Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast

The objective of this study was to determine whether tumor-infiltrating B cells (TIL-B) of infiltrating ductal carcinoma (IDC) of the breast represent a tumor-specific humoral immune response. Immunohistochemical analysis of three Her-2/neu-negative IDC tumors from geriatric patients showed that TIL-B cluster in structures similar to germinal centers containing CD20(+) B lymphocyte and CD3(+) T lymphocyte zones with interdigitating CD21(+) follicular dendritic cells, suggesting an in situ immune response. A total of 29, 31, and 58 IgG1 H chain clones was sequenced from the three IDC tumors, respectively. Intratumoral oligoclonal expansion of TIL-B was demonstrated by a preponderance (45-68%) of clonal B cells. In contrast, only 7% of tumor-draining lymph node and 0% of healthy donor PBL IgG H chains were clonal, consistent with the larger repertoires of node and peripheral populations. Patterns and levels of TIL-B IgG H chain somatic hypermutation suggested affinity maturation in intratumoral germinal centers. To examine the specificity of TIL-B Ig, a phage-displayed Fab library was generated from the TIL-B of one IDC tumor. Panning with an allogeneic breast cancer cell line enriched Fab binding to breast cancer cells, but not nonmalignant cell lines tested. However, panning with autologous tumor tissue lysate increased binding of Fab to both tumor tissue lysate and healthy breast tissue lysate. These data suggest an in situ Ag-driven oligoclonal B cell response to a variety of tumor- and breast-associated Ags.     

Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires

Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (V(H) and V(L)) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the V(H)1 and V(H)4 families, for example, V(H)1-46, V(H)4-31, and V(H)4-61. This gene segment bias differed markedly from the V(H)3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant V(H) segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2-11 mo and those of adults were highly related when compared by V(H), D, J(H), V(L), and J(L) segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.     

Molecular characterization of the anti-idiotypic immune response of a relapse-free neuroblastoma patient following antibody therapy: a possible vaccine against tumors of neuroectodermal origin?

Neuroblastoma treatment with chimeric antidisialoganglioside GD2 Ab ch14.18 showed objective antitumor responses. Production of anti-idiotypic Abs (Ab2) against ch14.18 (Ab1) in some cases was positively correlated with a more favorable prognosis. According to Jerne's network theory, a subset of anti-idiotypic Abs (Ab2beta) carries an "internal image" of the Ag and induces Abs (Ab3) against the original Ag. The molecular origin of an anti-idiotypic Ab response in tumor patients was not investigated previously. To clone anti-idiotypic Abs, B cells of a ch14.18-treated neuroblastoma patient with Ab2 serum reactivity were used to construct Ab phage display libraries. After repeated biopannings on ch14.18 and its murine relative, anti-GD2 mAb 14G2a, we selected 40 highly specific clones. Sequence analysis revealed at least 10 of 40 clones with different Ig genes. Identities to putative germline genes ranged between 94.90 and 100% for V(H) and between 93.90 and 99.60% for V(L). An overall high rate of replacement mutations suggested a strong Ag-driven maturation of the anti-idiotypic Abs. Two clones that were analyzed further, GK2 and GK8, inhibited binding of ch14.18 to GD2 just as the patient's serum did. GK8 alone inhibited >80% of the patient's anti-idiotypic serum Abs in binding to ch14.18. Rabbits vaccinated with GK8 or GK2 (weaker) produced Ab3 against the original target Ag GD2. GK8 may be useful as a tumor vaccine for GD2-positive [corrected] tumors.     

Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the V(H)1-69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of V(H)1-69(+) cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of V(H)1-69(+) B cells: naive (small, IgM(high)IgD(high)CD38(+)CD27(-)CD21(high)CD95(-)CD5(-)), "early memory" (medium-sized, IgM(high)IgD(low)CD38(-)CD27(+)CD21(low)CD95(+)CD5(+)), and "late memory" (large-sized, IgM(low)IgD(low-neg)CD38(-)CD27(low)CD21(low-neg)CD5(-)CD95(-)). The B cells expanded in cryoglobulinemia patients have a "memory" phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated V(H)1-69(+) B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal V(H)1-69(+) B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a V(H)1-69(+) natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.     

Inhibitor of glutamine metabolism V9302 promotes ROS-induced autophagic degradation of B7H3 to enhance antitumor immunity

Despite the enormous successes of anti-PD-1/PD-L1 immunotherapy in multiple other cancer types, the overall response rates of breast cancer remain suboptimal. Therefore, exploring additional immune checkpoint molecules for potential cancer treatment is crucial. B7H3, a T-cell coinhibitory molecule, is specifically overexpressed in breast cancer compared with normal breast tissue and benign lesions, making it an attractive therapeutic target. However, the mechanism by which B7H3 contributes to the cancer phenotype is unclear. Here we show that the expression of B7H3 is negatively related to the number of CD8⁺ T cells in breast tumor sites. In addition, analysis of the differentially expressed B7H3 reveals that it is inversely correlated to autophagic flux both in breast cancer cell lines and clinical tumor tissues. Furthermore, block of autophagy by bafilomycin A1 (Baf A1) increases B7H3 levels and attenuates CD8⁺ T cell activation, while promotion of autophagy by V9302, a small-molecule inhibitor of glutamine metabolism, decreases B7H3 expression and enhances granzyme B (GzB) production of CD8⁺ T cells via regulation of reactive oxygen species (ROS) accumulation. We demonstrate that combined treatment with V9302 and anti-PD-1 monoclonal antibody (mAb) enhances antitumor immunity in syngeneic mouse models. Collectively, our findings unveil the beneficial effect of V9302 in boosting antitumor immune response in breast cancer and illustrate that anti-PD-1 together with V9302 treatment may provide synergistic effects in the treatment of patients insensitive to anti-PD-1 therapy.     

Selection of individual VH genes occurs at the pro-B to pre-B cell transition

B cells are subjected to selection at multiple checkpoints during their development. The selection of Ab H chains is difficult to study because of the large diversity of the CDR3. To study the selection of individual Ab H chain V region genes (V(H)), we performed CDR3 spectratyping of ～ 75-300 rearrangements per individual V(H) in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame in B cell DNA. We demonstrate that individual V(H)s have different fractions of in-frame rearrangements (IF fractions) ranging from 10 to 90% and that these IF fractions are reproducible in different mice. For most V(H)s, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high in-frame (IF) V(H) usage increased in cycling pre-B cells compared with that in pro-B cells, whereas this did not occur for low IF V(H)s. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by L chain usage for most V(H). High IF clan II/III V(H)s share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual V(H)s are subjected to differential selection, that V(H) IF fraction is mainly established through pre-BCR-mediated selection, that it may operate differently in clan I versus II/III V(H)s, and that it has a lasting influence on the Ab repertoire.     

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.     

B7-1 antigen expression in tumor cells from cancerous human tissues

OBJECTIVE: To explore the expression of B7-1 antigen (Ag), the most important costimulatory molecule for T-lymphocyte activation, on tumor cells from cancerous human tissues. STUDY DESIGN: The study group consisted of 82 biopsies taken from a variety of cancerous human tissues. Ten normal cervical specimens, used as controls, were taken from uteri removed for leiomyomas or leiomyomatosis. B7-1 molecule expression was identified using monoclonal antibody (Ab) with CD80 and the SABC technique. RESULTS: In most cases (50/82), the tumor cells were totally or partially B7-1 Ag positive. The positive products were distributed mainly on the cell surface and, in some cases, also in the cytoplasm. Although in transitional cell carcinoma of the bladder, all tumor cells in all three cases stained strongly with CD80, in squamous cell carcinoma and adnocarcinoma, the B7-1 expression in cancer cells was detected in 3/56 and 15/23 cases, respectively. The intensity and distribution of B7-1 expression from case to case were heterogeneous: half the cases had relatively homogeneous and stronger B7-1 expression in cancer cells. Additionally, there were B7-1+ dendritic cells and lymphocytes scattered or densely infiltrating interstitial tissue. In some cases, the endothelia of small vessels were proved to be B7-1+. There was one esophageal cancer specimen containing normal-looking mucosa in which B7-1 molecule expression was also demonstrated in the squamous and glandular epithelia. CONCLUSION: Our findings demonstrate that the tumor cells in cancerous human tissues do not all fail to express B7-1. The mechanism of the failure of hosts to reject tumors can be attributed not only to the lack of costimulatory molecules in tumor cells but also to events after the Ag-presenting process.     

The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.     

Staphylococcal protein a deletes B-1a and marginal zone B lymphocytes expressing human immunoglobulins: an immune evasion mechanism

Protein A (SpA) of Staphylococcus aureus is endowed with the capacity to interact with the H chain variable region (V(H)) of human Abs and to target >40% of B lymphocytes. To investigate whether this property represents a virulence factor and to determine the in vivo consequences of the confrontation of SpA with B lymphocytes, we used transgenic mice expressing fully human Abs. We found that administration of soluble SpA reduces B-1a lymphocytes of the peritoneal cavity and marginal zone B lymphocytes of the spleen, resulting in a markedly deficient type 2 humoral response. Single-cell PCR analysis and sequencing of the Ab V(H) gene repertoire revealed a significant reduction of V(H)3+ marginal zone B cells. Since the two B lymphocyte subsets targeted are involved in innate immune functions, our data suggest that crippling of humoral immunity by S. aureus represents an immune evasion mechanism that may aggravate recurrent infections.     

Transitional B cells lose their ability to receptor edit but retain their potential for positive and negative selection

Ligation of B cell receptors on immature bone marrow B cells, either by an endogenous Ag or by an anti-B cell receptor Ab induces secondary V(D)J gene rearrangements, termed receptor editing. Whether the same signal induces receptor editing in transitional B cells is not clear. In this study, we examined the responses of immature and transitional B cells from V(H)12Vkappa1A Ig transgenic mice to stimulation with an anti-Igbeta Ab. Our results demonstrated that immature B cells stimulated with a low concentration of anti-Igbeta Ab, mimicking Ag stimulation, underwent receptor editing both in vivo and in vitro, as evidenced by the detection of dsDNA breaks at Jkappa recombination signal sequences, whereas transitional B cells did not. The lack of dsDNA breaks in transitional B cells contrasts with their increased expression of RAG1 and RAG2, suggesting a novel mechanism that may prevent rearrangements. Furthermore, treatment of transitional B cells with high concentrations of anti-Igbeta Abs induced apoptosis, whereas low concentrations induced differentiation. Our results support the idea that transitional B cells lose the capacity to edit, but are sensitive to positive and negative selection.     

GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma – assessing tumor and T cell interaction

A novel disialoganglioside 2 (GD2)-specific chimeric antigen receptor (CAR)-modified T cell therapy against retinoblastoma (RB) were generated. GD2-CAR consists of a single-chain variable fragment (scFv) derived from a monoclonal antibody, hu3F8, that is linked with the cytoplasmic signaling domains of CD28, 41BB, a CD3ζ, and an inducible caspase 9 death fusion partner. GD2 antigen is highly expressed in Y79RB cell line and in several surgical RB tumor specimens. In vitro co-culture experiments revealed the effective killing of Y79RB cells by GD2-CAR T cells, but not by control CD19-CAR T cells. The killing activities of GD2-CAR T cells were diminished when repeatedly exposed to the tumor, due to an attenuated expression of GD2 antigen on tumor cells and upregulation of inhibitory molecules of the PD1 and PD-L1 axis in the CAR T cells and RB tumor cells respectively. This is the first report to describe the potential of GD2-CAR T cells as a promising therapeutic strategy for RB with the indication of potential benefit of combination therapy with immune checkpoint inhibitors.     

Tolerance to DNA in (NZB x NZW)F1 mice that inherit an anti-DNA V(H) as a conventional micro H chain transgene but not as a V(H) knock-in transgene

Lupus-prone (NZB x NZW)F(1) (BWF(1)) mice were made transgenic (Tg) for an anti-DNA Ab inherited either as a conventional V(H)3H9- micro H chain Tg (3H9- micro ) with or without a conventional V(kappa)8-kappa Tg, or a V(H)3H9 V(H) knock-in Tg allele (3H9R) with or without a V(kappa)4 V(kappa) knock-in Tg allele (V(kappa)4R). V(H)3H9 yields an anti-DNA Ab with most L chains including an anti-ssDNA with the V(kappa)8 Tg and an anti-dsDNA with the V(kappa)4 Tg. BWF(1) mice that inherited the conventional 3H9- micro had normal serum IgM, little to none of which was encoded by 3H9- micro, and only a small percentage of those mice had serum anti-DNA, none of which was transgene encoded. B cells expressing the conventional 3H9- micro Tg were anergic. BWF(1) mice that inherited the knock-in 3H9R Tg allele also had normal serum IgM, one-half of which was encoded by 3H9R, and produced anti-DNA encoded by the Tg allele. Most B cells expressing the knock-in 3H9R Tg also had an anergic phenotype. The results indicate that autoimmune-prone BWF(1) mice initially develop effective B cell tolerance to DNA through anergy, and anergy was sustained in 3H9- micro Tg peripheral B cells but not in 3H9R Tg B cells. B cells expressing the 3H9R knock-in Tg allele were able to achieve an activation threshold that B cells expressing the 3H9- micro conventional Tg could not. The maintenance of B cell tolerance to DNA in autoimmune-prone BWF(1) mice appears to differ from both normal mice and autoimmune-prone MRL(lpr/lpr) mice.     

Analysis of marginal zone B cell development in the mouse with limited B cell diversity: role of the antigen receptor signals in the recruitment of B cells to the marginal zone

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had approximately 2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V(H)T/lambda2 B cells significantly predominated over V(H)T/lambda1 B cells in MZ-(V(H)T/lambda1:V(H)T/lambda2 approximately 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V(H)T/lambda2 B cells were further augmented by doubling the V(H)T gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V(H)T/lambda1 and V(H)T/lambda2 IgM mAbs revealed a polyreactive nature of the V(H)T/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.     

DNA binding by the VH domain of anti-Z-DNA antibody and its modulation by association of the VL domain

mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.     

Tetrameric and homodimeric camelid IgGs originate from the same IgH locus

In addition to producing conventional tetrameric IgGs, camelids have the particularity of producing a functional homodimeric IgG type lacking L (light) chains and only made up of two H (heavy) chains. This nonconventional IgG type is characterized by variable and constant regions referred to as V(H)H and C(H)H, respectively, and which differ from conventional V(H) and C(H) counterparts. Although the structural properties of homodimeric IgGs have been well investigated, the genetic bases involved in their generation are still largely unknown. In this study, we characterized the organization of genes coding for the H chains of tetrameric and homodimeric IgGs by constructing an alpaca (Lama pacos) genomic cosmid library. We showed that a single IgH locus in alpaca chromosome 4 contains all of the genetic elements required for the generation of the two types of Igs. The alpaca IgH locus is composed of a V region that contains both V(H)H and V(H) genes followed by a unique D(H)-J(H) cluster and C region genes, which include both C(H)H and C(H) genes. Although this general gene organization greatly resembles that of other typical mammalian V(n)-D(n)-J(n)-C(n) translocon IgH loci, the intermixed gene organization within the alpaca V and C regions reveals a new type of translocon IgH locus. Furthermore, analyses of cDNA coding for the membrane forms of IgG and IgM present in alpaca peripheral blood B cells are most consistent with the notion that the development of a B cell bearing homodimeric IgG passes through an IgM(+) stage, similar to the case for conventional IgG.     

Histone Demethylase KDM6B Promotes Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a critical event that occurs in embryonic development, tissue repair control, organ fibrosis, and carcinoma invasion and metastasis. Although significant progress has been made in understanding the molecular regulation of EMT, little is known about how chromatin is modified in EMT. Chromatin modifications through histone acetylation and methylation determine the precise control of gene expression. Recently, histone demethylases were found to play important roles in gene expression through demethylating mono-, di-, or trimethylated lysines. KDM6B (also known as JMJD3) is a histone demethylase that might activate gene expression by removing repressive histone H3 lysine 27 trimethylation marks from chromatin. Here we report that KDM6B played a permissive role in TGF-β-induced EMT in mammary epithelial cells by stimulating SNAI1 expression. KDM6B was induced by TGF-β, and the knockdown of KDM6B inhibited EMT induced by TGF-β. Conversely, overexpression of KDM6B induced the expression of mesenchymal genes and promoted EMT. Chromatin immunoprecipitation (ChIP) assays revealed that KDM6B promoted SNAI1 expression by removing histone H3 lysine trimethylation marks. Consistently, our analysis of the Oncomine database found that KDM6B expression was significantly increased in invasive breast carcinoma compared with normal breast tissues. The knockdown of KDM6B significantly inhibited breast cancer cell invasion. Collectively, our study uncovers a novel epigenetic mechanism regulating EMT and tumor cell invasion, and has important implication in targeting cancer metastasis.     

Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.     

Anti-ganglioside anti-idiotypic monoclonal antibody-based cancer vaccine induces apoptosis and antiangiogenic effect in a metastatic lung carcinoma

Anti-idiotype monoclonal antibody (mAb) 1E10 was generated by immunizing BALB/c mice with an Ab1 mAb which recognizes NeuGc-containing gangliosides, sulfatides and some tumor antigens. 1E10 mAb induces therapeutic effects in a primary breast carcinoma and a melanoma model. However, the tumor immunity mechanisms have not been elucidated. Here we show that aluminum hydroxide-precipitated 1E10 mAb immunization induced anti-metastatic effect in the 3LL-D122 Lewis Lung carcinoma, a poorly immunogenic and highly metastatic model in C57BL/6 mice. The therapeutic effect was associated to the increment of T cells infiltrating metastases, the reduction of new blood vessels formation and the increase of apoptotic tumor cells in lung nodules. Interestingly, active immunization does not induce measurable antibodies to the 1E10 mAb, the NeuGc-GM3 or tumor cells, which may suggest a different mechanism which has to be elucidated. These findings may support the relevance of this target for cancer biotherapy.