Nina Nagel is gratefully acknowledged for technical assistance during the video shoot and experimental preparation. We thank Eva-Maria Weiss for help with the intracellular FOXP3 staining protocol and Elisabeth Suri-Payer and Nina Oberle for establishing the nTreg isolation protocol. Matilda Eriksson and Peri Noori are acknowledged for laboratory management. 
Funding: A.S. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme, the Dr. &#197;ke Olsson Foundation and KI research foundations; A.S. and J.T. were supported by a CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) grant, J.T. was supported by Vetenskapsr&#229;det Medicine and Health (Dnr 2011-3264), Torsten S&#246;derberg Foundation, FP7 STATegra, AFA Insurance and Stockholm County Council.

<ol>
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The authors have nothing to disclose.

The described protocol enables the robust induction of human CD4+FOXP3+ iTregs from human na&#239;ve CD4+ T cells. It includes a new protocol that we described recently, using a combination of TGF-&#946;, ATRA and rapamycin, for induction of iTregs with superior in vitro suppressive function22. Compared to other published protocols, another advantage is the induction of different iTreg populations in parallel by different protocols, which enables the direct comparison of effects of certain iTreg-inducing factors, along with control cells that are activated in the presence of IL-2 alone. The described protocols enable reproducible induction of FOXP3 with low donor variation. Na&#239;ve CD4+ T cells in this protocol are isolated by magnetic-activated cell sorting, but fluorescence-activated cell sorting is also possible. The expected yield of na&#239;ve CD4+ T cells with this protocol is typically between 5-10%, but strongly depends on the donor (age) and appears also lower when high fractions of erythrocytes are present. If an estimate is needed, PBMCs can be stained (see step 1.4) during the monocyte depletion step. Typically, the yield of na&#239;ve CD4+ T cells is about half of the &#34;percentage na&#239;ve CD4+ T cells of the lymphocyte gate&#34; in the PBMC stain. This protocol uses limited amounts of CD25 beads to obtain CD25-high (nTreg) cells29. However, these are not pure Tregs, but these are just enriched in Tregs to be used as positive control. If pure Tregs are needed, other kits should be used (such as combined with CD4 enrichment and CD127-depletion) or alternatively, CD25+ cells pre-enriched with 8 &#181;l CD25 beads per 107 cells and stained and sorted by fluorescence-activated cell sorting with stringent CD4+CD25++ gating. Also inclusion of other markers, such as CD127 exclusion, should be considered.
It is important to consider that FOXP3 is necessary, but not sufficient to confer Treg identity30. While iTregs can be used to study certain aspects of, for example, FOXP3 regulation, it is important however to note that iTregs differ from nTregs in several aspects. It is therefore crucial to culture nTregs (ideally derived from the same donor) in parallel to iTregs in all assays for comparison. The difference between nTregs and iTregs is exemplified by only partial overlap of the nTreg and iTreg transcriptome as measured in murine iTregs31. Other Treg signature genes in addition to FOXP3 should be measured for this reason, and to ensure discrimination from activation-induced FOXP3 expression in human cells. For example, iTregs should display higher expression of CD25, CTLA-4 and EOS compared to activated T cells while expression of IFN-&#947; and SATB1 should be low in nTregs and iTregs induced by the protocols described here, as published previously22. Another important major difference between nTregs and iTregs is the lack of stable FOXP3 expression in iTregs, which most likely corresponds to methylation of the TSDR region in the FOXP3 locus in iTregs13. Also on a genome-wide scale, it was described that epigenetic patterns of DNA methylation and histone modifications in murine iTregs do not reflect the patterns found in nTregs30. We previously described that iTregs induced by the here described protocols, in contrast to nTregs, did not exhibit TSDR demethylation. Accordingly, iTregs lost FOXP3 when restimulated, but maintained FOXP3 expression when further cultured in the presence of IL-2 and without restimulation22. Interestingly, iTregs induced by an alternative protocol using M2 macrophage supernatants displayed enhanced FOXP3 stability, despite lack of TSDR demethylation and TGF-&#946; being causative for FOXP3 induction27.
Modification of iTreg-inducing protocols by addition of compounds (such as Vitamin C or hydrogen sulfide) that influence Ten-eleven Translocation (TET) methylcytosine dioxygenase enzymes, as described very recently32-34, may add in stabilizing FOXP3 by affecting DNA methylation. Also, the stimulation strength and timing has an influence on FOXP3 expression and stability35. Along these lines, the use of bead-coupled CD3/CD28 antibodies instead of plate-bound antibodies was shown to increase murine iTreg in vivo suppressive function and stability albeit independent of TSDR demethylation36. Another factor that needs to be considered as a potential source of variation is the use of serum, which contains undefined factors including TGF-&#946; which even from bovine source is 100% cross-reactive with human cells. Also, the source and activity of IL-2 can drastically influence the results of FOXP3 induction.
An important feature of Tregs is their suppressive ability, which needs to be tested subsequently with iTregs generated in this protocol. It should be noted that suppression assays with iTregs are not trivial and the literature about suppressive abilities of iTregs is controversial. Several methods and protocols to assess suppressive function of human Tregs have been published elsewhere22,37-41, with in vitro proliferation assays based on flow cytometry readouts such as dilution of Carboxyfluorescein succinimidyl ester (CFSE) being the most commonly used. It is important to wash and rest iTregs before use in suppression assays, and it needs to be considered that iTregs, in contrast to nTregs, may not be anergic but proliferate themselves during suppression assays. We consider it extremely important to use `mock&#180; stimulated T cells as control suppressor cells to identify the degree of unspecific suppression (such as through CTLA-4 expression, IL-2 consumption and culture overgrowth by activated T cells) that is unrelated to FOXP3+ Treg-specific effects, and the frequent lack of this control may contribute to some controversies in the literature. Using this control, we defined that only TGF-&#946;/ATRA/Rapa-induced iTregs displayed suppressive activity in vitro22. Thus, we conclude that regarding suppressive activity (albeit not highest fraction of FOXP3+ cells), TGF-&#946;/ATRA/Rapa is the best combination of factors of the described protocols to induce iTregs. Nevertheless, suppressive activity in vitro does not necessarily reflect suppressive activity in vivo, and indeed we determined that human iTregs generated by these protocols did not suppress in a xenogeneic graft-versus-host disease model at least under the conditions tested22. This may be related to instable FOXP3 expression which was lost in iTregs upon restimulation, in line with a lack of TSDR demethylation22.
Depending on which aspect of iTreg features (high fraction of FOXP3+ cells, superior suppressive activity, FOXP3 stability) is most important for a particular research question, different protocols may be most suitable to study these questions, rendering it difficult to define the generally &#39;best&#39; protocol for Treg induction. Furthermore, several above-described subtle experimental differences can influence results and may contribute to controversies with respect to phenotype and suppressive function of TGF-&#946;-induced iTregs that appear between reports even with apparently similar protocols for iTreg generation42. For example, even within one laboratory, we observed that iTregs induced with TGF-&#946; and IL-2 in serum-containing RPMI medium displayed some suppressive activity compared to control cells27, while TGF-&#946;/IL-2-induced iTregs generated in defined, serum-free T cell culture medium did not22, despite similar levels of FOXP3.
Future applications based on these protocols should strive to further optimize Treg induction conditions to achieve a phenotype with stable FOXP3 expression, TSDR demethylation, stable phenotype without conversion to cytokine-producing effector T cells and optimal suppressive activity. Further development of iTreg induction protocols may be useful for adoptive transfer approaches in the future, in which therapy by Treg transfer is highly promising for the potential treatment of autoimmune and inflammatory diseases.

CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance, preventing autoimmunity and excessive inflammation1. The importance of Tregs is exemplified by the human disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), in which loss of Tregs due to mutations in the `master&#180; Treg transcription factor forkhead box P3 (FOXP3) leads to severe systemic autoimmune disease, lethal at an early age. However, Tregs act as a double-edged sword in the immune system as they can also hamper anti-tumor immunity in certain settings2. Therapeutic manipulation of Treg number and function is therefore subject to numerous clinical investigations. In cancer, depletion of Tregs can be desirable and some success of clinical approaches encourages further research3. In autoimmune and inflammatory diseases, in addition to therapeutic effects of Tregs in several mouse disease models, recent first in-man trials of adoptive Treg transfer to prevent graft-versus-host disease (GvHD)4-7 and to assess safety in treating type 1 diabetes8 showed very promising outcomes.
Naturally occurring Tregs (nTregs) comprise thymic-derived tTregs and peripherally induced pTregs, with non-redundant essential functions in maintaining health9-11. However, nTreg numbers are limited, encouraging the complementary approach of inducing Tregs (iTregs) in vitro from na&#239;ve T cell precursors12. Still stability of iTregs, presumably due to lack of demethylation in the so-called Treg-specific demethylated region (TSDR) in the FOXP3 gene locus13, remains a concern and several studies indicate that in vivo induced Tregs are more stable14.
To date, FOXP3 remains the best protein marker for Tregs but it is not absolutely specific because human conventional CD4+CD25- T cells transiently express intermediate levels of FOXP3 upon activation15,16. Although significant progress has been made in elucidating the regulation of FOXP3 expression, much remains to be discovered regarding the induction, stability and function of FOXP3 particularly in human cells. Despite differences to nTregs, in vitro induced FOXP3+ CD4+ T cells can be used as a model system to study molecular mechanisms of FOXP3 induction and as a starting point to develop protocols in the future that allow for generation of iTregs that are more similar to in vivo generated Tregs, which could be applicable for adoptive transfer strategies in the future.
There is no `gold standard&#180; protocol to induce human iTregs, and current protocols have been developed based on mimicking Treg-inducing conditions in vivo: interleukin 2 (IL-2) and transforming growth factor &#946; (TGF-&#946;) signaling are crucial for FOXP3 induction in vivo17, and all-trans retinoic acid (ATRA) &#8212; which is produced in vivo by gut-associated dendritic cells &#8212; is frequently used to enhance FOXP3 induction in vitro18-21. We have developed additional human Treg-inducing protocols using butyrate22, a gut microbiota-derived short-chain fatty acid that was recently shown to augment murine Treg induction23,24. We also recently established a new protocol for generation of iTregs with superior suppressive function in vitro by using a combination of TGF-&#946;, ATRA and rapamycin22, the latter being a clinically approved mammalian target of rapamycin (mTOR) inhibitor that is known to promote FOXP3 maintenance during human Treg expansion25,26.
This method describes the reproducible in vitro generation of human CD4+FOXP3+ iTregs using a set of different conditions, and their subsequent phenotyping by flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to reveal protocol-specific patterns of expression of FOXP3 and other Treg signature molecules such as CD25, CTLA-4, EOS, as well as repression of IFN-&#947; and SATB1 expression22. The generated cell populations can be used for functional assays regarding suppressive activity or for molecular studies, either concerning general FOXP3 regulators or to study effects specific to certain compounds such as butyrate or rapamycin. Further understanding of molecular mechanisms driving Treg differentiation is highly relevant for future therapeutic approaches in autoimmunity or cancer to specifically target molecules involved in Treg generation and function.

<table><tbody><tr><td>All-trans retinoic acid</td><td>Sigma Aldrich</td><td>R2625-50MG&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>anit-human Foxp3-APC clone 236A/E7</td><td>eBioscience</td><td>17-4777-42</td><td></td></tr><tr><td>anti-human CD25 microbeads</td><td>Miltenyi Biotec</td><td>130-092-983</td><td></td></tr><tr><td>anti-human CD25-PE</td><td>Miltenyi Biotec</td><td>130-091-024</td><td></td></tr><tr><td>anti-human CD28 antibody, LEAF Purified&amp;nbsp;</td><td>Biolegend</td><td>302914</td><td></td></tr><tr><td>anti-human CD3 Antibody, LEAF Purified&amp;nbsp;</td><td>Biolegend</td><td>317315</td><td></td></tr><tr><td>anti-human CD45RA, FITC</td><td>Miltenyi Biotec</td><td>130-092-247</td><td></td></tr><tr><td>anti-human CD45RO PE clone UCHL1</td><td>BD Biosciences</td><td>555493</td><td></td></tr><tr><td>anti-human CD4-PerCP clone SK3; mIgG1</td><td>BD Biosciences</td><td>345770</td><td></td></tr><tr><td>anti-human CD8-eFluor 450 (clone OKT8), mIgG2a</td><td>eBioscience</td><td>48-0086-42&amp;nbsp;</td><td></td></tr><tr><td>anti-human CTLA-4 (CD152), clone BNI3, mIgG2ak, Brilliant violet 421</td><td>BD Biosciences</td><td>562743</td><td></td></tr><tr><td>anti-human IFN-g FITC clone 4S.B3; mIgG1k</td><td>eBioscience</td><td>11-7319-81&amp;nbsp;</td><td></td></tr><tr><td>Brefeldin A-containing solution: GolgiPlug</td><td>BD Biosciences</td><td>555029</td><td></td></tr><tr><td>cDNA synthesis kit: SuperScript VILO(Reverse transcriptase) cDNA Synthesis Kit</td><td>Invitrogen</td><td>11754-250</td><td></td></tr><tr><td>Density centrifugation medium: Ficoll-Paque</td><td>GE healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>DMSO 99.7%</td><td>Sigma Aldrich</td><td>D2650-5X5ML</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>Invitrogen</td><td>10082-147</td><td></td></tr><tr><td>Fixable Viability Dye, eFluor 780&amp;nbsp;&amp;nbsp;</td><td>eBioscience</td><td>65-0865-14 or 65-0865-18&amp;nbsp;</td><td></td></tr><tr><td>Foxp3 Staining Buffer Set</td><td>eBioscience</td><td>00-5523-00</td><td>Caution: contains paraformaldehyde. Can be also bought in combined kit with antibody; 77-5774-40 Anti-Human Foxp3 Staining Set APC Clone: 236A/E7 Set</td></tr><tr><td>GlutaMAX (200 mM L-alanyl-L-glutamine)</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>human naive CD4 T cell isolation kit II</td><td>Miltenyi Biotec</td><td>130-094-131</td><td></td></tr><tr><td>Human serum albumin 50 g/L</td><td>Baxter</td><td>1501057</td><td></td></tr><tr><td>Ionomycin from Streptomyces conglobatus &gt;98%</td><td>Sigma Aldrich</td><td>I9657-1MG</td><td></td></tr><tr><td>MACS LS-columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>mouse IgG1 K Isotype Control APC Clone: P3.6.2.8.1</td><td>eBioscience</td><td>17-4714-42</td><td></td></tr><tr><td>mouse IgG1 K Isotype Control FITC 50 &amp;#956;g</td><td>eBioscience</td><td>11-4714-81&amp;nbsp;</td><td></td></tr><tr><td>mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173</td><td>BD Biosciences</td><td>563464</td><td></td></tr><tr><td>Pasteur pipet plastic, individually packed</td><td>Sarstedt</td><td>86.1172.001&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>PMA Phorbol 12-myristate 13-acetate</td><td>Sigma Aldrich</td><td>P1585-1MG&amp;nbsp;</td><td></td></tr><tr><td>Rapamycin</td><td>EMD (Merck)</td><td>553210-100UG</td><td></td></tr><tr><td>Recombinant Human IL-2, CF</td><td>R&amp;amp;D</td><td>202-IL-050/CF</td><td></td></tr><tr><td>Recombinant Human TGF-beta 1, CF</td><td>RnD</td><td>240-B-010/CF</td><td></td></tr><tr><td>RNA isolation kit: RNAqueous-Micro Kit</td><td>Ambion</td><td>AM1931&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>RPMI 1640 Medium&amp;nbsp;</td><td>Invitrogen</td><td>72400-054&amp;nbsp;</td><td></td></tr><tr><td>Sodium butyrate</td><td>Sigma Aldrich</td><td>B5887-250MG&amp;nbsp;</td><td></td></tr><tr><td>T cell culture medium: X-Vivo 15 medium, with gentamicin+phenolred</td><td>Lonza</td><td>04-418Q</td><td></td></tr><tr><td>TaqMan Gene Expression Assay, FOXP3 (Best Coverage)&amp;nbsp;</td><td>Applied Biosystems</td><td>4331182; assay ID: Hs01085834_m1</td><td>Caution: contains paraformaldehyde</td></tr><tr><td>TaqMan Gene Expression Assay, RPL13A (Best Coverage)</td><td>Applied Biosystems</td><td>4351370; assay ID: Hs04194366_g1&amp;nbsp;&amp;nbsp;</td><td>Caution: contains paraformaldehyde</td></tr><tr><td>TaqMan Gene Expression Master mix</td><td>Applied Biosystems</td><td>4369514</td><td></td></tr></tbody></table>

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.

Figure 1 shows a scheme of the experimental setup. Figure 2 shows a representative purity control staining for magnetically isolated na&#239;ve CD4+ T cells and nTregs.
Figure 3A shows the flow cytometry gating strategy and Figure 3B shows representative FOXP3 and CD25 flow cytometry stainings on day 6 of culture under the indicated iTreg or control conditions. Upon in vitro stimulation, most cells upregulate CD25, which is reduced in the presence of rapamycin. Only under addition of iTreg-inducing factors, a clear population of FOXP3+ cells becomes apparent, which is also enriched within CD25+ cells under iTreg conditions. Figure 3C depicts the phenotypic appearance of iTreg cultures in the microscope with proliferating cells seen as dark clusters. Each iTreg condition shows a specific and reproducible pattern of cell proliferation: Under these culture conditions, TGF-&#946; increases proliferation slightly, and ATRA further increases proliferation. At the same time, the inhibition of proliferation by rapamycin is apparent by the strongly reduced cluster size. The microscopic appearance of proliferation strength also corresponds to total cell counts (data not included).
Figure 4 displays representative results of qRT-PCR analyses of FOXP3 mRNA induction in iTreg (and control) cultures at different time points. As for FOXP3 protein, FOXP3 mRNA expression is higher in all iTregs compared to control stimulated cells, with rapamycin-treated iTregs having relatively low levels compared to other iTregs. FOXP3 mRNA in nTregs is higher than in iTregs.
<img alt="Figure 1" src="/files/ftp_upload/55015/55015fig1.jpg" /> 
Figure 1: Experimental scheme for iTreg induction, nTreg isolation, and Treg analysis. Human na&#239;ve CD4+ T cells were isolated from buffy coats and stimulated in serum-free medium with anti-CD3 and anti-CD28 antibodies plus 100 U/ml IL-2 for up to 6 days, either in the absence (`mock control cells&#180;) or presence of different Treg-inducing factors (`iTreg&#180;) as indicated. nTregs are isolated and assayed in parallel. Phenotypic analysis can be done by flow cytometry and qRT-PCR. Modified from Schmidt, A. et al. 201622. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55015/55015fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55015/55015fig2.jpg" /> 
Figure 2: Cell purity of starting populations. Na&#239;ve human CD4+ T cells were isolated by the Na&#239;ve CD4 T Cell Isolation Kit II, human. Upper panel: Na&#239;ve CD4+ T cell purity, based on CD4, CD45RA and CD45RO, was 94 to 98% and the purity for a representative donor of more than 30 is shown (`Tna&#239;ve&#180;). Ex vivo Tregs (`nTreg&#180;) were isolated by using limited amounts of CD25 microbeads and used as a positive control for iTreg experiments. nTreg purity of a representative donor, based on CD25 and CD4, is shown. Lower panel: Intracellular FOXP3 staining (performed as in Figure 3 and pre-gated on live CD4+ cells) is shown for na&#239;ve CD4+ T cells and nTregs respectively, using cells from the same donor as in the upper panel. Modified from Schmidt, A. et al. 201622. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55015/55015fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55015/55015fig3.jpg" /> 
Figure 3: Phenotypic appearance of iTregs and nTregs. Human na&#239;ve CD4+ T cells were cultured in serum-free medium under the indicated conditions. T cells were stimulated with anti-CD3 and anti-CD28 antibodies plus 100 U/ml IL-2 (`Stim.&#180;). Where indicated, TGF-&#946;1 (`TGF&#180;), rapamycin (`Rapa&#180;), all-trans retinoic acid (`ATRA&#180;) or butyrate were added. nTregs (ex vivo isolated peripheral blood CD25++ cells) were left unstimulated (`unstim.&#180;) and used as positive control. Unstimulated na&#239;ve T cells were used as negative control. (A) On day 6 of culture, cells were stained with surface antibodies including anti-CD25 and anti-CD4, then stained with fixable viability dye and subsequently fixed/permeabilized and stained intracellularly with anti-FOXP3 and anti-CTLA-4 or isotype control antibodies. Acquisition and compensation was performed on a Cyan ADP flow cytometer and the data were analyzed with the FlowJo software. The gating strategy is indicated by the red arrows, and the shown example is an iTreg sample induced with TGF+ATRA. (B) Cells were stained as in (A), and FOXP3 and CD25 expression in control T cells and iTregs induced by different protocols is shown. The pseudocolor plots show representative FOXP3 and CD25 stainings for one donor, pre-gated on singlet, live CD4+ cells as in (A). The isotype example is shown for an iTreg (stim.+TGF+ATRA) sample. (C) Representative microscopy images (40X magnification) of individual 96U-plate wells of cells cultured for 4 days. Dark clusters of cells represent proliferating cells. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55015/55015fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55015/55015fig4.jpg" /> 
Figure 4: FOXP3 mRNA expression upon use of different protocols for iTreg differentiation. iTregs were generated as in Figure 3 under the indicated conditions, and at the given time points, cell samples were taken and RNA was extracted. Unstimulated na&#239;ve T cells, and unstimulated nTregs, were sampled on day 0. Samples were analyzed by qRT-PCR, and FOXP3 mRNA expression was normalized to RPL13A expression for each sample. FOXP3 mRNA expression in unstimulated na&#239;ve T cells was set to 1, and fold change of FOXP3 mRNA was calculated. Shown are mean values and range of PCR replicate wells for a representative donor. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55015/55015fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Stock concentration</td>
			<td>Endconcentration</td>
			<td>4x premix concentration</td>
			<td>Stock dilution to obtain 4x premix 
			(dilute stock in T cell culture medium)</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>400 000 IU/mL&#160;</td>
			<td>100 IU/mL</td>
			<td>400 IU/mL IL-2&#160;</td>
			<td>&#160;1:1000</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD28</td>
			<td>1 mg/ml</td>
			<td>1 &#181;g/mL</td>
			<td>4 &#181;g/mL&#160;</td>
			<td>&#160;1:250</td>
			<td>Add within IL-2 4x premix</td>
		</tr>
		<tr>
			<td>TGF-&#946;1</td>
			<td>100 &#181;g/mL</td>
			<td>5 ng/mL</td>
			<td>20 ng/mL&#160;</td>
			<td>&#160;1:5000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>ATRA</td>
			<td>10 mM&#160;</td>
			<td>10 nM</td>
			<td>40 nM&#160;</td>
			<td>&#160;1:250000</td>
			<td>Pre-dilute stock solution first to enable pipettable volumes&#160;</td>
		</tr>
		<tr>
			<td>Rapamycin</td>
			<td>1 mg/mL&#160;</td>
			<td>100 ng/mL&#160;</td>
			<td>400 ng/mL&#160;</td>
			<td>&#160;1:2500&#160;</td>
			<td>If used together with ATRA, dilute Rapamycin within the 4x ATRA stimulation mix</td>
		</tr>
		<tr>
			<td>Butyrate</td>
			<td>0.908 M&#160;</td>
			<td>0.1 mM</td>
			<td>0.4 mM&#160;</td>
			<td>&#160;1:2270&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1

Watch this Scientific Journal Video about In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃ¯ve CD4+ T Cells Using a TGF-Î²-containing Protocol at JoVE.com

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

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 ...

in-vitro-differentiation-human-cd4-foxp3-induced-regulatory-t-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.2K Views.  Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory.  Dit protocol beschrijft de reproduceerbare productie en fenotypering van mens veroorzaakte regulerende T-cellen (iTregs) van naïeve CD4 + T-cellen in vitro. Verschillende protocollen voor FOXP3 inductie mogelijk maken voor de studie van specifieke iTreg fenotypes verkregen met de respectieve protocollen. 

Video: In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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&#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 Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

Th17 Cell Differentiation from Naive CD4+ T-Cells Using Flow Cytometry

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

Author Spotlight: Achieving High-Purity In Vitro Differentiation of Th17 Cells Using Cytokine Concentration Modulation

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

In Vitro Differentiation of Human CD4⁺FOXP3⁺ Induced Regulatory T Cells (iTregs) from Naïve CD4⁺ T Cells Using a TGF-β-containing Protocol

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