, named TALL-103/2, with a T lymphoid phenotype (CD3+, CD8+, TCR-gamma delta +, CD7+). The TALL-103/2 cells strictly require IL-2 for growth, are irreversibly committed to the lymphoid lineage, and cannot survive in the presence of any other hemopoietic growth factor tested so far. In contrast, the IL-3-dependent TALL-103/3 cells could be adapted to grow in synthetic (serum-free) medium also in the presence of either GM-CSF or IL-5, in which they retain a myeloid phenotype. Interestingly, after 18 mo in culture in IL-3, the TALL-103/3 cells can still be phenotypically converted to the lymphoid lineage upon addition of IL-2, thus maintaining its bipotentiality. Despite the marked phenotypic differences, the TALL-103/2 and TALL-103/3 cell lines show the same karyotypes with multiple abnormalities present in the primary malignant clone and have identical rearrangements of the TCR-gamma and -delta loci, thus confirming their derivation from a common precursor cell. Together, these findings indicate that the phenotype of immature T-ALL cells can be drastically modified by the presence of specific hemopoietic growth factors in the environment, leading to either lymphoid or myeloid lineage commitment while leaving their karyotype and genotype intact.     

Correlation of cell division and specific protein production during the course of lymphoid cell differentiation

Mice were immunized with horseradish peroxidase and injected with [³H] thymidine. Popliteal lymph node cells were submitted to electron microscopic immunocytochemistry and high resolution autoradiography in order to correlate antibody production and ability to undergo cell division at various stages of lymphoid cell differentiation. Antibody synthesis started in blast cells and increased steadily until the mature plasma cell stage was reached. Thymidine incorporation was highest in blastoid cells and decreased continuously afterwards. Chromatin dispersion was found to be paralleled by thymidine incorporation. This observation and data of other authors seem to indicate that chromatin dispersion is a prerequisite for replication. Almost all mature plasma cells were devoid of thymidine incorporation. This confirms that they are end cells apparently unable to divide.     

COUP-TFI controls Notch regulation of hair cell and support cell differentiation

The orphan nuclear receptor COUP-TFI (Nr2f1) regulates many aspects of mammalian development, but little is known about its role in cochlear hair cell and Deiter's support cell development. The COUP-TFI knockout (COUP-TFI(-/-)) has a significant increase in hair cell (HC) number in the mid-to-apical turns. The total number of hair cells is not increased over wild type, perhaps because of displaced hair cells and a shortened cochlear duct. This implicates a defect of convergent-extension in the COUP-TFI(-/-) duct. In addition, excess proliferation in the COUP-TFI(-/-) sensory epithelium indicates that the origin of the extra HCs in the apex is complex. Because loss-of-function studies of Notch signaling components have similar phenotypes, we investigated Notch regulation of hair cell differentiation in COUP-TFI(-/-) mice and confirmed misregulation of Notch signaling components, including Jag1, Hes5 and in a manner consistent with reduced Notch signaling, and correlated with increases in hair cell and support cell differentiation. The disruption of Notch signaling by a gamma-secretase inhibitor in an in vitro organ culture system of wild-type cochleae resulted in a reduction in expression of the Notch target gene Hes5 and an increase in hair cell differentiation. Importantly, inhibition of Notch activity resulted in a greater increase in hair cell differentiation in COUP-TFI(-/-) cochlear cultures than in wild-type cultures, suggesting a hypersensitivity to Notch inactivation in COUP-TFI(-/-) cochlea, particularly at the apical turn. Thus, we present evidence that reduced Notch signaling contributes to increases in hair cell and support cell differentiation in COUP-TFI(-/-) mice, and suggest that COUP-TFI is required for Notch regulation of hair cell and support cell differentiation.