RESUMO
Recent advances in sequencing, mass spectrometry, and cytometry technologies have enabled researchers to collect multiple 'omics data types from a single sample. These large datasets have led to a growing consensus that a holistic approach is needed to identify new candidate biomarkers and unveil mechanisms underlying disease etiology, a key to precision medicine. While many reviews and benchmarks have been conducted on unsupervised approaches, their supervised counterparts have received less attention in the literature and no gold standard has emerged yet. In this work, we present a thorough comparison of a selection of six methods, representative of the main families of intermediate integrative approaches (matrix factorization, multiple kernel methods, ensemble learning, and graph-based methods). As non-integrative control, random forest was performed on concatenated and separated data types. Methods were evaluated for classification performance on both simulated and real-world datasets, the latter being carefully selected to cover different medical applications (infectious diseases, oncology, and vaccines) and data modalities. A total of 15 simulation scenarios were designed from the real-world datasets to explore a large and realistic parameter space (e.g. sample size, dimensionality, class imbalance, effect size). On real data, the method comparison showed that integrative approaches performed better or equally well than their non-integrative counterpart. By contrast, DIABLO and the four random forest alternatives outperform the others across the majority of simulation scenarios. The strengths and limitations of these methods are discussed in detail as well as guidelines for future applications.
Assuntos
Biologia Computacional , Humanos , Biologia Computacional/métodos , Algoritmos , Genômica/métodos , Genômica/estatística & dados numéricos , MultiômicaRESUMO
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.
Assuntos
Quimera/fisiologia , Células-Tronco Hematopoéticas/fisiologia , MicroRNAs/metabolismo , Receptores de Antígenos/metabolismo , Linfócitos T/metabolismo , Timo/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Linhagem da Célula , Células-Tronco Hematopoéticas/citologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Receptores de Antígenos/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , TransgenesRESUMO
Nucleotide synthesis inhibitors are currently used in neoplastic diseases or as immunosuppressive agents for the prevention of acute rejection in organ transplantation and the treatment of autoimmune disorders. We have previously described that these inhibitors interfere with proliferation and survival of primary T cells in vitro. However, the precise effects of nucleotide restriction on effector and memory functions have not been elucidated. In this study, we investigated the impact of nucleotide synthesis inhibition on CD8 T cell differentiation by using TCR transgenic mice (F5) specific for the influenza virus nucleoprotein 68 peptide presented on the H-2Db molecule. Our results show that methotrexate and 5-fluorouracil prevent the acquisition of effector functions, such as IFN-gamma, granzyme B expression, and cytotoxic function following antigenic stimulation of naive cells. Surprisingly, in the presence of mycophenolate mofetil, activated F5 cells are still able to produce granzyme B and to kill target cells but to a lesser extent compared with control. All three inhibitors interfere with the differentiation of naive cells into memory CD8 T cells. In contrast, the drugs are unable to inhibit the development of improved cytotoxic functions displayed by memory CD8 T cells.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Fluoruracila/farmacologia , Memória Imunológica , Metotrexato/farmacologia , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , Nucleotídeos/biossíntese , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Doença Enxerto-Hospedeiro/prevenção & controle , Imunização , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos C57BLRESUMO
CpG oligodeoxynucleotide (ODN) promotes maturation of APCs in vivo and induces strong type 1 T cell responses in mice. In this study, we have investigated the ability of CpG1826 to modulate peptide-specific CD8 T cell responses in a context where CD4 T cells are likely to play a minor role. The effects of CpG1826 were evaluated in a system where a population of NP68-specific F5 TCR transgenic CD8 T cells is diluted into a polyclonal host following adoptive transfer into C57BL/10 syngeneic recipients. Using this approach, we found that CpG1826 enhanced the ability of F5 CD8 T cells to undergo multiple divisions in vivo, to express IFN-gamma ex vivo, and to up-regulate memory-associated cell surface markers such as CD122 (IL-2Rbeta) and Ly-6C. Moreover, CpG1826 greatly increased in vivo cytotoxic activity. Using tetramer detection, we found that CpG1826 promoted long-term survival of Ag-specific CD8 T cells after immunization while no NP68-specific cells were detected when the cognate peptide was injected alone. These results indicate that CpG1826 acts as an adjuvant which increases CD8 T cell effector responses and promotes long-term survival of NP68 peptide-specific cells in vivo. They also suggest that this adjuvant can modulate CD8 T cell responses in a system which is likely to be independent of CD4 T cell help.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Ilhas de CpG/imunologia , DNA/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Transferência Adotiva , Animais , Antígenos Ly/biossíntese , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Injeções Intraperitoneais , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Peptídeos/farmacologia , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfócitos T/genética , Receptores de Interleucina-2/biossíntese , Linfócitos T Citotóxicos/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologia , Receptor fas/fisiologiaRESUMO
The existence of distinct subsets of memory CD8 T cells with different characteristics is now well established. In this work, we describe two subsets of mouse CD8 T cells with memory characteristics that coexist in primed thymectomized TCR-transgenic F5 mice and that share some properties with the human central and effector memory cells. The first subset corresponds to CD8 T cells generated following nucleoprotein 68 peptide priming which are CD44(int)CD122(-)nucleoprotein 68/H-2D(b) tetramer(+) and express high levels of CCR7 mRNA. In contrast, CD8 T cells in the second subset are CD44(high)CD122(+), are heterogeneous in terms of Ag specificity, and express low levels of CCR7 mRNA. We have studied the functional characteristics and the persistence of these two subsets in thymectomized mice. CD44(int) CD8 T cells persist like naive cells; i.e., they are slowly lost with time. However, surviving cells maintain their phenotype and memory characteristics for the entire life span of the animal. In contrast, CD44(high) CD8 T cells are persistent and accumulate in thymectomized but not euthymic mice. This is correlated with an increased in vivo proliferative and survival potential of these cells. These results show that acquisition of enhanced functional characteristics and long-term persistence by memory T cells are independent. This may have important consequences for the design of specific vaccine.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Receptores de Hialuronatos/biossíntese , Memória Imunológica , Receptores de Interleucina-2/biossíntese , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Apoptose/genética , Apoptose/imunologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Cruzamentos Genéticos , Proteínas de Ligação a DNA/biossíntese , Humanos , Imunidade Inata/genética , Memória Imunológica/genética , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptores CCR7 , Receptores de Quimiocinas/biossíntese , Proteína de Replicação C , Subpopulações de Linfócitos T/citologia , Proteína bcl-XRESUMO
Strong memory T cell responses result partly from the selection of Ag-specific clones during immunization. In this study, we show that a monoclonal CD8 T cell population expressing a unique TCR is heterogeneous in terms of clonogenic potential following activation under optimal conditions. More importantly, the frequency of clonogenic cells is strongly increased among Ag-experienced cells, indicating that these cells were either generated or selected during the in vivo primary response. Moreover, strong proliferative responses of primed cells result from this enhanced frequency, as proliferating naive and primed cells display the same cycling parameters, i.e., lag time and intermitotic interval. Hence, these results suggest that the clonogenic potential of individual cells is imprinted before Ag encounter and that clonogenic precursors are selected or generated following in vivo activation.