In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Hongzheng Bi; Samiksha Wasnik; David  J. Baylink; Chenfan Liu; Xiaolei Tang

doi:10.3791/60585

JoVE Journal > Immunology and Infection

Immunologie und Infektion

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Published: January 22, 2020

doi:

10.3791/60585

Hongzheng Bi^1,2, Samiksha Wasnik¹, David J. Baylink¹, Chenfan Liu^1,3,4, Xiaolei Tang^1,3

¹Division of Regenerative Medicine, Department of Medicine,Loma Linda University, ²Zhengzhou University, ³Department of Veterinary Biomedical Sciences, College of Veterinary Medicine,Long Island University, ⁴Jinan Infectious Disease Hospital,Shandong University

Summary

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]₂D) and the active vitamin A (retinoic acid or RA) de novo.

Abstract

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]₂D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)₂D and RA based on previous findings showing that 1,25(OH)₂D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1α-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)₂D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Introduction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier¹^,². For this reason, currently available U.S. Food and Drug Administration (FDA)-approved drugs inhibit the functions of inflammatory mediators or block the homing of immune cells into the gut. However, the inflammatory mediators and immune cells that are targeted are also necessary for immune defenses. As a result, the inflammatory mediator inhibitors compromise systemic immune defense and the immune cell homing blockers weaken gut immune defense, both of which can lead to severe consequences³^,⁴. In addition, the immune cell homing blockers can also block the homing of regulatory T (Treg) cells into the gut and hence can worsen the already compromised gut immune tolerance in IBD patients. Furthermore, blocking of Treg cell homing into the gut may also lead to systemic immune suppression due to the accumulation of Treg cells in the blood⁵. Finally, inhibitors and blockers function transiently and, thereby, require frequent administrations. Frequent administration of these inhibitors and blockers may further exacerbate the untoward side effects.

Recently, we proposed a novel strategy that can potentially mitigate or even eliminate the side effects associated with current drugs for IBD treatment⁶. This strategy augments the induction of gut-homing Treg cells in peripheral lymphoid tissues⁶. The rationale of this strategy is that gut-homing Treg cells specifically home to the gut and hence will not compromise systemic immune defenses. In addition, since Treg cells can potentially form memory⁷^,⁸, gut-homing Treg cells can potentially provide a stable control of the chronic gut inflammation in IBD patients and, thereby, treatment should not need to be administered as frequently. Furthermore, since this strategy augments the induction of gut-homing Treg cells in vivo, it does not have the concern of in vivo instability in a highly proinflammatory environment that is associated with adoptive transfer of in vitro generated Treg cells⁹^,¹⁰. In this regard, in vitro generated Treg cells are one of the proposed strategies for the treatment of autoimmune diseases¹¹^,¹²^,¹³ and transplant rejection¹⁴^,¹⁵. Finally, in this strategy, dendritic cells (DCs) are engineered to produce locally high concentrations of two molecules de novo: active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]₂D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)₂D and RA because 1,25(OH)₂D can induce the expression of regulatory molecules (e.g., forkhead box P3 [foxp3] and interleukin-10 [IL-10])¹⁶^,¹⁷ and that RA can stimulate the expression of gut-homing receptors in T cells¹⁸. Because both 1,25(OH)₂D and RA can also tolerize DCs²⁸^,²⁹, we reason that the engineered DCs will be stably maintained in a tolerogenic status in vivo and hence circumvent the in vivo instability concerns that are associated with in vitro generated tolerogenic DCs (TolDCs)¹⁹^,²⁰^,²¹. In this respect, TolDCs are also one of the proposed strategies for in vivo augmentation of Treg cell functions¹⁹^,²⁰^,²¹. To support our reasoning, we have shown that the engineered DCs, upon in vivo delivery, can augment the induction of gut-homing Treg cells in peripheral lymphoid tissues⁶.

An additional advantage of our proposed strategy is that 1,25(OH)₂D also has other functions that can potentially benefit IBD patients. These other functions include the ability of 1,25(OH)₂D to stimulate the secretion of antimicrobials²² and to suppresses carcinogenesis²³. Infections and cancers are frequently associated with IBD²⁴^,²⁵.

To generate the DCs that can produce locally high concentrations of both 1,25(OH)₂D and RA de novo, we use a lentiviral vector to engineer DCs to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 (CYP27B1) that encodes 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase), which physiologically catalyzes the synthesis of 1,25(OH)₂D. The other gene is the aldehyde dehydrogenase 1 family member A2 (ALDH1a2) that encodes retinaldehyde dehydrogenase 2 (RALDH2), which physiologically catalyzes the synthesis of RA⁶.

Because in vivo augmentation of gut-homing Treg cell induction is potentially important in the treatment of IBD, in the following protocol we will detail the procedures for the generation of the 1α-hydroxylase-RALDH2-overexpressing DCs (DC-CYP-ALDH cells) that can be used for the future investigation of gut-homing Treg cells in vivo.

Protocol

All in vivo animal study protocols were reviewed and approved by the Loma Linda University Institutional Animal Care and Use Committee (IACUC) as well as the Animal Care and Use Review Office (ACURO) of the US Army Medical Research and Materiel Command (USAMRMC) of the Department of Defense.

1. Preparation of the Lentivirus that Expresses both 1α-hydroxylase and RALDH2 (lenti-CYP-ALDH Virus)

Day 0: In the early morning, prepare 5 x 10⁵ cells/mL of 293T cells in the CM-10-D cell culture medium.
Seed 20 mL/plate in 150 mm x 25 mm culture dishes. Culture the cells at 37 °C and 5% CO₂ for 24 h. Confluency will reach ~80–90% after 24 h.
Day 1: Make 2x HBS (50 mM HEPES, 280 mM NaCl, and 1.5 mM Na₂HPO₄, pH 7.1). Aliquot and store at -20 °C.
Make 2 M CaCl₂ and store at room temperature.
For each 150 mm x 25 mm culture dish, prepare a DNA precipitation mixture in a sterile 50 mL culture tube. First, add 1,620 µL of 2x HBS, 9.5 µg of PMD2G (VSVG), 17.5 µg of pCMVR8.74 (Capsid), 27 µg of Lenti-CYP-ALDH plasmid (a lentiviral vector that carries both CYP27B1 and ALDH1a2). Second, add H₂O to a final volume of 3,037.5 µL.
In the last, add 202.5 µL of 2 M CaCl₂ dropwise while mixing the solution to final concentrations of 1x HBS and 125 mM CaCl₂. CaCl₂ must be the last to be added. Leave the transfection mixtures at room temperature for 20 min with occasional mixing. For multiple 150 mm x 25 mm culture dishes, scale up accordingly.
Add the 3,240 µL of DNA precipitation mixture dropwise to one 150 mm x 25 mm culture dish containing the 293T cells. Gently swirl the culture dish side to side while adding the DNA precipitation mixture. Incubate the dish at 37 °C and 5% CO₂ for 24 h.
Day 2: Remove the calcium phosphate transfection solution, wash gently with 1x PBS, and refeed the cell culture with CM-4-D cell culture medium. Incubate the cells at 37 °C and 5% CO₂ for 24 h.
Day 3: Harvest the supernatants in sterile storage bottles and store at 4–8 °C. Replenish the cell culture with fresh CM-4-D cell culture medium. Culture the cells at 37 °C and 5% CO₂ for another 24 h.
Day 4: Harvest the supernatants into sterile storage bottles. Filter the supernatants from Day 3 and Day 4 through a 0.45 µm filter. To concentrate the VSVG pseudotype virus, transfer the supernatants into centrifuge tubes and spin at 4,780 x g for 24 h at 4 °C.
Day 5: Pour the supernatants off the pellets (the pellet is often visible) and allow the tubes to drain on a paper towel in a sterile biosafety cabinet for several minutes.
Resuspend the pellets by pipetting with sterile PBS containing 5% glycerol. The volume for resuspension will be 33.3 µL per 150 mm x 25 mm culture dish.
Aliquot 200 µL/tube and store at -80 °C. Make a separate aliquot of 50 µL/vial for titration purposes. Titers should be within the range of 10⁸–10⁹ transducing units (TUs)/mL.
Add bleach into the culture dishes and discard them.
NOTE: These transfected cells are the biggest safety concern in the protocol since all lentiviral elements are expressed in the cells.

2. Generation of Bone Marrow Derived DCs (BMDCs)

Harvest tibias and femurs from 4–5 Balb/c mice into a 50 mL sterile polypropylene tube containing the culture medium⁶.
Transfer the tibias and femurs into a 100 mm x 20 mm culture dish containing the culture medium. Remove tissues such as muscles from the tibias and femurs using dissecting scissors and forceps.
Cut both ends of the tibias and femurs to expose the bone marrow cavity.
Draw 10 mL culture medium in a 10 mL syringe attached to a 30 G needle.
Carefully insert the needle into the bone marrow cavity and flush the bone marrow into a clean 50 mL sterile polypropylene tube. Repeat this procedure if necessary, to ensure that the bone marrow has been completely flushed out.
Pellet the bone marrow cells by centrifugation (400 x g) at 2–8 °C for 5 min. Aspirate the supernatant.
Resuspend the pellet with 5 mL of 1x red blood cell lysis buffer.
Incubate the cells at room temperature for 4–5 min with occasional shaking.
Stop the reaction by adding 30 mL of culture medium.
Pellet the cells by centrifugation (400 x g) at 2–8 °C for 5 min. Resuspend the cells in 10 mL of CM-10-R cell culture medium. If needed, the cells can be passed through a 40 µm cell strainer to remove debris.
Perform a cell count and adjust the cells to 1 x 10⁶ cells/mL using CM-10-R cell culture medium.
Add recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF, final concentration = 100 U/mL) and murine interleukin-4 (IL-4, final concentration = 10 U/mL).
NOTE: The stock solutions of GM-CSF and IL-4 are stored at -80 °C at 100,000 U/mL (1,000x) and 10,000 U/mL (1,000x), respectively.
Distribute the cells into 6 well culture plates at 4 mL/well and culture the cells at 37 °C and 5% CO₂ for 48 h.
Remove nonadherent cells with gentle pipetting.
Add fresh CM-10-R cell culture medium containing the GM-CSF (100 U/mL) and IL-4 (10 U/mL). Culture the cells for another 48 h.
Harvest nonadherent cells (BMDCs) for transduction by the lenti-CYP-ALDH virus.

3. Transduction of DCs with lenti-CYP-ALDH Virus to Generate DC-CYP-ALDH Cells

Prepare 1 x 10⁶ DCs/well in a total volume of 0.5 mL of CM-10-R cell culture medium containing 50 µL of virus and 8 µg/mL protamine in a 6 well culture plate.
Culture the cells at 37 °C and 5% CO₂ for 24 h.
Replace the medium with fresh CM-10-R cell culture medium and culture the cells at 37 °C and 5% CO₂ for another 24 h. At this time point, the enzymatic activities of 1α-hydroxylase and RALDH2 can be evaluated (see step 4). If necessary, repeat steps 3.1–3.3 for a second transduction.
Add lipopolysaccharide (100 ng/mL) and culture the cells for 24 h.
Harvest the cells (DC-CYP-ALDH cells) for experiments.

4. Evaluation of the Overexpressed 1α-hydroxylase and RALDH2 in DC-CYP-ALDH Cells

Evaluation of the overexpressed 1α-hydroxylase in DC-CYP-ALDH cells
1. Seed 1.0 x 10⁶ DC-CYP-ALDH cells/well in 2 mL of CM-10-R cell culture medium into 12 well culture plates.
2. Add 25(OH)D to the final concentration of 2.5 μM. Incubate the DC-CYP-ALDH cells for 24 h.
3. Harvest the supernatants and store at -20 °C.
4. Assay for 1,25(OH)₂D concentrations in the supernatants using a commercially available radioimmunoassay (RIA) (see Table of Materials).
5. Determine the expression of 1α-hydroxylase in DC-CYP-ALDH cells by fluorescence activated cell sorting (FACS) using an anti-CYP27B1 antibody.
Evaluation of the overexpressed RALDH2 in DC-CYP-ALDH cells
NOTE: The expression and activity of the overexpressed RALDH2 are determined using a commercial aldehyde dehydrogenase (ALDH) detection kit (see Table of Materials).
1. Seed 1 mL of DC-CYP-ALDH or control cells (1 x 10⁵ cells/mL in assay buffer) in each of two 5 mL tubes (one labeled as "Control" and the other labeled as "Test").
2. Add 10 µL of diethylaminobenzaldehyde (DEAB) solution (1.5 mM in 95% ethanol) to each of the control tubes and mix immediately. DEAB is a RALDH2 inhibitor.
3. Add 5 µL of activated RALDH fluorescence substrate (300 µM) to the Control and Test tubes and mix immediately.
4. Incubate the tubes for 45 min.
5. Pellet the cells by centrifugation at 400 x g at 2–8 °C for 5 min. Aspirate the supernatants.
6. Reconstitute the cells in 200 µL of assay buffer.
7. Keep the cells on ice and proceed for analysis by FACS.

Representative Results

DC-CYP-ALDH cells expressed significantly increased amount of 1α-hydroxylase. To determine whether DC-CYP-ALDH cells generated from BMDCs expressed a significantly increased amount of 1α-hydroxylase, BMDCs were transduced with the lenti-CYP-ALDH virus to produce bone-marrow-derived DC-CYP-ALDH cells (BMDC-CYP-ALDH cells). Subsequently, the BMDC-CYP-ALDH cells were examined for the expression of 1α-hydroxylase by FACS. Our data showed that the BMDC-CYP-ALDH cells, when compared to the parental BMDCs, displayed enhanced expression of the 1α-hydroxylase (Figure 1A). We also determined the enzymatic activity of the 1α-hydroxylase in the BMDC-CYP-ALDH cells. To do so, 1.0 x 10⁶ BMDC-CYP-ALDH cells in 2 mL of CM-10-R cell culture medium were added into 12 well culture plates. 25(OH)D was then added to the cell culture at the final concentration of 2.5 μM. The cells were cultured at 37 ˚C and 5% CO₂ for 24 h and the supernatants were harvested for the measurement of 1,25(OH)₂D using a radioimmunoassay (RIA). Our data showed that 1,25(OH)₂D concentrations in the culture supernatants of the BMDC-CYP cells (BMDCs transduced with lenti-CYP-GFP virus) and the BMDC-CYP-ALDH cells were each approximately 20x higher than those of the parental BMDCs and the BMDC-ALDH cells (BMDCs transduced with lenti-ALDH virus) (Figure 1B).

DC-CYP-ALDH cells expressed significantly increased amount of RALDH2. To determine whether BMDC-CYP-ALDH cells expressed a significantly increased amount of RALDH2, a RALDH2 substrate was added into the cell cultures in the presence or absence of the RALDH2 inhibitor diethylaminobenzaldehyde (DEAB) (15 µM). The fluorescent product retained inside the cells was analyzed by FACS. Our data showed that mean fluorescence intensities (MFIs) of the BMDC-CYP-ALDH cells were approximately 6x higher than those of the parental BMDCs, suggesting that the BMDC-CYP-ALDH cells, when compared to the parental BMDCs, expressed significantly enhanced RALDH2 enzymatic activity (Figure 2A,B).

DC-CYP-ALDH cells augmented the induction of foxp3⁺CCR9⁺ gut-homing Treg cells in vitro. To investigate whether DC-CYP-ALDH cells were able to augment the induction of gut-homing Treg cells in vitro, we transduced DC2.4 cells (a bone marrow-derived DC line²⁶^,²⁷^,²⁸^,²⁹), with lenti-CYP-ALDH virus and generated DC2.4-CYP-ALDH cells. Subsequently, we determined whether the DC2.4-CYP-ALDH cells were able to augment the induction of gut-homing Treg cells in vitro. Accordingly, naive CD4⁺ T cells were purified from C57BL/6 mice. Purified naive CD4⁺ T cells at 5 x 10⁵ cells/well were then cocultured with either the parental DC2.4 cells (1 x 10⁵ cells/well) or the DC2.4-CYP-ALDH cells (1 x 10⁵ cells/well) in 24 well culture plates in a serum-free medium in the presence of an anti-CD3 monoclonal antibody (5 µg/mL) and recombinant human IL-2 (50 U/mL). In addition, 25(OH)D and retinol at various concentrations were also added into the cultures. The cells were incubated at 37 ˚C and 5% CO₂. Five days later, the cells were analyzed by FACS for the expressions of foxp3 and c-c chemokine receptor type 9 (CCR9). Our data showed that in the presence of the substrates, the DC2.4 cells did not significantly change the abundance of foxp3⁺CCR9⁺ cells in the CD4⁺ T cell populations (Figure 3A,B). In contrast, the DC2.4-CYP-ALDH cells significantly enhanced the abundance of foxp3⁺CCR9⁺ cells among CD4⁺ T cells. In addition, the more 25(OH)D added, the greater the ability of the DC2.4-CYP-ALDH cells to increase the abundance of foxp3⁺CCR9⁺ cells among CD4⁺ T cells. Therefore, our data support that the DC-CYP-ALDH cells can augment the induction of gut-homing Treg cells in vitro.

DC-CYP-ALDH cells augmented the induction of foxp3⁺CCR9⁺ gut-homing Treg cells in vivo. To determine whether DC-CYP-ALDH cells could augment the induction of gut-homing Treg cells in vivo, we intraperitoneally administered one of the following cells into Balb/c mice: the parental DC2.4 cells, the DC2.4-CYP cells (DC2.4 cells transduced with lenti-CYP-GFP virus), and the DC-2.4-CYP-ALDH cells. Four days after the cell administration, mesenteric lymph nodes were examined by FACS (Figure 4A). Our data showed that the DC2.4-CYP-ALDH cells, when compared to the controls, significantly increased the abundance of foxp3⁺CCR9⁺ cells among CD3⁺ T cells (Figure 4B,C). Based on these results, we conclude that the DC-CYP-ALDH cell administration significantly augments the induction of foxp3⁺CCR9⁺ T cells in peripheral lymphoid tissues.

Figure 1: DC-CYP-ALDH cells expressed significantly increased amount of 1α-hydroxylase. (A) BMDC-CYP-ALDH cells were generated and analyzed by FACS. A representative FACS plot shows the expression of 1α-hydroxylase in the parental BMDCs and the BMDC-CYP-ALDH cells (gated on live cells). (B) 1α-hydroxylase substrate (i.e., 25(OH)D) was added into the DC cultures. 24 h later, the supernatants were collected and 1,25(OH)₂D concentrations were measured. The data show concentrations of 1,25(OH)₂D in the cultures of the parental BMDCs, the BMDC-CYP cells, the BMDC-ALDH cells, and the BMDC-CYP-ALDH cells. **p < 0.01. ANOVA test. n = 4. This figure is adapted from Xu et al.⁶. Copyright 2019. The American Association of Immunologist, Inc. Please click here to view a larger version of this figure.

Figure 2: DC-CYP-ALDH cells expressed significantly increased amount of RALDH2. (A) BMDC-CYP-ALDH cells were generated and analyzed as described in the protocol. Representative overlaid FACS plots show the BODIPY aminoacetate fluorescence in the BMDCs and the BMDC-CYP-ALDH cells in the presence (+DEAB) or absence (-DEAB) of the RALHD2 inhibitor diethylaminobenzaldehyde (DEAB). (B) Mean fluorescence intensities (MFIs) of BODIPY aminoacetate in the BMDCs and the BMDC-CYP-ALDH cells in the absence of DEAB. *p < 0.05; t-test; n = 4. This figure is adapted from Xu et al.⁶. Copyright 2019. The American Association of Immunologist, Inc. Please click here to view a larger version of this figure.

Figure 3: DC-CYP-ALDH cells augmented the induction of foxp3⁺CCR9⁺ gut-homing Treg cells in vitro. (A) CD4⁺ naive T cells were isolated from C57BL/6 mouse spleens. The CD4⁺ T cells were then activated in cultures by an anti-CD3 mAb (5 µg/mL) in the presence of either the parental DC2.4 cells or the DC2.4-CYP-ALDH cells. Additionally, the cultures were added with the indicated concentrations of 25(OH)D and retinol. Five days later, the cells were collected and analyzed by FACS for the expressions of foxp3 and CCR9 in CD3⁺CD4⁺ T cell population. Representative FACS plots show the expressions of foxp3 and CCR9 in the CD3⁺CD4⁺ T cell populations. (B) Cumulative data from (A). *p < 0.05; ANOVA test; n = 4. This figure is adapted from Xu et al.⁶. Copyright 2019. The American Association of Immunologist, Inc. Please click here to view a larger version of this figure.

Figure 4: DC-CYP-ALDH cells augmented the induction of foxp3⁺CCR9⁺ gut-homing Treg cells in vivo. (A) Balb/c mice intraperitoneally (i.p.) received one of the following DC transfers (Transfer): no DC transfer (No transfer), parental DC2.4 cells, DC2.4-CYP cells, and DC2.4-CYP-ALDH cells. Four days later, mesenteric lymph nodes (MLNs) were analyzed by FACS. (B) Representative FACS plots show the expressions of foxp3 and CCR9 in CD3⁺ T cell population. (C) Cumulative data from (B) show the percentage of foxp3⁺CCR9⁺ cells in the CD3⁺ T cell population. Cells were gated on CD3⁺ T cells for all the analyses. Where applicable, the data presented are means ± SEM. *p < 0.05; ANOVA test; n = 4–6. This figure is adapted from Xu et al.⁶. Copyright 2019. The American Association of Immunologist, Inc. Please click here to view a larger version of this figure.

Discussion

In this article we describe the use of DC-CYP-ALDH cells, for augmenting the induction of gut-homing Treg cells in peripheral lymphoid tissues. Our data have shown that the DC-CYP-ALDH cells can synthesize locally high concentrations de novo of both 1,25(OH)₂D and RA in vitro in the presence of corresponding substrates (i.e., 25[OH]D and retinol, respectively). Because sufficient blood concentrations of 25(OH)D and retinol can be easily achieved through vitamin D and A supplementations respectively in patients who have deficiencies³⁰^,³¹, we reason that the DC-CYP-ALDH cells can augment the induction of gut-homing Treg cells in peripheral lymphoid tissues when normal blood concentrations of 25(OH)D and retinol are present. To support this reasoning, our data demonstrates that in normal healthy animals that do not have vitamin D and vitamin A deficiencies, the DC-CYP-ALDH cells augment the induction of Treg cells that express both regulatory molecules (i.e., foxp3 and IL-10) and a gut-homing receptor (i.e., CCR9). Therefore, this technology can be used for further investigation of gut-homing Treg cells for the treatment of IBD.

One critical step of this protocol is the production of lenti-CYP-ALDH virus with high titers. The preferred virus titers should be 10⁸–10⁹ TUs/mL. A high titer of the lenti-CYP-ALDH virus is necessary for a high transduction efficiency in DCs.

Another critical step of this protocol is the transduction efficiency in DCs. Because DC-CYP-ALDH cells are not tolerized in vitro in this technology, it is essential that the transduction rate be more than 90% to ensure that the DC-CYP-ALDH cells can efficiently augment the induction of gut-homing Treg cells in vivo. In addition, the DC-CYP-ALDH cells can be further purified by FACS before in vivo administration³².

A unique advantage of this protocol is that the DC-CYP-ALDH cells do not need to be tolerized in vitro before in vivo administration. It is expected that the DC-CYP-ALDH cells, as a result of the combined actions of 1,25(OH)₂D and RA, will be maintained in a tolerogenic status in vivo because both 1,25(OH)₂D and RA have been shown to tolerize DCs³³^,³⁴. Therefore, we anticipate that the DC-CYP-ALDH cells will not have instability concerns in an in vivo proinflammatory environment.

Currently, we have only demonstrated that DC-CYP-ALDH cells can increase the frequency (number) of gut-homing Treg cells in peripheral lymphoid tissues and intestines⁶. Consequently, regulatory function in the intestines as a whole is enhanced because the percent of Treg cells in the intestines is increased. Figure 4 shows that when compared to those with control treatments, intraperitoneal treatment with DC-CYP-ALDH cells significantly increased the percentage of CCR9⁺foxp3⁺ Treg cells in the mesenteric lymph nodes, meaning that more Treg cells in the mesenteric lymph nodes are able to specifically home into the intestinal tissues. Figure 4 further shows that most foxp3⁺ T cells in mesenteric lymph nodes are negative for CCR9 and therefore do not have gut-homing capacity. However, whether DC-CYP-ALDH cells can also enhance the regulatory function of each Treg cell per se (such as enhanced expression levels of foxp3 and/or IL-10) requires further investigation.

The reagents and materials described here are for animal studies only. However, the protocol is applicable for human studies by using corresponding human reagents and materials, except that DCs will be generated from peripheral blood monocytes in humans. The ultimate goal of this protocol is the generation of clinical grade DC-CYP-ALDH cells for the treatment of IBD.

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program under Award No. W81XWH-15-1-0240 (XT). Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This work was also partially supported by Research Innovation Grants from the Department of Medicine at Loma Linda University (681207-2967 [XT and GG], 681205-2967 [XT], and 325491 [DJB]).

Materials

10 mL syringes	ThermoFisher Scientific	Cat# 03-377-23
100 mm x 20 mm culture dishes	Sigma-Aldrich	Cat# CLS430167
12-well culture plates	ThermoFisher Scientific	Cat# 07-200-82
150 mm x 25 mm culture dishes	Sigma-Aldrich	Cat# CLS430559
25-hydroxycholecalciferol (25[OH]D)	Sigma-Aldrich	Cat# H4014
293T cells	ATCC	CRL-3216
2-mercaptoethanol	ThermoFisher Scientific	Cat#: 21985023
6-well culture plates	ThermoFisher Scientific	Cat# 07-200-83
ALDEFLUOR kit	Stemcell Technologies	Cat# 01700
Anti-CYP27B1	Abcam	Cat# ab95047
BD FACSAria II	BD Biosciences	N/A
CaCl2	Sigma-Aldrich	Cat# C1016
CM-10-D cell culture medium			DMEM medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.
CM-10-R cell culture medium			RPMI 1640 medium (no glutamine) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.
CM-4-D cell culture medium			DMEM medium containing 4% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 0.055 mM 2-mercaptoethanol (2-ME), 1 mM sodium pyruvate, 0.1 mM nonessential amino acid, and 2 mM L-glutamine.
Corning bottle-top vacuum filters, 0.22 mM, 500 mL	Sigma-Aldrich	Cat# CLS430513
Corning bottle-top vacuum filters, 0.45 mM, 500 mL	Sigma-Aldrich	Cat# CLS430514
Dissecting scissor	ThermoFisher Scientific	Cat# 08-940
DMEM medium	ThermoFisher Scientific	Cat# 11960044
Fetal bovine serum	ThermoFisher Scientific	Cat# 16000044
Forceps	ThermoFisher Scientific	Cat# 22-327379
Gibco ACK lysing buffer	ThermoFisher Scientific	Cat# A1049201
Glycerol	Sigma-Aldrich	Cat# G5516
Goat anti-rabbit IgG	Abcam	Cat# ab205718
HEPES	Millipore	Cat# 391340
Lenti-CYP-ALDH	Custom-made	1.6-kb mouse CYP27B1 and ALDH1a2 cDNAs were amplified by PCR using a plasmid containing the CYP27B1 cDNA and a plasmid containing the ALDH1a2 cDNA respectively (GeneCopoeia). The amplified CYP27B1 cDNA fragment with a 5' KOZAK ribosome entry sequence was cloned into the pRRL-SIN.cPPt.PGKGFP.WPRE lentiviral vector (Addgene). The resulting construct was designated as lenti-CYP-GFP. The amplified ALDH1a2 cDNA fragment was cloned into the lenti-CYP-GFP to replace the GFP and was designated as lenti-CYP-ALDH. This bicistronic plasmid expresses CYP27B1 controlled by SFFV promoter and ALDH1a2 controlled by PGK promoter.
L-glutamine	ThermoFisher Scientific	Cat#25030081
Lipopolysaccharide	Sigma-Aldrich	Cat# L3755
Murine GM-CSF	Peprotech	Cat# 315-03
Murine IL-4	Peprotech	Cat# 214-14
Na2HPO4	Sigma-Aldrich	Cat# NIST2186II
NaCl	Sigma-Aldrich	Cat# S9888
Needles	ThermoFisher Scientific	Cat# 14-841-02
Nonessential Amino Acids	ThermoFisher Scientific	Cat#: 11140076
pCMVR8.74	Addgene	Plasmid# 22036
Penicillin/Streptomycin	ThermoFisher Scientific	Cat#15140148
Phoshate Balanced Solution (PBS)	ThermoFisher Scientific	Cat#: 20012027
PMD2G	Addgene	Plasmid# 12259
Polypropylene tube, 15 mL	ThermoFisher Scientific	Cat# AM12500
Polypropylene tube, 50 mL	ThermoFisher Scientific	Cat# AM12502
Protamine sulfate	Sigma-Aldrich	Cat# P3369
Rabbit polycloncal IgG isotype control	Abcam	Cat# ab171870
Radioimmunoassay for 1,25(OH)₂D measurement	Heartland Assays
RPMI 1640 medium, no glutamine	ThermoFisher Scientific	Cat# 21870076
Sodium pyruvat	ThermoFisher Scientific	Cat#: 11360070
Sorvall Legend XTR Centrifuge	ThermoFisher Scientific	Cat# 75004521
Sterile Cell strainers, 40 mm	ThermoFisher Scientific	Cat# 07-201-430
Sterile storage bottles, 500 mL	ThermoFisher Scientific	Cat# CLS431432

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Bi, H., Wasnik, S., Baylink, D. J., Liu, C., Tang, X. In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction. J. Vis. Exp. (155), e60585, doi:10.3791/60585 (2020).