Background
T regulatory (Treg) lymphocytes are a cellular component of the immune system responsible for suppressing immune responses of effector cells. As a result of their ability to control immune responses and to sustain systemic immune balance, they have the potential to prevent allergic, autoimmune and inflammatory disorders [
1‐
10] as well as to be an adjuvant therapy for chronic and acute graft versus host disease [
11].
The safety profile of Treg cells has been established in phase I clinical trials [
11,
12] demonstrating that Treg cells are a suitable candidate for therapeutic purposes. However a major limitation to the clinical use of natural Treg (nTreg) cells is their low availability as they represent only a small percentage of the peripheral circulating CD4
+ T cell population. In order to overcome this issue, several groups have developed methods to expand nTreg cells
in vitro when keeping their functionality. Generally the technologies are complex, time-consuming and the plasticity of nTreg cell lineage in artificial environment during
ex vivo expansion can lead to loss of their suppressive activity [
13]. Furthermore their stage of differentiation makes their expansion
in vitro a difficult process [
14].
In vitro induced T regulatory (iTreg) cells represent a good alternative to nTreg cells since they have been reported to have similar functionality in
in vivo setup. Additionally iTreg cells show advantages over nTreg cells in terms of increased number of starting population and greater potential to proliferate [
15]. Therefore it is of importance to investigate and explore new approaches to generate iTreg cells.
Based on the activating and stimulating properties of phorbol myristate acetate (PMA)/ionomycin and anti-CD3 on T cells [
16,
17] we developed a new method to generate iTreg cells
in vitro, which we refer to as ‘TregPMA’ cells. The functionality of the TregPMA cells was assessed
in vivo in a mouse model of experimental colitis.
Methods
Mice
BALB/C and C.B.-17 SCID mice (8–10 weeks) were obtained from Harlan and maintained in specific pathogen-free conditions. Mouse experiments were approved by the local animal welfare committee (University of Amsterdam).
Generation of regulatory T lymphocytes (Treg PMA cells)
Splenocytes were isolated from BALB/C mice. CD4+ CD25- T cells were isolated from splenocytes by means of negative selection using the mouse “CD4+ T Cell Isolation Kit” followed by “CD25 MicroBead Kit” (Miltenyi Biotec). Cells were seeded at day 0 at 1x105/well into anti-CD3e (0.5 μg/well, clone 145-2C11, eBioscience) coated 96-well flat bottom plates (Costar) and cultured in X-VIVO 15 medium (Lonza) at 37°C in a 5% CO2 incubator. On day 1, cells were activated with PMA (10 ng/ml) and ionomycin (250 ng/ml) and at day 2, 3 and 4 supplemented with 20 U/well of IL-2 (eBioscience). On day 5, prior to use, cells were harvested and treated with “Dead Cell Removal Kit” (Miltenyi Biotec).
Flow cytometry analysis
Cells were analyzed using flow cytometry (FACSCalibur, BD Biosciences). For Treg cell staining the mouse “Regulatory T cell Staining Kit” was used which consisted of anti-mouse CD4 FITC (clone RM4-5), anti-mouse CD25 APC (clone PC61.5) and anti-mouse/rat Foxp3 PE (clone FJK16). Additional monoclonal anti-mouse flow cytometry antibodies used in this study were as follows: anti-mouse CD152 (CTLA-4) APC (clone UC10-4B9), anti-mouse GARP PE (clone YGIC86), anti-mouse/rat CD278 (ICOS) FITC (clone C398.4A) and anti-mouse CD134 (OX-40) APC (clone OX86). All antibodies were obtained from eBioscience.
Induction of CD45RB transfer colitis model and treatment with TregPMA cells
Chronic colitis was induced in C.B.-17 SCID mice by intraperitoneal (IP) injection of CD45RBhigh cells (4x 105) isolated from normal BALB/C mice splenocytes (positive control group). Mice that received CD45RBhigh cells in combination with CD45RBlow cells (2x105) were protected from disease development (negative control group). The treatment group received CD45RBhigh cells in combination with in vitro generated TregPMA cells (TregPMA cell treated group). The amount of TregPMA cells injected per mouse was 1.2x106.
Monitoring development of colitis
The primary read-out to assess the development of colitis was the body weight loss. Mice were weighed three times a week. Body weight loss was determined by percentage of weight loss from base line body weight.
After sacrifice, colon was excised and longitudinally divided into 2 parts both of which were rolled up: one was used for preparation of paraffin embedded samples while the other was snap frozen in liquid nitrogen. Disease activity index (DAI) was calculated by combining the scores applied to weight loss (0: <1%; 1: 1-5%; 2: 5-10%; 3: 10-15%; 4: >15%), stool consistency at sacrifice (0: normal; 1: loose droppings; 2: loose stools, colon filled with feces; 3: loose stool, feces only near caecum, 4: empty bowel) and rectal bleeding (0: negative; 2: positive; 3: gross bleeding) divided by three.
Histology
The half divided colon tissue was fixed in 4% paraformaldehyde and embedded in paraffin. The colon tissue was cut in 5 μm sections and stained with hematoxylin and eosin for histological scoring. An experienced pathologist blinded to experiment inspected microscopically all sections and graded them on a scale 0 to 4 looking at: mononuclear and polymorphonuclear infiltrate, goblet cell depletion, crypt loss, epithelial hyperplasia, presence of ulcerations and manifestations of crypt abscesses.
Cytokine analysis
Frozen colon tissue was crushed with the use of CryoPrep™ System (Covaris) and resuspended in ice-cold PBS (pH 7.2) containing complete protease inhibitor (Roche). Homogenates were then centrifuged at 15000 x g for 5 min at 4°C and the supernatants were stored at −80°C until the cytokine assay. Prior to cytokine analysis total concentration of the protein in supernatants was determined with the use of Bradford assay (Biorad). Colonic tissue and plasma levels of IL-1β, IL-6, IL-10, IL-17 and TNF-α were analyzed using a mouse cytokine magnetic bead-based multiplex assay (Biorad) according to the manufacturer’s instructions. Additionally TGF-β levels were analyzed with Bio-Plex Pro TGF-β assay (Biorad) according to manufacturer’s instructions.
Statistical analysis
Data were analyzed statistically and graphed using Prism 5.0 (GraphPad Software). Changes in mice weekly body weight, body weight at sacrifice, disease activity index (DAI), histological score and cytokine levels are shown as mean ± standard deviation (SD) and analyzed by one-way ANOVA, followed by Bonferroni’s Multiple Comparison Test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Discussion
In this article we describe the generation of iTreg cells (TregPMA cells)
in vitro from CD4
+CD25
- cells by PMA/ionomycin/anti-CD3 activation based method. Several other groups have explored different strategies to generate iTreg from naive T cells
in vitro, including use of TGF-β, all-trans-retinoic acid and the recently discovered regulatory cytokine IL-35 [
36,
37]. However, all these methods are more or less impeded with their own limitations [
16]. Therefore, there is need for improvement in this field and our group designed and explored a new method of
in vitro iTreg cells (TregPMA) generation which we report in this manuscript.
Mechanistically, PMA/ionomycin provides a potent stimulation allowing us to bypass the T cell receptor activation essential for Treg cell development and it prevents the emergence of CD4
-CD8
+ cells in the culture [
17]. PMA activates protein kinase C [
38] while ionomycin is a Ca
2+ mobilizing agent [
39]. This combination has been shown to up-regulate CD25 on T lymphocytes [
17] and a high CD25 expression is a marker of the Treg cell phenotype [
40]. Additionally, it was demonstrated that Ca
2+ signaling is required for the development and function of Treg cells [
41,
42]. Therefore we expected that raising the intracellular levels of Ca
2+ in naive T cells using ionomycin would have a synergistic effect with PMA in starting a regulatory developmental program in CD4
+CD25
- cells. Additionally a low dose of IL-2 was used in cell culture since IL-2 is important for Treg cell homeostasis and maintaining their suppressive survival program [
43] while anti-CD3 which is also present in the cell culture has been shown to maintain and expand Treg cells [
44], however use of only anti-CD3 and IL-2 for
in vitro CD4
+CD25
- cell stimulation resulted in low cell viability and poor regulatory markers expression which indicates that additional stimulation with PMA/ionomycin is needed.
The TregPMA cells up-regulate CD25, and ~30% of the CD4
+CD25
+ T cells up-regulate CTLA-4, GARP, ICOS, OX-40 and Foxp3 which are important markers associated with regulatory cell phenotype and function [
18‐
22,
45,
46]. Interestingly, the level of Foxp3 expression in TregPMA cells is quite low. Although Foxp3 expression is considered to be a main characteristic of Treg cells, Treg cells are recognized to be a heterogeneous cell population. Especially, it has been reported that CD4
+CD25
+ Foxp3
- cell population also can present regulatory function
in vitro and
in vivo[
23].
The functionality of the TregPMA cells has been demonstrated
in vivo by their potential to ameliorate the disease phenotype in a CD45RB transfer colitis mouse model. It cannot be fully excluded that the amelioration of the disease phenotype observed in the group of mice treated with the TregPMA cells does not partially involve cell types, other than regulatory, present in the cell suspension injected. Indeed, high numbers of CD45RB
high cells (6x10
6/mouse) have been shown to reduce the severity of the disease in a mouse model of colitis [
47]. However, in our study, the total number of cells injected (1,2x10
6/mouse) was significantly lower and the amelioration of the severity of the disease was observed in all of the treated animals (n = 10) against 3 out of 5 in the mentioned study. All together, these data suggest that the observed protective effect is mostly related to TregPMA cells.
The disease amelioration after TregPMA treatment was accompanied by a decrease of the pro-inflammatory IL-1 β levels in the colon tissue which reflect the severity of experimental colitis [
28]. Such a decrease in IL-1β was previously reported by Hirano and Kudo after treatment of CD45RB transfer colitis mouse model with both dexamethasone and anti-tumor necrosis factor-α (anti-TNF-α).
IL-1β stimulates the production of the pro-inflammatory cytokine IL-6 [
48] which has been described to be up-regulated in mouse model of IBD [
49] and in human ulcerative colitis or Crohn’s disease patients [
50]. Simultaneously with the decrease of IL-1β in our study, IL-6 levels were found to be reduced in both colonic homogenates and plasma samples after treatment with TregPMA cells.
The concentration of IL-17 was found to be more elevated in colon homogenates of TregPMA cell treated mice than in the positive control group. Although the role of this cytokine in the initiation or pathogenesis of IBD is controversial, a protective function of IL-17 in the gut of CD45RB transfer colitis model has been previously noted [
30,
33,
51]. Several groups have reported that both IL-17−/− CD45RB
high and IL-17R−/− CD45RB
high cells induce a more aggressive disease phenotype than wild type CD45RB
high cells [
30,
51]. Accordingly, our results show that an increased IL-17 level also correlated with less severe intestinal inflammation and lack of ulcerations.
IL-10 and TGF-β cytokine level in both mouse plasma and colon homogenates show no significant differences between experimental groups at the time of experiment termination (week 7). However, it does not exclude the possible influence of those cytokines on prevention of the colitis induction at earlier time point.
The cells that have been found mostly responsible for pathogenesis of CD45RB transfer model of IBD are Th1 cells. Recently it has been shown that IL-17 blocks the Th1 cell development
in vivo via IL-17R on naive CD4
+ T cells [
30,
51]. In the TregPMA cell treated group we also observe elevated level of IL-17 which correlates with disease amelioration. IL-17 can be produced by many types of cells among which are also T regulatory cells [
52]. Therefore we are hypothesizing that
in vivo disease amelioration by TregPMA cells is IL-17 mediated. As follow-up to this study, physiological relevance of TregPMA cells and their exact molecular mechanism of action needs to be determined.
Competing interests
Three of the authors are employees of uniQure (AM, HP and VF), SM currently works in the laboratory of uniQure and SJD is a board member of uniQure.
Authors’ contributions
AM carried out experiments and analyzed the data. AM and VF performed literature searches and wrote the article. SM and AAV participated in the animal experiment, SLM performed histological analysis of colon section, AM, VF, SM, AAV, SLM, HP and SJD contributed conceptually to the work, reviewed the manuscript and assisted with the editing of the paper. All authors read and approved the final manuscript.