UBD, a Downstream Element of FOXP3, Allows the Identification of LGALS3, a New Marker of Human Regulatory T Cells

UBD, a downstream element of FOXP3, allows the identification of LGALS3, a new marker of human regulatory T cells

Frank Ocklenburg1,*, Darius Moharregh-Khiabani1,*, Robert Geffers2, Viktoria Janke1, Susanne Pfoertner2, Henk Garritsen3, Lothar Groebe2, Juergen Klempnauer1, Kurt EJ Dittmar4, Siegfried Weiss5, Jan Buer2,6 and Michael Probst-Kepper1

1Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany; 2Mucosal Immunity Research Group, German Research Centre for Biotechnology (GBF), Braunschweig, Germany; 3Institute for Clinical Transfusion Medicine, Sta¨dtisches Klinikum Braunschweig gGmbH, Braunschweig, Germany; 4Department of Molecular Biotechnology, German Research Centre for Biotechnology (GBF), Braunschweig, Germany; 5Department fo Molecular Immunology, German Research Centre for Biotechnology (GBF), Braunschweig, Germany and 6Insitute of Medical Microbiology, Hannover Medical School, Hannover, Germany

Here, we report the identification of the ubiquitin-like gene UBD as a downstream element of FOXP3 in human þ hi activated regulatory CD4 CD25 T cells (Treg). Retroviral transduction of UBD in human allo-reactive effector þ CD4 T helper (Th) cells upregulates CD25 and mediates downregulation of IL4 and IL5 expression similar to

overexpression of FOXP3. Moreover, UBD impairs Th cell proliferation without upregulation of FOXP3 and

impairs calcium mobilization. In the presence of ionomycin, overexpression of UBD in Th cells leads to the

induction of IL1R2 that resemble FOXP3-transduced Th cells and naturally derived Treg cells. A comparison of

the transcriptome of FOXP3- and UBD-transduced Th cells with Treg cells allowed the identification of the gene LGALS3. However, high levels of LGALS3 protein expression were observed only in human CD4 þ CD25hi

derived Treg cells and FOXP3-transduced Th cells, whereas little was induced in UBD-transduced Th cells. Thus, UBD contributes to the anergic phenotype of human regulatory T cells and acts downstream in FOXP3 induced regulatory signaling pathways, including regulation of LGALS3 expression. High levels of LGALS3 expression represent a FOXP3-signature of human antigen-stimulated CD4 þ CD25hi derived regulatory T cells. Laboratory Investigation (2006) 86, 724–737. doi:10.1038/labinvest.3700432; published online 15 May 2006

Keywords: CTLA4; FOXP3; LGALS3; regulatory T cells; UBD; CD4 þ CD25hi

þ þ Regulatory CD4 CD25 T(Treg) cells have emerged been illustrated in different human disease settings, as a unique population of suppressor T cells that are that is, altered numbers and impairment of suppres- important for the maintenance of peripheral toler- sor activity of Treg cells has been demonstrated in 1,2 ance. In humans, Treg cells are characterized by patients with Hodgkin’s lymphomas, autoimmune high levels of CD25 expression (CD25hi) and repre- polyglandular syndrome type II, multiple sclerosis, sent 1–3% of all CD4 þ T cells in peripheral blood.3 myasthenia gravis, diabetes, psoriasis, patients with Typically, such Treg cells are anergic upon stimula- graft-versus-host-disease, and infectious diseases tion via their T-cell receptor and exhibit suppressive such as malaria and HIV.4–12 2 activity towards effector T-cell function. The clin- Development and function of Treg cells critically þ hi ical relevance of CD4 CD25 Treg cells has recently depend on the forkhead transcription factor FOXP3 that specifies the regulatory T cell lineage.13 As a result, humans and mice deficient in FOXP3 lack Correspondence: Dr M Probst-Kepper, MD, Department of Visceral þ þ and Transplant Surgery, Hannover Medical School, Building K25, CD4 CD25 Treg cells and suffer from multiorgan 2nd Floor, Room 1140, Carl Neuberg Strasse 1, D-30625 Hannover, autoimmune disease.2 Germany. Treg cells normally develop in the thymus. How- E-mail: [email protected] ever, they also may arise in a FOXP3-dependent *These authors contributed equally to this work. þ À 14,15 Received 25 January 2006; revised 31 March 2006; accepted 10 manner from peripheral CD4 CD25 T cells. The April 2006; published online 15 May 2006 antigen-specificity of Treg cells is currently a matter UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 725 of debate, although self-antigens most likely repre- b-galactoside binding protein LGALS3. High levels sent the dominating specificity.16–19 of LGALS3 represent a FOXP3-signature of human þ hi Mechanisms of immunosuppression by Treg cells antigen-stimulated CD4 CD25 derived Treg cells as comprise cell-contact dependent suppression, it is not upregulated in nonregulatory CD4 þ CD25À mainly observed in vitro, and cytokine-mediated derived effector Th cells despite the expression of suppression of effector T cells, mainly observed in CD25 and FOXP3. vivo.20 Cytokines most commonly found are IL10 and TGF-b, which also modulate Treg cell function in vivo and Treg cell development from nonregulatory Materials and methods þ À 14,17,21,22 CD4 CD25 T cells in vitro. However, IL10 Purification and Cultivation of Human CD4 þ T Cells and TGF-b are not essential for the development and 23,24 þ function of Treg cells. CD4 T cells were prepared from peripheral blood CTLA4 also functions as an important mediator of healthy donors by centrifugation over Ficoll- 20 and modulator of Treg cell activity. This member of Hypaque gradients (Biochrom AG, Berlin, Germany) the CD28-family is constitutively expressed at high and MACS isolation using the CD4 þ T cell isolation þ þ levels in CD4 CD25 Treg cells in mouse and man. kit and AutoMACS technology (Miltenyi Biotech, þ þ À In the human system, CTLA4 Treg cells display a Bergisch Gladbach, Germany). CD4 CD25 T cells À þ hi stronger suppressive activity in vitro than CTLA4 and CD4 CD25 Treg cells were then isolated by cell 25 Treg cells. However, CTLA4 is not essential for the sorting on a MoFlo (DakoCytomation, Fort Collins, þ hi development and function of Treg cells, since its loss CO, USA) to a purity of 498%. CD4 CD25 Treg could be compensated by increased secretion of cells were stimulated once with plate-bound anti- À þ þ 26 IL10 and TGF-b by CTLA4 CD4 CD25 Treg cells. CD3 (TR66, 1 mg/ml), soluble anti-CD28 (CD28.2, The conversion of murine naı¨ve nonregulatory 1 mg/ml, BD), and 50 U/ml recombinant human IL2 þ À CD4 CD25 T cells into Treg cells by retroviral (Proleukin, provided by P Wagner, Chiron Corpora- overexpression of FOXP3 underlines the master tion, Emeryville, CA, USA), and thereafter weekly control function of this transcription factor.27,28 with irradiated allogeneic EBV-transformed B cells Consequently, the use of FOXP3 as a tool for (LG2-EBV, provided by T Boon, LICR, Brussels, þ À potential clinical Treg cell therapy has been demon- Belgium). CD4 CD25 T cells were used to establish strated recently in mice. Retroviral transduction of long-term, allo-reactive effector Th cell lines against FOXP3 in polyclonal or antigen-specific naı¨ve LG2-EBV cells. Culture medium was IMDM, with þ À CD4 CD25 T cells generated Treg cells that 10% FCS, 100 U/ml penicillin/streptomycin, and suppressed the development of inflammatory nonessential amino acids (PAA Laboratories, Linz, bowl disease, autoimmune gastritis, diabetes or Austria). Human peripheral blood was obtained contact allergy, and induced transplant tolerance after informed consent according to the MHH in vivo.27,29–31 Despite these encouraging results in guidelines. mice, debate exists about the efficacy of FOXP3 to reprogram human naı¨ve CD4 þ T cells towards a regulatory phenotype.32–34 The most likely reason for Antibodies this ambiguity arises from the fact that unlike For immunostaining PE-, FITC-, APC-, and Cy- murine naı¨ve and effector CD4 þ T helper cells, Chrom-conjugated Abs against CD4 (RPA-T4), human nonregulatory CD4 þ T cells do express CD25 (M-A251), CD28 (CD28.2), TCRab (T10B9.1A- FOXP3.35 31), CTLA4 (BNI3), LGALS3 (B2C10; all from BD, Thus, the fundamental role of FOXP3 is well Bioscience, San Jose, CA, USA), and FOXP3 established, but target genes involved in the control (PCH101, eBioscine Inc., San Diego, CA, USA) and of the FOXP3-dependent regulatory phenotype still the respective isotype controls were used. For require defining.36 However, precise knowledge of FOXP3 staining, murine hybridoma T cell trans- this, including considerations of the differences duced with human FOXP3 or GFP were included as between FOXP3 in mice and men,35 is essential for further control. Anti-CD3e (TR66, produced from the effective manipulation of regulatory T cells as hybridoma supernatants) and anti-CD28 (CD28.2, immunomodulatory therapy. Hence, we compared BD) were used for T-cell stimulation. the genetic program of human CD4 þ CD25hi derived

Treg cells, which were expanded in vitro by allogeneic stimulation and IL2, with that of human allo- Analysing T-Cell Functions reactive effector Th cells, which were reprogrammed by retroviral transduction of FOXP3. Diubiquitin T-cell proliferation and suppressor activity was (UBD) was identified as a gene that is upregulated assessed by stimulating 3 Â 104 T cells in triplicate by FOXP3 overexpression in Th cells similar to Treg with irradiated LG2-EBV B cells or 1 mg/ml plate- cells. UBD overexpression in Th cells demonstrated bound anti-CD3 (TR66) with or without IL2, or 1 mM that it contributes in part to the FOXP3-dependent ionomycin (Sigma, St Louis, MO, USA), in 96-well regulatory phenotype without upregulation of flat-bottom microtiter plates (Nunc, Wiesbaden,

FOXP3, and led to the identification of the Germany). For transwell experiments, Th cells were

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 726 stimulated in 96 flat-bottom plates separated by under the control of an IRES sequence.37 The 0.2 mm-pore transwell inserts (Greiner bio-one, amphotropic PT67 packaging cell line (provided by Frickenhausen, Germany) from the T cells above M Wirth, GBF) was transfected as described else- the transwell. Cells were pulsed with 1 mCi/well of where.37 Filtered (0.45 mm) virus-containing super- [3H]thymidine after 72 h for the final 16 h. Statistical natant supplemented with 8 mg/ml sequabrene analysis was performed using two-sided Student’s (Sigma) was applied to differentiated allo-reactive t-test as indicated. TCR downmodulation upon Th cells, established as described above, at day 2 anti-CD3 stimulation (plate-bound, 1 mg/ml) was after allogeneic stimulation by centrifugation at assessed by FACS analysis (FACS Calibur or FACS 5000 g for 60 min at room temperature. Cells were Scan, BD) using PE-coupled anti-TCR antibodies. expanded thereafter with 50 U/ml IL2, and GFP expressing cells were sorted 1 to 2 weeks later using a FACS-Vantage (BD). Sorted cells were kept for up Cytokine Detection to 3 months using the restimulation procedure described above and were tested repeatedly for Stimulated cytokine release of IFNg and IL10 was stable functional, phenotypic, and transcriptional detected using ELISA sets of the CytoSet series changes. (IFNg, IL10; Biosource, Camarillo, CA, USA) and OptEIA series (IFNg, and IL10, BD), according to the manufacturer’s instructions. Supernatants of stimu- Quantitative and Semiquantitative RT-PCR lated T cells were harvested at 72 h after stimulation immediately before the [3H]thymidine pulse. Total RNA was isolated from CD4 þ T cells using RNAeasy (Qiagen, Hilden, Germany) or nucleospin RNA-II (Macherey Nagel, Du¨ ren, Germany). cDNA Calcium Mobilization Assays synthesis was performed using oligo-dT primers and Superscript II Reverse Transcriptase (Invitrogen). Calcium mobilization was measured by flow cyto- Quantitative Real-time PCR was performed on an metry and confocal microscopy. In brief, T cells were ABI PRISM 7000 cycler (Applied Biosystems, Foster loaded with 2 mM INDO-1 (Molecular Probes, City, CA, USA) using the SYBR Green PCR kit Eugene, OR, USA) for 30–45 min at 371C, extensively (Stratagene, La Jolla, CA, USA), and primers specific washed, and loaded with 1 mg/ml anti-CD3 (TR66) for FOXP3 (50-GAA CGC CAT CCG CCA CAA CCT for 20 min at 41C. Samples were applied to a MoFlo GA-30 and 50-CCC TGC CCC CAC CAC CTC TGC-30), for 30 to 60 s at 371C to measure the basal LGALS3 (50-CGG AGC ACC TGC ACC TGG AGT intracellular calcium concentration. Cells were CTA-30 and 50-TGA AGC GTG GGT TAA AGT GGA stimulated by crosslinking cell-bound CD3 anti- AGG-30), and RPS9 (50-CGC AGG CGC AGA CGG bodies with a 1/30 dilution of goat-anti-mouse IgG TGG AAG C-30 and 50-CGA AGG GTC TCC GCG GGG antiserum or 10 mg/ml goat-anti-mouse IgGg (Biomol TCA CAT-30) as described.37 For semiquantitative International LP, Plymouth Meeting, PA, USA). The RT-PCR, three-fold dilutions of cDNA samples time-dependent intracellular Ca2 þ was measured as starting with the first dilution, normalized to the the ratio of calcium-bound (405 nm) to calcium-free expression of RPS9 (50-CGC AGG CGC AGA CGG (515 nm) INDO-1 fluorescence, analyzed using TGG AAG C-30 and 50-CGT AGG AGA GCG CAG FlowJo software (Tree Star, San Carlos, CA, USA). AGA GAA GTC A-30) were amplified with primers For single cell analysis, INDO-1 labeled cells (1 mM) for FOXP3 (50-TCA ACG GTG GAT GCC CAC GC-30 were stimulated in coverslip-chambers (Nunc) pre- and 50-TCA GGG GCC AGG TGT AGG GT-30), UBD coated with 10 mg/ml anti-CD3 (TR66) using con- (50-CCC AAT GCT TCC TGC CTC TGT G-30 and 50- focal microscopy (BIO-Rad, Hercules, CA, USA). GAC CCC TGC CAA CAC CCC AT-30), IL4 (50-AAC ACA ACT GAG AAG GAA ACC TTC TGC-30 and 50-CTC TCT CAT GAT CGT CTT TAG CCT TTC-30), Retroviral Transduction of Human Effector CD4 þ IL5 (50-GAG GAT GCT TCT GCA TTT GAG TTT G-30 T Cells and 50-GTC AAT GTA TTT CTT TAT TAA GGA CAA G-30), GATA3 (50-ACC TGT CAG ACC ACC ACA The cDNAs encoding human FOXP3, CTLA4, and ACC ACA C-30 and 50-GGA TGC CTT CCT TCT TCA UBD were amplified from cDNA of T cells using reg TAG TCA GG-30), IL1R2 (50-AAA TGA CTC TGC high fidelity PFU polymerase (Promega) and speci- TAG GAC GGT CCC-30 and 50-TCT TCC AGG GCC fic primers (FOXP3: 50-GAC AAG GAC CCG ATG ACA TCG TG-30) and LGALS3 (50-CCC ATC TTC CCC A-30 and 50-TCA GGG GCC AGG TGT AGG GT- TGG ACA GCC AAG TGC-30 and 50-GCA TCA TTC 30; CTLA4: 50-TAC ATA TCT GGG ATC AAA GC-30 ACT GCA ACC-30). and 50-ATT TGG GTT CCG CAT CCA-30; UBD: 50- TGC AGA GAT GGC TCC CAA TG-30 and 50-GAC 0 CCC TGC CAA CAC CCC AT-3 ). PCR products were GeneChip Assays and Microarray Data Analysis cloned into pCR4.1 TOPO (Invitrogen, Carlsbad, CA, USA), sequenced, and inserted into a pMSCV-based For the screening of similarly regulated genes in Treg retroviral vector encoding an enhanced GFP (GFP) versus Th cells compared to ThFOXP3 versus ThGFP

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 727 cells, we analyzed two independent experiments ex vivo.39 In contrast to the latter report, our human 2 þ using Human Genome U133 A arrays (Affymetrix, Treg cells showed signs of sustained Ca -flux upon Santa Clara, CA, USA) and MAS 5.0 software anti-CD3 stimulation. This was reflected in the (www.affymetrix.com/support/technical/manual- prolonged time required to reach the lower plateau s.affx: Microarray Suite User’s Guide, Version 5.0). level, following the initial Ca2 þ -rise, and was also

Selection criteria for potential FOXP3-dependent higher than that found for Th cells (Supplementary genes in Treg and ThFOXP3 cells were: (1) a signal Figure 2a). Consistent results were obtained at the with a fold-change of more than 3 in all four single cell level (Supplementary Figure 2b and c). comparisons, or (2) a similar increase (I, including Differences between Treg and Th cells were also marginal increase, MI) or decrease (D, including observed in the extent of anti-CD3 induced TCR marginal decrease, MD) of the signal change in all downmodulation, which was strongly impaired in four experiments. GeneChip analysis of ThUBD Treg cells (Supplementary Figure 2d). This finding is compared to ThGFP cells was performed using in line with the observation that the extent of TCR Human Genome U133 PLUS 2.0 arrays (Affymetrix). downmodulation following ligand binding corre- 40 All data from ThFOXP3, ThUBD, and Treg cells were lates with the induction of effector function. compared to each other, according to the criteria (1) In summary, the Treg cell lines established by our a signal fold-change of more than 2, or (2) a similar protocol showed all the phenotypic and functional signal change as either I and MD or D and MD in all characteristic of bona fide regulatory CD4 þ CD25hi six comparisons. Selected differences of one cell Treg cells and thus can be used to establish the type were identified by (1) a signal fold-change of mechanisms of FOXP3-dependent gene regulation. less than 1, and (2) an opposite signal change or no signal change (NC) in the respective T-cell population. All microarray data have been deposited FOXP3 Overexpression in Human Effector Th Cells at GEO (http://www.ncbi.nlm.nih.gov/geo/; GEO Induces a Partial Regulatory Phenotype accession GSE4527). All T cells used for GeneChip analysis have been established for more than 2 to 3 To identify relevant FOXP3-dependent genes asso- months. ciated with the regulatory phenotype, we used retroviral overexpression of FOXP3 in human allo- antigen specific effector Th cells (ThFOXP3). Suc- Results cessfully transduced Th cells were sorted for GFP þ, Characterization of Human CD4 þ CD25hi Derived reintroduced into culture, and tested repeatedly over a period of up to 3 months. Treg Cells FOXP3-transduced Th cells were impaired in their For the generation and differentiation of human proliferative response upon antigen-specific stimu- allo-reactive Th cells and Treg cells, sorted lation using allogeneic EBV-transformed B cells or CD4 þ CD25À and CD4 þ CD25hi T cells were stimu- stimulation with anti-CD3, reversed by the addition lated with allogeneic EBV-transformed B cells and of IL2 (Figure 1a). In addition, ThFOXP3 cells þ hi IL2. CD4 CD25 derived Treg cells expanded by this acquired suppressor activity towards effector Th protocol could be grown for a long period of time cells stimulated by allogeneic EBV-transformed B (up to 6 months), but remained anergic to anti-CD3 cells, but this was not observed in control Th cells stimulation when IL2 was omitted (Supplementary transduced with GFP (ThGFP) or CTLA4 (ThCTLA4) Figure 1a). These Treg cells consistently suppressed (Figure 1b). Thus, FOXP3 was able to specifically proliferation of established allo-reactive Th cell lines reprogram differentiation of helper T cells towards a activated by anti-CD3 (Supplementary Figure 1a) or regulatory phenotype. However, compared to natu- allogeneic EBV-transformed B cells (Supplementary rally occurring Treg cells, this regulatory phenotype Figure 1b). The suppressive activity of such Treg cells was less pronounced (Figure 1a and b). was overcome by the addition of more than 10 U/ml In contrast, overexpression of CTLA4 did not

IL2 (Supplementary Figure 1b) and required cell- induce a regulatory phenotype in Th cells, although contact since it was blocked by a transwell mem- the levels of intracellular CTLA4 expression were brane (Supplementary Figure 1c). Phenotypical similar to Treg cells (Figure 1c). A possible contribu- analysis revealed stable expression of high levels tion of CTLA4 to the FOXP3-dependent transcrip- of CD25, CD28, and intracellular CTLA4 (data not tional control is therefore unresolved at this point. shown) as well as FOXP3 mRNA independent of We then analyzed the anti-CD3 induced TCR their activation status (Supplementary Figure 1d). downmodulation. Similar to Treg cells, ThFOXP3 þ hi Thus, CD4 CD25 derived Treg cells sorted and cells showed an impaired downmodulation of TCR expanded by our protocol maintained high level of expression that was not observed in ThGFP or sustained FOXP3 expression associated with pheno- ThCTLA4 cells (Figure 1d). Interestingly, this im- typic and functional stability. pairment of TCR downmodulation appears to be a Ca2 þ metabolism is essential for the regulation general effect of FOXP3 as it was even observed in of anergy38 and has recently been shown to be murine hybridoma T cells transduced with FOXP3. þ þ impaired in murine CD4 CD25 Treg cells analyzed However, such hybridoma T cells did not acquire an

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 728

Figure 1 FOXP3 is able to reprogram human allo-reactive effector Th cells towards a regulatory phenotype. (a)Treg cells and Th cells, retrovirally transduced with FOXP3 plus GFP (ThFOXP3), CTLA4 and GFP (ThCTLA4), and GFP alone (ThGFP), were stimulated with allogeneic LG2-EBV B cells or anti-CD3 without or with 100 U/ml IL2. Proliferation was assessed at day 3. (b)ThGFP cells as in (a) were stimulated by allogeneic LG2-EBV B cells in the presence of equal numbers of indicated T cells. Only Treg and ThFOXP3 cells significantly suppressed ThGFP cell proliferation; NS ¼ not significant. Similar results were obtained with normal effector Th cells instead of ThGFP cells. (c)Th cells as in (a) were tested for intracellular CTLA4 expression in flow cytometry. Shaded area: isotype control, bold line: intracellular anti-CTLA4 staining. (d) Transduced Th cells as in (a) were tested for downmodulation of TCR expression following anti- CD3 stimulation at the times indicated. Remaining surface TCR expression was measured with anti-TCRab antibodies by flow cytometry

(MFI: mean fluorescence intensity). (e) Sustained overexpression of FOXP3 in ThFOXP3 compared to Th GFP cells. Real-time RT-PCR was carried out at the times indicated after stimulation with plate-bound anti-CD3 plus IL2. (f) Intranuclear FOXP3 protein expression of

ThFOXP3 cells compared to Treg and ThGFP cells was measured by flow cytometry. Gates were set according to the control staining using murine hybridoma T cells transduced with GFP alone (negative control; gray shaded) or with human FOXP3 plus GFP (thin line). Percent of positive cells is indicated.

anergic phenotype, although they showed upregula- also showed reduced expression of the co-trans- tion of CD25 and impaired NFAT function (data not duced IRES-driven GFP. As a result, re-sorting of

shown). Therefore, a high level of sustained FOXP3 long-term cultured ThFOXP3 cells for high expres- mRNA (Figure 1e) and protein expression in sion of GFP re-established their regulatory pheno- ThFOXP3 cells (Figure 1f) induced most of the type (data not shown). characteristics of the FOXP3-dependent regulatory In addition, ThFOXP3 cells did not reach the same phenotype. high levels of CD25 expression (Figure 2a) while It should be noted that in long-term cultures, CD28 was present at similar levels in both types of ThFOXP3 cells were able to escape the FOXP3- cells (data not shown). We, therefore, analyzed the dependent control. Most likely, the appearance of T functional properties of ThFOXP3 cells expressing cells with lower expression levels of FOXP3 was different levels of CD25 (Figure 2b). CD25hi responsible for this phenomenon, since such T cells ThFOXP3 cells showed a more pronounced regula-

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 729 similarly regulated in both Treg and ThFOXP3 cells compared to Th and ThGFP cells. According to our stringent selection criteria, 39 genes were defined that were similarly regulated in both regulatory T-cell populations. As expected, known markers of

Treg cells were identified, that is CD25, CTLA4, TNFRSF1B, and CCR7. In addition, we found genes that were not previously associated with FOXP3- dependent transcriptional control, for instance the ubiquitin-like gene diubiquitin (UBD) and the b-galactoside binding lectin LGALS3 (Table 1). Proteins involved in ubiquitinylation play an important role in the regulation of T-cell anergy.41 Hence, we analyzed the potential contribution of UBD to the FOXP3-dependent transcriptional control in more detail.

UBD is Involved in the Regulation of CD4 þ T-Cell Anergy UBD is expressed at high levels in CD4 þ CD25hi derived Treg cells and consistently upregulated in FOXP3-overexpressing Th cells compared to ThGFP and ThCTLA4 cells (Figure 3a). Unlike FOXP3, which is expressed at sustained high levels, UBD expression is transiently downregulated following T-cell activation using anti-CD3/-CD28 antibodies and IL2 (Figure 3a). Overexpression of UBD in Th cells (ThUBD) via Figure 2 Differences in the regulatory phenotype between CD25hi retroviral transduction induced upregulation of lo and CD25 Th cells transduced with FOXP3. (a)Th cells CD25 without affecting CD28 expression (Figure transduced with FOXP3 plus GFP (ThFOXP3) and GFP alone 3b). In long-term ThUBD cell lines, significant (ThGFP) were tested for CD25 expression and compared with Treg impairment of proliferation upon TCR stimulation and Th cells. FOXP3 transduction never resulted in CD25 was observed, which was reversed by the addition of expression comparable to bona fide Treg cells. (b) Reanalysis of hi lo ThFOXP3 cells sorted for CD25 and CD25 expression is shown. IL2 (Figure 3c). Similar to FOXP3, overexpression of hi lo (c)ThFOXP3 cells sorted for CD25 and CD25 expression, Th and UBD in effector Th cells did not suppress IL10 and ThGFP cells were stimulated with allogeneic LG2-EBV B cells IFNg secretion (Figure 3d). Importantly, FOXP3 hi with or without 100 U/ml IL2. CD25 ThFOXP3 cells are more expression was not upregulated in T UBD cells impaired in their proliferative capacity than CD25lo T FOXP3 h h (Figure 4a and b) suggesting that UBD is a key player cells. (d)ThFOXP3 cells of (c) were added to Th cells in equal numbers and stimulated with allogeneic LG2-EBV B cells. in the regulation of anergy in Treg cells downstream Suppressive activity was significantly more pronounced of FOXP3. hi lo (*Po0.02) in CD25 ThFOXP3 than CD25 ThFOXP3 cells. Like ThFOXP3 cells, mRNA of IL5 and IL4 was significantly downregulated in ThUBD cells (Figure 4a). Whereas GATA3, regulating T 2 specific IL5 and tory phenotype than CD25lo T FOXP3 cells in terms h h IL4 cytokine expression, was unaltered (Figure 4a),42 of their proliferative impairment and suppressor LGALS3, which has been described to downregulate function (Figure 2c and d). Again, CD25 expression expression of IL4 and IL5 mRNA,43 was only slightly decreased in long-term culture despite the presence induced (Figure 4a and c). Thus, UBD appears to be of FOXP3 (data not shown). This suggests that the T h responsible for the IL4/IL5 downregulation as a cell specific regulation of CD25 expression is only downstream effector of FOXP3. transiently overcome by FOXP3 overexpression in We then analyzed the influence of UBD over- differentiated effector T cells. h expression on TCR downmodulation and Ca2 þ - mobilization. Interestingly, UBD did not impair the anti-CD3 induced TCR downmodulation observed Gene Expression Profiles of Treg Cells and T FOXP3 Cells with Treg cells and ThFOXP3 cells (data not shown). h However, Ca2 þ -mobilization was influenced (Figure The data presented above provide a biological 4d). Whereas FOXP3 overexpression induced a framework for the identification of genes associated slightly elevated steady-state phase of Ca2 þ -flux with the FOXP3-dependent regulatory phenotype. compared to ThGFP cells, this state was lower in Th Using GeneChip analysis, we screened for genes cells overexpressing UBD (Figure 4d).

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 730

Table 1 Genes expressed similarly in ThFoxp3 and Treg cells detected by microarray analysis

Probset Gene ThFOXP3/ThGFP Treg/Th

Fold changea Changeb Fold changea Changeb

Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2 Exp 1 Exp 2

206942_s_at PMCH 9.9 2.4 I I 2.6 5.3 I I 210299_s_at FHL1 7.4 14.8 NC I 3.3 11.0 NC NC 205890_s_at UBD 6.5 12.7 NC I 3.5 4.4 NC NC 216144_at — 5.0 4.4 NC NC 5.3 3.6 NC NC 208029_s_at LAPTM4B 4.2 13.2 MI I 10.5 30.9 I I 210930_s_at ERBB2 3.8 4.7 NC NC 4.4 7.6 NC NC 219456_s_at RIN3 3.7 5.1 NC NC 9.4 7.3 I I 206337_at CCR7 3.7 2.2 I I 1.9 10.5 I I 208949_s_at LGALS3 2.7 4.1 I I 2.8 7.4 I I 211559_s_at CCNG2 1.9 1.7 I I 1.9 2.0 I I 211269_s_at IL2RA 1.8 3.2 I I 1.7 11.6 I I 203508_at TNFRSF1B 1.6 1.9 I I 2.4 3.2 I I 213620_s_at ICAM2 1.4 1.3 I I 1.4 3.0 I I 221331_x_at CTLA4 1.3 1.5 I I 2.2 2.2 I I 210538_s_at BIRC3 1.3 1.5 I I 1.8 2.5 I I 205159_at CSF2RB 1.3 3.9 I I 1.7 3.5 I I 38149_at — À1.2 À1.2 D D À1.6 À1.6 D D 219869_s_at SLC39A8 À1.3 À1.3 D D À1.5 À4.9 D D 1405_i_at CCL5 À1.3 À1.6 D D À16.2 À119.5 D D 219033_at PARP8 À1.4 À1.4 D D À1.7 À1.9 D D 207735_at RNF125 À1.4 À2.0 D D À2.0 À3.8 D D 210038_at PRKCQ À1.4 À1.4 D D À1.7 À2.6 D D 210046_s_at IDH2 À1.4 À1.5 D D À1.6 À3.3 D D 211795_s_at FYB À1.4 À1.8 D D À2.8 À2.5 D D 209732_at CLEC2B À1.4 À1.2 D D À1.8 À8.4 D D 218805_at GIMAP5 À1.5 À1.7 D D À1.9 À3.3 D D 210140_at CST7 À1.5 À1.5 D D À2.4 À10.7 D D 213618_at CENTD1 À1.5 À1.5 D D À2.1 À2.5 D D 201061_s_at STOM À1.5 À1.3 D D À29.5 À3.5 D D 219243_at GIMAP4 À1.7 À2.1 D D À2.0 À2.4 D D 205495_s_at GNLY À1.8 À2.6 D D À17.5 À13.5 D D 219520_s_at KIAA1280 À1.8 À2.0 D D À1.2 À1.0 D MD 204009_s_at KRAS À1.8 À1.5 D D À2.0 À2.8 D D 213958_at CD6 À2.2 À1.7 D D À1.7 À1.5 D D 216920_s_at TARP À2.8 À1.8 D D À3.9 À1.6 D D 213915_at NKG7 À3.0 À2.0 D D À24.0 À81.0 D D 204157_s_at KIAA0999 À3.0 À6.7 NC D À6.1 À6.6 NC NC 205798_at IL7R À3.9 À8.5 D D À6.0 À1.6 D D 206666_at GZMK À5.7 À1.8 D D À21.8 À510.9 D D

a Signal fold-change between ThFOXP3 and ThGFP cells (ThFOXP3/ThGFP) or Treg vs Th cells (Treg/Th) as determined by MAS 5.0 software of two independent experiments (Exp 1 and Exp 2). bChange of signal differences as determined by MAS 5.0 software (I, increased; MI, marginal increased; NC, no change; D, decreased; MD, marginal decreased).

Since ionomycin is known to induce ubiuqitin- UBD and in parallel increases the levels of TCR- ligases associated with anergy,38 we investigated the stimulated Ca2 þ -flux, both UBD together with an effect of ionomycin treatment on UBD expression in elevated Ca2 þ -flux seem to cooperatively regulate Th cells. As predicted, ionomycin induced upregu- the expression of IL1R2. On the other hand, lation of UBD mRNA (Figure 4e), but at the same ionomycin did not improve the suppressive activity

time, FOXP3 mRNA expression was downregulated of Th cells overexpressing UBD (data not shown), despite overexpression of UBD (Figure 4e). This indicating that high levels of FOXP3 are required to suggests that the ionomycin-induced anergic state is obtain a suppressive phenotype. independent of FOXP3 and confirms that UBD does not induce the expression of FOXP3. Interestingly, similar to overexpression of FOXP3 in Th cells High LGALS3 Protein Expression Specifies a (Figure 4f), overexpression of UBD together with FOXP3-Dependent Signature of Human T Cells ionomycin-induced sustained Ca2 þ -flux upregu- reg lated IL1R2 mRNA expression (Figure 4e). As We extended our GeneChip expression analysis of

FOXP3 overexpression leads to the upregulation of ThFOXP3 and Treg cells to ThUBD cells. Regulated

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 731 derived Treg cells, and our FOXP3 and UBD engineered effector Th cells, respectively. Since upregulation of LGALS3 mRNA was com- mon to Treg cells and Th cells overexpressing FOXP3 and UBD, respectively, we analyzed LGALS3 protein expression in these cells. Interestingly, high sustained levels of LGALS3 protein expression were detected only in Treg cells (Figure 5b). Whereas significant induction of LGALS3 protein was also observed in ThFOXP3 cells, UBD overexpression in Th cells only minimally affected LGALS3 protein expression (Figure 5b). Contrary to mice,44 LGALS3 protein was not significantly induced in human nonregulatory effector T cells following stimulation with allogeneic EBV-transformed B cells and IL2, although upregulation of intranuclear FOXP3, cell surface CD25, and intracellular CTLA4 protein expression was observed in such cells (Figure 5c). As LGALS3 protein expression is restricted at low levels to nonactivated human peripheral blood CD4 þ CD25 þ T cells (Figure 5e), high level of LGALS3 protein expression represents a Treg-specific FOXP3 signature of antigen-stimulated human þ hi CD4 CD25 -derived Treg cells. LGALS3 has been described to induce T-cell apoptosis in human T cells via a lactose-dependent binding to CD7 and CD29.45 Therefore, we analyzed the suppressor function of Treg cells in the presence or absence of 50 mM lactose, which potently impairs the apoptotic function of LGALS3.45 But no impairment of Treg cell suppressor function was observed suggesting that LGALS3 is not directly involved in Treg cell suppression (Figure 5f).

Discussion

Figure 3 UBD contributes to the transcriptional and functional In the present study we compared the genetic þ hi control of FOXP3. (a)Th cells transduced as in Figure 1a were program of human CD4 CD25 derived Treg cells tested for UBD mRNA expression by semiquantitative RT-PCR with that of allo-reactive effector Th cells repro- after stimulation with plate-bound anti-CD3, soluble anti-CD28, and 100 U/ml IL2. cDNA was tested in three-fold dilutions grammed towards a partial regulatory phenotype by starting with the first dilution; RPS9 was used as house-keeping retroviral transduction of FOXP3. The reason for control. (b)Th cells transduced with UBD plus GFP (ThUBD), such an approach was to unmask FOXP3-dependent FOXP3 plus GFP (ThFOXP3), and GFP alone (ThGFP) were tested genes by mimicking sustained high levels of FOXP3 for surface CD25 and CD28 expression in flow cytometry. (c)Treg expression in Th cells and to reduce the size of cells and Th cells as in b were stimulated with LG2-EBV B cells alone or in the presence of 100 U/ml IL2. Proliferation was differentially expressed genes to the most significant assessed at day 3. (d) The same T cells as in c were analyzed for set. Critical FOXP3-dependent target genes were IFNg and IL10 cytokine secretion following stimulation with LG2- identified. EBV B cells. One of the genes consistently upregulated was the ubiquitin-like gene diubiquitin (UBD, also known as FAT10). UBD has been initially cloned as a gene exclusively expressed in dendritic cells and mature genes could be classified into four groups (Figure B cells.46 It was reported to be involved in protein 5a). (i) Genes regulated in all three T-cell popula- degradation, apoptosis, and control of spindle tions. These included UBD and LGALS3. (ii) Genes assembly.47,48 Moreover, UBD expression can be expressed similarly in ThFOXP3 and ThUBD cells induced by IFNg, whereas another IFNg inducible only, such as CD9. (iii) Genes expressed similarly in gene, NEDD8 ultimate buster-1L (NUB1L), has been 49 ThUBD and Treg cells only, such as CBL-B. (iv) Genes reported to accelerate the degradation of UBD. A expressed similarly in ThFOXP3 and Treg cells only, potential function of UBD in the physiology of such as CCR7, FYB, and CCNG2. These data reflect human Treg cells has not been addressed so far. This the congruencies and differences between naturally is most likely due to the fact that UBD expression is

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 732 found at significant levels only after in vitro directly induced by FOXP3. Rather, UBD transcrip- activation as demonstrated in this work. tion should be indirectly regulated, for example, by Since FOXP3 is a transcriptional repressor,36,50 downregulating transcriptional repressors of UBD. upregulation of UBD expression should not be This is supported by preliminary experiments in

Figure 4 UBD expression impairs T-cell proliferation independent of FOXP3. (a) Effector Th cells transduced as described in Figure 3b were tested by semiquantitative RT-PCR as described in Figure 3a for mRNA of genes known to be regulated in FOXP3 transduced Th cells. Note that FOXP3 is not upregulated in ThUBD cells. (b) Expression of high levels of FOXP3 protein in ThFOXP3 cells, but not in ThGFP and ThUBD cells, determined by flow cytometry as described in Figure 1f. Percent of positive cells is indicated. (c) Quantification 2 þ of LGALS3 mRNA by Real-time RT-PCR in Treg and ThFOXP3 cells compared to ThGFP cells. (d) Anti-CD3 induced Ca -mobilization in ThFOXP3, ThUBD, and ThGFP cells was determined on a MoFlo as described in Supplementary Figure 2a. (e) ThGFP and ThUBD cells were tested for upregulation of IL1R2, FOXP3, and UBD mRNA. Ionomycin (Io) or vehicle (DMSO) was added. Determination was carried

out after 24 h by semiquantitative RT-PCR as described in Figure 3a. (f) IL1R2 specific mRNA was determined in ThFOXP3, ThUBD, and ThGFP cells of Figure 3b by semiquantitative RT-PCR. Note that no signal is obtained in ThUBD as in ThGFP cells. The slight discrepancy between e and f in this respect is most likely due to differences between the individual donors of Th cells.

þ hi Figure 5 LGALS3 protein expression represents a FOXP3-signature of CD4 CD25 derived Treg cells. (a) Transcriptome analysis of ThUBD, ThFOXP3, and Treg cells. Four groups were defined when analyzing significantly regulated genes. ThUBD ¼ ThFOXP3 ¼ Treg: genes regulated similarly in all three regulatory types of T cells including UBD and LGALS3. ThUBD ¼ Treg: genes regulated similarly in regulatory T cells and UBD transduced Th cells. ThUBD ¼ ThFOXP3: genes regulated similarly in UBD and FOXP3 transduced Th cells. ThFOXP3 ¼ Treg: genes regulated similarly in regulatory T cells and FOXP3 transduced Th cells. The signal fold change is represented as red (upregulated) or green color (downregulated) indicated below the panels. (b)Treg and Th cells were tested for intracellular LGALS3 expression in flow cytometry. Thin line: isotype control; bold line: anti-LGALS3 staining. (c) LGALS3 expression is not induced

following T-cell stimulation. Th and Treg cells were tested for the expression of FOXP3, CD25, LGALS3, and CTLA4 at day 3 following allogeneic stimulation using LG2-EBV B cells and IL2. (d) Specificity of FOXP3 staining was confirmed using murine hybridoma T cells transduced with GFP alone (GFP) and human FOXP3 and GFP (FOXP3) as described in Figure 1f. Shaded area: isotype control, bold line: antibody staining. Percent of positive cells is indicated. (e) Surface staining of CD4 and CD25 on human peripheral blood lymphocytes of a healthy donor (left panel). Regions and percentage of CD4 þ T cells expressing high, intermediary, and no CD25 expression, respectively, correspond to the colors in the right panel (CD25hi ¼ red, CD25int ¼ green, CD25neg ¼ blue curve), which depicts the

corresponding intracellular LGALS3 staining. (f)Th cells were stimulated by allogeneic LG2-EBV B cells in the presence of Treg cells at the indicated ratios with or without 50 mM lactose. Proliferation was assessed at day 3.

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 733 which a 1.5 kb genomic DNA fragment preceding the became apparent, as we could show that UBD transcriptional start of UBD was tested in a reporter overexpression in Th cells interfered with prolifera- system. No reporter activity was induced by FOXP3 tion and upregulated cell surface expression of (unpublished data). CD25. In addition, expression of IL4 and IL5 mRNA The contribution of high levels of UBD expression was downregulated in ThUBD cells comparable to Th to the FOXP3-dependent regulatory phenotype cells overexpressing FOXP3, although FOXP3 was

a b T T UBD T FOXP3 GFP reg T GFP h h h h GFP h h vs T vs UBD vs T UBD vs T vs FOXP3 h h reg T T T T UBD = T FOXP3 = T h h reg NM_000049 ASPA BC0002915 DLG5 AF177761 ERBB2 LGALS3 AU146580 HEMBB1 NM_006398 UBD BC001120 LGALS3 T UBD = T T T h reg c reg h NM_003265 TLR3 NM_014766 SCRN1 GFP FOXP3 NM_004351 CBLB 93.7% d AJ302559 OR2J2 26.1% AI979276 MGC33887 AL535113 PLCB4 AK000353 --- 0.9% 91.4% AA931929 --- AL359599 ATBF1 NM_031294 DFKZP586M1120 BC002660 TMOD1 U85962 CREBBP FOXP3 NM_017681 FLJ20130 AF038181 APC NM_006737 KIR3DL2 BC003064 DAB2 FOXP3 AL569804 PDZRN3 BG397856 HLA-DQA1 AV711904 LYZ/// LILRB1 93.0% BF059313 KIF5C 44.3% AI968085 WNT5A T UBD = T FOXP3 h h e NM_005932 IMPEP CD25hi N58524 INADL int NM_024508 ZBED2 CD25 NM_001769 CD9 CD25 0.25 CD25neg AI356398 ZFP36L2 W74620 HNRPD AE000659 --- AU157140 LOC153914 1.59 AA431984 R30953_1 92.41 BG340670 IGHD 6.4% 93.2% CD25 T FOXP3 = T h reg CD4 LGALS3 D55696 LGMN NM_002674 PMCH AF063002 FHL1 NM_004974 KCNA2 AI978623 KIAA0657 AL137457 SRGAP3 NM_001838 CCR7 LGALS3 AF312918 MONDOA NM_016279 CDH9 KO3122 IL2RA T15777 LAPTM4B AW134535 CCNG2 51.9% AA126728 ICAM2 NM_005214 CTLA4 AV756141 CSF2RB 92.9% D29642 --- NM_022154 SLC39A8 M21121 CCL5 NM_024615 PARP8 NM_017831 RNF125 AL137145 PRKCQ U52144 IDH2 AF198052 FYB CTLA4 NM_002985 --- BC005254 CLEC2B NM_018384 GIMAP5 AF031824 CST7 AB011152 CENTD1 f T /T (1:0.25) M81635 STOM h reg NM_018326 GIMAP4 T /T (1:0.5) NM_006433 GNLY h reg 50 mM lactulose NM_018458 KIAA1280 T /T (1:1) W80678 KRAS h reg AW134823 CD6 T /T (1:0.25) M27331 TRGV9 /// TARP h reg NM_005601 NKG7 T /T (1:0.5) BG546884 CRIM1 h reg NM_018700 TRIM36 T /T (1:1) NM_002104 GZMK h reg M36118 GZMH T /T (1:1) h h T h 3.0 1:1 -3.0 T background reg T h

0 20000 40000 60000 H -incorporation (cpm) 3

Laboratory Investigation (2006) 86, 724–737 UBD and LGALS3 represent FOXP3-dependent genes F Ocklenburg et al 734 not induced by UBD. In contrast, genes normally phosphorylation and dephosporylation at position 6 induced upon FOXP3-overexpression, for example of LGALS3 by casein kinase 1 and protein phospha- CD28 and CTLA4, were not affected by UBD tase 1, respectively, controls the carbohydrate bind- transduction. This indicates that UBD acts down- ing capability, nuclear localization, transcriptional stream in some of the FOXP3-induced regulatory modulation, and antiapoptotic function of signaling branches, and, therefore, only a partial LGALS3.58,63,64 Diversified functions of LGALS3 regulatory phenotype is induced by UBD alone as it have also been reported for cells of the immune does not upregulate FOXP3. A clear confirmation of system. LGALS3 has been detected in monocytes this phenomenon was obtained, when ionomycin and macrophages, dendritic cells, mast cells, murine 65 was added to Th cells overexpressing UBD. Only the T cells, and granulocytes. Several important ionomycin-mediated higher Ca2 þ -levels together processes and functions are modulated by LGALS3 with overexpression of UBD mimicked the FOXP3- in these cells including thymocyte migration,66 induced transcriptional control of IL1R2. In line innate immunity against infections,67 inflamma- with this was the observation that UBD overexpres- tion,68 and T-cell apoptosis.45,69 Owing to the non- sion only minimally induced LGALS3 protein differential expression of LGALS3 in activated þ þ 44 expression in Th cells, although LGALS3 mRNA murine CD8 and CD4 T cells, it was unexpected was upregulated similar to Th cells overexpressing that LGALS3 might act as a Treg-specific marker. FOXP3. However, only low levels of LGALS3 protein were How UBD mediates transcriptional control is detectable in human peripheral blood CD4 þ CD25 þ questionable. It is known that E3-ubiquitin ligases T cells, but sustained high levels were induced in þ hi such as Itch, Cbl-b, and Grail play an essential role naturally derived human CD4 CD25 Treg cells in the induction of T-cell anergy. Their mode of following antigenic stimulation in vitro. Similarly, action is to target key TCR signaling molecules for upregulation of LGALS3 protein expression was 38,51 degradation that are localized in the cytoplasm. observed in FOXP3-transduced effector Th cells UBD has similarly been reported to target proteins reprogrammed towards a partial regulatory pheno- for degradation but not in the context of TCR signal type. This indicates that LGALS3 protein expression transduction. However, UBD has also been found to in fact represents a FOXP3-signature of the regula- be localized in the nucleus.52 Therefore, its main tory program. effect might be a transcriptional modulation, as The potential contribution of LGALS3 to the the activity of many signal transduction path- function of activated Treg cells is speculative. The ways and transcription factors are regulated by anti-apoptotic activity of LGALS3 might protect Treg ubiquitin-mediated proteolytic and nonproteolytic cells from apoptotic signals through CD95,70 which 53,54 mechanisms. is expressed at high levels on Treg cells. Alterna- Whatever the mechanisms, our extended trans- tively, the activation and modulation of specific criptome analysis of Treg cells compared to ThFOXP3 transcription factors, like AP1, NFAT, SP1, and 61,62 and ThUBD cells suggested that LGALS3 might play CREB, might contribute to the FOXP3-depen- a central role for some of the overlapping functional dent transcriptional control. However, more direct and phenotypic changes observed in these cells. effects of LGALS3 on Treg cell functions have to be LGALS3 represents a unique member of the considered. In murine T cells, a direct association of family of b-galactoside-binding proteins. It is com- LGALS3 with the TCR complex, restricting TCR posed of a small N-terminal domain, which controls signaling, has been reported. This interaction its subcellular targeting, a repetitive collagen-like dependents on N-glycosylation mediated by the region that serves as substrate for matrix metallo- enzyme beta-1,6 N-acetylglucosaminyltransferase V proteinases, and a C-terminal carbohydrate-recogni- (Mgat5).71 Therefore, mice deficient in Mgat5 show a tion domain.55 Expression of LGALS3 has been lowered T-cell activation threshold with enhanced observed in different tissues and cells types. It recruitment of TCRs to agonist-coated beads, and displays pleiotropic biological functions depending actin microfilament re-organization.71 In contrast to þ hi on subcellular localization. Extracellular LGALS3 the latter, in human CD4 CD25 derived Treg cells, binds to cells through glycosylated receptors, there- that express LGALS3, we have observed a dynamic by triggering or modulating cellular responses such impairment of actin remodeling compared to as mediator release, apoptosis, cell adhesion, and CD4 þ CD25À T cells.72 migration.45,56 Intracellular LGALS3 has been re- The addition of soluble LGALS3 to Jurkat T cells ported to inhibit apoptosis57 and the cell cycle.58 has been reported to trigger a sustained influx of Interestingly, upregulation of LGALS3 has been extracellular Ca2 þ that can be blocked by lactose.73 2 þ observed during neoplastic transformation in sev- As a sustained Ca flux in Treg cells following TCR eral human malignancies.59 Moreover, LGALS3 has stimulation was observed in our case, LGALS3 been found in the nucleus, where it is reported to be released from the cells might act in an autocrine 60 2 þ involved in pre-mRNA splicing, and the regulation manner and modulate the Ca homeostasis of Treg of gene expression (eg cyclinD and Muc2) through cells. However, similar to T cells of mice,44 we did the activation of specific transcription factors, not detect significant cell surface bound LGALS3 61,62 including AP1, NFAT, SP1, and CREB. Serine in resting or activated Treg cells. We, therefore,

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Supplementary Information accompanies the paper on the Laboratory Investigation website (http:// www.nature.com/labinvest).

Laboratory Investigation (2006) 86, 724–737