Alteration of interleukin-10-producing Type 1 regulatory cells in autoimmune diseases.

PURPOSE OF REVIEW: This review highlights findings describing the role of interleukin (IL)-10-producing Type 1 regulatory T (Tr1) cells in controlling autoimmune diseases and possible approaches to restore their function and number. RECENT FINDINGS: Reduced frequency and/or function of cell subsets playing a role in Tr1 cell induction (e.g., DC-10 and Bregs), was found in patients with autoimmunity and may impact on Tr1 cell frequency. SUMMARY: IL-10 is a pleiotropic cytokine with fundamental anti-inflammatory functions acting as negative regulator of immune responses. IL-10 is critically involved in the induction and functions of Tr1 cells, a subset of memory CD4+ T cells induced in the periphery to suppress immune responses to a variety of antigens (Ags), including self-, allogeneic, and dietary Ags. Alterations in IL-10-related pathways and/or in the frequency and activities of Tr1 cells have been associated to several autoimmune diseases. We will give an overview of the alterations of IL-10 and IL-10-producing Tr1 cells in Multiple Sclerosis, Type 1 Diabetes, and Celiac Disease, in which similarities in the role of these tolerogenic mechanisms are present. Current and future approaches to overcome Tr1 cell defects and restore tolerance in these diseases will also be discussed.

Corrigendum: Generation of Powerful Human Tolerogenic Dendritic Cells by Lentiviral-Mediated IL-10 Gene Transfer.

[This corrects the article DOI: 10.3389/fimmu.2020.01260.].

Generation of Powerful Human Tolerogenic Dendritic Cells by Lentiviral-Mediated IL-10 Gene Transfer.

The prominent role of dendritic cells (DC) in promoting tolerance and the development of methods to generate clinical grade products allowed the clinical application of tolerogenic DC (tolDC)-based therapies for controlling unwanted immune responses. We established an efficient method to generate tolerogenic human DC, producing supra-physiological levels of IL-10, by genetically engineering monocyte-derived DC with a bidirectional Lentiviral Vector (bdLV) encoding for IL-10 and a marker gene. DCIL-10 are mature DC, modulate T cell responses, promote T regulatory cells, and are phenotypically and functionally stable upon stimulation. Adoptive transfer of human DCIL-10 in a humanized mouse model dampens allogeneic T cell recall responses, while murine DCIL-10 delays acute graft-vs.-host disease in mice. Our report outlines an efficient method to transduce human myeloid cells with large-size LV and shows that stable over-expression of IL-10 generates an effective cell product for future clinical applications in the contest of allogeneic transplantation.

Forkhead box P3: the peacekeeper of the immune system.

Ten years ago Forkhead box P3 (FOXP3) was discovered as master gene driving CD4(+)CD25(+) T cell regulatory (Treg) function. Since then, several layers of complexity have emerged in the regulation of its expression and function, which is not only exerted in Treg cells. While the mechanisms leading to the highly selective expression of FOXP3 in thymus-derived Treg cells still remain to be elucidated, we review here the current knowledge on the role of FOXP3 in the development of Treg cells and the direct and indirect consequences of FOXP3 mutations on multiple arms of the immune response. Finally, we summarize the newly acquired knowledge on the epigenetic regulation of FOXP3, still largely undefined in human cells.

Lentiviral vector gene transfer is limited by the proteasome at postentry steps in various types of stem cells.

The isolation of human embryonic and somatic stem cells of different types has made it possible to design novel gene and cell replacement therapies. Vectors derived from retro/lentiviruses are used to stably introduce genes into stem cells and their progeny. However, the permissivity to retroviral infection varies among cell types. We previously showed that hematopoietic stem cells are poorly permissive to human immunodeficiency virus (HIV)-derived vectors and that pharmacological inhibition of the proteasome strongly enhances gene transfer. Here we report that the proteasome limits lentiviral gene transfer in all stem cell types tested, including embryonic, mesenchymal, and neural, of both human and mouse origin. Remarkably, this inhibitory activity was sharply reduced upon differentiation of the stem cells, suggesting that it represents a novel feature of the stem cell/immature progenitor phenotype. Proteasome-mediated inhibition was specific for lentiviral vectors and occurred at a postentry infection step. It was not mediated by activation of nuclear factor-kappaB, a major signaling pathway modulated by the proteasome, and did not correlate with high proteasome activity. Interaction of the virion core with cyclophilin A was required to maximize the effect of proteasome inhibitor on the infection pathway. These findings are relevant to uncover new mediators of HIV gene transfer and help in designing more effective protocols for the genetic modification of stem cells. Disclosure of potential conflicts of interest is found at the end of this article.

Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction.

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture, they display low permissivity to the vector, requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation, we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays, we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer, allowing the reach of very high levels of vector integration in their progeny in vivo. Thus, LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly, cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors, highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy.

Molecular evidence of lentiviral vector-mediated gene transfer into human self-renewing, multi-potent, long-term NOD/SCID repopulating hematopoietic cells.

A major challenge in gene therapy is to achieve efficient transduction of hematopoietic stem cells (HSC). It has previously been shown that lentiviral vectors (LV) transduce efficiently human cord blood-derived NOD/SCID mouse repopulating cells (SRC). Here we studied the effect of cytokines during the short ex vivo incubation with vector. Although SRC transduction was efficient without stimulation, the presence of cytokines significantly improved it. The treatment did not affect the engraftment level or the SRC frequency, but seemed to enhance SRC susceptibility to LV. SRC transduced in both conditions repopulated primary and secondary recipients, maintaining stable multi-lineage transgene expression. Using linear amplification-mediated PCR, we then analyzed vector integration in the bone marrow and CFC of the engrafted mice to monitor the clonal activity of the transduced SRC in vivo. We showed polyclonal engraftment, multi-lineage differentiation, and propagation to secondary recipients of individual SRC. We observed multiple integrations in most clones. These results provide the first formal demonstration that primitive human HSC with self-renewal and multi-lineage repopulation capacities were transduced by LV. Our findings are relevant for the design of clinical protocols that exploit this system to reach significant engraftment by genetically modified HSC in the absence of in vivo selection or strong conditioning regimens.