Medicine (Austin & Northern Health) - Research Publications
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ItemComplementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis.(Rockefeller University Press, 2003-11-17)Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Ralpha, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Ralpha and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL-/- x IL-7Ralpha-/- BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes. Furthermore, in contrast to IL-7Ralpha-/- mice, FL-/- x IL-7Ralpha-/- mice also lack mature B cells and detectable committed B cell progenitors during fetal development. Thus, signaling through the cytokine tyrosine kinase receptor flt3 and IL-7Ralpha are indispensable for fetal and adult B cell development.
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ItemFas transduces activation signals in normal human T lymphocytes.(Rockefeller University Press, 1993-12-01)The Fas gene encodes a cell surface molecule that is a member of the the nerve growth factor/tumor necrosis factor receptor family of proteins and can mediate programmed cell death (apoptosis) in certain transformed cell lines. To characterize further the biological function of Fas, particularly with regard to its function in normal cells, a panel of monoclonal antibodies (mAbs) was generated against the extracellular portion of human Fas. Some of these mAbs induced apoptosis in transformed cell lines expressing Fas, but only when immobilized on the culture vessel. One of the new Fas mAbs (M38) was used for studies on normal lymphoid cells and found to stimulate the proliferation of purified human T cells and thymocytes when immobilized on culture wells along with CD3 antibody. T cell proliferation induced by Fas mAb was largely interleukin 2 independent and was demonstrated to be due to a direct effect on the precursor T cell. Thus, the data demonstrate that in addition to a role in the induction of apoptosis in certain transformed cell lines, the Fas protein may also play an important role in the activation and proliferation of normal T cells.
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ItemEarly lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.(Rockefeller University Press, 1994-11-01)Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.
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ItemPrevention of peripheral tolerance by a dendritic cell growth factor: flt3 ligand as an adjuvant.(Rockefeller University Press, 1998-12-07)Injections of soluble proteins are poorly immunogenic, and often elicit antigen-specific tolerance. The mechanism of this phenomenon has been an enduring puzzle, but it has been speculated that tolerance induction may be due to antigen presentation by poorly stimulatory, resting B cells, which lack specific immunoglobulin receptors for the protein. In contrast, adjuvants, or infectious agents, which cause the release of proinflammatory cytokines such as tumor necrosis factor alpha and interleukin 1beta in vivo are believed to recruit and activate professional antigen-presenting cells to the site(s) of infection, thereby eliciting immunity. Here we show that administration of Flt3 ligand (FL), a cytokine capable of inducing large numbers of dendritic cells (DCs) in vivo, (a) dramatically enhances the sensitivity of antigen-specific B and T cell responses to systemic injection of a soluble protein, through a CD40-CD40 ligand-dependent mechanism; (b) influences the class of antibody produced; and (c) enables productive immune responses to otherwise tolerogenic protocols. These data support the hypothesis that the delicate balance between immunity and tolerance in vivo is pivotally controlled by DCs, and underscore the potential of FL as a vaccine adjuvant for immunotherapy in infectious disease and other clinical settings.
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ItemEx vivo restimulation of human PBMC expands a CD3+CD4-CD8- γδ+ T cell population that can confound the evaluation of CD4 and CD8 T cell responses to vaccination.(Hindawi Limited, 2013)The measurement of vaccine-induced humoral and CD4(+) and CD8(+) cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3(+)CD4(-)CD8(-) γδ (+) T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3(+)CD4(-)CD8(-) γδ (+) T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4(+) and CD8(+) immune responses following vaccination, the CD3(+)CD4(-)CD8(-) γδ (+) T cells are either excluded or separately enumerated from the overall frequency determination.
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ItemDramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.(Rockefeller University Press, 1996-11-01)Dendritic cells (DC) are the most efficient APC for T cells. The clinical use of DC as vectors for anti-tumor and infectious disease immunotherapy has been limited by their trace levels and accessibility in normal tissue and terminal state of differentiation. In the present study, daily injection of human Flt3 ligand (Flt3L) into mice results in a dramatic numerical increase in cells co-expressing the characteristic DC markers-class II MHC, CD11c, DEC205, and CD86. In contrast, in mice treated with either GM-CSF, GM-CSF plus IL-4, c-kit ligand (c-kitL), or G-CSF, class II+ CD11c+ cells were not significantly increased. Five distinct DC subpopulations were identified in the spleen of Flt3L-treated mice using CD8 alpha and CD11b expression. These cells exhibited veiled and dendritic processes and were as efficient as rare, mature DC isolated from the spleens of untreated mice at presenting allo-Ag or soluble Ag to T cells, or in priming an Ag-specific T cell response in vivo. Dramatic numerical increases in DC were detected in the bone marrow, gastro-intestinal lymphoid tissue (GALT), liver, lymph nodes, lung, peripheral blood, peritoneal cavity, spleen, and thymus. These results suggest that Flt3L could be used to expand the numbers of functionally mature DC in vivo for use in clinical immunotherapy.
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ItemInterleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells.(Rockefeller University Press, 2000-09-18)Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell-inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell-derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon gamma effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.
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ItemDendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor.(Rockefeller University Press, 1996-12-01)The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8 alpha or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti-GM-CSF antibody or the use of precursors from GM-CSF-deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.
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ItemQuantitative HLA-class-II/factor VIII (FVIII) peptidomic variation in dendritic cells correlates with the immunogenic potential of therapeutic FVIII proteins in hemophilia A(WILEY, 2020-01)BACKGROUND: Plasma-derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti-FVIII-antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic. Since inhibitor development is T-helper-cell-dependent, human leukocyte antigen (HLA)-class-II (HLAcII) molecules constitute an important early determinant. OBJECTIVES: Use dendritic cell (DC)-protein processing/presentation assays with mass-spectrometric and peptide-proteomic analyses to quantify the DP-bound, DQ-bound, and DR-bound FVIII-derived peptides in individual HLAcII repertoires and compare the immunogenic potential of six distinct FVIIIs based on their measured peptide counts. PATIENTS/METHODS: Monocyte-derived DCs from normal donors and/or PWHA were cultured with either: Mix-rFVIII, a VWF-free equimolar mixture of a full-length (FL)-rFVIII [Advate® (Takeda)] and four distinct B-domain-deleted (BDD)-rFVIIIs [Xyntha® (Pfizer), NovoEight® (Novo-Nordisk), Nuwiq® (Octapharma), and Afstyla® (CSL Behring GmBH)]; a pdFVIII + pdVWF [Beriate® (CSL Behring GmBH)]; Advate ± pdVWF; Afstyla ± pdVWF; and Xyntha + pdVWF. RESULTS: We showed that (i) Beriate had a significantly lower immunogenic potential than Advate ± pdVWF, Afstyla - pdVWF, and Mix-rFVIII; (ii) distinct FVIIIs differed significantly in their immunogenic potential in that, in addition to (i), Afstyla + pdVWF had a significantly lower immunogenic potential than Beriate, while the immunogenic potential of Beriate was not significantly different from that of Xyntha + pdVWF; and (iii) rFVIIIs with pdVWF had significantly lower immunogenic potentials than the same rFVIIIs without pdVWF. CONCLUSIONS: Our results provide HLAcII peptidomic level explanations for several important clinical observations/issues including the differential immunogenicity of distinct FVIIIs and the role of HLAcII genetics in inhibitor development.
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ItemCancer testis antigens expression in mesothelioma: role of DNA methylation and bioimmunotherapeutic implications(NATURE PUBLISHING GROUP, 2002-03-18)Recent evidences suggest that malignant mesothelioma may be sensitive to immunotherapy; however, little is known about malignant mesothelioma-associated tumour antigens. Focusing on cancer/testis antigens, the expression of well-characterised immunogenic tumour-associated antigens was investigated in malignant mesothelioma cells. At variance with MAGE-4 and NY-ESO-1, malignant mesothelioma cells frequently expressed MAGE-1, -2 and -3, GAGE 1-2, GAGE 1-6, SSX-2 and SSX 1-5, and distinct malignant mesothelioma cells concomitantly expressed at least four cancer/testis antigens. Additionally, the tumour-associated antigens RAGE-1 was expressed at high levels in both benign and malignant mesothelial cells. Lastly, treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the expression of the cancer/testis antigen examined in malignant mesothelioma cells. Overall, these findings strongly suggest that cancer/testis antigens-based immunotherapy may represent a suitable therapeutic approach to malignant mesothelioma, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemo-immunotherapeutic strategies in malignant mesothelioma patients.