This work was supported by the American Heart Association and the Children&#8217;s Cardiomyopathy Foundation (SDG2462390204001), and the National Institutes of Health (HL114669). CM is a recipient of a postdoctoral research fellowship grant awarded by the Myocarditis Foundation, NJ.

Ethics Statement:
All animal procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and approved by the University of Nebraska-Lincoln, Lincoln, NE.
1. Induction of EAE
<ol>
	<li>To induce EAE, prepare the peptide/complete Freund&#8217;s adjuvant (CFA) emulsion as described in 11 with the following modifications: Step 1.1: prepare PLP 139-151 at a concentration of 1 mg/ml in 1x phosphate buffered saline (PBS). Step 1.4: use 200 &#181;l of CFA emulsion containing 100 &#181;g of PLP 139-151 per animal. Step 1.4.1: to immunize ten animals, prepare 2 ml of emulsion by mixing 1 ml of PLP 139-151 (1 mg/ml in 1x PBS) and an equal volume of CFA. Step 1.5: inject 200 &#956;l of emulsion in split doses subcutaneously in the two inguinal regions (~75 &#956;l each) and sternal region (~50 &#956;l) of five-to-six-week-old female SJL/J mice. Steps 1.7 and 1.8 are not applicable for induction of EAE.</li>
</ol>
2. Clinical Scoring of EAE Mice and Harvesting of Cerebrums
<ol>
	<li>Monitor the animals for appearance of clinical signs, and score the disease-severity daily from day 9 postimmunization as follows: 0 - healthy; 1 - limp tail or hind limb weakness but not both; 2 - limp tail and hind limb weakness; 3 - partial paralysis of hind limbs; 4 - complete paralysis of hind limbs; 5 - moribund or dead 12.</li>
	<li>Euthanize the mice when they reach a neurological score of 3 or 4 using CO2. To euthanize the mice, pre-fill the euthanasia chamber first with CO2 by turning the regulator of the CO2 tank not exceeding 6 psi and allow animals to stay within the chamber for approximately 5 min to achieve complete asphyxia. Soak the euthanized animals in 70% alcohol. Immediately, place the animals on an absorbent pad and using a pair of sterile forceps and scissors expose the hearts by cutting through the thoracic cavity. Puncture the right auricles and perfuse by injecting 10 ml of cold 1x PBS into the left ventricles of the hearts.</li>
	<li>Excavate the skull by snipping the cranium in a circular fashion using a pair of curved scissors, and carefully, flip the skull bones with forceps. Harvest the brain by blunt dissection and using a clean scalpel, separate cerebrum from the cerebellum. Place the cerebrum in a tube containing cold 1x PBS and leave the tube on ice until processing.</li>
</ol>
3. In Situ Staining of Cerebral Sections with MHC Class II (IAs)/PLP 139-151 Dextramers
<ol>
	<li>Prepare 4% PBS-buffered low-melting agarose, melt by heating at 50 &#176;C and leave it in a 40 &#176;C water bath until use.</li>
	<li>Place the cerebrum in a disposable deep base mold histology cassette kept over ice, and pour molten (40 &#176;C) agarose. Immediately, press the cerebrum gently with forceps to submerge the tissue. Leave the cassette on ice for 15-20 min until the solidification is complete.</li>
	<li>Fill the vibratome bath with chilled 1x PBS. Fill the space around vibratome bath with ice cubes to maintain the temperature of PBS between 0 and 2 &#176;C. Fix a clean razor blade into the vibratome and set the blade angle to 15&#176;.</li>
	<li>Placement of tissue in vibratome. Trim excess agarose from all the sides of the embedded tissue and slightly expose the ventral surface of the cerebrum.
	<ol>
		<li>Smear vibratome tissue block with super glue and transfer the agarose-embedded tissue over the glued surface by placing the exposed ventral surface of the cerebrum over the glue for transverse sectioning. Do not move the tissue, once the tissue is set over the block.</li>
		<li>Place the vibratome block containing the agarose-embedded tissue on ice, and allow the glue to dry for 15 min.</li>
	</ol>
	</li>
	<li>In the meantime, place the tissue chambers one per well of a 24-well plate. Fill the wells containing the tissue chambers with 1 ml of cold 1x PBS, and leave the plate on ice.
	<ol>
		<li>Prepare the tissue chambers by attaching a thin nylon mesh to the cut-end of a 25 ml polypropylene pipette using super glue; after trimming the excess mesh around the pipette, cut the pipette to a length of 1 cm from the mesh.</li>
	</ol>
	</li>
	<li>Lower the vibratome blade to the level of the top surface of tissue; set the vibratome speed to 0.22 mm/sec and start sectioning with a forward movement to make 200 &#181;m thick sections. Transfer one cerebral section per well using a watercolor soft brush into the tissue chambers placed within the wells of a 24-well plate. Keep the plate on ice until sectioning is complete to prevent tissue degradation. Note: Follow these steps to obtain in total, three paired sections from three different regions of the brain dorsal to the ventral surface (Figure&#160;2). One section from each pair (in total three sections) placed in three individual tissue chambers are designated for staining with specific dextramers (IAs/PLP 139-151) and anti-CD4. Likewise, another set of three sections that are placed in three individual tissue chambers are designated for staining with control dextramers (IAs/TMEV 70-86) and anti-CD4.</li>
	<li>Transfer the three tissue chambers containing the sections designated for staining with specific dextramers into three wells in a 24-well plate with one chamber per well in which, 300 &#181;l of a cocktail containing allophycocyanin (APC)-conjugated PLP 139-151 dextramers (2.5 &#956;g/ml) and anti-CD4-phycoerythrin (PE; 5 &#181;g/ml) in blocking solution (1x PBS with 2% normal goat serum) was previously added. Cover the wells with a lid.</li>
	<li>Similarly, transfer the three tissue chambers containing the sections designated for staining with control dextramers into three wells in a 24-well plate with one chamber per well in which, 300 &#181;l of a cocktail containing APC-conjugated TMEV 70-86 dextramers (2.5 &#956;g/ml) and anti-CD4- PE (5 &#181;g/ml) in blocking solution was previously added. Cover the wells with a lid. Note: Refer to Massilamany et al., 8 for preparation of dextramers, and the dextran molecules to prepare dextramers were kindly provided by Immudex, Copenhagen, Denmark.</li>
	<li>Label the wells, and wrap the plate with aluminum foil and place the plate on a rocking platform set to gentle rotations for 1.5 hr in the dark at RT.</li>
	<li>After the incubation period, wash tissue sections on the rocking platform, by transferring the tissue chambers to a fresh well containing 1 ml of cold 1x PBS, and avoid dribbling the contents of one well into the other. Repeat washing thrice, allowing at least 10 min per wash. Ensure that the tissue sections do not get dried as they can stick to the sides of the chamber.</li>
	<li>Fix the sections by transferring the tissue chambers into a fresh well in 24-well plate containing 1ml of filtered 4% PBS-buffered paraformaldehyde (pH 7.4) on a rocking platform for 2 hr with gentle rotations.</li>
	<li>Repeat washing thrice as described in step 3.10.</li>
	<li>Place the stained and fixed sections on a clean microscopic slide using a watercolor soft brush. Wipe excess water without touching the section, and mount the section using mounting medium. Cover with a microscopic coverslip and allow the slides to dry at RT O/N&#160;in the dark room.</li>
	<li>Store the slides in a light-proof slide storage box at 4 &#176;C.</li>
</ol>
4. Induction of EAM and Collection of Hearts
<ol>
	<li>Induce EAM as described in reference 11.</li>
	<li>Euthanize the mice on day 21 postimmunization using clean chamber fitted with CO2 gas cylinder, and soak the animals in 70% alcohol. Immediately, place the animals on an absorbent pad and using a pair of forceps and scissors expose the hearts by cutting through the thoracic cavity. Puncture the right auricles and perfuse by injecting 10 ml of cold 1x PBS into the left ventricles of the hearts. Collect the hearts and place in tubes containing cold 1x PBS.</li>
</ol>
5. In Situ Staining of Heart Sections with MHC Class II (IAk)/Myhc 334-352 Dextramers
<ol>
	<li>Prepare 4% PBS-buffered low-melting agarose, melt by heating at 50 &#176;C and leave it in a 40&#160;&#176;C water bath until use.</li>
	<li>Place the heart in a disposable deep base mold histology cassette kept on ice, and pour molten 40&#160;&#176;C agarose until the tissues are completely submerged as described in step 3.2.</li>
	<li>Follow step 3.3.</li>
	<li>Trim excess agarose from all the sides of the embedded tissue and slightly expose the base of the heart. Smear vibratome tissue block with super glue and transfer the agarose-embedded tissue over the glued surface by placing the exposed surface of the heart over the glue. Do not move the tissue, once the tissue is set over the block.
	<ol>
		<li>Place the vibratome block containing the agarose-embedded tissue on ice, and allow the glue to dry for 15 min.</li>
	</ol>
	</li>
	<li>Follow steps 3.5 and 3.6.</li>
	<li>Transfer the heart sections using a watercolor soft brush into the tissue chambers placed within the wells of a 24-well plate. Keep the plate on ice until sectioning is complete to prevent degradation of tissue. Note: Follow these steps to obtain in total, eight paired sections from the apex to base of the heart (Figure&#160;2). Place all sections in tissue chambers, allocating up to 3 sections per chamber. One section from each pair (in total eight sections) is designated for staining with specific dextramers (IAk/Myhc 334-352) and anti-CD4. Likewise, another set of eight sections is designated for staining with control dextramers (IAk/RNase 43-56) and anti-CD4.</li>
	<li>Transfer the three tissue chambers containing the sections designated for staining with specific dextramers into three wells in a 24-well plate with one chamber per well in which, 300 &#181;l of a cocktail containing APC-conjugated Myhc 334-352 dextramers (2.5 &#956;g/ml) and anti-CD4-PE (5 &#181;g/ml) in blocking solution was previously added. Cover the wells with a lid.
	<ol>
		<li>Similarly, transfer the three tissue chambers containing the sections designated for staining with control dextramers into three wells in a 24-well plate with one chamber per well in which, 300 &#181;l of a cocktail containing APC-conjugated RNase 43-56 dextramers (2.5 &#956;g/ml) and anti-CD4- PE (5 &#181;g/ml) in blocking solution was previously added. Cover the wells with a lid.</li>
	</ol>
	</li>
	<li>Label the wells, and wrap the plate with aluminum foil and place the plate on a rocking platform set to gentle rotations for 1.5 hr in the dark at RT.</li>
	<li>Follow steps, 3.10 to 3.11</li>
	<li>Repeat washing thrice as described in step 3.10.</li>
	<li>Place the stained and fixed sections (up to three sections/slide) on a clean microscopic slide using a watercolor soft brush. Wipe excess water without touching the sections, and mount the sections using mounting medium. Cover with a microscopic coverslip and allow the slides to dry at RT O/N in the dark room.</li>
	<li>Store the slides in a light-proof slide storage box at 4 &#176;C.</li>
</ol>
6. Analysis of Dextramer+ (dext+) CD4+ T Cells by Laser Scanning Confocal Microscope (LSCM) (Common to Both Brain and Heart Sections)
<ol>
	<li>Use Nikon A1-Eclipse 90i confocal microscope system for routine scanning and acquisition of images. To start Click open, the NIS Elements AR software (version 4.13).
	<ol>
		<li>Choose &#8220;TRITC&#8221; and &#8220;Cy5&#8221; channels from the panel. The excitation/emission wavelengths respectively for TRITC and Cy5 channels, 561.5 nm / 553-618 nm and 640.7 nm / 663-738 nm appear by default.</li>
		<li>Assign the pseudocolors &#8220;green&#8221; and &#8220;red&#8221; for the TRITC (CD4-PE) and Cy5 (dextramer-APC) channels, respectively.</li>
		<li>Place the slide to be examined on the microscopic stage and focus manually under 100X magnification. Set the &#8220;HV&#8221; to 110 and offset to &#8220;0&#8221;. Click &#8220;Focus&#8221; and adjust the &#8220;voltage&#8221; to optimize the lasers.</li>
		<li>Click &#8220;Focus&#8221;; scan the section from top to bottom, and set the &#8216;Z&#8217; level for image acquisition. Click &#8220;CH series&#8221; and acquire the image sequentially. Identify dext+ CD4+ T cells appearing as yellow punctate cells based on co-localization of signals generated from both the red (dextramers) and green (CD4) channels.</li>
	</ol>
	</li>
	<li>Use Olympus FV500-BX60 confocal microscope for easy quantitative enumeration of dext+ CD4+ T cells. To begin click open, the FLUOVIEW software (version 4.3).
	<ol>
		<li>Select the dyes &#8220;Cy3&#8221; and &#8220;Cy5&#8221; from the drop-down menu, and click &#8220;apply&#8221; to load the respective lasers. Note: the excitation/emission wavelengths for Cy3 (543 nm/pseudocolored &#8220;green&#8221;; CD4-PE) and Cy5 (633 nm/pseudocolored &#8220;red&#8221;; dextramer-APC) channels.</li>
		<li>Place the slide to be examined on the microscopic slide, and focus it manually under low magnification (4X or 10X). Click &#8220;Focus&#8221; while the Cy3 laser is on, and focus the inflammatory focus.</li>
		<li>Change the objective to 100X magnification. Set the file size to &#8220;512/512&#8221; from the drop-down menu. Click &#8220;Focus&#8221; and adjust the values for &#8220;PMT&#8221;, &#8220;Gain&#8221; and &#8220;Offset&#8221; parameters to optimize the lasers separately for Cy3 and Cy5.</li>
		<li>Click &#8220;Z stage&#8221; and then click &#8220;Focus&#8221; while both the lasers are on. Set the thickness of the &#8220;Z stage&#8221; by clicking the &#8220;up&#8221; and &#8220;down&#8221; arrows. Set the &#8220;Z&#8221; interval to 2 &#181;m.</li>
		<li>Click &#8220;Stop&#8221;, and choose &#8220;Seq123&#8221;. Then, click &#8220;XYZ&#8221; and start acquiring &#8220;Z&#8221; series images for the selected area within the inflammatory focus. Save the &#8220;Z serial images&#8221; as AVI files. To save image files, select the image from the &#8220;Z serial image&#8221;, and then save as &#8220;tiff&#8221; format.</li>
	</ol>
	</li>
	<li>Examine three paired sections from the cerebrum where one set of three sections was stained with IAs/PLP 139-151 dextramers/anti-CD4 and the other set of three sections with IAs/TMEV 70-86 dextramers/anti-CD4 to perform quantitative analysis.
	<ol>
		<li>Similarly, analyze eight paired sections for heart, in which, one set of eight sections was stained with IAk/Myhc 334-352 dextramers/anti-CD4 and another set of eight sections with RNase 43-56 dextramers/anti-CD4.</li>
	</ol>
	</li>
	<li>Examine 10 to 15 inflammatory foci per section in cerebrum, and in each focus, acquire a set of 15 to 25 serial images sequentially.
	<ol>
		<li>Examine a total of 20 inflammatory foci from all the eight sections of heart and from each focus acquire 10 to 15 serial images sequentially. Take the &#8216;Z&#8217; serial images by scanning the slides in a &#8216;horizontal-vertical up-horizontal-vertical down&#8217; movement so that repetition of &#8216;Z&#8217; series within the same focus is avoided.</li>
		<li>Once &#8216;Z&#8217; serial images are captured, name the files by identifying the slide numbers, name of the sections, and the number of the &#8216;Z&#8217; serial images taken.
		<ol>
			<li>Use &#8216;ImageJ&#8217; software to count dext+ CD4+ T cells in relation to the total number of CD4+ T cells. Select the &#8216;type of counter&#8217; in &#8216;ImageJ, check &#8216;show all&#8217; and start counting by clicking the center of each cell and continue counting in different &#8216;Z&#8217; serial images by using side arrows. After counting all the CD4 T cells, choose another type of counter and count the CD4+ dext+ T cells. Click the results tab to get the total number of cells counted with the two different counters.</li>
			<li>To obtain the total number, add the number of cells in all the &#8216;Z&#8217; serial images representing all the three cerebral sections or all the eight heart sections.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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No conflicts of interests were declared.

Unlike MHC class I tetramers, the use of MHC class II tetramers for routine applications continues to be challenging, especially to enumerate the precursor frequencies of low-affinity autoreactive CD4 T cells, due in part to their activation dependency 6,15-17. Recently, this problem was circumvented by creating the next generation of tetramers, called &#8220;dextramers,&#8221; permitting us to detect antigen-specific CD4 T cells for a number of autoantigens, such as PLP 139-151, MOG 35-55 and Myhc 334-352 <su...

The use of major histocompatibility complex (MHC) class II tetramers for detection of antigen-specific CD4 T cells has been a challenge 1-7. To enhance the detection sensitivity of MHC class II tetramers, the research team has created next-generation tetramers, designated &#8220;dextramers&#8221; 8. Dextramers contain dextran backbones in which multiple streptavidin-fluorophore moieties are conjugated, such that several peptide-tethered, biotinylated MHC monomers can be attached to one dextran molecule9. Thus, large aggregates of MHC dextramers that contain several MHC-peptide complexes can interact with multiple T cell receptors (TCRs).
This group recently generated dextramers for various self-antigens and demonstrated that the detection sensitivity of dextramers was approximately five-fold more than tetramers 8. The experimental systems include experimental autoimmune encephalomyelitis (EAE) induced with proteolipid protein (PLP) 139-151 in SJL mice; EAE induced with myelin oligodendrocyte glycoprotein (MOG) 35-55 in C57Bl/6 mice; and experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain-&#945; (Myhc) 334-352 in A/J mice. This study demonstrates the use of PLP 139-151- and Myhc 334-352 dextramers to detect antigen-specific CD4 T cells in situ with specificity by developing a direct staining protocol, thereby eliminating the need to amplify the signals from using fluorophore antibodies, an approach commonly employed with the use of tetramers. In addition, a comprehensive method of evaluating tissues has also been devised to reliably enumerate the frequencies of antigen-specific CD4 T cells in situ 10. Detection of antigen-specific T cells in situ permits delineation of events caused by specific antigenic stimuli from those caused by bystander activation. Likewise, the in situ technique also provides opportunities to track the kinetics of antigen-specific T cell infiltrations in the target organs.

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			<td height="25" style="height:25px;width:619px;">CFA</td>
			<td style="width:364px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:219px;">5881</td>
			<td style="width:167px;">Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MTB&#160; H37Rv extract&#160;</td>
			<td>Difco Laboratories, Detroit, MI</td>
			<td>231141</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PT</td>
			<td>List Biologicals Laboratories, Campbell, CA</td>
			<td>181</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1x PBS&#160;</td>
			<td>Corning, Manassas, VA</td>
			<td>21-040-CV</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female A/J mice&#160;</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female SJL/J mice</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>686</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 1 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 3 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 18 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 27 1/2 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way stopcock&#160;</td>
			<td>Smiths Medical ASD, Inc. Dublin, OH</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kerlix gauze bandage rolls&#160;</td>
			<td>Covidien, Mansfield, MA</td>
			<td>6720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes</td>
			<td>Kimberly-Clark Professional, Roswell, GA</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose, Low Melting Point, Analytical Grade</td>
			<td>Promega corporation, Madison, WI</td>
			<td style="width:219px;">V2111</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Immunopure normal goat serum</td>
			<td>Thermo Scientific, Waltham, MA</td>
			<td style="width:219px;">31873</td>
			<td>Store at -20 &#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Anti-mouse CD4 conjugated with PE dye</td>
			<td style="width:364px;">eBioscience, San Diego, CA&#160;</td>
			<td style="width:219px;">12004185</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td style="width:219px;">19202</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Faramount Aqueous Mounting Medium</td>
			<td>Dako, Carpinteria, CA</td>
			<td style="width:219px;">10073021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Conical tube (15ml)</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td style="width:219px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical tube (50 ml)</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td style="width:219px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well flat bottomed tissue culture plates</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>356775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water color #2 brushes</td>
			<td>Charles Leonard, Inc. Hauppauge, NY</td>
			<td style="width:219px;">73502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plain Microscope slides, size: 3&#34; x 1&#34; x 1.2 mm</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:219px;">1255010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Cover glass, 22 x 22-1</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:219px;">12548B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable deep base mold histology cassettes</td>
			<td>Laboratory prodcust, Rochester, NY</td>
			<td style="width:219px;">M-475-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite Super glue</td>
			<td>Henkel Corporation, Westlake, OH</td>
			<td style="width:219px;">1471879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Gillette SuperSilver</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PLP 139-151 (HSLGKWLGHPDKF)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MHC class II/IAs PLP 139-151 or TMEV 70-86 dextramers conjugated with APC dye</td>
			<td></td>
			<td></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHC class II/IAk Myhc 334-352 or&#160; or RNase 40-56 dextramers conjugated with APC dye</td>
			<td></td>
			<td></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dextran conjugated SA-APC</td>
			<td>&#160;Immundex, Copenhagen, Demark</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical scissors and forceps</td>
			<td style="width:364px;">INOX tool Corporation</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro oven</td>
			<td style="width:364px;">GE Healthcare, Pittsuburgh, PA</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica VT 1200 Vibratome</td>
			<td>Leica Microsystems, Inc. Buffalo Grove, IL</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus BX 60&#160; Laser scanning confocal microscope</td>
			<td>Olympus America, Inc. Center Valley, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1-Eclipse 90i confocal microscope</td>
			<td>Nikon Inc. Melville, NY</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 this report, in situ detection of antigen-specific, autoreactive CD4 T cells by direct staining with MHC class II dextramers is described. Freshly cut cerebral sections were derived from EAE mice and stained with cocktails containing PLP 139-151 (specific) or TMEV 70-86 (control) dextramers and anti-CD4. The top panels in Figure 1 shows LSCM analysis of cerebral sections costained with PLP 139-151 dextramers (red) and CD4 antibody (green), where the double-positive cells appeared yellow. Such...

Watch this Scientific Journal Video about In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers at JoVE.com

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.

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 ...

in-situ-detection-autoreactive-cd4-t-cells-brain-heart-using-major

Research

JoVE Journal

Immunology and Infection

11.6K Views.  University of Nebraska, Lincoln. 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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

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 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

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 ...

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.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &amp;#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.

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

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" of a T cell needs to be assessed by function, and higher function is associated with more powerful immune protection. T cells that can simultaneously produce two or more cytokines or chemokines at the single-cell level are called polyfunctional T cells (TPFs), which mediate immune responses through a variety of molecular mechanisms to express degranulation markers (CD107a) and secrete interferon (IFN)-&#947;, tumor necrosis factor (TNF)-&#945;, interleukin (IL)-2, or macrophage inflammatory protein (MIP)-1&#945;. There is increasing evidence that TPFs are closely related to the maintenance of long-term immune memory and protection and that their increased proportion is an important marker of protective immunity and is important in the effective control of viral infection and reactivation. This evaluation applies not only to specific immune responses but also to the assessment of cross-reactive immune responses. Here, taking the Japanese encephalitis virus (JEV) as an example, the detection method and flow cytometry color scheme of JEV-specific TPFs produced by peripheral blood mononuclear cells of children vaccinated against Japanese encephalitis were tested to provide a reference for similar studies.

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (TPF)&#160;profiles in peripheral blood mononuclear cells (PBMCs) within Japanese encephalitis virus (JEV)-vaccinated children. The detection method and flow cytometry color scheme of JEV-specific TPFs were tested to provide a reference for similar studies.

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" ...