Characterizing the BCR repertoire during lymphocyte reduction and recovery mediated by cyclophosphamide and granulocyte-macrophage colony-stimulating factor.

Leukopenia is a common manifestation of many diseases, including global outbreak SAS-CoV-2 infection. Granulocyte-macrophage colony-stimulating factor (GM -CSF) has been proved to be effective in promoting lymphocyte regeneration, but adverse immunological effects have also emerged. This study aim to investigate the effect of GM -CSF on BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration. Cyclophosphamide (CTX) and GM -CSF were used to inhibit and stimulate bone marrow hematopoiesis, respectively. High throughput sequencing was applied to detect the characteristics of BCR CDR3 repertoire in controls, CTX group and GM -CSF group. The white blood cells (WBCs) were quickly reduced (P < 0.05) with lymphocytes decreasing causing by CTX, and the WBCs and lymphocytes returned to the level of controls after GM -CSF treatment. The diversity of BCR heavy chain CDR3 repertoire was also significantly decreased in CTX group. Although there is still a big gap from the controls, the diversity was picked up after GM -CSF treatment. The expression of IGHD01-01, IGHD02-14 and IGHJ04-01 with high-frequency usage regularly and significantly changed in three groups, and many genes with low-frequency usage lost in CTX group and did not reappear in GM -CSF group. Moreover, two shared sequences and accounted for the highest proportion in GM -CSF group have been detected in animal model of chronic lymphocytic leukemia. These results revealed that GM -CSF can partially restore changes in the BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration, but it may also lead to rearrangement, proliferation and activation of abnormal B cells, which can provide a basis for further study on the adverse immunological effects and mechanism of GM -CSF treatment.

The Proteolytic Cleavage of Therapeutic Monoclonal Antibody Hinge Region: More Than a Matter of Subclass.

The hinge region of immunoglobulin G (IgG) is involved in C1q and FcγRIIIA-expressing natural killer (NK) cell recruitment. Both heavy chains (HCs) of the hinge region can be cleaved sequentially by several proteases of the tumor/inflammatory/infectious microenvironment, including matrix metalloproteinase 12 (MMP12), or immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS), impairing Fc-mediated functions. The cleavage of therapeutic monoclonal antibodies (TmAbs), which are based on a human IgG1, IgG2 or IgG4 structure, has been poorly investigated, although it may represent an escape mechanism to these treatments. Therefore, we used non-reducing SDS-PAGE to compare the cleavage kinetics of five IgG1 TmAbs (trastuzumab, rituximab, cetuximab, infliximab, ipilimumab), one IgG2 TmAb (panitumumab), and two IgG4 TmAbs (nivolumab and pembrolizumab) by MMP12 and IdeS, which were found to cleave the first and second HCs with different kinetics. Panitumumab was more protease-resistant than IgG1 and IgG4 TmAbs. The latter were usually more protease-sensitive, whereas IgG1 TmAbs were usually cleaved with intermediate kinetics. However, we observed intra-subclass variability among IgG4 and IgG1 TmAbs. Nivolumab and pembrolizumab were cleaved similarly by MMP12, whereas pembrolizumab was more IdeS-resistant. Ipilimumab was more IdeS-sensitive and MMP12-resistant than the other IgG1 TmAbs, regardless of G1m allotype. In addition the Fc fragment of IgG1 TmAbs were highly resistant to cleavage by MMP12, whereas their cleavage kinetic by IdeS was very similar to that observed with the intact forms (excluding ipilimumab). Importantly, the cleavage kinetic of ipilimumab Fc fragment by IdeS was superimposable to that of trastuzumab, cetuximab and infliximab Fc fragment, showing that the variability observed for intact ipilimumab is unrelated to its Fc portion. We propose that the variability in the cleavage sensitivity/resistance balance among TmAbs of IgG1 and IgG4 subclasses results partially, from TmAb characteristics related to and/or located in the Fab region. Finally, with ELISA and flow cytometry, we observed that a single cleavage of IgG1 TmAbs greatly decreased their affinity for FcγRIIIA and C1q and their ability to induce FcγRIIIA-dependent functional responses of NK cells. Overall, our results indicate that the cleavage of the hinge region should be considered with TmAbs treatment and in the development of new molecules.

Skewed primary Igκ repertoire and V-J joining in C57BL/6 mice: implications for recombination accessibility and receptor editing.

Previous estimates of the diversity of the mouse Ab repertoire have been based on fragmentary data as a result of many technical limitations, in particular, the many samples necessary to provide adequate coverage. In this study, we used 5'-coding end amplification of Igκ mRNAs from bone marrow, splenic, and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and nonfunctional Vκ repertoire. To evaluate the potential effects of receptor editing, we also compared V/J associations and usage in bone marrows of mouse mutants under constitutive negative selection or an altered ability to undergo secondary recombination. To focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of ~90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functional Vκ genes are used but at frequencies ranging from ~0.001 to ~10%. Usage of seven Vκ genes made up >40% of the repertoire. A small class of transcripts from apparently nonfunctional Vκ genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 Vκs × 4 Jκs), 398 (98.5%) were found at least once in our sample. For most Vκ transcripts, all Jκs were used, but V-J association biases were common. Usage patterns were remarkably stable in different selective conditions. Overall, the primary κ repertoire is highly skewed by preferred rearrangements, limiting Ab diversity, but potentially facilitating receptor editing.

PI3Kß plays a critical role in neutrophil activation by immune complexes.

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the ß isoform of phosphoinositide 3-kinase (PI3Kß) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kß alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kß and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kß by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the ß isoform of PI3K. PI3Kß-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kß and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kß as a potential therapeutic target in inflammatory disease.

Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample.

OBJECTIVE: Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes. DESIGN, PATIENTS AND MEASUREMENTS: Two heterohybridoma cell lines secreting TSHR autoantibodies were isolated using standard techniques from the lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies (160 units/l by inhibition of TSH binding). The ability of the two new monoclonal antibodies (MAbs; K1-18 and K1-70) to bind to the TSHR and compete with TSH or TSHR antibody binding was analysed. Furthermore, the effects of K1-18 and K1-70 on cyclic AMP production in Chinese hamster ovary cells (CHO) cells expressing the TSHR were investigated. RESULTS: One MAb (K1-18) was a strong stimulator of cyclic AMP production in TSHR-transfected CHO cells and the other (K1-70) blocked stimulation of the TSHR by TSH, K1-18, other thyroid-stimulating MAbs and patient serum stimulating type TSHR autoantibodies. Both K1-18 (IgG1 kappa) and K1-70 (IgG1 lambda) bound to the TSHR with high affinity (0.7 x 10(10) l/mol and 4 x 10(10) l/mol, respectively), and this binding was inhibited by unlabelled K1-18 and K1-70, other thyroid-stimulating MAbs and patient serum TSHR autoantibodies with stimulating or blocking activities. V region gene analysis indicated that K1-18 and K1-70 heavy chains used the same V region germline gene but different D and J germline genes as well as having different light chains. Consequently, the two antibodies have evolved separately from different B cell clones. CONCLUSIONS: This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.

T-lineage cells require the thymus but not VDJ recombination to produce IL-17A and regulate granulopoiesis in vivo.

IL-17A and IL-17F regulate granulopoiesis and are produced by memory T cells. Rag1(-/-) recombinase-activating gene-deficient mice cannot produce mature T cells but maintain normal neutrophil counts. Athymic nude mice are neutropenic or have near-normal neutrophil counts, depending on the prevailing intestinal flora, and do not produce IL-17A. By contrast, thymi from Rag1(-/-) mice contain as much IL-17A as those from wild-type (WT) mice. IL-17A-producing cells are found in the double negative DN1 compartment of the Rag1(-/-) thymus and express intracellular CD3. These cells colonize the spleen and mesenteric lymph node and secrete IL-17A in vitro following stimulation with IL-23 at a level similar to that of WT splenocytes. Adoptively transferred Rag1(-/-) or WT thymocytes correct neutrophil counts in neutropenic nude mice. We conclude that the development of IL-17A-producing T-lineage cells requires an intact thymic epithelium, but not V(D)J recombination.

Cutting edge: developmental stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci during B lymphocyte development.

Contraction of the large Igh and Igkappa loci brings all V genes, spanning >2.5 Mb in each locus, in proximity to DJ(H) or J(kappa) genes. CCCTC-binding factor (CTCF) is a transcription factor that regulates gene expression by long-range chromosomal looping. We therefore hypothesized that CTCF may be crucial for the contraction of the Ig loci, but no CTCF sites have been described in any V loci. Using ChIP-chip, we demonstrated many CTCF sites in the V(H) and V(kappa) regions. However, CTCF enrichment in the Igh locus, but not the Igkappa locus, was largely unchanged throughout differentiation, suggesting that CTCF binding alone cannot be responsible for stage-specific looping. Because cohesin can colocalize with CTCF, we performed chromatin immunoprecipitation for the cohesin subunit Rad21 and found lineage and stage-specific Rad21 recruitment to CTCF in all Ig loci. The differential binding of cohesin to CTCF sites may promote multiple loop formation and thus effective V(D)J recombination.

Developmental stage-specific regulation of TCR-alpha-chain gene assembly by intrinsic features of the TEA promoter.

The TCR delta- and alpha-chain genes lie in a single complex locus, the TCRalpha/delta locus. TCRdelta-chain genes are assembled in CD4(-)CD8(-) (double negative (DN)) thymocytes and TCRalpha-chain genes are assembled in CD4(+)CD8(+) (double positive) thymocytes due, in part, to the developmental stage-specific activities of the TCRdelta and TCRalpha enhancers (Edelta and Ealpha), respectively. Edelta functions with TCRdelta promoters to mediate TCRdelta-chain gene assembly in DN thymocytes. However, Edelta is unable to function with TCRalpha promoters such as the TEA promoter to drive TCRalpha-chain gene assembly in these cells. This is important, because the premature assembly of TCRalpha-chain genes in DN thymocytes would disrupt alphabeta and gammadelta T cell development. The basis for TEA inactivity in DN thymocytes is unclear, because Edelta can activate the Vdelta5 gene segment promoter that lies only 4 kb upstream of TEA promoter. In this study, we use gene targeting to construct a modified TCRalpha/delta locus (TCRalpha/delta(5DeltaT)) in which the TEA promoter lies in the same location as the Vdelta5 gene segment on the wild-type TCRalpha/delta allele. Remarkably, the TEA promoter on this allele exhibits normal developmental stage-specific regulation, being active in double positive thymocytes but not in DN thymocytes as is the case with the Vdelta5 promoter. Thus, the inactivity of the TEA promoter in DN thymocytes is due primarily to intrinsic developmental stage-specific features of the promoter itself and not to its location relative to other cis-acting elements in the locus, such as Edelta.

Clonal expansion of IgA-positive plasma cells and axon-reactive antibodies in MS lesions.

Immunoglobulin A (IgA), the predominant immunoglobulin class in mucosal secretions, has been found in the cerebrospinal fluid of patients with multiple sclerosis (MS). In this study we examined the infiltration of clonally expanded IgA plasma cells in lesions of MS brains. Sequences of complementarity-determining region 3 of IgA variable heavy chain (V(H)) genes demonstrated the clonal expansion of IgA-bearing plasma cells in MS lesions. Somatic mutations and ongoing intra-clonal mutations occurred in their V(H) genes. Immunohistochemical study demonstrated infiltration of dimer and polymer IgA1- and A2-positive plasma cells in perivascular spaces, in the parenchyma of MS lesions, and in the adjacent white matter. Double immunofluorescence staining showed binding of IgA antibody on axons and walls of microvessels in the areas of chronic active and inactive demyelination. Bielshowsky's silver impregnation revealed axonal damage in these areas. These findings suggest that IgA in the CNS are localized on axons in lesions and may contribute to axonal damage in MS.

Shark Ig light chain junctions are as diverse as in heavy chains.

We have characterized a small family of four genes encoding one of the three nurse shark Ig L chain isotypes, called NS5. All NS5 cDNA sequences are encoded by three loci, of which two are organized as conventional clusters, each consisting of a V and J gene segment that can recombine and one C region exon; the third contains a germline-joined VJ in-frame and the fourth locus is a pseudogene. This is the second nurse shark L chain type where both germline-joined and split V-J organizations have been found. Since there are only two rearranging Ig loci, it was possible for the first time to examine junctional diversity in defined fish Ig genes, comparing productive vs nonproductive rearrangements. N region addition was found to be considerably more extensive in length and in frequency than any other vertebrate L chain so far reported and rivals that in H chain. We put forth the speculation that the unprecedented efficiency of N region addition (87-93% of NS5 sequences) may be a result not only of simultaneous H and L chain rearrangement in the shark but also of processing events that afford greater accessibility of the V or J gene coding ends to terminal deoxynucleotidyltransferase.

Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination.

The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.

V(D)J recombination frequencies can be profoundly affected by changes in the spacer sequence.

Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two V(H)S107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is approximately 9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.

DNA-Rag protein interactions in the control of selective D gene utilization in the TCR beta locus.

Ordered assembly of Ag receptor genes by VDJ recombination is a key determinant of successful lymphocyte differentiation and function. Control of gene rearrangement has been traditionally viewed as a result of complex reorganization of the nucleochromatin mediated by several nuclear factors. Selective recombination of the variable (V) genes to the diversity (D), but not joining (J), gene segments within the TCRbeta locus has been shown to be controlled by recombination signal (RS) sequences that flank the gene segments. Through ex vivo and in vitro recombination assays, we demonstrate that the Rag proteins can discriminate between the RS of the D and J genes and enforce selective D gene incorporation into the TCRbeta variable domain in the absence of other nuclear factors or chromatin structure. DNA binding studies indicate that discrimination is not simply caused by higher affinity binding of the Rag proteins to the isolated 12RS of the D as opposed to the J genes. Furthermore, we also demonstrate that the 12RS within the TCRbeta locus is functionally inferior to the consensus 12RS. We propose that selective gene segment usage is controlled at the level of differential assembly and/or stability of synaptic RS complexes, and that evolutionary "deterioration" of the RS motifs may have been important to allow the VDJ recombinase to exert autonomous control over gene segment use during gene rearrangement.

A new model of sheep Ig diversification: shifting the emphasis toward combinatorial mechanisms and away from hypermutation.

The current model of Ig repertoire development in sheep focuses on the rearrangement of a small number (approximately 20) of Vlambda gene segments. It is believed that this limited combinatorial repertoire is then further diversified through postrearrangement somatic hypermutation. This process has been reported to introduce as many as 110 mutations/1000 nucleotides. In contrast, our data have that indicated somatic hypermutation may diversify the preimmune repertoire to a much lesser extent. We have identified 64 new Vlambda gene segments within the rearranged Ig repertoire. As a result, many of the unique nucleotide patterns thought to be the product of somatic hypermutation are actually hard-coded within the germline. We suggest that combinatorial rearrangement makes a much larger contribution, and somatic hypermutation makes a much smaller contribution to the generation of diversity within the sheep Ig repertoire than is currently acknowledged.

The kappa B transcriptional enhancer motif and signal sequences of V(D)J recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study.

BACKGROUND: The ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N) and the carboxyl-DNA binding domain (ZAS-C) of a representative family member, named kappaB DNA binding and recognition component (KRC), were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains. RESULTS: Both fusion proteins selected sequences that were similar to the kappaB motif or the canonical elements of the V(D)J recombination signal sequences (RSS) from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS. CONCLUSIONS: The RSS are cis-acting DNA motifs which are essential for V(D)J recombination of antigen receptor genes. Due to its specific binding affinity for RSS and kappaB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(D)J recombination.

Effect of environmental antigens on the Ig diversification and the selection of productive V-J joints in the bursa.

In chickens, a single set of unique functional segments of both Ig H and L chain genes is rearranged during early embryogenesis to generate a pool of B cell progenitors that will be diversified in the bursa by gene conversion, forming the preimmune repertoire. After hatching, bursal cells are exposed to environmental Ags in the bursal lumen. We prepared B cells from each single bursal follicle and used PCR-directed Ig L chain gene analysis to study the differentiation of B cells and the effect of antigenic stimulation from the bursal lumen on the neonatal chicken B cell repertoire formation. Selective amplification of B cell clones with a productive V-J joint was observed during the late embryonic stage, possibly by the interaction with ligands expressed on the bursal stroma and further accelerated in the neonatal chicken. Administration of the artificial Ags into the bursal lumen before the isolation of bursa by bursal duct ligation in the embryo caused a significant increase in lymphocytes with a productive V-J joint in the neonatal chicken bursa compared with the isolated bursa. Intra- and interclonal diversity of a complementarity-determining region measured by an evolutionary distance increased during bursal development. Clonal diversification did not require stimulation by artificial Ags from the bursal lumen. Thus, the preimmune repertoire in the bursa is generated by gene conversion during Ag-independent B cell proliferation, and antigenic stimulation from the bursal epithelium to bursal B cells plays roles in the selection of clones with a productive V-J joint.

The RAG proteins in V(D)J recombination: more than just a nuclease.

V(D)J recombination is the process that generates the diversity among T cell receptors and is one of three mechanisms that contribute to the diversity of antibodies in the vertebrate immune system. The mechanism requires precise cutting of the DNA at segment boundaries followed by rejoining of particular pairs of the resulting termini. The imprecision of aspects of the joining reaction contributes significantly to increasing the variability of the resulting functional genes. Signal sequences target DNA recombination and must participate in a highly ordered protein-DNA complex in order to limit recombination to appropriate partners. Two proteins, RAG1 and RAG2, together form the nuclease that cleaves the DNA at the border of the signal sequences. Additional roles of these proteins in organizing the reaction complex for subsequent steps are explored.

Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase.

V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.

Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage.

The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (mu(0)) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F(1) (DQ52(+/-)) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52(-) allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM(+) B cells of bone marrow and spleen used the DQ52(-) allele less frequently. On DJ joints of the DQ52(-) allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52(-) allele. In splenic B cells of the DQ52(-/-) mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3' JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.

Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination.

DNA-dependent protein kinase (DNA-PK) is utilized in both DNA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these processes is unknown. To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombination in the V(D)J recombinase inducible B cell line HDR37. Wortmannin radiosensitized HDR37, but had no affect on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wild-type human DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstrated that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombination. These apparently contradictory results were reconciled by analyses of cell lines varying in their expression of recombinant wild-type human DNA-PKcs. These studies establish that minimal DNA-PKcs protein levels are sufficient to support V(D)J recombination, but insufficient to confer resistance to ionizing radiation.