Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Podsumowanie

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4⁺ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Streszczenie

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-α, IFN-γ, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Wprowadzenie

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, progressive invasiveness with 0.5–1% incidence^1,2. The underlying pathogenesis is attributed to the abnormal proliferation of autoreactive CD4⁺ and CD8⁺ T cells, manifested by an increase in the proportion of CD4⁺IFN-γ⁺ and CD4⁺IL-17A⁺ T cells, and the reduced number of CD4⁺IL-4⁺ and CD4⁺CD25⁺FoxP3⁺ T cells. Therefore, the secretion of inflammatory cytokines is increased, and an excessive inflammatory reaction destroys the native balance and tolerance function of the body's immune system. Moreover, the helper T lymphocyte (Th) 1 cells that penetrate the joint aggravate the inflammatory response and joint damage. Therefore, the inhibition of excessive inflammatory response and restoration of immune tolerance and immune balance are key to the treatment of RA^3,4.

The iNKT cells have both NK cell and T cell functions and characteristics. The iNKT cells harbor a distinct, invariant T cell receptor (TCR) α-chain with limited TCR β-chain repertoires⁵ and recognize the glycolipid antigen presented by the major histocompatibility complex (MHC) class I molecule CD1d on the surface of the antigen-presenting cells. Mitsuo et al.⁶ detected a large number of iNKT cells and functional defects in many autoimmune diseases, including RA. Aurore et al.⁷ demonstrated that iNKT cells have a positive effect on maintaining autoimmune tolerance and that when the number and function of iNKT cells are restored, the disease is alleviated. In addition, Miellot-Gafsou et al.⁸ found that iNKT cells not only abrogated the disease but also increased the progression of the disease. These contradictory results suggest that iNKT cells are heterogeneous T cells, and the function of different subsets may be reversed. In a clinical study of RA, the frequency of iNKT cells correlated with the score of the disease activity⁹. The results also confirmed that the frequency of iNKT was decreased in RA patients, the number of CD4⁺IFN-γ⁺ T cell subsets increased, and the secretory levels of inflammatory cytokines IFN-γ and TNF-α increased^10,11. In addition, Sharif et al.¹² investigated type 1 diabetes (T1D) and found that selective infusion of iNKT cells upregulated the expression of the inflammatory cytokine IL-4, maintained immune tolerance, and prevented the development of type 1 diabetes. Therefore, adoptive infusion of specific iNKT cells or targeted activation of iNKT cells increases the level of iNKT cells in RA patients, which can be a breakthrough in RA treatment.

Cellular immunotherapy is currently of great interest and has been widely used in cancer therapy. However, iNKT cells are rare, heterogeneous immunoregulatory cells (only 0.3% of the total number of PBMCs)¹³, which limits potential clinical applications. These cells are mainly divided into three subpopulations: 1) iNKT1 cells, which have a high expression of promyelocytic leukemia zinc-finger protein (PLZF) and T-box transcription factor (T-bet); 2) iNKT2 cells with intermediate expression of PLZF and GATA binding protein 3 (GATA3); 3) iNKT17 cells with low expression of PLZF and retinoid-related orphan nuclear receptor (ROR)-γt that secrete IFN-γ, IL-4, and IL-17¹⁴. Activated iNKT cells secrete Th1, Th2, and Th17-like cytokines, which determine the different immunomodulatory effects of iNKT cells¹⁵. The immunomodulatory and immunotherapeutic effects of specific activation of various subpopulations of iNKT cells are different. Therefore, the selection of specific phenotypes of iNKT cells (mainly iNKT2) with anti-inflammatory functions to regulate the immune response of the body can correct the immune imbalance and immune disorders in RA.

The establishment of an ideal animal model is of great significance for the treatment and study of RA pathogenesis. Presently, the most commonly used and mature animal models include collagen-induced arthritis, adjuvant arthritis, zymosan-induced arthritis, and polysaccharide-induced arthritis^16–17. However, there is no model that can fully simulate all the features of human RA. Type II collagen-induced arthritis (CIA) is a classic arthritis model. The CIA is induced by immunization of mice with type II collagen-specific monoclonal antibodies, reflecting the antibody dependence of this disease model. Benurs et al. described a model with a systemic immune response to glucose-6-phosphate isomerase (G6PI), which induces peripheral symmetric polyarthritis in susceptible mouse strains^18,19. In this model, the development of arthritis depends on T cells, B cells, and innate immunity^18,19,20. Horikoshi²¹ found that RA models resulting from immunization of DBA/1 mice with G6PI polypeptide fragments are more similar to human RA in terms of CD4⁺ T cells and cytokines (i.e., IL-6 and TNF-α) than the CIA models. In order to increase the stimulating effect on the TCR recognition site, the mixed polypeptide fragments of G6PI (hGPI325-339 and hGPI469-483) were used to immunize DBA/1 mice to construct the RA mouse model. The success rate of this approach can high because hGPI325-339 and hGPI469-483 are immunodominant for I-A q-restricted T cell responses. Therefore, this model can simulate the overproliferation of CD4⁺ T cells and iNKT cell defects in RA patients²². The basic research of RA immunopathology laid the foundation for our further in-depth investigation.

Protokół

All experimental mice (150 in total) were healthy male DBA/1 mice, 6–8 weeks old (20.0 ± 1.5 g), reared in a specific pathogen-free (SPF) environment. There is no special treatment before modeling. The experiment was divided into a healthy control group (15 mice), a model control group (15 mice), and a cell therapy group (55 mice). This study was approved by the Animal Welfare and Ethical Committee of Hebei University.

1. Constructing the Disease Model

1. Duplicating the RA animal model
   1. Weigh 1.75 mg of both hG6PI 325-339 and hG6PI 469-483 fragments and dissolve them in 5.25 mL of 4 °C triple distilled water.
   2. Dissolve complete Freund's adjuvant (CFA) in a 50 °C water bath, draw 5.25 mL into another 10 mL centrifuge tube, and cool it for use.
   3. Put the mixture of hG6PI solution and CFA solution in an artificial emulsification unit with two glass syringes connected.
   4. Push the syringe at a constant speed and frequency of 10–20x per min to completely emulsify the mixed peptide solution and CFA solution. Perform the operation in an ice bath, and keep the emulsion droplets in the water for 10 min after the completion of the emulsification without dispersing.
   5. Inject 150 µL of emulsified hG6PIs into the mouse's tail root subcutaneously.
   6. Inject 200 mg of Pertussis toxin into the mouse intraperitoneally at 0 h and 48 h after the hG6PI injection.
2. Experimental verification of the RA model
   1. Measure the thickness of the mouse's paw with a Vernier caliper (2x a day).
   2. Observe and mark the degree of redness and swelling of the foot. Use the following scoring criteria: 1) toes with mild swelling; 2) dorsum pedis and foot pad with clear red swelling; 3) ankle with red swelling.
   3. Euthanize the mice under deep anesthesia by intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg body weight) 14 days after modeling and remove the paws for HE staining.
   4. Determine the secretion levels of serum IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and IFN-γ using a commercial cytometric bead array (CBA) assay according to the manufacturer's protocol.

2. Obtaining iNKT Cells with Adoptive Cellular Therapy

1. Directional induction of iNKT cells
   1. Inject normal mice intraperitoneally with α-GalCer (0.1 mg/kg of body weight).
2. Isolation of iNKT cells
   1. Three days after modeling, inject mice intraperitoneally with 1% sodium pentobarbital (50 mg/kg of body weight) for anesthetization. Adequately anesthetized mice do not show a hind paw withdrawal response to a toe pinch.
   2. Isolate the spleen of a DBA/1 mouse after injecting it intraperitoneally with α-GalCer. Prepare a single cell suspension by cutting and grinding the spleen in a 200 mesh sieve.
   3. Wash the cell suspension with PBS, centrifuge at 200 x g for 5 min, and discard the supernatant. Repeat.
   4. Resuspend the cells with 1 mL of whole blood and tissue dilution solution. Add 3 mL of mouse lymphocyte separation medium, and then centrifuge the cells for 20 min at 300 x g at room temperature.
   5. Collect the layer of milky white lymphocytes (i.e., the second layer from the top), wash it 2x with PBS, and count with an automated cell counter.
3. Magnetic activated cell sorting (MACS)positive selection strategy for purification of iNKT cells
   NOTE: For the pretreatment of CD1d tetramers, 1 mg/mL of α-Galcer was diluted to 200 μg/mL with 0.5% of Tween-20 and 0.9% of NaCl, and 5 µL of the resulting solution was added to 100 µL of the CD1d tetramer solution. The mixture was incubated for 12 h at room temperature and placed at 4 °C for use. TCR β was diluted 80x with deionized water. All other antibodies were used as a stock solution.
   1. Resuspend 10⁷ cells with 100 µL of 4 °C PBS, add 10 µL of α-GalCer-loaded CD1d Tetramer-PE, and incubate them at 4 °C for 15 min in the dark.
   2. Wash the cells 2x with PBS and resuspend them in 80 µL of PBS.
   3. Add 20 µL of anti-PE-MicroBeads and incubate them at 4 °C for 20 min in the dark.
   4. Wash them 2x with PBS and resuspend the cells with 500 µL of PBS.
   5. Place the sorting column in the magnetic field of the MACS sorter and rinse with 500 µL of PBS.
   6. Add the cell suspension from step 2.3.4 to the sorting column, collect the flowthrough, and rinse 3x with PBS buffer.
   7. Remove the magnetic field and collect the cells from the sorting column. At this point, add 1 mL of PBS buffer to the sorting column, and quickly push the plunger at a constant pressure to drive the labeled cells to the collection tube and obtain purified iNKT cells. Count with an automated cell counter.
4. Identification of the iNKT cell phenotype
   1. Take 1 x 10⁶ cells from steps 2.2.5 and 2.3.7, respectively, and resuspend them in 50 µL of PBS.
   2. Antibody incubation: Do not add the antibody to the negative control tube, add 0.5 µL of α-GalCer-PE-CD1d Tetramer or 10 µL of FITC-TCR β in the single positive control tube. Add 0.5 µL of α-GalCer-PE-CD1d tetramer and 10 µL of FITC-TCR β in the sample tube. Incubate them at 4 °C for 30 min in the dark.
   3. Wash the cells in PBS and then centrifuge at 200 x g for 5 min.
   4. Discard the supernatant, add 1 mL of Foxp3 Foxation/Permeabilization working solution, and incubate the cells for 45 min at 4 °C in the dark.
   5. Add 1 mL of 1x Permeabilization Buffer working solution and centrifuge the cells at room temperature for 500 x g at room temperature for 5 min.
   6. Discard the supernatant. Add 1 μL of Alexa Fluor 647 mouse Anti-PLZF and 1 μL of PerCP-Cy 5.5 mouse anti-T-bet (or 1 μL of PerCP-Cy 5.5 mouse anti-RORΥt) for 30 min at room temperature in the dark.
   7. Add to 2 mL of Permeabilization Buffer working solution for cleaning.
   8. Discard the supernatant, resuspend the cells in 500 µL of PBS, and measure by flow cytometry.
5. Functional identification of iNKT cells
   1. Take 3 x 10⁶ iNKT cells from step 2.3.7 and resuspend them in 12 well plates with 1.5 mL of RPMI-1640 incomplete medium (i.e., without serum).
   2. Add phorbol ester (PMA, 50 ng/mL) and ionomycin calcium (IO, 1 μg/mL) and place in a CO₂ incubator for 24 h.
   3. Collect the cell supernatant and detect the secretion levels of IL-2, IL-17A, TNF-α, IL-6, IL-4, IFN-γ, and IL-10 using a commercial CBA assay according to the manufacturer's protocol.
6. Experimental study on the migration pathway of iNKT cells in RA mice
   1. Dissolve DiR dye (2.5 mg/mL) in DMSO.
   2. Resuspend iNKT cells in 6 well plates with RPMI-1640 incomplete medium. The density is 1 × 10⁶ cells/mL.
   3. Add DiR (5 μg/mL) solution and incubate in a CO₂ incubator for 25 min.
   4. Wash with PBS and resuspend the cells (3 x 10⁶/300 µL) to obtain DiR-labeled iNKT cells (DiR-iNKT).
   5. Inject 1% sodium pentobarbital (50 mg/kg body weight) intraperitoneally to anesthetize the mice. Adequately anesthetized mice do not show a hind paw withdrawal to toe pinch. Apply veterinary ointment to the mouse eyes to prevent dryness while under anesthesia for imaging.
   6. Inject DiR-iNKT cells 3 x 10⁶ per mouse into the tail vein with the RA model for 8 days. Monitor the iNKT cells in mice postinjection for 0 min, 10 min, 30 min, 60 min, and day 0 (after 3 h), 1, 3, 6, 12, 26, 34, 38, and 42 days using a small animal in vivo imaging system (IVIS). The excitation wavelength used was 748 nm, the emission wavelength was 780 nm, and the exposure time was automatic.
   7. Place each mouse in a separate cage after each observation and maintain sternal recumbency. Observe until recovery from anesthesia.

3. Evaluation of Adoptive Immunotherapy of RA Mice with iNKT Cells

1. iNKT cell adoptive immunotherapy for RA mice
   1. Inject 3 x 10⁶ cells iNKT cells per mouse through the tail vein. Randomly select 15 mice that were modeled 8 days prior and obtain iNKT cells without DiR labeling from step 2.3.7 by the tail vein infusion.
2. Evaluate the efficacy of adoptive immunotherapy for iNKT cells.
   1. Measure the thickness of the paw of the mouse, quantify the swelling of the ankle joint, and systematically score after the infusion of iNKT cells as described in steps 1.2.1–1.2.2.
   2. Observe the inflammatory cell infiltration and joint changes of the mouse joints as described in step 1.2.3.
   3. Determine the secretion levels of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and IFN-γ as described in step 1.2.4.
3. Determine the frequency of iNKT cells and subsets.
   1. Isolate the mouse thymus and prepare a single cell suspension.
   2. Separate the lymphocytes with lymphocyte separation fluid.
   3. Determine the iNKT cell frequency and subgroup frequency as described in step 2.4.
4. Statistical analysis
   NOTE: All data are presented as mean ± SD. Values of P < 0.05 were considered statistically significant.
   1. Use one-factor analysis of variance (ANOVA). If the variance is satisfied, use the LSD test for further comparison.
   2. If the variance is not uniform, use the nonparametric test. Use the Kruskal-Wallis H test for further comparison³¹.

Wyniki

The arthritis index score and paw thickness increased after modeling. Compared with the control group, the toes of the RA model group began to show red swelling at 6 days after modeling, with gradual aggravation. At 14 days, the red swelling in the ankle joint peaked, followed by gradual relief. The thickness of the paw changed similarly (P < 0.05) (Figure 1).

The inflammatory cell infiltration increased significantly after modeling. The pathological ...

Dyskusje

iNKT cells are special T cells that bridge innate and adaptive immunity and are mainly developed from CD4⁺⁺/CD8⁺ thymocytes. iNKT cells have diverse immunoregulatory functions and interact with other immune cells by direct contact and secretion of different cytokines²³, affecting dendritic cells (DCs), macrophages, neutrophils, B cells, T cells, and NK cell differentiation and development²⁴. α-GalCer...

Materiały

Name	Company	Catalog Number	Comments
Alexa Fluor 647 Mouse Anti-PLZF	BD	563490	America
Anti-PE MicroBeads	Miltenyi	130-048-801	Germany
Columns	Miltenyi	MS	Germany
Cryogenic Centrifuge	Beckman	Allegra® X-15R	America
DiR	Thermo Fisher Scientific	D12731	America
Embedding Center	Tianjin Aviation Electromechanical Co., Ltd.	BMJ-1	China
FITC Hamster Anti-Mouse TCR β Chain	BD	553170	America
Flow cytometer	BD	Accuri C6	America
Freund's complete adjuvant	Sigma	F5881	America
hGPI325-339 (IWYINCFGCETHAML)	Karebay Biochem	18062202	China
hGPI469-483 (EGNRPTNSIVFTKLT)	Karebay Biochem	18062203	China
In Vivo Imaging System	PerkinElmer	caliper IVIS lumina II	America
Ionomycin Calcium	Cayman	10004974	America
KRN7000	AdipoGen	AG-CN2-0013	America
Mouse CD1d Tetramer-PE	MBL	TS-MCD-1	Japan
Mouse percoll	Solarbio	P8620	China
Optical Microscope	Olympus	Olympus-II	Japan
PerCP-CyTM5.5 Mouse anti-ROR-ϒt	BD	562683	America
PerCP-CyTM5.5 Mouse anti-T-bet	BD	561316	America
Pertussis toxin	Sigma	P7208	America
phorbol esters	Cayman	10008014	America
Red Blood Cell Lysis Buffer	BD	555899	America
RPMI-1640	Biological Industries	01-100-1ACS	Israel
Th1/Th2/Th17 cytokines kit	BD	560485	America
Ultramicrotome	Leica	Leica EM UC6	Germany