Name: adoptive immunotherapy of inkt cells in glucose-6-phosphate isomerase (g6pi)-induced ra mice
Uploaded: 2020-01-31T12:30:00
Description: Watch this Scientific Journal Video about Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice at JoVE.com

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...

<ol>
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D., Ulloa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+neuroimunomodulation+to+bioelectronic+treatment+of+rheumatoid+arthritis.">From neuroimunomodulation to bioelectronic treatment of rheumatoid arthritis.</a> Bioelectronics in Medicine. 1 (2), 151-165 (2018).</li><li>Brennan, P. J., Brigl, M., Brenner, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+natural+killer+T+cells:+an+innate+activation+scheme+linked+to+diverse+effector+functions.">Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions.</a> Nature Reviews Immunology. 13 (2), 101-117 (2013).</li><li>Bianca, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+Natural+Killer+T-Cells+Development.">Unraveling Natural Killer T-Cells Development.</a> Frontiers in Immunology. 8, 1950 (2018).</li><li>Mitsuo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+CD161+CD8++T+cells+in+the+peripheral+blood+of+patients+suffering+from+rheumatic+diseases.">Decreased CD161+CD8+ T cells in the peripheral blood of patients suffering from rheumatic diseases.</a> Rheumatology. 45 (12), 1477-1484 (2006).</li><li>Miellot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+invariant+NK+T+cells+protects+against+experimental+rheumatoid+arthritis+by+an+IL-10-dependent+pathway.">Activation of invariant NK T cells protects against experimental rheumatoid arthritis by an IL-10-dependent pathway.</a> European Journal of Immunology. 35 (12), 3704-3713 (2005).</li><li>Miellot-Gafsou, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+activation+of+invariant+natural+killer+T+cells+in+a+rheumatoid+arthritis+model+and+application+to+disease+treatment.">Early activation of invariant natural killer T cells in a rheumatoid arthritis model and application to disease treatment.</a> Immunology. 130 (2), 296-306 (2010).</li><li>Tudhope, S. J., Delwig, A. V., Falconer, J., Pratt, A., Ng, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+invariant+natural+killer+t-cell+deficiency+in+inflammatory+arthritis.">Profound invariant natural killer t-cell deficiency in inflammatory arthritis.</a> Annals of the Rheumatic Diseases. 69 (10), 1873-1879 (2010).</li><li>Ming, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+on+immunoregulation+of+iNKT+cells+in+RA+by+novel+synthetic+immunostimulator+CH1b.">Effects on immunoregulation of iNKT cells in RA by novel synthetic immunostimulator CH1b.</a> Chinese Journal of Immunology. 32 (02), 218-222 (2016).</li><li>Ming, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+correlation+between+the+percentage+of+iNKT+cells+and+the+ratio+of+IFN-&#947;/IL-4+in+patients+with+rheumatoid+arthritis.">Study of the correlation between the percentage of iNKT cells and the ratio of IFN-&#947;/IL-4 in patients with rheumatoid arthritis.</a> Chinese Journal of Microbiology Immunology. 35 (3), 213-218 (2015).</li><li>Sharif, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+natural+killer+T+cells+by+&#945;-galactosylceramide+treatment+prevents+the+onset+and+recurrence+of+autoimmune+Type+1+diabetes.">Activation of natural killer T cells by &#945;-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes.</a> Nature Medicine. 7, 1057-1062 (2010).</li><li>Gapin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+invariant+natural+killer+T+cells.">Development of invariant natural killer T cells.</a> Current Opinion in Immunology. 39, 68-74 (2016).</li><li>Kwon, D. I., Lee, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+Differentiation+Program+of+Invariant+Natural+Killer+T+Cells.">Lineage Differentiation Program of Invariant Natural Killer T Cells.</a> Immune Network. 17 (6), (2017).</li><li>Thapa, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differentiation+of+ROR-&#947;t+expressing+iNKT17+cells+is+orchestrated+by+Runx1.">The differentiation of ROR-&#947;t expressing iNKT17 cells is orchestrated by Runx1.</a> Scientific Reports. 7 (1), 7018 (2017).</li><li>Schurgers, E., Billiau, A., Matthys, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-induced+arthritis+as+an+animal+model+for+rheumatoid+arthritis:+focuson+interferon-&#947;.">Collagen-induced arthritis as an animal model for rheumatoid arthritis: focuson interferon-&#947;.</a> Interferon Cytokine Research. 31 (12), 917-926 (2011).</li><li>Van Haalen, H. G. M., Severens, J. L., Tran-Duy, A., Boonen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+cost-effectiveness+A+systematic+review+and+stepwise+approach+for+selecting+a+transferable+health+economic+evaluation+rheumatoid+arthritis.">How cost-effectiveness A systematic review and stepwise approach for selecting a transferable health economic evaluation rheumatoid arthritis.</a> Pharmacoeconomics. 32 (5), 429-442 (2014).</li><li>Schubert, D., Maier, B., Morawietz, L., Krenn, V., Kamradt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+glucose-6-phosphate+isomerase+induces+T+cell-dependent+peripheral+polyarthritis+in+genetically+unaltered+mice.">Immunization with glucose-6-phosphate isomerase induces T cell-dependent peripheral polyarthritis in genetically unaltered mice.</a> Journal of Immunology. 172, 4503-4509 (2004).</li><li>Bockermann, R., Schubert, D., Kamradt, T., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a+B-cell-dependent+chronic+arthritis+with+glucose-6-phosphate+isomerase.">Induction of a B-cell-dependent chronic arthritis with glucose-6-phosphate isomerase.</a> Arthritis Research, Therapy. 7, 131613-131624 (2005).</li><li>Kamradt, T., Schubert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+clinical+implications+of+G6PI+in+experimental+models+of+rheumatoid+arthritis.">The role and clinical implications of G6PI in experimental models of rheumatoid arthritis.</a> Arthritis Research, Therapy. 7, 20-28 (2005).</li><li>Horikoshi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Invariant+NKT+Cells+with+Glycolipid+Ligand+&#945;-Galactosylceramide+Ameliorates+Glucose-6-Phosphate+Isomerase+Peptide-Induced+Arthritis.">Activation of Invariant NKT Cells with Glycolipid Ligand &#945;-Galactosylceramide Ameliorates Glucose-6-Phosphate Isomerase Peptide-Induced Arthritis.</a> PlosOne. 7 (12), 51215 (2012).</li><li>Zhang, X. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+mixed+peptides+derived+from+glucose-6-phosphate+isomerase+induces+rheumatoid+arthritis+in+DBA+/1+mice.">Immunization with mixed peptides derived from glucose-6-phosphate isomerase induces rheumatoid arthritis in DBA /1 mice.</a> Chinese Journal of Pathophysiology. 32 (3), 569-576 (2016).</li><li>Motohashi, S., Nakayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+natural+killer+T+cell-based+immunotherapy+for+cancer.">Invariant natural killer T cell-based immunotherapy for cancer.</a> Immunotherapy. 1 (1), 73 (2017).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+of+natural+killer+T-cells+in+tolerogenic+APCs-mediated+suppression+of+collagen-induced+arthritis.">The requirement of natural killer T-cells in tolerogenic APCs-mediated suppression of collagen-induced arthritis.</a> Experimental and Molecular Medicine. 42 (8), 547-554 (2010).</li><li>Luc, V. K., Lan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+Potential+of+Invariant+Natural+Killer+T+Cells+in+Autoimmunity.">Therapeutic Potential of Invariant Natural Killer T Cells in Autoimmunity.</a> Frontiers in Immunology. 9, 519-526 (2018).</li><li>Chiba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+collagen-induced+arthritis+by+natural+killer+T+cell+activation+with+OCH,+a+sphingosine-truncated+analog+of+&#945;-galactosylceramide.">Suppression of collagen-induced arthritis by natural killer T cell activation with OCH, a sphingosine-truncated analog of &#945;-galactosylceramide.</a> Arthritis, Rheumatism. 50 (1), 305-313 (2004).</li><li>Tudhope, S. J., Delwig, A. V., Falconer, J., Pratt, A., Ng, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profound+invariant+natural+killer+t-cell+deficiency+in+inflammatory+arthritis.">Profound invariant natural killer t-cell deficiency in inflammatory arthritis.</a> Annals of the Rheumatic Diseases. 69 (10), 1873-1879 (2010).</li><li>Bruns, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+an+immunodominant+self-peptide+derived+from+glucose-6-phosphate+isomerase+induces+arthritis+in+DBA/1+mice.">Immunization with an immunodominant self-peptide derived from glucose-6-phosphate isomerase induces arthritis in DBA/1 mice.</a> Arthritis Research, Therapy. 11 (4), (2009).</li><li>Parietti, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rituximab+treatment+overcomes+reduction+of+regulatory+iNKT+cells+in+patients+with+rheumatoid+arthritis.">Rituximab treatment overcomes reduction of regulatory iNKT cells in patients with rheumatoid arthritis.</a> Clinical Immunology. 134 (3), 331-339 (2010).</li><li>Yoshida, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+mechanism(s)+of+the+inhibition+of+disease+progression+by+combination+treatment+with+fingolimod+plus+pathogenic+antigen+in+a+glucose-6-phosphate+isomerase+peptide-induced+arthritis+mouse+model.">Functional mechanism(s) of the inhibition of disease progression by combination treatment with fingolimod plus pathogenic antigen in a glucose-6-phosphate isomerase peptide-induced arthritis mouse model.</a> Biological, Pharmaceutical Bulletin. 38 (8), 1120-1125 (2015).</li><li>Chen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+adoptive+immunotherapy+on+rheumatoid+arthritis+with+Thymus-derived+invariant+natural+killer+T+cells.">Study of the adoptive immunotherapy on rheumatoid arthritis with Thymus-derived invariant natural killer T cells.</a> International Immunopharmacology. 67, 427-440 (2019).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#174; 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 &#946; 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&#39;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-&#978;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 ...

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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 ...

Our protocol describes a method to build an RA model. Its success rate is over 93 percent and its pathogenesis is depends on T cells, B cells, and innate immunity, which allows researchers to better study the pathogenesis of RA from a global immune perspective. Then we use INKT cells with immunomodulatory effects for their treatment.INKT cells have good effect on the treatment by in vivo introduction which provide basic data support for the clinical application of INKT cells and truly explodes the clinical application value of INKT cells. The greatest advantage of this technology is that it is simple to operate and easy to copy. Demonstrating the procedure will be Chen Shengde, Gao Xiang, and Zhang Jingnan, graduate student from my laboratory.To begin, weigh 1.75 milligrams of both HGPI325 to 339 and HGPI469 to 483 fragments and dissolve them in 5.25 milliliters of 4 degrees Celsius triple distilled water. Dissolve Complete Freund's Adjuvant in a 50 degree Celsius water bath. Draw 5.25 milliliters into another 10 milliliter centrifuge tube and cool it for use.Put the mixture of HG6PI solution and Complete Freund's Adjuvant solution in an artificial emulsification unit with two glass syringes, connected. In an ice bath, push the syringe at a constant speed and frequency of ten to twenty times per minute to completely emulsify the mixture peptide solution and complete Freund's Adjuvant solution. Then, keep the emulsion droplets in the water for ten minutes.Next, inject 150 microliters of the emulsified HG6PIs into the mouse's tail root subcutaneously. Immediately and after 48 hours, inject 200 milligrams of pertussis toxin into the mouse intraperineally. Now, inject normal DBA1 mouse intraperineally without the gausser at the dosage of 0.1 milligrams per kilogram of body weight.Three days after modeling, after anesthetization, isolate the spleen of the mouse. Prepare a single cell suspension by cutting and grinding the spleen in a 200 mesh sieve. Wash the cell suspension with PBS.Centrifuge at 200 times g for five minutes and discard the supernatant. Re suspend the cells with one milliliter of whole blood tissue solution. Add three milliliters of mouse lymphocyte separation medium.And then centrifuge the cells for 20 minutes at 300 times g at room temperature. To purify the INKT cells, resuspend 10 to the seventh cells with 100 microliters of four degree Celsius PBS, add 10 microliter of alpha gausser loaded CD1 tetramer PE and incubate them at 4 degrees Celsius for fifteen minutes in the dark. Wash the cells twice with PBS and resuspend them in 80 microliters of PBS.Add 20 microliters of anti-PE microbeads and incubate them at four degrees Celsius for twenty minutes in the dark. Wash them twice with PBS and resuspend the cells with 500 microliters of PBS. Place the sorting column in the magnetic field of the MACS sorter and rinse with 500 microliters of PBS.Add the prepared cell suspension to the sorting column, collect the flow through, and rinse three times with PBS buffer. Remove the magnetic field and add one milliliter of PBS buffer to the sorting column. Quickly push the plunger at a constant pressure to drive the labeled cells into the collection tube.And obtain the purified INKT cells. Count with an automated cell counter. To identify the INKT cell phenotype, first, take one times ten to the sixth cells from the purified INKT cells and resuspend them in 50 microliters of PBS.Add 0.5 microliters of alpha gausser PE CD1 tetramer, or 10 microliters of FITC TCR beta in the single positive control tube. Add 0.5 microliters of alpha gausser PE CD1 tetramer, Add 10 microliters of FITC TCR beta in the sample tube. Incubate them at four degrees Celsius for thirty minutes in the dark.After that, wash the cells in PBS and then centrifuge at 200 times g for five minutes. Discard the supernatant and add one milliliter of FoxP3 fixation permeabilization working solution. And incubate the cells for 45 minutes at four degrees Celsius in the dark.Then, add one milliliter of 1x permeabilization working solution and centrifuge the cells at 500 times g at room temperature for five minutes. Discard the supernatant. Add one microliter of AlexaFluor 647 mouse anti-PLZF and one microliter of PerCP Cy5.5 mouse anti-Tbet and incubate for thirty minutes at room temperature in the dark.Next, add two microliters of permeabilization buffer working solution and centrifuge at 200 times g for five minutes for cleaning. Discard the supernatant. Resuspend the cells in 500 microliters of PBS and measure by flow cytometry.In this study, compared with the control group, the toes of the RA model group began to show red swelling after modeling with gradual aggravation. At 14 days, the red swelling in the ankle joint peaked followed by gradual relief. The pathological results show that the infiltration degree of inflammatory cells in the ankle synovial tissue of the RA model mice was different at different stages.Peak inflammation occurred on day 14 post modeling. In the RA model group, the serum levels of pro-inflammatory cytokines significantly increased 11 days and 14 days after modeling. While anti-inflammatory cytokines significantly decreased.This figure shows the rate of INKT2 in normal mice was around five percent. The rate of INK2 was about 82 percent after in vivo induction. The rate of INK2 was more than 92 percent after MACS purification.Here we show a method to emulsify the major polypeptide we see in RA.The emulsified droplets are kept in the water for 10 minutes. Results is dispersing. This protocol is a new add-on for the treatment of RA raised special NKT cells.Which can be applied towards cell immunotherapy or other autoimmune disease and tumors. This model can stimulate the overall proliferation of CD4 T cells and NKT cells deficits in RA patients, which lays the foundation for in depth study of RA immune pathways. In this protocol, pertussis toxin is injected the interperitoneally during modeling.Please wear experimental clothes and disposable gloves. And carry all the standards of operation so as to prevent stab wound an infections.

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