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1.
Immunology ; 154(3): 465-475, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29338084

RESUMO

We previously showed that congenic bone marrow transplantation (BMTx) post myeloablation augmented tissue allograft survival in association with increased regulatory T (Treg) cells of both host and bone marrow donor origin. Regulatory B (Breg) cells can also modulate T-cell immunity and B cells may be implicated in the development of Treg cells. Accordingly, we explored the effect of B-cell depletion in vivo on augmented graft survival post BMTx. C57BL/6 mice received BALB/c skin allografts followed 7 days later by myeloablation using cyclophosphamide and busulphan. Mice then received T-cell-depleted bone marrow from CD45.1 congenic donors, and ongoing immunosuppression with rapamycin (to day 28 after BMTx). Control mice received cyclophosphamide and busulphan followed by rapamycin, but not congenic bone marrow. At different times post BMTx, mice received B-cell-depleting antibody treatment, and the effect on both skin graft survival, and induction of Treg cells was assessed. BMTx resulted in significantly prolonged skin graft survival versus control mice, in association with attenuated donor-specific alloreactivity relative to controls, increased splenic Treg cells and significantly diminished anti-donor IgG. In mice receiving infusion of B-depleting antibodies for 12 days from day 15 post BMTx, both graft survival and Treg cell activity were diminished, particularly for functional Treg cells of donor origin. Adoptive transfer of Breg cells from mice harvested at 15 days post BMTx prolonged survival in naive transplanted mice and increased Treg cell levels. Thus, autologous BMTx augmentation of graft survival is dependent in part upon a population of Breg cells that can modulate the function of donor-derived Treg cells.


Assuntos
Linfócitos B/imunologia , Transplante de Medula Óssea , Comunicação Celular/imunologia , Sobrevivência de Enxerto/imunologia , Imunomodulação , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/metabolismo , Biomarcadores , Citotoxicidade Imunológica , Imunoglobulina G/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos , Transplante de Pele , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Transplante Homólogo
2.
Pharmacogenomics J ; 18(4): 546-555, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29302041

RESUMO

Injections of a crude fetal sheep liver extract (FSLE) containing fetal hemoglobin, MPLA, and glutathione (GSSH) reversed cytokine changes in aged mice. To investigate the role of fetal hemoglobin we derived mice with homzygous deletions for either of the two major ßchains, HgbßmaKO or HgbßmiKO. Hgbßmi is the most prominent fetal Hgbß chain, with Hgbßma more prominent in adult mice. Mice lacking another fetal Hgb chain, HgbεKO, died in utero. CHO cells transfected with cloned Hgb chains were used to produce proteins for preparation of rabbit heteroantibodes. Splenocytes from HgbßmaKO mice stimulated in vitro with Conconavalin A showed a higher IL-2:IL-4 ratio than cells from HgbßmiKO mice. Following immunization in vivo with ovalbumin in alum, HgbßmaKO mice produced less IgE than HgbßmiKO mice, suggesting that in the absence of HgbßmiKO mice had a predeliction to heightened allergic-type responses. Using CHO cells transfected with cloned Hgb chains, we found that only the fetal Hgb chain, Hgbε, was secreted at high levels. Secretion of Hgbßma or Hgbßmi chains was seen only after genetic mutation to introduce the two N-linked glycosylation sites present in Hgbε, but absent in the Hgbß chains. We speculated that a previously unanticipated biological function of a naturally secreted fetal Hgb chain may be partly responsible for the effects reported following injection of animals with fetal, not adult, Hgb. Mice receiving injections of rabbit anti-Hgbε but not either anti-Hgbßma or anti-Hgbßmi from day 14 gestation also showed a bias towards the higher IL-2:IL-4 ratios seen in HgbßmiKO mice.


Assuntos
Citocinas/imunologia , Hemoglobina Fetal/imunologia , Hemoglobinas/imunologia , Imunidade Inata , Animais , Células CHO , Cricetinae , Cricetulus , Hemoglobina Fetal/administração & dosagem , Feto/imunologia , Glutationa/imunologia , Hemoglobinas/genética , Humanos , Extratos Hepáticos/administração & dosagem , Extratos Hepáticos/imunologia , Camundongos , Camundongos Knockout , Ovinos/imunologia , Baço/citologia
3.
Immunology ; 150(4): 418-431, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27859243

RESUMO

Significant barriers to transplantation exist for individuals who are pre-sensitized to donor antigen and have high titres of donor-reactive antibody. We report the effect of autologous bone marrow transplantation (BMTx) after myeloablation in pre-sensitized mice along with the use of monoclonal antibodies (mAbs) to tumour necrosis factor-receptor super family 25 (TNFRSF25), expressed on regulatory T (Treg) cells. C57BL/6 mice, which had been sensitized earlier with BALB/c skin allografts, received secondary BALB/c grafts after the primary grafts had been rejected. Subsequently, recipient mice underwent myeloablation with cyclophosphamide and busulphan and were injected with T-cell-depleted bone marrow from CD45.1 congenic donors (BMTx). Recipient mice underwent immunosuppressive treatment with rapamycin. A subgroup of mice was also treated with mAbs to TNFRSF25. Control mice were pre-sensitized mice that received cyclophosphamide and busulphan followed by rapamycin. BMTx-treated mice had significantly prolonged skin graft survival versus control mice. These mice also showed attenuated donor-specific mixed lymphocyte co-culture responses relative to controls, increased splenic Treg cells and markedly diminished serum anti-donor IgG. Infusion of anti-TNFRSF25 mAbs further augmented graft survival and increased graft-infiltrating Treg cells. These mAbs also expanded murine and human Treg cells in vitro with the capacity to attenuate mixed lymphocyte co-cultures using fresh peripheral blood mononuclear cells. Overall, this study delineates the roles of autologous BMTx and anti-TNFRSF25 mAbs in expanding Treg cells and attenuating alloimmune responses in pre-sensitized mice.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Transplante de Medula Óssea , Rejeição de Enxerto/prevenção & controle , Imunoterapia/métodos , Linfócitos T Reguladores/imunologia , Aloenxertos/imunologia , Animais , Células Cultivadas , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto , Humanos , Tolerância Imunológica , Isoanticorpos/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Membro 25 de Receptores de Fatores de Necrose Tumoral/imunologia , Transplante Homólogo
4.
Breast Cancer Res Treat ; 162(2): 255-266, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28160157

RESUMO

PURPOSE: We investigated whether miRNAs in exosomes from EMT6 or 4THM tumor-bearing mice played a role in regulating inflammatory cytokine expression and/or metastasis in WT mice injected with EMT6 and/or 4THM tumor cells. METHODS: EMT6 tumors in BALB/c CD200R1KO mice resolve following surgical resection of localized tumor and immunization with irradiated EMT6 cells along with CpG as adjuvant. Wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. DLNs from CD200R1KO mice contain no tumor cells detectable at limiting dilution. In contrast, 4THM tumor cells injected into CD200R1KO show increased metastasis compared with WT mice. Transfer of serum exosomes from 4THM tumor-bearing mice to WT animals increased metastasis of EMT6 tumors, an effect attenuated by anti-IL-6 antibody. We compared miRNA expression in exosomes from the serum of 4THM/EMT6 WT or CD200R1KO tumor-bearing mice, and the effects of antagomirs to miRNAs on tumor growth. RESULTS: Complex changes in miRNA expression were observed in the isolated exosomes. Some miRNAs, including miR155, have been reported to potentiate inflammatory responses and augment inflammatory cytokine expression. Expression of miR155 increased in exosomes from 4THM relative to EMT6 tumor bearers, and antagomirs to miR155 attenuated tumor growth and metastasis, and improved survival, following infusion into WT mice. Antagomirs to the miR205 family were thought to affect metastasis by targeting epithelial-to-mesenchymal transition (EMT), increased growth and metastasis in both 4THM and EMT6 tumor-bearing mice, and decreased survival, with some modulation of inflammatory cytokine production. CONCLUSIONS: Multiple pathways are implicated in differential metastasis of EMT6/4THM, and targeting these may have clinical utility in human breast cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , MicroRNAs/genética , Receptores de Orexina/deficiência , Animais , Neoplasias da Mama/sangue , Linhagem Celular Tumoral , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Exossomos/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/sangue , Metástase Neoplásica , Transcriptoma
5.
Blood Adv ; 8(12): 3013-3026, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38547431

RESUMO

ABSTRACT: Allogeneic double-negative T cells (DNTs) are a rare T-cell subset that effectively target acute myeloid leukemia (AML) without inducing graft-versus-host disease in an allogeneic setting. A phase 1 clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy among patients with relapsed AML. However, the molecular mechanisms of DNT-mediated cytotoxicity against AML remain elusive. Thus, we used a flow cytometry-based high throughput screening to compare the surface molecule expression profile on DNTs during their interaction with DNT-susceptible or -resistant AML cells and identified a tumor necrosis factor α (TNFα)-dependent cytotoxic pathway in DNT-AML interaction. TNFα secreted by DNTs, upon encountering susceptible AML targets, sensitized AML cells to DNT-mediated killing, including those otherwise resistant to DNTs. Mechanistically, TNFα upregulated ICAM-1 on AML cells through a noncanonical JAK1-dependent pathway. DNTs then engaged with AML cells more effectively through an ICAM-1 receptor, lymphocyte function-associated antigen 1, leading to enhanced killing. These results reveal a TNFα-JAK1-ICAM-1 axis in DNT-mediated cytotoxicity against AML to improve therapeutic efficacy.


Assuntos
Molécula 1 de Adesão Intercelular , Janus Quinase 1 , Leucemia Mieloide Aguda , Fator de Necrose Tumoral alfa , Humanos , Leucemia Mieloide Aguda/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Janus Quinase 1/metabolismo , Citotoxicidade Imunológica , Transdução de Sinais , Linhagem Celular Tumoral , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia
6.
Breast Cancer Res Treat ; 142(2): 271-82, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24166280

RESUMO

In previous studies, we observed that regulation of expression of CD200, both on cells of a transplantable breast cancer, EMT6, and of the host, as well as of the receptor, CD200R in host mice, regulated local tumor growth and metastasis in immunocompetent animals. This in turn led to an improved ability to document immunity to EMT6 in CD200R1KO mice. In the current study, we have explored the ability to cure BALB/c CD200KO or CD200R1KO mice of tumors ≤1 cm(3) in size by surgical resection of localized tumor, followed by immunization with irradiated EMT6 cells along with CpG as adjuvant. While control animals treated in this fashion developed significant pulmonary and liver metastases within 30 days of surgery, significant protection was seen in both CD200KO or CD200R1KO mice, with no macroscopic lung/liver metastases observed in CD200R1KO mice on sacrifice at day 300. Following surgical resection and immunization, draining lymph nodes from control mice contained tumor cells cloned at limiting dilution in vitro even before pulmonary and hepatic metastasis was seen. In contrast, within the limits of detection of the assay used (sensitivity ~1 in 10(7) cells), no tumor cells were detected at limiting dilution in similarly treated CD200R1KO mice, and significant reductions were seen in CD200KO mice. Infusion of anti-CD4, but less so anti-CD8, mAb into surgically treated and immunized CD200R1KO mice attenuated protection from both macroscopic (liver/lung) and microscopic (assayed by limiting dilution of DLN) metastasis. Adoptive transfer of lymphocytes from treated CD200R1KO mice to surgically treated control mice also attenuated metastatic growth of tumor, which was abolished by pretreatment of transferred cells with anti-CD4 mAb. Our data suggest that CD200:CD200R attenuates a potentially tumor-protective CD4 host response to breast cancer.


Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Glicoproteínas de Membrana/metabolismo , Adjuvantes Imunológicos/farmacologia , Transferência Adotiva , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/genética , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/radioterapia , Neoplasias da Mama/cirurgia , Antígenos CD4/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Linfonodos/patologia , Metástase Linfática , Linfócitos/fisiologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oligodesoxirribonucleotídeos/imunologia , Transdução de Sinais/genética
7.
J Immunother Cancer ; 11(9)2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37678917

RESUMO

BACKGROUND: Patients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous T-cell products and fratricide of CAR-T cells targeting T-lineage antigens. Recently, allogeneic double-negative T cells (DNTs) have been shown to be safe as an off-the-shelf adoptive cell therapy and to be amendable for CAR transduction. Here, we explore the antitumor activity of allogeneic DNTs against T-cell malignancies and the potential of using anti-CD4-CAR (CAR4)-DNTs as adoptive cell therapy for T-cell malignancies. METHODS: Healthy donor-derived allogeneic DNTs were ex vivo expanded with or without CAR4 transduction. The antitumor activity of DNTs and CAR4-DNTs against T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) were examined using flow cytometry-based cytotoxicity assays and xenograft models. Mechanisms of action were investigated using transwell assays and blocking assays. RESULTS: Allogeneic DNTs induced endogenous antitumor cytotoxicity against T-ALL and PTCL in vitro, but high doses of DNTs were required to attain therapeutic effects in vivo. The potency of DNTs against T-cell malignancies was significantly enhanced by transducing DNTs with a third-generation CAR4. CAR4-DNTs were manufactured without fratricide and showed superior cytotoxicity against CD4+ T-ALL and PTCL in vitro and in vivo relative to empty-vector transduced-DNTs. CAR4-DNTs eliminated T-ALL and PTCL cell lines and primary T-ALL blasts in vitro. CAR4-DNTs effectively infiltrated tumors, delayed tumor progression, and prolonged the survival of T-ALL and PTCL xenografts. Further, pretreatment of CAR4-DNTs with PI3Kδ inhibitor idelalisib promoted memory phenotype of CAR4-DNTs and enhanced their persistence and antileukemic efficacy in vivo. Mechanistically, LFA-1, NKG2D, and perforin/granzyme B degranulation pathways were involved in the DNT-mediated and CAR4-DNT-mediated killing of T-ALL and PTCL. CONCLUSIONS: These results demonstrate that CAR4-DNTs can effectively target T-ALL and PTCL and support allogeneic CAR4-DNTs as adoptive cell therapy for T-cell malignancies.


Assuntos
Carcinoma , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Linfócitos T , Linfócitos , Leucócitos , Antígenos Virais de Tumores
8.
Sci Immunol ; 7(70): eabl3642, 2022 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-35452255

RESUMO

The development of autologous chimeric antigen receptor T (CAR-T) cell therapies has revolutionized cancer treatment. Nevertheless, the delivery of CAR-T cell therapy faces challenges, including high costs, lengthy production times, and manufacturing failures. To overcome this, attempts have been made to develop allogeneic CAR-T cells using donor-derived conventional CD4+ or CD8+ T cells (Tconvs), but severe graft-versus-host disease (GvHD) and host immune rejection have made this challenging. CD3+CD4-CD8- double-negative T cells (DNTs) are a rare subset of mature T cells shown to fulfill the requirements of an off-the-shelf cellular therapy, including scalability, cryopreservability, donor-independent anticancer function, resistance to rejection, and no observed off-tumor toxicity including GvHD. To overcome the challenges faced with CAR-Tconvs, we evaluated the feasibility, safety, and efficacy of using healthy donor-derived allogeneic DNTs as a CAR-T cell therapy platform. We successfully transduced DNTs with a second-generation anti-CD19-CAR (CAR19) without hampering their endogenous characteristics or off-the-shelf properties. CAR19-DNTs induced antigen-specific cytotoxicity against B cell acute lymphoblastic leukemia (B-ALL). In addition, CAR19-DNTs showed effective infiltration and tumor control against lung cancer genetically modified to express CD19 in xenograft models. CAR19-DNT efficacy was comparable with that of CAR19-Tconvs. However, unlike CAR19-Tconvs, CAR19-DNTs did not cause alloreactivity or xenogeneic GvHD-related mortality in xenograft models. These studies demonstrate the potential of using allogeneic DNTs as a platform for CAR technology to provide a safe, effective, and patient-accessible CAR-T cell treatment option.


Assuntos
Doença Enxerto-Hospedeiro , Imunoterapia Adotiva , Neoplasias Pulmonares , Receptores de Antígenos Quiméricos , Antígenos CD19 , Linfócitos T CD8-Positivos , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Neoplasias Pulmonares/terapia , Receptores de Antígenos Quiméricos/genética
9.
Breast Cancer Res Treat ; 130(1): 49-60, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21165772

RESUMO

Previous studies have confirmed that levels of CD200 expression on the cells of the transplantable EMT6 mouse breast cancer line are increased markedly during growth in immunocompetent mice, unlike the persistent low levels of expression observed in NOD-SCID.IL-2(γr-/-) mice or mice with generalized over-expression of a CD200 transgene (CD200(tg) mice). Faster tumor growth occurs in both of these latter mice, with decreased evidence for a host immune reaction in lymph nodes draining the tumor (DLN). We now report evidence for a role for CD200 expression (by the host and/or tumor cells) in increased seeding of tumor cells to DLN in immunocompromised (CD200(tg) or NOD-SCID.IL-2(γr-/-)) vs immunocompetent mice, by limiting dilution cloning of tumor cells from DLN (vs contralateral lymph nodes, CLN), using control and GFP-tagged EMT6 cells. Neutralization of expressed CD200 by anti-CD200mAbs decreased the tumor metastasis at the same time as increasing detection of cytotoxic anti-tumor immune cells in DLN. Infusion of either anti-CD4 to deplete T-effector cells, or anti-TGFß antibody, increased metastasis to DLN, as did indeed the infusion of EMT6 cells selected for the loss of TGFßRII expression. It is concluded that the increased CD200 expression by breast cancer cells (and/or host tissue) may be an important variable involved in determining the risk of metastasis.


Assuntos
Antígenos CD/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Antígenos CD/imunologia , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linfonodos/patologia , Metástase Linfática/genética , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Receptores de Fatores de Crescimento Transformadores beta/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Carga Tumoral/genética
10.
J Immunol ; 183(3): 1560-8, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19592654

RESUMO

CD200, a type 2 transmembrane molecule of the Ig supergene family, can induce immunosuppression in a number of biological systems, as well as promote increased graft acceptance, following binding to its receptors (CD200Rs). Skin and cardiac allograft acceptance are readily induced in transgenic mice overexpressing CD200 under control of a doxycycline-inducible promoter, both of which are associated with increased intragraft expression of mRNAs for a number of genes associated with altered T cell subset differentiation, including GATA-3, type 2 cytokines (IL-4, IL-13), GITR, and Foxp3. Interestingly, some 12-15 days after grafting, induction of transgenic CD200 expression can be stopped (by doxycycline withdrawal), without obvious significant effect on graft survival. However, neutralization of all CD200 expression (including endogenous CD200 expression) by anti-CD200 mAb caused graft loss, as did introduction of an acute inflammatory stimulus (LPS, 10 microg/mouse, delivered by i.p. injection). We conclude that even with apparently stably accepted tissue allografts, disruption of the immunoregulatory balance by an intense inflammatory stimulus can cause graft loss.


Assuntos
Antígenos CD/genética , Transplante de Coração/imunologia , Tolerância Imunológica/genética , Transplante de Pele/imunologia , Transgenes/genética , Animais , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Rejeição de Enxerto/etiologia , Inflamação/etiologia , Camundongos , Camundongos Endogâmicos , Transplante Homólogo
11.
Cancers (Basel) ; 13(20)2021 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-34680188

RESUMO

The double negative T cell (DNT) is a unique subset of T cells with potent anti-leukemic potential. Previously, DNT therapy has been shown to effectively target AML cells in patient-derived xenograft (PDX) models. Further, a recently completed phase I/IIa clinical study demonstrated the safety, feasibility, and potential efficacy in AML patients that relapsed after allogeneic hematopoietic stem cell transplantation. However, the persistence and durability of DNT-mediated anti-leukemic response is less well understood. In this study, we characterized the in vivo persistence of DNTs in PDX models. Further, we improved the efficacy and durability of DNT-mediated activity with phosphoinositide 3-kinase delta (PI3Kδ) inhibition. Mechanistically, DNTs treated with the PI3Kδ inhibitor, Idelalisib (Ide), exhibited early memory phenotype with superior viability and proliferative capacity but less cell exhaustion. Collectively, the findings from this study support the use of Ide-treated DNTs to improve its therapeutic outcome.

12.
Breast Cancer Res Treat ; 123(2): 405-15, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19953316

RESUMO

CD200 has been characterized as an important immunoregulatory molecule, increased expression of which can lead to decreased transplant rejection, autoimmunity, and allergic disease. Elevated CD200 expression has been reported to be associated with poor prognosis in a number of human malignancies. We have found that cells of the transplantable EMT6 mouse breast cancer line growing in vitro express low levels of CD200, but levels increase markedly during growth in immunocompetent mice. Similar increased in vivo expression does not occur in NOD-SCID.IL-2(gammar-/-) mice or mice with generalized over-expression of a CD200 transgene. In both mice, tumor growth occurs faster. Altered CD200 expression in control versus transgenic mice is accompanied by reproducible changes in tumor-infiltrating host cells, and altered cell composition in lymph nodes draining the tumor (DLN). Neutralization of expressed CD200 by anti-CD200mAbs leads to decreased tumor growth in immunocompetent mice, with improved detection of cytotoxic anti-tumor immune cells in DLN. Finally, we report that tumor growth in vivo can be monitored by levels of soluble CD200 (sCD200) in serum of tumor-bearing animals.


Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos CD/sangue , Antígenos CD/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Células Clonais , Citocinas/imunologia , Citotoxicidade Imunológica , Feminino , Imunocompetência , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Linfonodos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Fatores de Tempo , Carga Tumoral
13.
Exp Gerontol ; 43(8): 771-81, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18555631

RESUMO

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.


Assuntos
Envelhecimento/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Imunidade nas Mucosas , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Fígado/imunologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Nódulos Linfáticos Agregados/imunologia , Reação em Cadeia da Polimerase/métodos , Ovinos , Extratos de Tecidos/imunologia
14.
Med Chem ; 4(6): 624-31, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18991748

RESUMO

Our laboratory and others have documented in some detail the immunological consequences which follow from interaction of the ubiquitously expressed molecule CD200 with its receptor(s) CD200R (expressed predominantly on cells of myeloid and lymphoid origin). In particular, there is evidence that these interactions lead to immunosuppressive signals which modulate graft rejection responses; decrease the manifestations of arthritis in rodent models; diminish mast cell mediator release in models of allergic disease; and favour the growth of tumors in both mice and humans. The development of small molecular weight agonists (and/or antagonists) of these interactions would thus likely have significant clinical importance. The data reported herein characterizes several such molecules in a number of rodent models.


Assuntos
Antígenos CD/efeitos dos fármacos , Imunossupressores/síntese química , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Feminino , Rejeição de Enxerto/prevenção & controle , Imunossupressores/química , Imunossupressores/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Peso Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Transplante de Pele/imunologia , Baço/citologia , Baço/efeitos dos fármacos
15.
Leuk Res ; 68: 40-47, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29525600

RESUMO

Chronic Lymphocytic Leukemia B cells (CLL) are malignant cells which retain at least some functions of normal B cells. Paramount amongst the latter is that when such cells are appropriately stimulated, they are able to present antigens, including any potential tumor antigens, making them excellent choices as a candidate tumor vaccine. We show that following stimulation of CLL cells with Phorbol myristic acetate, IL-2, the TLR7 agonist imiquimod (P2I) and ionomycin (P2Iio), markedly increased expression of CD54 and CD83 was seen, indicative of B cell activation and a transition to antigen-presenting cells. However, this occurred in the context of augmented expression of the known immunoregulatory molecule, CD200. Accordingly we explored the effect of stimulation of CLL cells with P2Iio, followed by coating of cells with a non-depleting anti-CD200mAb, on the ability of those cells to immunize PBL in vitro to become cytotoxic to CLL cells, or to protect NOD-SCIDγcnull (NSG) mice from subsequent CLL tumor challenge. Our data indicate that this protocol is effective in inducing CD8+ CTL able to lyse CLL cells in vitro, and decrease tumor burden in vivo in spleen and marrow of mice injected with CLL cells. Pre-treatment of mice with a CD8 depleting antibody before vaccination with P2Iio/anti-CD200 coated cells abolished any protection seen. These data suggest a potential role for blockade of CD200 expression on CLL cells as a component of a tumor vaccination strategy.


Assuntos
Vacinas Anticâncer/uso terapêutico , Leucemia Linfocítica Crônica de Células B/terapia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD/biossíntese , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Humanos , Ionomicina/farmacologia , Leucemia Linfocítica Crônica de Células B/imunologia , Depleção Linfocítica , Camundongos Endogâmicos NOD , Camundongos SCID
16.
Leuk Res ; 69: 72-80, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29698858

RESUMO

In previous studies we had reported that the immunosuppressive cell membrane bound molecule CD200 is released from the cell following cleavage by matrix metalloproteases, with the released soluble CD200 acting as an immunosuppressant following binding to, and signaling through, its cognate receptor CD200R expressed on target cells. We now show that although the intracellular cytoplasmic tail (CD200C-tail) of CD200 has no consensus sites for adapter molecules which might signal the CD200+ cell directly, cleavage of the CD200C-tail from the membrane region of CD200 by a consensus γ-secretase, leads to nuclear translocation and DNA binding (identified by chromatin immunoprecipitation followed by sequencing, Chip-sequencing) of the CD200C-tail. Subsequently there occurs an altered expression of a limited number of genes, many of which are transcription factors (TFs) known to be associated with regulation of cell proliferation. Altered expression of these TFs was also prominent following transfection of CD200+ B cell lines and fresh patient CLL cells with a vector construct containing the CD200C-tail. Artificial transfection of non CD200+ Hek293 cells with this CD200C-tail construct resulted in altered expression of most of these same genes. Introduction of a siRNA for one of these TFs, POTEA, reversed CD200C-tail regulation of altered cell proliferation.


Assuntos
Antígenos CD/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Espaço Extracelular/metabolismo , Transdução de Sinais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Transporte Biológico , Western Blotting , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Imunoprecipitação da Cromatina , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia
17.
Mol Ther Nucleic Acids ; 12: 350-358, 2018 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-30195773

RESUMO

Functional aptamers displaying agonistic or antagonistic properties are showing great promise as modulators of immune responses. Here, we report the development of a polyethylene glycol-modified (PEGylated) DNA aptamer as a cross-species (murine and human) CD200R1 agonist that modulates inflammatory responses in vivo. Specifically, DNA aptamers were discovered by performing independent SELEX searches on recombinant murine and human CD200R1. Aptamer motifs identified by next generation sequencing (NGS) were subsequently compared, leading to the discovery of motifs common to both targets. The CD200R1 DNA aptamer CCS13 displayed the highest agonistic activity toward CD200R1 in terms of suppressing the induction of cytotoxic T-lymphocytes (CTLs) in both human and murine allogeneic-mixed lymphocyte cultures (allo-MLCs). A 20-kDa polyethylene glycol (PEG) chain was covalently attached to the 5' end of this aptamer, and the resulting conjugate was shown to block inflammatory responses in murine models of skin graft rejection and house-dust-mite-induced allergic airway inflammation. Importantly, this agonistic aptamer does not suppress CTL induction in 5-day allo-MLCs with responder cells derived from CD200R1-/- mice, indicating that its mode of action is directly linked to CD200R1 activation. This study suggests that one can derive agonistic DNA aptamers that can be verified as immuno-modulators in murine models with outcomes potentially translatable to the treatment of human conditions.

18.
PLoS One ; 12(2): e0171586, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28234914

RESUMO

Cell-surface CD200 expression by mouse EMT6 breast tumor cells increased primary tumor growth and metastasis to the draining lymph nodes (DLN) in normal (WT) BALB/c female recipients, while lack of CD200R1 expression in a CD200R1-/- host negated this effect. Silencing CD200 expression in EMT6siCD200 tumor cells also reduced their ability to grow and metastasize in WT animals. The cellular mechanisms responsible for these effects have not been studied in detail. We report characterization of tumor infiltrating (TILs) and draining lymph node (DLN) cells in WT and CD200-/- BALB/c mice, receiving WT tumor cells, or EMT6 lacking CD200 expression (EMT6siCD200 cells). Our data show an important correlation with augmented CD8+ cytotoxic T cells and resistance to tumor growth in mice lacking exposure (on either host cells or tumor) to the immunoregulatory molecule CD200. Confirmation of the importance of such CD8+ cells came from monitoring tumor growth and characterization of the TILs and DLN cells in WT mice challenged with EMT6 and EMT6siCD200 tumors and treated with CD8 and CD4 depleting antibodies. Finally, we have assessed the mechanisms(s) whereby addition of metformin as an augmenting chemotherapeutic agent in CD200-/- animals given EMT6 tumors and treated with a previously established immunotherapy regime can increase host resistance. Our data support the hypothesis that increased autophagy in the presence of metformin increases CD8+ responses and tumor resistance, an effect attenuated by the autophagy inhibitor verteporfin.


Assuntos
Antígenos CD/imunologia , Imunização Passiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Glândulas Mamárias Animais/imunologia , Neoplasias Mamárias Experimentais/genética , Receptores de Orexina/imunologia , Animais , Anticorpos Antineoplásicos/farmacologia , Antígenos CD/genética , Autofagia/efeitos dos fármacos , Autofagia/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Inativação Gênica , Humanos , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Depleção Linfocítica/métodos , Linfócitos do Interstício Tumoral/patologia , Linfócitos do Interstício Tumoral/transplante , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Orexina/deficiência , Receptores de Orexina/genética , Porfirinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Verteporfina
19.
PLoS One ; 11(4): e0152073, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27111430

RESUMO

We have previously reported the existence of a soluble form of CD200 (sCD200) in human plasma, and found sCD200 to be elevated in the plasma of Chronic Lymphocytic Leukemia (CLL) patients. CLL cells release CD200 at a constitutive level, which could be attenuated partially by ADAM28 silencing. In this study, we further explored mechanisms of CD200 shedding beyond that of ADAM28, and performed biochemical analysis of sCD200 using materials derived from purified CLL cells and Hek293 cells stably transfected with CD200, and antibodies generated specifically against either the extracellular or cytoplasmic regions of CD200. CD200 shedding was enhanced by PMA stimulation, and the loss of cell surface CD200 could be monitored as a reduction in CD200 cell surface expression by flow cytometry, in parallel with an increase in the detection of sCD200 in the supernatant. Western blot analyses and functional studies using CD200R1 expressing Hek293 cells showed that the shed CD200 detected in CLL and Hek293-hCD200 supernatants lacked the cytoplasmic domain of CD200 but retained the functional extracellular domain required for binding to, and phosphorylation of, CD200R. These data confirms that a functionally active CD200 extracellular moiety can be cleaved from the surface of CD200 expressing cells following ectodomain shedding.


Assuntos
Antígenos CD/metabolismo , Proteínas ADAM/genética , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Inativação Gênica , Células HEK293 , Humanos
20.
Biochem J ; 378(Pt 1): 207-12, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14572308

RESUMO

Rat intestinal mucin Muc3 (rMuc3), like its human homologue (MUC3) and several other membrane mucins, contains a C-terminally located SEA (sea urchin sperm protein, enterokinase and agrin) module, with an intrinsic proteolytic site sequence G downward arrow SIVV (where G downward arrow S is the glycine serine cleavage site). As shown previously [Wang, Khatri and Forstner (2002) Biochem. J. 366, 623-631], expression of the C-terminal domain of rMuc3 in COS-1 cells yields a V5 epitope-tagged N-terminal glycopeptide of 30 kDa and a Myc- and His epitope-tagged C-terminal glycopeptide of 49 kDa. The present study shows that the 49 kDa membrane-anchored fragment undergoes a further cleavage reaction which decreases its size to 30 kDa. Western blotting, pulse-chase metabolic incubations, immunoprecipitation and deglycosylation with N-glycosidase F were used to detect and identify the proteolytic products. Both the first and second cleavages are presumed to facilitate solubilization of Muc3 at the apical surface of enterocytes and/or enhance the potential for Muc3 to participate in ligand-receptor and signal transduction events for enterocyte function in vivo.


Assuntos
Mucinas/química , Mucinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Células CACO-2 , Chlorocebus aethiops , Epitopos/análise , Humanos , Cinética , Mucina-3 , Mucina-4 , Fragmentos de Peptídeos/química , Estrutura Terciária de Proteína , Ratos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
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