The many roles of IL-7 in B cell development; mediator of survival, proliferation and differentiation.

Interleukin-7 (IL-7) plays several important roles during B cell development including aiding in; the specification and commitment of cells to the B lineage, the proliferation and survival of B cell progenitors; and maturation during the pro-B to pre-B cell transition. Regulation and modulation of IL-7 receptor (IL-7R) signaling is critical during B lymphopoiesis, because excessive or deficient IL-7R signaling leads to abnormal or inhibited B cell development. IL-7 works together with E2A, EBF, Pax-5 and other transcription factors to regulate B cell commitment, while also functions to regulate Ig rearrangement by modulating FoxO protein activation and Rag enhancer activity. Suppressor of cytokine signaling (SOCS) proteins are inhibitors of cytokine activation and, in B cells, function to fine tune IL-7R signaling; ensuring that appropriate IL-7 signals are transmitted to allow for efficient B cell commitment and development.

Seasonal H1N1 influenza virus infection induces cross-protective pandemic H1N1 virus immunity through a CD8-independent, B cell-dependent mechanism.

During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.

Modulation of IL-7 thresholds by SOCS proteins in developing B lineage cells.

During B lymphopoiesis, IL-7 induces survival, proliferation, and differentiation signals that are important during the pro-B to pre-B cell transition. We showed that murine small pre-B stage cells do not signal or proliferate in response to IL-7, yet they maintain IL-7R surface expression. Loss of proliferative responsiveness to IL-7 is mediated by suppressor of cytokine signaling protein 1 (SOCS-1), the expression of which is regulated during B lymphopoiesis, with the highest levels observed in small pre-B cells. SOCS-1 inhibits IL-7 responses in pre-B cell lines and ex vivo B lineage cells. SOCS-1 expression and, thus, responsiveness to IL-7, can be regulated by IL-7 itself, as well as IFN-γ and IL-21. Additionally, the transcriptional repressor Gfi-1b enhances the proliferative responsiveness of B cell lines to IL-7. We demonstrated that these molecules act together to form a SOCS-mediated "rheostat" that controls the level of IL-7R signaling in developing murine B lineage cells.

Targeted deletion of the tachykinin 4 gene (TAC4-/-) influences the early stages of B lymphocyte development.

Hemokinin-1 (HK-1), encoded by the TAC4 gene, is a tachykinin peptide that is predominantly expressed in non-neuronal cells, such as immune cells. We have disrupted the mouse TAC4 gene to obtain a better understanding of the actions of HK-1 during hematopoiesis. We demonstrate here that TAC4(-/-) mice exhibit an increase of CD19(+)CD117(+)HSA(+)BP.1(-) "fraction B" pro-B cells in the bone marrow, whereas pre-B, immature, and mature B cells are within the normal range. We show that in vitro cultures derived from TAC4(-/-) bone marrow, sorted "fraction B" pro-B cells or purified long-term reconstituting stem cells, contain significantly higher numbers of pro-B cells compared with controls, suggesting an inhibitory role for HK-1 on developing B cells. Supporting this idea, we show that addition of HK-1 to cultures established from long-term reconstituting stem cells and the newly described intermediate-term reconstituting stem cells leads to a significant decrease of de novo generated pro-B cells. Based on our studies, we postulate that HK-1 plays an inhibitory role in hematopoiesis, and we hypothesize that it may be part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells.

Generation and characterization of stromal cell independent IL-7 dependent B cell lines.

In-vitro B cell cultures have played a significant role in the study of B cell development. Their utility in developmental and biochemical studies, however, has been limited by the challenges associated with obtaining and maintaining adequate cell numbers of pure and/or rare populations. Although B cell lines allow for circumvention of some of these issues, they have traditionally been generated via viral infection or genetic transformation and are thus less representative of in-vivo cells. In order to avoid such alterations in cell state, we have designed a procedure for the creation of B cell lines directly from murine bone marrow. In this study, we describe the generation and characterization of these IL-7 dependent cell lines. Our lines, established from both wild type and mutant mice, do not require stromal cell support for generation or maintenance. In addition, clones survive repeated freeze/thaw cycles and, in the presence of IL-7, can be kept in culture indefinitely. Phenotypically, our lines resemble pro/pre-B cells and exhibit IL-7 and preBCR signaling profiles that mimic ex-vivo B cells. These lines promise to be useful in the study of the signaling pathways that regulate B cell development.

Homeodomain-interacting protein kinase (HIPK)-1 is required for splenic B cell homeostasis and optimal T-independent type 2 humoral response.

The homeodomain-interacting protein kinase (HIPK) family is comprised of four highly related serine/threonine kinases originally identified as co-repressors for various homeodomain-containing transcription factors. The HIPKs have been shown to be involved in growth regulation and apoptosis, with numerous studies highlighting HIPK regulation of the tumor suppressor p53. In this study, we have discovered a B cell homeostatic defect in HIPK1-deficient (HIPK1(-/-)) mice. Lymphopoietic populations within the thymus and bone marrow of HIPK1(-/-) mice appeared normal based upon FACS analysis; however, the spleen exhibited a reduced number of total B cells with a significant loss of transitional-1 and follicular B cell populations. Interestingly, the marginal zone B cell population was expanded in HIPK1(-/-) mice, yielding an increased frequency of these cells. HIPK1(-/-) B cells exhibited impaired cell division in response to B cell receptor cross-linking in vitro based upon thymidine incorporation or CFSE dilution; however, the addition of CD40L rescued HIPK1(-/-) proliferation to wild-type levels. Despite the expanded MZ B cell population in the HIPK1(-/-) mice, the T-independent type 2 humoral response was impaired. These data identify HIPK1 as a novel kinase required for optimal B cell function in mice.

Heparan sulfate and heparin enhance ERK phosphorylation and mediate preBCR-dependent events during B lymphopoiesis.

As B lineage cells develop, they interact with cells, proteins, and extracellular matrix components of the surrounding microenvironment. In vitro, one critical checkpoint for developing cells occurs as they lose responsiveness to IL-7. These cells require contact with either stromal cells or other B lineage cells to mature. Our results demonstrate that heparan sulfate and heparin are able to promote this transition when added exogenously to the culture system or when heparan sulfate-bearing cell lines are cocultured with primary B cell progenitors. Addition of heparan sulfate or heparin to LPS-stimulated cultures of primary B cell progenitors resulted in more IgM secreted compared with untreated cultures. Heparan sulfate has been reported to be a ligand for the pre-B cell receptor (preBCR). Extending this observation, we found that treatment of preBCR+ cells with heparan sulfate before anti-micro stimulation leads to increased phosphorylation of ERK1/2. Consequently, preBCR+ cells proliferate more in the presence of IL-7 and heparan sulfate, whereas preBCR- cells are unaffected, suggesting that in these experiments, heparan sulfate is not directly affecting IL-7 activity. Heparin treatment of cultures induces many of the same biological effects as treatment with heparan sulfate, including elevated pERK levels in preBCR+ cells. However, heparin reduces the proliferation of cells expressing only the preBCR (opposed to both the preBCR and BCR) possibly due to internalization of the preBCR. Heparan sulfates are present on stromal cells and B lineage cells present in hemopoietic tissues and may provide stimulation to preB cells testing the signaling capacity of the preBCR.

Cutting edge: transplant tolerance induced by anti-CD45RB requires B lymphocytes.

Selective interference with the CD45RB isoform by mAb (anti-CD45RB) reliably induces donor-specific tolerance. Although previous studies suggest participation of regulatory T cells, a mechanistic understanding of anti-CD45RB-induced tolerance is lacking. We report herein the unexpected finding that tolerance induced by this agent is not established in B cell-deficient mice but can be recovered by preemptive B lymphocyte transfer to B cell-deficient hosts. Using B cells from genetically modified donors to reconstitute B cell-deficient recipients, we evaluate the role of B lymphocyte-expressed CD45RB, T cell costimulatory molecules, and the production of Abs in this novel tolerance mechanism. Our data document an Ab-induced tolerance regimen that is uniquely B lymphocyte-dependent and suggest mechanistic contributions to tolerance development from the B cell compartment through interactions with T cells.