Tumor Necrosis Factor α and Lymphotoxin α Are Not Required for Induction of Acute Experimental Autoimmune Encephalomyelitis

Immunization of mice with myelin components results in experimental autoimmune encephalomyelitis (EAE), which is mediated by myelin-specific CD4⁺ T cells and anti-myelin antibodies. Tumor necrosis factor α (TNF-α) and lymphotoxin α (LT-α) are thought to be involved in the events leading to inflammatory demyelination in the central nervous system. To ascertain this hypothesis 129 × C57BL/6 mice with an inactivation of the tnf and lta genes (129 × C57BL/6^−/−) and SJL/J mice derived from backcrosses of the above mentioned mutant mice (SJL^−/−) were immunized with mouse spinal cord homogenate (MSCH) or proteolipid protein. Both 129 × C57BL/6^−/− mice and SJL^−/− mice developed EAE. In SJL^−/− mice immunized with MSCH, a very severe form of EAE with weight loss, paralysis of all four limbs, and lethal outcome was observed. The histologic hallmark was an intense perivascular and parenchymal infiltration with predominantly CD4⁺ T cells and some CD8⁺ T cells associated with demyelination in both brain and spinal cord. These results indicate that TNF-α and LT-α are not essential for the development of EAE.     

NF-κB RelA-deficient Lymphocytes: Normal Development of T Cells and B Cells, Impaired Production of IgA and IgG1 and Reduced Proliferative Responses

To investigate the function of NF-κB RelA (p65), we generated mice deficient in this NF-κB family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the SCID mice receiving relA−/− fetal liver transplants, similar to the relA+/+ and +/− cases. T cells were found to mature to Thy-1⁺/TCRαβ⁺/CD3⁺/CD4⁺ or CD8⁺, while B cells had the ability to differentiate to IgM⁺/B220⁺ and to secrete immunoglobulins. However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3+anti-CD28, LPS, anti-IgM, and PMA+calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.     

CD40 Ligand Is Not Essential for Induction of Type 1 Cytokine Responses or Protective Immunity after Primary or Secondary Infection With Histoplasma capsulatum

The induction of type 1 immune responses (interleukin [IL]-12, interferon [IFN]-γ) has been shown to be important in mediating protection against many intracellular infections including Histoplasma capsulatum. Costimulatory molecules such as CD40 ligand (CD40L) have been shown to be a central regulator of type 1 responses in vivo. To study the role of CD40L in mediating protection against infection with H. capsulatum, CD40L-deficient (CD40L^−/−) and CD40L^+/+ mice were infected with H. capsulatum and assessed for various parameters. After a lethal challenge of H. capsulatum, CD40L^−/− mice were not substantially different from CD40L^+/+ mice in terms of mortality, fungal burden, or production of IFN-γ, IL-12, nitric oxide, or tumor necrosis factor α. Moreover, CD40L^−/− mice treated with anti–IFN-γ or anti–IL-12 at the time of infection had accelerated mortality, providing further evidence that IL-12 and IFN-γ are produced in vivo in the absence of CD40L. In addition, CD40L^−/− mice infected with a sublethal dose of H. capsulatum survived infection, whereas all mice infected with the same dose and treated with anti–IFN-γ had accelerated mortality, demonstrating that IFN-γ but not CD40L was essential for primary immunity to H. capsulatum infection. Interestingly, depletion of either CD4⁺ or CD8⁺ T cells resulted in accelerated mortality in CD40L^−/− mice, suggesting a critical role for these cells in response to infection. Finally, CD40L^−/− mice initially infected with a sublethal dose of H. capsulatum were protected from secondary infection with a lethal dose of H. capsulatum, demonstrating that CD40L is not required for the maintenance of memory immunity.     

Identification of a Committed T Cell Precursor Population in Adult Human Peripheral Blood

Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4⁺CD3⁺αβ⁺ mature T cells. These cells, which represent 0.1–0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor α (pTα) gene, CD3-γ, CD-δ and CD-ε, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-β locus (D–J). Moreover, low levels of CD3ε protein, but not of TCR-β chain, can be detected in their cytoplasm. Our results suggest that CD4⁺CD3⁻ cells identified in peripheral blood are different from CD3⁻CD4⁺CD8⁻ thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.     

TAP-1 indirectly regulates CD4+ T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-gamma production

To investigate if transporter associated with antigen processing (TAP)–1 is required for CD8⁺ T cell–mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP-1^−/−, CD8^−/−, and wild-type (WT) mice to infection with the parasite. Unexpectedly, TAP-1^−/− mice displayed greater susceptibility than CD8^−/−, β₂-microglobulin^−/− (β₂m^−/−), or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP-1^−/− mice correlated with a reduction in the frequency of activated (CD62L^low CD44^hi) and interferon (IFN)-γ–producing CD4⁺ T cells. Interestingly, infected TAP-1^−/− mice also showed reduced numbers of IFN-γ–producing natural killer (NK) cells relative to WT, CD8^−/−, or β₂m^−/− mice, and after NK cell depletion both CD8^−/− and WT mice succumbed to infection with the same kinetics as TAP-1^−/− animals and displayed impaired CD4⁺ T cell IFN-γ responses. Moreover, adoptive transfer of NK cells obtained from IFN-γ^+/+, but not IFN-γ^−/−, animals restored the CD4⁺ T cell response of infected TAP-1^−/− mice to normal levels. These results reveal a role for TAP-1 in the induction of IFN-γ–producing NK cells and demonstrate that NK cell licensing can influence host resistance to infection through its effect on cytokine production in addition to its role in cytotoxicity.     

A Role for Fas in Negative Selection of Thymocytes In Vivo

To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1–100 μg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vβ8⁺ cells at the level of the semi-mature population of HSA^hi CD4⁺ 8⁻ cells found in the thymic medulla; deletion of CD4⁺ 8⁺ cells was minimal. SEB injection also caused marked elimination of Vβ8⁺ HSA^hi CD4⁺ 8⁻ thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (≤1 μg) but not by high doses (100 μg). Similar findings applied when T cell receptor transgenic mice were injected with specific peptide. These findings suggest that clonal elimination of semi-mature medullary T cells is Fas independent at low doses of antigen but Fas dependent at high doses. Previous reports documenting that negative selection is not obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level.     

Spontaneous Autoimmune Diabetes in Monoclonal T Cell Nonobese Diabetic Mice

It has been established that insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a CD4⁺ and CD8⁺ T cell–dependent autoimmune process directed against the pancreatic beta cells. The precise roles that beta cell–reactive CD8⁺ and CD4⁺ T cells play in the disease process, however, remain ill defined. Here we have investigated whether naive beta cell–specific CD8⁺ and CD4⁺ T cells can spontaneously accumulate in pancreatic islets, differentiate into effector cells, and destroy beta cells in the absence of other T cell specificities. This was done by introducing K^d– or I-A^g7–restricted beta cell–specific T cell receptor (TCR) transgenes that are highly diabetogenic in NOD mice (8.3- and 4.1-TCR, respectively), into recombination-activating gene (RAG)-2–deficient NOD mice, which cannot rearrange endogenous TCR genes and thus bear monoclonal TCR repertoires. We show that while RAG-2^−/− 4.1-NOD mice, which only bear beta cell–specific CD4⁺ T cells, develop diabetes as early and as frequently as RAG-2⁺ 4.1-NOD mice, RAG-2^−/− 8.3-NOD mice, which only bear beta cell–specific CD8⁺ T cells, develop diabetes less frequently and significantly later than RAG-2⁺ 8.3-NOD mice. The monoclonal CD8⁺ T cells of RAG-2^−/− 8.3-NOD mice mature properly, proliferate vigorously in response to antigenic stimulation in vitro, and can differentiate into beta cell–cytotoxic T cells in vivo, but do not efficiently accumulate in islets in the absence of a CD4⁺ T cell–derived signal, which can be provided by splenic CD4⁺ T cells from nontransgenic NOD mice. These results demonstrate that naive beta cell– specific CD8⁺ and CD4⁺ T cells can trigger diabetes in the absence of other T or B cell specificities, but suggest that efficient recruitment of naive diabetogenic beta cell–reactive CD8⁺ T cells to islets requires the assistance of beta cell–reactive CD4⁺ T cells.     

T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells

IgM⁺IgD⁺CD27⁺ B cells from peripheral blood have been described as circulating marginal zone B cells. It is still unknown when and where these cells develop. These IgM⁺IgD⁺CD27⁺ B cells exhibit somatic hypermutations (SHMs) in their B cell receptors, but the exact nature of the signals leading to induction of these SHMs remains elusive. Here, we show that IgM⁺IgD⁺CD27⁺ B cells carrying SHMs are observed during human fetal development. To examine the role of T cells in human IgM⁺IgD⁺CD27⁺ B cell development we used an in vivo model in which Rag2^−/−γ_C^−/− mice were repopulated with human hematopoietic stem cells. Using Rag2^−/−γ_C^−/− mice on a Nude background, we demonstrated that development and induction of SHMs of human IgM⁺IgD⁺CD27⁺ B cells can occur in a T cell–independent manner.     

The RAG1 N-terminal region regulates the efficiency and pathways of synapsis for V(D)J recombination

Immunoglobulin and T cell receptor gene assembly depends on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1–383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo is poorly understood. We generated mice in which RAG1 lacks ubiquitin ligase activity (P326G), the major site of autoubiquitination (K233R), or its first 215 residues (Δ215). While few abnormalities were detected in R1.K233R mice, R1.P326G mice exhibit multiple features indicative of reduced recombination efficiency, including an increased Igκ⁺:Igλ⁺ B cell ratio and decreased recombination of Igh, Igκ, Igλ, and Tcrb loci. Previous studies indicate that synapsis of recombining partners during Igh recombination occurs through two pathways: long-range scanning and short-range collision. We find that R1Δ215 mice exhibit reduced short-range Igh and Tcrb D-to-J recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by multiple pathways, including control of the balance between short- and long-range recombination.     

Infected Cell Protein (ICP)47 Enhances Herpes Simplex Virus Neurovirulence by Blocking the CD8+ T Cell Response

The herpes simplex virus (HSV) infected cell protein (ICP)47 blocks CD8⁺ T cell recognition of infected cells by inhibiting the transporter associated with antigen presentation (TAP). In vivo, HSV-1 replicates in two distinct tissues: in epithelial mucosa or epidermis, where the virus enters sensory neurons; and in the peripheral and central nervous system, where acute and subsequently latent infections occur. Here, we show that an HSV-1 ICP47⁻ mutant is less neurovirulent than wild-type HSV-1 in mice, but replicates normally in epithelial tissues. The reduced neurovirulence of the ICP47⁻ mutant was due to a protective CD8⁺ T cell response. When compared with wild-type virus, the ICP47⁻ mutant expressed reduced neurovirulence in immunologically normal mice, and T cell–deficient nude mice after reconstitution with CD8⁺ T cells. However, the ICP47⁻ mutant exhibited normal neurovirulence in mice that were acutely depleted of CD8⁺ T cells, and in nude mice that were not reconstituted, or were reconstituted with CD4⁺ T cells. In contrast, CD8⁺ T cell depletion did not increase the neurovirulence of an unrelated, attenuated HSV-1 glycoprotein (g)E⁻ mutant. ICP47 is the first viral protein shown to influence neurovirulence by inhibiting CD8⁺ T cell protection.     

Subsets of Transgenic T Cells That Recognize CD1 Induce or Prevent Murine Lupus: Role of Cytokines

T cells with T cell receptor (TCR) transgenes that recognized CD1 on syngeneic B cells stimulated B cells to secrete immunoglobulins in vitro. The CD4⁺, CD8⁺, or CD4⁻CD8⁻ T cells from the spleen of the TCR transgenic BALB/c donors induced lupus with anti–double stranded DNA antibodies, proteinuria, and immune complex glomerulonephritis in irradiated BALB/c nude mice reconstituted with nude bone marrow. Injection of purified CD4⁻CD8⁻ T cells from the marrow of transgenic donors prevented the induction of lupus by the transgenic T cells. Transgenic T cells that induced lupus secreted large amounts of interferon (IFN)-γ and little interleukin (IL)-4, and those that prevented lupus secreted large amounts of IL-4 and little IFN-γ or IL-10.     

The Spliceosomal Phosphopeptide P140 Controls the Lupus Disease by Interacting with the HSC70 Protein and via a Mechanism Mediated by γδ T Cells

The phosphopeptide P140 issued from the spliceosomal U1-70K snRNP protein is recognized by lupus CD4⁺ T cells, transiently abolishes T cell reactivity to other spliceosomal peptides in P140-treated MRL/lpr mice, and ameliorates their clinical features. P140 modulates lupus patients' T cell response ex vivo and is currently included in phase IIb clinical trials. Its underlying mechanism of action remains elusive. Here we show that P140 peptide binds a unique cell-surface receptor, the constitutively-expressed chaperone HSC70 protein, known as a presenting-protein. P140 induces apoptosis of activated MRL/lpr CD4⁺ T cells. In P140-treated mice, it increases peripheral blood lymphocyte apoptosis and decreases B cell, activated T cell, and CD4⁻CD8⁻B220⁺ T cell counts via a specific mechanism strictly depending on γδ T cells. Expression of inflammation-linked genes is rapidly regulated in CD4⁺ T cells. This work led us to identify a powerful pathway taken by a newly-designed therapeutic peptide to immunomodulate lupus autoimmunity.     

A human postnatal lymphoid progenitor capable of circulating and seeding the thymus

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34⁺CD10⁺ progenitor population and which is distinct from B cell–committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin⁻CD34⁺CD10⁺CD24⁻ progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor α, and CD3ε. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.     

Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV–deficient B cells

Immunoglobulin heavy chain class switch recombination (CSR) is believed to occur through the generation and repair of DNA double-strand breaks (DSBs) in the long and repetitive switch regions. Although implied, the role of the major vertebrate DSB repair pathway, nonhomologous end joining (NHEJ), in CSR has been controversial. By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR. Disruption of the Lig4 gene in CH12F3 cells severely inhibits the initial rate of CSR and causes a late cell proliferation defect under cytokine stimulation. However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4-null cells. The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.DNA double-strand breaks (DSBs) are considered by many to be the most severe form of DNA damage, and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). NHEJ is the primary DSB repair pathway in mammalian cells (1). A characteristic feature of NHEJ is the lack of long stretches of homology at the site of joining (junction), although a few base pairs of microhomology (nucleotides that can be assigned to either of the two DNA ends) are seen at 10–40% of junctions (2). The core components of the NHEJ pathway have been well characterized over the last decade. These include DNA end-binding complex Ku70–Ku80, DNA-dependent protein kinase (DNA-PKcs), Artemis, and the ligase complex x-ray cross complementation (XRCC) 4–DNA ligase IV (Lig4). Several accessory factors, including an XRCC4-like factor (known as XLF or Cernunnos) and polymerases μ and λ, were identified in the following years. However, when NHEJ is disrupted by mutations to its core components, many forms of DSBs can still be joined, suggesting the existence of an alternative route for end joining.During lymphocyte development, physiological DSBs arise as critical intermediates in V(D)J and class switch recombination (CSR). V(D)J recombination is a site-specific DNA recombination that assembles the antigen-binding domain of Ig and T cell receptor genes. The RAG protein complex recognizes recombination signal sequences (RSS) next to the V, D, or J coding segments and cleaves the DNA at the RSS-coding sequence junction. A concerted cleavage of a pair of RSS generates four DNA ends that are joined to form a coding joint and a signal joint, respectively. CSR changes the constant domain of the Ig heavy chain, which allows a B cell to switch from expressing IgM to another isotype (IgG, IgA, or IgE). CSR is directed by kilobase-long repetitive switch regions (Sμ, Sγ, Sα, and Sε) lying upstream of each constant region and requires activation-induced cytidine deaminase (AID). The preponderant experimental evidence is consistent with a cut-and-paste mechanism in a way similar to that of V(D)J recombination (3). First, the deleted region is circularized before its disposal. Second, DSBs were detected in switching B cells by ligation-mediated PCR. Third, phosphorylated histone H2AX (a marker for chromosomal breaks) foci were detected in switching B cells at the IgH locus in an AID-dependent manner. Finally, switch junctions show little or no homology, consistent with direct joining of switch region breaks through NHEJ. Although the exact mechanism for switch region breaks remains unclear, the current view is that AID catalyzes cytidine deamination (converts cytidines to uracils) in the switch regions, followed by the repair of uracils that ultimately results in DSBs (3).In contrast to V(D)J recombination, which absolutely requires NHEJ, direct testing of the role of NHEJ in CSR has met with considerable challenge. One obvious obstacle is that NHEJ-deficient animals cannot generate mature lymphocytes necessary for a CSR assay because the V(D)J recombination defect blocks T and B cell development. To circumvent the developmental block on B cells, Ku- or DNA-PKcs–deficient mice have been crossed with mice harboring preassembled Ig heavy and light chain genes to generate monoclonal NHEJ-deficient B cells (4–7). By this method, CSR was found completely abolished in the absence of Ku70 or Ku80 (4, 5). However, this conclusion must be interpreted with caution, because Ku may have non-NHEJ–related functions (e.g., telomere maintenance) and Ku deficiency causes defective cell proliferation (4, 5). As for DNA-PKcs, controversial results were obtained between a point mutation that inactivates DNA-PKcs and a complete deletion of the gene (6, 7). Although catalytically inactive DNA-PKcs allows normal CSR (7), deletion of the DNA-PKcs gene inhibited CSR to all isotypes except IgG1 (6). A caveat of this approach is that there are no T cells in the B cell–reconstituted mice. As a result, these mice have greatly reduced B cell numbers (4–6) and the B cells are inactive (8). The importance of T cell regulation was demonstrated by a later study showing restored CSR in DNA-PKcs–null mice upon T cell transplantation from a B cell–deficient host (8).In contrast to Ku and DNA-PKcs, XRCC4 and Lig4 have no identified function outside NHEJ and their inactivation results in the most severe form of NHEJ deficiency. Therefore, XRCC4 and Lig4 are the most suitable and specific targets for abolishing NHEJ. However, the B cell reconstitution strategy is not feasible for XRCC4 or Lig4 because deletion of either gene in mice results in embryonic lethality (9, 10). Although p53 deficiency can extend the life of XRCC4 and Lig4 knockout mice, they all succumb to pro–B cell lymphomas shortly after birth (11, 12). Recently, two independent studies used conditional knockout methods to delete XRCC4 in mature B cells (13, 14). Both studies found that CSR was reduced but not abolished in the absence of XRCC4. It is known that XRCC4-deficient cells have a very low level of Lig4. However, it was also reported that even a very low level of Lig4 is still sufficient for NHEJ (15).To unequivocally determine the role of NHEJ in CSR while avoiding the complications associated with some of the animal models, we disrupted the Lig4 gene in a B cell line (CH12F3) capable of highly efficient cytokine-induced CSR in vitro (16). Like its parental line CH12.LX (17, 18), CH12F3 has a mostly stable diploid genome and is thus suitable for gene targeting. We chose to disrupt the Lig4 gene not only because of its central role in NHEJ but also because it is dispensable for somatic cell growth (19). We found that deletion of Lig4 in CH12F3 cells mildly reduced CSR after 3 d of cytokine stimulation, which is qualitatively and quantitatively similar to what was observed with XRCC4-deficient B cells, as well as the Lig4^−/− p53^−/− B cells (13, 14). However, we also found a proliferation defect of Lig4-null CH12F3 cells in the presence of cytokine stimulation. This led us to another important finding that the initial rate of CSR is severely inhibited in Lig4-null cells. Thus, the initially observed mild reduction appears to be an underestimation of a marked CSR defect, which is masked by an enrichment of switched cells. These observations reveal the kinetics of a fast-acting NHEJ and a slow-acting alternative end joining during CSR.     

Effect of Ku80 Deficiency on Mutation Frequencies and Spectra at a LacZ Reporter Locus in Mouse Tissues and Cells

Non-homologous end joining (NHEJ) is thought to be an important mechanism for preventing the adverse effects of DNA double strand breaks (DSBs) and its absence has been associated with premature aging. To investigate the effect of inactivated NHEJ on spontaneous mutation frequencies and spectra in vivo and in cultured cells, we crossed a Ku80-deficient mouse with mice harboring a lacZ-plasmid-based mutation reporter. We analyzed various organs and tissues, as well as cultured embryonic fibroblasts, for mutations at the lacZ locus. When comparing mutant with wild-type mice, we observed a significantly higher number of genome rearrangements in liver and spleen and a significantly lower number of point mutations in liver and brain. The reduced point mutation frequency was not due to a decrease in small deletion mutations thought to be a hallmark of NHEJ, but could be a consequence of increased cellular responses to unrepaired DSBs. Indeed, we found a substantial increase in persistent 53BP1 and γH2AX DNA damage foci in Ku80^−/− as compared to wild-type liver. Treatment of cultured Ku80-deficient or wild-type embryonic fibroblasts, either proliferating or quiescent, with hydrogen peroxide or bleomycin showed no differences in the number or type of induced genome rearrangements. However, after such treatment, Ku80-deficient cells did show an increased number of persistent DNA damage foci. These results indicate that Ku80-dependent repair of DNA damage is predominantly error-free with the effect of alternative more error-prone pathways creating genome rearrangements only detectable after extended periods of time, i.e., in young adult animals. The observed premature aging likely results from a combination of increased cellular senescence and an increased load of stable, genome rearrangements.     

DNA-dependent protein kinase activity is not required for immunoglobulin class switching

Class switch recombination (CSR), similar to V(D)J recombination, is thought to involve DNA double strand breaks and repair by the nonhomologous end-joining pathway. A key component of this pathway is DNA-dependent protein kinase (DNA-PK), consisting of a catalytic subunit (DNA-PKcs) and a DNA-binding heterodimer (Ku70/80). To test whether DNA-PKcs activity is essential for CSR, we examined whether IgM(+) B cells from scid mice with site-directed H and L chain transgenes were able to undergo CSR. Although B cells from these mice were shown to lack DNA-PKcs activity, they were able to switch from IgM to IgG or IgA with close to the same efficiency as B cells from control transgenic and nontransgenic scid/+ mice, heterozygous for the scid mutation. We conclude that CSR, unlike V(D)J recombination, can readily occur in the absence of DNA-PKcs activity. We suggest nonhomologous end joining may not be the (primary or only) mechanism used to repair DNA breaks during CSR.     

AID expression levels determine the extent of cMyc oncogenic translocations and the incidence of B cell tumor development

Immunoglobulin (Ig) isotype switching is a recombination event that changes the constant domain of antibody genes and is catalyzed by activation-induced cytidine deaminase (AID). Upon recruitment to Ig genes, AID deaminates cytidines at switch (S) recombination sites, leading to the formation of DNA breaks. In addition to their role in isotype switching, AID-induced lesions promote Igh-cMyc chromosomal translocations and tumor development. However, cMyc translocations are also present in lymphocytes from healthy humans and mice, and thus, it remains unclear whether AID directly contributes to the dynamics of B cell transformation. Using a plasmacytoma mouse model, we show that AID^+/− mice have reduced AID expression levels and display haploinsufficiency both in the context of isotype switching and plasmacytomagenesis. At the Ig loci, AID^+/− lymphocytes show impaired intra- and inter-switch recombination, and a substantial decrease in the frequency of S mutations and chromosomal breaks. In AID^+/− mice, these defects correlate with a marked decrease in the accumulation of B cell clones carrying Igh-cMyc translocations during tumor latency. These results thus provide a causality link between the extent of AID enzymatic activity, the number of emerging Igh-cMyc–translocated cells, and the incidence of B cell transformation.     

人白血病细胞株重组激活基因表达及T细胞受体基因重排的检测

背景:淋巴细胞特异性重组激活基因编码的重组激活基因1与重组激活基因2蛋白是参与V(D)J重排机制的重要的重组酶。除参与V(D)J重排以外,近年的研究结果表明重组激活基因介导的转位作用可能与染色体易位及淋巴性恶性肿瘤的发生有关,但迄今尚未有明确定论。目的:检测重组激活基因、DNA修复因子Ku70/Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴瘤细胞株的发生情况。设计:重复测量实验。单位:南方医科大学生物技术学院分子免疫研究所。材料:T淋巴白血病细胞株Jurkat和6T-CEM购自上海细胞生物研究所;T淋巴白血病细胞株Molt-4,皮肤T细胞淋巴瘤细胞株HuT102,Burkitt’s淋巴瘤细胞株Raji和Daudi以及原髓细胞白血病细胞株HL-60和慢性髓原白血病细胞株K562均由本实验室保存。细胞用含有体积分数0.1胎牛血清的RPMI1640培养基于37℃,体积分数0.05CO2条件下培养。方法:实验于2005-10/2006-01在南方医科大学生物技术学院分子免疫研究所完成。采用反转录聚合酶链反应检测重组激活基因1,重组激活基因2,非同源末端连接装置途径中的DNA修复因子Ku70/Ku80,以及末端脱氧核苷转移酶mRNA表达;采用巢式、半巢式聚合酶链反应、连接介导的聚合酶链反应等方法检测T细胞受体重排删除DNA环和T细胞受体β链重组信号序列两端的断裂点。了解参与V(D)J重排过程的基因表达和T细胞受体基因重排中间体的产生情况。主要观察指标:重组激活基因、DNA修复因子Ku70/Ku80和末端脱氧核苷转移酶mRNA表达以及T细胞受体基因重排在人白血病和淋巴瘤细胞株的发生情况。结果:反转录聚合酶链反应检测结果显示:重组激活基因1mRNA在4种T细胞株中均被检测到,在两种B细胞株和两种髓性白血病细胞株中未检测到;重组激活基因2和末端脱氧核苷转移酶mRNA表达仅在Jurkat,Molt-4和6T-CEM3种T细胞株中检测到,但在6T-CEM表达较弱;除HL-60细胞未检测到Ku80表达外,所有细胞株均检测到Ku70和Ku80表达。对4种T细胞株T细胞受体重排中间体检测结果表明:仅在Jurkat细胞中检测到Dβ2-Jβ2sjTRECs与Dβ25’端和3’RSS断点,表明Jurkat细胞发生T细胞受体基因重排。同时发现JurkatTCRDβ2-Jβ2重排删除环结合区具有明显的多样性特征。结论:重组激活基因可能与T细胞白血病具有更为密切的关系。Jurkat细胞有可能成为研究重组激活基因与T细胞淋巴性肿瘤的一个潜在的细胞模型。     

Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination

V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice.