Browsing by Subject "B-Lymphocytes"
Now showing 1 - 20 of 39
Results Per Page
Sort Options
Item Open Access A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.(PloS one, 2016-01) Yu, Yen-Rei A; O'Koren, Emily G; Hotten, Danielle F; Kan, Matthew J; Kopin, David; Nelson, Erik R; Que, Loretta; Gunn, Michael DFlow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.Item Open Access A Randomized Phase II Crossover Study of Imatinib or Rituximab for Cutaneous Sclerosis after Hematopoietic Cell Transplantation.(Clin Cancer Res, 2016-01-15) Arai, Sally; Pidala, Joseph; Pusic, Iskra; Chai, Xiaoyu; Jaglowski, Samantha; Khera, Nandita; Palmer, Jeanne; Chen, George L; Jagasia, Madan H; Mayer, Sebastian A; Wood, William A; Green, Michael; Hyun, Teresa S; Inamoto, Yoshihiro; Storer, Barry E; Miklos, David B; Shulman, Howard M; Martin, Paul J; Sarantopoulos, Stefanie; Lee, Stephanie J; Flowers, Mary EDPURPOSE: Cutaneous sclerosis occurs in 20% of patients with chronic graft-versus-host disease (GVHD) and can compromise mobility and quality of life. EXPERIMENTAL DESIGN: We conducted a prospective, multicenter, randomized, two-arm phase II crossover trial of imatinib (200 mg daily) or rituximab (375 mg/m(2) i.v. weekly × 4 doses, repeatable after 3 months) for treatment of cutaneous sclerosis diagnosed within 18 months (NCT01309997). The primary endpoint was significant clinical response (SCR) at 6 months, defined as quantitative improvement in skin sclerosis or joint range of motion. Treatment success was defined as SCR at 6 months without crossover, recurrent malignancy or death. Secondary endpoints included changes of B-cell profiles in blood (BAFF levels and cellular subsets), patient-reported outcomes, and histopathology between responders and nonresponders with each therapy. RESULTS: SCR was observed in 9 of 35 [26%; 95% confidence interval (CI); 13%-43%] participants randomized to imatinib and 10 of 37 (27%; 95% CI, 14%-44%) randomized to rituximab. Six (17%; 95% CI, 7%-34%) patients in the imatinib arm and 5 (14%; 95% CI, 5%-29%) in the rituximab arm had treatment success. Higher percentages of activated B cells (CD27(+)) were seen at enrollment in rituximab-treated patients who had treatment success (P = 0.01), but not in imatinib-treated patients. CONCLUSIONS: These results support the need for more effective therapies for cutaneous sclerosis and suggest that activated B cells define a subgroup of patients with cutaneous sclerosis who are more likely to respond to rituximab.Item Open Access Adenovirus F protein as a delivery vehicle for botulinum B.(BMC Immunol, 2010-07-07) Clapp, Beata; Golden, Sarah; Maddaloni, Massimo; Staats, Herman F; Pascual, David WBACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.Item Open Access An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.(J Exp Med, 2005-03-07) Ait-Azzouzene, Djemel; Verkoczy, Laurent; Peters, Jorieke; Gavin, Amanda; Skog, Patrick; Vela, José Luis; Nemazee, DavidUnderstanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.Item Open Access Analysis of Epstein-Barr virus-regulated host gene expression changes through primary B-cell outgrowth reveals delayed kinetics of latent membrane protein 1-mediated NF-κB activation.(Journal of virology, 2012-10) Price, Alexander M; Tourigny, Jason P; Forte, Eleonora; Salinas, Raul E; Dave, Sandeep S; Luftig, Micah AEpstein-Barr virus (EBV) is an oncogenic human herpesvirus that dramatically reorganizes host gene expression to immortalize primary B cells. In this study, we analyzed EBV-regulated host gene expression changes following primary B-cell infection, both during initial proliferation and through transformation into lymphoblastoid cell lines (LCLs). While most EBV-regulated mRNAs were changed during the transition from resting, uninfected B cells through initial B-cell proliferation, a substantial number of mRNAs changed uniquely from early proliferation through LCL outgrowth. We identified constitutively and dynamically EBV-regulated biological processes, protein classes, and targets of specific transcription factors. Early after infection, genes associated with proliferation, stress responses, and the p53 pathway were highly enriched. However, the transition from early to long-term outgrowth was characterized by genes involved in the inhibition of apoptosis, the actin cytoskeleton, and NF-κB activity. It was previously thought that the major viral protein responsible for NF-κB activation, latent membrane protein 1 (LMP1), is expressed within 2 days after infection. Our data indicate that while this is true, LCL-level LMP1 expression and NF-κB activity are not evident until 3 weeks after primary B-cell infection. Furthermore, heterologous NF-κB activation during the first week after infection increased the transformation efficiency, while early NF-κB inhibition had no effect on transformation. Rather, inhibition of NF-κB was not toxic to EBV-infected cells until LMP1 levels and NF-κB activity were high. These data collectively highlight the dynamic nature of EBV-regulated host gene expression and support the notion that early EBV-infected proliferating B cells have a fundamentally distinct growth and survival phenotype from that of LCLs.Item Open Access Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.(PLoS One, 2010-01-20) Corti, Davide; Langedijk, Johannes PM; Hinz, Andreas; Seaman, Michael S; Vanzetta, Fabrizia; Fernandez-Rodriguez, Blanca M; Silacci, Chiara; Pinna, Debora; Jarrossay, David; Balla-Jhagjhoorsingh, Sunita; Willems, Betty; Zekveld, Maria J; Dreja, Hanna; O'Sullivan, Eithne; Pade, Corinna; Orkin, Chloe; Jeffs, Simon A; Montefiori, David C; Davis, David; Weissenhorn, Winfried; McKnight, Aine; Heeney, Jonathan L; Sallusto, Federica; Sattentau, Quentin J; Weiss, Robin A; Lanzavecchia, AntonioBACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.Item Open Access B cells in rheumatoid synovitis.(Arthritis Res Ther, 2005) Weyand, Cornelia M; Seyler, Thorsten M; Goronzy, Jörg JIn rheumatoid arthritis, T cells, B cells, macrophages, and dendritic cells invade the synovial membranes, establishing complex microstructures that promote inflammatory/tissue destructive lesions. B cell involvement has been considered to be limited to autoantibody production. However, recent studies suggest that B cells support rheumatoid disease through other mechanisms. A critical element of rheumatoid synovitis is the process of ectopic lymphoid neogenesis, with highly efficient lymphoid architectures established in a nonlymphoid tissue site. Rheumatoid synovitis recapitulates the pathways of lymph node formation, and B cells play a key role in this process. Furthermore, studies of rheumatoid lesions implanted in immunodeficient mice suggest that T cell activation in synovitis is B cell dependent, indicating the role played by B cells in presenting antigens and providing survival signals.Item Open Access B-lymphocyte effector functions in health and disease.(2010) DiLillo, David JohnB cells and humoral immunity make up an important component of the immune system and play a vital role in preventing and fighting off infection by various pathogens. B cells also have been implicated in the pathogenesis of autoimmune disease. However, the various functions that B cells perform during the development and maintenance of autoimmune conditions remain unclear. Therefore, the overall goal of this dissertation was to determine what roles B cells play during autoimmune disease. In the Chapter 3 of this dissertation, the function of B cells was assessed during tumor immunity, a model of immune system activation and cellular immunity. To quantify B cell contributions to T cell-mediated anti-tumor immune responses, mature B cells were depleted from wild type adult mice using CD20 monoclonal antibody (mAb) prior to syngeneic B16 melanoma tumor transfers. Remarkably, subcutaneous (s.c.) tumor volume and lung metastasis were increased two-fold in B cell-depleted mice. Effector-memory and interferon (IFN)γ or tumor necrosis factor (TNF)α-secreting CD4+ and CD8+ T cell induction was significantly impaired in B cell-depleted mice with tumors. Tumor antigen (Ag)-specific CD8+ T cell proliferation was also impaired in tumor-bearing mice that lacked B cells. Thus, B cells were required for optimal T cell activation and cellular immunity in this in vivo non-lymphoid tumor model. In Chapter 4 of this dissertation, the roles that B cells play during immune responses elicited by different allografts were assessed, since allograft rejection is thought to be T cell-mediated. The effects of B cell-depletion on acute cardiac rejection, chronic renal rejection, and skin graft rejection were compared using CD20 or CD19 mAbs. Both CD20 and CD19 mAbs effectively depleted mature B cells, while CD19 mAb treatment depleted plasmablasts and some plasma cells. B cell depletion did not affect acute cardiac allograft rejection, although CD19 mAb treatment prevented allograft-specific IgG production. Nonetheless, CD19 mAb treatment significantly reduced renal allograft rejection and abrogated allograft-specific IgG development, while CD20 mAb treatment did not. By contrast, B cell depletion exacerbated skin allograft rejection and augmented the proliferation of adoptively transferred alloAg-specific CD4+ T cells, demonstrating that B cells can also negatively regulate allograft rejection. Thereby, B cells can either positively or negatively regulate allograft rejection depending on the nature of the allograft and the intensity of the rejection response. Serum antibody (Ab) is, at least in part, responsible for protection against pathogens and tissue destruction during autoimmunity. In Chapter 5 of this dissertation, the mechanisms responsible for the maintenance of long-lived serum Ab levels were examined, since the relationship between memory B cells, long-lived plasma cells, and long-lived humoral immunity remains controversial. To address the roles of B cell subsets in the longevity of humoral responses, mature B cells were depleted in mice using CD20 mAb. CD20+ B cell depletion prevented humoral immune responses and class switching, and depleted existing and adoptively-transferred B cell memory. Nonetheless, B cell depletion did not affect serum Ig levels, Ag-specific Ab titers, or bone marrow (BM) Ab-secreting plasma cell numbers. Co-blockade of LFA-1 and VLA-4 adhesion molecules temporarily depleted long-lived plasma cells from the BM. CD20+ B cell depletion plus LFA-1/VLA-4 mAb treatment significantly prolonged Ag-specific plasma cell depletion from the BM, with a significant decrease in Ag-specific serum IgG. Collectively, these results indicate that BM plasma cells are intrinsically long-lived. Further, these studies now demonstrate that mature and memory B cells are not required for maintaining BM plasma cell numbers, but are required for repopulation of plasma cell-deficient BM. Thereby, depleting mature and memory B cells does not have a dramatic negative effect on pre-existing Ab levels. Collectively, the studies described in this dissertation demonstrate that B cells function through multiple effector mechanisms to influence the course and intensity of normal and autoreactive immune responses: the promotion of cellular immune responses and CD4+ T cell activation, the negative regulation of cellular immune responses, and the production and maintenance of long-lived Ag-specific serum Ab titers. Therefore, each of these three B cell effector mechanisms can contribute independently or in concert with the other mechanisms to clear pathogens or cause tissue damage during autoimmunity.Item Open Access Combined HIV-1 Envelope Systemic and Mucosal Immunization of Lactating Rhesus Monkeys Induces a Robust Immunoglobulin A Isotype B Cell Response in Breast Milk.(Journal of virology, 2016-05) Nelson, Cody S; Pollara, Justin; Kunz, Erika L; Jeffries, Thomas L; Duffy, Ryan; Beck, Charles; Stamper, Lisa; Wang, Minyue; Shen, Xiaoying; Pickup, David J; Staats, Herman F; Hudgens, Michael G; Kepler, Thomas B; Montefiori, David C; Moody, M Anthony; Tomaras, Georgia D; Liao, Hua-Xin; Haynes, Barton F; Ferrari, Guido; Fouda, Genevieve GA; Permar, Sallie RUnlabelled
Maternal vaccination to induce anti-HIV immune factors in breast milk is a potential intervention to prevent postnatal HIV-1 mother-to-child transmission (MTCT). We previously demonstrated that immunization of lactating rhesus monkeys with a modified vaccinia Ankara (MVA) prime/intramuscular (i.m.) protein boost regimen induced functional IgG responses in milk, while MVA prime/intranasal (i.n.) boost induced robust milk Env-specific IgA responses. Yet, recent studies have suggested that prevention of postnatal MTCT may require both Env-specific IgA and functional IgG responses in milk. Thus, to investigate whether both responses could be elicited by a combined systemic/mucosal immunization strategy, animals previously immunized with the MVA prime/i.n. boost regimen received an i.n./i.m. combined C.1086 gp120 boost. Remarkably, high-magnitude Env-specific IgA responses were observed in milk, surpassing those in plasma. Furthermore, 29% of vaccine-elicited Env-specific B cells isolated from breast milk were IgA isotype, in stark contrast to the overwhelming predominance of IgG isotype Env-specific B cells in breast milk of chronically HIV-infected women. A clonal relationship was identified between Env-specific blood and breast milk B cells, suggesting trafficking of that cell population between the two compartments. Furthermore, IgA and IgG monoclonal antibodies isolated from Env-specific breast milk B cells demonstrated diverse Env epitope specificities and multiple effector functions, including tier 1 neutralization, antibody-dependent cellular cytotoxicity (ADCC), infected cell binding, and inhibition of viral attachment to epithelial cells. Thus, maternal i.n./i.m. combined immunization is a novel strategy to enhance protective Env-specific IgA in milk, which is subsequently transferred to the infant via breastfeeding.Importance
Efforts to increase the availability of antiretroviral therapy to pregnant and breastfeeding women in resource-limited areas have proven remarkably successful at reducing HIV vertical transmission rates. However, more than 200,000 children are infected annually due to failures in therapy implementation, monitoring, and adherence, nearly half by postnatal HIV exposure via maternal breast milk. Intriguingly, in the absence of antiretroviral therapy, only 10% of breastfed infants born to HIV-infected mothers acquire the virus, suggesting the existence of naturally protective immune factors in milk. Enhancement of these protective immune factors through maternal vaccination will be a critical strategy to reduce the global pediatric AIDS epidemic. We have previously demonstrated that a high magnitude of HIV Env-specific IgA in milk correlates with reduced risk of infant HIV acquisition. In this study, we describe a novel HIV vaccine regimen that induces potent IgA responses in milk and therefore could potentially protect against breast milk HIV MTCT.Item Open Access Costimulation blockade alters germinal center responses and prevents antibody-mediated rejection.(Am J Transplant, 2014-01) Kim, EJ; Kwun, J; Gibby, AC; Hong, JJ; Farris III, AB; Iwakoshi, NN; Villinger, F; Kirk, AD; Knechtle, SJDe novo donor-specific antibody (DSA) after organ transplantation promotes antibody-mediated rejection (AMR) and causes late graft loss. Previously, we demonstrated that depletion using anti-CD3 immunotoxin combined with tacrolimus and alefacept (AMR regimen) reliably induced early DSA production with AMR in a nonhuman primate kidney transplant model. Five animals were assigned as positive AMR controls, four received additional belatacept and four received additional anti-CD40 mAb (2C10R4). Notably, production of early de novo DSA was completely attenuated with additional belatacept or 2C10R4 treatment. In accordance with this, while positive controls experienced a decrease in peripheral IgM(+) B cells, bela- and 2C10R4-added groups maintained a predominant population of IgM(+) B cells, potentially indicating decreased isotype switching. Central memory T cells (CD4(+) CD28(+) CD95(+)) as well as PD-1(hi) CD4(+) T cells were decreased in both bela-added and 2C10R4-added groups. In analyzing germinal center (GC) reactions in situ, lymph nodes further revealed a reduction of B cell clonal expansion, GC-follicular helper T (Tfh) cells, and IL-21 production inside GCs with additional belatacept or 2C10R4 treatment. Here we provide evidence that belatacept and 2C10R4 selectively suppresses the humoral response via regulating Tfh cells and prevents AMR in this nonhuman primate model.Item Open Access Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+/H+ antiport in human T cells.(J Cell Biol, 1986-08) Rosoff, PM; Terres, GThe cyclic undecapeptide cyclosporine A (CsA) is a potent immunosuppressive agent that inhibits the initial activation of T lymphocytes. This agent appears to be most effective in blocking the action of mitogens such as concanavalin A and the calcium ionophore A23187, which cause an influx of Ca2+, but not those that may act by alternate mechanisms. These observations suggest that CsA may block a Ca2+-dependent step in T cell activation. We have shown that stimulation of the T3-T cell receptor complex-associated Ca2+ transporter activates the Na+/H+ antiport (Rosoff, P. M., and L. C. Cantley, 1985, J. Biol. Chem., 260: 14053-14059). The tumor-promoting phorbol esters, which are co-mitogenic for T cells, activate the exchanger by a separate pathway which is mediated by protein kinase C. Both the rise in intracellular Ca2+ and intracellular pH may be necessary for the successful triggering of cellular activation. In this report we show that CsA blocks the T3-T cell receptor-stimulated, Ca2+ influx-dependent activation of Na+/H+ exchange, but not the phorbol ester-mediated pathway in a transformed human T cell line. CsA inhibited mitogen-stimulation of interleukin-2 production in a separate cell line. CsA also inhibited vasopressin stimulation of the antiporter in normal rat kidney fibroblasts, but had no effect on serum or 12-O-tetradecanoyl phorbol 13-acetate stimulation. CsA did not affect serum or vasopressin or serum stimulation of normal rat kidney cell proliferation. CsA also had no effect on lipopolysaccharide or phorbol ester stimulation of Na+/H+ exchange activity or induction of differentiation in 70Z/3 pre-B lymphocytes in which these events are initiated by the protein kinase C pathway. These data suggest that mechanisms of activation of Na+/H+ exchange that involve an elevation in cytosolic Ca2+ are blocked by CsA but that C kinase-mediated regulation is unaffected. The importance of the Na+/H+ antiport in the regulation of growth and differentiation of T cells is discussed.Item Open Access Donor apoptotic cell-based therapy for effective inhibition of donor-specific memory T and B cells to promote long-term allograft survival in allosensitized recipients.(American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2020-10) Dangi, Anil; Yu, Shuangjin; Lee, Frances T; Burnette, Melanie; Knechtle, Stuart; Kwun, Jean; Luo, XunrongAllosensitization constitutes a major barrier in transplantation. Preexisting donor-reactive memory T and B cells and preformed donor-specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long-term immunosuppression-free allograft survival. We demonstrate that donor-specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti-CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen-presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti-CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long-term islet allograft survival in such recipients.Item Open Access Epstein-Barr virus induces global changes in cellular mRNA isoform usage that are important for the maintenance of latency.(Journal of virology, 2013-11) Homa, Nicholas J; Salinas, Raul; Forte, Eleonora; Robinson, Timothy J; Garcia-Blanco, Mariano A; Luftig, Micah AOncogenic viruses promote cell proliferation through the dramatic reorganization of host transcriptomes. In addition to regulating mRNA abundance, changes in mRNA isoform usage can have a profound impact on the protein output of the transcriptome. Using Epstein-Barr virus (EBV) transformation of primary B cells, we have studied the ability of an oncogenic virus to alter the mRNA isoform profile of its host. Using the algorithm called SplicerEX with two complementary Affymetrix microarray platforms, we uncovered 433 mRNA isoform changes regulated by EBV during B-cell transformation. These changes were largely orthogonal with the 2,163 mRNA abundance changes observed during transformation, such that less than one-third of mRNAs changing at the level of isoform also changed in overall abundance. While we observed no preference for a mechanistic class of mRNA isoform change, we detected a significant shortening of 3' untranslated regions and exclusion of cassette exons in EBV-transformed cells relative to uninfected B cells. Gene ontology analysis of the mRNA isoform changes revealed significant enrichment in nucleic acid binding proteins. We validated several of these isoform changes and were intrigued by those in two mRNAs encoding the proteins XBP1 and TCF4, which have both been shown to bind and activate the promoter of the major EBV lytic trans-activator BZLF1. Our studies indicate that EBV latent infection promotes the usage of mRNA isoforms of XBP1 and TCF4 that restrict BZLF1 activation. Therefore, characterization of global changes in mRNA isoform usage during EBV infection identifies a new mechanism for the maintenance of latent infection.Item Open Access Genotypic analysis of B cell colonies by in situ hybridization. Stoichiometric expression of three VH families in adult C57BL/6 and BALB/c mice.(J Exp Med, 1987-07-01) Schulze, DH; Kelsoe, GThe filter paper disc method for cloning inducible lymphocytes was used to census the splenic B cell population of C57BL/6 and BALB/c mice for the expression of three VH gene-families, VH X-24, -Q52, and -J558. B cell colonies, arising from single founder lymphocytes, were identified by in situ hybridization with VH family- and C mu-specific cDNA probes. Some 6.7 X 10(4) C mu+ colonies were screened. Among C57BL/6- or BALB/c-derived colonies, approximately 3% were VH X-24+, approximately 19% were VH Q52+, and approximately 54% were VH J558+. These frequencies are consistent with a process of equiprobable expression for individual VH segments, and provide direct evidence that normal splenic B lymphocytes use a process of random genetic combinatorics to generate the antibody repertoire.Item Open Access Germinal center entry not selection of B cells is controlled by peptide-MHCII complex density.(Nature communications, 2018-03-02) Yeh, Chen-Hao; Nojima, Takuya; Kuraoka, Masayuki; Kelsoe, GarnettB cells expressing high affinity antigen receptors are advantaged in germinal centers (GC), perhaps by increased acquisition of antigen for presentation to follicular helper T cells and improved T-cell help. In this model for affinity-dependent selection, the density of peptide/MHCII (pMHCII) complexes on GC B cells is the primary determinant of selection. Here we show in chimeric mice populated by B cells differing only in their capacity to express MHCII (MHCII+/+ and MHCII+/-) that GC selection is insensitive to halving pMHCII density. Alone, both B cell types generate identical humoral responses; in competition, MHCII+/+ B cells are preferentially recruited to early GCs but this advantage does not persist once GCs are established. During GC responses, competing MHCII+/+ and MHCII+/- GC B cells comparably accumulate mutations and have indistinguishable rates of affinity maturation. We conclude that B-cell selection by pMHCII density is stringent in the establishment of GCs, but relaxed during GC responses.Item Open Access Identification and utilization of arbitrary correlations in models of recombination signal sequences.(Genome Biol, 2002) Cowell, Lindsay G; Davila, Marco; Kepler, Thomas B; Kelsoe, GarnettBACKGROUND: A significant challenge in bioinformatics is to develop methods for detecting and modeling patterns in variable DNA sequence sites, such as protein-binding sites in regulatory DNA. Current approaches sometimes perform poorly when positions in the site do not independently affect protein binding. We developed a statistical technique for modeling the correlation structure in variable DNA sequence sites. The method places no restrictions on the number of correlated positions or on their spatial relationship within the site. No prior empirical evidence for the correlation structure is necessary. RESULTS: We applied our method to the recombination signal sequences (RSS) that direct assembly of B-cell and T-cell antigen-receptor genes via V(D)J recombination. The technique is based on model selection by cross-validation and produces models that allow computation of an information score for any signal-length sequence. We also modeled RSS using order zero and order one Markov chains. The scores from all models are highly correlated with measured recombination efficiencies, but the models arising from our technique are better than the Markov models at discriminating RSS from non-RSS. CONCLUSIONS: Our model-development procedure produces models that estimate well the recombinogenic potential of RSS and are better at RSS recognition than the order zero and order one Markov models. Our models are, therefore, valuable for studying the regulation of both physiologic and aberrant V(D)J recombination. The approach could be equally powerful for the study of promoter and enhancer elements, splice sites, and other DNA regulatory sites that are highly variable at the level of individual nucleotide positions.Item Open Access Identification of autoantigens recognized by the 2F5 and 4E10 broadly neutralizing HIV-1 antibodies.(J Exp Med, 2013-02-11) Yang, Guang; Holl, T Matt; Liu, Yang; Li, Yi; Lu, Xiaozhi; Nicely, Nathan I; Kepler, Thomas B; Alam, S Munir; Liao, Hua-Xin; Cain, Derek W; Spicer, Leonard; VandeBerg, John L; Haynes, Barton F; Kelsoe, GarnettMany human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance because mice expressing the V(H) and V(L) regions of 2F5 have a block in B cell development that is characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. We identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes an epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but they retain the SF3B3 4E10 epitope. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motifs shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1.Item Open Access Immune checkpoint modulation enhances HIV-1 antibody induction.(Nature communications, 2020-02-19) Bradley, Todd; Kuraoka, Masayuki; Yeh, Chen-Hao; Tian, Ming; Chen, Huan; Cain, Derek W; Chen, Xuejun; Cheng, Cheng; Ellebedy, Ali H; Parks, Robert; Barr, Maggie; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Bouton-Verville, Hilary; Santra, Sampa; Wiehe, Kevin; Lewis, Mark G; Ogbe, Ane; Borrow, Persephone; Montefiori, David; Bonsignori, Mattia; Anthony Moody, M; Verkoczy, Laurent; Saunders, Kevin O; Ahmed, Rafi; Mascola, John R; Kelsoe, Garnett; Alt, Frederick W; Haynes, Barton FEliciting protective titers of HIV-1 broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development, but current vaccine strategies have yet to induce bnAbs in humans. Many bnAbs isolated from HIV-1-infected individuals are encoded by immunoglobulin gene rearrangments with infrequent naive B cell precursors and with unusual genetic features that may be subject to host regulatory control. Here, we administer antibodies targeting immune cell regulatory receptors CTLA-4, PD-1 or OX40 along with HIV envelope (Env) vaccines to rhesus macaques and bnAb immunoglobulin knock-in (KI) mice expressing diverse precursors of CD4 binding site HIV-1 bnAbs. CTLA-4 blockade augments HIV-1 Env antibody responses in macaques, and in a bnAb-precursor mouse model, CTLA-4 blocking or OX40 agonist antibodies increase germinal center B and T follicular helper cells and plasma neutralizing antibodies. Thus, modulation of CTLA-4 or OX40 immune checkpoints during vaccination can promote germinal center activity and enhance HIV-1 Env antibody responses.Item Open Access In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations.(J Exp Med, 1991-05-01) Jacob, J; Kassir, R; Kelsoe, GAfter primary immunization with an immunogenic conjugate of (4-hydroxy-3-nitrophenyl)acetyl, two anatomically and phenotypically distinct populations of antibody-forming cells arise in the spleen. As early as 2 d after immunization, foci of antigen-binding B cells are observed along the periphery of the periarteriolar lymphoid sheaths. These foci expand, occupying as much as 1% of the splenic volume by day 8 of the response. Later, foci grow smaller and are virtually absent from the spleen by day 14. A second responding population, germinal center B cells, appear on day 8-10 and persist at least until day 16 post-immunization. Individual foci and germinal centers represent discrete pauciclonal populations that apparently undergo somatic evolution in the course of the primary response. We suggest that foci may represent regions of predominantly interclonal competition for antigen among unmutated B cells, while germinal centers are sites of intraclonal clonal competition between mutated sister lymphocytes.Item Open Access In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers.(J Exp Med, 1992-09-01) Jacob, J; Kelsoe, GIn the genetically restricted response that follows immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to protein carriers, two distinct populations of B cells are observed in the spleens of C57BL/6 mice. By 48 h postimmunization, foci of antigen-binding B cells appear along the periphery of the periarteriolar lymphoid sheaths. These foci expand to contain large numbers of antibody-forming cells that neither bind the lectin, peanut agglutinin, nor mutate the rearranged immunoglobulin variable region loci. Germinal centers containing peanut agglutinin-positive B cells can be observed by 96-120 h after immunization. Although specific for the immunizing hapten, these B cells do not produce substantial amounts of antibody, but are the population that undergoes somatic hypermutation and affinity-driven selection. Both focus and germinal center populations are pauciclonal, founded, on average, by three or fewer B lymphocytes. Despite the highly specialized roles of the focus (early antibody production) and germinal center (higher affinity memory cells) B cell populations, analysis of VH to D to JH joins in neighboring foci and germinal centers demonstrate that these B cell populations have a common clonal origin.