Our study was supported by the National Natural Science Foundation of China (NSFC) (81771755), Colleges and university&#39;s science and technology key research project of Hebei province (ZD2017009) and the Animal Lab of Medical Experiment Center, Hebei University. We are grateful for their support.

All experimental mice (150 in total) were healthy male DBA/1 mice, 6&#8211;8 weeks old (20.0 &#177; 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
<ol>
	<li>Duplicating the RA animal model
	<ol>
		<li>Weigh 1.75 mg of both hG6PI 325-339 and hG6PI 469-483 fragments and dissolve them in 5.25 mL of 4 &#176;C triple distilled water.</li>
		<li>Dissolve complete Freund&#39;s adjuvant (CFA) in a 50 &#176;C water bath, draw 5.25 mL into another 10 mL centrifuge tube, and cool it for use.</li>
		<li>Put the mixture of hG6PI solution and CFA solution in an artificial emulsification unit with two glass syringes connected.</li>
		<li>Push the syringe at a constant speed and frequency of 10&#8211;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.</li>
		<li>Inject 150 &#181;L of emulsified hG6PIs into the mouse&#39;s tail root subcutaneously.</li>
		<li>Inject 200 mg of Pertussis toxin into the mouse intraperitoneally at 0 h and 48 h after the hG6PI injection.</li>
	</ol>
	</li>
	<li>Experimental verification of the RA model
	<ol>
		<li>Measure the thickness of the mouse&#39;s paw with a Vernier caliper (2x a day).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Determine the secretion levels of serum IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-&#945;, and IFN-&#947; using a commercial cytometric bead array (CBA) assay according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>
2. Obtaining iNKT Cells with Adoptive Cellular Therapy
<ol>
	<li>Directional induction of iNKT cells
	<ol>
		<li>Inject normal mice intraperitoneally with &#945;-GalCer (0.1 mg/kg of body weight).</li>
	</ol>
	</li>
	<li>Isolation of iNKT cells
	<ol>
		<li>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.</li>
		<li>Isolate the spleen of a DBA/1 mouse after injecting it intraperitoneally with &#945;-GalCer. Prepare a single cell suspension by cutting and grinding the spleen in a 200 mesh sieve.</li>
		<li>Wash the cell suspension with PBS, centrifuge at 200 x g for 5 min, and discard the supernatant. Repeat.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Magnetic activated cell sorting (MACS)positive&#8194;selection&#8194;strategy for purification of iNKT cells 
	NOTE: For the pretreatment of CD1d tetramers, 1 mg/mL of &#945;-Galcer was diluted to 200 &#956;g/mL with 0.5% of Tween-20 and 0.9% of NaCl, and 5 &#181;L of the resulting solution was added to 100 &#181;L of the CD1d tetramer solution. The mixture was incubated for 12 h at room temperature and placed at 4 &#176;C for use. TCR &#946; was diluted 80x with deionized water. All other antibodies were used as a stock solution.
	<ol>
		<li>Resuspend 107 cells with 100 &#181;L of 4 &#176;C PBS, add 10 &#181;L of &#945;-GalCer-loaded CD1d Tetramer-PE, and incubate them at 4 &#176;C for 15 min in the dark.</li>
		<li>Wash the cells 2x with PBS and resuspend them in 80 &#181;L of PBS.</li>
		<li>Add 20 &#181;L of anti-PE-MicroBeads and incubate them at 4 &#176;C for 20 min in the dark.</li>
		<li>Wash them 2x with PBS and resuspend the cells with 500 &#181;L of PBS.</li>
		<li>Place the sorting column in the magnetic field of the MACS sorter and rinse with 500 &#181;L of PBS.</li>
		<li>Add the cell suspension from step 2.3.4 to the sorting column, collect the flowthrough, and rinse 3x with PBS buffer.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Identification of the iNKT cell phenotype
	<ol>
		<li>Take 1 x 106 cells from steps 2.2.5 and 2.3.7, respectively, and resuspend them in 50 &#181;L of PBS.</li>
		<li>Antibody incubation: Do not add the antibody to the negative control tube, add 0.5 &#181;L of &#945;-GalCer-PE-CD1d Tetramer or 10 &#181;L of FITC-TCR &#946; in the single positive control tube. Add 0.5 &#181;L of &#945;-GalCer-PE-CD1d tetramer and 10 &#181;L of FITC-TCR &#946; in the sample tube. Incubate them at 4 &#176;C for 30 min in the dark.</li>
		<li>Wash the cells in PBS and then centrifuge at 200 x g for 5 min.</li>
		<li>Discard the supernatant, add 1 mL of Foxp3 Foxation/Permeabilization working solution, and incubate the cells for 45 min at 4 &#176;C in the dark.</li>
		<li>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.</li>
		<li>Discard the supernatant. Add 1 &#956;L of Alexa Fluor 647 mouse Anti-PLZF and 1 &#956;L of PerCP-Cy 5.5 mouse anti-T-bet (or 1 &#956;L of PerCP-Cy 5.5 mouse anti-ROR&#933;t) for 30 min at room temperature in the dark.</li>
		<li>Add to 2 mL of Permeabilization Buffer working solution for cleaning.</li>
		<li>Discard the supernatant, resuspend the cells in 500 &#181;L of PBS, and measure by flow cytometry.</li>
	</ol>
	</li>
	<li>Functional identification of iNKT cells
	<ol>
		<li>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).</li>
		<li>Add phorbol ester (PMA, 50 ng/mL) and ionomycin calcium (IO, 1 &#956;g/mL) and place in a CO2 incubator for 24 h.</li>
		<li>Collect the cell supernatant and detect the secretion levels of IL-2, IL-17A, TNF-&#945;, IL-6, IL-4, IFN-&#947;, and IL-10 using a commercial CBA assay according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Experimental study on the migration pathway of iNKT cells in RA mice
	<ol>
		<li>Dissolve DiR dye (2.5 mg/mL) in DMSO.</li>
		<li>Resuspend iNKT cells in 6 well plates with RPMI-1640 incomplete medium. The density is 1 &#215; 106 cells/mL.</li>
		<li>Add DiR (5 &#956;g/mL) solution and incubate in a CO2 incubator for 25 min.</li>
		<li>Wash with PBS and resuspend the cells (3 x 106/300 &#181;L) to obtain DiR-labeled iNKT cells (DiR-iNKT).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Place each mouse in a separate cage after each observation and maintain sternal recumbency. Observe until recovery from anesthesia.</li>
	</ol>
	</li>
</ol>
3. Evaluation of Adoptive Immunotherapy of RA Mice with iNKT Cells
<ol>
	<li>iNKT cell adoptive immunotherapy for RA mice
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Evaluate the efficacy of adoptive immunotherapy for iNKT cells.
	<ol>
		<li>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&#8211;1.2.2.</li>
		<li>Observe the inflammatory cell infiltration and joint changes of the mouse joints as described in step 1.2.3.</li>
		<li>Determine the secretion levels of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-&#945;, and IFN-&#947; as described in step 1.2.4.</li>
	</ol>
	</li>
	<li>Determine the frequency of iNKT cells and subsets.
	<ol>
		<li>Isolate the mouse thymus and prepare a single cell suspension.</li>
		<li>Separate the lymphocytes with lymphocyte separation fluid.</li>
		<li>Determine the iNKT cell frequency and subgroup frequency as described in step 2.4.</li>
	</ol>
	</li>
	<li>Statistical analysis 
	NOTE: All data are presented as mean &#177; SD. Values of P &#60; 0.05 were considered statistically significant.
	<ol>
		<li>Use one-factor analysis of variance (ANOVA). If the variance is satisfied, use the LSD test for further comparison.</li>
		<li>If the variance is not uniform, use the nonparametric test. Use the Kruskal-Wallis H test for further comparison31.</li>
	</ol>
	</li>
</ol>

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The authors declare no funding or conflicts of interest.

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. &#945;-GalCer...

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, progressive invasiveness with 0.5&#8211;1% incidence1,2. 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-&#947;+ 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&#39;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 RA3,4.
The iNKT cells have both NK cell and T cell functions and characteristics. The iNKT cells harbor a distinct, invariant T cell receptor (TCR) &#945;-chain with limited TCR &#946;-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-&#947;+ T cell subsets increased, and the secretory levels of inflammatory cytokines IFN-&#947; and TNF-&#945; increased10,11. 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)-&#947;t that secrete IFN-&#947;, 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 arthritis16&#8211;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 strains18,19. In this model, the development of arthritis depends on T cells, B cells, and innate immunity18,19,20. 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-&#945;) 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.

<table><tbody><tr><td>Alexa Fluor 647 Mouse Anti-PLZF</td><td>BD</td><td>563490</td><td>America</td></tr><tr><td>Anti-PE MicroBeads</td><td>Miltenyi</td><td>130-048-801</td><td>Germany</td></tr><tr><td>Columns</td><td>Miltenyi</td><td>MS</td><td>Germany</td></tr><tr><td>Cryogenic Centrifuge</td><td>Beckman</td><td>Allegra&amp;reg; X-15R</td><td>America</td></tr><tr><td>DiR</td><td>Thermo Fisher Scientific</td><td>D12731</td><td>America</td></tr><tr><td>Embedding Center</td><td>Tianjin Aviation Electromechanical Co., Ltd.</td><td>BMJ-1</td><td>China</td></tr><tr><td>FITC Hamster Anti-Mouse TCR &amp;beta; Chain</td><td>BD</td><td>553170</td><td>America</td></tr><tr><td>Flow cytometer</td><td>BD</td><td>Accuri C6</td><td>America</td></tr><tr><td>Freund&#039;s complete adjuvant</td><td>Sigma</td><td>F5881</td><td>America</td></tr><tr><td>hGPI325-339 (IWYINCFGCETHAML)</td><td>Karebay Biochem</td><td>18062202</td><td>China</td></tr><tr><td>hGPI469-483 (EGNRPTNSIVFTKLT)</td><td>Karebay Biochem</td><td>18062203</td><td>China</td></tr><tr><td>In Vivo Imaging System</td><td>PerkinElmer</td><td>caliper IVIS lumina II</td><td>America</td></tr><tr><td>Ionomycin Calcium</td><td>Cayman</td><td>10004974</td><td>America</td></tr><tr><td>KRN7000</td><td>AdipoGen</td><td>AG-CN2-0013</td><td>America</td></tr><tr><td>Mouse CD1d Tetramer-PE</td><td>MBL</td><td>TS-MCD-1</td><td>Japan</td></tr><tr><td>Mouse percoll</td><td>Solarbio</td><td>P8620</td><td>China</td></tr><tr><td>Optical Microscope</td><td>Olympus</td><td>Olympus-II</td><td>Japan</td></tr><tr><td>PerCP-CyTM5.5 Mouse anti-ROR-&amp;upsih;t</td><td>BD</td><td>562683</td><td>America</td></tr><tr><td>PerCP-CyTM5.5 Mouse anti-T-bet</td><td>BD</td><td>561316</td><td>America</td></tr><tr><td>Pertussis toxin</td><td>Sigma</td><td>P7208</td><td>America</td></tr><tr><td>phorbol esters</td><td>Cayman</td><td>10008014</td><td>America</td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>BD</td><td>555899</td><td>America</td></tr><tr><td>RPMI-1640</td><td>Biological Industries</td><td>01-100-1ACS</td><td>Israel</td></tr><tr><td>Th1/Th2/Th17 cytokines kit</td><td>BD</td><td>560485</td><td>America</td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>Leica EM UC6</td><td>Germany</td></tr></tbody></table>

adoptive immunotherapy of inkt cells in glucose-6-phosphate isomerase (g6pi)-induced ra mice

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-&#945;, IFN-&#947;, 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.

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 &#60; 0.05) (Figure 1).
The inflammatory cell infiltration increased significantly after modeling. The pathological ...

Watch this Scientific Journal Video about Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice at JoVE.com

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

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.

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

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

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Research

JoVE Journal

Immunology and Infection

6.8K Views.  Medical School of Hebei University. Ons protocol beschrijft een methode om een RA-model te bouwen. Het slagingspercentage is meer dan 93 procent en de pathogenese is afhankelijk van T-cellen, B-cellen en aangeboren immuniteit, waardoor onderzoekers de pathogenese van RA beter kunnen bestuderen vanuit een mondiaal immuunperspectief. Vervolgens gebruiken we INKT-cellen met immunomodulerende effecten voor hun behandeling.INKT cellen hebben een goed effect op de behandeling door in vivo introductie die basisgegevens onderste...

Video: Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Biology

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Injecting Human HSPCs into the Femur of Sub-Lethally Irradiated Mice

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. 
This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.

Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell ...

Isolating Invariant Natural Killer T Cells from a Mouse Model

Source: Meng, M., et al. <a href="https://app.jove.com/v/60048">Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice.</a>&#160;J. Vis. Exp.&#160;(2020)
This video illustrates a method for isolating invariant natural killer T cells, also known as iNKT cells. The procedure begins with injecting a synthetic glycolipid into a mouse to stimulate the activation and proliferation of iNKT cells in the spleen. Subsequently, single-cell suspensions are prepared from the spleen and incubated with iNKT-cell-recognizing complexes labeled with fluorophores, followed by magnetic bead-based separation, to isolate the iNKT cells.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

A Cell-Based Therapy for Autoimmune Arthritis in a Murine Model

Source: Haque, M. et al., <a href="https://app.jove.com/v/54720">Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity.</a>&#160;J. Vis. Exp. (2016)
This video showcases a cell-based therapy for autoimmune arthritis in mice. iPSC-derived T regulatory cells expressing ovalbumin-specific receptors are injected with methylated bovine serum albumin (mBSA) emulsified in an adjuvant. Murine antigen-presenting cells (APCs) process and present the mBSA to T cells, prompting T cell activation and migration to the knee joint&#39;s synovial membrane. Subsequent injections involve mBSA alone in the left knee and a combination of mBSA and ovalbumin in the right knee. In the left knee, the immune response to mBSA triggers arthritis, while in the right knee, T-regulatory cells interacting with ovalbumin mitigate inflammation, improving arthritis symptoms.

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

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