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Expression Profiling of Murine Double-Negative Regulatory T Cells Suggest Mechanisms for Prolonged Cardiac Allograft Survival¹

Boris P.-L. Lee^*,, Elaine Mansfield, Szu-Chuan Hsieh, Tina Hernandez-Boussard^¶, Wenhao Chen^*,, Christopher W. Thomson^*,, Megan S. Ford^*,, Steven E. Bosinger^*, Sandy Der, Zhu-xu Zhang^*,, Meixia Zhang, David J. Kelvin^*,¶, Minnie M. Sarwal^2, and Li Zhang^2,3,*,,

^* Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada; Departments of Laboratory Medicine and Pathobiology and Immunology, University of Toronto, Toronto, Ontario, Canada; and Departments of Pediatrics and ^¶ Genetics, Stanford University, Stanford, CA 94305

	Abstract

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Recent studies have demonstrated that both mouse and human TCR⁺CD3⁺NK1.1^–CD4^–CD8^– double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8⁺ and CD4⁺ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8⁺ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 ± 11.1 days) compared with untreated controls (22.8 ± 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including FcRI subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.

	Introduction

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Regulatory T (Treg)⁴ cells play an important role in modulating various diseases, including autoimmune diseases (1, 2), transplantation (3), allergy (4), infections (5), and cancer (6). Many types of Treg cells are known to be present in various animal and human disease models. The most extensively studied Treg cell populations are CD4⁺, which include CD4⁺CD25⁺ cells (7), Tr1 cells (8), CD4⁺CD103⁺ T cells (9), and CD4⁺CD25^– Treg cells (10). In addition to CD4⁺ T cells, CD8⁺CD28^– cells (11), T cells (12), NK T cells (13), and TCR⁺CD3⁺NK1.1^–CD4^–CD8^– double-negative (DN) T cells (14, 15, 16, 17, 18) have also been shown to have potent immunoregulatory function. Currently, identification of Treg cells is based primarily on nonspecific cell surface markers such as CD25 and CD62L. However, these markers are present only on certain subsets of Treg cells, as well as on nonregulatory cells.

Global expression analysis using cDNA microarray technology is a very powerful tool for high-throughput comparison of gene expression profiling. This technique allows for the identification of genes that are expressed cooperatively or are coregulated by a given treatment or stimulus. Microarray analysis has been successfully used to study many aspects of transplantation, including graft rejection (19, 20) and early transplant rejection prognosis (21). Recently, several groups have used this approach in an attempt to identify specific markers for CD4⁺CD25⁺ Treg cells. By comparing CD4⁺CD25⁺ Tr cells to CD4⁺CD25^– T cells (9, 10, 22, 23), it was discovered that CD4⁺CD25⁺ Treg cells have a unique expression profile of various genes, including suppressors of cytokine signaling and glucocorticoid-induced TNFR family-related receptor, a member of the TNFR superfamily (22). Furthermore, differences in the regulation of apoptosis, cell cycle, cytokine receptor, cell-cell interaction, and stress pathway genes were also observed (23). In addition, levels of _e7 integrin appears to correlate with presence of highly suppressive CD4⁺CD25⁺ Treg cells (9). Expression of the transcription factor scurfin, encoded by the Foxp3 gene, has been correlated to CD4⁺CD25⁺ and CD8⁺CD25⁺ Treg cells (24, 25). Foxp3 and neuropilin-1 are currently accepted as the best candidate markers for these Treg cells (26). However, it is not known whether the genes that are preferentially expressed in CD4⁺CD25⁺ Treg cells are also expressed in other Treg cells. Furthermore, the molecular mechanisms by which Treg cells control immune responses remain elusive.

DN Treg cells comprise 1–3% of peripheral T lymphocytes in rodents (15). These cells can down-regulate syngeneic CD8⁺ T cell-mediated immune responses to self-Ags, suggesting a role for DN Treg cells in regulation of autoimmunity (27). We have previously shown that DN Treg cells isolated from mice that have permanently accepted allo- or xenografts can specifically suppress and kill syngeneic anti-donor CD4⁺ and CD8⁺ T cells in vitro (16, 17, 28). DN Treg cells expanded in vitro upon stimulation with allogeneic donor lymphocytes can specifically suppress proliferation of syngeneic CD4⁺ and CD8⁺ T cells in vitro and prolong donor-specific allogeneic skin graft survival when infused into syngeneic naive mice (15, 17). Furthermore, injection of DN Treg clones can also attenuate graft vs host disease caused by infusion of allogeneic CD8⁺ T cells, suggesting a role in in vivo suppression of anti-host CD8⁺ T cells (14). Recently, human DN Treg cells have been isolated and characterized. These cells compose 0.8–1% of total human peripheral blood CD3⁺ T cells and 2.5% of lymph note T cells (18). Similar to what has been found in murine models, human DN Treg cells can also suppress immune responses mediated by syngeneic CD8⁺ T cells in an Ag-specific and dose-dependent manner (15, 18). Interestingly, both mouse and human DN Treg cells are cytotoxic to syngeneic CD8⁺ T cells that express the same TCR specificity as DN Treg cells (15, 18). Although these findings suggest that DN Treg cells are important in regulating immune responses both in vitro and in vivo, the molecular mechanisms behind their effects are poorly understood.

The goal of this study was to analyze global gene expression patterns that differentiate DN Treg clones that have potent immunoregulatory functions in vitro and in vivo from nonfunctional mutant cell lines derived from these clones to gain insight into the molecular mechanisms that contribute to DN Treg cell function. By comparing the expression profiles of DN Treg clones with those from the nonregulatory mutant strains, we identified 1099 highly differentially expressed genes revealing increased expression of genes involved in cell proliferation, immune regulation, and chemotaxis and an associated decrease in expression of genes involved in Ag presentation, apoptosis, and protein phosphatases involved in signal transduction pathways. A subset of overexpressed and underexpressed genes were validated using quantitative real-time PCR (QRT-PCR) or FACS analysis. The potential involvement of these molecules in controlling DN Treg cell function is discussed.

	Materials and Methods

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Mice

C57BL/6 x BALB/c (CB6F₁) and BALB/c-H-2-dm2 (dm2) were purchased from The Jackson Laboratory. A breeding stock of 2C-transgenic mice (on C57BL/6 background) was provided by Dr. D. Y. Loh (Nippon Research Center, Kanagawa, Japan). The 2C mice were bred with dm2 mice to obtain anti-L^d-transgenic TCR⁺ (2Cxdm2)F₁ (H-2^b/d, L^{d –}) mice. The anti-L^d-transgenic TCR can be detected by a specific mAb 1B2 (29). All of the animals were kept in the animal facility at the University Health Network. All protocols were approved by the University Health Network Animal Care Committee, which meets the guidelines of the Canadian Council on Animal Care.

Generating and maintaining DN Treg and mutant clones

Generation of DN Treg clones was performed using previously described methods (15). In brief, splenocytes were collected from either naive or tolerant (2Cxdm2)F₁ mice that permanently accepted CB6F₁ skin allografts following donor lymphocyte infusion and stimulated with irradiated L^d+ CB6F₁ splenocytes in the presence of 30 U/ml recombinant human IL-2 and 30 U/ml rIL-4 for 10 days. Activated 1B2⁺ T cells were subsequently cultured in 96-well tissue culture plates at limiting dilutions of 0.5 cells/well. Additionally, cells arising from these wells were subcloned at dilutions of 0.5 cells/well to ensure clonality. To maintain the T cell clones, 5 x 10⁴ cells were cultured in a 24-well plate containing 5 x 10⁵ irradiated L^d+ CB6F₁ splenocytes as stimulators in -MEM supplemented with 10% FBS, 0.1% 2-ME, and 30 U/ml recombinant human IL-2 and 30 U/ml rIL-4. The cells were incubated at 37°C with 5% CO₂. Cells were restimulated in the same way every 3–4 days.

Cytotoxicity assays

(2Cxdm2)F₁ splenocytes were stimulated with irradiated (20 Gy) allogeneic spleen cells from L^d+ CB6F₁ mice for 3 days. Activated L^d-specific 1B2⁺CD8⁺ T cells then were labeled with [³H]TdR at a concentration of 10 µCi/ml with 10 µg/ml con A and 50 U/ml IL-2 for 18 h, then washed three times in -MEM and used as targets. DN Treg clones (control naive (CN)4 and tolerant CD4/CD8 negative (TN)12) and mutants (CN4.8 and TN12.8) were used as effector cells at day 3–4 poststimulation and plated in serial dilutions in a round-bottom 96-well tissue culture plate in the presence of 50 U/ml rIL-2, 30 U/ml rIL-4, and irradiated CB6F₁ splenocytes as stimulator cells. After coculture with effector cells at 37°C for 18 h, the cells were harvested and analyzed using a TOP COUNT (Packard Instruments) plate reader. Percent-specific cell killing was calculated using the equation: percentage of specific killing = (S – E)/S x 100, where E (experimental) is cpm of the retained DNA in the presence of effector cells, and S (spontaneous) is cpm of retained DNA in the absence of effector cells.

Mouse cardiac transplantation

Naive 8- to 10-wk-old (2Cxdm2)F₁ mice were preinfused with 10⁷/mouse of either the regulatory clone TN12, the mutant clone TN12.8, or left untreated 1 day pretransplantation. Heart grafts from 6- to 8-wk-old CB6F₁ mice were transplanted heterotopically into the abdomen of the mice as described previously (16). Graft survival was monitored daily by palpation. Grafts were considered rejected when heartbeats could no longer be detected and confirmed by autopsy. Graft survival was expressed as mean survival time ± SD.

cDNA microarray and data analysis

Total RNA was isolated from regulatory and mutant clones using the standard TRIzol reagent protocol (Invitrogen Life Technologies) 3–4 days poststimulation. Before RNA isolation, all clones were tested for their ability to kill activated CD8⁺ T cells in vitro as described above. Five of six regulatory clones showed at least 30% cytotoxicity toward alloreactive syngeneic CD8⁺ T cells and one (TN12 clone) showed reduced but detectable cytotoxicity. None of the mutant clones showed cytotoxicity toward activated syngeneic CD8⁺ T cells.

The cDNA microarrays were processed using protocols as previously described by our laboratory (19) using 100 µg of total RNA in each channel. Briefly, a 39,000 element cDNA microarray (mouse cDNA array Mouse Microarray Consortium developed at the Stanford Microarray Core Facility http://microarray. org/sfgf/jsp/home.jsp) was used for global gene expression profiling. Each cDNA microarray contains 35,000 cDNA clones with Unigene cluster identifiers representing 20,182 unique putative genes based on these identifiers (Unigene database build 141). Total RNA (100 µg) was obtained from 12 independent cell cultures of DN Treg cell lines (CN4 and TN12) and noncytotoxic mutant lines (CN4.8 and TN12.8). The DN Treg cell lines (CN4 and TN12) were labeled with Cy3 (green) and mutant cell lines (CN4.8 and TN12.8) labeled with Cy5 (red) and cohybridized in DN Treg: mutant cell pairs to six cDNA microarrays. Hybridized microarrays were scanned using GenePix 4000 (Axon Instruments), and fluorescent images were analyzed with the GenePix Pro software package.

Defective spots were flagged, and data for the six arrays in this study were stored on Stanford Microarray Database (30). Gene lists filtered at retrieval and green:red log₂ normalized ratio collected (positive values represent higher expression in the DN Treg cell lines relative to their respective mutations). Low-stringency retrieval settings (80% representative data, signal:noise ratio of expression measurements > 1.5, signal > 200 in both channels) yields 28,142 cDNA clones. Of these, 1099 clones were found to be differentially expressed (using expression cutoff of 2-fold in one array) and subjected to additional statistical analysis. Cluster (v3.0) generated hierarchical clusters of the samples measuring similarity of expression of the genes and similarity across arrays, which are visualized with the TreeView program (Ref. 31 ; www.microarrays.org/software).

Array data were centered before statistical analysis and significant gene expression differences between the DN Treg cells and mutants identified using a false discovery rate threshold of q <0.025% and one-class response in Statistical Analysis of Microarray (SAM) (32). Individual genes are ranked by a significance score (the q value) that is the estimate of the percentage of false positives in a set of differentially expressed genes that have the same or larger T statistic. The Predication Analysis of Microarray program (33) was used to identify the minimum gene set that differentiated the two parental cell lines.

Two approaches were used to assess the functional composition of genes assessed as significantly associated with phenotype: first, Expression Analysis Systematic Explorer (EASE; http://apps1.niaid.nih.gov/david/; Ref. 34) was used to identify significant enrichment of cellular functional gene classes identified to be over- or underexpressed using SAM. EASE provides statistical significance of gene families identified using standardized Kyoto Encylopedia of Genes and Genomes or Genome Ontology databases terms. A normalized gene enrichment score and Fisher t test are reported for each functional category. Representative clones from these functional areas were identified using an independent set of 718 cDNA clones selected using high-stringency unsupervised gene filtering (less than four observed values with a minimum 2-fold expression difference on arrays derived from among five paired samples with greatest cytotoxicity (three CN4 and two TN12)). Average linkage clustering was applied to the expression data using Genelinker Platinum version 4.5 (Predictive Patterns).

QRT-PCR

QRT-PCR analysis was performed with the ABI Prizm 7900HT thermocycler (Applied Biosystems) using SYBR Green detection. Briefly, two of the RNA sample sets used for microarray analysis were used initially to validate QRT-PCR. Later, when these samples were depleted, RNA from independent cultures of CN4, TN12, CN4.8, and TN12.8 were obtained as described above reverse transcribed using random hexamers. Each reaction was performed in a 10 µL reaction containing 3 mM MgCl₂, 50 nM dNTP, 20 nM primers, 40 ng of cDNA, 1x Rox reference dye (Invitrogen Life Technologies), 1x SYBR green reagent, and 0.125 U/µL Jumpstart Taq polymerase (Sigma-Aldrich). The following protocol was used: denaturation program (95°C for 3 min), followed by the amplification and quantification program (95°C for 10 s, 60°C for 15 s, 72°C for 20 s) repeated for 40 cycles, with one cycle of a finishing program (72°C for 1 min). Amplification was followed by melting curve analysis (95°C for 15 s, 60°C for 15 s, and 95°C for 15 s) to ensure the presence of a single PCR product. The expression of -actin was used to normalize starting cDNA concentrations. The primers used for all QRT-PCR are listed in Table I. A standard curve consisting of five 3-fold dilutions of cDNA from a pool of all four samples (1:3:9:27:81) was used for linear regression analysis of all samples.

Regulatory and mutant clone cells were collected at day 3 poststimulation and incubated with fluorescence-conjugated mAbs CD8-FITC, CD44-FITC, TLR4-FITC, and CD80-PE from eBioscience, biotinylated CXCR5 (BD Biosciences), or subject to intracellular detection using Cytofix/Cytoperm Plus (BD Biosciences) along with FcRI from Upstate Biotechnology followed by goat anti-rabbit-PE (Cedarlane Laboratories). Data were acquired and analyzed on an EPICS XL-MCL flow cytometer.

	Results

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Generation and characterization of DN Treg and mutant clones

As DN Treg cells make up only a small percentage of total CD3⁺ cells, it was necessary to generate clonal cell lines to provide sufficient materials for molecular characterization. More than 20 DN Treg clones were generated from CN (2Cxdm2)F₁ mice but only four survived subsequent culturing (designated CN2, CN3, CN4, and CN5). Twenty-eight DN Treg clones were derived from mice that permanently accepted donor-specific skin allografts after pretreatment with donor lymphocyte infusion and two were successfully maintained. Hence, these clones were referred to as TN cells (designated TN2 and TN12). All the DN Treg clones express an anti-L^d-transgenic TCR, which can be detected by mAb 1B2. They are TCR⁺CD3⁺NK1.1^–CD4^–CD8^–, as well as being negative for CD44 and CD28 (Table II) by flow cytometric analysis. Long-term cultivation of the DN Treg clones CN4 and TN12 resulted in the generation of two phenotypic variants (CN4.8 from CN4 and TN12.8 from TN12), which significantly underexpressed their TCR and simultaneously acquired CD8 expression (Table II). During the subsequent cultures, the phenotype of these cells remained stable and never reverted to that of parental regulatory clones; therefore, we designated these variants as mutant clones.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Regulatory capacity of DN Treg clones and their natural mutants in vitro and in vivo. A, Two Ld-specific DN Treg clones TN12 () and CN4 () and their natural mutants TN12.8 () and CN4.8 (), respectively, were collected 3 days after stimulation and used as effector cells. In vitro-activated H-2d-specific (2Cxdm2)F1 CD8+ T cells that are syngeneic to the DN T cell clones were labeled with [3H]TdR and used as targets at E:T ratios as indicated in cytotoxicity assays as described previously (15 ). The data are expressed as mean percent killing of CD8 cells from three replicate cultures. The experiment was repeated three times, and representative data is shown. B, (2Cxdm2)F1 mice were i.v. infused with (1 x 107/mouse) either DN Treg clones (n = 6) or its mutant clone TN12.8 (n = 5) or left untreated (n = 5). All mice were transplanted with a heart allograft from CB6F1 mice. Graft survival was monitored by daily palpation. The data show percentage graft survival.

To further examine whether DN Treg and mutant clones differ in their function in vivo, we compared the ability of DN Treg clones to prolong allograft survival with that of the mutant clones. (2Cxdm2)F₁ mice were infused with either syngeneic DN Treg clone TN12 or its natural mutant TN12.8 clone cells followed by transplantation of cardiac allografts from CB6F₁ mice. A group of (2Cxdm2)F₁ mice received cardiac transplantation without pretransplant infusion of clone cells as a control. Fig. 1B shows the graft survival in the control, TN12.8-treated, and TN12-treated (2Cxdm2)F₁ mice. The allogeneic heart grafts survived over 100 days in all the animals that were preinfused with TN12 regulatory clones. In contrast, infusion of mutant clones had no beneficial effect on allograft survival (19.5 ± 11.1 days) and no significant difference in mean graft survival time compared with untreated controls (22.8 ± 10.5 days survival). These data clearly demonstrate that DN Treg clones have a potent regulatory function, whereas their natural mutants have lost the regulatory function both in vitro and in vivo (p < 0.001). The availability of DN Treg clones and their natural mutants provides us with unique tools to further identify the molecules that are expressed by DN Treg cells and are important for their immunoregulatory function.

Microarray analysis reveals differences in gene expression between DN Treg and mutant clones

To characterize the genetic changes associated with the change in function from DN Treg cells to nonregulatory mutants, we used cDNA microarray technology for global gene expression profiling. Unsupervised clustering of 10,000 genes differentially expressed between the DN Treg and mutant clones reveals parallel expression analysis across both regulatory cell lines (Fig. 2A), despite the fact that 12 independent RNA preparations were used on each array. Consistency in the informative expression profile was confirmed using one-class response in SAM (32) to identify a subset of 1099 cDNAs significantly over- or underexpressed in the DN Treg clones (Fig. 2B; q = 0.021% and 90^th percentile false positive rate < 4.5% or 49 clones). To better understand the functional pathways predominantly impacted by these differences, gene ontology annotations of the known unique genes (187 underexpressed and 356 overexpressed genes) in this list were analyzed using the EASE-online (http://apps1.niaid.nih.gov/david/; Ref. 34), and results are summarized in Table III. Redundant pathways are represented clearly on this intrinsic gene list, and consequently, genes that are differentially expressed between DN Treg and mutant clones can be categorized into eight functional groups, including TCR associated, Ag presentation, chemotaxis, IFN-inducible genes, survival and proliferation markers, cytotoxicity related, apoptosis, and immunomodulators. The average expression change in DN Treg cells relative to mutant cell lines is summarized in Table IV column A, and representative genes from each of these functional classifications were selected for confirmatory studies. Far more cDNAs (1099 vs 22) were found to have similarities in expression changes between the two DN Treg cell lines than to differentiate them. Only 10 known genes (mapping to the 22 differentiating cDNAs) were found to segregate the CN4 and TN12 DN Treg cell lines (Fig. 2D). Qualitative differences in the expression of an apoptosis marker (annexin 4A), proliferation/growth-related genes, myeloid-associated differentiation marker and insulin-like growth factor (IGF)-2R, and two H2 class II genes A1 and DM are among this differentiating gene set.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Genes differentially expressed between DN Treg clones and their mutants identified by cDNA microarray analysis. Six DN Treg:mutant clones are compared by hierarchical clustering of 10,199 differentially expressed transcripts (A). Array experiments consist of three DN Treg CN4 clones (blue) and three TN12 clones (turquoise) each labeled in Cy5 (red) that have been cohybridized with mutant clones CN4.8 and TN12.8, respectively, labeled in Cy3 (green). The relative expression scale ranged ± 2log8 or 256-fold, and missing data are displayed in gray. Thus, genes displayed in red are expressed in higher levels in the parental DN-Treg lines, whereas those displayed in green are expressed in higher levels in the noncytotoxic mutant lines. B, Supervised analysis across the six arrays using SAM across the genes in A reveals that 10% (or 1099 transcripts) show highly significant differential expression between the DN Treg clones relative to the noncytotoxic mutant clones. The false positive rate beyond the 95% confidence interval in the SAM plot was observed to be <0.025% or under one gene. Unsupervised clustering was used to identify candidate genes among the five arrays with highest level of cytotoxicity (the array for clone TN12# was excluded) and 718 differentially expressed transcripts identified (representative data and 2-fold expression difference across four of five samples). Samples were clustered on the basis of the array expression results (C). These genes were all either over- or underexpressed in parallel between the two parental lines, as well as in TN12#. Overall similarity in the global expression profiles in the CN4 and TN12 clones was confirmed using Predictive Analysis of Microarray class prediction. A set of only 22 cDNA clones representing 10 known genes can differentiate the two parental DN Treg cell lines (D).

To validate the differences in gene expression observed by cDNA microarray screen, 57 representative genes (33 overexpressed and 24 underexpressed in both DN Treg cell lines) were selected for confirmation using QRT-PCR assays. Table IV column B shows fold increase and decrease of genes in regulatory clones compared with their mutant progenies. Similar expression differences in the two DN Treg strains were observed using QRT-PCR as determined by cDNA microarrays (Fig. 3).

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Validation of cDNA microarray gene expression data by QRT-PCR. The relative gene expression measurements for 57 candidate genes identified by cDNA array screening from Fig. 2C is graphed for two DN Treg cell lines (A). The average fold change between expression CN4 relative to CN4.8 of 24 underexpressed genes and 33 overexpressed genes are graphed (). Relative abundances comparing TN12 with TN12.8 mutant clones () show similar pattern of expression. The expression level relative to -actin as a normalizing gene was determined by QRT-PCR and relative expression in the two cell lines graphed (B). Independent RNA preparations were used in the array and QRT-PCR experiments and genes are ordered by fold differences in the TN12/TN12.8 cell lines. A significant positive correlation was obtained by the two independent assay methods as evidenced by log-log plot correlation of R2 = 0.76. Measurements for TCR-v13 were eliminated from the analysis because it falls below the limit of detection for QRT-PCR in both mutant cell lines.

Genes that showed underexpression in DN Treg cells could also be classified into significant functional groups: 1) Ag presentation genes histocompatibility 2, class II Ags E1 (H2-E1) and D1 (H2-Dm1); 2) chemotaxis genes vimentin (Vim) and homing-associated cellular adhesion molecule (CD44); 3) IFN-associated genes IRF4, IFI transmembrane protein 3-like (IFITM3l), IFI proteins 202a (IFI202a) and 204 (IFI204), and IFN consensus sequence-binding protein 1 (Icsbp1); 4) survival and proliferation genes IGF-1 and cell division cycle-like kinase 4 (Clk4); 5) cytotoxic genes lymphotoxin , TNF-, GzmA, GzmB, and GzmC; 6) the apoptosis-associated gene RAR; and 7) immunomodulatory genes Campath-1 (CD52), CD8 subunit (CD8), Notch gene homologue 1 (Notch1), CD80, CD48, and IL-4.

Flow cytometry correlates surface protein expression to cDNA microarray data

As both cDNA microarray and QRT-PCR detect only RNA transcript levels, it is also desirable to determine whether these gene expression patterns also reflect changes in protein expression. To test this, flow cytometric analysis of FcRI, TLR4, CXCR5, CD8, CD44, and CD80 was performed to validate expression at the protein levels (Fig. 4). FcRI, TLR4, and CXCR5 protein were overexpressed in both regulatory clone strains relative to their mutant progeny, whereas CD8, CD44, and CD80 were noticeably underexpressed in the regulatory clones. These data suggest that the gene expression patterns observed using cDNA microarray and QRT-PCR are consistent with their corresponding protein expression for this subset of the genes.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Detection of protein expression on DN Treg and mutant clones. The DN Treg clones TN12 (dotted lines) and mutant clone TN12.8 (dashed lines) were compared using flow cytometry for expression of the genes TLR4, FcRI, CXCR5, CD8, CD44, and CD80. The negative control is shown as solid lines. Data are representative of at least four experiments.

	Discussion

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We and others (15, 17, 18, 27) have shown that both human and mouse DN Treg cells and clones down-regulate immune responses, at least in part, through killing of activated syngeneic CD8⁺ and CD4⁺ T cells. T cells mediate cytotoxicity through two independent mechanisms, including expression of perforin/Gzm (35) and Fas-FasL interaction (as reviewed by Hahn and Erb (36)). We have previously shown that DN Treg cells do not mediate killing of syngeneic CD8⁺ T cells through the perforin/Gzm pathway (15). Rather, these cells kill activated CD4⁺ and CD8⁺ T cells at least partially through Fas-FasL-mediated interaction (15, 27). Blocking Fas-FasL interaction significantly decreases DN Treg cell cytotoxicity (15, 17). Our examination of cytotoxicity-associated genes revealed significant increases in the expression of Egr-1, FasL, and GzmM in DN Treg clones. In contrast, lymphotoxin , TNF-, GzmA, GzmB, and GzmC were underexpressed. Egr-1 has previously been shown to up-regulate expression of FasL in both lymphoid and nonlymphoid cells (37, 38). Although FasL is widely accepted as an inducer of cell death upon ligation with the Fas receptor (36), this interaction may also be a mechanism by which immune privilege is achieved in certain sites (39). These results validate previous observations concerning Fas-FasL-mediated cytotoxicity being the primary mechanism by which DN Treg cells act upon activated syngeneic CD8⁺ T cells (15, 17, 27).

Both mouse and human DN Treg cells produce high levels of IFN- (15, 18), and constitutive IFN- expression has been demonstrated in CD4⁺CD25⁺ Treg cells (40). Additionally, we demonstrated that neutralization of secreted IFN- abrogates the cytotoxic capacity of DN Treg cells (J. Pun and L. Zhang, unpublished data). The mechanism by which IFN- regulates DN Treg-mediated cytotoxicity is not clear. Several IFN-regulated genes (TGTP, IGTP, IRF1, IFI47, and IFI30) were found to be overexpressed in this study, whereas others (IRF4, IFI204, IFI202a, IFITM3l, and Icsbp1) were underexpressed. Whether these IFN-regulated genes are involved in DN Treg cell-mediated suppression requires further study.

It has been shown that both human and murine DN Treg cells are able to acquire allo-MHC-peptides from APCs, and this process is critical for DN Treg cell-mediated Ag-specific suppression (15, 18). Furthermore, we have shown previously that the TCR on DN Treg cells is required for the acquisition of allo-MHC-peptide and that blocking the interaction between the TCR and allo-MHC-peptide abrogates DN Treg cell-mediated cytotoxicity to CD8⁺ T cells (15). However, the molecules that are involved in the acquisition remain unclear. Two genes that are associated with TCR (FcRI and TCR-v13) were identified to be differentially expressed between DN Treg and mutant cell lines. FcRI was identified originally as a subunit of the high-affinity IgE receptor (FcRI) and is expressed on a variety of cells, such as mast cells, basophils, neutrophils, macrophages, NK cells, and some T cells (41). More recently, FcRI has been demonstrated to be an alternate component of the TCR complex in place of CD3 (42). FcRI is functionally and structurally very similar to CD3 that is associated as a homodimer within the TCR of the majority of peripheral T cells. FcRI was found to be the most significantly overexpressed molecule in both functional DN Treg clones relative to the nonfunctional mutant clones (100-fold higher in DN Treg clones relative to their mutants by cDNA array analysis and over 220-fold higher by QRT-PCR) (Table IV). Furthermore, intracellular staining of FcRI also showed a significantly higher protein expression in the functional DN Treg clones. It is possible that DN Treg cells preferentially use FcRI in place of CD3 as part of the TCR complex to acquire allo-MHC-peptides. Alternatively, expression of FcRI may indicate an alternate method of TCR signaling in DN Treg cells (43). Increased expression of TCR-v13 may correlate with the observed increased expression of TCR in DN Treg cells relative to mutants as indicated in Table II and may represent a general increase in genes associated with Ag detection.

Recent studies indicate that CD4⁺ Treg cells accumulate in autoimmune diseased organs (2), accepted allografts (44), and at tumor sites (6). We have previously shown that DN Treg cells preferentially accumulate in accepted skin allografts (28). Furthermore, DN Treg clones, but not mutant clones, are able to migrate to cardiac allografts and induce tolerance.⁵ However, the mechanisms by which these cells migrate to the graft are as yet unclear. In the present study, we report that CXCR5 gene expression is increased 50-fold in both of the DN Treg clones compared with their mutant progeny. CXCR5 was described initially in Burkitt’s lymphoma as a G protein-coupled receptor (45) and subsequently found in B cells and a subset of CD4⁺ T cells (46). More recently, CXCR5 expression has also been shown in DN T cells from MRL-Lpr mice (47). CXCR5 responds to its exclusive ligand CXCL13 by initiating chemotaxis toward an increasing gradient in vitro and to B cell zones in lymph nodes that express CXCL13 (47, 48, 49). Several reports indicated no expression of CXCL13 in rejecting allografts (50). However, it is not known whether tolerant allografts express this chemokine or whether this expression occurs at early stages of posttransplantation. It is possible that grafts expressing elevated CXCL13 may preferentially attract DN Treg cells expressing high levels of CXCR5, thereby homing these cells into donor tissue. Our recent studies have indicated that graft-infiltrating DN Treg cells are able to suppress anti-graft CD8⁺ T cells that could otherwise destroy the graft (28). We are currently testing this hypothesis in a model of transplant tolerance. In addition to CXCR5, regulatory clones also showed overexpression of other genes associated with chemotaxis, including the chemokine receptors CCR2 and CCR3. The ligands of CCR2 and CCR3, such as MCP-1, MCP-2, and MCP-3, Eotaxin, and RANTES, have been detected in transplanted allografts in both humans and animal models (51, 52, 53, 54). This suggests that DN Treg cells could home in to the transplanted graft through CCR2 and/or CCR3, possibly in conjunction with CXCR5. Moreover, we found increased expression of genes that are involved in cell mobility, such as Gsn, Ptger2, RhoC, and Itg1. These molecules may facilitate the mobility of DN Treg cells. Although they may aid in chemotaxis, it is necessary to determine under what conditions they function. Furthermore, whether they respond exclusively to the stimulation of a specific receptor or whether they represent a general increase in migratory capacity remains to be tested.

As previously described, DN Treg cells do not appear to undergo activation-induced cell death in response to TCR cross-linking (55, 56). Consequently, we focused on genes involved in cell survival and proliferation. We found that DN Treg cells overexpressed CD55 and VEGF, whereas mutant clones did not show significant increases in expression of any survival genes. It has been demonstrated that CD55 prevents cell death through complement activation (57, 58), whereas VEGF has been implicated in cell proliferation (59, 60). This supports the observation that DN Treg cells are highly resistant to activation induced cell death (55, 56). Although this data suggests that CD55 and VEGF may contribute to this DN Treg cell resistance to activation-induced cell death, additional studies are still required.

In nature, DN Treg cells are anergic, much like CD4⁺ Treg cells (7, 61). An important mechanism for anergy in DN Treg cells may result from a finding of potentially altered Csk-Lck interaction. There is increased expression of Csk, the COOH-terminal Src kinase (data not shown), which is a strong repressor of TCR signaling and is known to directly inhibit TCR-induced tyrosine protein phosphorylation and lymphokine production (62). Csk is important for dephosphorylating and inactivating the protein tyrosine kinases responsible for T cell activation, such as Lck (63). Lck is responsible for proximal TCR signal transduction (63) and is down-regulated 16-fold on the array in DN Treg cells compared with their mutants (data not shown). TCR-based signaling is required at several stages of T cell development, including pre-TCR signaling, positive selection, peripheral maintenance of naive T cells, and lymphopenia-induced proliferation. Lck seems to be the major contributor to TCR-based signaling (64). The significantly reduced expression of Lck, accompanied by an increased expression of Csk in regulatory DN cells, thus suggests an important mechanism for Lck-Csk-mediated DN Treg cell anergy.

Recently, several genes have been put forth as possible regulatory cell markers. Foxp3 has been shown to be expressed in CD4⁺CD25⁺ and CD8⁺CD25⁺ Treg cells (24, 25) and may play a role in their regulatory function (65, 66, 67). Using QRT-PCR and Western blot analysis, we found that Foxp3 is expressed at low levels in DN Treg clones and its expression is increased in both of the mutant clones (data not shown). This suggests that although expression and function of this gene is important in some subsets of regulatory T cells, it may not be a general marker for all regulatory cells. Other studies of CD4⁺CD25⁺ Treg cells demonstrated over expression of RAR and FasL (68), as well as CCR2 and Itg1 (9). In the present study, we demonstrate that each of these genes are also overexpressed in DN Treg cells, suggesting possible common mechanisms used by Treg cells during the course of immune regulation.

We report here that a large percentage of genes (10% of the transcriptome) are differentially expressed between regulatory DN T cell clones and nonregulatory mutants. Furthermore, many of these genes can be associated with various functional properties of DN Treg cells. The gene expression differences identified in this study may underlie the molecular mechanisms involved in DN Treg cell-mediated immune regulation and potentially also for Treg cells in general.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Acknowledgments

 
We gratefully acknowledge technical assistance from Betty Joe and Olivia Wilkins. We also acknowledge the assistance from staff at the Stanford Microarray Database and Drs. Balasubramanian Narasimhan and Mei-Sze Chua. A complete web supplement is available online http://microarray-pubs.stanford.edu/DN_Treg/.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work is supported by Canadian Institutes of Health Research Grants MOP 14431 (to L.Z.) and HRP 52447 (to D.J.K., L.Z.) and National Cancer Institute of Canada Grant 15067 (to L.Z.), and further funding was provided by Wyeth-Ayerst Canada (to L.Z.). Additional support for this work was funded by National Institutes of Health Grants NIH5P3-05 and NIH3P3-05S1 (to M.S.), the Clinical Center for Immunological Studies at Stanford University, the Packard Foundation, and Roche Pharmaceuticals.

² L.Z. and M.M.S. are senior authors of this manuscript.

³ Address correspondence and reprint requests to Dr. Li Zhang, Toronto General Research Institute, University Health Network, 621 University Avenue, NU-G-001, Toronto, Ontario, M5G 2C4 Canada. E-mail address: lzhang{at}transplantunit.org

⁴ Abbreviations used in this paper: Treg, regulatory T; DN, double negative; DN Treg, CD4^–CD8^– double-negative regulatory T cell; QRT-PCR, quantitative real-time PCR; CN, control naïve; TN, tolerant CD4/CD8 negative; SAM, Statistical Analysis of Microarray; EASE, Expression Analysis Systematic Explorer; IGF, insulin-like growth factor; IRF, IFN regulatory factor; IFI, IFN- inducible; VEGF, vascular endothelial growth factor; Egr-1, early growth response-1; FasL, Fas ligand; RAR, retinoic acid receptor.

⁵ Lee, B. P.-L., W. Chen, R. Forster, and L. Zhang. CXCR5 is important for migration of double-negative regulatory T cells.

Received for publication December 8, 2004. Accepted for publication February 1, 2005.

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