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.

B-cell tolerance defects in the B6.Aec1/2 mouse model of Sjögren's syndrome.

PURPOSE: Primary Sjögren's syndrome (SjS) is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, B-cell clonal expansions and an increased risk of lymphoma. In order to understand the role of B cells in this disorder, the antibody repertoire and B-cell maturation were studied in a mouse model of SjS called B6.Aec1/2. METHODS: B6.Aec1/2 serum was analyzed for antibodies by ELISA and immunoprecipitation, B-cell development by flow cytometry, and antibody gene rearrangements by CDR3 spectratyping and quantitative PCR. In order to test the functional consequences of the observed defects, B6.Aec1/2 mice were crossed with anti-dsDNA antibody heavy chain knock-in mice (B6.56R). RESULTS: B6.Aec1/2 mice exhibit B-cell clonal expansions, have altered serum immunoglobulin levels and spontaneously produce multireactive autoantibodies. B6.Aec1/2 mice also have decreased numbers of bone marrow pre-B cells and decreased frequencies of kappa light chain gene deletion. These findings suggest that B6.Aec1/2 mice have a defective early B-cell tolerance checkpoint. B6.56R.Aec1/2 mice unexpectedly had lower anti-dsDNA antibody levels than B6.56R mice and less salivary gland infiltration than B6.Aec1/2 mice. CONCLUSIONS: These data suggest that the early tolerance checkpoint defect in B6.Aec1/2 mice is not sufficient to promulgate disease in mice with pre-formed autoantibodies, such as B6.56R. Rather, B6.Aec1/2 mice may require a diverse B-cell repertoire for efficient T-B-cell collaboration and disease propagation. These findings imply that therapies aimed at reducing B-cell diversity or T-B interactions may be helpful in treating SjS.

Live tumor imaging shows macrophage induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.

Hematogenous Dissemination of Breast Cancer Cells From Lymph Nodes Is Mediated by Tumor MicroEnvironment of Metastasis Doorways.

In primary breast tumors, cancer cells hematogenously disseminate through doorways in the vasculature composed of three-cell complexes (known as Tumor MicroEnvironment of Metastasis) comprising a perivascular macrophage, a tumor cell overexpressing the actin-regulatory protein Mammalian Enabled (Mena), and an endothelial cell, all in direct physical contact. It has been previously shown that once tumor cells establish lymph node metastases in patients, TMEM doorways form in the metastatic tumor cell nests. However, it has not been established if such lymph node-TMEM doorways actively transit tumor cells into the peripheral circulation and on to tertiary sites. To address this question in this short report, we used a mouse model of lymph node metastasis to demonstrate that TMEM doorways: (1) exist in tumor-positive lymph nodes of mice, (2) are restricted to the blood vascular endothelium, (3) serve as a mechanism for further dissemination to peripheral sites such as to the lungs, and (4) their activity can be abrogated by a pharmaceutical intervention. Our data suggest that cancer cell dissemination via TMEM doorways is a common mechanism of breast cancer cell dissemination to distant sites and thus the pharmacological targeting of TMEM may be necessary, even after resection of the primary tumor, to suppress cancer cell dissemination.

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism.

Breast cancer cells disseminate through TIE2/MENACalc/MENAINV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENAINV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENA gene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.