This study was approved by the institutional review board at Sheba Medical Center, and informed written consent was obtained from all participating subjects.
1. DNA isolation and quantification
<ol>
	<li>Digestion and cell lysis of intestinal biopsies
	<ol>
		<li>Retrieve intestinal biopsies, either freshly collected or those stored at -20 &#176;C or -80 &#176;C. If using frozen biopsies, thaw on ice.</li>
		<li>Add 600 &#181;L of nuclei lysis solution to a sterile 1.7 mL micro-centrifuge tube, chilled on ice.</li>
		<li>Place biopsy into a microcentrifuge tube with lysis solution and incubate at 65 &#176;C for 15-30 min.</li>
		<li>Add 17.5 &#181;L of 20 mg/mL proteinase K and incubate overnight at 55 &#176;C, with gentle shaking.</li>
		<li>Allow sample to cool to room temperature for 5 min before proceeding to step 1.3.</li>
	</ol>
	</li>
	<li>Cell lysis of whole blood
	<ol>
		<li>Obtain 3 mL of whole blood in EDTA-, heparin-, or citrate-containing tube.</li>
		<li>Gently rock tube until thoroughly mixed, and transfer blood to a sterile 15 mL centrifuge tube containing 9 mL of cell lysis solution. Invert several times during a 10 min incubation period at room temperature.</li>
		<li>Centrifuge at 2,000 x g for 10 min at room temperature, and then discard as much supernatant as possible without disturbing the visible white pellet. Approximately 50&#8211;100 &#956;L of residual liquid will remain.</li>
		<li>Vortex tube vigorously for 15 s until completely resuspended. Add 3 mL of nuclei lysis solution and pipet several times. The solution should become viscous.</li>
	</ol>
	</li>
	<li>Protein precipitation
	<ol>
		<li>Add 200 &#181;L of protein precipitation buffer to the tissue, or 1 mL to the blood. Vortex vigorously for 20 s and incubate on ice for 5 min. Small protein clumps may be visible after vortexing.</li>
		<li>Centrifuge at 16,000 x g for 4 min. A tight pellet containing precipitated proteins should form.</li>
		<li>Carefully transfer supernatant, without disturbing the pellet, to a new 1.7 mL tube. 
		NOTE: If pellet is not tight, consider another centrifuge at 16,000 x g for 4 min.</li>
	</ol>
	</li>
	<li>DNA precipitation and rehydration
	<ol>
		<li>Add 1 mL of 2-propanol to tube containing the supernatant, and gently mix by inverting. DNA should become visible as white floating substance.</li>
		<li>Centrifuge at 16,000 x g for 1 min. Remove the supernatant completely using a pipette.</li>
		<li>Add 1 mL of freshly prepared 70% ethanol, and invert tube several times. Centrifuge at 16,000 x g for 1 min at room temperature. Discard supernatant carefully.</li>
		<li>Repeat step 1.4.3.</li>
		<li>Place open tube inverted on clean absorbent paper for 10-15 min. Re-invert tube and confirm complete hydration. If needed, leave to air-dry for additional 10-15 min. 
		NOTE: It is crucial for pellet to dry completely.</li>
		<li>Add 50 &#181;L of ultra-pure water (UPW). If needed, DNA can be diluted more after quantification.</li>
		<li>Incubate for 1 h on heat block at 65 &#176;C, for DNA rehydration.</li>
	</ol>
	</li>
	<li>DNA quantification 
	NOTE: DNA was quantified using a fluorometer and designated reagents, as specified in table of materials.
	<ol>
		<li>Bring standards to room temperature and prepare the kit, including 0.5 mL designated tubes.</li>
		<li>Prepare buffer and dye mix in a 15 mL tube, according to number of samples. Add 200 &#181;L of buffer and 1 &#181;L of dye per sample. Include 2 samples for standards. It is recommended to calculate an additional sample in order to avoid that the buffer will not be sufficient.
		<ol>
			<li>For the 2 standards, place 190 &#181;L of the above-prepared mix in 0.5 mL tube. Add 10 &#181;L of the standard.</li>
			<li>For each sample, place 199 &#181;L of the above-prepared mix in 0.5 mL tube. Add 1 &#181;L of the DNA.</li>
		</ol>
		</li>
		<li>Read the samples. The result indicates DNA concentration of the sample.</li>
		<li>If samples are over the limit, dilute the DNA 1:10 with UPW, and repeat read.</li>
	</ol>
	</li>
</ol>
2. Library preparation
NOTE: The current protocol utilizes a multiplex, PCR-based assay kit compatible with NGS sequencers. The kit contains 24 different indices each targeting conserved regions within the V&#946; and the J&#946; regions. This enables a one-step PCR reaction and pooling of different samples. See table of materials for details.
<ol>
	<li>Prepare DNA samples. Obtain 150 ng of DNA and add UPW for a total of 5 &#181;L. 
	NOTE: It is possible to use less than 150 ng of DNA for library preparation, with minimum concentration of 10 ng/&#181;L.</li>
	<li>Place pipettes and tips in hood with UV light for 15 min to break down residual DNA. Meanwhile, allow different index tubes (for library preparation) and DNA polymerase to thaw on ice.</li>
	<li>In a biological hood, add 45 &#181;L from the different index tubes into PCR tubes. To follow, add 0.2 &#181;L of the DNA polymerase and the 5 &#181;L of DNA prepared in step 2.1 into PCR tubes.</li>
	<li>Run PCR reaction using a pre-set program, according to the manufacturer&#39;s instructions.</li>
</ol>
3. Amplicon purification and quantification
<ol>
	<li>Use magnetic beads for removal of excess primers, nucleotides, and enzymes.
	<ol>
		<li>Warm the beads at least 30 min to room temperature. Prepare enough fresh 80% ethanol for 400 &#181;L per sample.</li>
		<li>Add 50 &#181;L (IGH) or 35 &#181;L (TCR&#946;) of the beads to each PCR tube and mix by pipetting 10 times. Incubate 10 min at room temperature.</li>
		<li>Place the mixed sample on the magnetic stand for 5 min.</li>
		<li>Aspirate 95 &#181;L (IGH) or 80 &#181;L (TCR&#946;) of the clear liquid and discard. Aspirate remaining liquid using a 10 &#181;L tip.</li>
		<li>Add 200 &#181;L of 80% ethanol to each sample. Incubate 30 s. Aspirate 195 &#181;L of ethanol and discard. Aspirate remaining liquid using 10 &#181;L tip.</li>
		<li>Repeat step 3.1.5. Open the caps of the tubes and let air dry for 5 min.</li>
		<li>Immediately after the 5 min, remove from magnetic stand and add 25 &#181;L of elution buffer (10 mM Tris-HCl, pH 8.0). Mix by pipetting until homogeneous. Incubate at room temperature for 2 min.</li>
		<li>Place the tubes on the magnetic stand for 5 min. Transfer 22 &#181;L to a new PCR tube. Measure the DNA concentration (step 1.5).</li>
	</ol>
	</li>
	<li>Amplicon quantification 
	NOTE: The current protocol uses an automated electrophoresis machine for quality control of DNA concentration, size, and integrity. See table of materials for details.
	<ol>
		<li>Place reagents and screentape at room temperature for at least 30 min.</li>
		<li>In a new 1.7 mL micro-centrifuge tube, make a 1 ng/&#181;L dilution of the DNA samples quantified in step 3.1.8 with UPW for a total volume of at least 3 &#181;L.</li>
		<li>Vortex diluted DNA before use. Place 2 &#181;L of buffer, together with 2 &#181;L of the diluted DNA in the pre-labeled specialized PCR strips (provided in the kit) and place the caps. Place on shaker for 1 min, followed by spin down of PCR strips. 
		NOTE: Due to the viscosity of buffer, ensure excess buffer is removed from the tip before transfer to the sample tubes.</li>
		<li>Place in the machine, the screentape and PCR strips without the caps. Open the lid of tip box inside the machine. Assign the position with appropriate labels. Start the machine. 
		NOTE: The output histogram should be a single peak at the desired size of the amplicons (Figure 1A). In samples of poor quality, additional peaks will be observed (Figure 1B), indicating poor bead clean-up or formation of primer dimers.</li>
	</ol>
	</li>
</ol>
4. Next-generation sequencing
<ol>
	<li>Quantification and pooling of library
	<ol>
		<li>Calculate final DNA concentration in nM, using DNA value from step 3.1.8, and the size in base pairs (bp) of the product&#8217;s peak, obtained from electrophoresis machine results (see Figure 1).</li>
		<li>For each sample, calculate DNA volume to take for a final concentration of 4 nM, in a final volume of 10-20 &#181;L elution buffer.</li>
		<li>Prepare a new dilution mix for each sample and take 2 &#181;L from it to a new pool mix. 
		NOTE: For samples that have less than 4 nM, add the maximum amount of sample possible.</li>
	</ol>
	</li>
	<li>NGS run of library 
	NOTE: The current protocol uses a bench-top sequencer platform. See table of materials for details.
	<ol>
		<li>Prepare a fresh solution of 0.2 N NaOH. Add 10 &#181;L of 0.2 N NaOH to the diluted library prepared in step 4.1.3. Add 5% PhiX to tube and vortex briefly. Spin down the tube to ensure all the solution has settled to the bottom of the tube.</li>
		<li>Incubate for 5 min at room temperature to denature the double stranded DNA. Add 980 &#956;L of pre-chilled buffer from the kit to the tube containing DNA and vortex briefly.</li>
		<li>Place the diluted library on ice, and prepare the library as follows. For a mix of 17 pM, add 425 &#181;L of DNA from step 4.2.2 and 575 &#181;L of buffer. Invert library several times and spin down. Load 600 &#181;L of the final library onto the designated cartridge, and place samples in the machine.</li>
		<li>Start the run following the software instructions.</li>
	</ol>
	</li>
</ol>
5. Sequencing analysis
<ol>
	<li>Download sequencing metrics from sequencer, and verify that sequencing data is within the following ranges (specific for the kit used in the current protocol). Samples not meeting these criteria are subject to repeat run: 
	Cluster Density (Density (K/mm2)): 945K &#8211; 1800K 
	Percent of reads passing filter (Clusters PF (%)): &#62;90% 
	Quality scores (%&#62;=Q30): &#62;85% 
	PhiX alignment (Aligned %): 1.5% - 10% 
	PhiX Error Rate (%): &#60;1.0%</li>
</ol>

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All authors have nothing to disclose.

Changes in abundance and function of B and T lymphocytes are often encountered in different malignancies18, chronic inflammatory disorders (e.g., ulcerative colitis and rheumatoid arthritis)10,19, and in various immunodeficiencies17,20. The current method utilizes NGS to facilitate an in-depth view of TCR and BCR repertoires, enabling detection of subtle changes in T and B cell clonality, sharing of clones, V(D)J gene usage, and information on the degree of somatic hyper-mutations in the case of B cells.
The method of DNA isolation was described for intestinal biopsies and blood samples. However, with modifications of lysis and DNA extraction, the method can be applied to other tissues such as tumors, lymph nodes, synovial fluid, etc. It is important during library preparation not to cross-contaminate the different primer sets. At bead clean-up, care should be taken to leave tubes on the magnet at all times of incubation. For DNA samples of low concentrations, stocks can be concentrated at 65 &#176;C for 10-20 minutes. Moreover, amplicons can be eluted from beads in as little as 20 &#181;L elution buffer.
Different techniques used in the past to characterize the landscape of T and B cell composition provided only a superficial overview of the immune repertoire. One of the biggest advantages of NGS for immune repertoire analysis is the ability to identify unique clones, and consequently track them in different anatomical sites (e.g., intestine vs. blood vs. lymph node) or in different individuals. The clinical implications of such a technique are significant, and go beyond enhancing the understanding of the role of T or B cells in different immune-mediated diseases or malignancies21. In oncology, NGS is used to identify specific clones that can be predictive biomarkers for the patients who will most likely benefit from current immunotherapies22,23,24. In leukemia, NGS is used to confirm full eradication of cancerous cells by detection of residual leukemic clones that remain in a patient after treatment25.
One of the main limitations of immune repertoire analysis of whole tissue or blood samples is the inability to identify repertoire changes in less frequent populations. For example, if regulatory T cells, which comprise only a few percentages of total CD4+ T cells, express a unique repertoire profile, this would be missed if conducting these studies on whole blood. This problem could be addressed by conducting these studies on sorted immune populations. Alternatively, in recent years technology has developed to facilitate coupling of single cell RNA data with TCR or IGH repertoire profiles26. This provides another level of functionality of the T or B cells, since the transcriptional landscape of each clone can be characterized. As an example, Zemmour et al. used scRNAseq TCRseq to demonstrate that regulatory T cells from human blood, display broad heterogeneity with an activated subpopulation that is transcriptionally related to conventional T cells27. Similarly, in patients with hepatocellular carcinoma this method identified 11 distinct T cell subsets that infiltrate the tumor, each one with a unique transcriptional and repertoire profile28. Studies like these will be helpful especially when studying rare populations, and will provide important functional information of specific clones.
NGS of immune repertoire is a new research field that provides novel insights about adaptive immune function and the role of T and B cells in various diseases. In addition, it is rapidly entering the clinical world in different disciplines, by tracking disease associated clonotypes. There are currently several available kits for immune repertoire. These are based on similar methodology, regardless of whether they use RNA or DNA as the source. They use similar techniques for calibration, bias corrections, and analysis tools; and thus need to be properly chosen according to specific demands. One of the great advantages of the current kit is that it is a ready-to-use and optimized for human immune repertoire, without need for calibration. In the future we will see more studies using repertoire features as biomarkers for different disease, potentially also leading to development of targeted therapies against these clones. We believe researchers should consider applying these methods for studying the adaptive immune landscape in different disorders.

The adaptive immune system, comprised of T and B lymphocytes, utilizes immunological memory to recognize a previously encountered antigen and initiate a rapid response. Lymphocytes are generated in the bone marrow and mature in the thymus (T cells) or bone marrow (B cells). Both the T cell receptor (TCR) and B cell receptor (BCR) display unique configurations that allow recognition of specific antigens. In homeostasis, T and B cells constantly circulate and survey the trillions of different peptides presented on antigen-presenting cells. TCR or BCR ligation of a specific antigen with high affinity, together with appropriate co-stimulation, leads to cell activation, resulting in cytokine secretion, clonal expansion and generation of antibodies, in the case of B cells.
The enormous array of the different T or B cells is collectively termed immune repertoire, enabling recognition of countless of different epitopes. In order to generate such a vast repertoire, a complex process of random assembly of different gene segments takes place, creating nearly endless combinations of receptors that can bind unique antigens1. This process, called V(D)J recombination, includes rearrangements of different variable (V), diversity (D) and joining (J) genes, accompanied by random deletions and insertions of nucleotides in the junctions2.
The architecture of the adaptive immune system has interested scientists in different fields for many decades. In the past, Sanger sequencing, complementary determining region 3 (CDR3) spectratyping, and flow cytometry were used to characterize the immune repertoire, but provided low resolution. In the last decade, advances in next-generation sequencing (NGS) methods enabled in-depth insight into the characteristics and composition of an individual&#8217;s TCR and BCR repertoires3,4. These high-throughput systems (HTS) sequence and process millions of rearranged TCR or BCR products simultaneously and permit a high-resolution analysis of specific T and B cells at the nucleotide or amino acid level. NGS provides a new strategy to study the immune repertoire in both health and disease. Studies utilizing HTS demonstrated altered TCR and BCR repertoires in autoimmune diseases5, primary immunodeficiencies6,7, and malignancies, such as in acute myeloid leukemia8. Using NGS, we and others have shown oligoclonal expansion of specific T and B cell clones, in patients with inflammatory bowel disease (IBD), including ulcerative colitis and Crohn&#8217;s disease9,10,11,12,13,14. Overall, studies from different fields suggest changes in the repertoire have a crucial role in the pathogenesis of immune-mediated disorders.
The current protocol describes a method for isolation of DNA from intestinal biopsies and blood, generation of TCR&#946; and IGH PCR libraries for NGS, and performance of sequencing run. We also provide basic steps in immune repertoire data analysis. This protocol can be applied for the generation of TCR&#945;, TCR&#947;, and IGL libraries as well. The method is also compatible with other organs (e.g., lymph nodes, tumors, synovial fluid, fat tissue, etc.) as long as tissue-specific digestion protocols are used.

<table>
	<tbody>
		<tr>
			<td>2-propanol</td>
			<td>Sigma</td>
			<td>I9516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL micro-centrifuge tubes</td>
			<td>Axygen</td>
			<td>8187631104051</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Greiner</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Merck</td>
			<td>1.08543.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplitaq Gold</td>
			<td>Thermo Fisher</td>
			<td>N8080241</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP Beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Bioer</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 Sample Buffer</td>
			<td>Agilent</td>
			<td>5067-5603</td>
			<td>For Tapestation</td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 ScreenTape</td>
			<td>Agilent</td>
			<td>5067-5584</td>
			<td>For Tapestation. Tubes sold seperately</td>
		</tr>
		<tr>
			<td>Lymphotrack Assay kit</td>
			<td>Invivoscribe</td>
			<td>TRB: 70-91210039 IGH: 70-92250019</td>
			<td>Each includes 24 indexes</td>
		</tr>
		<tr>
			<td>MiSeq Reagent Kit v2 (500 cycle)</td>
			<td>Illumina</td>
			<td>MS-102-2003</td>
			<td>Includes standard flow cell type and all reagents required</td>
		</tr>
		<tr>
			<td>MiSeq Sequencer</td>
			<td>Illumina</td>
			<td>SY-410-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR strips</td>
			<td>4titude</td>
			<td>4ti-0792</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Invitrogen</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer</td>
			<td>Thermo Fisher</td>
			<td>Q33226</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>Q32854</td>
			<td>Includes buffer, dye, standards, and specialized tubes</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Biosan</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapestation 2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>ultra pure water</td>
			<td>Bio-lab</td>
			<td>7501</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard DNA isolation kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td>Includes cell lysis solution, nuclei lysis solution, and protein precipitation buffer</td>
		</tr>
	</tbody>
</table>

t and b cell receptor immune repertoire analysis using next-generation sequencing

Immunological memory, the hallmark of adaptive immunity, is orchestrated by T and B lymphocytes. In circulation and different organs, there are billions of unique T and B cell clones, and each one can bind a specific antigen, leading to proliferation, differentiation and/or cytokine secretion. The vast heterogeneity in T and B cells is generated by random recombination of different genetic segments. Next-generation sequencing (NGS) technologies, developed in the last decade, enable an unprecedented in-depth view of the T and B cell receptor immune repertoire. Studies in various inflammatory conditions, immunodeficiencies, infections and malignancies demonstrated marked changes in clonality, gene usage, and biophysical properties of immune repertoire, providing important insights about the role of adaptive immune responses in different disorders.
Here, we provide a detailed protocol for NGS of immune repertoire of T and B cells from blood and tissue. We present a pipeline starting from DNA isolation through library preparation, sequencing on NGS sequencer and ending with basic analyses. This method enables exploration of specific T and B cells at the nucleotide or amino-acid level, and thus can identify dynamic changes in lymphocyte populations and diversity parameters in different diseases. This technique is slowly entering clinical practice and has the potential for identification of novel biomarkers, risk stratification and precision medicine.

Herein, we describe a method for DNA isolation from intestinal tissue and blood, preparation of libraries for NGS, and basic steps of a sequencing run for immune repertoire sequencing. The run will generate fastq files, which can be further converted to fasta files for use in the international ImMunoGeneTics (IMGT)/HighV-QUEST platform. This HTS performs and manages many analyses of tens of thousands of rearranged TCR&#946; and IGH sequences, at the nucleotide level15. IMGT/HighV-QUEST enables analysis of different TCRs and IGH repertoires in both health and disease. This can lead to identification of new &#34;disease-specific&#34; clones, analysis of clonal expansion and diversity parameters, delineation of differential V(D)J usage, analysis of somatic hyper-mutations, and more. The IMGT/HighV-QUEST provides CSV files, which contain specific sequences and their abundance. Using genomic DNA as a starting material yields sequence numbers that are representative of cell numbers. Thus, if original DNA quantity is equal, percentage of T cells in a given sample can also be calculated.
We include a basic analysis from representative, autologous blood and rectal samples of a patient with IL10 receptor deficiency and history of severe infantile-onset IBD, resulting from a deleterious IL10RA mutation. Samples were assayed for both TCR&#946; and IGH repertoire. The intestinal TCR&#946; sample yielded a total 12,450 sequences, of which 9,050 sequences were unique. The blood TCR&#946; sample had a total 54,880 sequences, of which 35,110 were unique. In the intestinal IGH sample, a total of 49,070 sequences were obtained, of which 23,670 were unique. In the blood IGH sample, a total of 13,710 sequences were obtained, of which 13,540 were unique. All clones can be identified by their unique sequence both at the nucleotide or amino acid level. These sequences can be compared between different patients, in search for shared clones, or in the same patient between different anatomical sites (e.g., blood vs. intestine). We present for each of the samples the 5 most frequent clones (Table 1).
To quantify the degree of clonal expansion different indices can be used, including Shannon&#8217;s H, Gini-Simpson, entropy and clonality. As an example, Shannon&#39;s H, which takes into account the number of unique sequences (richness of the repertoire) and how evenly they are distributed was found to be decreased in the patient&#39;s intestinal IGH vs. blood (8.3 vs. 9.5), suggesting clonal expansion of B cells in the inflamed gut.
For a broad overview of the repertoire, Treemap images (www.treemap.com) were generated (Figure 2). Each colored square represents a different clone, and the size correlates with its frequency. These Treemap images demonstrate clonal expansion in the intestinal IGH repertoire compared with the blood. In contrast, in the TCR&#946; repertoire, marked clonal expansion is observed in the blood, in comparison to autologous intestine.
At the gene level, NGS provides information regarding V- D- and J- usage at the level of either gene, family, or allele, as shown in Table 1. Moreover, specific V(D)J combinations can be inferred from the data for TCR&#946; and IGH repertoires (Figure 3A,B), which can reveal differential gene usage patterns in various conditions. Biophysical properties of the CDR3 region such as length (Table 1) or hydrophobicity can also be analyzed. Importantly, CDR3 length distribution is altered in different immune-mediated disorders10,16,17. For example, in IL10R deficient patients, blood-derived T cells, but not B cells, have shorter CDR3 length, and differential hydrophobicity, in comparison to healthy controls7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61792/61792fig01.jpg" /> 
Figure 1: Representative bioanalyzer data.&#160;Representative images of intestinal TCR&#946; samples showing optimal results (A) with the desired peak at 400 bp. An example of a low-quality library (B) with additional peaks at 179bp and 114bp (marked *). Peaks seen at ends are upper and lower markers of the electrophoresis strip. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61792/61792fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61792/61792fig02.jpg" /> 
Figure 2: Overview of T and B cell immune repertoire.&#160;Treemap diagrams of TCR&#946; and IGH blood and intestinal samples. Each colored square represents a different clone, and the size correlates with its frequency. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61792/61792fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61792/61792fig03.jpg" /> 
Figure 3: Representative graphs of TCR&#946; and IGH V-J usage.&#160;A representative intestinal sample depicting total number of sequences for each V-J combination in TCR&#946; (A) or IGH (B). Data is not shown for D usage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61792/61792fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing at JoVE.com

T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

The current protocol describes a method for DNA isolation from blood samples and intestinal biopsies, generation of TCR&#946; and IGH PCR libraries for next-generation sequencing, performance of a NGS run and basic data analysis.

Immunological memory, the hallmark of adaptive immunity, is orchestrated by T and B lymphocytes. In circulation and different organs, there are billions ...

t-b-cell-receptor-immune-repertoire-analysis-using-next-generation

Nanyang Technological University

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JoVE Core

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Anatomy and Physiology

Immunology and Infection

Unit 1

The Lymphatic and Immune System

SCIENTISTS IN ACTION

8.1K Views.  Sheba Medical Center. The current protocol describes a method for DNA isolation from blood samples and intestinal biopsies, generation of TCRβ and IGH PCR libraries for next-generation sequencing, performance of a NGS run and basic data analysis.

Video: T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.