Induction of CD4+ CD25+ Foxp3+ T regulatory cells by dendritic cells derived from ILT3 lentivirus-transduced human CD34+ Cells
Highlights
► ILT3 transduced CD34+ HPSCs by LVs, could be induced to dendritic cells. ► These DCs expressed high levels of ILT3, and kept strong tolerogenic properties. ► Down-regulation of NF-κB. was associated with expression of ILT3 ► These DCs reduced to stimulate T cells, and enhance inducing regulatory T cells.
Introduction
Dendritic cells (DCs), which arise from CD34+ hematopoietic stem/progenitor cells (HSPCs) [1], [2], play a pivotal role in the induction of both immunity and tolerance. Recently, the processing and presentation of antigens by different types of DCs have been appreciated as the initial events involved in determining immune responses [3], [4]. When exposed to inflammatory conditions, tolerogenic DCs, which have an immature phenotype, inhibit T cell responses and favor regulatory T cells (Tregs) induction/expansion [5]. Tregs expressing CD4, high level of CD25, and forkhead box P3 (Foxp3) are a specialized subpopulation of T cells that act to suppress the activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens [6]. Recently, it was widely used as a key indicator to reflect the level of immune tolerance. The establishment of immuno tolerance has been a major goal in the fields of transplantation and autoimmunity, especially to promote acceptance of allogeneic organ/tissue transplants or for the treatment and prevention of autoimmune diseases.
Different approaches including the use of inhibitory drugs, cytokines and CD8+CD28+ T suppressor cells to enhance the tolerogenic properties of DCs have been studied [7], [8]. A popular approach to generate tolerogenic DCs is to down-regulate the expression of co-stimulatory molecules, such as CD80 and CD86. The immuno-deviated DCs demonstrated effects of T cell immunosuppression and induced transplant tolerance as observed in different transplantation models [9], [10]. However, to date the results are far from satisfactory. One important factor is that the tolerogenic DCs are prone to undergo further maturation in vivo. It is believed that the immune tolerance induced by immature DCs is short-lived.
Therefore, stable gene transfer of inhibitory molecules may be a good means to generate long-term tolerogenic DCs. However, as professional antigen-presenting cells (APCs), DCs are very sensitive to exogenous danger signals. A previous report showed that ex vivo transfection or transduction of DCs can effectively trigger their maturation and therefore neutralize their capacity to induce T cell tolerance [11]. In addition, transfer of ex vivo-manipulated DCs is not conducive for long-term treatments due to the restricted DC life-span after transfer [12]. HSPCs have the unique capability of repopulating the entire hematopoietic system, including the immune system, due to their self-renewal and pluripotent differentiation potentials. Lentiviral vectors can be used to efficiently transduce HSPCs, which result in permanent integration of the transgene into the host genome [13], [14]. We hypothesized that large numbers of tolerogenic DCs may be generated from HSPCs by lentiviral transduction with inhibitory molecules, such as inhibitory molecule immunoglobulin-like transcript 3 (ILT3).
ILT3 belongs to a family of inhibitory receptors with cytoplasmic immunoreceptor tyrosine based inhibitory motifs (ITIMs). Numerous studies have reported that increased ILT3 expression is associated with the tolerogenic properties of APCs including dendritic cells (DCs) and then used to modulate immunity. In the present study, human CD34+ HSPCs were transduced with self-inactivating (SIN) lentiviral vectors carrying the ILT3 gene, expanded and then induced to differentiate into DCs. The genetically modified DCs derived from lentiviral transduced HSPCs avoided viral components stimulating maturation while stably expressing high level of ILT3. The expression profile and functional analysis suggest the ILT3high DCs are similar to tolerogenic DC with respect to the expression of surface markers, cytokines, and transcription factors. Its immunomodulatory phenotype was also validated by its ability to induce CD4+CD25+Foxp3+ Tregs and suppress T cell proliferation.
Section snippets
Purification of human umbilical cord blood (UCB) CD34+HPSCs
UCB samples were collected after informed consent had been obtained in writing. The study protocol was approved by the ethics committees of the First Affiliated Hospital, Medical College, Xi'an Jiaotong University.UCB samples from healthy, full-term neonates were collected into 84-ml blood donor sets containing 16 ml of the citrate-phosphate-adenine-1(CPD-A) anticoagulant. Nucleated cells were separated by sedimentation with 1.2% (final, wt/vol) hydroxyethyl starch in 0.9% sodium chloride (HES),
Generating ILT3high DCs from transduced UCB CD34+ HPSCs
Lentiviral transduction of HSPCs was optimized using the centrifugal method in the presence of polybrene. About 31% of the cells show high expression of ILT3 after three consecutive spin-infections. The ratio of ILT3 positive CD34+ HSPCs was increased to 95% after selection with blasticidin (Fig. 1B). The RT-PCR data showed that ILT3 RNA transcription levels were higher in DCs derived from ILT3-modified HSPCs (Fig. 1C), indicating that DCs differentiated from ILT3-modified HSPCs expressed high
Discussion
This study addresses two important points. Firstly, human CD34+ HSPCs can be efficiently transduced with self-inactivating lentiviral vectors carrying the ILT3 gene, expanded, induced to differentiate into DCs. Importantly, these HSPCs induced DCs persistently express high levels of ILT3 without the adverse effect of viral vector stimulated DCs maturation. Secondly, ILT3high DCs displayed a tolerogenic phenotype characterized by down-regulated costimulatory molecules and HLA-DR expression.
Acknowledgments
This research was supported in part by National Nature Science Foundation of China (No. 30872578).
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These authors (Guanqun Ge, Puxun Tian and Hongbao Liu) contributed equally to this work.