Richard R Hardy

Publications of Richard R Hardy

• NEDD9 Promotes Oncogenic Signaling in Mammary Tumor Development.

  Authors: Eugene Izumchenko, Mahendra K Singh, Olga V Plotnikova, Nadezhda Tikhmyanova, Joy L Little, Ilya G Serebriiskii, Sachiko Seo, Mineo Kurokawa, Brian L Egleston, Andres Klein-Szanto, Elena N Pugacheva, Richard R Hardy, Marina Wolfson, Denise C Connolly, Erica A Golemis

  Cancer research. 10/2009;

  In the past 3 years, altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has emerged as contributing to cancer metastasis in multiple cancer types. However, whereas some studies haveIn the past 3 years, altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has emerged as contributing to cancer metastasis in multiple cancer types. However, whereas some studies have identified elevated NEDD9 expression as prometastatic, other work has suggested a negative role in tumor progression. We here show that the Nedd9-null genetic background significantly limits mammary tumor initiation in the MMTV-polyoma virus middle T genetic model. Action of NEDD9 is tumor cell intrinsic, with immune cell infiltration, stroma, and angiogenesis unaffected. The majority of the late-appearing mammary tumors of MMTV-polyoma virus middle T;Nedd9(-/-) mice are characterized by depressed activation of proteins including AKT, Src, FAK, and extracellular signal-regulated kinase, emphasizing an important role of NEDD9 as a scaffolding protein for these prooncogenic proteins. Analysis of cells derived from primary Nedd9(+/+) and Nedd9(-/-) tumors showed persistently reduced FAK activation, attachment, and migration, consistent with a role for NEDD9 activation of FAK in promoting tumor aggressiveness. This study provides the first in vivo evidence of a role for NEDD9 in breast cancer progression and suggests that NEDD9 expression may provide a biomarker for tumor aggressiveness. [Cancer Res 2009;69(18):7198-206].

• Mice Heterozygous for Germ-line Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-Cell Lymphoma.

  Authors: Yuwaraj Kadariya, Bu Yin, Baiqing Tang, Susan A Shinton, Eoin P Quinlivan, Xiang Hua, Andres Klein-Szanto, Tahseen I Al-Saleem, Craig H Bassing, Richard R Hardy, Warren D Kruger

  Cancer research. 07/2009;

  Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whetherLarge homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (Mtap(lacZ)) have an embryonic lethal phenotype dying around day 8 postconception. Mtap/Mtap(lacZ) heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and fluorescence-activated cell sorting analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma-infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein in addition to unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF. [Cancer Res 2009;69(14):5961-9].

• A Role for cFLIP in B Cell Proliferation and Stress MAPK Regulation.

  Authors: Haibing Zhang, Stephen Rosenberg, Francis J Coffey, You-Wen He, Timothy Manser, Richard R Hardy, Jianke Zhang

  Journal of immunology (Baltimore, Md. : 1950). 02/2009; 182(1):207-15.

  Fas/Apo-1 signals through the FADD (Fas-associated death domain) adaptor protein, which recruits and activates the apical caspase 8 and leads to apoptosis. Cellular FLIP (cFLIP) is a homolog ofFas/Apo-1 signals through the FADD (Fas-associated death domain) adaptor protein, which recruits and activates the apical caspase 8 and leads to apoptosis. Cellular FLIP (cFLIP) is a homolog of caspase 8 and is also capable of binding to FADD. Previous studies suggest that cFLIP could either enhance or inhibit apoptosis and lead to NF-kappaB and Erk1/2 activation. Like FADD or caspase 8 deficiency, a lack of cFLIP disrupts embryogenesis and T cell proliferation. It has been demonstrated that B cells lacking either FADD or caspase 8 were defective in both Fas-induced apoptosis and TLR-induced proliferation, which indicates that these death-inducing proteins have an additional role in regulating innate immunity. To analyze the function of cFLIP in B cells, conditional deletion of cFLIP was induced by using CD19(Cre). The resulting B cell-specific cFLIP-deficient mice were found to have reduced numbers of peripheral B cells that were hypersensitive to Fas-induced apoptosis and impaired in proliferation induced by TLRs and the BCR. Furthermore, there was aberrant expression of costimulatory proteins and activation markers in cFLIP-deficient B cells. Whereas LPS-induced activation of NF-kappaB and Erk1/2 appears to be unaffected, p38 and Jnk were spontaneously activated and hyperinduced in cFLIP-deficient B cells. Therefore, these data revealed novel functions of cFLIP in B cells.

• Biallelic, ubiquitous transcription from the distal germline Ig{kappa} locus promoter during B cell development.

  Authors: Rupesh H Amin, Dragana Cado, Hector Nolla, Dan Huang, Susan A Shinton, Yan Zhou, Richard R Hardy, Mark S Schlissel

  Proceedings of the National Academy of Sciences of the United States of America. 01/2009;

  Allelic exclusion of Ig gene expression is necessary to limit the number of functional receptors to one per B cell. The mechanism underlying allelic exclusion is unknown. Because germlineAllelic exclusion of Ig gene expression is necessary to limit the number of functional receptors to one per B cell. The mechanism underlying allelic exclusion is unknown. Because germline transcription of Ig and TCR loci is tightly correlated with rearrangement, we created two novel knock-in mice that report transcriptional activity of the Jkappa germline promoters in the Igkappa locus. Analysis of these mice revealed that germline transcription is biallelic and occurs in all pre-B cells. Moreover, we found that the two germline promoters in this region are not equivalent but that the distal promoter accounts for the vast majority of observed germline transcript in pre-B cells while the activity of the proximal promoter increases later in development. Allelic exclusion of the Igkappa locus thus occurs at the level of rearrangement, but not germline transcription.

• B cell receptor basal signaling regulates antigen-induced Ig light chain rearrangements.

  Authors: Brian R Schram, Lina E Tze, Laura B Ramsey, Jiabin Liu, Lydia Najera, Amanda L Vegoe, Richard R Hardy, Keli L Hippen, Michael A Farrar, Timothy W Behrens

  Journal of immunology (Baltimore, Md. : 1950). 05/2008; 180(7):4728-41.

  BCR editing in the bone marrow contributes to B cell tolerance by orchestrating secondary Ig rearrangements in self-reactive B cells. We have recently shown that loss of the BCR or a pharmacologicBCR editing in the bone marrow contributes to B cell tolerance by orchestrating secondary Ig rearrangements in self-reactive B cells. We have recently shown that loss of the BCR or a pharmacologic blockade of BCR proximal signaling pathways results in a global "back-differentiation" response in which immature B cells down-regulate genes important for the mature B cell program and up-regulate genes characteristic of earlier stages of B cell development. These observations led us to test the hypothesis that self-Ag-induced down-regulation of the BCR, and not self-Ag-induced positive signals, lead to Rag induction and hence receptor editing. Supporting this hypothesis, we found that immature B cells from xid (x-linked immunodeficiency) mice induce re-expression of a Rag2-GFP bacterial artificial chromosome reporter as well as wild-type immature B cells following Ag incubation. Incubation of immature B cells with self-Ag leads to a striking reversal in differentiation to the pro-/pre-B stage of development, consistent with the idea that back-differentiation results in the reinduction of genes required for L chain rearrangement and receptor editing. Importantly, Rag induction, the back-differentiation response to Ag, and editing in immature and pre-B cells are inhibited by a combination of phorbol ester and calcium ionophore, agents that bypass proximal signaling pathways and mimic BCR signaling. Thus, mimicking positive BCR signals actually inhibits receptor editing. These findings support a model whereby Ag-induced receptor editing is inhibited by BCR basal signaling on developing B cells; BCR down-regulation removes this basal signal, thereby initiating receptor editing.

• Autoreactive B-1 B cells: constraints on natural autoantibody B cell antigen receptors.

  Authors: Ben Rowley, Lingjuan Tang, Susan Shinton, Kyoko Hayakawa, Richard R Hardy

  Journal of autoimmunity. 01/2008; 29(4):236-45.

  B-1 B-cells constitute a distinctive population of cells that are enriched for self-reactive B cell receptors (BCRs). These BCRs are encoded by a restricted set of heavy and light chains, includingB-1 B-cells constitute a distinctive population of cells that are enriched for self-reactive B cell receptors (BCRs). These BCRs are encoded by a restricted set of heavy and light chains, including heavy chains that lack nontemplated nucleotide additions at the V-D and D-J joining regions. One prototype natural autoantibody produced by B-1 B cells binds to a cryptic determinant exposed on senescent red blood cells that includes the phosphatidylcholine (PtC) moiety. The V(H)11Vkappa9 BCR, which accounts for a large fraction of the anti-PtC specificity, is underrepresented in other B-cell populations, including newly formed B cells in bone marrow, and the transitional B cells, follicular B cells, and marginal zone B cells in spleen. Previous work has shown that V(H)11 heavy chains pair ineffectively with surrogate light chain (SLC) and so do not promote development in bone marrow, but instead allow fetal liver maturation because of a fetal preference for weaker pre-BCR signaling. Such inefficient SLC pairing constitutes one constraint on the maturation of B cells containing V(H)11 rearrangements that biases their generation to fetal development. Here, we examine another possible bottleneck to the B1 cell repertoire: light chain pairing with V(H)11 heavy chain, finding very significant preferences.

• The protean nature of cells in the B lymphocyte lineage.

  Authors: Richard R Hardy, Paul W Kincade, Kenneth Dorshkind

  Immunity. 07/2007; 26(6):703-14.

  The subdivision of bone marrow (BM) with surface markers and reporter systems and the use of multiple culture and transplantation assays to assess differentiation potential have led to extraordinaryThe subdivision of bone marrow (BM) with surface markers and reporter systems and the use of multiple culture and transplantation assays to assess differentiation potential have led to extraordinary progress in defining stages of B lymphopoiesis between the hematopoietic stem cell and B cell receptor (BCR)-expressing lymphocytes. Despite the lack of standard nomenclature and a series of technical issues that still need to be resolved, there seems to be a general consensus regarding the major route to becoming a B cell. Nevertheless, evidence that additional, minor pathways through which B lineage cells are generated exists, and a new appreciation that lymphoid progenitors are protean and able to alter their differentiation potential during embryogenesis and after birth in response to infections suggests that a full understanding of B cell development and how it is regulated has not yet been attained.

• Ablation of ribosomal protein L22 selectively impairs alphabeta T cell development by activation of a p53-dependent checkpoint.

  Authors: Stephen J Anderson, Jens Peter Holst Lauritsen, Matthew G Hartman, Ann Marie Digeorge Foushee, Juliette M Lefebvre, Susan A Shinton, Brenda Gerhardt, Richard R Hardy, Tamas Oravecz, David L Wiest

  Immunity. 07/2007; 26(6):759-72.

  The alphabeta and gammadelta T lineages are thought to arise from a common precursor; however, the regulation of separation and development of these lineages is not fully understood. We report hereThe alphabeta and gammadelta T lineages are thought to arise from a common precursor; however, the regulation of separation and development of these lineages is not fully understood. We report here that development of alphabeta and gammadelta precursors was differentially affected by elimination of ribosomal protein L22 (Rpl22), which is ubiquitously expressed but not essential for translation. Rpl22 deficiency selectively arrested development of alphabeta-lineage T cells at the beta-selection checkpoint by inducing their death. The death was caused by induction of p53 expression, because p53 deficiency blocked death and restored development of Rpl22-deficient thymocytes. Importantly, Rpl22 deficiency led to selective upregulation of p53 in alphabeta-lineage thymocytes, at least in part by increasing p53 synthesis. Taken together, these data indicate that Rpl22 deficiency activated a p53-dependent checkpoint that produced a remarkably selective block in alphabeta T cell development but spared gammadelta-lineage cells, suggesting that some ribosomal proteins may perform cell-type-specific or stage-specific functions.

• B-1 B cells: development, selection, natural autoantibody and leukemia.

  Authors: Richard R Hardy

  Current opinion in immunology. 11/2006; 18(5):547-55.

  B-1 (CD5+) B cells constitute a phenotypic and functionally distinct population of B cells in mouse that show enriched expression of autoreactive B-cell antigen receptors and that produce severalB-1 (CD5+) B cells constitute a phenotypic and functionally distinct population of B cells in mouse that show enriched expression of autoreactive B-cell antigen receptors and that produce several types of natural autoantibodies. Recently, there has been much progress in this field of research. Evidence has appeared for the existence of distinctive B-cell precursors that preferentially generate B-1 B cells, and the crucial requirement for strong B-cell antigen receptor signaling in the maturation of B-1 B cells has been established. Other work focuses on a phenotypically similar population that lacks CD5, termed 'B-1b', which shows similarities and differences from most CD5+ B cells in both development and function. The relationship of normal B-1 cells with B-cell lymphomas and leukemias continues to be a subject of interest and debate.

• B-1 B cell development.

  Authors: Richard R Hardy

  Journal of immunology (Baltimore, Md. : 1950). 10/2006; 177(5):2749-54.

  CD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatalCD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatal precursors, but inefficiently from precursors in adult. One model proposed to explain this difference is that their production occurs through a distinctive developmental process, termed B-1, that enriches pre-B cells with novel germline VDJs and that requires positive selection of newly formed B cells by self-Ag. In contrast, follicular B cells are generated throughout adult life in a developmental process termed B-2, selecting VDJs that pair well with surrogate L chain, and whose maturation appears relatively independent of antigenic selection. In the present study, I focus on processes that shape the repertoire of mouse CD5+ B cells, describing the differences between B-1 and B-2 development, and propose a model encompassing both in the generation of functional B cell subpopulations.

• Lineage specification and plasticity in CD19- early B cell precursors.

  Authors: Lynn L Rumfelt, Yan Zhou, Benjamin M Rowley, Susan A Shinton, Richard R Hardy

  The Journal of experimental medicine. 04/2006; 203(3):675-87.

  We describe here three CD19- B cell precursor populations in mouse bone marrow identified using 12-color flow cytometry. Cell transfer experiments indicate lineage potentials consistent withWe describe here three CD19- B cell precursor populations in mouse bone marrow identified using 12-color flow cytometry. Cell transfer experiments indicate lineage potentials consistent with multilineage progenitor (MLP), common lymphoid progenitor (CLP), and B lineage-restricted pre-pro-B (Fr. A), respectively. However, single cell in vitro assays reveal lineage plasticity: lymphoid/myeloid lineage potential for CLP and B/T lineage potential for Fr. A. Despite myeloid potential, recombination activating gene 2 reporter activation is first detected at low levels in most MLP cells, with 95% of CLPs showing 10-fold increased levels. Furthermore, single cell analysis shows that half of CLP and 90% of Fr. A cells contain heavy chain DJ rearrangements. These data, together with expression profiles of lineage-specific genes, demonstrate progressive acquisition of B lineage potential and support an asynchronous view of early B cell development, in which B lineage specification initiates in the MLP/CLP stage, whereas myeloid potential is not lost until the pre-pro-B (Fr. A) stage, and B/T lymphoid plasticity persists until the CD19+ pro-B stage. Thus, MLP, CLP, and Fr. A represent progressively B lineage-specified stages in development, before the CD19+ B lineage-committed pro-B stage.

• Evidence of marginal-zone B cell-positive selection in spleen.

  Authors: Lijun Wen, Joni Brill-Dashoff, Susan A Shinton, Masanao Asano, Richard R Hardy, Kyoko Hayakawa

  Immunity. 10/2005; 23(3):297-308.

  Antigen receptor-mediated signaling is critical for the development and survival of B cells. However, it has not been established whether B cell development requires a signal from self-ligandAntigen receptor-mediated signaling is critical for the development and survival of B cells. However, it has not been established whether B cell development requires a signal from self-ligand engagement at the immature stage, a process known as "positive selection." Here, using a monoclonal B cell receptor (BCR) mouse line, specific for the self-Thy-1/CD90 glycoprotein, we demonstrate that BCR crosslinking by low-dose self-antigen promotes survival of immature B cells in culture. In spleen, an increase in BCR signaling strength, induced by low-dose self-antigen, directed naive immature B cells to mature, not into the default follicular B cell fate, but instead into the marginal-zone B cell subset. These data indicate that positive selection can occur in developing B cells and that BCR signal strength is a key factor in deciding between two functionally distinct mature B cell compartments in the microenvironment of the spleen.

• Association of B-1 B cells with follicular dendritic cells in spleen.

  Authors: Lijun Wen, Susan A Shinton, Richard R Hardy, Kyoko Hayakawa

  Journal of immunology (Baltimore, Md. : 1950). 07/2005; 174(11):6918-26.

  Although CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult dueAlthough CD5(+) B-1 B cells have been recognized as an infrequent B cell subset in mice for many years, attempts to identify their histologic location in normal mouse spleen have proven difficult due to both their paucity and low level expression of CD5. In this study we have studied V(H)11/D(H)/J(H) gene-targeted mice, V(H)11t, that develop elevated numbers of CD5(+) V(H)11/V(k)9 B cells with an anti-phosphatidylcholine (anti-PtC) autoreactive specificity, allowing B-1 B cell detection by anti-PtC Id-specific Abs in spleen section staining. Using this approach we found that anti-PtC B-1 cells first appear within the white pulp in neonates, expand in association with follicular dendritic cells (FDC), and localize more centrally than other (non-B-1) IgD(high) follicular B cells in adults. Among neonatal B cells, CD5(+) B-1 cells in both normal and V(H)11t mouse spleen and peritoneal cavity express the highest levels of CXCR5, which is important for FDC development. Injection of purified spleen or peritoneal B-1 cells into RAG knockout mice resulted in B-1 cell follicle formation in spleen, inducing FDC development and plasma cell generation. These results indicate that B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of FDC.

• Development of B cells producing natural autoantibodies to thymocytes and senescent erythrocytes.

  Authors: Richard R Hardy, Kyoko Hayakawa

  Springer seminars in immunopathology. 04/2005; 26(4):363-75.

  Natural antibodies produced by CD5+ B-1 B cells include those with specificity for senescent erythrocytes (anti-BrMRBC, anti-PtC) and for thymocytes (anti-thymocyte autoantibody, ATA). Here weNatural antibodies produced by CD5+ B-1 B cells include those with specificity for senescent erythrocytes (anti-BrMRBC, anti-PtC) and for thymocytes (anti-thymocyte autoantibody, ATA). Here we describe work from our laboratories studying two prototypic examples, V(H)11Vkappa9-encoded anti-BrMRBC and V(H)3609Vkappa21c-encoded ATA. Using V(H)11-mu transgenic mice, we discovered that certain natural autoantibodies utilize V(H) genes that are selected against in bone marrow B cell development, but not fetal liver, effectively restricting their generation to fetal/neonatal life. Studies with ATA-mu transgenic mice demonstrated a critical requirement for self antigen in the accumulation of B cells with this specificity and for the production of high levels of serum ATA. Finally, analysis of B cell development in ATA-mu kappa transgenic mice revealed two distinct responses by B cells to expression of this B cell receptor (BCR): most developing B cells in spleen of adult mice were blocked at an immature stage and only escaped apoptosis by editing their BCR to eliminate the ATA specificity; nevertheless, high levels of serum ATA were observed, indicating that some B cells differentiated to antibody-forming cells without altering their specificity. Thus, our studies reveal mechanisms for restricting the generation of B cells producing natural autoantibodies, demonstrate a key positive selection step in their development, and show that most developing B cells in adult mice bearing such specificities fail to reach a mature stage.

• Basal immunoglobulin signaling actively maintains developmental stage in immature B cells.

  Authors: Lina E Tze, Brian R Schram, Kong-Peng Lam, Kristin A Hogquist, Keli L Hippen, Jiabin Liu, Susan A Shinton, Kevin L Otipoby, Peter R Rodine, Amanda L Vegoe, Manfred Kraus, Richard R Hardy, Mark S Schlissel, Klaus Rajewsky, Timothy W Behrens

  PLoS biology. 04/2005; 3(3):e82.

  In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-BIn developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking "back-differentiation" of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.

• Early B cell factor cooperates with Runx1 and mediates epigenetic changes associated with mb-1 transcription.

  Authors: Holly Maier, Rachel Ostraat, Hua Gao, Scott Fields, Susan A Shinton, Kay L Medina, Tomokatsu Ikawa, Cornelis Murre, Harinder Singh, Richard R Hardy, James Hagman

  Nature immunology. 11/2004; 5(10):1069-77.

  Cd79a (called mb-1 here) encodes the Ig-alpha signaling component of the B cell receptor. The early B cell-specific mb-1 promoter was hypermethylated at CpG dinucleotides in hematopoietic stem cellsCd79a (called mb-1 here) encodes the Ig-alpha signaling component of the B cell receptor. The early B cell-specific mb-1 promoter was hypermethylated at CpG dinucleotides in hematopoietic stem cells but became progressively unmethylated as B cell development proceeded. The transcription factor Pax5 activated endogenous mb-1 transcription in a plasmacytoma cell line, but could not when the promoter was methylated. In this context, early B cell factor (EBF), a transcription factor required for B lymphopoiesis, potentiated activation of mb-1 by Pax5. EBF and the basic helix-loop-helix transcription factor E47 each contributed to epigenetic modifications of the mb-1 promoter, including CpG demethylation and nucleosomal remodeling. EBF function was enhanced by interaction with the transcription factor Runx1. These data suggest a molecular basis for the hierarchical dependence of Pax5 function on EBF and E2A in B lymphocyte development.

• Selection during development of VH11+ B cells: a model for natural autoantibody-producing CD5+ B cells.

  Authors: Richard R Hardy, Chi-Ju Wei, Kyoko Hayakawa

  Immunological reviews. 03/2004; 197:60-74.

  Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from aNatural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CD5. Here, we describe our studies with a representative CD5+ B-cell-derived natural autoantibody, the VH11Vkappa9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5-10% of the CD5+ B-cell subset, with a large portion accounted for by two novel BCRs, VH11Vkappa9 and VH12Vkappa4. We have found that the development of B-lineage cells with a VH11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i). one of the most common VH11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii). cells containing VH11- micro chains lacking N-addition are favored for progression to the B-cell stage, eliminating most bone marrow VH11 rearrangements; and (iii). only a subset of Vkappa-light chains combine with VH11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain VH11 generation to fetal development and may favor production of B cells with the prototype VH11Vkappa9 BCR.

• Characterization of B lymphopoiesis in mouse bone marrow and spleen.

  Authors: Richard R Hardy, Susan A Shinton

  Methods in molecular biology (Clifton, N.J.). 02/2004; 271:1-24.

  This chapter provides information on the application of flow cytometry for analysis of B-cell development, describing in detail the particular surface proteins that can serve as markers forThis chapter provides information on the application of flow cytometry for analysis of B-cell development, describing in detail the particular surface proteins that can serve as markers for recognizing distinct stages in this process. These cell fractions range from just prior to initial heavy chain rearrangement, the germline pro-B stage, through D-J rearranged pro-B and heavy chain expressing pre-B stages, to the maturing surface BCR positive B-cell stages. It also outlines assays for the characterization of these cells, including procedures for testing functional lineage restriction, determination of rearrangement status, analyses of gene expression at the ribonucleic acid (RNA) and protein level, and assessment of cell cycle state.

• Isolation of Ly-1+/CD5+ B cells by cell sorting.

  Authors: Richard R Hardy

  Current protocols in immunology / edited by John E. Coligan ... [et al.]. 09/2003; Chapter 3:Unit 3.5B.

  Ly-1/CD5 is a 68-kDa glycoprotein that was originally thought to mark the helper subset of T cells. Later it was shown to be present on all T cells and, more recently, on a subset of B cells.Ly-1/CD5 is a 68-kDa glycoprotein that was originally thought to mark the helper subset of T cells. Later it was shown to be present on all T cells and, more recently, on a subset of B cells. Although its function remains the subject of speculation, CD5 expression serves as a useful marker for a functionally distinct population of B cells that has attracted a considerable amount of interest from investigators of both murine and human immune systems. Of critical importance in much of this work is the isolation of pure populations of CD5+ B cells along with appropriate control populations. A flow cytometry technique is presented in this unit, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules.

• B-cell commitment: deciding on the players.

  Authors: Richard R Hardy

  Current opinion in immunology. 05/2003; 15(2):158-65.

  Considerable progress has been made over the past few years in determining the key molecular players that regulate the commitment and development of early B-lineage cells in mouse bone marrowConsiderable progress has been made over the past few years in determining the key molecular players that regulate the commitment and development of early B-lineage cells in mouse bone marrow (transcription factors, cytokines and their receptors). However, confusion remains as to the precise stages, surface phenotypes and lineage restrictions that are observed early in B-cell development. Increasingly powerful flow cytometry analysis of genetically altered animals (knockouts and transgenics) combined with sensitive functional assays may provide an answer, a "scorecard" for the cell stages.