Identification of Circulating Human -Reactive CD4 +FOXP3+ Natural Regulatory T Cells

This information is current as Nicolle H. R. Litjens, Karin Boer and Michiel G. H. Betjes of September 27, 2021. J Immunol 2012; 188:1083-1090; Prepublished online 21 December 2011; doi: 10.4049/jimmunol.1101974 http://www.jimmunol.org/content/188/3/1083 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2011/12/21/jimmunol.110197 Material 4.DC1

References This article cites 24 articles, 7 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/188/3/1083.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Identification of Circulating Human Antigen-Reactive CD4+ FOXP3+ Natural Regulatory T Cells

Nicolle H. R. Litjens, Karin Boer, and Michiel G. H. Betjes

Circulating human CD4+CD25++CD1272FOXP3+ T cells with a persistent demethylated regulatory (Treg)-specific deme- thylated region Foxp3 gene are considered natural Tregs (nTregs). We have shown that it is possible to identify functional Ag- reactive nTregs cells for a range of different common viral and vaccination Ags. The frequency of these Ag-reactive nTregs within the nTreg population is strikingly similar to the frequency of Ag-reactive T effector cells within the CD4+ T cell population. The Ag-reactive nTregs could be recognized with great specificity by induction of CD154 expression. These CD154+ Ag-reactive nTregs showed a memory phenotype and shared all phenotypical and functional characteristics of nTregs. The isolated CD154+ nTregs could be most efficiently expanded by specific antigenic stimulation, while their Ag-reactive suppressive activity was maintained.

After an in vivo booster Ag challenge, the ratio of Ag-reactive T cells to Ag-reactive Tregs increased substantially, which could be Downloaded from attributed to the rise in effector T cells but not Tregs. In conclusion, the nTreg population mirrors the effector T cell population in the frequency of Ag-reactive T cells. Isolation and expansion of functional Ag-reactive nTregs is possible and of potential benefit for specific therapeutic goals. The Journal of Immunology, 2012, 188: 1083–1090.

egulatory CD4+ T cells (Tregs) are considered to be high levels of CD25 and low levels of CD127 (FOXP3+CD25++ 2 + crucial for the suppression of excessive T cell responses CD127 CD4 T cells) (7). Recently, it has been shown that this http://www.jimmunol.org/ R that may cause damage to the organism. FOXP3 is a key cell population is composed of Tregs with a naive and memory molecule for identification of suppressive function of Tregs. De- T cell phenotype based on differential expression of the CD45 ficiency or dysfunction of FOXP3+ Tregs in humans and Foxp3 isoforms CD45RO (memory phenotype) and CD45RA (naive Tregs in rodents is associated with T cell-mediated autoimmune phenotype) (8, 9). The memory Tregs behave similar to memory diseases, allergies, and inflammatory bowel disease. Additionally, cells of effector CD4+ T cells (Teff), as they have short telomeres Tregs may be important in establishing tolerance to organ trans- and a remarkably high turnover rate (10). Sequence-based T cell plants (1). clonotype tracing of human peripheral blood and transfer of The ontogeny of Tregs may involve several differentiation path- CD45RA+ Tregs into NOD/SCID/common g-chain–deficient mice ways, with thymus-derived and induced Tregs (iTregs) being the showed conversion of these CD45RA+ Tregs into CD45RA2 by guest on September 27, 2021 main ones. Tregs generated within the thymus are called natural Foxp3+ cells (8). Tregs (nTregs) (2). Additionally, at least in vitro Tregs may be An unresolved issue that remains is whether Ag-reactive Tregs induced from a FOXP32 T cell population using the appropriate are present within the human nTreg population, as such cells cannot milieu, including TGF-b. These iTregs are phenotypi- be directly identified. Previous studies have indicated the existence cally indistinguishable from nTregs, are FOXP3+, and can be of HIV and CMV Ag-reactive Tregs in the peripheral blood but generated as Ag-reactive T cells as opposed to nTregs (4). Re- failed to show their existence on the single cell level. It has been cently, demethylation at a highly conserved region within the postulated that these Ag-reactive Tregs may originate from acti- FOXP3 gene (Treg-specific demethylated region [TSDR]) was vated CD4+ Teff and are in fact iTregs (7). found to discriminate between natural Tregs (highly demethylated Alternatively, Ag-reactive Tregs could stem from conversion of TSDR) and induced Tregs (highly methylated TSDR) (5, 6). naive FOXP3+ nTregs into memory nTregs, in a similar fashion Within the peripheral blood of healthy humans a relative stable as the Ag-reactive memory CD4+ Teff develop from FOXP32 population of Tregs can be recognized that comprise ∼5–10% of naive CD4+ T cells (11). Identification on the single cell level of the total CD4+ T cell population. These FOXP3+ cells express Ag-reactive nTregs would allow for tracking Ag-reactive respon- ses. Isolation and propagation of such Ag-reactive nTregs could be Division of Nephrology and Transplantation, Department of Internal Medicine, Eras- highly useful for immunotherapy, as this would avoid any un- mus Medical Center, 3000 CA Rotterdam, The Netherlands wanted generalized suppression of immune responses (12). Pre- Received for publication July 8, 2011. Accepted for publication November 18, 2011. viously, we have used membrane-bound CD154 (CD40L) expres- Address correspondence and reprint requests to Dr. Michiel G.H. Betjes, Division of sion in a live cell assay to detect and isolate human Ag-reactive Nephrology, Department of Internal Medicine, Erasmus Medical Center, H438, P.O. CD4+ Teff with high sensitivity and specificity (13, 14). In this Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail address: m.g.h.betjes@ erasmusmc.nl study we followed this strategy to show the presence and char- acteristics of Ag-reactive nTregs. N.H.R.L. designed and conducted the experiments and wrote the paper; K.B. con- ducted experiments; and M.G.H.B. designed the experiments and wrote the paper. The online version of this article contains supplemental material. Materials and Methods Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; HBs Ag, hepatitis Blood samples B surface Ag; iTreg, induced regulatory T cell; MFI, mean fluorescence intensity; nTreg, natural regulatory T cell; Teff, effector T cell; Treg, regulatory T cell; TSDR, PBMCs were isolated from healthy donor-derived buffy coats obtained from regulatory T cell-specific demethylated region; VZV, varicella-zoster virus. the local blood bank (Sanquin Blood Bank, Rotterdam, The Netherlands), as described before in detail (14). The vaccination of healthy volunteers was Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 performed with standard doses of tetanus toxoid vaccine and hepatitis B www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101974 1084 Ag-REACTIVE HUMAN Tregs surface Ag (HBs Ag) with weekly isolation of PBMCs thereafter, using the CD4+ Teff in individuals that were tested seropositive for CMVAg (34 mg/ protocols published previously (15, 16). All individuals included gave ml), varicella-zoster virus (VZV) Ag (30 mg/ml; Microbix Systems), or informed consent, and the local Medical Ethical Committee approved the were previously immunized against hepatitis B virus (5 mg/ml HBs Ag; Bio- study. It was conducted according to the principles of the Declaration of connect, Huissen, The Netherlands) or tetanus toxoid (37.5 Lf/ml; Dutch Helsinki and in compliance with International Conference on Harmonization/ National Institute for Public Health and the Environment, Bilthoven, The Good Clinical Practice regulations. Netherlands). Similar as described above, the percentages of Ag-reactive CD154-expressing Tregs and CD4+ Teff were corrected for the background Cell sorting experiments signal. PBMCs were enriched for T lymphocytes using the Pan T cell isolation Expansion of sorted fractions (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched T lymphocytes were prepared for sorting experiments using the following mAbs: AmCyan- Tregs or the CD154-enriched and CD154-depleted Tregs (at 1 3 104/well labeled CD3 (BD Pharmingen, Erembodegem, Belgium), Pacific Blue- for polyclonal expansion and 2.5 3 104/well for Ag-reactive expansion) labeled CD4 (BD Pharmingen), PeCy7-labeled CD25 (epitope A; BD were expanded for 7–10 d using either CD3/CD28 T cell expander Pharmingen), PE-labeled CD127 (BD Pharmingen), and inclusion of a live/ dynabeads (Invitrogen Dynal, Oslo, Norway) at a 1:1 ratio or CMV Ag- dead marker ViaProbe (7-aminoactinomycin D [7-AAD]; BD Pharmingen). loaded (34 mg/ml) irradiated (30 Gy) autologous PBMCs (1 3 105/well). After staining, the cells were washed and resuspended at 20–25 3 106/ml Expansion was performed in standard culture medium supplemented with and sorted (BD FACSAria II SORP; BD Biosciences) into CD3+CD4+ (in 25 IU/ml recombinant human IL-2 (Alloga, Veghel, The Netherlands) and some cases, in particular for proliferation experiments, we selected for 10 ng/ml recombinant human IL-15 (PeproTech, Rocky Hill, NJ). Every CD3+ only) T cells, Tregs (CD3+CD4+CD25++CD1272), and Teff depleted 3 d, fresh culture medium supplemented with was added to the for Tregs (CD4+ Teff). For proliferation experiments, we used CD3+ wells. T cells depleted of Tregs (CD3+ Teff). The isolated fractions were tested for purity, that is, CD4+ T cells (99.7 6 0.07%), Tregs (97.6 6 0.34%), and Suppression assay + + 6 Downloaded from CD3 (CD4 )Teff (98.7 0.33%), and were either used immediately or, Suppressive capacity was tested using the freshly isolated, the Ag-reactive, following an overnight rest at 37˚C in standard culture medium containing and/or the polyclonal expanded Treg fractions (unseparated or CD154- 20% of heat-inactivated pooled human serum, frozen until further use in enriched and CD154-depleted). Briefly, Tregs were transferred in tripli- standard medium containing a final concentration of 10% of DMSO. cate to the wells and used at different ratios (i.e., 1:5 to 1:2560). Subse- quently, 5 3 104 CD3+ Teff (responder cells) were added in triplicate to the Phenotype of isolated fractions wells. Irradiated (40 Gy) autologous PBMCs either loaded with the dif- To compare the phenotype of Tregs with that of CD4+ Teff, we stained ferent Ags—that is, CMV Ag (34 mg/ml), VZV Ag (30 mg/ml), HBs Ag (5 http://www.jimmunol.org/ intracellularly for FOXP3 using the FOXP3 PE-staining kit (Bender mg/ml), Tet-Tox (37.5 Lf/ml), and PHA (1 mg/ml; Roche Diagnostics, MedSystems, Vienna, Austria) according to the manufacturer’s instruction. Mannheim, Germany)—or not were used as stimulators and used at a 4 3 We also dissected Tregs and CD4+ Teff into the different T cell subsets, concentration of 5 3 10 /well. At day 5, [ H]thymidine was added and that is, naive (CD45RO2CCR7+), central memory (CD45RO+CCR7+), and following 16–18 h incorporation, the plates were harvested, and radioac- effector memory (CD45RO+CCR72) T cells (17), using allophycocyanin- tivity was counted using a liquid scintillation counter. The cpm or pro- labeled CD45RO (BD Pharmingen) and FITC-labeled CCR7 (R&D Sys- liferation in presence of Tregs was related to proliferation in the absence of tems Europe, Abingdon, U.K.). Tregs and depicted as percentages. Methylation analysis for TSDR FOXP3 Cytokine-producing capacity Genomic DNA from total PBMCs and FACS-sorted fractions was isolated Percentages of cytokine-producing cells were determined by stimulating + by guest on September 27, 2021 using the QIAamp DNA Micro kit (Qiagen Benelux, Venlo, The Nether- both Tregs and CD4 Teff either in the presence of costimulation only lands) following the protocol for small volumes of blood. Bisulfite treatment (anti-CD28 and anti-CD49d both at 1 mg/ml) or with the combination of using the EZ DNA Methylation-Direct kit (Zymo Research from BaseClear, PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO) and ionomycin (1 mg/ml; Leiden, The Netherlands) according to the manufacturer’s instructions Sigma-Aldrich) for 24 h, of which the last 12 h were in the presence of converted unmethylated cytosines into uracil, whereas methylated cyto- GolgiPlug (BD Biosciences). Subsequently, the cells were harvested and sines remained unchanged. The TSDR of FOXP3 was amplified by quan- the cell surface was stained using Abs directed against CD3, CD4, and a titative PCR using the StepOnePlus real-time PCR system (Applied Bio- viability marker (7-AAD). Following fixation and permeabilization, in- systems Europe, Nieuwerkerk aan den IJssel, The Netherlands) with tracellular staining was performed using mAbs directed against FOXP3 methylation- and demethylation-specific primers and probes (6). The (Bender MedSystems), IL-2, IFN-g, or TNF-a (the last three from BD percentage of TSDR demethylation was calculated as the ratio of unme- Pharmingen) (18, 19). Percentages of cytokine-producing cells within the thylated DNA copy numbers and the sum of methylated and unmethylated Tregs were performed in the context of FOXP3 expression. DNA copy numbers. Statistical analyses + Kinetics of CD154 and CD137 expression on Tregs and CD4 For comparisons between groups, the t test, Mann–Whitney U test, one- Teff upon CMV Ag stimulation way ANOVA, or Kruskal–Wallis test was used, as appropriate. Post hoc analysis was performed using the Bonferroni test for multiple comparisons + 3 5 Isolated Tregs and CD4 Teff (1 10 ) were stimulated in triplicate/ or the Mann–Whitney U test. A p value of ,0.05 for two sides was duplicate in 96-well plates for 6, 12, 18, 24, 30, 36, and 48 h with au- considered statistically significant. tologous PBMCs (1 3 105) either loaded with CMV Ag (34 mg/ml; Microbix Biosystems, Toronto, ON, Canada) or not (background). Stim- ulation was done in the presence of costimulation, that is, Abs directed Results against CD28 (1 mg/ml; Serotec/Morphosys, Duesseldorf, Germany) and CD49d (1 mg/ml; BD Pharmingen). Anti-CD40 (1 mg/ml, clone 14G7; Ag-reactive Tregs can be detected by CD154 expression in Sanquin, Amsterdam, The Netherlands) was added when CD154 was a live cell assay stained on the cell surface. The autologous PBMCs that were used as APCs Typically, Ag-reactive T cells are found at very low frequencies were labeled with 5 mM CFSE (Molecular Probes Europe, Leiden, The Netherlands) for 10 min at 37˚C. At the different time points, cells were (1:10,000 or less of total circulating T cells) in the peripheral blood harvested and stained to determine percentages of CD154- and CD137- of healthy individuals. The exception to this rule is CMV Ag- expressing cells, respectively. Percentages of CD154 or CD137, induced reactive T cells that can be detected at a much higher frequency by CMV Ag-loaded autologous CFSE-labeled PBMCs, were corrected for of 1:1000 to 1:100. Therefore, we started our study with CMVAg, the background (i.e., autologous CFSE-labeled PBMCs not loaded with Ags) (14). as this allowed for relatively easy detection and isolation of Ag- reactive T cells, using the CD154 live cell assay. PBMCs were + CD154 signal on Tregs and CD4 Teff upon stimulation with enriched for T cells and the viable CD4+T cells were sorted in different viral and vaccination Ags a Teff (CD4+ Teff/CD3+CD4+CD252/dimCD127+/2) and Treg + + bright 2 Upon a 24-h stimulation with different viral and vaccination Ags, we (CD3 CD4 CD25 CD127 ) population based on the differ- determined percentages of Ag-reactive (CD154-expressing) Tregs and ential expression of CD25 and CD127 (Fig. 1). This resulted in The Journal of Immunology 1085

FIGURE 1. Phenotype of sorted CD4+ Teff and Tregs. PBMCs were enriched for T cells that were la- beled with Abs directed against CD3, CD4, CD25 epitope A, CD127, and a live/dead marker (7- AAD). The lymphocytes were gated (A) and viable CD3+ (B) and sub- sequently CD4+ T cells (C) were selected. After FACS sorting, we obtained a highly enriched (.98%) fraction of viable CD4+ T cells (D), CD4+ T cells depleted of CD4+ CD25++CD1272, termed CD4+ Teff (E) and Tregs (F). Immediately fol- lowing sorting, the CD4+ Teff (G) and Tregs (H) were stained intra- cellularly for FOXP3 (the open his- Downloaded from togram resembles the isotype control staining in each of the fractions). Additionally, the percentage of demethylated TSDR FOXP3 was also determined for the different fractions (I). Results are a typical http://www.jimmunol.org/ example from 10–15 experiments except for I, which represents mean and SEM for each fraction.

a Treg population with on average 95 6 2.4% FOXP3-expressing stimulation in the sorted Tregs (Fig. 2M). However, in contrast to 6 +

cells (mean fluorescence intensity [MFI], 4475 220) (Fig. 1H) the CD154 live cell assay, the expression in time of CD137 Ag- by guest on September 27, 2021 as compared with CD4+ Teff that contained on average 7 6 1% reactive T cells was similar for CD4+ Teff and Tregs and reached FOXP3-expressing cells with an overall MFI of 318 6 157 (Fig. the maximum at 24 h (Fig. 2N,2O). We continued the experiments 1G). Moreover, the Tregs showed highly demethylated TSDR with the CD154 live assay, as CD137 background signals were in FOXP3 as compared with CD4+ Teff that contained predomi- general higher and could vary considerably among donors. nantly methylated TSDR FOXP3 (Fig. 1I). In the presence of + + CD154 Tregs have a memory phenotype and express CMV Ag, CD154 T cells were readily and consistently detected inflammatory cytokines after polyclonal stimulation in both T cell populations in CMV-seropositive donors (Fig. 2). In + contrast, CMV-seronegative donors did not have a CD154 signal Within the CD4 Teff population, cells can be classified in subsets above background (data not shown) (14). The background was based on the expression of CCR7 and CD45RO isoform on their , cell membrane. This leads to a division into three major subsets— very low in both populations ( 0.01%) when freshly isolated + 2 + T cells were used. Kinetic analysis of Ag-reactive CD154+ T cells naive T cells (CCR7 CD45RO ), central memory T cells (CCR7 CD45RO+), and effector memory T cells (CCR72CD45RO+)— showed a typical difference between CD4+ Teff and Tregs (Fig. that correlate with increasing T cell differentiation (16). Within 2A–F). The CD4+ Teff (Fig. 2A,2B,2E) showed a rapid but bi- the Treg population a similar subset distribution was observed as phasic response (maximum at 6 and 24 h) of cell surface CD154 for the CD4+ Teff population (Fig. 3B and 3A, respectively). The expression, whereas in the Tregs (Fig. 2C,2D,2F) only a few CD154+CD4+ Teff and CD154+ Tregs were predominantly found CD154+ cells were observed at 6 h. However, sorting of both within the memory compartment (Fig. 3C). populations was necessary to allow for discrimination of the As expected, substantially lower numbers of Tregs secreting IL-2 CD154+CD4+ Teff and CD154+ Tregs at 24 h because the acti- (6.4 6 2.4%), IFN-g (1.5 6 0.6%), or TNF-a (8.7 6 2.8%) were vated CD154+CD4+ Teff increased their expression of CD25 (Fig. found compared with the CD4+ Teff population (IL-2+, 30.3 6 2I) and downregulated expression of CD127. This resulted in an + 3.3%; IFN-g , 9.9 6 2%; and TNF-a, 22.8 6 6.6%) after poly- average 30–50% overlap between both populations of Ag-reactive clonal stimulation with PMA/ionomycin (Fig. 3H–K and 3D–G, T cells at 24 h. It is known that activated CD4+ Teff may become + + respectively). More than 98% of the cytokine-producing Tregs FOXP3 (20), but at 24 h the CD154 Tregs still showed a sig- coexpressed FOXP3 (Fig. 3I–K). nificant higher FOXP3 expression (Fig. 2G). Additionally, the per- + centage demethylated TSDR FOXP3 remained high in CD154+ CD154 Tregs can be expanded in an Ag-specific manner and Tregs as compared with CD154+CD4+ Teff (Fig. 2H), even after are potent suppressors expansion of .80% of the TSDR FOXP3 of CD154+Tregs was Next, the CMV Ag-specific CD154+ Tregs were sorted (Fig. 4A– demethylated (data not shown). C) and compared with CD154-depleted Tregs for their suppres- Membrane-bound CD137 has been used to detect Ag-reactive sive function either immediately (Fig. 4D,4E) or following ex- T cells (21), and CD137+ cells could be detected after CMV Ag pansion (Fig. 4I, where only results of CMV Ag-expanded 1086 Ag-REACTIVE HUMAN Tregs Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. CD154 and CD137 expression following CMV Ag stimulation in CD4+ Teff and Tregs. CD4+ Teff and Tregs were stimulated with CFSE- labeled autologous PBMCs, either loaded with CMVAg or not, in the presence of costimulation (anti-CD28 and anti-CD49d mAbs) and anti-CD40 to stain for CD154 on the cell surface (A–F) or without anti-CD40 to stain for CD137 (J–O). At 6 and 24 h CMV Ag stimulation, CD154 (A–D) or CD137 (J–M) expression (black dots) was determined within CD4+ Teff and Tregs. The kinetics of CD154- and CD137-expressing cells within CD4+ Teff (E, N) and Tregs (F, O) was determined immediately following isolation and upon stimulation with or without CMV Ag for different time points. One of three representative examples is shown. MFI of FOXP3 within total and CD154-expressing CD4+ Teff and Tregs following stimulation with CMVAg for 24 h (G) was analyzed as well as the percentage of demethylated TSDR FOXP3 (H) and MFI of CD25 (I). Results are means 6 SEM of three to five independent experiments. *p , 0.05.

CD154-expressing Tregs are displayed). The CD154+ Tregs were although both cell populations showed a similar efficacy in sup- significantly better suppressors of CMV Ag-induced T cell pro- pressing polyclonal PHA-induced T cell proliferation (Fig. 4E, liferation compared with the CD154-depleted Tregs (Fig. 4D,4F), 4F). The Journal of Immunology 1087 Downloaded from http://www.jimmunol.org/

FIGURE 3. Subset and cytokine expression of CD4+ Teff and Tregs. Immediately following sorting, CD4+ Teff (A) and Tregs (B) were dissected into subsets using CCR7 and CD45RO mAbs. Next, CD154+CD4+ Teff and CD154+ Tregs (C, right) were dissected into naive and memory subsets upon a 24-h CMV Ag stimulation. Additionally, CD4+ Teff and Tregs were incubated for 24 h, of which the last 12 h was with brefeldin A, in the presence of co- stimulation combined with PMA and ionomycin (D–G and H–K, respectively), to determine percentages of IL-2–, IFN-g–, or TNF-a–producing cells in the context of CD4 and FOXP3, respectively. The signal of the isotype control for the mAbs directed against cytokines is given in D and H.

It was possible toexpandunseparatedorCD154-expressing(CMV Treg population (Fig. 5A). Independent of the frequency of Ag- Ag-reactive) Tregs of a CMV-seropositive donor in the presence of reactive T cells, a strikingly similar ratio for the percentage of by guest on September 27, 2021 CMV Ag, IL-2, and IL-15. No proliferation of Tregs was observed CD154+CD4+ Teff/percentage of CD154+ Tregs was found for the when CMV Ag expansion was performed without IL-2 and IL-15 different Ags tested (Fig. 5B). This ratio was on average 10:1, and (data not shown) or when cells of a seronegative individual were similar to the amount of Tregs in the circulation, when tested in chosen for expansion (Fig. 4G). These results could be replicated by the unstimulated healthy individual. using VZV Ag in cultures of Tregs derived from VZV-seropositive After challenging the memory T cells with a booster vaccination donors, but again not in VZV-seronegative donors (data not shown). of HBs Ag or Tet-Tox (Fig. 5C), a time-dependent increase of Ag- The CD154-enriched Tregs could be expanded more efficiently reactive (CD154+) T cells was observed for CD4+ Teff. The fre- (yielding higher cell numbers) when compared with unseparated quency of circulating Ag-reactive CD154+ Tregs did not signifi- Tregs (Fig. 4G) using CMV Ag. In contrast, CD154-depleted Treg cantly change, leading to a temporarily increase of the CD154+ cultures showed no significant expansion in response to CMV Ag, CD4+ Teff/CD154+ Treg ratio with a maximum at 2 wk (Fig. 5D). and expansion was comparable to cytokine-only conditions. Ex- panded CMVAg-specific CD154+ Tregs showed stable high FOXP3 expression (Fig. 4I); that is, .95% of these Tregs still expressed Discussion FOXP3. These CMV Ag-expanded CD154-expressing Tregs were In this study we show the presence of human Ag-reactive nTregs in potent suppressors of CMVAg-induced T cell proliferation (Fig. 4I). the peripheral blood. Reactivity for CMV Ag was initially studied, Additionally, they showed a modest suppression of T cell prolifer- as the frequency of CMV Ag-reactive T cells is remarkably high, ation to other common Ags but only at the highest ratios of Tregs/ thereby facilitating the identification and isolation of Ag-reactive + CD3+ Teff (Fig. 4I). These results indicate that CMV Ag-reactive Tregs. Activated CD4 Teff or iTregs with (temporarily) increased CD154+ Tregs, when expanded in an Ag-specific manner, remain FOXP3 expression and increased CD25 and decreased CD127 predominantly Ag-reactive in their suppressor function. expression may mimick nTregs (8, 20). However, there are a Proliferation in cpm is given in Supplemental Fig. 1A–D. Addi- number of arguments in favor of the conclusion that Ag-reactive + ++ 2 + tionally, a kinetic analysis did not reveal a shift in the proliferative CD154 cells within the sorted CD25 CD127 CD4 T cell response of CD3+ Teff to CMVAg in the presence of 1:5 CMVAg- population are true nTregs. expanded CD154+ Tregs. The strong suppression observed at day 6 First, the expression in time of CD154 on Tregs was different from + was therefore unlikely to be caused by exhaustion of this culture CD4 Teff, as the rapid biphasic expression pattern (14) was not (Supplemental Fig. 1E). observed. Instead, similar to Ag-specific induction of CD137 on the + + + cell surface, the expression was detectable after 6 h and reached the Ratio of Ag-reactive CD154 Tregs to CD154 CD4 Teff maximal level after 24 h stimulation. This implies that after 6 h Ag- Not only CMV Ag, but a number of other common Ags such as specific stimulation all CD154+ T cells are found within the CD4+ VZV Ag, HBsAg, and Tet-Tox showed a CD154+ response in the Teff, which potentially offers the possibility to discriminate Ag- 1088 Ag-REACTIVE HUMAN Tregs Downloaded from http://www.jimmunol.org/

+

FIGURE 4. Functional analysis of CMV Ag-stimulated Tregs. Upon a 24-h stimulation with CMV Ag, Tregs (A, 0.21% CD154 ) of a CMV-seropositive by guest on September 27, 2021 donor were sorted into a CD154-enriched (B, .90%) and -depleted (C, .95%) fraction. The total CMV Ag-stimulated Tregs were also sorted again (sham treated) to be able to compare with CD154-enriched and -depleted Tregs. The function of these different Treg fractions was analyzed immediately following sorting (D, E). F, Summary (means 6 SEM) of five experiments with respect to the proliferation of CD3+ Teff, in presence of a 1:5 ratio of the different Tregs, to CMV Ag and PHA Ag, respectively. Additionally, the different Treg fractions were also expanded using CMV Ag or CD3/CD28 beads (poly- clonal). The fold increase of Ag-specific and polyclonal expansion of the different Treg fractions is depicted in G. As a negative control, the Ag-specific expansion protocol was applied to Tregs of a CMV-seronegative individual. A typical example of the FOXP3 expression at day 9 CMV Ag-expanded CD154-expressing Tregs is given in H (the open histogram resembles the isotype control staining, whereas the closed one resembles the FOXP3 staining). CMV Ag-expanded CD154-enriched Tregs were also tested for their suppressive capacity. The suppressive capacity of the different Treg fractions was tested using a proliferation assay with [3H]thymidine incorporation as a readout, and proliferation in presence of Tregs was compared with that without Tregs (no Tregs). For this purpose, CD3+ Teff were stimulated with irradiated autologous PBMCs only or combined with CMV Ag (D) or PHA Ag (E). Different numbers of the various Treg fractions were added to the CD3+ Teff stimulated with CMVAg or PHA Ag. In F, the CMVAg- and PHA Ag-induced relative proliferation of responders (means 6 SEM, five experiments) at a ratio of 1:5 (Tregs/CD3+ Teff) is depicted. *p , 0.05 when CD154-enriched Tregs are compared with CD154-depleted Tregs. In I, the proliferation of CD3+ Teff induced by different Ags in presence of CMV Ag-expanded CD154-enriched Tregs is depicted. Results are a typical example from three to five experiments, except for F and G, which represent mean 6 SEM for each value. reactive CD4+ Teff from Tregs. Unfortunately, at 24 h the differen- Fourth, the Treg population we isolated expressed low levels of tiation between CD154+ Tregs and CD154+ Teff could not be made inflammatory cytokines after polyclonal activation with PMA/iono- without previous sorting, as both CD127 and CD25 expression of mycin, as has been reported before (8, 9). CD154+CD4+ Teff substantially overlapped with Tregs at that The final and most compelling argument is the observation that point. freshly isolated Ag-reactive CD154+ Tregs showed Ag-reactive Second, the CD154+ Tregs were found in a homogeneous T cell suppression and could be efficiently expanded in vitro by population of Tregs characterized by the simultaneous expression Ag-specific stimulation combined with exogenous IL-2 and IL-15. of CD127 at low levels, as well as CD25 and FOXP3 at high These Ag-expanded CD154+ Tregs were highly suppressive in an levels. Additionally, the FOXP3 expression in CD154+ Tregs was Ag-specific fashion while still displaying a nonspecific, but much still significantly higher at 24 h compared with the unchanged less effective suppression when tested in an assay of polyclonal- FOXP3 expression in CD154+CD4+ Teff. stimulated T cells or other recall Ags. Third, the unchanged highly demethylated TSDR FOXP3 within Based on these findings we conclude that Ag-reactive CD154+ CD154+ Tregs was in favor of the development of CMV Ag- cells within the sorted CD4+CD25++CD1272FOXP3+ Tcell reactive Tregs from natural occurring Tregs instead of a develop- population are indeed nTregs and not activated CD4+ Teff or ment from CD4+ Teff. iTregs. This finding was not specific for CMV Ag, as we could The Journal of Immunology 1089 Downloaded from

FIGURE 5. CD154 signal in CD4+ Teff and Tregs for different Ags. The CD154 signal was determined for different Ags in CD4+ Teff and Tregs upon a 24-h stimulation with autologous CFSE-labeled PBMCs (ratio 1:1) with anti-CD40 and costimulation only or supplemented with CMV Ag, VZV Ag,

HBs Ag, or Tet-Tox. Individuals were tested seropositive for CMV Ag and VZV Ag and immunized against HBV (HBs Ag) or tetanus (Tet-Tox), re- http://www.jimmunol.org/ spectively. Percentages of CD154-expressing cells were corrected for the background (i.e., autologous CFSE-labeled PBMCs, anti-CD40, and costimu- lation only). In A, the ratio of CD154 signal for different Ags in CD4+ Teff per Tregs is corrected for the percentage of Tregs within peripheral blood and the percentage of Tregs present within the circulation. In B, the CD154 signal for the different Ags within CD4+ Teff is plotted against that of Tregs. In C, the CD154 signal within Tregs and CD4+ Teff at several time points following vaccination against tetanus is shown. Subsequently, the ratio of the CD154 signal within the CD4+ Teff over Tregs is corrected for the percentage of Tregs within peripheral blood and is depicted for the different time points following vaccination against tetanus and HBV and, as a control, CMV Ag ratios over time are given (D). One out of four representative experiments is shown. by guest on September 27, 2021 subsequently show similar results using different Ags, including fection and autoimmunity is required to understand the relationship both Ags involved in natural infections and vaccination Ags. between circulating Ag-reactive Tregs and disease activity. The + A remarkable stable and similar ratio of Ag-reactive CD154 present data indicate that findings for Tregs within the peripheral + + CD4 Teff and CD154 Tregs was found for the Ags tested. The blood may not accurately reflect the actual Treg response at the site formation of Tregs in a fixed ratio, irrespective of the Ag, indicates of immune reaction and should be interpreted with caution. that these cells may actually be the brake on every Teff response. A major conclusion of our study is that by inference of the results Such an intimate relation between Teff and Tregs was not ob- with several unrelated Ags, the CD4+CD25++CD1272FOXP3+ served within the peripheral blood after booster vaccination, as the + Treg compartment is filled with Ag-specific T cells as a mirror frequency of CD154 Tregs did not significantly change. How- + + + + image of the memory CD4 Teff population. This in fact supports ever, this led to a highly increased CD154 CD4 Teff/CD154 the hypothesis, based on the recent work by Mayari et al. (8) that, Treg ratio that eventually returned to baseline, but only after 6–8 + similar to Teff, the natural Treg compartment consists of thymus- wk. The difference in kinetics between CD154 Tregs and derived naive T cells that will differentiate into memory T cells CD154+CD4+ Teff also argues against the induction of Tregs from + + after appropriate antigenic stimulation. Our findings are also in the CD154 CD4 Teff. line with the results from TCR analysis of Teff and Tregs, which Our data obtained in human individuals are in accordance with the did not show any evidence for affinity for autoreactive Ag (23), data from in vivo tracking of color-coded Teff and Treg in the al- and Vb T cell repertoire analysis, which showed that human CD4+ lograft response against pancreatic islets transplants in the mouse CD25++ Tregs share equally complex and comparable repertoires (22). Using this elegant technique, it was shown that natural Tregs with CD4+CD252 counterparts (24). In the latter study it was also and Teff accumulate at the same pace within the transplant. This a remarkable finding that CMV seropositivity gave rise to a simi- result is very similar to the observations made on the local T cell lar shift in Vb repertoire in Teff and Tregs. immune response after cutaneous inoculation with tuberculin-puri- The possibility of direct recognition of Ag-reactive Tregs will fied derivative in humans (11). However, similar to our facilitate further research in this direction, aiming a more complete vaccination data, numbers of Tregs within the peripheral blood understanding of the significance and interaction of Tregs in hu- hardly change after islet transplantation and do not show the steep mane disease and immune responses. Additionally, isolation and increase as observed for Teff (22). Therefore, after vaccination it propagation of Ag-reactive T cells identified on the single cell level may be a possibility that Ag-reactive Tregs, having a memory is of potential benefit for future immunotherapy with Tregs. lymphoid homing profile (CD45RO+CCR7+), are retained and ac- tive within the lymphoid organs in the weeks following antigenic Disclosures exposure. Further research in different clinical settings such as in- The authors have no financial conflicts of interest. 1090 Ag-REACTIVE HUMAN Tregs

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