Joseph J. Dolence

Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B-cell precursors.

The generation of B‐cell precursors (BCP) from lymphohematopoietic progenitors (LHP) in bone marrow is dependent on signals provided by the receptor tyrosine kinase Flt3 and its ligand, Flt3‐ligand (FL). Mice deficient in FL exhibit striking reductions in LHP and BCP. Currently, the mechanism by which Flt3 regulates lymphoid lineage/B‐cell development is unknown. Here, we show that haploinsufficiency of FL (FL+/−) reduced the numbers of LHP, common lymphoid progenitors, and pro‐B cells, suggesting that FL levels set a threshold for B lymphopoiesis. Limiting dilution analysis confirmed reduced BCP frequency in FL+/− mice. Real‐time PCR of LHP from FL+/− animals showed increased transcripts of the B lineage inhibitor id1. However, targeted deletion of id1 did not restore the lymphoid/B lineage deficiencies in FL−/− mice, supporting Id1‐independent mechanisms. BrdU incorporation studies established that FL is not essential for the proliferation of Flt3+ multipotential progenitors. Analysis of FL−/− progenitors expressing low levels of Flt3 revealed decreased levels of the pro‐survival factor Mcl1. Consequently, the Flt3+ LHP progeny of Flt3low LSK+ cells exhibited increased Annexin V staining. Together, these data suggest that Flt3 signaling initiates a cascade of events in Flt3low precursors that promote the survival of LHP from which BCP are derived.

Hoxa9 and Flt3 Signaling Synergistically Regulate an Early Checkpoint in Lymphopoiesis

Hoxa9 and Flt3 signaling are individually important for the generation of lymphoid lineage precursors from multipotent hematopoietic progenitors (MPP) in bone marrow. Mice deficient for Hoxa9, Flt3, or Flt3 ligand (FL) have reduced numbers of lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP), and B/T cell precursors. Hoxa9 regulates lymphoid development, in part, through transcriptional regulation of Flt3. However, it was unclear whether Hoxa9 has functions in lymphopoiesis independent of, or alternatively, synergistically with Flt3 signaling. In this study, we show that Hoxa9−/−Flt3l−/− mice have more severe deficiencies in all B lineage cells, CLP, LMPP, and total Flt3+ MPP in bone marrow than the single knockouts. Although LMPP and Flt3+ CLP contain precursors for NK and dendritic cell lineage cells, no deficiencies in these lineages beyond that in Flt3l−/− mice was found. Thymocyte cellularity was significantly reduced in the compound knockout, although peripheral T cell numbers mirrored Flt3l−/− mice. Analysis of the hematopoietic progenitor compartment revealed elevated numbers of CD150+hiCD34−CD41+ myeloid–biased stem cells in Hoxa9−/−Flt3l−/− mice. In contrast, CD150− MPP enriched for lymphoid potential were synergistically reduced, suggesting Hoxa9 and Flt3 signaling function coordinately to regulate lymphopoiesis at a very early stage. Real-time PCR analysis of CD150−Flt3+ cells from wild-type control, Hoxa9−/−, and Flt3l−/− single knockouts revealed decreased lymphoid transcripts, corroborating the importance of these regulators in lymphoid development. Taken together, these studies reveal a very early checkpoint in lymphopoiesis dependent on the combinatorial activities of Hoxa9 function and Flt3 signaling.

Flt3 signaling regulates the proliferation, survival, and maintenance of multipotent hematopoietic progenitors that generate B cell precursors.

Flt3 signaling plays a crucial role in regulating the survival and differentiation of lymphoid progenitors into B cell precursors (BCPs) in bone marrow. To define further the role of Flt3 signaling in lymphoid progenitor survival, mice deficient in Flt3 ligand that also expressed a Bcl2 transgene (Eμ-bcl2tg flt3l(-/-)) were generated. Intracellular flow cytometry established transgene expression in primitive hematopoietic progenitors, including lineage-negative Sca-1(+) c-kit(+) (LSK(+)) CD27(-) cells enriched for functional hematopoietic stem cells. Compared with flt3l(-/-) mice, Eμ-bcl2tg flt3l(-/-) mice had significantly increased multipotential progenitors (MPPs), IL-7R(+) common lymphoid progenitors, and B cell precursors. To determine whether forced expression of Bcl2 was sufficient to restore lymphoid priming in the absence of Flt3 signaling Eμ-bcl2tg flt3l(-/-)rag1-gfp(+) mice were generated. Analysis of Eμ-bcl2tg flt3l(-/-)rag1-gfp(+) mice revealed that the Bcl2 transgene had no effect on lymphoid priming before CD19 expression. Thus, forced expression of a survival gene can bypass the requirement for threshold levels of Flt3 signaling requisite for lymphoid priming. Temporal Flt3 ligand (FL) replacement therapy in flt3l(-/-) mice revealed specific requirements for Flt3 signaling in the expansion and maintenance of Flt3(+hi) MPP and Flt3(+) all lymphoid progenitors, but not Flt3(+) B lymphoid progenitors (BLPs), the immediate precursors of BCPs. BCPs were restored after temporal in vivo FL treatment, albeit with delayed kinetics. Together, these results show that Flt3 regulates the proliferation, survival, and maintenance of developmental stage-specific hematopoietic progenitors that give rise to BCPs.

Differential requirement for Hoxa9 in the development and differentiation of B, NK, and DC-lineage cells from Flt3+ multipotential progenitors.

Hoxa9 is a homeodomain transcription factor important for the generation of Flt3+hiIL-7R- lymphoid biased-multipotential progenitors, Flt3+IL-7R+ common lymphoid progenitors (CLPs), and B cell precursors (BCP) in bone marrow (BM). In addition to B-cell, Flt3+IL-7R+ CLPs possess NK and DC developmental potentials, although DCs arise from Flt3+IL-7R- myeloid progenitors as well. In this study, we investigated the requirement for Hoxa9, from Flt3+ or Flt3- progenitor subsets, in the development of NK and DC lineage cells in BM. Flt3+IL-7R+Ly6D- CLPs and their Flt3+IL-7R+Ly6D+ B lineage-restricted progeny (BLP) were significantly reduced in hoxa9−/− mice. Interestingly, the reduction in Flt3+IL-7R+ CLPs in hoxa9−/− mice had no impact on the generation of NK precursor (NKP) subsets, the differentiation of NKP into mature NK cells, or NK homeostasis. Similarly, percentages and numbers of common dendritic progenitors (CDP), as well as their plasmacytoid or conventional dendritic cell progeny in hoxa9−/− mice were comparable to wildtype. These findings reveal distinct requirements for Hoxa9 or Hoxa9/Flt3 molecular circuits in regulation of B versus NK and DC development in BM.

Airway exposure initiates peanut allergy by involving the IL-1 pathway and T follicular helper cells in mice

Background Little is currently known regarding the immunologic mechanism(s) that initiate peanut allergy. Notably, peanut proteins have been detected in house dust, and their levels correlate with peanut allergy prevalence. Objective This study aimed to develop a new mouse model for peanut allergy and to investigate the immunologic mechanisms involved in peanut allergen sensitization. Methods To mimic environmental exposure, naive mice were exposed to peanut flour by inhalation for up to 4 weeks. We then analyzed serum levels of IgE antibody and challenged mice with peanut proteins. Immunological mechanisms involved in sensitization were analyzed using cytokine reporter mice, an adoptive cell transfer model, and gene knockout mice. Results When exposed to peanut flour by inhalation, both BALB/c and C57BL/6 mice developed peanut allergy, as demonstrated by the presence of peanut‐specific IgE antibodies and manifestation of acute anaphylaxis on challenge. A large number of follicular helper T (Tfh) cells were also detected in draining lymph nodes of allergic mice. These cells produced IL‐4 and IL‐21, and they more robustly promoted peanut‐specific IgE production than Th2 cells did. Genetic depletion of Tfh cells decreased IgE antibody levels and protected mice from anaphylaxis, without affecting Th2 cells. Furthermore, peanut flour exposure increased lung levels of IL‐1&agr; and IL‐1&bgr;, and mice deficient in the receptor for these cytokines showed a significant decrease in Tfh cells compared with in wild‐type mice. Conclusions Tfh cells play a key role in peanut allergy, and the IL‐1 pathway is involved in the Tfh response to peanut allergen exposure. Graphical abstract Figure. No Caption available.

Cell extrinsic alterations in splenic B cell maturation in Flt3-ligand knockout mice

B lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3‐ligand (FL‐/‐) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL‐/‐ mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL‐/‐ mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF‐R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL‐/‐ mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL‐/‐ mice. A Bcl2 transgene did not rescue TS cells in FL‐/‐ mice, uncoupling FL‐deficiency to Bcl2‐dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL‐/‐ mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.

Proceedings of the 2015 WAO Symposium on Food Allergy and the Microbiome

Table of contentsA1 Characterization of the immunoallergic profile towards the proteins of the wheat flour in Cuban populationRaúl Lázaro Castro Almarales, Mary Carmen Reyes Zamora, Beatriz Tamargo, Damaris Torralba Averoff, Raysa Cruz, Yunia Oliva Diaz, Mirta Alvarez Castello, Alexander Ciria, Alexis Labrada, Maytee MateoA2 Are peanuts causing food allergy in Cuba?Maytee Mateo, Damaris Torralba Averoff, Raysa Cruz, Yunia Oliva Diaz, Mirta Alvarez Castello, Alexander Ciria, Mary Carmen Reyes Zamora, Beatriz Tamargo, Alexis LabradaA3 Prick test and immunoallergic profile to soy allergens in Cuban populationOmar Herrera, Maytee Mateo, Raysa Cruz, Mirta Alvarez Castello, Alexander Ciria, Raúl Lázaro Castro Almarales, Mary Carmen Reyes Zamora, Alexis LabradaA4 Skin sensitization and immunoallergic profile to hens egg in Cuban populationJosé Severino Rodríguez Canosa, Raysa Cruz, Maytee Mateo, Mirta Alvarez Castello, Alexander Ciria, Raúl Lázaro Castro Almarales, Mary Carmen Reyes Zamora, Alexis LabradaA5 Sensitization to three domestic mites in patients with adverse food events to shellfishMirta Alvarez Castello, Raúl Lázaro Castro Almarales, Alexis Labrada, BiocenA6 Diagnostic efficacy by skin prick test with allergenic extracts of legumes in Cuban patientsYamilet Ibizate Novales, Ilonka Estruch Fajardo, Alexis Labrada, Maytee Mateo, Armando GinardA7 Baked egg goods without wheat flour carry an increased risk of reactionBruce Lanser, Anna Faino, Erwin Gelfand, Pia HaukA8 Prevalence, incidence and associated risk factors of adverse reaction to food in Cuban infants - a population-based prospective studySilvia Venero Fernández, Julia Urbina, Mirta Alvarez Castello, Raúl Lázaro Castro Almarales, Ramón Suárez Medina, Hermes Fundora Hernández, John Britton, Andrew William FogartyA9 Microbiome in ice machines and assessing the plasma nanotechnology in breaking the biofilm and improving air qualityNabarun Ghosh, Clinton Ross Bell, Chandini Revanna, Constantine Saadeh, Jeff Bennert, Danius Bouyi, Mitsy Veloz, Nelofar SheraliA10 Characteristics of patients with food allergy in health public serviceMagna CoelhoA11 Allergic rhinitis and asthma index increased in Texas panhandle and AHPCO and plasma nanotechnology as solutionsNabarun Ghosh, Jeff Bennert, Danius Bouyi, Constantine Saadeh, Clinton Ross Bell, Mitsy Veloz, Chandini Revanna, Nelofar SheraliA12 Antigen-specific T follicular helper cells mediate peanut allergy in miceJoseph J. Dolence, Takao Kobayashi, Koji Iijima, Hirohito Kita, Hirohito Kita, Ashli Moore, James KrempskiA13 Production of recombinant Mal d 3, a major apple allergen, in Pichia Pastoris, to investigate the impact of the food matrix and post-translational modifications on Mal d 3 immuno-reactivityRoberta Aina, Riccardo Asero, Sabine Pfeifer, Pawel Dubiela, Merima Bublin, Christian Radauer, Piotr Humeniuk, Karin Hoffmann-SommergruberA14 Reaction to sports drink: no whey! Whey allergy in absence of clinical cow’s milk allergyFrank Eidelman, Ves Dimov, Charl KhalilA15 Food allergy on Tumblr: focus on teenage audience may increase educational impactVes Dimov, Frank Eidelman, Charl KhalilA16 Changes in IgE levels following one-year immunizations in two children with food allergyAlice E. W. Hoyt, Peter Heymann, Alexander Schuyler, Scott Commins, Thomas Platts-MillsA17 IgE and IgG4 antibodies to cows milk components in children with eosinophilic esophagitis: higher specific IgG4 antibodies and IgG4:IgE ratios compared with subjects with IgE-mediated food allergyAlexander Schuyler, Patrice Kruszewski, John Russo, Lisa Workman, Thomas Platts-Mills, Elizabeth Erwin, Anubha TripathiA18 Frequency of Sensitization to Food Allergens in Patients with Rhinitis and Asthma in the National Medical Center La Raza “Dr. Antonio Fraga Mouret”, Mexico CityGabriela Yvette Castellanos, Elizabeth Mendieta, Martín Becerril-Angeles