Influence of surrogate L chain on DHJH-reading frame 2 suppression in mouse precursor B cells

DHJH rearrangements start in progenitor and precursor B cells and occur in three reading frames (rf). A strong bias for rf I has been noticed in murlne and chicken antibodies, while the representation of rf II has been found suppressed both in peripheral as well as in precursor B cells. H chain gene loci DHJH rearranged in rf II are potentially capable of expressing a truncated DnJHC, protein on the cell surface. Mice incapable of expressing this protein on the surface have previously been shown to have all reading frames represented in near equal frequency, suggesting that membrane-bound DHJHCμ protein is involved in the suppression of rf II. In this paper we show that suppression of rf II Is not yet established in c-kit+ CD43+ IL-7/stromal cell-reactive pre-B I cells of fetal liver at day 15 of gestation, but becomes established when such precursor cell populations are expanded in vitro on stromal cells in the presence of IL-7. H chain gene loci using the DQ52 segment for rearrangements (which contains a stop codon in rf II, thus being unable to make DHJHCμ protein) do not show rf II suppression under these conditions. The same type of fetal liver-derived pro B-l cells from λ5 deficient mice also do not show rf II suppression after in vitro expansion. Bone marrow-derived pre B-I cells from normal mice assayed ex vivo and expanded in vivo show rf II suppression, while the corresponding pre-B I cells from λ5T mice do not. Collectively these experiments suggest that surrogate L chain Is involved in rf II suppression. This may happen by inhibition of proliferation of pre-B cells expressing a complex of DHJHCμ protein and surrogate L chai

Induction of sterile transcription from the kL chain gene locus in V(D)J recombinasedeficient progenitor B cells

B cell development in RAG-2-deficient (RAG-2T) mice is impeded at an early stage, due to the inability of these animals to rearrange their endogenous lg gene loci. Expression of an Eμ-bci-2 transgene in these mice did not change this phenotype. However, stromal cell/IL-7-reactive B cell progenitors (pro-B cells) were found in fetal liver and bone marrow of RAG-2T and RAG-2T/Eμ-bci-2 transgenic mice in numbers comparable to normal mice. Like cells from normal mice they are c-kit+, surrogate L chain+ and CD25−, and can proliferate in vitro for long periods of time. Upon IL-7 deprivation, they can be induced to differentiate into c-kit−, surrogate L chain+ and CD25− cells that are no longer clonable on stromal cells and IL-7. Furthermore, sterile transcription from the kL.chain gene loci is induced. The latter was also observed with pro-B cells directly isolated ex vivo from the bone marrow of RAG-2-deficient animals. The results suggest that progenitor B celldifferentiation can occur in cells from V(D)J recombinase-deficient mice to the stage where KL chain gene rearrangements would normally be initiated. It further indicates that some molecular programs of early B cell differentiation can take place in the absence of lg gene rearrangement

The status of Ig loci rearrangements in single cells from different stages of B cell development

Differential expression of c-kit, CD25 (TAC), surrogate L chain and cytoplasmic μH chain, and surface expression of IgM and IgD allows the separation of B220 (CD45+)B cell subpopulations. PCR analyses with DNA of single cells developed by others and by us have been used to monitor the conformation of the Ig H and L chain gene loci in these different B lineage subpopulations. The results of these analyses indicate that B220+/c-kit+/CD25− cells are the precursors of large B220+/CD25+/slgM− which, in turn, are the precursors of small B220+/CD25+/slgM− cells. The majority of B220+/c-kit+/CD25− cells are DHJH-rearranged, with L chain loci in germline configuration and are thus pre-B I cells. More than 90% of all large B220+/CD25+/slgM− cells have at least one H chain locus VHDHJH rearranged; half of them have also the second locus VHDHJH rearranged and are thus large pre-B II cells. Rearrangements of at least one allele of the kL chain loci become detectable in 65% of the small B220+/CD25+/slgM− cells, 67% of the immature B and >75% of the mature B cells. The ratio of kL to λL gene rearrangements in all three subpopulations is ˜10:1, indicating that the kL/λL ratio is established as soon as rearrangements are mad

Rearrangement and expression of χ light chain genes can occur without μ heavy chain expression during differentiation of pre-B cells

The kinetics of χ light (χL) chain gene rearrangement and expression on mRNA and protein level has been studied with four stromal cell/IL-7 reactive, long-term in vitro proliferating pre-B cell lines and clones, two from fetal liver of normal mice and two from fetal liver of EμH-bcl-2 transgenic (bcl-2-tg) mice. These pre-B cell lines and clones are DJH-rearranged on both H chain alleles. Two of the clones harbor H chain rearrangements which do not allow the expression of VHDJH rearranged H chain genes as μH chain proteins. Upon removal of IL-7 from the pre-B cell cultures all four cell lines rearrange VH-DJH and VL-JL gene segments, loose the surface expression of c-kit, CD43, and surrogate light chain, as well as the capacity to be clonable on stromal cells in the presence of IL-7. Pre-B cells from normal mice die by apoptosis during differentiation, while those from bcl-2-tg mice do not. All four lines and clones express comparable levels of mRNA for μH and μL chains with the same time kinetics during 3 days of differentiation. However, only two of the four pre-B cell lines and clones express μH chain protein, whereas all four pre-B cell lines and clones express μL chain protein at comparable levels between 2×105 and 1.40×106 μL chain molecules per cell. These results suggest that μH chain expression is not mandatory for rearrangement and normal expression of μL chain genes when pre-B cells differentiate to B cell

IL-2 receptor α chain (CD25JAC) expression defines a crucial stage in pre-B cell development

The analysis of the expression of the a chain of the IL-2 receptor (CD25.TAC) on the surface of B lineage cells In mouse bone marrow reveals that it is a useful marker to distinguish pre-B-I from pre-B-II cells. CD25 Is not expressed on CD45R(B220)+ c-kit+ CD43+ TdT+ λ5+ Cμ− slg− lgH chain locus DJH-rearranged pre-B-I cells of mouse bone marrow. It is expressed on large cycling CD45R(B220)+ c-kit+ CD43+ TdT+ λ5+ Cμ− sig− and on small resting CD45R(B220)+ c-kit+ CD43− TdT+ λ5+ Cμ− sig− sig- IgH chain locus VHDJH-rearranged pre-B-II cells. Therefore, the transition from pre-B-I to large pre-B-II cells is marked by the downregulation of c-kit and terminal deoxynucleotldyl transferase (TdT), and by the upregulatton of CD25. SCID, RAG-2T, μMT and γ6T mutant mice do have normal, If not elevated numbers of pre-B-I cells but lack all CD25+ pre-B-II cells in their bone marrow. The expression of a transgenic H chain under control of the μH chain enhancer in RAG-2T bone marrow B lineage precursors allows the development of large and small CD25+ pre-B-II cells. The results suggest that the differentiation of pre-B-I to pre-B-II cells in mouse bone marrow requires the expression of μH chains and surrogate L chains in membranes, probably on the surface of precursor B cell

Frequencies of Multiple IgL Chain Gene Rearrangements in Single Normal or κL Chain–Deficient B Lineage Cells

AbstractPCR analyses of the κL chain locus in single B-lineage cells of wild-type, Cκ-, or JCκ-deficient homozygous or heterozygous mice often detect multiple in- and out-of-frame rearrangements at the κL and λL loci. They are most frequent in small pre-BII cells and equally so in wild-type and κL chain–deficient cells. Hence, κL chain production appears not to inhibit secondary rearrangements. Around 20% of all small preBII cells express IgL chains in their cytoplasm. Cells with a first productive rearrangement on one allele are favored to enter the immature B cell compartment. Thus, allelic exclusion might be secured by control of accessibility of IgL chain loci for rearrangement and by rapid selection of cells with a fitting over those with a nonfitting IgL chain

The Cytokine Flt3-Ligand in Normal and Malignant Hematopoiesis

The cytokine Fms-like tyrosine kinase 3 ligand (FL) is an important regulator of hematopoiesis. Its receptor, Flt3, is expressed on myeloid, lymphoid and dendritic cell progenitors and is considered an important growth and differentiation factor for several hematopoietic lineages. Activating mutations of Flt3 are frequently found in acute myeloid leukemia (AML) patients and associated with a poor clinical prognosis. In the present review we provide an overview of our current knowledge on the role of FL in the generation of blood cell lineages. We examine recent studies on Flt3 expression by hematopoietic stem cells and its potential instructive action at early stages of hematopoiesis. In addition, we review current findings on the role of mutated FLT3 in leukemia and the development of FLT3 inhibitors for therapeutic use to treat AML. The importance of mouse models in elucidating the role of Flt3-ligand in normal and malignant hematopoiesis is discussed

Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, V(preB), recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for D(H)J(H) and V(K)J(K) V(K)J(K)K((de)) and V(K)K((de)) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+V(preB)+RAG-1+TdT+, D(H)J(H)-rearranged, KL germline cycling pre-B I cells → CD34- V(preB)+μH chain+ (pre-B receptor+) RAG- 1+ TdT, V(H)D(H)J(H)-rearranged, KL germline, cycling pre-B II cells → CD34- V(preB)-, intracytoplasmic μH chain+ (pre-B receptor) RAG-1+ TdT, V(H)D(H)J(H)-rearranged, mainly KL germline cycling pre-B II cells → CD34+ V(preB)- intracytoplasmic μH chain+, RAG-1+ TdT, V(H)D(H)J(H)-rearranged, V(K)J(K)-rearranged, IgM-, resting pre-B II cells → CD34+ V(preB)-, sIgM+, RAG-1+ TdT-, V(H)D(H)J(H)- and V(K)J(K)-rearranged IgM+ immature B cells → CD34+, CD10- sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.We thank Drs. Rod Ceredig and Thomas Winkler for critical reading of this manuscript. We are grateful to Marcus Dessing for his outstanding skill at the FACSÒ sorter and his extraordinary help during long, unusual hours. We thank Prof. A. Gratwohl, Dr. E. Signer, and Dr. U. Ramenghi for providing the bone marrow samples and Prof. F. Caligaris Cappio for continuous encouragement and discussions. We gratefully acknowledge Ms. Nadia Straube’s technical experience in DNA sequencing. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland. E. Sanz was supported by contracts from the CSIC and grant CAM92/126, and A. de la Hera was supported by grants SAF-93-0925 and SAF-96-0201 from the CICY.Peer reviewedPeer Reviewe

Molecular characterization of the β chain of the murine interleukin 5 receptor

Interleukin 5 (IL-5) is a multifunctional cytokine that regulates the proliferation and differentiation of hematopoietlc cells Including B cells and eosinophlls. The murine IL-5 acts on target cells via an IL-5 specific high-affinity receptor (Kd ≃ 150 pM) that has been proposed to be composed of at least two membrane polypeptide chains. The p60 component recognized by anti-murine IL-5 receptor mAbs H7 and T21 binds IL-5 with low affinity (Kd ≃ 10 nM). The other component is p130, detectable by following cross-linking experiments with IL-5. Using H7, T21, and R52.120 mAbs specific to murine IL-5 receptor, we characterized the molecular nature of the p130 of the high affinity receptor for murine IL-5. R52.120 mAb did not recognize the IL-5 binding recombinant p60 expressed on COS7 cells, but reacted with p130/140 on IL-5-dependent cell lines. R52.120 mAb showed partial inhibition of the IL-5-induced proliferation of the IL-5-dependent early B cell line Y16 at high IL-5 concentrations. Addition of R52.120 mAb together with H7 or T21 mAb caused more striking inhibition of the IL-5-dependent proliferation than that caused by either of them alone. R52.120 mAb down-regulated the number and dissociation constant of IL-5 binding sites with high affinity without affecting the levels of these with low-affinity. It also preferentially inhibited the formation of the cross-linked complex of p130 with radlolabeledIL-5. These results Indicate that p130/p140, recognized by R52.120 mAb, Is indispensable, together with p60, for the formation of high affinity IL-5 receptor. We propose to designate p60 and p130/p140 as the α and β chain of IL-5 receptor, respectivel