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1.
EMBO Mol Med ; 14(4): e14297, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35298086

RESUMO

T cells are the current choice for many cell therapy applications. They are relatively easy to access, expand in culture, and genetically modify. Rapamycin-conditioning ex vivo reprograms T cells, increasing their memory properties and capacity for survival, while reducing inflammatory potential and the amount of preparative conditioning required for engraftment. Rapamycin-conditioned T cells have been tested in patients and deemed to be safe to administer in numerous settings, with reduced occurrence of infusion-related adverse events. We demonstrate that ex vivo lentivirus-modified, rapamycin-conditioned CD4+ T cells can also act as next-generation cellular delivery vehicles-that is, "micropharmacies"-to disseminate corrective enzymes for multiple lysosomal storage disorders. We evaluated the therapeutic potential of this treatment platform for Fabry, Gaucher, Farber, and Pompe diseases in vitro and in vivo. For example, such micropharmacies expressing α-galactosidase A for treatment of Fabry disease were transplanted in mice where they provided functional enzyme in key affected tissues such as kidney and heart, facilitating clearance of pathogenic substrate after a single administration.


Assuntos
Lentivirus , Doenças por Armazenamento dos Lisossomos , Sirolimo , Linfócitos T , Animais , Modelos Animais de Doenças , Lentivirus/genética , Doenças por Armazenamento dos Lisossomos/terapia , Lisossomos , Camundongos , Sirolimo/uso terapêutico , Linfócitos T/efeitos dos fármacos , alfa-Galactosidase/uso terapêutico
2.
Cytotherapy ; 20(8): 1001-1012, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30076069

RESUMO

BACKGROUND: Mesenchymal stromal cells (MSCs) promote wound healing, including after radiotherapy (RT) and surgery. The use of MSCs in regenerative medicine in the context of malignancy, such as to enhance wound healing post-RT/surgery in patients with soft tissue sarcomas (STSs), requires safety validation. The aim of this study was to determine the effects of human MSCs on STS growth in vitro and local recurrence and metastasis in vivo. METHODS: Human primary STS and HT-1080 fibrosarcoma lines were transduced to express luciferase/eGFP (enhanced green fluorescent protein). Sarcoma cells were co-cultured or co-injected with bone marrow-derived MSCs for growth studies. Xenograft tumor models were established with STS lines in NOD/SCID/γcnull mice. To emulate a clinical scenario, subcutaneous tumors were treated with RT/surgery prior to MSC injection into the tumor bed. Local and distant tumor recurrence was studied using histology and bioluminescence imaging. RESULTS: MSCs did not promote STS proliferation upon co-culture in vitro, which was consistent among MSCs from different donors. Co-injection of MSCs with sarcoma cells in mice exhibited no significant tumor-stimulating effect, compared with control mice injected with sarcoma cells alone. MSC administration after RT/surgery had no effect on local recurrence or metastasis of STS. DISCUSSION: These studies are important for the establishment of a safety profile for MSC administration in patients with STS. Our data suggest that MSCs are safe in STS management after standard of care RT/surgery, which can be further investigated in early-phase clinical trials to also determine the efficacy of MSCs in reducing morbidity and to mitigate wound complications in these patients.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Radioterapia , Sarcoma/patologia , Sarcoma/terapia , Procedimentos Cirúrgicos Operatórios , Adulto , Animais , Técnicas de Cocultura , Terapia Combinada , Células HEK293 , Xenoenxertos , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Recidiva Local de Neoplasia/etiologia , Recidiva Local de Neoplasia/patologia , Radioterapia/efeitos adversos , Radioterapia/métodos , Procedimentos Cirúrgicos Operatórios/efeitos adversos , Procedimentos Cirúrgicos Operatórios/métodos , Células Tumorais Cultivadas , Cicatrização , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Immunity ; 49(2): 247-263.e7, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30054205

RESUMO

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.


Assuntos
Cisteína Endopeptidases/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Colite/imunologia , Colite/patologia , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/citologia , Células Th1/citologia
4.
Haematologica ; 102(8): 1446-1456, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28473623

RESUMO

CD4+ T-helper subsets drive autoimmune chronic graft-versus-host disease, a major complication after allogeneic bone marrow transplantation. However, it remains unclear how specific T-helper subsets contribute to chronic graft-versus-host disease. T-helper type 1 cells are one of the major disease-mediating T-cell subsets and require interferon-γ signaling and Tbet expression for their function. Regulatory T cells on the other hand can inhibit T-helper type 1 cell-mediated responses. Using an established murine model that isolates the autoimmune component of graft-versus-host disease, we hypothesized that T-helper type 1 cells would restrict FoxP3-driven regulatory T cells. Upon transfer into immune-deficient syngeneic hosts, alloreactive Tbx21-/-CD4+ T cells led to marked increases in FoxP3+ cells and reduced clinical evidence of autoimmunity. To evaluate whether peripheral induction contributed to regulatory T-cell predominance, we adoptively transferred Tbx21-/- T cells that consisted of fate mapping for FoxP3: recipients of flow-purified effector cells that were Foxp3- and Tbx21-/- had enhanced T-regulatory-cell predominance during autoimmune graft-versus-host disease. These data directly demonstrated that peripheral T-regulatory-cell induction was inhibited by Tbet. Finally, Tbx21-/- T-regulatory cells cross-regulated autoimmune wild-type T-effector-cell cytokine production in vivo The Tbet pathway therefore directly impairs T-regulatory-cell reconstitution and is consequently a feasible target in efforts to prevent autoimmune graft-versus-host disease.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Proteínas com Domínio T/imunologia , Animais , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Camundongos , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética , Subpopulações de Linfócitos T , Linfócitos T Reguladores/imunologia
5.
J Exp Med ; 214(6): 1663-1678, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28490441

RESUMO

Group 2 innate lymphoid cells (ILC-2s) regulate immune responses to pathogens and maintain tissue homeostasis in response to cytokines. Positive regulation of ILC-2s through ICOS has been recently elucidated. We demonstrate here that PD-1 is an important negative regulator of KLRG1+ ILC-2 function in both mice and humans. Increase in KLRG1+ ILC-2 cell numbers was attributed to an intrinsic defect in PD-1 signaling, which resulted in enhanced STAT5 activation. During Nippostrongylus brasiliensis infection, a significant expansion of KLRG1+ ILC-2 subsets occurred in Pdcd1-/- mice and, upon adoptive transfer, Pdcd1-/- KLRG1+ ILC-2s significantly reduced worm burden. Furthermore, blocking PD-1 with an antibody increased KLRG1+ ILC-2 cell number and reduced disease burden. Therefore, PD-1 is required for maintaining the number, and hence function, of KLRG1+ ILC-2s.


Assuntos
Imunidade Inata , Lectinas Tipo C/metabolismo , Linfócitos/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos/metabolismo , Transativadores/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Contagem de Linfócitos , Subpopulações de Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Nippostrongylus/fisiologia , Parasitos/fisiologia , Receptor de Morte Celular Programada 1/deficiência , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Infecções por Strongylida/imunologia , Infecções por Strongylida/patologia
6.
Biomedicines ; 4(1)2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-28536373

RESUMO

Anti-cancer immunotherapy is emerging from a nadir and demonstrating tangible benefits to patients. A variety of approaches are now employed. We are invoking antigen (Ag)-specific responses through direct injections of recombinant lentivectors (LVs) that encode sequences for tumor-associated antigens into multiple lymph nodes to optimize immune presentation/stimulation. Here we first demonstrate the effectiveness and antigen-specificity of this approach in mice challenged with prostate-specific antigen (PSA)-expressing tumor cells. Next we tested the safety and efficacy of this approach in two cohorts of rhesus macaques as a prelude to a clinical trial application. Our vector encodes the cDNA for rhesus macaque PSA and a rhesus macaque cell surface marker to facilitate vector titering and tracking. We utilized two independent injection schemas demarcated by the timing of LV administration. In both cohorts we observed marked tissue-specific responses as measured by clinical evaluations and magnetic resonance imaging of the prostate gland. Tissue-specific responses were sustained for up to six months-the end-point of the study. Control animals immunized against an irrelevant Ag were unaffected. We did not observe vector spread in test or control animals or perturbations of systemic immune parameters. This approach thus offers an "off-the-shelf" anti-cancer vaccine that could be made at large scale and injected into patients-even on an out-patient basis.

7.
Stem Cells ; 33(1): 91-100, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25330775

RESUMO

The high risk of insertional oncogenesis reported in clinical trials using integrating retroviral vectors to genetically modify hematopoietic stem and progenitor cells (HSPCs) requires the development of safety strategies to minimize risks associated with novel cell and gene therapies. The ability to ablate genetically modified cells in vivo is desirable, should an abnormal clone emerge. Inclusion of "suicide genes" in vectors to facilitate targeted ablation of vector-containing abnormal clones in vivo is one potential safety approach. We tested whether the inclusion of the "inducible Caspase-9" (iCasp9) suicide gene in a gamma-retroviral vector facilitated efficient elimination of vector-containing HSPCs and their hematopoietic progeny in vivo long-term, in an autologous non-human primate transplantation model. Following stable engraftment of iCasp9 expressing hematopoietic cells in rhesus macaques, administration of AP1903, a chemical inducer of dimerization able to activate iCasp9, specifically eliminated vector-containing cells in all hematopoietic lineages long-term, suggesting activity at the HSPC level. Between 75% and 94% of vector-containing cells were eliminated by well-tolerated AP1903 dosing, but lack of complete ablation was linked to lower iCasp9 expression in residual cells. Further investigation of resistance mechanisms demonstrated upregulation of Bcl-2 in hematopoietic cell lines transduced with the vector and resistant to AP1903 ablation. These results demonstrate both the potential and the limitations of safety approaches using iCasp9 to HSPC-targeted gene therapy settings, in a model with great relevance to clinical development.


Assuntos
Caspase 9/genética , Genes Transgênicos Suicidas , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Apoptose/fisiologia , Caspase 9/biossíntese , Terapia Genética , Vetores Genéticos , Células-Tronco Hematopoéticas/enzimologia , Macaca mulatta
8.
Stem Cells ; 33(4): 1200-12, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25532725

RESUMO

The use of bone marrow-derived mesenchymal stromal cells (BMSC) in the treatment of alloimmune and autoimmune conditions has generated much interest, yet an understanding of the therapeutic mechanism remains elusive. We therefore explored immune modulation by a clinical-grade BMSC product in a model of human-into-mouse xenogeneic graft-versus-host disease (x-GVHD) mediated by human CD4(+) Th1 cells. BMSC reversed established, lethal x-GVHD through marked inhibition of Th1 cell effector function. Gene marking studies indicated BMSC engraftment was limited to the lung; furthermore, there was no increase in regulatory T cells, thereby suggesting a paracrine mechanism of BMSC action. BMSC recipients had increased serum CD73 expressing exosomes that promoted adenosine accumulation ex vivo. Importantly, immune modulation mediated by BMSC was fully abrogated by pharmacologic therapy with an adenosine A2A receptor antagonist. To investigate the potential clinical relevance of these mechanistic findings, patient serum samples collected pre- and post-BMSC treatment were studied for exosome content: CD73 expressing exosomes promoting adenosine accumulation were detected in post-BMSC samples. In conclusion, BMSC effectively modulate experimental GVHD through a paracrine mechanism that promotes adenosine-based immune suppression.


Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Células-Tronco Mesenquimais/imunologia , Transdução de Sinais/imunologia , Células Th1/imunologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Técnicas de Cocultura , Doença Enxerto-Hospedeiro/imunologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução de Sinais/efeitos dos fármacos , Células Th1/efeitos dos fármacos
9.
Virol J ; 10: 240, 2013 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-23870437

RESUMO

BACKGROUND: Dendritic cells (DCs) are promising mediators of anti-tumor immune responses due to their potent antigen-presentation capacity. Unfortunately, cancer cells can often disarm differentiated DCs by rendering them incapable of maturation or by promoting their apoptosis. DC vaccine regimens attempt to generate functional DCs and preload them with Tumor-Associated Antigens (TAAs) to target various malignancies. Despite these efforts, the efficacy of DC vaccines in clinical trials is still rather disappointing to date. In addition to undergoing cancer-induced apoptosis, it is well established that DCs are intrinsically short-lived cell types. It is likely that a significant portion of infused DCs undergo apoptosis prior to locating and activating naïve TAA-reactive T cells. METHODS: In our current study, we constructed and investigated novel bicistronic lentivectors (LVs) encoding the cDNA for the xeno-TAA, rat HER-2/neu (rHER-2), along with five candidate mouse DC survival factors (c-FLIPS, c-FLIPL, Bcl-XL, M11L, and AKT-1) that operate in both the extrinsic and intrinsic cycles of apoptosis. The murine DC cell line, DC2.4 was transduced separately with each novel LV construct. Infected cells were enriched via flow cytometric methods based on rHER-2 expression. Transduced DC2.4 cell lines were then exposed to Fetal Calf Serum (FCS) withdrawal and to specific pharmacological apoptosis-inducing agents. DC2.4 cell death was assayed based on Annexin V and PI double-positive staining via flow cytometry. The phenotype and function of transduced DC2.4 cells and primary bone marrow-derived DCs were then assessed via expression and secretion of DC markers and cytokines, respectively. RESULTS: DC2.4 cells transduced with LVs encoding cDNAs for c-FLIPS, c-FLIPL, Bcl-XL, and M11L were protected from apoptosis when exposed to low FCS-containing culture media. When treated with an anti-CD95 antibody, only DC2.4 cells transduced with LVs encoding c-FLIPS and c-FLIPL were protected from apoptosis. In contrast, only DC2.4 cells transduced with LVs encoding Bcl-XL and M11L were protected from effects of staurosporine (STS) treatment. Also, LV-modified DCs maintained their original phenotype and function. CONCLUSIONS: We present evidence that by employing novel recombinant bicistronic LVs we can simultaneously load DCs with a relevant TAA and block apoptosis; thereby confirming the usage of such LVs in the modulation of DC lifespan and function.


Assuntos
Antígenos de Neoplasias/imunologia , Apoptose , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Engenharia Genética/métodos , Vetores Genéticos , Lentivirus/genética , Animais , Antígenos de Neoplasias/genética , Sobrevivência Celular , Células Dendríticas/virologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Transdução Genética
10.
Autophagy ; 9(7): 1069-79, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23633667

RESUMO

We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides "cell-fate control" due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.


Assuntos
Autofagia , Linhagem da Célula , Terapia Genética , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Antígenos CD19/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lentivirus/metabolismo , Camundongos , Núcleosídeo-Fosfato Quinase/metabolismo , Fenótipo , Sirolimo/farmacologia , Transdução Genética , Transgenes/genética , Zidovudina/farmacologia
11.
Exp Parasitol ; 130(1): 48-57, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22019418

RESUMO

Lesion development in tegumentary leishmaniasis is markedly influenced by the inoculation site and the type and number of injected infective forms. This and the yet unclear contribution of Th2 cytokines as susceptibility factors to Leishmania amazonensis infection prompted us to investigate the roles of IL-4, IL-13 and IL-10 on C57BL/6 and BALB/c mice infected in the footpad (paw) or rump with low-dose L. amazonensis purified-metacyclics. Wild-type (WT) mice of either strain developed, in the rump, a single large ulcerated lesion whereas paw lesions never ulcerated and were much smaller in C57BL/6 than in BALB/c mice. However, rump-inoculated IL-4-deficient (IL-4(-/-)) C57BL/6 mice did not develop any visible lesions although parasites remained in the dermis and lymph nodes, even after systemic IL-10-receptor blocking. By comparison, all IL-4(-/-) BALB/c mice developed rump ulcers. Strikingly, only 30% of rump-infected IL-4Rα(-/-) BALB/c mice developed lesions. IL-4(-/-) mice had higher IFN-γ and lower IL-10 and IL-13 levels than WT mice. Paw-infected IL-4Rα(-/-) BALB/c mice developed minimal paw lesions. While other factors contributing to L. amazonensis susceptibility cannot be discounted, our results indicate that absent signalling by IL-4 or by IL-4/IL-13 have more intense attenuating effects on rump than on paw lesions but do not eradicate parasitism.


Assuntos
Interleucina-13/fisiologia , Interleucina-4/fisiologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais/administração & dosagem , Dorso , Feminino , Pé/parasitologia , Pé/patologia , Interleucina-4/deficiência , Interleucina-4/genética , Leishmania mexicana/fisiologia , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Interleucina-10/imunologia , Pele/parasitologia , Pele/patologia , Baço/parasitologia
12.
Sci Transl Med ; 3(111): 111ra120, 2011 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-22133721

RESUMO

Immune surveillance by T helper type 1 (T(H)1) cells is not only critical for the host response to tumors and infection, but also contributes to autoimmunity and graft-versus-host disease (GVHD) after transplantation. The inhibitory molecule programmed death ligand 1 (PDL1) has been shown to anergize human T(H)1 cells, but other mechanisms of PDL1-mediated T(H)1 inhibition such as the conversion of T(H)1 cells to a regulatory phenotype have not been well characterized. We hypothesized that PDL1 may cause T(H)1 cells to manifest differentiation plasticity. Conventional T cells or irradiated K562 myeloid tumor cells overexpressing PDL1 converted TBET(+) T(H)1 cells into FOXP3(+) regulatory T (T(reg)) cells in vivo, thereby preventing human-into-mouse xenogeneic GVHD (xGVHD). Either blocking PD1 expression on T(H)1 cells by small interfering RNA targeting or abrogation of PD1 signaling by SHP1/2 pharmacologic inhibition stabilized T(H)1 cell differentiation during PDL1 challenge and restored the capacity of T(H)1 cells to mediate lethal xGVHD. PD1 signaling therefore induces human T(H)1 cells to manifest in vivo plasticity, resulting in a T(reg) phenotype that severely impairs cell-mediated immunity. Converting human T(H)1 cells to a regulatory phenotype with PD1 signaling provides a potential way to block GVHD after transplantation. Moreover, because this conversion can be prevented by blocking PD1 expression or pharmacologically inhibiting SHP1/2, this pathway provides a new therapeutic direction for enhancing T cell immunity to cancer and infection.


Assuntos
Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Doença Enxerto-Hospedeiro/imunologia , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Modelos Biológicos , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/enzimologia , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/enzimologia
13.
PLoS One ; 6(2): e16700, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21304898

RESUMO

Gamma delta T cells (GDTc) lyse a variety of hematological and solid tumour cells in vitro and in vivo, and are thus promising candidates for cellular immunotherapy. We have developed a protocol to expand human GDTc in vitro, yielding highly cytotoxic Vgamma9/Vdelta2 CD27/CD45RA double negative effector memory cells. These cells express CD16, CD45RO, CD56, CD95 and NKG2D. Flow cytometric, clonogenic, and chromium release assays confirmed their specific cytotoxicity against Ph(+) cell lines in vitro. We have generated a fluorescent and bioluminescent Ph(+) cell line, EM-2eGFPluc, and established a novel xenogeneic leukemia model. Intravenous injection of EM-2eGFPluc into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice resulted in significant dose-dependent bone marrow engraftment; lower levels engrafted in blood, lung, liver and spleen. In vitro-expanded human GDTc injected intraperitoneally were found at higher levels in blood and organs compared to those injected intravenously; GDTc survived at least 33 days post-injection. In therapy experiments, we documented decreased bone marrow leukemia burden in mice treated with GDTc. Live GDTc were found in spleen and bone marrow at endpoint, suggesting the potential usefulness of this therapy.


Assuntos
Imunoterapia Adotiva/métodos , Leucemia/terapia , Cromossomo Filadélfia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/transplante , Animais , Proliferação de Células , Células Cultivadas , Citotoxicidade Imunológica/fisiologia , Modelos Animais de Doenças , Humanos , Células K562 , Leucemia/genética , Leucemia/imunologia , Leucemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/fisiologia , Transplante Heterólogo
14.
Cytotherapy ; 13(6): 753-64, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21314241

RESUMO

BACKGROUND AIMS: There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets. METHODS: GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry. RESULTS: Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1). CONCLUSIONS: Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.


Assuntos
Leucemia Linfocítica Crônica de Células B/terapia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Anexina A5 , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Concanavalina A/farmacologia , Citometria de Fluxo , Humanos , Imunoterapia , Leucemia Linfocítica Crônica de Células B/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo
15.
Biol Blood Marrow Transplant ; 17(5): 620-31, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21130889

RESUMO

We describe a novel animal model of nonmyeloablative bone marrow transplantation (BMT) using the purine analog pentostatin. Other cohorts of mice received another purine analog, fludarabine, which we and others have previously evaluated in nonmyeloablative murine models. We evaluated pentostatin for its ability to (1) operate synergistically with cyclophosphamide to induce host T cell depletion; (2) induce host T cell suppression, as defined by modulation of cytokine secretion in vitro and abrogation of host-versus-graft reactivity in vivo; (3) constrain host T cell recovery post-therapy; and (4) prevent the rejection of T cell-depleted, fully major histocompatibility complex-mismatched bone marrow allografts. Relative to single-agent regimens, combination regimens with pentostatin and cyclophosphamide (PC) and with fludarabine and cyclophosphamide (FC) worked synergistically to deplete host CD4(+) and CD8(+) T cells. PC and FC regimens were developed that yielded similar levels of host T cell and myeloid cell depletion. In the setting of these generally comparable states of host T cell and myeloid cell depletion, the PC regimen was found to be highly immunosuppressive, as evidenced by a reduced host T cell capacity to secrete interleukin-2 and interferon-γ in vitro, to mediate host-versus-graft reactivity in vivo, and to recover numerically and functionally during a 2-week observation period after chemotherapy. Finally, using B6 hosts treated with the 14-day chemotherapy regimens, the PC regimen more consistently prevented the rejection of BALB/c T cell-depleted allografts compared with the FC regimen (rate of alloengraftment, 14/15 [93%] of PC-treated recipients vs 8/14 [57%] of FC-treated recipients; P < .05); similar results were observed using an 8-day conditioning regimen. These data suggest that host T cell suppression, distinct from T cell depletion, may be a critical determinant of engraftment after purine analog-based regimens and also may be preferentially attained by the use of pentostatin.


Assuntos
Transplante de Medula Óssea/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Doença Enxerto-Hospedeiro/prevenção & controle , Imunossupressores/administração & dosagem , Pentostatina/administração & dosagem , Quimeras de Transplante/imunologia , Animais , Antineoplásicos/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ciclofosfamida/administração & dosagem , Sinergismo Farmacológico , Feminino , Sobrevivência de Enxerto/imunologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Interferon gama/análise , Interferon gama/biossíntese , Interleucina-2/análise , Interleucina-2/biossíntese , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Transplante Homólogo , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados
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