This study was supported by the open fund of state key laboratory of Pharmaceutical Biotechnology, Nan-jing University, China (Grant no. KF-GN-201905), the National Natural Science Foundation of China (grants 81971501). William J. Liu is supported by the Excellent Young Scientist Program of the NSFC (81822040) and Beijing New-star Plan of Science and Technology (Z181100006218080).

1. Preparation of expression constructs
<ol>
	<li>Retrieve the sequences of MHC class I genes (including predicted genes) from bats from the NCBI database.</li>
	<li>Retrieve higher mammal MHC I heavy chain sequences from the Immuno Polymorphism Database (IPD) (www.ebi.ac.uk/ipd/mhc) and the UniProt database (www.uniprot.org).</li>
	<li>To obtain soluble MHC complexes, mutagenize the sequences to remove the cytosolic and transmembrane regions.</li>
	<li>Clone the genes encoding the ectodomains of the bat Ptal-N*01:01 (GenBank no. KT98792919) (residues 1&#8211;277) and bat &#946;2m (GenBank no. XP_006920478.1) (residues 1&#8211;98) into pET-28a vectors (Novagen, Beijing, China), respectively.</li>
	<li>Insert the optimized (for Escherichia coli) sequences were inserted between the NcoI and EcoR1 sites of a modified pET-28a vector.</li>
	<li>Construct human &#946;2m expression plasmids as previously described20.</li>
</ol>
2. Peptide synthesis
<ol>
	<li>Peptides prediction
	<ol>
		<li>As described previously10, to screen for peptides that may bind to Ptal-N*01:01, predict candidate peptides using the protein bodies of bat-related viruses hendra virus (HeV). To obtain high affinity peptides based on the structural model from the online server, NetMHCpan 4.0 server (http://www.cbs.dtu.dk/services/NetMHCpan/)21 and Rosetta FlexPepDock22 predicted potential binding in the selected peptide fraction. In addition, other software and databases including BIMAS, the Immune Epitope Database (IEDB), NetCTL 1.2, and SYFPEITHI can also be used, all of them integrating prediction of peptide&#8211;MHC class I binding affinity, transporter associated with antigen processing (TAP) transport efficiency, proteasomal C-terminal cleavage and half-time of dissociation of peptide&#8211;HLA class I molecules in their readouts.</li>
		<li>To predict high-binding peptides, use both the NetMHCpan and Rosetta FlexPepDock server. 
		NOTE: Unfortunately, neither produced predictions matched the experimental data. This may indicate that current MHC binding peptide predictions were not suitable for non-human and non-mouse mammals such as bats, which may have a different manner of peptide binding. Therefore, we need to combine various prediction software and experimental results for comprehensive analysis to obtain high affinity peptides.</li>
	</ol>
	</li>
	<li>Peptides preparation and preservation
	<ol>
		<li>Rather than generating custom peptides, use specialized service providers. Purchase all peptides commercially following peptide prediction tools, which have a higher binding score. The peptide purity was determined to be &#62;95% by HPLC and mass spectrometry.</li>
		<li>Store all purchased peptides as freeze-dried powder at &#8722;80 &#176;C and dissolve in dimethyl sulfoxide (DMSO) before use.</li>
	</ol>
	</li>
</ol>
3. Purification of inclusion bodies
<ol>
	<li>Transformation of E. coli
	<ol>
		<li>Transform 10 ng of plasmid containing bat MHC I Ptal-N*01:01 H chain, or human &#946;2m or bat &#946;2m into 100 &#181;L of BL21(DE3) E. coli. Bathe in ice for 30 min.</li>
		<li>Heat shock at 42 &#176;C for 90 s, followed by bathing in ice for 2 min.</li>
		<li>Add 800 &#181;L of lysogeny broth (LB: yeast extract, tryptone and NaCl; see the Table of Materials) to step 3.1.2 and shake at 200 rotations per minute (rpm) on a rocking platform at 37 &#176;C for 20 min.</li>
		<li>Apply 100 &#181;L of bacterial suspension to the plate containing the corresponding antibiotic resistance (ampicillin: 100 ng/mL), which are same as previous constructs.</li>
	</ol>
	</li>
	<li>Inoculate cultures
	<ol>
		<li>Pick a single recently transformed bacterial clone into 3 mL of LB with antibiotic medium (ampicillin: 100 ng/mL) and culture at 37 &#176;C, while shaking at 200 rpm on a rocking platform to activate the bacteria. Cultures can be inoculated late at night, to be ready for induction the next morning.</li>
		<li>One night in advance, transfer 500 &#181;L of the activated bacterial stock into 50 mL of LB with antibiotic medium (ampicillin: 100 ng/mL) and incubate overnight at 37 &#176;C, shaking at 200 rpm. For convenience when preparing the next inoculate, freeze the remaining activated bacterial stock in 1 mL aliquots (500 &#956;L of culture with 500 &#956;L of 40% glycerol) at &#8722;80 &#176;C until the next preparation is needed.</li>
		<li>To generate large amounts of recombinant protein, transfer the bacterial preparation into 2 L of LB with antibiotic medium (ampicillin: 100 ng/mL) at a ratio of 1:100, incubate at 37 &#176;C while shaking at 200 rpm. Culture until an absorbance of 0.6 at 600 nm is achieved. Add 1 mM isopropyl-1-thio-&#946;-D-galactopyranoside (IPTG) (vary the concentration of IPTG for different MHCs, mostly between 0.1 mM-1 mM) to the flask for induction of protein expression.</li>
	</ol>
	</li>
	<li>Harvest bacteria
	<ol>
		<li>Transfer the bacteria to centrifuge bottles and centrifuge at 5000 x g for 20 min at 4 &#176;C. All steps from now on should be performed at 4 &#176;C.</li>
		<li>Resuspend the bacteria in 60 mL of phosphate buffer saline (PBS) and liberate the expressed recombinant protein by ultrasonic cell disruptor. 
		NOTE: In general, the program we set on this machine is ultrasonic 6S, interval 12S, 300 W, 99 times).</li>
	</ol>
	</li>
	<li>Purification of inclusion bodies
	<ol>
		<li>Centrifuge the sonicated bacterial suspension at 12,000 x g for 30 min. Discard the supernatant and resuspend the pellet in an appropriate volume of washing buffer (0.5% Triton-100, 50 mM Tris pH 8.0, 300 mM NaCl, 10 mM EDTA, 10 mM DTT). Repeat this process once more.</li>
		<li>Centrifuge at 12,000 x g for 10&#8211;20 min. Discard the supernatant and resuspend the inclusion bodies in resuspension buffer (50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA, 10 mM DTT). Remove a 20 &#181;L sample (inclusion bodies in resuspension buffer) for SDS-PAGE to test the purity of inclusion bodies.</li>
		<li>Centrifuge the remaining preparation at 12,000 x g for 10&#8211;20 min. Discard the supernatant. Weigh the pellet containing the inclusion bodies and add dissolution buffer (6 M Gua-HCl, 10% glycerin, 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA) to a final concentration of 30 mg/mL. Stir slowly using a magnetic stirrer at 4 &#176;C until the inclusion bodies are dissolved in the dissolution buffer.</li>
		<li>Centrifuge 12,000 x g for 10&#8211;20 min. Discard the precipitates.&#160;Store the inclusion bodies at &#8722;20 &#176;C or &#8722;80 &#176;C. 
		NOTE: Before proceeding with the purification of inclusion bodies, confirm that the induction and expression of recombinant protein were successful. Run samples from each culture (taken before and after IPTG induction) on a protein gel; 15% SDS-PAGE (SDS-PAGE concentrated gum: H2O, 30% acrylamide, 1 M Tris-HCl pH 6.8, 10% SDS, 10% APS and TEMED; SDS-PAGE separation gel: H2O, 30% acrylamide, 1.5 M Tris-HCl pH 8.8, 10% SDS, 10% APS and TEMED; see the Table of Materials ) for the &#946;2m, 10% SDS-PAGE for the H chain.</li>
	</ol>
	</li>
</ol>
4. Refolding of MHC complex
NOTE: The efficiency of inclusion bodies refolded will affect the yield of protein obtained. Folding competes with polymerization, so it is generally accepted that refolding at low protein concentrations is the most successful method. In this paper, the inclusion bodies concentration is 30 mg/mL.
<ol>
	<li>Prepare refolding buffer.
	<ol>
		<li>Vary the composition of the refolding buffer depending on the protein. For example, the pH, ionic strength, redox conditions and the presence of ligands will affect the refolding results. The most common is to use Tris or HEPES-based buffers at a neutral pH with the NaCl concentration of 50-500 mM, but this also depends on the target protein. The following is the refolding buffer formula used in this article.</li>
		<li>Prepare the folding buffer with 100 mM Tris-HCl pH 8.0, 400 mM L-arginine, 2 mM EDTA-2Na.</li>
		<li>Cool the buffer to 4 &#176;C in a 250&#8211;300 mL flask, and then add 5 mM reduced glutathione (GSH) and 0.5 mM oxidized glutathione (GSSG). Stir slowly using a magnetic stirrer at 4 &#176;C for a further 10&#8211;20 min before adding the inclusion bodies and peptides.</li>
	</ol>
	</li>
	<li>Injection and dilution of MHC H chain and &#946;2m 
	NOTE: The temperature at which refolding is performed may vary, although generally, in order minimize aggregation, 4 &#176;C is best. We use dilution to refold the proteins.
	<ol>
		<li>Using a needle from a 1 mL syringe, inject 1 mL of h&#946;2m inclusion bodies or b&#946;2m inclusion bodies into two refolding buffers (1 liter each), respectively. Inject near the vigorously rotating stirring rod to obtain fast and efficient dilution. &#946;2m refolds relatively easy and remains stable even in the absence of the H chain.</li>
		<li>After the &#946;2m has been dissolved in refolding solution, dissolve peptides (5 mg/mL) in DMSO and quickly inject 200 &#181;L into the refolding solution. Stir slowly for 10&#8211;20 min before adding the H chain.</li>
		<li>Inject the H chain with the same procedure described above for &#946;2m. The H chain is very unstable; therefore, the order of injection is quite important. Inject 3 mL of H chain inclusion bodies into two different refolding buffers (1 liter each) with h&#946;2m or b&#946;2m, respectively. The consecutive injection of small aliquots instead of the single application of the whole amount of protein increases the yield of refolded MHC. Allow refolding to proceed at 4 &#176;C for 8&#8211;10 h.</li>
	</ol>
	</li>
	<li>Concentration of refolded protein
	<ol>
		<li>Use ultrafiltration in a pressurized chamber with 10 kDa MMCO membrane for concentration of the refolding proteins. This method is convenient and can be combined with buffer exchange. Add exchange buffer (20 mM Tris-HCl, 50 mM NaCl pH 8.0) to the chamber and concentrate to a final volume of 30&#8211;50 mL.</li>
		<li>Transfer the refolding solution to a centrifuge tube, spin at 12,000 x g for 15 min at 4 &#176;C to remove precipitates.</li>
		<li>Carefully transfer the supernatant and concentrate further to a final volume of ~1 mL.</li>
		<li>Centrifuge at 12,000 xg for 10&#8211;20 min. Transfer the supernatant to a sterile tube and purify the proteins using a 10/300 GL size-exclusion column.</li>
		<li>Collect the samples at the peak and analyze them using SDS&#8211;PAGE (15% polyacrylamide gel).</li>
		<li>Collect the MHC complex peak and concentrate to a final concentration of 15 mg/mL.</li>
		<li>Dilute the complex to 7.5 mg/mL and 15 mg/mL for crystallization.</li>
	</ol>
	</li>
</ol>
5. Crystallization, data collection, and processing
<ol>
	<li>Perform crystallization of complexed MHC and peptide using the sitting drop vapor diffusion technique.</li>
	<li>Screen the Ptal-N*01:01/peptide complexes with commercial crystal kits (e.g., Crystal Screen kit I/II, Index Screen kit, PEGIon kit I/II, and the PEGRx kit).</li>
	<li>Seal the resulting solution and equilibrate against 100 &#181;L of reservoir solution at 4 or 18 &#176;C.</li>
	<li>Observe the crystal growth in culture for 3 days, 1 week, 2 weeks, 1 month, 3 months and 6 months. Use a microscope to see if each drop in the crystal plate has crystals. 
	NOTE: Ptal-N*01:01/HeV1(b&#946;2m) crystals were observed in 0.2 M NaCl, 0.1 M Bis-Tris (pH 5.5), and 25% (w/v) polyethylene glycol 3,350 at a concentration of 7.5 mg/mL. Ptal-N*01:01/HeV1(h&#946;2m) crystals were observed in 0.1 M HEPES, pH 7.0, 2% w/v polyethylene glycol 3,350 at a concentration of 7.5 mg/mL.</li>
	<li>For cryogenic protection, transfer the crystals to a storage solution containing 20% glycerol and then cool rapidly in a 100 K gaseous nitrogen stream.</li>
	<li>Collect X-ray diffraction data from the beamline BL19U of the Shanghai synchrotron radiation facility (Shanghai, China).</li>
</ol>
6. Structure determination and analyses
<ol>
	<li>With high-resolution structural data, process and scale the strength of the collection using the Denzo program and the HKL2000 software package&#160;(https://hkl-xray.com/)23.</li>
	<li>After choosing a better initial model in the Protein Data Bank (PDB), determine the structures using molecular replacement with the program Phaser MR in CCP424 (http://www.ccp4.ac.uk/). The model used was the structure coordinates with Protein Data Bank (PDB) code 5F1I.</li>
	<li>Use the refined X-ray model for the initial joint refinement. Use Phenix refine alone and joint modes for the X-ray alone and the joint neutron and X-ray refinement, respectively.</li>
	<li>After every round of refinement, manually check the model against Fo &#8722; Fc and 2Fo &#8722; Fc positive nuclear density maps in Coot25 (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/). This facilitates the proper placement of waters and certain D atoms. All figures of structures were created by PyMOL (http://www.pymol.org/).</li>
</ol>

<ol>
	<li>Vyas, J. M., Van der Veen, A. G., Ploegh, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+known+unknowns+of+antigen+processing+and+presentation.">The known unknowns of antigen processing and presentation.</a> Nature Reviews Immunology. 8 (8), 607-618 (2008).</li><li>Bjorkman, P. 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=Structure+of+the+human+class+I+histocompatibility+antigen,+HLA-A2.">Structure of the human class I histocompatibility antigen, HLA-A2.</a> Nature. 329 (6139), 506-512 (1987).</li><li>Seong, R. H., Clayberger, C. A., Krensky, A. M., Parnes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+Daudi+cell+HLA+expression+by+transfection+of+the+mouse+beta+2-microglobulin+gene.">Rescue of Daudi cell HLA expression by transfection of the mouse beta 2-microglobulin gene.</a> Journal of Experimental Medicine. 167 (2), 288-299 (1988).</li><li>Zijlstra, 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=Beta+2-microglobulin+deficient+mice+lack+CD4-8++cytolytic+T+cells.">Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells.</a> Nature. 344 (6268), 742-746 (1990).</li><li>Gao, G. F., 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=Crystal+structure+of+the+complex+between+human+CD8alpha(alpha)+and+HLA-A2.">Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2.</a> Nature. 387 (6633), 630-634 (1997).</li><li>Bjorkman, P. J., Parham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function,+and+diversity+of+class+I+major+histocompatibility+complex+molecules.">Structure, function, and diversity of class I major histocompatibility complex molecules.</a> Annual Review of Biochemistry. 59, 253-288 (1990).</li><li>Achour, 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=Structural+basis+of+the+differential+stability+and+receptor+specificity+of+H-2Db+in+complex+with+murine+versus+human+beta+2-microglobulin.">Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta 2-microglobulin.</a> Journal of Molecular Biology. 356 (2), 382-396 (2006).</li><li>Kubota, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+serum+beta+2-microglobulin+with+H-2+class+I+heavy+chains+on+the+surface+of+mouse+cells+in+culture.">Association of serum beta 2-microglobulin with H-2 class I heavy chains on the surface of mouse cells in culture.</a> Journal of Immunology. 133 (6), 3203-3210 (1984).</li><li>Bernabeu, C., van de Rijn, M., Lerch, P. G., Terhorst, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+2-microglobulin+from+serum+associates+with+MHC+class+I+antigens+on+the+surface+of+cultured+cells.">Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells.</a> Nature. 308 (5960), 642-645 (1984).</li><li>Lu, 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=Peptide+presentation+by+bat+MHC+class+I+provides+new+insight+into+the+antiviral+immunity+of+bats.">Peptide presentation by bat MHC class I provides new insight into the antiviral immunity of bats.</a> PLoS Biology. 17 (9), 3000436 (2019).</li><li>Wu, 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=Structural+basis+of+diverse+peptide+accommodation+by+the+rhesus+macaque+MHC+class+I+molecule+Mamu-B*17:+insights+into+immune+protection+from+simian+immunodeficiency+virus.">Structural basis of diverse peptide accommodation by the rhesus macaque MHC class I molecule Mamu-B*17: insights into immune protection from simian immunodeficiency virus.</a> Journal of Immunology. 187 (12), 6382-6392 (2011).</li><li>Chu, F., 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=First+glimpse+of+the+peptide+presentation+by+rhesus+macaque+MHC+class+I:+crystal+structures+of+Mamu-A*01+complexed+with+two+immunogenic+SIV+epitopes+and+insights+into+CTL+escape.">First glimpse of the peptide presentation by rhesus macaque MHC class I: crystal structures of Mamu-A*01 complexed with two immunogenic SIV epitopes and insights into CTL escape.</a> Journal of Immunology. 178 (2), 944-952 (2007).</li><li>Liu, 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=Diverse+peptide+presentation+of+rhesus+macaque+major+histocompatibility+complex+class+I+Mamu-A*02+revealed+by+two+peptide+complex+structures+and+insights+into+immune+escape+of+simian+immunodeficiency+virus.">Diverse peptide presentation of rhesus macaque major histocompatibility complex class I Mamu-A*02 revealed by two peptide complex structures and insights into immune escape of simian immunodeficiency virus.</a> Journal of Virology. 85 (14), 7372-7383 (2011).</li><li>Liu, W. 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=Protective+T+cell+responses+featured+by+concordant+recognition+of+Middle+East+respiratory+syndrome+coronavirus-derived+CD8++T+cell+epitopes+and+host+MHC.">Protective T cell responses featured by concordant recognition of Middle East respiratory syndrome coronavirus-derived CD8+ T cell epitopes and host MHC.</a> Journal of Immunology. 198 (2), 873-882 (2017).</li><li>Mitaksov, V., Fremont, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+definition+of+the+H-2Kd+peptide-binding+motif.">Structural definition of the H-2Kd peptide-binding motif.</a> Journal of Biological Chemistry. 281 (15), 10618-10625 (2006).</li><li>Xiao, 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=Diversified+anchoring+features+the+peptide+presentation+of+DLA-88*50801:+first+structural+insight+into+domestic+dog+MHC+class+I.">Diversified anchoring features the peptide presentation of DLA-88*50801: first structural insight into domestic dog MHC class I.</a> Journal of Immunology. 197 (6), 2306-2315 (2016).</li><li>Li, X., 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=Two+distinct+conformations+of+a+rinderpest+virus+epitope+presented+by+bovine+major+histocompatibility+complex+class+I+N*01801:+a+host+strategy+to+present+featured+peptides.">Two distinct conformations of a rinderpest virus epitope presented by bovine major histocompatibility complex class I N*01801: a host strategy to present featured peptides.</a> Journal of Virology. 85 (12), 6038-6048 (2011).</li><li>Yao, 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=Structural+illumination+of+equine+MHC+class+I+molecules+highlights+unconventional+epitope+presentation+manner+that+is+evolved+in+equine+leukocyte+antigen+alleles.">Structural illumination of equine MHC class I molecules highlights unconventional epitope presentation manner that is evolved in equine leukocyte antigen alleles.</a> Journal of Immunology. 196 (4), 1943-1954 (2016).</li><li>Wynne, J. W., 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=Characterization+of+the+antigen+processing+machinery+and+endogenous+peptide+presentation+of+a+bat+MHC+class+I+molecule.">Characterization of the antigen processing machinery and endogenous peptide presentation of a bat MHC class I molecule.</a> Journal of Immunology. 196 (11), 4468-4476 (2016).</li><li>Zhang, 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=Structural+basis+of+cross-allele+presentation+by+HLA-A*0301+and+HLA-A*1101+revealed+by+two+HIV-derived+peptide+complexes.">Structural basis of cross-allele presentation by HLA-A*0301 and HLA-A*1101 revealed by two HIV-derived peptide complexes.</a> Molecular Immunology. 49 (1-2), 395-401 (2011).</li><li>Hoof, I., 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=NetMHCpan,+a+method+for+MHC+class+I+binding+prediction+beyond+humans.">NetMHCpan, a method for MHC class I binding prediction beyond humans.</a> Immunogenetics. 61 (1), 1-13 (2009).</li><li>Raveh, B., London, N., Zimmerman, L., Schueler-Furman, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosetta++FlexPepDock+ab-initio:+simultaneous+folding,+docking+and+refinement+of+peptides+onto+their+receptors.">Rosetta FlexPepDock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors.</a> PLoS One. 6 (4), 18934 (2011).</li><li>Otwinowski, Z., Minor, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+X-ray+diffraction+data+collected+in+oscillation+mode.">Processing of X-ray diffraction data collected in oscillation mode.</a> Methods in Enzymology. 276, 307-326 (1997).</li><li>Brunger, A. T., 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=Crystallography+&amp;+NMR+system:+A+new+software+suite+for+macromolecular+structure+determination.">Crystallography &amp; NMR system: A new software suite for macromolecular structure determination.</a> Acta Crystallographica Section D: Biological Crystallography. 54, 905-921 (1998).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallographica Section D: Biological Crystallography. 66, 486-501 (2010).</li><li>Glithero, 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=Crystal+structures+of+two+H-2Db/glycopeptide+complexes+suggest+a+molecular+basis+for+CTL+cross-reactivity.">Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.</a> Immunity. 10 (1), 63-74 (1999).</li><li>Tungatt, K., 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=Induction+of+influenza-specific+local+CD8+T-cells+in+the+respiratory+tract+after+aerosol+delivery+of+vaccine+antigen+or+virus+in+the+Babraham+inbred+pig.">Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig.</a> PLoS Pathogens. 14 (5), 1007017 (2018).</li><li>McCoy, W. H. t., Wang, X., Yokoyama, W. M., Hansen, T. H., Fremont, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanism+of+ER+retrieval+of+MHC+class+I+by+cowpox.">Structural mechanism of ER retrieval of MHC class I by cowpox.</a> PLoS Biology. 10 (11), 1001432 (2012).</li><li>Altman, J. 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=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274 (5284), 94-96 (1996).</li><li>Zhao, 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=Heterosubtypic+protections+against+human-infecting+avian+influenza+viruses+correlate+to+biased+cross-T-cell+responses.">Heterosubtypic protections against human-infecting avian influenza viruses correlate to biased cross-T-cell responses.</a> mBio. 9 (4), (2018).</li><li>Zhao, L., Cao, 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=Engineered+T+cell+therapy+for+cancer+in+the+clinic.">Engineered T cell therapy for cancer in the clinic.</a> Search Results. 10, 2250 (2019).</li><li>Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Higgins, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CLUSTAL_X+windows+interface:+flexible+strategies+for+multiple+sequence+alignment+aided+by+quality+analysis+tools.">The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.</a> Nucleic Acids Research. 25 (24), 4876-4882 (1997).</li><li>Gouet, P., Robert, X., Courcelle, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESPript/ENDscript:+Extracting+and+rendering+sequence+and+3D+information+from+atomic+structures+of+proteins.">ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins.</a> Nucleic Acids Research. 31 (13), 3320-3323 (2003).</li></ol>

The authors have nothing to declare.

The construction of a hybrid protein complex through heterologous substitution from different taxa is a common strategy for functional and structural investigations when the homologous complex is not available, such as in the MHC I and its ligands. However, there is limited summarization regarding the methodology and the technology. Herein, the structure of bat MHC I, Ptal-N*01:01, stabilized by b&#946;2m or h&#946;2m was analyzed. The key amino acids of &#946;2m binding to Ptal-N*01:01 w...

The major histocompatibility complex (MHC) exists in all vertebrates and is a set of genes that determines the cell-mediated immunity to infectious pathogens. MHC class I presents endogenous peptides, such as viral components produced upon virus infection, to T cell receptors (TCR) on the surface of CD8+ T cells to mediate cellular immunity and participate in immune regulation1. A structural study of MHC I binding to peptides provides information regarding peptide binding motifs and presentation features by MHC I molecules, which plays vital roles in evaluation of CD8+ T cell immune responses and vaccine development.
Since the first crystallization and structural determination of MHC I molecular by Bjorkman et al.2, the crystal structure analysis of MHC I molecules has greatly promoted the understanding of how peptides bind to MHC I molecules, and helps to understand the interaction of light chains with heavy chains and peptides. A series of follow-up studies indicated that although the genes encoding the light chain is not associated with the MHC, the light chain is a key protein for the assembly of MHC I molecules3,4. It interacts with the three domains of MHC class I molecules on multiple surfaces. When the light chain is absent, MHC class I molecules cannot be correctly expressed on the surface of antigen-presenting cells and cannot interact with TCR to exert their immunological functions.
MHC I is comprised of a heavy chain (H chain) and light chain (i.e., &#946;2-microglobulin (&#946;2m)), and is assembled through binding to a suitable peptide5. The extracellular segment of the H chain consists of &#945;1, &#945;2 and &#945;3 domains6. The &#945;1 and &#945;2 domains form the peptide binding groove (PBG). The &#946;2m chain acts as a structural subunit of the assembly complex in MHC I, stabilizing the conformation of the complex, and is a molecular chaperone for MHC I H chain folding7,8,9. A series of studies have shown that MHC I H chains from various mammals such as bat (Chiroptera) (Ptal-N*01:01)10, rhesus macaque (Primates) (Mamu-B*17)11 (Mamu-A*01)12 (Mamu-A*02)13, mouse (Rodentia) (H-2Kd)14,15, dog (Carnivora) (DLA-88*50801)16, cattle (Artiodactyla) (BoLA-A11)17 and equine (Perissodactyla) (Eqca-N*00602 and Eqca-N*00601)18 can combine with heterologous &#946;2m (Table 1). These hybrid molecules are often used in structural and functional studies. However, the methodology for the functional and structural study of the hybrid MHC I with heterologous &#946;2m is not yet summarized. Meanwhile, the structural basis for the interchanged &#946;2m between different taxa remain unclear.
Herein, the procedure for MHC I expression, refolding, crystallization, crystal data collection and structure determination are summarized. In addition, potential substitutions of &#946;2m from different species are analyzed through comparing the structural conformation of MHC I stabilized by homologous and heterologous &#946;2m. These methods will be helpful for further MHC I structural study and CD8+ T cell immune response evaluation in cancer and infectious disease.

<table>
	<tbody>
		<tr>
			<td>10 kDa MMCO membrane</td>
			<td>Merck millipore</td>
			<td>PLGC07610</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide</td>
			<td>LABLEAD</td>
			<td>A3291-500ml*5</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#215;Protein SDS Loading</td>
			<td>Novoprotein</td>
			<td>PM099-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>AMICON ULTRA-15 15ML-10 KDa cutoff</td>
			<td>Merck millipore</td>
			<td>UFC901096</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Inalco</td>
			<td>1758-9314</td>
			<td></td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Sigma</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3) strain</td>
			<td>TIANGEN</td>
			<td>CB105-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>MP</td>
			<td>219605580</td>
			<td>Wear suitable gloves and eye/face protection. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Solarbio</td>
			<td>D1070</td>
			<td>Gloves and goggles should be worn and operated in a ventilated kitchen. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.</td>
		</tr>
		<tr>
			<td>EDTA-2Na</td>
			<td>KeyGEN BioTECH</td>
			<td>KGT515500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>HUSHI</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>GSH</td>
			<td>Amresco</td>
			<td>0399-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>GSSG</td>
			<td>Amresco</td>
			<td>0524-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Amresco</td>
			<td>E424-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>h&#946;2m</td>
			<td>our lab</td>
			<td></td>
			<td>Zhang, S. et al. Structural basis of cross-allele presentation by HLA-A*0301 and HLA-A*1101 revealed by two HIV-derived peptide complexes. Mol Immunol. 49 (1-2), 395-401, (2011).</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Inalco</td>
			<td>1758-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine Hydrochloride</td>
			<td>Amresco</td>
			<td>0877-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Solarbio</td>
			<td>S8210</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Marker</td>
			<td>Fermentas</td>
			<td>26614</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Boao Rui Jing</td>
			<td>A112130</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex Increase 200 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Thermo</td>
			<td>17919</td>
			<td>Gloves and goggles should be worn and operated in a ventilated kitchen.</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Amresco</td>
			<td>0497-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bioruler</td>
			<td>RH30056-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

stability and structure of bat major histocompatibility complex class i with heterologous &beta;2-microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous &#946;2m is not available. Meanwhile, it is indicated that mammalian &#946;2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m). Herein, methods to evaluate the feasibility of heterologous &#946;2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.

Previous work reported that the HeV-derived HeV1 (DFANTFLP) peptide was presented by Ptal-N*01:0110,19. Herein, the binding capacity of this peptide to Ptal-N*01:01 with homologous bat &#946;2m (b&#946;2<font face="Helvetica Neue...

Watch this Scientific Journal Video about Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous ÃŽÂ²2-Microglobulin at JoVE.com

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &#946;2-Microglobulin

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential &#946;2-microglobulin (&#946;2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous &#946;2m were investigated.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...

stability-structure-bat-major-histocompatibility-complex-class-i-with

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Immunology and Infection

6.2K Views.  Wenzhou Medical University. The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential β2-microglobulin (β2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous β2m were investigated.

Video: Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous β2-Microglobulin

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.

This protocol describes a method for culturing primary hippocampal neurons from embryonic mouse brain. The expression of major histocompatibility complex class I on the extracellular surface of cultured neurons is then assessed by flow cytometric analysis.

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...