The work was supported by a grant from Leukaemia UK to ETB and MJA.

Blood samples were obtained from normal subjects (NS) (n = 12) as well as patients with marginal zone lymphoma (MZL) (n = 7) and other types of B-cell non-Hodgkin&#39;s lymphoma (BNHL) (6 FL patients, 2 lymphoplasmacytic lymphoma patients and 1 low-grade B-cell non-Hodgkin&#39;s lymphoma not otherwise specified patient). Patients were recruited from the hematology clinics at Leicester Royal Infirmary after having given informed, written consent, with ethical approval in place for all studies. Ethical approval was obtained by Leicestershire, Northamptonshire and Rutland Research Ethics Committee 1, reference 06/Q2501/122 for patient samples and the Health Research Authority (HRA) NRES Committee East Midlands-Derby, reference 14/EM/1176 for normal subjects.
1. Isolation of CD4+ T-cells from Whole Peripheral Blood
<ol>
	<li>Take fresh peripheral blood (2 to 15 mL) into K2EDTA tubes by standard venipuncture and process as soon as possible to maintain maximum viability. 
	NOTE: Use standard laboratory personal protective equipment (coat, gloves, glasses) and carry out the work in a class II Biosafety Cabinet.</li>
	<li>Bring the blood and all necessary reagents (CD4+ enrichment cocktail, 2% fetal bovine serum/phosphate buffered saline (FBS/PBS), density gradient media, and freezing medium if cryopreserving cells) to room temperature.</li>
	<li>Mix the blood with a commercially available cocktail of antibodies for the enrichment of human CD4+ T Cells. Use 50 &#181;L of the reagent for every 1 mL of blood. Use a 50 mL conical polypropylene tube for 5 to 15 mL of blood. 
	&#8203;NOTE: If using 2 to 4 mL of blood, perform this step in a 14 mL conical polypropylene tube.</li>
	<li>Incubate the mixture at room temperature for 20 min.</li>
	<li>Dilute the mixture with an equal volume of 2% FBS/PBS</li>
	<li>Layer this diluted mixture on top of the density gradient media slowly to avoid disturbing the interface and proceed to centrifugation immediately to avoid diffusion of the blood into the density gradient medium.
	<ol>
		<li>For 2 to 3 mL of blood, use 3 mL of density gradient medium in a 14 mL conical polypropylene tube, but for 4 mL of blood, use 4 mL of density gradient medium in a 14 mL conical polypropylene tube and for 5 to 15 mL blood, use 15 mL of density gradient medium in a 50 mL conical polypropylene tube.</li>
	</ol>
	</li>
	<li>Centrifuge the layered mixture for 20 min at 1,200 x g with the brake off at 20 &#176;C. 
	NOTE: A temperature below ambient may result in the contamination with red blood cells and granulocytes. A lower speed will cause the CD4+ isolation process to fail. Leaving the break engaged will decrease cell yield. Use a bench top centrifuge with rotors that can be closed to avoid aerosols.</li>
	<li>Remove the enriched CD4+ cell layer from the interface using a Pasteur pipette. 
	NOTE: The enriched cell layer will resemble a standard &#34;buffy coat&#34; of white cloudy cells, but as only CD4+ cells are present, this will naturally be smaller and more difficult to see.</li>
	<li>Add 2% FBS/PBS to the CD4+ cells up to a total volume of 10 mL and then wash twice with 2% FBS/PBS by centrifuging for 10 min at 400 x g during each wash.</li>
	<li>Resuspend the pellet in 2% FBS/PBS and count the cells stained with a vital dye (trypan blue) to determine the cell numbers and viability.</li>
	<li>Optional step: resuspend 5 x 105 to 1 x 106&#160;cells in freezing medium (10% dimethyl sulfoxide in FBS) in 1 mL aliquots. 
	NOTE: Levels of some surface markers may be altered by cryo-preservation and thawing. It is, therefore, very important that all samples are processed consistently. In order to minimize the differences in analytical variables, the samples were cryo-preserved and batched for analysis in this study.</li>
</ol>
2. Flow Cytometry
<ol>
	<li>Thaw the cryopreserved CD4+ cells quickly in a water bath at 37 &#176;C and wash once in pre-warmed medium (RPMI 1640 + L-glutamine supplemented with 10% FBS and 1x penicillin-streptomycin).</li>
	<li>Add the cells to 9 mL of medium, centrifuge for 5 min at 400 x g and remove the supernatant.</li>
	<li>Resuspend the cells in 1% bovine serum albumin (BSA) in PBS (50 &#181;L) so that each condition to be tested uses between 5 x 105 and 1 x 106 cells.</li>
	<li>Mix the cells with Staining Buffer (50 &#181;L). 
	NOTE: Brilliant Stain Buffer prevents various staining artefacts that may interfere with data analysis when two or more BV or BUV stains are used simultaneously owing to inherent chemical properties of these dyes. In single or unstained conditions, the 50 &#181;L of brilliant stain buffer can be substituted for 50 &#181;L of 1% BSA/PBS.</li>
	<li>Add the antibodies to cells (as per the &#34;volume used&#34; column in Table 1) and incubate on ice in the dark for 30 min.</li>
	<li>Wash the cells twice in 1% BSA/PBS by centrifuging for 3 min at 600 x g during each wash.</li>
	<li>Resuspend in 1% BSA/PBS (400 &#181;L) and transfer to a flow cytometry acquisition tube.</li>
	<li>Vortex the cells gently before acquiring data on a flow cytometer:</li>
</ol>
3. Flow Cytometry Controls and Set-up
<ol>
	<li>Using an unstained sample, set photodiode voltages so that lymphocytes can be separated from obvious debris and dead cells (events with high side scatter (SSC) area and low forward scatter (FSC) area)16. Using single stained samples, set photomultiplier (PMT) voltages so that positive fluorescence can be discerned from background fluorescence while making sure that all events fall within the detectable scale. 
	NOTE: An improvement would be to plot CV against PMT voltage for each PMT using dimly fluorescent beads to find the minimum voltage for optimum resolution (The &#34;Peak 2&#34; method19).</li>
	<li>Account for spectral overlap by generating a compensation matrix using single stains with capture beads. Add commercially available compensation beads (60 &#181;L), and negative control beads (60 &#181;L) to 1% BSA/PBS (100 &#181;L), and vortex. 
	NOTE: The capture beads used must match the host species and IgG isotype of the antibody being used. The compensation matrix should be re-calculated if the lot number of any tandem dyes changes, as they can display considerable variability in their emission spectra between lots.</li>
	<li>Add a single antibody (20 &#181;L) and vortex.</li>
	<li>Incubate at room temperature in the dark for 30 min.</li>
	<li>Centrifuge at 200 x g for 10 min and discard the supernatant.</li>
	<li>Resuspend in 1%BSA/PBS (500 &#181;L) and vortex.</li>
	<li>Acquire the sample with a flow cytometer using the designated compensation matrix generator in the acquisition software according to the manufacturer&#39;s instructions.</li>
	<li>Use FMO controls to guide the placement of gates for positive marker fluorescence to account for dye spill over, which is the main source of background fluorescence in experiments using &#8805;4 colours16. Follow the general cell-surface staining protocol as described in Step 3, but for each condition, omit one of the fluorophores from the staining step (where CD45RA is omitted, also omit the live/dead stain). Acquire 100,000 lymphocytes for each condition.</li>
	<li>Set the gate positivity at &#8804;0.5% of cells. See Figure 2 for an illustrated example of FMO controls.</li>
</ol>
4. Data Analysis
<ol>
	<li>Employ the flow cytometer&#39;s acquisition software to set a threshold of 5,000 units on the FSC parameter to exclude very small debris.</li>
	<li>Use a stopping gate to acquire 10,000 cTfh cells (CD4+ CD45RA- CXCR5+). 
	NOTE: The individual user can collect more than 10,000 cells. This number was a compromise between collecting enough cells to provide meaningful results and the time taken for collection.</li>
	<li>Using an FSC-Area / SSC-Area dot plot, draw an ellipse or polygon gate to select the population of lymphocytes whilst excluding debris and overtly dead cells (events with a high SSC-Area and low FSC-Area).</li>
	<li>Using an FSC-Area / FSC-Width dot plot, draw a polygon gate to select single cells while excluding doublets (doublets have an increased area but similar width to single cells).</li>
	<li>Using an FSC-Area / CD45RA and viability marker dot plot, draw a rectangular gate to select live, CD45RA- cells (cells with a low fluorescence for this marker).</li>
	<li>Using an FSC-Area / CD4 dot plot, draw a rectangular gate to select CD4+ cells (cells with a high fluorescence for this marker).</li>
	<li>Using a CD4 / CXCR5 dot plot, draw a rectangular gate to select CXCR5+ (i.e., cTfh) cells (cells with a high fluorescence for this marker).</li>
	<li>Using a CXCR3 / CCR6 dot plot, place a quad gate to subdivide cTfh cells into cTfh1,2 and 17 cells (cells that are high and low for each marker). 
	NOTE: Cells with the phenotype CD4+ CXCR5+ CXCR3+ CCR6+ are poorly characterized. We refer to these cells as cTfh1/1718 because conventional helper T-cells (CD4+ CXCR5-) that are transitioning between Th17 and Th1 show expression of CXCR3 and CCR6 and have been described as cTfh1/1719.</li>
	<li>Using a PD-1 histogram, use the range tool to subdivide cTfh cells (or if preferred, the individual cTfh1,2,17 or 1/17 subsets) into PD-1-/+ or hi populations. 
	NOTE: The distinction between PD-1+ and PD-1hi is not well defined in the literature. The threshold was set as the same PD-1 intensity required to detect bona-fide Tfh in human tonsil lymphocyte suspensions using flow cytometry with the same antibody panel. Alternatively, a marker for ICOS can be added to the antibody panel, as only PD1hi cells are ICOS+.</li>
</ol>

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl6+Mediates+the+Development+of+T+Follicular+Helper+Cells.">Bcl6 Mediates the Development of T Follicular Helper Cells.</a> Science. 325 (5943), 1001-1005 (2009).</li><li>Breitfeld, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+B+Helper+T+Cells+Express+Cxc+Chemokine+Receptor+5,+Localize+to+B+Cell+Follicles,+and+Support+Immunoglobulin+Production.">Follicular B Helper T Cells Express Cxc Chemokine Receptor 5, Localize to B Cell Follicles, and Support Immunoglobulin Production.</a> The Journal of Experimental Medicine. 192 (11), 1545-1552 (2000).</li><li>Linterman, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+helper+T+cells+are+required+for+systemic+autoimmunity.">Follicular helper T cells are required for systemic autoimmunity.</a> The Journal of Experimental Medicine. 206 (3), 561-576 (2009).</li><li>Bindea, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+Dynamics+of+Intratumoral+Immune+Cells+Reveal+the+Immune+Landscape+in+Human+Cancer.">Spatiotemporal Dynamics of Intratumoral Immune Cells Reveal the Immune Landscape in Human Cancer.</a> Immunity. 39 (4), 782-795 (2013).</li><li>Gu-Trantien, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++follicular+helper+T+cell+infiltration+predicts+breast+cancer+survival.">CD4+ follicular helper T cell infiltration predicts breast cancer survival.</a> The Journal of Clinical Investigation. 123 (7), 2873-2892 (2013).</li><li>Am&#233;-Thomas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intratumoral+follicular+helper+T+cells+in+follicular+lymphoma:+role+in+the+survival+of+malignant+B+cells.">Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells.</a> Leukemia. 26 (5), 1053-1063 (2012).</li><li>Bossaller, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICOS+Deficiency+Is+Associated+with+a+Severe+Reduction+of+CXCR5+CD4+Germinal+Center+Th+Cells.">ICOS Deficiency Is Associated with a Severe Reduction of CXCR5+CD4 Germinal Center Th Cells.</a> The Journal of Immunology. 177 (7), 4927-4932 (2006).</li><li>Morita, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Blood+CXCR5+CD4++T+Cells+Are+Counterparts+of+T+Follicular+Cells+and+Contain+Specific+Subsets+that+Differentially+Support+Antibody+Secretion.">Human Blood CXCR5+CD4+ T Cells Are Counterparts of T Follicular Cells and Contain Specific Subsets that Differentially Support Antibody Secretion.</a> Immunity. 34 (1), 108-121 (2011).</li><li>Locci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+circulating+PD-1+CXCR3-CXCR5++memory+Tfh+cells+are+highly+functional+and+correlate+with+broadly+neutralizing+HIV+antibody+responses.">Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses.</a> Immunity. 39 (4), 758-769 (2013).</li><li>He, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Precursor+CCR7loPD-1hi+CXCR5++CD4++T+Cells+Indicate+Tfh+Cell+Activity+and+Promote+Antibody+Responses+upon+Antigen+Reexposure.">Circulating Precursor CCR7loPD-1hi CXCR5+ CD4+ T Cells Indicate Tfh Cell Activity and Promote Antibody Responses upon Antigen Reexposure.</a> Immunity. 39 (4), 770-781 (2013).</li><li>Schmitt, N., Bentebibel, S. -. E., Ueno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+and+functions+of+memory+Tfh+cells+in+human+blood.">Phenotype and functions of memory Tfh cells in human blood.</a> Trends in Immunology. 35 (9), 436-442 (2014).</li><li>Simpson, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+circulating+T+cells+resembling+follicular+helper+T+cells+is+a+fixed+phenotype+that+identifies+a+subset+of+severe+systemic+lupus+erythematosus.">Expansion of circulating T cells resembling follicular helper T cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus.</a> Arthritis and Rheumatism. 62 (1), 234-244 (2010).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequencies+of+activated+B+cells+and+T+follicular+helper+cells+are+correlated+with+disease+activity+in+patients+with+new-onset+rheumatoid+arthritis.">High frequencies of activated B cells and T follicular helper cells are correlated with disease activity in patients with new-onset rheumatoid arthritis.</a> Clinical and Experimental Immunology. 174 (2), 212-220 (2013).</li><li>Ueno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Circulating+T+Follicular+Helper+Cell+Subsets+in+Health+and+Disease.">Human Circulating T Follicular Helper Cell Subsets in Health and Disease.</a> Journal of Clinical Immunology. 36, 34-39 (2016).</li><li>Maecker Holden, T., Trotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+controls,+instrument+setup,+and+the+determination+of+positivity.">Flow cytometry controls, instrument setup, and the determination of positivity.</a> Cytometry Part A. 69 (9), 1037-1042 (2006).</li><li>Jia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+Function+of+CD4++T+Follicular+Helper+(Tfh)+Cells+Associated+with+Hepatocellular+Carcinoma+Progression.">Impaired Function of CD4+ T Follicular Helper (Tfh) Cells Associated with Hepatocellular Carcinoma Progression.</a> PLOS ONE. 10 (2), 0117458 (2015).</li><li>Byford, E. T., Carr, M., Ladikou, E., Ahearne, M. J., Wagner, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Tfh1+(cTfh1)+cell+numbers+and+PD1+expression+are+elevated+in+low-grade+B-cell+non-Hodgkin's+lymphoma+and+cTfh+gene+expression+is+perturbed+in+marginal+zone+lymphoma.">Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin's lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma.</a> PLOS ONE. 13 (1), 0190468 (2018).</li><li>Annunziato, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+features+of+human+Th17+cells.">Phenotypic and functional features of human Th17 cells.</a> Journal of Experimental Medicine. 204 (8), 1849-1861 (2007).</li><li>Duan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+and+function+of+CXCR5+CD45RA-CD4++T+cells+were+altered+in+HBV-related+hepatocellular+carcinoma+and+elevated+serum+CXCL13+predicted+better+prognosis.">Phenotype and function of CXCR5+CD45RA-CD4+ T cells were altered in HBV-related hepatocellular carcinoma and elevated serum CXCL13 predicted better prognosis.</a> Oncotarget. 6 (42), 44239-44253 (2015).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+CXCR5+CD4+helper+T+cells+in+systemic+lupus+erythematosus+patients+share+phenotypic+properties+with+germinal+center+follicular+helper+T+cells+and+promote+antibody+production.">Circulating CXCR5+CD4+helper T cells in systemic lupus erythematosus patients share phenotypic properties with germinal center follicular helper T cells and promote antibody production.</a> Lupus. 24 (9), 909-917 (2015).</li><li>Sage, P. 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The authors have nothing to disclose.

This protocol represents a simple and efficient way to analyze peripheral blood cTfh cells, enabling the detection of all relevant subsets identified in the literature thus far. Blood samples can be easily and efficiently obtained as part of standard out-patient clinics and serial samples can be collected in parallel with clinical data. In turn, this enables prospective studies evaluating cTfh subsets as biomarkers for disease progression or response to treatment. These studies would be particularly warranted in disease ...

T-follicular helper cells (Tfh) are a CD4+ T-cell subset that was initially characterized in lymphoid tissues1. These cells express PD-1 and CXCR5 surface receptors, secrete IL-21 and IL-4 and show nuclear expression of the transcription factor, BCL-62,3. As their name suggests, they are found in germinal centers and are essential for high affinity antibody production1.
Dysregulated Tfh responses have been implicated in disease pathogenesis, most notably autoimmune disease, where they promote the expansion of autoreactive B cells4. They also play a role in the tumor microenvironment of both solid5,6 and lymphoid cancers7. Conversely, genetic defects of surface proteins essential for Tfh function such as inducible T-cell costimulator (ICOS) result in human immunodeficiency syndromes8. CD4+ CXCR5+ cells in human peripheral blood are termed circulating T-follicular helper cells (cTfh) and are believed to be the memory compartment of Tfh cells in tissues9. The purpose of the method described here is the analysis of cTfh subsets following CD4+ cell isolation from peripheral blood samples.
Several cTfh subsets have been defined and the efficiency with which they provide B-cell help differs from one subset to another9,10,11,12. The relative proportions of these subsets are altered in a number of diseases, most prominently autoimmune disease in which there is almost always a relative increase in the more functional PD-1+/hi and/or cTfh2 or cTfh17 subsets in comparison to the less functional PD-1- and/or cTfh1 subsets12. The extent of these changes frequently associate with clinical parameters including disease activity and autoantibody titers, indicating a potential role of cTfh subset distribution as a prognostic biomarker in disease, which may reflect the activity of Tfh in lymphoid tissues9,12,13,14. Additionally, taking blood samples from participants is quick, safe and acceptable, and so allows serial monitoring for the analysis of disease progression or response to therapy.
The use of isolated CD4+ T-cells over traditional peripheral blood mononuclear cell (PBMNC) suspensions enables higher throughput flow cytometry experiments by reducing the time required to acquire a substantial number of cTfh cells for analysis. This is particularly helpful when sorting cells from rare cTfh subsets using flow activated cell sorting (FACS). To aid the efficiency, these suspensions can be cryopreserved to enable &#34;batching&#34; of samples to be used in the flow cytometry experiment. On testing, the CD4+ purity was not reduced by cryopreservation.
While different laboratories used different markers to categorise cTfh cells in the early stages of their discovery, the method presented here makes use of a unified scheme of two groups of cell-surface markers as proposed by Schmidt et al.12,15 to enable the simultaneous identification of cTfh and their nine recognized subsets in a single flow cytometry experiment.
As only cell surface markers are used, the cells do not require fixation or permeabilization, and thus can remain alive for downstream functional studies. This could be facilitated by cell sorting using FACS with the same antibody panel. This panel could be expanded to include other markers, allowing for the restrictions of the flow cytometer being used.
The analysis of multi-color flow cytometry experiments can be challenging due to the inherently subjective nature of gating on 2-dimensional dot plots, especially when cell populations do not have a clear bi-modal distribution in marker fluorescence, as is the case for cTfh cells and their subsets. For this reason, it is imperative to set up effective controls to reduce the artefacts to enable better resolution of populations and to set gating strategies confidently. As such, antibody panel design and the set-up of basic controls for a flow cytometry experiment, i.e., using compensation and FMO controls are outlined in Step 3.4.2 and 3.4.3,respectively.
All cTfh cells are defined as CD4+ CXCR5+ CD45RA-. The level of expression of the characteristic Tfh activation marker PD-1 can then be determined to identify the subsets of PD-1-, PD-1+ or PD-1hi cTfh cells. Then, using a combination of the chemokine receptors CXCR3 and CCR6, which are differentially expressed by traditional Th1,2 or 17 cells, cTfh can be characterized as cTfh1,2 or 17-like by a profile of CXCR3+ CCR6-, CXCR3- CCR6-, and CXCR3- CCR6+, respectively.
The antibody panel used by our laboratory is displayed in Table 1. The user may have to adapt their fluorophore selection to account for the laser and light filter configuration available on their local flow cytometer.
The following considerations influence the choice of fluorophores. Use bright fluorophores where possible. In particular, use the brightest available fluorophores on the dimmest (less highly expressed) markers. Dimmer markers include PD-1, CXCR3 and CCR6, and to a lesser extent, CXCR5. We specifically made use of the newer BB, BV and BUV fluorophores which provide excellent brightness and thus enable easier resolution of distinct populations of the cells.
Spread the fluorophore selection across the emission spectra as much as possible to minimize spectral overlap and thus the level of compensation required. A free, online tool that can be used to assist designing a flow cytometry panel can be found here: http://www.bdbioscienes.com/us/s/spectrumviewer. To save space on the emission spectrum, we employed a &#34;dump channel&#34; by using a viability dye with an emission wavelength that overlaps with that of APC-H7 (conjugated to CD45RA) to enable the detection (and exclusion) of both dead and/or CD45RA+ using a single detector.
Here, a protocol is presented for the isolation of peripheral blood CD4+ T-cells and their subsequent analysis by flow cytometry to determine the proportions of the different and recently described circulating subsets.

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		<tr height="20">
			<td height="20" style="height:20px;width:485px;">RosetteSep Human CD4+ T Cell Enrichment Cocktail</td>
			<td style="width:179px;">STEMCELL TECHNOLOGIES</td>
			<td style="width:119px;">15062</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ficoll-Paque PLUS</td>
			<td style="width:179px;">GE Healthcare Life Sciences</td>
			<td>17144003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">BUV395 Mouse Anti-Human CD183&#160;Clone 1C6/CXCR3</td>
			<td style="width:179px;">BD Horizon</td>
			<td>565223</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">BV711 Mouse Anti-Human CD196 (CCR6)&#160;Clone 11A9</td>
			<td style="width:179px;">BD Horizon</td>
			<td>563923</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">BV421 Rat Anti-Human CXCR5 (CD185)&#160;Clone&#160;RF8B2</td>
			<td style="width:179px;">BD Horizon</td>
			<td>562747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">BB515 Mouse Anti-Human CD4&#160;Clone &#160;RPA-T4</td>
			<td style="width:179px;">BD Horizon</td>
			<td>564419</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">PE Mouse Anti-Human CD279&#160;Clone MIH4</td>
			<td style="width:179px;">BD Pharmingen</td>
			<td>557946</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">APC-H7 Mouse Anti-Human CD45RA Clone&#160; HI100</td>
			<td style="width:179px;">BD Pharmingen</td>
			<td>560674</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">LIVE/DEAD Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation</td>
			<td style="width:179px;">ThermoFisher Scientific</td>
			<td style="width:119px;">L34973</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Brilliant Stain Buffer</td>
			<td style="width:179px;">BD Horizon</td>
			<td>563794</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td style="width:179px;">BD CompBead</td>
			<td>552843</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">FACS Aria II Flow Cytometer</td>
			<td style="width:179px;">BD Biosciences</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">FACSDiva 6.1.3</td>
			<td style="width:179px;">BD Biosciences</td>
			<td>643629</td>
			<td>Flow Cytometer Acquisition Software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">FlowJo 10.2</td>
			<td style="width:179px;">Treestar Inc.</td>
			<td style="width:119px;"></td>
			<td>Flow Cytometry Data Analysis Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CXCR3 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">565223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CCR6 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">563923</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CXCR5 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">562747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CD4 antibody</td>
			<td style="width:179px;">BD Horizon&#160;</td>
			<td style="width:119px;">564419</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-PD-1 antibody</td>
			<td style="width:179px;">BD Pharmingen&#160;</td>
			<td style="width:119px;">557946</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Anti-CD45RA antibody</td>
			<td style="width:179px;">BD Pharmingen&#160;</td>
			<td style="width:119px;">560674</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Viability Marker</td>
			<td style="width:179px;">ThermoFisher Scientific&#160;</td>
			<td style="width:119px;">L34973</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cd4+ t-cells and analysis of circulating t-follicular helper (ctfh) cell subsets from peripheral blood using 6-color flow cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

High CD4+ purity was achieved using the CD4+ isolation protocol, which was reliable across all blood samples tested by us (mean: 96.6%, SD: 2.38, n=31) (Figure 3).
Identification of cTfh (CD4+ CXCR5+ cells) in a representative normal subject is presented (Figure 4A). The proportion of total cTfh cells within C...

Watch this Scientific Journal Video about Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry at JoVE.com

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

This method can help to demonstrate the differences between circulating T-follicular helper cell subsets in healthy and diseased states.The main advantages of this technique are that it's fast, that the samples can be batch-processed, and that the cells remain alive for downstream functional analysis.This technique facilitates the evaluation of circulating T-follicular helper subsets as biomarkers for disease progression as they correlate with markers of clinical severity.Helping with the demonstration of this procedure will be Dr.Lucia Pi

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JoVE Journal

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12.0K Views.  University of Leicester. This method can help to demonstrate the differences between circulating T-follicular helper cell subsets in healthy and diseased states.The main advantages of this technique are that it's fast, that the samples can be batch-processed, and that the cells remain alive for downstream functional analysis.This technique facilitates the evaluation of circulating T-follicular helper subsets as biomarkers for disease progression as they correlate with markers of clinical severity.Helping with the dem...