Receptor Editing Occurs Frequently during Normal B Cell Development

Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that λ light chain genes usually rearrange after κ genes. This allowed us to analyze κ loci in IgMλ⁺ cells to determine how frequently in-frame κ genes fail to suppress λ gene rearrangements. To do this, we analyzed recombined VκJκ genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the κ locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged κ loci, as nearly half (47%) of the RS-inactivated VκJκ joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.     

Regulation of B Cell Development by Variable Gene Complexity in Mice Reconstituted with Human Immunoglobulin Yeast Artificial Chromosomes

The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered with yeast artificial chromosomes (YACs) containing different sized fragments of the human heavy (H) chain and κ light (L) chain loci. Each of the two H and the two κ chain fragments encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique V_H (5 versus 66) or Vκ genes (3 versus 32). Although each of these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected. Whereas the YACs with great V gene complexity restored efficient development throughout all the different recombination and expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues. Our analysis identified four key checkpoints regulated by V_H and Vκ gene complexity: (a) production of functional μ chains at the transition from the pre B-I to the pre B-II stage; (b) productive VκJκ recombination at the small pre B-II stage; (c) formation of surface Ig molecules through pairing of μ chains with L chains; and (d) maturation of B cells. These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell lineage.     

Rearrangement of λ Light Chain Genes in Mature B Cells In Vitro and In Vivo. Function of Reexpressed Recombination-activating Gene (RAG) Products

V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J recombinase. DNA excision product reflecting Vλ1 to Jλ1 rearrangement was generated in parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar λ chain gene rearrangement was observed in the draining lymph node of immunized mice. Further, B cells that underwent λ gene rearrangement were shown by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.     

Mutations in the Human λ5/14.1 Gene Result in B Cell Deficiency and Agammaglobulinemia

B cell precursors transiently express a pre–B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of λ5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igα and Igβ. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine λ5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for λ5/14.1. The maternal allele carried a premature stop codon in the first exon of λ5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the λ5/14.1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant λ5/14.1. These findings indicate that expression of the functional λ5/14.1 is critical for B cell development in the human.     

Clinical responses with T lymphocytes targeting malignancy-associated κ light chains

BACKGROUND. Treatment of B cell malignancies with adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan–B cell marker, also depletes normal B cells and causes severe hypogammaglobulinemia. Here, we developed a strategy to target B cell malignancies more selectively by taking advantage of B cell light Ig chain restriction. We generated a CAR that is specific for the κ light chain (κ.CAR) and therefore recognizes κ-restricted cells and spares the normal B cells expressing the nontargeted λ light chain, thus potentially minimizing humoral immunity impairment.METHODS. We conducted a phase 1 clinical trial and treated 16 patients with relapsed or refractory κ⁺ non-Hodgkin lymphoma/chronic lymphocytic leukemia (NHL/CLL) or multiple myeloma (MM) with autologous T cells genetically modified to express κ.CAR (κ.CARTs). Other treatments were discontinued in 11 of the 16 patients at least 4 weeks prior to T cell infusion. Six patients without lymphopenia received 12.5 mg/kg cyclophosphamide 4 days before κ.CART infusion (0.2 × 10⁸ to 2 × 10⁸ κ.CARTs/m²). No other lymphodepletion was used.RESULTS. κ.CART expansion peaked 1–2 weeks after infusion, and cells remained detectable for more than 6 weeks. Of 9 patients with relapsed NHL or CLL, 2 entered complete remission after 2 and 3 infusions of κ.CARTs, and 1 had a partial response. Of 7 patients with MM, 4 had stable disease lasting 2–17 months. No toxicities attributable to κ.CARTs were observed.CONCLUSION. κ.CART infusion is feasible and safe and can lead to complete clinical responses.TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00881920","term_id":"NCT00881920"}}NCT00881920.FUNDING. National Cancer Institute (NCI) grants 3P50CA126752 and 5P30CA125123 and Leukemia and Lymphoma Society (LLS) Specialized Centers of Research (SCOR) grant 7018.     

Human Immunoglobulin (Ig)M+IgD+ Peripheral Blood B Cells Expressing the CD27 Cell Surface Antigen Carry Somatically Mutated Variable Region Genes: CD27 as a General Marker for Somatically Mutated (Memory) B Cells

Immunoglobulin (Ig)M⁺IgD⁺ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM⁺IgD⁺ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM⁺IgD⁺ B cells. IgM⁺IgD⁺CD27⁺ B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise ∼15% of PB B lymphocytes in healthy adults. Moreover, a very small population (<1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of ∼40% mutated memory B cells and 60% unmutated, naive IgD⁺CD27⁻ B cells (including CD5⁺ B cells). In the somatically mutated B cells, V_H region genes carry a two- to threefold higher load of somatic mutation than rearranged V_κ genes. This might be due to an intrinsically lower mutation rate in κ light chain genes compared with heavy chain genes and/or result from κ light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.     

Oncogenic transformation in the absence of Xrcc4 targets peripheral B cells that have undergone editing and switching

Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks (DSBs) during V(D)J recombination in developing lymphocytes and during immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) in peripheral B lymphocytes. We now show that CD21-cre–mediated deletion of the Xrcc4 NHEJ gene in p53-deficient peripheral B cells leads to recurrent surface Ig-negative B lymphomas (“CXP lymphomas”). Remarkably, CXP lymphomas arise from peripheral B cells that had attempted both receptor editing (secondary V[D]J recombination of Igκ and Igλ light chain genes) and IgH CSR subsequent to Xrcc4 deletion. Correspondingly, CXP tumors frequently harbored a CSR-based reciprocal chromosomal translocation that fused IgH to c-myc, as well as large chromosomal deletions or translocations involving Igκ or Igλ, with the latter fusing Igλ to oncogenes or to IgH. Our findings reveal peripheral B cells that have undergone both editing and CSR and show them to be common progenitors of CXP tumors. Our studies also reveal developmental stage-specific mechanisms of c-myc activation via IgH locus translocations. Thus, Xrcc4/p53-deficient pro–B lymphomas routinely activate c-myc by gene amplification, whereas Xrcc4/p53-deficient peripheral B cell lymphomas routinely ectopically activate a single c-myc copy.Ig heavy (IgH) and light (IgL) chain variable region exons are assembled from component V, D, and J segments in developing B lymphocytes. V(D)J recombination is initiated by the RAG1/2 endonuclease, which introduces DNA double-strand breaks (DSBs) between V, D, and J segments and flanking recombination signal sequences (RSs) (1). Subsequently, cleaved coding segments are joined to form V(D)J exons and RSs are joined to form RS joins (2). Both coding and RS joining are performed by classical nonhomologous end-joining (C-NHEJ), which is a major general DSB repair pathway in mammalian cells (3). Xrcc4 and DNA Ligase IV (Lig4) form a complex that is required for V(D)J recombination (4, 5). In their absence, coding or RS ends are joined at low frequency, usually with substantial sequence deletion from one or both partners (6, 7). In mice, Xrcc4 inactivation results in severe combined immune deficiency owing to inability to complete V(D)J recombination (6).In progenitor B (pro–B) cells in the mouse BM, productive assembly of variable region exons within the IgH locus (Igh) on chromosome 12 leads to production of IgH μ chains that signal differentiation to the precursor B (pre–B) cell stage in which IgL variable region exons are assembled (8). Mice, like humans, have two IgL families, termed Igκ and Igλ, which are encoded by Igκ and Igλ loci that, respectively, lie on chromosomes 6 and 16. Igκ and Igλ expression is “isotype” excluded, such that a given B cell usually expresses either Igκ or Igλ, but not both (9). In mice, ∼95% of mature B lymphocytes are Igκ⁺, with the remainder being Igλ⁺. In that context, Igκ assembly usually precedes that of Igλ (9). Thus, most Igκ⁺ B cells contain Igλ in germline configuration, with Igλ rearrangements occurring in cells in which both Igκ alleles are rearranged out-of-frame or that harbor deletions of the Jκ segments, κ enhancer, and/or Cκ exons (9). Such deletions usually occur via rearrangement of Vκs or an RS heptamer in the Jκ-Cκ intron (IRS) to a bona fide RS 25 kb downstream of Cκ (3′RS) (10). Recent analyses suggest that Igκ deletions via 3′RS rearrangements may play a role in progression to Igλ rearrangement (11).Expression of complete Ig (IgH/IgL) leads to IgM⁺ B lymphocytes, which ultimately down-regulate RAG expression to enforce allelic exclusion (1). However, newly generated BM IgM⁺ B lymphocytes that express autoreactive B cell receptors can maintain RAG expression and continue to rearrange IgL loci to generate new IgL chains in a tolerance process termed “receptor editing” (12–14). Receptor editing can replace rearranged Igκ loci with secondary productive Igκ rearrangements, as well as with nonfunctional Igκ rearrangements or Igκ deletions that may lead to Igλ rearrangement (12–14). Thus, Igλ⁺ B cells can be generated developmentally from pre–B cells with two nonproductive Igκ rearrangements or via receptor editing from immature Igκ⁺ B cells. Receptor editing is initiated in immature BM B cells (15, 16). Yet, several studies suggested IgL gene rearrangement, sometimes called “revision,” in mouse and human peripheral B cells, including germinal center B cells (17–21). However, many peripheral mouse RAG⁺ B lineage cells are pro– or pre–B cells that migrate to the periphery after immunization (22, 23), and knock-in reporter studies suggested that although RAG genes are expressed in B cells that have just migrated from the BM (24, 25), they are not reinduced in peripheral B cells once expression is terminated (25, 26).After antigen stimulation, mature IgM⁺ peripheral B cells can undergo IgH class switch recombination (CSR), a recombination/deletion process in which the IgH μ constant region exons (Cμ) are deleted and replaced by one of several sets of downstream C_H exons (e.g., Cγ, Cε, and Cα; referred to as C_H genes) (27), leading to switching from IgM to another Ig class (e.g., IgG, IgE, or IgA). The activation-induced cytidine deaminase (AID) initiates CSR (28) by deaminating cytidines in switch (S) regions (29), which are 1–10-kb repetitive sequences located 5′ of each C_H gene. AID-generated lesions within the donor Sμ and a downstream acceptor S region are processed into DSBs, which are end-joined to complete CSR (27). In contrast to V(D)J recombination, substantial CSR occurs in the absence of Xrcc4 or Lig4 (C-NHEJ) via an alternative end-joining (A-EJ) pathway strongly biased to use microhomology (30). However, CSR is significantly impaired in Xrcc4-deficient B cells owing to failure to join broken S regions because up to 20% of Xrcc4-deficient B cells activated for CSR in vitro have IgH chromosomal breaks, with a substantial portion participating in chromosomal translocations (30).Inactivation of Xrcc4 in mice results in impaired cellular proliferation and ionizing radiation sensitivity. Xrcc4 deficiency also results in extensive apoptosis of newly generated neurons and late embryonic death (6), both of which can be rescued by deficiency for the p53 tumor suppressor (31). In this context, p53 monitors the G1 cell cycle checkpoint, signaling apoptosis of certain cell types, such as neurons and progenitor lymphocytes, which harbor persistent DSBs (32). However, as p53 deficiency does not rescue defective NHEJ associated with Xrcc4 deficiency, Xrcc4/p53–double-deficient mice are still immunodeficient and inevitably succumb to pro–B cell lymphomas that harbor RAG-dependent complex translocations (33). These translocations usually join IgH on chromosome 12 to a region downstream of c-myc on chromosome 15, resulting in dicentric 12;15 translocations and c-myc amplification via breakage–fusion–bridge cycles (34). Such complex translocations are rare in human peripheral B cell lymphomas, which more frequently harbor reciprocal translocations that fuse IgH, or less frequently IgL loci, just upstream of c-myc, leading to ectopic c-myc activation (35).In the current study, we have asked whether inactivation of C-NHEJ in WT or p53-deficient peripheral B cells leads to peripheral B cell lymphoma with CSR or V(D)J recombination-associated IgH or IgL locus translocations.     

PRESENCE OF TWO TYPES OF L POLYPEPTIDE CHAINS IN GUINEA PIG 7S IMMUNOGLOBULINS

Guinea pig 7S immunoglobulins (γ₁ and γ₂) consist of two groups of molecules (K and L) bearing different types of L chains (κ and λ). Approximately one-third of the molecules in normal guinea pig γ₂-globulin bear the λ- type of L chains, and all or most of the others bear κ-chains. κ- and λ-chains have similar molecular weights.     

Assembly of Productive T Cell Receptor δ Variable Region Genes Exhibits Allelic Inclusion

The generation of a productive “in-frame” T cell receptor β (TCR β), immunoglobulin (Ig) heavy (H) or Ig light (L) chain variable region gene can result in the cessation of rearrangement of the alternate allele, a process referred to as allelic exclusion. This process ensures that most αβ T cells express a single TCR β chain and most B cells express single IgH and IgL chains. Assembly of TCR α and TCR γ chain variable region genes exhibit allelic inclusion and αβ and γδ T cells can express two TCR α or TCR γ chains, respectively. However, it was not known whether assembly of TCR δ variable regions genes is regulated in the context of allelic exclusion. To address this issue, we have analyzed TCR δ rearrangements in a panel of mouse splenic γδ T cell hybridomas. We find that, similar to TCR α and γ variable region genes, assembly of TCR δ variable region genes exhibits properties of allelic inclusion. These findings are discussed in the context of γδ T cell development and regulation of rearrangement of TCR δ genes.     

The Normal Counterpart of IgD Myeloma Cells in Germinal Center Displays Extensively Mutated IgVH Gene, Cμ–Cδ Switch, and λ Light Chain Expression

Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre–B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased λ light chain expression and a Cμ–Cδ isotype switch. Using surface markers, we have previously isolated a population of surface IgM⁻IgD⁺CD38⁺ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased λ light chain expression and a Cμ–Cδ isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs.     

One of the CD3ε Subunits within a T Cell Receptor Complex Lies in Close Proximity to the Cβ FG Loop

A recent crystal structure of the N15 α/β-T cell receptor (TCR) in complex with an Fab derived from the H57 Cβ-specific monoclonal antibody (mAb) shows the mAb fragment interacting with the elongated FG loop of the Cβ domain. This loop creates one side wall of a cavity within the TCR Ti-α/β constant region module (CαCβ) while the CD and EF loops of the Cα domain form another wall. The cavity size is sufficient to accommodate a single nonglycosylated Ig domain such as the CD3ε ectodomain. By using specific mAbs to mouse TCR-β (H57) and CD3ε (2C11) subunits, we herein provide evidence that only one of the two CD3ε chains within the TCR complex is located in close proximity to the TCR Cβ FG loop, in support of the above notion. Moreover, analysis of T cells isolated from transgenic mice expressing both human and mouse CD3ε genes shows that the heterologous human CD3ε component can replace the mouse CD3ε at this site. The location of one CD3ε subunit within the rigid constant domain module has implications for the mechanism of signal transduction throughout T cell development.     

AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY

In an attempt to account for antibody specificity and complementarity in terms of structure, human κ-, human λ-, and mouse κ-Bence Jones proteins and light chains are considered as a single population and the variable and constant regions are compared using the sequence data available. Statistical criteria are used in evaluating each position in the sequence as to whether it is essentially invariant or group-specific, subgroup-specific, species-specific, etc. Examination of the invariant residues of the variable and constant regions confirms the existence of a large number of invariant glycines, no invariant valine, lysine, and histidine, and only one invariant leucine and alanine in the variable region, as compared with the absence of invariant glycines and presence of three each of invariant alanine, leucine, and valine and two each of invariant lysine and histidine in the constant region. The unique role of glycine in the variable region is emphasized. Hydrophobicity of the invariant residues of the two regions is also evaluated. A parameter termed variability is defined and plotted against the position for the 107 residues of the variable region. Three stretches of unusually high variability are noted at residues 24–34, 50–56, and 89–97; variations in length have been found in the first and third of these. It is hypothesized that positions 24–34 and 89–97 contain the complementarity-determining residues of the light chain—those which make contact with the antigenic determinant. The heavy chain also has been reported to have a similar region of very high variability which would also participate in forming the antibody-combining site. It is postulated that the information for site complementarity is contained in some extrachromosomal DNA such as an episome and is incorporated by insertion into the DNA of the structural genes for the variable region of short linear sequences of nucleotides. The advantages and disadvantages of this hypothesis are discussed.     

Converting Tumor-specific Markers Into Reporters of Oncolytic Virus Infection

Preferential killing of transformed cells, while keeping normal cells and organs unharmed, is the main goal of cancer gene therapy. Genetically engineered trackable markers and imaging reporters enable noninvasive monitoring of transduction efficiency and pharmacokinetics of anticancer virotherapeutics. However, none of these reporters can differentiate between infection in the targeted tumors and that in the normal tissue. Thus, we constructed oncolytic measles virus (MV) armed with a human light immunoglobulin chain reporter gene for the treatment of multiple myeloma (MM). Excessive production of monoclonal immunoglobulin is a key characteristic and marker for diagnostics of MM. Once expressed in infected target cells, vector-encoded λ protein recombines with myeloma IgG-κ immunoglobulin creating a unique IgG-κ/λ. A modified immunoassay technique allows precise quantification of converted marker molecules. Only antibody producing cells were able to assemble this chimeric immunoglobulin molecule, whereas other cells secreted only free λ light chain. Human myeloma xenografts inoculated with λ chain expressing MV secreted converted IgG-κ/λ in the plasma of tumor bearing animals and elevated reporter levels correlated with response to the therapy. This is the first report of a gene therapy vector engineered to discriminate between infection in malignant and normal cells by molecular modification of a tumor-specific protein.     

The immune response of allophenic mice to the synthetic polymer L-glutamic acid, L-lysine, L-phenylalanine. II. Lack of gene complementation in two nonresponder strains

The genetic control of the immune response of inbred strains of mice to certain antigens has been demonstrated to be governed by a set of Ir genes linked to the major histocompatibility complex (H-2) of mice (1,2). Until recently, the control was thought to be governed by single, dominant genes, located within the I region of the H-2 complex. Merryman et al. (3) originally demonstrated that the immune response to the synthetic terpolymer L-glutamic acid, L-lysine, L-phenylaline (GL) is under dominant, H-2-linked Ir gene control (4-7). This was shown both by crossing two nonresponder parental strains to produce responder offspring in the F(1) generation, and by the analysis of appropriate recombinant stains of mice. The two complementing genes have been mapped in the IA and IC regions of the H-2 complex, and have been termed β and α, respectively (5,6). Thus, any strain of mouse may contain neither, one, or both genes. Only mice containing both genes are capable of responding to GL. It has been shown using F(1) hybrid and recombinant strains of mice, that the α- and β-genes can complement each other in either the cis (on the same chromosome) or in the trans (on different chromosomes) position (8). In this paper we report the results of studies aimed at answering the question of whether or not the α- and β- genes can complement each other when they are present in different lymphoid cells. To this end we have constructed allophenic mice composed of two nonresponder strains (A and C57BL/6), which show gene complementation in the F(1) generation. Allophenic mice are chimeras containing two cell types coexisting in a “normal” environment. The mice were tested for the specific cellular composition of the two parental cell types and were found to possess a complete range in the relative proportion of the two cell types. This report demonstrates that regardless of the mixture of cell types present in the allophenic mice, none of them were responders to GL. Thus no complementation of the α- and β-genes is seen when the two genes are present in different cells.     

Immunoglobulin and other surface antigens of cells of the immune system

Immunoglobulins (Ig) on cells of the immune system: The cytotoxicity test, with class-specific and type-specific anti-Ig sera, identifies κ and µ determinants on mouse lymphocytes. The proportion of κ⁺ cells is characteristic for each source of cells: 30% of bone marrow cells, 40% of cells from peripheral lymph nodes, 45% of lymphocytes from peripheral blood or peritoneal cavity, and 50% of spleen cells. No Ig was demonstrable on thymocytes or on leukemia cells (most of which arise from thymus-derived [T] cells). Cytotoxicity tests were performed on various myelomas secreting different Ig; the only positive reactions were given by κγ1 myelomas (all four κγ1 myelomas tested were sensitive to both anti-κ and anti-γ1). Hemolytic plaque-forming cells (PFC) of IgG type had no demonstrable surface Ig, but a proportion of IgM PFC were κ⁺µ⁺. Virtually all rosette-forming cells (RFC) have surface Ig, more than 90% of them being inhibited by anti-κ, 50% by anti-µ, and 10–30% by antisera to other heavy chains. Anti-λ sera gave no positive reactions with any cell type, which is in keeping with the low level of this light chain in mouse serum. Ig and other differentiation antigens as markers for T and B cells: Thymocytes are hallmarked by the alloantigens TL, θ, and the Ly series, and it is generally held that extrathymic lymphoid cells that bear them are derived from thymocytes. There is one alloantigen marker for the thymus-independent (B) cell, and that is PC, which appears late in differentiation. (The mouse-specific lymphocyte (MSLA) and mouse-specific bone marrow-derived lymphocyte (MBLA) antigens recognized by heteroantisera, not used in the present study, are other candidates for T and B cell markers.) Making use of antisera to these surface antigens to inhibit the function of cells that carry them, we find the following: Approximately 30% of RFC, 60% of IgM PFC, and 90% of IgG are PC⁺ and so are identified as B cells. No T markers were demonstrable on these cell populations. Thus if T cells do become RFC or PFC they presumably lose their T surface markers in the process (cf. the quantitative reduction of T markers accompanying the thymocyte → lymphocyte transition). Cells that have the potential to initiate graft-versus-host (GVH) reactions have the T cell surface phenotype θ⁺Ig^-. Adoptive transfer of thymus-dependent antibody-forming capacity (response to sheep erythrocytes) required θ⁺ cells but transfer of a thymus-independent immune response to Brucella antigen did not. Cells with surface Ig were involved in both types of adoptive transfers. Thus the presently available T markers do not provide evidence for T cells carrying surface Ig. Suppression of the Ig phenotype by antibody: antigenic modulation? A phenotypic change from Ig⁺ to Ig^- occurs when Ig⁺ lymphocytes or myeloma cells are incubated with anti-Ig sera in vitro in the absence of complement (C). As with antigenic modulation in the TL system, which it resembles, this phenomenon is temperature dependent and in the case of lymph node cells (LNC) can be inhibited by high doses of actinomycin D.     

A Transgenic Marker for Mouse B Lymphoid Precursors

Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5′ of the precursor (pre)–B cell–specific λ5 gene. All three strains express human CD25 in parallel to endogenous λ5 on pre–B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220⁺CD19⁻λ5⁺ precursor of the B220⁺CD19⁺λ5⁺ c-kit⁺ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19⁻ precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19⁺ pre–BI cells are predominantly DJ_H rearranged. The results indicate that random integration of the 722-bp 5′ region of the λ5 gene into the mouse genome confers tissue and differentiation stage–specific expression of a transgene.     

Arctigenin inhibits apoptosis,extracellular matrix degradation,and inflammation in human nucleus pulposus cells by up‐regulating miR‐483‐3p

BackgroundArctigenin (ATG) is the active ingredient of the Chinese herbal medicine Arctium lappa, with anti‐inflammatory and antioxidant effects. Excessive inflammation and cell apoptosis are important causes of intervertebral disc degeneration (IDD). Hence, this study probed into the possible role of ATG in IDD.MethodsInterleukin (IL)‐1β (10 ng/ml) was adopted to induce human nucleus pulposus cells (HNPCs) as a cell model for IDD. The effects of different concentrations of ATG (0, 2, 5, 10, 20, 50 μmol/L) on the viability of HNPCs and effects of ATG (10, 50 μmol/L) on the viability of IL‐1β‐induced HNPCs were detected by cell counting kit‐8 (CCK‐8). After IL‐1β‐induced HNPCs were transfected with miR‐483‐3p inhibitor and/or treated with ATG, cell viability and apoptosis were determined by CCK‐8 and flow cytometry; the expressions of miR‐483‐3p, extracellular matrix (ECM)‐related genes, and inflammation‐related genes were measured by quantitative real time polymerase chain reaction (qRT‐PCR), and expressions of ECM/apoptosis/NF‐κB pathway‐related proteins were quantified by Western blot.ResultsATG had no significant effect on the viability of HNPCs but could promote the viability of IL‐1β‐induced HNPCs. ATG inhibited apoptosis, ECM degradation, inflammation, and activation of NF‐κB pathway in HNPCs induced by IL‐1β, but promoted the expression of miR‐483‐3p. MiR‐483‐3p inhibitor reversed the above‐mentioned regulatory effects of ATG.ConclusionArctigenin suppresses apoptosis, ECM degradation, inflammation, and NF‐κB pathway activation in HNPCs by up‐regulating miR‐483‐3p.     

Regional location of T cell receptor gene Ti alpha on human chromosome 14

The chromosomal location of Ti alpha was determined by hybridization of a radiolabeled cDNA for the alpha chain of human T cell receptor with 12 human X mouse cell hybrid DNAs cleaved with BamHI. Seven hybrids contained human Ti alpha, while the remaining five lacked it. Only human chromosome 14 matched the distribution of human Ti alpha signal across the mapping panel. Hybrids segregating a chromosome 14 translocation were used to demonstrate that Ti alpha is in the region 14pter greater than 14q21. Thus, the alpha and beta chain genes that contribute structural components to the Ti moiety of the human T cell receptor lie on different chromosomes. In humans, the immunoglobulin heavy chain locus and Ti alpha are in different regions of chromosome 14, with Ti alpha more proximal and the immunoglobulin heavy chain locus more distal.