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.

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		<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>
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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 ...

adoptive-immunotherapy-inkt-cells-glucose-6-phosphate-isomerase-g6pi

Research

JoVE Journal

Immunology and Infection

6.7K Views.  Medical School of Hebei University. 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.

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

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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 ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.