Activation of dendritic cells from patients with epithelial ovarian cancer

Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing immune responses to tumor cells. Lin−HLA-DR + DC populations in peripheral blood mononuclear cells (PBMCs) and in ascites mononuclear leukocytes (MNLs) of patients with epithelial ovarian cancer (EOC) are phenotypically immature. Lin−HLA-DR + DCs from PBMCs of normal subjects and EOC patients and MNLs from ascites cells of patients were examined for specific cell surface markers or indicators of differentiation or activation. Separating Lin− HLA-DR+ DCs into subsets based on their HLA-DR intensity provided an additional method for identifying the two major lineages of DCs, myeloid and plasmacytoid. The activation potential of these DCs following exposure to the maturation agents CD40 ligand (CD40L) and lipopolysaccharide (LPS) was examined by measurement of IL-12 and IL-10 concentrations in DC culture supernatants in addition to their ability to stimulate allogeneic T cells. DCs from PBMCs of normal subjects and EOC patients and DCs isolated from ascites MNLs of EOC patients were separated into subsets based on CD11c and CD123 cell surface marker expression identifying the major DC types. These subsets were then compared with cells sorted on the basis of HLA-DR intensity. The in vivo behavior of DCs and DC subsets in peripheral blood and ascites following treatment of peritoneal carcinoma patients with the growth factor fins-like tyrosine kinase 3 ligand (Flt3L) was also examined. Increases in proportions and total numbers of DCs from peripheral blood and ascites were associated with increased secretion of IL-12 and IL-10 following in vitro activation of cultured DCs. There were differences between DCs from PBMCs and ascites and between DC subsets in expression of cell surface markers, cytokine profile, and the ability of Lin−HLA-DR + cells to stimulate proliferation of allogeneic T cells from EOC patients. These Lin−HLA-DR+ cells have certain functional properties that suggest that they could have the potential to facilitate an adaptive anti-tumor immune response. ^

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE Publication No.___________ Lisa Harn-Ging Shiue, B.S. Supervisory Professor: Madeleine Duvic, M.D. Extracorporeal photopheresis (ECP) is an effective, low-risk immunomodulating therapy for leukemic cutaneous T cell lymphoma (L-CTCL) and graft versus host disease (GVHD), but whether the mechanism(s) of action in these two diseases is (are) identical or different is unclear. To determine the effects of ECP in vivo, we studied regulatory T cells (T-regs), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) by immunofluorescence flow cytometry in 18 L-CTCL and 11 GVHD patients before and after ECP at Day 2, 1 month, 3 months, and 6 months. In this study, ECP was effective in 12/18 L-CTCL patients with a 66.7% overall response rate (ORR) and 6/11 GVHD patients with a 54.5% ORR. Prior to ECP, the percentages of CD4+Foxp3+ T cells in 9 L-CTCL patients were either lower (L-CTCL-Low, n=2) or higher (L-CTCL-High, n=7) than normal. Five of the 7 GVHD patients had high percentages of CD4+Foxp3+ T cells (GVHD-High). Six of 7 L-CTCL-High patients had >80% CD4+Foxp3+ T cells which were correlated with tumor cells, and were responders. Both L-CTCL-High and GVHD-High patients had decreased percentages of CD4+Foxp3+ and CD4+Foxp3+CD25- T cells after 3 months of treatment. CD4+Foxp3+CD25+ T cells increased in GVHD-High patients but decreased in L-CTCL-High patients after 3 months of ECP. In addition, numbers of CTLs were abnormal. We confirmed that numbers of CTLs were low in L-CTCL patients, but high in GVHD patients prior to ECP. After ECP, CTLs increased after 1 month in 4/6 L-CTCL patients whereas CTLs decreased after 6 months in 3/3 GVHD patients. Myeloid (mDCs) and plasmacytoid DCs (pDCs) were also low at baseline in L-CTCL and GVHD patients confirming the DC defect. After 6 months of ECP, numbers and percentages of mDCs and pDCs increased in L-CTCL and GVHD. MDCs were favorably increased in 8/12 L-CTCL responders whereas pDCs were favorably increased in GVHD patients. These data suggest that ECP is favorably modulating the DC subsets. In L-CTCL patients, the mDCs may orchestrate Th1 cell responses to overcome immune suppression and facilitate disease regression. However, in GVHD patients, ECP is favorably down-regulating the immune system and may be facilitating immune tolerance to auto-or allo-antigens. In both L-CTCL and GVHD patients, DCs are modulated, but the T cell responses orchestrated by the DCs are different, suggesting that ECP modulates depending on the immune milieu. _______________

Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma.

Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.

Análise fenotípica de células dendríticas e de linfócitos T efetores, polifuncionais e reguladores no líquen plano

INTRODUÇÃO: Líquen plano (LP) é uma doença mucocutânea de natureza inflamatória crônica de etiologia ainda desconhecida. A estimulação da imunidade inata via os receptores Toll-like (TLRs) podem influenciar as células dendríticas e direcionar a resposta de células T CD4+ e CD8+ efetoras, assim como também favorecer o estado inflamatório do LP. OBJETIVOS: Avaliar o perfil fenotípico de células dendríticas mielóides (mDCs) e plasmocitóides (pDCs) e de linfócitos T CD4+ e CD8+ após estímulo com agonistas de TLRs no sangue periférico de pacientes com LP. Além disto, avaliar a frequência, perfil de maturação e os subtipos de células T CD4+ e TCD8+ reguladores. MÉTODOS: Foram selecionados 18 pacientes com LP (15 mulheres, 3 homens), com 41,57 ± 4,73 anos de idade e um grupo controle com 22 indivíduos sadios (18 mulheres, 4 homens), com 43,92 ± 7,83 anos de idade. As células mononucleares (CMNs) de sangue periférico foram avaliadas por citometria de fluxo quanto à: 1) Produção de TNF-? em mDCs e de IFN-? em pDCs em CMNs ativadas por agonistas de TLR 4, 7, 7/8 e 9; 2) Análise de células T CD4+ e CD8+ monofuncionais e polifuncionais após estímulo com agonistas de TLR 4, 7/8, 9 e enterotoxina B de Staphylococcus aureus (SEB); 3) Avaliação de células Th17 e Th22/Tc22 em CMNs após estímulo com SEB; 4) Frequência, perfil de maturação e subtipos de células T CD4+ e CD8+ reguladoras. RESULTADOS: 1) Nos pacientes com LP foi demonstrado um aumento na frequência de mDCs TNF-alfa+ após estímulo com agonistas de TLR4/LPS e TLR7-8/CL097, mas com imiquimode/TLR7 houve diminuição da expressão de CD83. Já nas pDCs do grupo LP, o imiquimode foi capaz de diminuir a expressão de CD80 e o CpG/TLR9 diminuiu a expressão de CD83 no LP. 2) As células T CD4+ secretoras de IL-10 mostraram aumento da frequência nos níveis basais, que diminuiu após estímulo com LPS e SEB. Em contraste, a produção de IFN-y aumentou em resposta ao LPS enquanto diminuiu para CpG. As células T CD4+ polifuncionais, secretoras de 5 citocinas simultâneas (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) diminuíram no LP após estímulo com CL097 e CpG. Entretanto, na ausência de IL-10, houve aumento da frequência de células CD4+IL-17+IL-22+TNF+IFN-y+ em resposta ao LPS. Um aumento na polifuncionalidade foi observado em células TCD4+ que expressam CD38, marcador de ativação crônica e na ausência de IL-10. Similarmente, às TCD4+, uma diminuição de células T CD8+ IFN-y+ e TNF+ foram detectadas após estímulo com CpG. 3) As células Th22/Tc22 nos níveis basais e após estímulo com SEB se mostraram aumentadas. As células Th17 não mostraram diferenças entre os grupos. 4) A frequência das células T CD4+ e CD8+ reg totais (CD25+Foxp3+CD127low/-) está elevada no LP. Quanto aos perfis de maturação, há aumento na frequência de células TCD4+ de memória efetora enquanto que para as células T CD8+ há predomínio das células de memória central. Quanto aos subtipos, há aumento nas células T CD4+ regs periféricas (pT reg). CONCLUSÕES: O estado de ativação das mDCs após ativação das vias de TLRs 4 e 7/8 pode influenciar na geração de resposta T efetoras no LP. O perfil de resposta monofuncional e polifuncional aos estímulos TLRs reflete a ativação destas células no sangue periférico. Além disso, o aumento de Th22/Tc22 e das células T regs indicam uma relação entre regulação e células efetoras no sangue periférico evidenciando que existem alterações extracutâneas no LP

A population of HLA-DR+ immature cells accumulates in the blood dendritic cell compartment of patients with different types of cancer

Dendritic cell (DC) defects are an important component of immunosuppression in cancer. Here, we assessed whether cancer could affect circulating DC populations and its correlation with tumor progression. The blood DC compartment was evaluated in 136 patients with breast cancer, prostate cancer, and malignant glioma. Phenotypic, quantitative, and functional analyses were performed at various stages of disease. Patients had significantly fewer circulating myeloid (CD11c(+)) and plasmacytoid (CD123(+)) DC, and a concurrent accumulation of CD11c(-)CD123(-) immature cells that expressed high levels of HLA-DR+ immature cells (DR+IC). Although DR+IC exhibited a limited expression of markers ascribed to mature hematopoietic lineages, expression of HLA-DR, CD40, and CD86 suggested a role as antigen-presenting cells. Nevertheless, DR+IC had reduced capacity to capture antigens and elicited poor proliferation and interferon-gamma secretion by T-lymphocytes. Importantly, increased numbers of DR+IC correlated with disease status. Patients with metastatic breast cancer showed a larger number of DR+IC in the circulation than patients with local/nodal disease. Similarly, in patients with fully resected glioma, the proportion of DR+IC in the blood increased when evaluation indicated tumor recurrence. Reduction of blood DC correlating with accumulation of a population of immature cells with poor immunologic function may be associated with increased immunodeficiency observed in cancer.

The colony-stimulating factor 1 receptor is expressed on dendritic cells during differentiation and regulates their expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.

Cytokine expanded myeloid precursors function as regulatory antigen-presenting cells and promote tolerance through IL-10-producing regulatory T cells

The initiation of graft-vs-host disease (GVHD) after stem cell transplantation is dependent on direct Ag presentation by host APCs, whereas the effect of donor APC populations is unclear. We studied the role of indirect Ag presentation in allogenic T cell responses by adding populations of cytokine-expanded donor APC to hemopoietic grafts that would otherwise induce lethal GVHD. Progenipoietin-1 (a synthetic G-CSF/Flt-3 ligand molecule) and G-CSF expanded myeloid dendritic cells (DC), plasmacytoid DC, and a novel granulocyte-monocyte precursor population (GM) that differentiate into class II+,CD80/CD86(+),CD40(-) APC during GVHD. Whereas addition of plasmacytoid and myeloid donor DC augmented GVHD, GM cells promoted transplant tolerance by MHC class II-restricted generation of IL-10-secreting, Ag-specific regulatory T cells. Importantly, although GM cells abrogated GVHD, graft-vs-leukemia effects were preserved. Thus, a population of cytokine-expanded GM precursors function as regulatory APCs, suggesting that G-CSF derivatives may have application in disorders characterized by a loss of self-tolerance.

Higher-order CpG-DNA stimulation reveals distinct activation requirements for marginal zone and follicular B cells in lupus mice

Mouse follicular B cells express TLR9 and respond vigorously to stimulation with single-stranded CpG-oligodeoxynucleotides (ODN). Surprisingly, follicular B cells do not respond to direct stimulation with other TLR9 ligands, such as bacterial DNA or class A(D) CpG-ODN capable of forming higher-order structures, unless other cell types are present. Here, we show that priming with interferons or with B cell-activating factor, or simultaneous co-engagement of the B cell receptor for antigen (BCR), can overcome this unresponsiveness. The effect of interferons occurs at the transcriptional level and is mediated through an autocrine/paracrine loop, which is dependent on IRF-1, IL-6 and IL-12 p40. We hypothesize that the lack of bystander activation of follicular B cells with more complex CpG ligands may be an important safety mechanism for avoiding autoimmunity. This will prevent resting B cells from responding to foreign or self-derived hypomethylated double-stranded CpG ligands unless these ligands are either delivered through the B cell receptor or under conditions where B cells are simultaneously co-engaged by activated plasmacytoid dendritic cells or TH1 cells. A corollary is that the heightened responsiveness of lupus B cells to TLR9-induced stimulation cannot be ascribed to unprimed follicular B cells, but is rather mediated by hypersensitive marginal zone B cells.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

DNA Motifs Suppressing TLR9 Responses

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythernatosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleoti des (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.

Tratamiento del rechazo mediado por anticuerpos en pacientes trasplantados renales revisión sistemática

Antecedentes: El trasplante renal es la mejor alternativa terapéutica para la enfermedad renal crónica terminal. Los medicamentos inmunosupresores previenen el rechazo. El rechazo mediado por anticuerpos es frecuente y disminuye la función y duración del injerto. Objetivo: Evaluar sistemáticamente la evidencia disponible relacionada con la eficacia y seguridad del tratamiento para el rechazo mediado por anticuerpos en pacientes trasplantados renales. Metodologia: Revisión sistemática en bases de datos MEDLINE, EMBASE, Scopus y Biblioteca virtual de la salud. Literatura gris google scholar, google academico, www.clinicaltrialsregister.eu, and https://clinicaltrials.gov/. Búsqueda manual referencias artículos pre-seleccionados así como de revisiones previamente publicadas. Se siguieron las recomendacioes guia PRISMA para la identificacion de artículos potenciales, tamizaje y selección teniendo en cuenta los criterios de inclusion. Extracción datos de acuerdo a las variables, revisión calidad de los artículos elegidos utilizando evaluación riesgo de segos de Cochrane. Resultados: Se seleccionaron 9 ensayos clínicos publicados entre 1980 y 2016, incluyeron 222 pacientes (113 brazo de intervención y 109 en el control), seguimiento promedio 16 meses. Intervenciones evaluadas plasmaféresis, inmunoadsorción y rituximab. Hubo una amplia heterogeneidad en la definición de criterios de inclusión, criterios diagnósticos de rechazo y medidas de evaluación de eficacia de las intervenciones. Tres estudios encontraron diferencias estadísticamente significativas entre los grupos de tratamiento. Conclusiones: La evidencia sobre la eficacia de los tratamientos del rechazo mediado por anticuerpos en injertos renales es de baja calidad. Son necesarios ensayos clínicos controlados para poder definir el tratamiento óptimo de estos pacientes.