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1.
Heliyon ; 9(9): e19435, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37810095

RESUMO

Selective T-cell depletion prior to cell or organ transplantation is considered a preconditioning regimen to induce tolerance and immunosuppression. An immunotoxin consisting of a recombinant anti-CD3 antibody conjugated with diphtheria toxin was used to eliminate T-cells. It showed significant T-cell depletion activity in the peripheral blood and lymph nodes in animal models used in previous studies. To date, a comprehensive evaluation of T-cell depletion and CD3 proliferation for all lymphoid tissues has not been conducted. Here, two rhesus macaques were administered A-dmDT390-SCFBdb (CD3-IT) intravenously at 25 µg/kg twice daily for four days. Samples were collected one day prior to and four days post administration. Flow cytometry and immunofluorescence staining were used to evaluate treatment efficiency accurately. Our preliminary results suggest that CD3-IT treatment may induce higher depletion of CD3 and CD4 T-cells in the lymph nodes and spleen, but is ineffective in the colon and thymus. The data showed a better elimination tendency of CD4 T-cells in the B-cell zone relative to the germinal center in the lymph nodes. Further, CD3-IT treatment may lead to a reduction in germinal center T follicular helper CD4 cells in the lymph nodes compared to healthy controls. The number of proliferating CD3 T-cell indicated that repopulation in different lymphoid tissues may occur four days post treatment. Our results provide insights into the differential efficacy of CD3-IT treatment and T-cell proliferation post treatment in different lymphoid tissues. Overall, CD3-IT treatment shows potential efficacy in depleting T-cells in the periphery, lymph nodes, and spleen, making it a viable preconditioning regimen for cell or organ transplantation. Our pilot study provides critical descriptive statistics and can contribute to the design of larger future studies.

2.
Stem Cell Res Ther ; 12(1): 528, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620229

RESUMO

BACKGROUND: Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment. METHODS: Humanized bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias. RESULTS: A total of 897 HSPC clones were longitudinally tracked in hu-BLT mice-providing a first-ever demonstration of clonal dynamics and coordinated expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (~ 60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ± 1 kb of H3K36me3-enriched regions. CONCLUSIONS: Human repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Animais , Modelos Animais de Doenças , Hematopoese , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
3.
JCI Insight ; 6(1)2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33427210

RESUMO

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/terapia , Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva , Animais , Linhagem da Célula/imunologia , Modelos Animais de Doenças , Reservatórios de Doenças/virologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/patologia , Trato Gastrointestinal/virologia , Centro Germinativo/imunologia , Centro Germinativo/patologia , Centro Germinativo/virologia , Infecções por HIV/virologia , HIV-1 , Humanos , Imuno-Histoquímica , Macaca nemestrina , Masculino , Receptores de Antígenos Quiméricos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Transplante Homólogo
4.
Sci Adv ; 6(30): eaay9206, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32766447

RESUMO

Despite advances in hematopoietic stem/progenitor cell (HSPC) transplant for HIV-1-infected patients, the impact of a preexisting HIV-1 infection on the engraftment and clonal repopulation of HSPCs remains poorly understood. We have developed a long terminal repeat indexing-mediated integration site sequencing (LTRi-Seq) method that provides a multiplexed clonal quantitation of both anti-HIV-1 RNAi (RNA interference) gene-modified and control vector-modified cell populations, together with HIV-1-infected cells-all within the same animal. In our HIV-1-preinfected humanized mice, both therapeutic and control HSPCs repopulated efficiently without abnormalities. Although the HIV-1-mediated selection of anti-HIV-1 RNAi-modified clones was evident in HIV-1-infected mice, the organ-to-organ and intra-organ clonal distributions in infected mice were indistinguishable from those in uninfected mice. HIV-1-infected cells showed clonal patterns distinct from those of HSPCs. Our data demonstrate that, despite the substantial impact of HIV-1 infection on CD4+ T cells, HSPC repopulation remains polyclonal, thus supporting the use of HSPC transplant for anti-HIV treatment.


Assuntos
Infecções por HIV , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Animais , Infecções por HIV/genética , Infecções por HIV/terapia , HIV-1/genética , Células-Tronco Hematopoéticas , Humanos , Camundongos , Interferência de RNA
5.
J Vis Exp ; (124)2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28654067

RESUMO

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , Retroviridae/genética , Integração Viral/genética , Fluxo de Trabalho , Animais , Células Cultivadas , Células Clonais , Vetores Genéticos , Células-Tronco Hematopoéticas/citologia , Reprodutibilidade dos Testes , Análise de Sequência de DNA
6.
Cell Syst ; 4(3): 330-343.e5, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28237795

RESUMO

Combinatorial control of gene expression is presumed to be mediated by molecular interactions between coincident transcription factors (TFs). While information on the genome-wide locations of TFs is available, the genes they regulate and whether they function combinatorially often remain open questions. Here, we developed a mechanistic, rather than statistical, modeling approach to elucidate TF control logic from gene expression data. Applying this approach to hundreds of genes in 85 datasets measuring the transcriptional responses of murine fibroblasts and macrophages to cytokines and pathogens, we found that stimulus-responsive TFs generally function sequentially in logical OR gates or singly. Logical AND gates were found between NF-κB-responsive mRNA synthesis and MAPKp38-responsive control of mRNA half-life, but not between temporally coincident TFs. Our analyses identified the functional target genes of each of the pathogen-responsive TFs and prompt a revision of the conceptual underpinnings of combinatorial control of gene expression to include sequentially acting molecular mechanisms that govern mRNA synthesis and decay.


Assuntos
Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Animais , Simulação por Computador , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Humanos , Modelos Biológicos , Ligação Proteica , Fatores de Transcrição/genética , Ativação Transcricional/genética , Ativação Transcricional/fisiologia
7.
Proc Natl Acad Sci U S A ; 114(8): 1916-1921, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28167786

RESUMO

Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called "stripping." To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen-deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be "caught in the act of stripping" because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.


Assuntos
DNA/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/genética , Ativação Transcricional , Animais , Núcleo Celular/metabolismo , Células Cultivadas , DNA/genética , Fibroblastos , Imunofluorescência , Técnicas de Inativação de Genes , Humanos , Proteínas I-kappa B/genética , Camundongos , Simulação de Acoplamento Molecular , Mutação , Inibidor de NF-kappaB alfa/genética , NF-kappa B/genética , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Estresse Fisiológico/fisiologia , Fator de Transcrição RelA/genética
8.
J Theor Biol ; 386: 89-104, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26385832

RESUMO

Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif׳s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target HIV-1 accessory proteins and boost antiviral activity of host factors.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Sinergismo Farmacológico , Quimioterapia Combinada , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/química , HIV-1/fisiologia , Humanos , Carga Viral/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/análise , Replicação Viral/efeitos dos fármacos
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