Suppressive Role of B Cells in Chronic Colitis of T Cell Receptor α Mutant Mice

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-α^−/− mice was explored by creating double mutant mice (TCR-α^−/− × immunoglobulin (Ig)μ^−/−), which lack B cells. TCR-α^−/− × Igμ^−/− mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-α^−/− mice. Colitis was induced in recombination-activating gene-1 (RAG-1^−/−) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-α^−/− × Igμ^−/− mice. When purified B cells from TCR-α^−/− mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1^−/− mice. Administration of the purified Ig from TCR-α^−/− mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-α^−/− × Igμ^−/− mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-α^−/− × Igμ^−/− mice as compared to Igμ^−/− mice and TCR-α^−/− mice. Administration of the purified Ig from TCR-α^−/− mice into TCR-α^−/− × Igμ^−/− mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.     

The Immunoglobulin (Ig)α and Igβ Cytoplasmic Domains Are Independently Sufficient to Signal B Cell Maturation and Activation in Transgenic Mice

The B cell antigen receptor, composed of membrane immunoglobulin (Ig) sheathed by the Igα/Igβ heterodimer plays a critical role in mediating B cell development and responses to antigen. The cytoplasmic tails of Igα and Igβ differ substantially but have been well conserved in evolution. Transfection experiments have revealed that, while these tails share an esssential tyrosine-based activation motif (ITAM), they perform differently in some but not all assays and have been proposed to recruit distinct downstream effectors. We have created transgenic mouse lines expressing chimeric receptors comprising an IgM fused to the cytoplasmic domain of each of the sheath polypeptides. IgM/α and IgM/β chimeras (but not an IgM/β with mutant ITAM) are each independently sufficient to mediate allelic exclusion, rescue B cell development in gene-targeted Igμ⁻ mice that lack endogenous antigen receptors, as well as signal for B7 upregulation. While the (IgM/α) × (IgM/β) double-transgenic mouse revealed somewhat more efficient allelic exclusion, our data indicate that each of the sheath polypeptides is sufficient to mediate many of the essential functions of the B cell antigen receptor, even if the combination gives optimal activity.     

Regulation of B Lymphocyte Development by the Truncated Immunoglobulin Heavy Chain Protein Dμ

The development of B lymphocytes from progenitor cells is dependent on the expression of a pre–B cell–specific receptor made up by a μ heavy chain associated with the surrogate light chains, immunoglobulin (Ig)α, and Igβ. A variant pre–B cell receptor can be formed in which the μ heavy chain is exchanged for a truncated μ chain denoted Dμ. To investigate the role of this receptor in the development of B cells, we have generated transgenic mice that express the Dμ protein in cells of the B lineage. Analysis of these mice reveal that Dμ expression leads to a partial block in B cell development at the early pre–B cell stage, probably by inhibiting V_H to D_HJ_H rearrangement. Furthermore, we provide evidence that Dμ induces V_L to J_L rearrangements.     

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.     

Immunization of Mice with Urease Vaccine Affords Protection against Helicobacter pylori Infection in the Absence of Antibodies and Is Mediated by MHC Class II–restricted Responses

We examined the roles of cell- and antibody-mediated immunity in urease vaccine–induced protection against Helicobacter pylori infection. Normal and knockout mice deficient in major histocompatibility complex (MHC) class I, MHC class II, or B cell responses were mucosally immunized with urease plus Escherichia coli heat-labile enterotoxin (LT), or parenterally immunized with urease plus aluminum hydroxide or a glycolipid adjuvant, challenged with H. pylori strain X47-2AL, and H. pylori organisms and leukocyte infiltration in the gastric mucosa quantified. In an adjuvant/route study in normal mice, there was a direct correlation between the level of protection and the density of T cells recruited to the gastric mucosa. In knockout studies, oral immunization with urease plus LT protected MHC class I knockout mice [β₂-microglobulin (−/−)] but not MHC class II knockout mice [I-A^b (−/−)]. In B cell knockout mice [μMT (−/−)], vaccine-induced protection was equivalent to that observed in immunized wild-type (+/+) mice; no IgA⁺ cells were detected in the stomach, but levels of CD4⁺ cells equivalent to those in the wild-type strain (+/+) were seen. These studies indicate that protection of mice against H. pylori infection by immunization with the urease antigen is dependent on MHC class II–restricted, cell-mediated mechanisms, and antibody responses to urease are not required for protection.     

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.     

A Role for Tumor Necrosis Factor Receptor Type 1 in Gut-associated Lymphoid Tissue Development: Genetic Evidence of Synergism with Lymphotoxin β

Lymphotoxin α (LTα) signals via tumor necrosis factor receptors (TNFRs) as a homotrimer and via lymphotoxin β receptor (LTβR) as a heterotrimeric LTα₁β₂ complex. LTα-deficient mice lack all lymph nodes (LNs) and Peyer''s patches (PPs), and yet LTβ-deficient mice and TNFR-deficient mice have cervical and mesenteric LN. We now show that mice made deficient in both LTβ and TNFR type 1 (TNFR1) lack all LNs, revealing redundancy or synergism between TNFR1 and LTβ, acting presumably via LTβR. A complete lack of only PPs in mice heterozygous for both ltα and ltβ, but not ltα or ltβ alone, suggests a similar two-ligand phenomenon in PP development and may explain the incomplete lack of PPs seen in tnfr1 ^−/− mice.     

The Inositol Polyphosphate 5-Phosphatase Ship Is a Crucial Negative Regulator of B Cell Antigen Receptor Signaling

Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fcγ receptor IIB (FcγRIIB), a low affinity receptor for the Fc portion of immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor (BCR)–FcγRIIB coligation. The function of Ship in lymphocytes was investigated in Ship^−/− recombination-activating gene (Rag)^−/− chimeric mice generated from gene-targeted Ship^−/− embryonic stem cells. Ship^−/−Rag^−/− chimeras showed reduced numbers of B cells and an overall increase in basal serum Ig. Ship^−/− splenic B cells displayed prolonged Ca²⁺ influx, increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR–FcγRIIB coligation. These results demonstrate that Ship plays an essential role in FcγRIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca²⁺ flux and MAPK activation.     

Counterselection against Dμ Is Mediated through Immunoglobulin (Ig)α-Igβ

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (Dμ). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Igα-Igβ, whereas the mechanism for counterselection against Dμ has not been determined. We have examined the role of the Igα-Igβ signal transducers in counterselection against Dμ using mice that lack Igβ. We found that Dμ expression is not selected against in developing B cells in Igβ mutant mice. Thus, the molecular mechanism for counterselection against Dμ in pre-B cells resembles positive selection in that it requires interaction between mDμ and Igα-Igβ.     

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.     

Cytosolic Phospholipase A2α Protects against Ischemia/Reperfusion Injury in the Heart

Studies with sPLA₂ Group X, and cPLA₂α gene‐targeted mice suggest that absence of sPLA₂ Group X results in protection from ischemia/reperfusion (I/R) injury in the heart, and absence of cPLA₂α Group IV is protective in the brain. Although latter studies might suggest a similar deleterious role for cPLA₂α in I/R injury in the heart, the pathophysiology of stroke is intricately related to excitotoxicity and cannot necessarily be extrapolated to the heart. We report here that unlike findings in the brain, cPLA₂α^(−/−) mice have exaggerated injury following I/R in vivo. In contrast, there is no difference in injury induced by simulated ischemia in cardiomyocytes isolated from cPLA₂α^(−/−) versus cPLA₂α^(+/+) mice. This suggests that cPLA₂α does not have an important cardiomyocyte autonomous effect on ischemic injury. Prostaglandin E₂ (PGE₂) levels are significantly reduced in the hearts of the cPLA₂α^(−/−) mice, and the enhanced injury is ameliorated by treatment with the PGE analog, misoprostol. We demonstrate that cPLA₂α is cardioprotective in vivo, and this is likely via cPLA₂α‐mediated production of cardioprotective eicosanoids. These studies are the first to identify a protective role for cPLA₂ in I/R injury in any organ and raise concerns over long‐term inhibition of cPLA₂. Clin Trans Sci 2011; Volume 4: 236–242     

Intracellular time course, pharmacokinetics, and biodistribution of isoniazid and rifabutin following pulmonary delivery of inhalable microparticles to mice

Intracellular concentrations of isoniazid and rifabutin resulting from administration of inhalable microparticles of these drugs to phorbol-differentiated THP-1 cells and the pharmacokinetics and biodistribution of these drugs upon inhalation of microparticles or intravenous administration of free drugs to mice were investigated. In cultured cells, both microparticles and dissolved drugs established peak concentrations of isoniazid (~1.4 and 1.1 μg/10⁶ cells) and rifabutin (~2 μg/ml and ~1.4 μg/10⁶ cells) within 10 min. Microparticles maintained the intracellular concentration of isoniazid for 24 h and rifabutin for 96 h, whereas dissolved drugs did not. The following pharmacokinetic parameters were calculated using WinNonlin from samples obtained after inhalation using an in-house apparatus (figures in parentheses refer to parameters obtained after intravenous administration of an equivalent amount, i.e., 100 μg of either drug, to parallel groups): isoniazid, serum half-life (t_1/2) = 18.63 ± 5.89 h (3.91 ± 1.06 h), maximum concentration in serum (C_max) = 2.37 ± 0.23 μg·ml⁻¹ (3.24 ± 0.57 μg·ml⁻¹), area under the concentration-time curve from 0 to 24 h (AUC_0-24) = 55.34 ± 13.72 μg/ml⁻¹ h⁻¹ (16.64 ± 1.80 μg/ml⁻¹ h⁻¹), and clearance (CL) = 63.90 ± 13.32 ml·h⁻¹ (4.43 ± 1.85 ml·h⁻¹); rifabutin, t_1/2 = 119.49 ± 29.62 h (20.18 ± 4.02 h), C_max = 1.59 ± 0.01 μg·ml⁻¹ (3.47 ± 0.33 μg·ml⁻¹), AUC_0-96 = 109.35 ± 14.78 μg/ml⁻¹ h⁻¹ (90.82 ± 7.46 μg/ml⁻¹ h⁻¹), and CL = 11.68 ± 7.00 ml·h⁻¹ (1.03 ± 0.11 ml·h⁻¹). Drug targeting to the lungs in general and alveolar macrophages in particular was observed. It was concluded that inhaled microparticles can reduce dose frequency and improve the pharmacologic index of the drug combination.     

Structural transformation of methasterone with Cunninghamella blakesleeana and Macrophomina phaseolina

An anabolic-androgenic synthetic steroidal drug, methasterone (1) was transformed by two fungi, Cunninghamella blakesleeana and Macrophimina phaseclina. A total of six transformed products, 6β,7β,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (2), 6β,7α,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (3), 6α,17β-dihydroxy-2α,17α-dimethyl-5α-androstane-3,7-dione (4), 3β,6β,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-7-one (5), 7α,17β-dihydroxy-2α,17α-dimethyl-5α-androstane-3-one (6), and 6β,9α,17β-trihydroxy-2α,17α-dimethyl-5α-androstane-3-one (7) were synthesized. Among those, compounds 2–5, and 7 were identified as new transformed products. MS, NMR, and other spectroscopic techniques were performed for the characterization of all compounds. Substrate 1 (IC₅₀ = 23.9 ± 0.2 μg mL⁻¹) showed a remarkable anti-inflammatory activity against nitric oxide (NO) production, in comparison to standard LNMMA (IC₅₀ = 24.2 ± 0.8 μg mL⁻¹). Whereas, its metabolites 2, and 7 showed moderate inhibition with IC₅₀ values of 38.1 ± 0.5 μg mL⁻¹, and 40.2 ± 3.3 μg mL⁻¹, respectively. Moreover, substrate 1 was found to be cytotoxic for the human normal cell line (BJ) with an IC₅₀ of 8.01 ± 0.52 μg mL⁻¹, while metabolites 2–7 were identified as non-cytotoxic. Compounds 1–7 showed no cytotoxicity against MCF-7 (breast cancer), NCI-H460 (lung cancer), and HeLa (cervical cancer) cell lines.

Fungal transformation of methasterone resulted in six products (2–7). 2–5, and 7 were identified as new. Substrate 1 showed remarkable anti-inflammatory activity but was cytotoxic. Products 2 and 7 showed moderate activity but were non-cytotoxic.     

The Ataxia Telangiectasia mutated kinase controls Igκ allelic exclusion by inhibiting secondary Vκ-to-Jκ rearrangements

Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre–B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.Assembly of Ig and TCR genes from variable (V), diversity (D), and joining (J) segments is the pervasive means by which antigen receptor (AgR) diversity is generated (Brady et al., 2010). V(D)J recombination is initiated by the RAG1/RAG2 (RAG) endonuclease that induces DNA double-strand breaks (DSBs) adjacent to participating segments (Schatz and Ji, 2011) and completed by DSB repair factors that process V(D)J coding ends (CEs) into coding joins (CJs; Helmink and Sleckman, 2012). AgR assembly occurs during and is required for lymphocyte differentiation. IgH genes are assembled through D_H-to-J_H recombination, followed by V_H-to-DJ_H rearrangements on one allele at a time in pro–B cells (Rajewsky, 1996). IgH chains expressed from in-frame V_HDJ_H joins can bind λ5/Vpre–B chains to form pre-BCRs that signal inhibition of V_H rearrangements, proliferation, and differentiation into pre–B cells (Rajewsky, 1996). The two-thirds of cells that assemble out-of-frame V_HDJ_H joins can attempt to assemble in-frame V_HDJ_H joins on the second allele (Rajewsky, 1996). Igκ genes are assembled from Vκ and Jκ segments on one allele at a time in G1 phase pre–B cells (Rajewsky, 1996). Igκ chains expressed from VκJκ joins can bind IgH chains to form κ⁺ BCRs that are subject to selection (Rajewsky, 1996; Nemazee, 2006). Non-autoreactive BCRs signal inhibition of Igκ recombination and differentiation into B cells (Nemazee, 2006). Autoreactive BCRs induce additional Igκ rearrangements that replace VκJκ complexes, a process known as Igκ editing (Nemazee, 2006). Pre–B cells that assemble out-of-frame VκJκ joins can attempt to assemble in-frame VκJκ joins on the other allele (Rajewsky, 1996).Most lymphocytes express surface AgR chains from a single allele. Allelic exclusion is enforced through the ability of Ig and TCR chains expressed from one allele to signal feedback inhibition of V-to-(D)J rearrangements on the second allele (Brady et al., 2010; Vettermann and Schlissel, 2010). To achieve allelic exclusion, only one allele can initiate V-to-(D)J recombination in the time required for feedback inhibition. V-to-(D)J recombination requires CTCF-mediated looping between RAG accessible V segments and RAG-bound D/J segments (Guo et al., 2011; Schatz and Swanson, 2011). In pre–B cells, Igκ loci replicate asynchronously and the early replicating allele is preferentially rendered accessible and selected for Igκ recombination (Mostoslavsky et al., 2001). The time between replication of Igκ loci might be sufficient to enable Igκ chains from the first allele to prevent Igκ rearrangements on the second allele. Yet experiments that show Igκ allelic exclusion is enforced by asynchronous replication between Igκ alleles have not been reported.The feedback model for allelic exclusion hypothesized that V(D)J recombination could activate transient intracellular signals that inhibit recombination on the second allele (Alt et al., 1980). RAG DSBs activate DNA-dependent protein kinase (DNA-PK), which forms an endonuclease with Artemis that processes CEs (Ma et al., 2002). RAG DSBs also activate Ataxia Telangiectasia mutated (ATM), which phosphorylates proteins to coordinate the cellular DSB response (Bredemeyer et al., 2006, 2008). In pre–B cells, RAG DSBs signal through ATM to initiate a genetic program that controls differentiation (Bredemeyer et al., 2008). ATM promotes high-density histone H2AX phosphorylation along RAG-cleaved loci (Savic et al., 2009). H2AX phosphorylation creates binding sites for MDC1, which retains activated ATM kinases around DSBs (Lou et al., 2006). The pools of activated ATM bound and not bound to H2AX/MDC1 exhibit different signaling capabilities (Celeste et al., 2002; Lou et al., 2006). In G1 phase cells, ATM promotes CJ formation independent of H2AX and MDC1 (Bredemeyer et al., 2006; Yin et al., 2009; Helmink et al., 2011). H2AX phosphorylation is detectable over only one Igκ locus in most pre–B cells including those with paired Igκ alleles (Hewitt et al., 2009). The fraction of cells with H2AX phosphorylation over both Igκ loci is fivefold higher in Atm^−/− mice relative to wild-type mice (Hewitt et al., 2009), suggesting that Igκ recombination initiates on a single paired allele and ATM bound to this allele acts on the other allele to prevent bi-allelic rearrangements (Hewitt et al., 2009). Detection of bi-allelic Igκ chromosome breaks in Atm-deficient pre–B cell lines provided support for this model (Hewitt et al., 2009).Here, we show in mice that inactivation of ATM causes a higher frequency of B cells expressing surface Igκ chains from both alleles. We show in primary pre–B cells that DSBs induced during Igκ recombination signal through ATM, but not DNA-PK, to suppress further Igκ rearrangements. Neither H2AX nor MDC1 is required for the ability of ATM to enforce Igκ allelic exclusion or inhibit Igκ recombination. Upon activation in response to RAG DSBs, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by Igκ cleavage transduce transient H2AX/MDC1-independent signals that suppress further Igκ rearrangements and thereby enforce Igκ allelic exclusion.     

Essential and Partially Overlapping Role of CD3γ and CD3δ for Development of αβ and γδ T Lymphocytes

CD3γ and CD3δ are two highly related components of the T cell receptor (TCR)–CD3 complex which is essential for the assembly and signal transduction of the T cell receptor on mature T cells. In gene knockout mice deficient in either CD3δ or CD3γ, early thymic development mediated by pre-TCR was either undisturbed or severely blocked, respectively, and small numbers of TCR-αβ⁺ T cells were detected in the periphery of both mice. γδ T cell development was either normal in CD3δ^−/− mice or partially blocked in CD3γ^−/− mice. To examine the collective role of CD3γ and CD3δ in the assembly and function of pre-TCR and in the development of γδ T cells, we generated a mouse strain with a disruption in both CD3γ and CD3δ genes (CD3γδ^−/−). In contrast to mice deficient in either CD3γ or CD3δ chains, early thymic development mediated by pre-TCR is completely blocked, and TCR-αβ⁺ or TCR-γδ⁺ T cells were absent in the CD3γδ^−/− mice. Taken together, these studies demonstrated that CD3γ and CD3δ play an essential, yet partially overlapping, role in the development of both αβ and γδ T cell lineages.     

Impairment of T and B Cell Development by Treatment with a Type I Interferon

Type I interferons α and β, naturally produced regulators of cell growth and differentiation, have been shown to inhibit IL-7–induced growth and survival of B cell precursors in vitro. After confirming an inhibitory effect on B lymphopoiesis in an ex vivo assay, we treated newborn mice with an active IFN-α2/α1 hybrid molecule to assess its potential for regulating B and T cell development in vivo. Bone marrow and splenic cellularity was greatly reduced in the IFN-α2/α1–treated mice, and B lineage cells were reduced by >80%. The bone marrow progenitor population of CD43⁺B220⁺HSA⁻ cells was unaffected, but development of the CD19⁺ pro–B cells and their B lineage progeny was severely impaired. Correspondingly, IL-7–responsive cells in the bone marrow were virtually eliminated by the interferon treatment. Thymus cellularity was also reduced by >80% in the treated mice. Phenotypic analysis of the residual thymocytes indicated that the inhibitory effect was exerted during the pro–T cell stage in differentiation. In IFN-α/β receptor^−/− mice, T and B cell development were unaffected by the IFN-α2/α1 treatment. The data suggest that type I interferons can reversibly inhibit early T and B cell development by opposing the essential IL-7 response.     

Ficus racemosa Stem Bark Extract: A Potent Antioxidant and a Probable Natural Radioprotector

Ethanol extract (FRE) and water extract (FRW) of Ficus racemosa (family: Moraceae) were subjected to free radical scavenging both by steady state and time resolved methods such as nanosecond pulse radiolysis and stopped-flow spectrophotometric analyses. FRE exhibited significantly higher steady state antioxidant activity than FRW. FRE exhibited concentration dependent DPPH, ABTS^•−, hydroxyl radical and superoxide radical scavenging and inhibition of lipid peroxidation with IC₅₀ comparable with tested standard compounds. In vitro radioprotective potential of FRE was studied using micronucleus assay in irradiated Chinese hamster lung fibroblast cells (V79). Pretreatment with different doses of FRE 1h prior to 2 Gy γ-radiation resulted in a significant (P < 0.001) decrease in the percentage of micronucleated binuclear V79 cells. Maximum radioprotection was observed at 20 μg/ml of FRE. The radioprotection was found to be significant (P < 0.01) when cells were treated with optimum dose of FRE (20 μg/ml) 1 h prior to 0.5, 1, 2, 3 and 4 Gy γ-irradiation compared to the respective radiation controls. The cytokinesis-block proliferative index indicated that FRE does not alter radiation induced cell cycle delay. Based on all these results we conclude that the ethanol extract of F. racemosa acts as a potent antioxidant and a probable radioprotector.     

Essential Role of Nuclear Factor κB in the Induction of Eosinophilia in Allergic Airway Inflammation

The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)- κB are incapable of mounting eosinophilic airway inflammation compared with wild-type mice. This deficiency was not due to a block in T cell priming or proliferation in the p50^−/− mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50^−/− mice were the lack of production of the Th2 cytokine interleukin 5 and the chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment, respectively, of eosinophils into the asthmatic airway. Additionally, the p50^−/− mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1α and MIP-1β that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-κB in vivo in the expression of important molecules that have been implicated in the pathogenesis of asthma.     

Peripheral Expression of Jak3 Is Required to Maintain T Lymphocyte Function

The Jak family tyrosine kinase, Jak3, is involved in signaling through cytokine receptors that utilize the common γ chain (γ_c), such as those for IL-2, IL-4, IL-7, IL-9, and IL-15. Recent studies of Jak3-deficient mice and humans have demonstrated that Jak3 plays a critical role in B and T lymphocyte maturation and function. The T lymphocyte defects in Jak3-deficient mice include a small thymus, a decrease in peripheral CD8⁺ cells, an increase in the surface expression of activation markers, and a severe reduction in proliferative and cytokine secretion responses to mitogenic stimuli. To determine whether the peripheral T lymphocyte defects result from aberrant maturation in the thymus or from the absence of Jak3 protein in peripheral T cells, we generated reconstituted mice that express normal levels of Jak3 protein in the thymus but lose Jak3 expression in peripheral T cells. Jak3 expression in the thymus restores normal T cell development, including CD8⁺, γδ, and natural killer cells. However, the loss of Jak3 protein in peripheral T cells leads to the Jak3^−/− phenotype, demonstrating that Jak3 is constitutively required to maintain T cell function.