Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming

Aging; Epigenetic clocks; PluripotencyEnvelliment; Rellotges epigenètics; PluripotènciaEnvejecimiento; Relojes epigenéticos; PluripotenciaThe expression of the pluripotency factors OCT4, SOX2, KLF4, and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, partial and reversible reprogramming does not change cell identity but can reverse markers of aging in cells, improve the capacity of aged mice to repair tissue injuries, and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome, and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen, and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites and biomarkers altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic, and metabolomic changes toward a younger configuration in multiple tissues and in the serum

Pseudoalignment tools as an efficient alternative to detect repeated transposable elements in scRNAseq data

Transposable elements (TE) have played a major role in configuring the structures of mammalian genomes through evolution. In normal conditions, expression of these elements is repressed by different epigenetic regulation mechanisms such as DNA methylation, histone modification and regulation by small RNAs. TE re-activation is associated with stemness potential acquisition, regulation of innate immunity, and disease, such as cancer. However, the vast majority of current knowlededge in the field is based on bulk expression studies and very little is known on cell type- or state-specific expression of TE derived transcripts. Therefore, cost-efficient single cell-resolution TE expression analytical approaches are needed. We have implemented an analytical approach based on pseudoalignment to consensus sequences to incorporate TE expression information to scRNAseq data. All the data and code implemented is available as Supplementary data and in: https://github.com/jmzvillarreal/kallisto_TE_scRNAseq. Supplementary data are available at Bioinformatics online

Improved induction of anti-melanoma T cells by adenovirus-5/3 fiber modification to target human DCs

To mount a strong anti-tumor immune response, non T cell inflamed (cold) tumors may require combination treatment encompassing vaccine strategies preceding checkpoint inhibition. In vivo targeted delivery of tumor-associated antigens (TAA) to dendritic cells (DCs), relying on the natural functions of primary DCs in situ, represents an attractive vaccination strategy. In this study we made use of a full-length MART-1 expressing C/B-chimeric adenoviral vector, consisting of the Ad5 capsid and the Ad3 knob (Ad5/3), which we previously showed to selectively transduce DCs in human skin and lymph nodes. Our data demonstrate that chimeric Ad5/3 vectors encoding TAA, and able to target human DCs in situ, can be used to efficiently induce expansion of functional tumor-specific CD8⁺ effector T cells, either from a naïve T cell pool or from previously primed T cells residing in the melanoma-draining sentinel lymph nodes (SLN). These data support the use of Ad3-knob containing viruses as vaccine vehicles for in vivo delivery. "Off-the-shelf" DC-targeted Ad vaccines encoding TAA could clearly benefit future immunotherapeutic approaches

Natural killer cells act as an extrinsic barrier for <i>in vivo</i> reprogramming

The ectopic expression of transcription factors Oct4, Sox2, Klf4 and Myc (OSKM) enables reprogramming of differentiated cells into pluripotent embryonic stem cells. Methods based on partial and reversible in vivo reprogramming are a promising strategy for tissue regeneration and rejuvenation. However, little is known about the barriers that impair reprogramming in an in vivo context. We report that natural killer (NK) cells significantly limit reprogramming, both in vitro and in vivo. Cells and tissues at the intermediate states of reprogramming upregulate the expression of NK activating ligands, such as MULT1 and ICAM1. NK cells recognize and kill partially reprogrammed cells in a degranulation-dependent manner. Importantly, in vivo partial reprogramming is strongly reduced by adoptive transfer of NK cells, whereas it is significantly improved by depletion of NK cells. Notably, in the absence of NK cells, the pancreatic organoids derived from OSKM-expressing mice are remarkably large, suggesting the generation of cells with progenitor properties. We conclude that NK cells pose an important barrier for in vivo reprogramming, and this concept may apply to other contexts of transient cellular plasticity

Descifrando la reprogramación in vivo y su potencial de rejuvenecimiento

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-07-2020Esta tesis tiene embargado el acceso al texto completo hasta el 24-01-2022The manipulation of cell fates through reprogramming is one of the most exciting advances in recent years. The most striking breakthrough in the field occurred when Yamanaka first illustrated the possibility to convert differentiated cells into pluripotent stem cells by the ectopic expression of four transcription factors, Oct4, Sox2, Klf4 and c-Myc or OSKM. Our laboratory has demonstrated that transient expression of these four Yamanaka factors leads to teratoma formation in mice indicative of in vivo reprogramming. We were interested in deciphering the intermediate events of de-differentiation induced by OSKM in vivo before reaching pluripotency. For this purpose, we focused on the pancreas which we found to be the organ with the highest reprogramming efficiency in vivo. We demonstrate that in vivo reprogramming leads to the loss of pancreatic acinar identity and the acquisition of an atypical tubular morphology different from normal pancreatic ducts or from the metaplastic ducts produced by pancreatitis. Interestingly, the OSKM-induced atypical tubules are characterized by the presence of keratin 14 (KRT14), a protein that it is normally absent from pancreas, except in the context of rare squamous pancreatic cancers. Moreover, these atypical KRT14-positive cells emerge in many different tissues and primary cells upon OSKM activation, with Klf4 overexpression being the driving force in their emergence, at least in fibroblasts. Single cell RNA sequencing of partially reprogrammed pancreas confirms the presence of extensive changes in cell identity both in acinar and ductal cells. Finally, cells derived from partially reprogrammed pancreas efficiently form organoids in vitro with a transcriptional profile that resembles primitive embryonic stages. In a complementary experimental approach, we have investigated the potential of partial reprogramming to rejuvenate tissues. For this, OSKM was transiently induced and then switched off to allow the restoration of normal histology. Interestingly, we found that transient OSKM activation in old mice rejuvenates transcriptomic alterations associated to aging. Moreover, at an epigenetic level, OSKM transient expression reverses the methylation status of some age-associated differentially methylated regions, including promoters and enhancers, highlighting its beneficial window towards rejuvenation. Overall, unraveling the cellular plasticity induced by transient OSKM activation is of great importance considering that a shift in equilibrium could lead either to extensive tissue remodeling and tumor formation, or to rejuvenationLa manipulación de la identidad celular a través de la reprogramación ha sido uno de los avances científicos más relevantes de los últimos años. La aportación más destacada al campo fue la de Yamanaka, quien por primera vez obtuvo células madre pluripotentes inducidas a partir de células diferenciadas mediante la expresión ectópica de cuatro factores de transcripción, Oct4, Sox2, Klf4 y c-Myc, también conocidos como OSKM. Nuestro laboratorio ha demostrado que la expresión transitoria de estos cuatro factores de Yamanaka promueve la formación de teratomas en ratones, demostrando la existencia de reprogramación in vivo. Nuestro interés se centraba en discernir los pasos intermedios de desdiferenciación celular, inducidos por la expresión de OSKM in vivo con anterioridad a la adquisición de la pluripotencia. Para ello enfocamos el estudio en el páncreas, puesto que es el órgano con mayor eficiencia de reprogramación in vivo. Aquí demostramos que la reprogramación in vivo lleva a la pérdida de la identidad acinar en el páncreas y a la adquisición de una morfología tubular atípica que difiere de los ductos pancreáticos y de los ductos metaplásticos producidos durante una pancreatitis. Es destacable que los túbulos atípicos inducidos por OSKM se caracterizan por la presencia de keratina 14 (KRT14), una proteína que no está normalmente presente en el páncreas, a excepción de infrecuentes cánceres pancreáticos de células escamosas. Además, estas células atípicas positivas para KRT14 se observan en varios tejidos tras la expresión de OSKM, siendo Klf4 el principal propulsor de su aparición, al menos en fibroblastos. La secuenciación del transcriptoma de células individuales que han sido reprogramadas parcialmente confirma la presencia de múltiples cambios en identidad celular, tanto en células acinares como ductales. Finalmente, células derivadas de páncreas parcialmente reprogramados son capaces de formar organoides in vitro, cuyo perfil transcripcional se asemeja a estadios embrionarios primitivos. En un enfoque experimental similar, hemos investigado el potencial de células parcialmente reprogramadas para rejuvenecer tejidos. Para ello, la expresión de OSKM fue activada de manera transitoria y posteriormente inactivada para permitir la recuperación de la homeostasis histológica. Resulta interesante que la activación transitoria de OSKM en ratones envejecidos promueve el rejuvenecimiento de alteraciones transcripcionales asociadas al envejecimiento. Además, a nivel epigenético, la expresión transitoria de OSKM revierte los cambios en aquellas regiones, incluyendo promotores y enhancers, cuyo estado de metilación se altera con el envejecimiento, poniendo de manifiesto su efecto beneficioso en el rejuvenecimiento. En conclusión, entender la plasticidad celular inducida por la expresión transitoria de OSKM es de gran importancia, puesto que leves desequilibrios pueden inducir tanto a la remodelación tisular, como a la formación de tumores o bien al rejuvenecimient

High and Interrelated Rates of PD-L1+CD14+ Antigen-Presenting Cells and Regulatory T Cells Mark the Microenvironment of Metastatic Lymph Nodes from Patients with Cervical Cancer

A better understanding of the microenvironment in relation to lymph node metastasis is essential for the development of effective immunotherapeutic strategies against cervical cancer. In the present study, we investigated the microenvironment of tumor-draining lymph nodes of patients with cervical cancer by comprehensive flow cytometry-based phenotyping and enumeration of immune-cell subsets in tumor-negative (LN(-), n = 20) versus tumor-positive lymph nodes (LN(+), n = 8), and by the study of cytokine release profiles (n = 4 for both LN(-) and LN(+)). We found significantly lower CD4(+) and higher CD8(+) T-cell frequencies in LN(+) samples, accompanied by increased surface levels of activation markers (HLA-DR; ICOS; PD-1; CTLA-4) and the memory marker CD45RO. Furthermore, in LN(+), we found increased rates of a potentially regulatory antigen-presenting cell (APC) subset (CD11c(hi)CD14(+)PD-L1(+)) and of myeloid-derived suppressor cell subsets; the LN(+) APC subset correlated with significantly elevated frequencies of FoxP3(+) regulatory T cells (Treg). After in vitro stimulation with different Toll-like receptor (TLR) ligands (PGN; Poly-IC; R848), we observed higher production levels of IL6, IL10, and TNFα but lower levels of IFNγ in LN(+) samples. We conclude that, despite increased T-cell differentiation and activation, a switch to a profound immune-suppressive microenvironment in LN(+) of patients with cervical cancer will enable immune escape. Our data indicate that the CD14(+)PD-L1(+) APC/Treg axis is a particularly attractive and relevant therapeutic target to specifically tackle microenvironmental immune suppression and thus enhances the efficacy of immunotherapy in patients with metastasized cervical cancer. Cancer Immunol Res; 3(1); 48-58. ©2014 AAC

Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repai

Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming.

The expression of the pluripotency factors OCT4, SOX2, KLF4, and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, partial and reversible reprogramming does not change cell identity but can reverse markers of aging in cells, improve the capacity of aged mice to repair tissue injuries, and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome, and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen, and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites and biomarkers altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic, and metabolomic changes toward a younger configuration in multiple tissues and in the serum

Local delivery of low-dose anti-CTLA-4 to the melanoma lymphatic basin leads to systemic Treg reduction and effector T cell activation

Preclinical studies show that locoregional CTLA-4 blockade is equally effective in inducing tumor eradication as systemic delivery, without the added risk of immune-related side effects. This efficacy is related to access of the CTLA-4 blocking antibodies to tumor-draining lymph nodes (TDLNs). Local delivery of anti-CTLA-4 after surgical removal of primary melanoma, before sentinel lymph node biopsy (SLNB), provides a unique setting to clinically assess the role of TDLN in the biological efficacy of locoregional CTLA-4 blockade. Here, we have evaluated the safety, tolerability, and immunomodulatory effects in the SLN and peripheral blood of a single dose of tremelimumab [a fully human immunoglobulin gamma-2 (IgG2) mAb directed against CTLA-4] in a dose range of 2 to 20 mg, injected intradermally at the tumor excision site 1 week before SLNB in 13 patients with early-stage melanoma (phase 1 trial; NCT04274816). Intradermal delivery was safe and well tolerated and induced activation of migratory dendritic cell (DC) subsets in the SLN. It also induced profound and durable decreases in regulatory T cell (Treg) frequencies and activation of effector T cells in both SLN and peripheral blood. Moreover, systemic T cell responses against NY-ESO-1 or MART-1 were primed or boosted (N = 7), in association with T cell activation and central memory T cell differentiation. These findings indicate that local administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, our data demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and support TDLN-targeted delivery methods