Cell surface expression of surrogate light chain (ΨL) in the absence of μ on human pro-B cell lines and normal pro-B cells

Surrogate light chains (ΨL) encoded by λ-like (λ5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the μ chain. These mAb provide the first characterization of human pro-B cell lines expressing surface ΨL. We demonstrate that surface ΨL expression is considerably enhanced upon interleukin-7 stimulation and that the ΨL complex is formed independently of the Igα/Igβ heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34⁺ CD38⁺ ΨL⁺, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.     

B cell development in mice with a defective λ5 gene

The surrogate light chain encoded by the two pre-B cell-specific genes V_preB and λ₅ plays a critical role in B cell development of the mouse. It has been shown that targeted disruption of the λ ₅gene results in a depletion of B220⁺ CD43^? IgM^? pre-B cells in bone marrow, and in a delayed appearance both of CD5⁺ as well as CD5^? surface immunoglobulin (slg)⁺ B cells in the periphery. In this report we show that D_HJ_H-rearranged B220^? and B220⁺, CD43⁺, c-kit⁺, sIgM^? pro- and pre-B-I cells with long-term capacity to proliferate in vitro on stromal cells in the presence of interleukin-7 are present in normal numbers in the bone marrow of λ ₅T/λ.₅T mice at various ages. They express normal levels of V_preB mRNA but, in contrast to normal pre-B-I cells, do not express surrogate light chain on their surface. Pre-B-I cells from fetal liver and bone marrow of λ ₅T/λ ₅T mice differentiate with normal kinetics and in normal numbers to sIg⁺, mitogen-reactive B cells. These results suggest that the delayed generation of sIg⁺ B cells in the peripheral, mature compartments of CD5⁺ and CD5^? cells could be accounted for by the daily production of approximately 5 × 10⁵ sIg⁺ B cells from the pre-B-I cell pool in the absence of a normal pool of pre-B-II cells.     

Available λ B cell repertoire in the mouse: Evidence of positive selection by environmental factors

We have recently shown that, from two BALB/c mice treated with rabbit anti-Cλ2/Cλ3 antibodies coupled to lipopolysaccharide, variable heavy chain (V_H) family repertoires associated with λ2 or λ3 light chains can differ from one λ. subtype to another and from one individual mouse to another. Indeed, 4 out of 6 λ2 (VxJ2) hybridomas from one mouse preferentially expressed the V_H10 family while 3 out of 8 λ2 (V2J2) and 5 out of 8 λ2 (VxJ2) hybridomas from a second mouse preferentially expressed the S107 and VGAM3.8 V_H families, respectively. In this report, we describe the structural basis of such preferential pairings by sequence analysis of the 12 λ2 hybridomas. The sequence comparison of their V_H regions show that each preferential association of a VH family to one Vλ region is restricted to the use of a single member or very closely related members inside a V_H family and that a great variability of CDR3 of heavy chain is observed. We, therefore, suggest that environmental factors can modify the available XλB cell repertoire through a positive selection of particular VH/Vλ pairings. Moreover, our data support that this selection does not require clonal expansion and punctual somatic mutation.     

Diversity of immunoglobulin χ light chain gene rearrangements and evidence for somatic mutation in VχIV family gene segments in X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency disease in man, characterized by an arrest in B lymphocyte differentiation at the precursor B cell stage. The structure of expressed immunoglobulin (Ig) χ light (L) chain rearrangements of nine B lymphoblastoid cell lines from one XLA patient was investigated by amplification of cDNA by the polymerase chain reaction using 5′ Vχ family-specific primers and a 3′ χ constant region primer. Members of all four Vχ gene families were found to be utilized in Ig χ L chain rearrangements at frequencies that were consistent with random Vχ family usage. There was no preference for usage of any particular χ joining segment. Additional diversity was generated by deletions and random nucleotide insertions at the site of juxtaposition. Particular Vχ members seemed to be overrepresented in the sample. The observed homology of the VχI, VχII and VχIII region sequences, both to each other and to known germ-line Vχ sequence indicated the absence of somatic mutations in the majority of these expressed Ig genes. In contrast of the single-member VχIV family four different sequences were found to be expressed. That these sequences were mutated derivatives of a germ-line VχIV element was substantiated both by sequence analysis and oligonucleotide hybridization. This finding shows that the mutation process can occur in early stages of B cell development i.e. before H chain class switch has occurred. The presence of these mutations is probably independent of clonal expansion since XLA patients are unable to respond to antigen. We conclude that the differentiation arrest in XLA does not preclude early onset of somatic mutation events in Vχ gene segments.     

Pairing of VH gene families with the λ light chain: Evidence for a non-stochastic association

The frequencies at which four V_H gene families pair with the λ1 light (L) chain were determined by sequential hybridization of V_H- and λ1-specific DNA probes to mitogen-induced colonies of B cells. Analysis of pair frequencies indicates that the repertoire of λ L chain antibodies is generated by the stochastic pairing of smaller 3′-to-mid-locusV_H gene families (X-24, S107, Q52). However, the large 5′ V_H J558 family appeared to associate with the λ1L chain non-stochastically; the frequency of VhJ558/λ1⁺ colonies among all λ1⁺ colonies was significantly lower than the frequency of J558 expression among all (Cμ⁺) B cell colonies. This difference suggests that selection, either intrinsic at the level of rearrangement or heavy and L chain pairing, or extrinsic following surface immunoglobulin expression, may operate to shape the λ antibody repertoire prior to the introduction of exogenous antigen.     

Analysis of individual immunoglobulin λ light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenviroment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse λ light chain genes which arose in response to a specific antigen. Laboratory mice possess three Vλ subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single λ light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V_λ1 genes from these single cells for mutational analysis. None of the 32 V_λ1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other Vλ subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.     

Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus

B lymphocyte differentiation is characterized by an orderedseries of Ig gene assembly and expression events. In the majorityof normal B cells, assembly and expression of Ig heavy (H) chaingenes precedes that of light (L) chain genes. To determine therole of the Ig heavy chain protein in B cell development andL chain gene rearrangement, we have generated mice that cannotassemble Ig H chain genes as a result of targeted deletion ofthe J_H gene segments in embryonic stem cells. Mice homozygousfor this deletion are devoid of slg⁺ B cells in the bone marrowand periphery. B cell differentiation in these mice is blockedat the large, CD43⁺ precursor stage. However, these precursorB cells do assemble L chain genes at a low level in the absenceof µ H chain proteins. These data demonstrate that rearrangementand expression of the µ H chain gene is not absolutelyrequired for L chain gene rearrangement in vivo. Expressionof µ chains may facilitate either efficient L chain generearrangement or the survival of cells that have rearrangedlight chain genes by promoting the differentiation of large,CD43⁺ to small, CD43^– pre-B cells.     

Expression level of a transgenic λ2 chain results in isotype exclusion and commitment to B1 cells

Two new λ2 chain-transgenic mouse lines were established, both of which showed stable transgene expression during aging of the mice. The line L23, which expressed the transgene at low levels, exhibited normal B cell development, antibody responses and serum Ig levels. Most of the B cells in this mouse line co-expressed the transgenic λ2 chain together with an endogenous κ chain, thus showing poor allelic exclusion of endogenous L chains. On the other hand, high expression of the transgenic λ2 chain in the other mouse line, L2, resulted in nearly complete exclusion of endogenous L chain isotypes. In this line, the λ2 transgene was already detectable in the cytoplasm of all preB-II cells and some pro/preB-I cells. Its expression during these early phases obviously inhibited development of conventional B2 cells, since the B cells in the periphery of these mice were almost exclusively of the B1 type. This finding was confirmed by adoptive transfer of transgenic bone marrow into lethally irradiated recipients. Very few B cells were present in the spleen of such recipients. The serum IgM levels of L2 mice were close to normal and the majority of these IgM were associated with the transgenic λ2 chain. Antibody responses to thymus-dependent antigens in such mice were almost exclusively found to be of IgM class. Together, these findings indicate a developmental bias leading to a predominance of B1 cells in the L2 line.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β-/- mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre-TCR complex.     

人CXCR3B基因转染细胞株的构建、鉴定及生物学功能研究

目的:克隆人CXCR3B基因,并构建含有该目的基因的真核表达载体,获得稳定表达人CXCR3B分子的基因转染细胞株L929-huCXCR3B.方法:采用PCR方法从pMD19-T/huCXCR3A质粒中扩增人CXCR3B基因,通过双酶切装入真核表达载体pIRES2-EGFP中;脂质体法转染L929细胞,G418加压筛选阳性克隆;分别用RT-PCR方法与免疫荧光技术分析阳性克隆中人CXCR3B在mRNA和蛋白水平的表达.MTT分析基因转染细胞株 L929-huCXCR3B 在Mig( monokine induced by IFN-γ,IFN-γ诱导的单核因子)作用下的增殖能力.结果:成功克隆了人CXCR3B基因并构建了真核表达载体pIRES2-EGFP/huCXCR3B,转染该载体后获得了稳定表达人CXCR3B的基因转染细胞株L929-huCXCR3B,膜表面CX-CR3B分子的阳性表达率为93％.该基因转染细胞与其配体Mig共培养24、48及72 h,抑制率分别为41.44％、44.01％和24.80％.结论:L929-huCXCR3B细胞株的建立为研究CXCR3B信号转导及制备相应的单克隆抗体(mAb)奠定了基础.     

Expression of TIMP-3 gene by construction of a eukaryotic cell expression vector and its role in reduction of metastasis in a human breast cancer cell line

The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing arecombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 genefrom the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vectorby means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell lineMDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Thenthe expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correctconstruction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication andnucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3,indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructedsuccessfully.Our experiments further indicated that the potential of metastasis was significantly reduced forthe transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.     

Recombination pattern of the TCR γ locus in human peripheral T-cell lymphomas

The recombination events of the γ and β T-cell receptor (TCR) loci were analysed in a series of 39 peripheral T-cell lymphomas (PTCLs) in association with the expression of TCR chains. In TCR αβ PTCLs, 22/23 cases showed a γ-gene rearrangement while only 18/23 showed a concomitant β-gene rearrangement. The germline configuration of the β locus was found in angioimmunoblastic lymphadenopathy and lymphoepithelioid lymphomas. Three γδ PTCLs rearranged both γ and β genes. TCR silent PTCLs showed three different patterns of γ- and β-gene rearrangements. Three cases were in germline configuration for both loci; five cases had a rearranged γ and a germline β locus; and five cases had the two loci rearranged. Regarding the variable genes in the γ-rearranged alleles, members of the VγI subgroup were the most frequently presented (39/50), followed by VγII, VγIII, and VγIV (9/50, 1/50, and 1/50, respectively). Joining segment usage was as follows: J1 or J2 (32/50), JP1 or JP2 (17/50), and JP (1/50). Taken together, these data demonstrate that the γ locus is more frequently rearranged whatever the TCR expression. The γ-locus analysis provides a better diagnostic yield than the β locus in the study of PTCL clonality.     

DNA adducts,mutant frequencies,and mutation spectra in various organs of λlacZ mice exposed to ethylating agents

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, λlacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O⁶-ethylguanine (O⁶-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O⁶-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O⁶-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC → AT transitions, consistent with the O⁶-EtG lesion, and 28% TA → AT transversions, expected from O²-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC → AT mutations and 22% were TA → AT mutations. This suggests that in bone marrow O⁶-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O⁶-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O⁶- and N7-adducts were present. Environ. Mol. Mutagen. 31:18–31, 1998. © 1998 Wiley-Liss, Inc.