Immunology Letters 164 (2015) 117–124

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Complement receptor type 1 (CR1/CD35) expressed on activated human CD4+ T cells contributes to generation of regulatory T cells Katalin Török a , Balázs Dezso˝ b , András Bencsik a , Barbara Uzonyi a , Anna Erdei a,c,∗ a b c

MTA-ELTE Immunology Research Group, Budapest, Hungary Department of Pathology, Medical Center, University of Debrecen, Hungary Department of Immunology, Eötvös Loránd University, Budapest, Hungary

a r t i c l e

i n f o

Article history: Received 4 February 2015 Received in revised form 18 February 2015 Accepted 19 February 2015 Available online 2 March 2015 Keywords: CR1 (CD35) expression CD4+ human T cells Treg differentiation Synergism with MCP (CD46)

a b s t r a c t The role of complement in the regulation of T cell immunity has been highlighted recently by several groups. We were prompted to reinvestigate the role of complement receptor type 1 (CR1, CD3) in human T cells based on our earlier data showing that activated human T cells produce C3 (Torok et al. (2012) [48]) and also by results demonstrating that engagement of Membrane Cofactor Protein (MCP, CD46) induces a switch of anti-CD3-activated helper T cells into regulatory T cells (Kemper et al. (2003) [17]). We demonstrate here that co-ligation of CD46 and CD3, the two C3b-binding structures present on activated CD4+ human T cells significantly enhances CD25 expression, elevates granzyme B production and synergistically augments cell proliferation. The role of CR1 in the development of the Treg phenotype was further confirmed by demonstrating that its engagement enhances IL-10 production and reduces IFN␥ release by the activated CD4+ T cells in the presence of excess IL-2. The functional in vivo relevance of our findings was highlighted by the immunohistochemical staining of tonsils, revealing the presence of CD4/CD3 double positive lymphocytes mainly in the inter-follicular regions where direct contact between CD4+ T cells and B lymphocytes occurs. Regarding the in vivo relevance of the complement-dependent generation of regulatory T cells in secondary lymphoid organs we propose a scenario shown in the figure. The depicted process involves the sequential binding of locally produced C3 fragments to CD46 and CD3 expressed on activated T cells, which – in the presence of excess IL-2 – leads to the development of Treg cells. © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Some decades ago the complement system was described as a heat-sensitive serum component that is necessary to kill bacteria in the presence of specific antibodies. By now it is known that this system comprises more than 30 components – including not only serum factors, but several cell membrane regulators and various receptors, which are expressed on a wide range of different cell types. Involvement of complement in a great variety of biological functions is well accepted, including its involvement in the uptake of foreign structures and apoptotic cells as well as initiation and direction of adaptive immunity. Its role has also been demonstrated

Abbreviations: CR1 (CD35), complement receptor type 1; MCP (CD46), Membrane Cofactor Protein; C3, third component of complement. ∗ Corresponding author at: 1117 Budapest, Pázmány s. 1/c, Hungary. Tel.: +36 1 3812 175; fax: +36 1 3812 176. E-mail address: [email protected] (A. Erdei).

in the activation, survival and proliferation of lymphocytes, besides modulation of autoimmunity and transplant rejection. The most abundant component of complement is protein C3, of which the first cleavage products are the anaphylatoxic peptide C3a and the larger C3b fragment. Freshly generated C3b has the capacity to bind covalently to the activating surface, and besides being an indispensable element of the lytic route, it also serves as ligand for various cell membrane structures, including complement receptor type 1 (CR1, CD35) and Membrane Cofactor Protein (MCP, CD46). CR1 (CD3) is an integral membrane glycoprotein that binds C3b and iC3b as well as C4b [1]. Along with MCP and Decay Accelerating Factor (DAF, CD), CR1 belongs to the family of the RCA (Regulators of Complement Activation) proteins which are expressed on the surface of various cells and protects them from lysis by homologous complement [2]. In addition to the regulation of complement convertase activity, CR1 has several further important biological functions. Human CR1 expressed on erythrocytes plays a role in immune complex clearance [3–5], on macrophages, monocytes and neutrophil granulocytes it mediates the uptake of complementopsonized particles and immune complexes [6,7]. On the surface

http://dx.doi.org/10.1016/j.imlet.2015.02.009 0165-2478/© 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

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of follicular dendritic cells (FDC), CR1 keeps opsonized antigens in native, accessible form for a long time which is important for the maintenance of immunological memory [6,7]. A further important function of CR1 has been described by Jozsi et al. [8] and Kremlitzka et al. [9]. Namely, it has been demonstrated that crosslinking CD3 on human B cells inhibits BCR mediated activation and differentiation to antibody producing cells. Although CR1 possesses an intracellular tail containing 38 amino acid residues with a casein-kinase II phosphorylation site which might mediate intracellular signaling events [6,7], so far signaling via CR1 has not been demonstrated. The expression and role of CR1 on human T lymphocytes is still controversial, although its appearance has been first demonstrated in 1983 [10]. Most authors agree that CR1 expression is upregulated during T cell activation and the extent of increase is donor dependent [11–14]. The distribution of CR1 on CD4+ and CD8+ T cells has been studied so far only employing mitogen-activated human T cells, and the expression of this complement receptor was found significantly increased in the CD4+ population [13]. Regarding the function of CR1, earlier it had been shown that its ligation on CD4+ human T cells enhances HIV replication [15]. Furthermore, Wagner et al. demonstrated that proliferation and cytokine synthesis (IFN␥ and IL-2) is inhibited when T cells are treated with antibodies to CR1 while neither the expression of the early genes nor intracellular Ca2+ levels are affected [16]. MCP is also a type 1 transmembrane glycoprotein that binds the complement activation product C3b, and it is present on all nucleated cells. Traditionally, it has been considered a cell membrane regulator of complement activation, as well as a receptor for various pathogens (including pathogenic Neisseria spp. and Streptococcus pyogenes, measles virus, herpesvirus 6, adenoviruses). It has been shown by Kemper et al. that co-ligation of the TCR and the complement regulator CD46 on CD4+ T cells induces a Treg specific cytokine phenotype in the presence of excess IL-2 [17]. Furthermore, these IL-10 producing T cells were shown to proliferate strongly and suppressed the activation of bystander T cells. These experiments were done employing CD3 and CD46 specific antibodies. Later, it had been suggested that engagement of the TCR induces C3b production by the activated T cells, which thus provide locally the natural ligand for CD46 [18]. Since C3b is also the major ligand of CR1, we hypothesized that this complement receptor might also be involved in the differentiation of Th cells, most probably in cooperation with CD46. Here we report that engagement of CR1 by its natural ligand or by CR1-specific antibodies induces the expression of Treg markers, enhances IL-10 production and reduces IFN␥ release by activated CD4+ T cells, similarly to the process mediated by CD46. We also show that the anti-CD3 induced proliferation of CD4+ T cells is synergistically enhanced upon the concomitant ligation of CD3 and CD46 on these cells. Finally, we demonstrate that the CD4+ CD3+ T cells localize in the germinal center (GC) and the mantle zone of folliculi in human tonsils, where these cells may interact with the B lymphocytes.

anti-CD14-microbeads on LS Columns (Miltényi) or by sorting using anti-CD8-PE, anti-CD16-PE, anti-CD19-APC and anti-CD14FITC (BD-Pharmingen) antibodies and FACSAria III instrument. The percentage of T cells was always higher than 96% when applying MACS, and was 99% when T cells were sorted by the FACSAria III instrument. 2.2. T cell proliferation assay Isolated T cells (105 cells/well) were activated in RPMI medium (Sigma–Aldrich) supplemented with 10% FCS (Gibco) for three days by culturing in microwells coated with 1 ␮g/ml anti-CD3 (BD Pharmingen) or pooled normal mouse IgG (nmIgG) for control, and/or the given combination of the following mAbs or ligands of CR1 and MCP: anti-CD3 (To5; 5 ␮g/ml, Santa Cruz Biotechnology), anti-CD46 (MEM-258, 5 ␮g/ml, Immunotools), C3b (5 ␮g/ml, Calbiochem), aggregated C3 or “C3b-like C3” (5 ␮g/ml). In certain experiments 80 U recombinant human IL-2 (Immunotools) was added, as indicated. Cell proliferation was measured after pulsing the cells with 1 ␮Ci/well H3 -thymidine (NEN, Boston, MA) for the last 18 h of the three-day culture period. Incorporated radioactivity was measured with a Wallac 1409 liquid scintillation beta counter (Wallac, Allerod, Denmark). The results are expressed as mean counts per minute (cpm) ± standard error of the mean (SEM) of 5 independent measurements. 2.3. Flow cytometry For the identification of the cells the following antibodies were used: anti-CD3-FITC (MEM-57), anti-CD3-PE (MEM-57), mIgG1-FITC, mIgG1-PE, anti-CD8-PE (MEM-31), anti-CD4-APC (MEM-241), anti-CD25-APC (MEM-185), anti-granzyme-B-FITC (Immunotools); anti-CD35-FITC (To-5), anti-CD35-PE (To-5), mIgG-FITC, mIgG-PE (BD Pharmingen), anti-C3-FITC (F(ab’)2 , Cappel), anti-CD11a-PE (BioLegend). Cells were stained in PBS containing 1% FCS and 0.1% sodium azide (FACS buffer) for 30 min on ice. Intracellular staining was performed after permeabilization with 0.1% saponine (Fluka) and fixing in FACS buffer containing 3% paraformaldehyde. 10,000 cells were routinely measured on a FACSCalibur cytometer (Beckton Dickinson) using the CellQuest software and data were analyzed with the Flowing Software 2.1. 2.4. Isolation and aggregation of human C3 Human C3 was isolated from pooled normal human serum by fast protein liquid chromatography as described by Basta and Hammer [19]. To minimalize IgG contamination, C3 solution was incubated with Protein G beads (ThermoScientific, Rockford). To obtain C3b-like C3 the pure protein was heat-aggregated by incubation at 63 ◦ C for 20 min [8]. 2.5. Cytokine measurements

2. Materials and methods 2.1. Cells For the isolation of T cells, tonsils of children aged 2–8 and buffy coats of healthy donors were used, based on ethical permission. Informed consent was provided according to the Declaration of Helsinki, and the use of tonsils was implemented by the acquiescent declaration of the donors’ parents. Mononuclear cells were isolated by density-gradient centrifugation on Ficoll-PaqueTM Plus (GE Healthcare Bio-Sciences AB). T cells were negatively isolated by Vario MACS Separator using anti-CD19-, anti-CD16 and

Concentration of IFN␥ and IL-10 cytokines was determined from the supernatant of activated Th cells by Flow cytomix kits (BenderMedsystems) according to the manufacturer’s instructions. 2.6. Immunohistochemistry on lymphoid tissues and cytospin preparations For immunofluorescent (IF) labeling, normal human lymphoepithelial tissues were used obtained from routine tonsillectomy on children with no active tonsillitis or systemic infective or autoimmune diseases that were confirmed both clinically and

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Fig. 1. CR1 expression is elevated on in vitro activated human T cells derived from blood and tonsil. T cells were separated from human tonsils (A) and blood (B) by MACS and cultured for three days in microwells coated with anti-CD3 and normal mouse IgG (nmIgG). CR1 expression was assessed by flow cytometry using the To5 mAb. MFI was calculated from the geometric mean of fluorescence intensity values. Results of eight independent experiments + SEM are shown.

histologically. For the preparation of cryostat sections portions of tonsils were snap-frozen in liquid nitrogen. Double immunofluorescence was performed on acetone-fixed 5 ␮m cryo-sections according to standard techniques as described earlier in detail [20]. Staining was carried out using fluorochrome-conjugated mAbs and/or tyramide-taged FITC (fluorescein isothiocyanate; green fluorescent) or TMR (tetramethylrhodamine; red fluorescent) reporter conjugates (both from Perklin-Elmer) for visualization. Briefly, the sections were incubated with monoclonal rabbit anti-human-CD4 (AN722; Biogenex, Fremont, CA, USA), followed by peroxidase conjugated goat anti-rabbit-IgG (DAKO, Glostrup, Denmark) and tyramide-FITC to strengthen fluorescence. After washing and blocking with 5% mouse serum, mouse anti-humanCD3-phycoerythrin (PE) (Santa Cruz) or mAb to CD35 (DAKO) was added to the sections followed by biotinylated secondary antibody and streptavidin-TMR or tyramide-TMR treatments with thorough washing in each step. For negative controls, each IF-run included parallel slides that had isotype-matched control antibodies (DAKO) in place of the primaries. Also, the immuno-staining procedures were carried out on consecutive (identical) sections with single labeling methods for each antibody to confirm the cellular distributions of CD4 and CD3positive cells separately using standard peroxidase-based or IF methods. These served as positive control-references which confirmed that no reduction of labeled cells and no significant cross reactions by the antibodies occurred during double labeling (not shown). Samples were then digitalized in a computer-guided Panoramic slide-scanner (3DHistech, Budapest, Hungary) equipped with excitation filters for green (FITC), red (PE, TMR), blue (DAPI) fluorescent channels, respectively, detected by a high resolution Hitachi 3ccd Sx6A camera for photo-documentation, or analyzed by Olympus Fluowiev 00 CLSM with the ImageJ software. Cytospin-samples were stained with the same antibodies described above and analyzed by Thermo Scientific Shandon Cytocentrifuge Cytospin 4. 2.7. Statistical analysis Statistical differences were assessed by pairwise comparisons of relevant groups using permutation tests. Briefly, values from the groups to be compared were randomly reassigned to two groups and the differences between the group means were calculated. Distribution of 5000 randomizations was drawn and the two-tailed p value corresponding to the real sample assignments was determined. The arithmetic mean of 50 such p values was accepted as the probability of ˛-error. Wilcoxon’s signed rank test was made to compare the values of two related, non-parametric data sets.

Values of p ≤ 0.05 were considered significant, and were indicated as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. All the error bars mean SEM. 3. Results 3.1. CR1 (CD35) expression is enhanced on TCR-activated human T cells derived from blood and tonsil The expression of CR1 on the surface of human T cells has earlier been examined employing either T cell lines or mitogen-stimulated T cells [10,12,13,21], but so far it has not been studied on isolated TCR-activated CD4+ lymphocytes. Since data are still controversial, and the role of complement in T cell functions is being intensively studied recently [16–18,22–40], we have decided to re-investigate the expression and function of this complement receptor on human T cells in the light of the new findings. First we studied the cell surface expression of CR1 on T lymphocytes isolated from human blood and tonsil before and after activation via the TCR. The results of FACS analysis clearly show that both blood- and tonsil-derived T cells cultured in anti-CD3 coated micro wells express significantly more CR1 than non-activated cells (Fig. 1). These data strengthen and extend earlier results published by Rodgaard et al. [12,13] and Yaskanin and Waxman [11]. Next we set out to investigate whether the enhanced expression is restricted to the CD4+ or CD8+ subpopulation of human T cells. Our data shown in Fig. 2 demonstrate that both subpopulations of the antiCD3 stimulated cells express significantly elevated levels of CR1. In our present study we decided to focus on CR1-expressing CD4+ T cells, since recent data indicate that complement is strongly involved in the switch of Th1 cells toward a regulatory phenotype [22,41]. 3.2. Ligation of CR1 on TCR-stimulated T cells induces expression of Treg markers Earlier data show that co-engagement of TCR and MCP in the presence of excess amounts of IL-2 results in Treg differentiation. It had also been suggested that locally produced, T-cell derived C3b plays a role in this process [18]. Since in addition to binding to MCP, C3b is also the major natural ligand of CR1, we hypothesized that this complement receptor might also contribute to the C3b-mediated induction of Treg cells. Therefore we set up cultures of CD3-activated CD4+ human T cells, where cell surface CD3 and CD46 molecules were engaged separately or concomitantly by specific antibodies, as well as by multimeric C3b. After three days we assessed the appearance of Treg cells by investigating the expression of CD2 and granzyme-B.

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cells which have a strongly activated, CD11a++ phenotype. In good agreement with the published results we also found that CD11a expression is higher on the CD2+ cells (not shown). Granzyme B expression showed a similar pattern; it was elevated in the CD11a++/CD25+ double positive population of T cells when CR1 and MCP were engaged either by specific antibodies or by the crosslinking ligand (Fig. 4). 3.3. Engagement of CD35 on activated CD4+ T cells enhances IL-10 and reduces IFN production

Fig. 2. Expression of CR1 is enhanced both on CD4+ and CD8+ human T cells upon activation. T cells were separated from human tonsils by MACS and cultured for three days in anti-CD3 coated microwells. CR1 expression of CD4+ and CD8+ cells was measured by flow cytometry. Results of one representative experiment of three are shown.

As shown in Fig. 3, the expression of CD2 is significantly enhanced in all cases when CR1 and/or MCP are engaged either by their natural ligand (aggregated C3) or by specific mAbs. Since it has been shown earlier that CD11a expression is elevated on Treg cells [42,43], we compared CD25 expression on the population of the

Cardone et al. showed earlier that MCP-induced Treg cells produce more IL-10 and less IFN␥ than activated bystander T cells [22]. After proving that CR1 ligation also leads to the appearance of the Treg phenotype, we set out to examine if CR1 could as well modulate cytokine production by the activated T cells. Measuring the release of the cytokines after engagement of CR1 on TCR-activated CD4+ T cells we found that production of IFN␥ is significantly reduced, while the release of IL-10 is significantly enhanced after three days of culture (Fig. 5A and B). This is well in accordance with the appearance of Treg markers on these cells, and further supports the involvement of CR1 in the differentiation of CD4+ T cells into Treg cells. 3.4. Concomitant engagement of CD35 and CD46 synergistically enhances the anti-CD3 induced proliferation of CD4+ T Next we aimed to clarify whether ligation of CR1 alone and along with MCP can influence the anti-CD3 induced proliferation of

Fig. 3. Induction of CD25 expression by stimuli exerted via CD35 and CD46 in activated human T cells. CD4+ T cells were activated for three days in anti-CD3 coated microwells in the presence of excess IL-2 (80 U/ml). CD3 and CD46 specific antibodies, aggregated C3 or normal mouse IgG (nmIgG) were also present in the cultures, as indicated in the figure. CD25 expression was assessed by flow cytometry. Data were normalized to the percentage of CD25+ cells in the anti-CD3 activated samples, which was taken 100%. Statistical analysis was done by permutation test. Average values ± SEM obtained in four independent experiments are shown.

Fig. 4. Ligation of CD35 and CD46 enhances expression of granzyme B in anti-CD3 activated T cells. CD4+ T cells were activated employing CD35 and CD46 specific antibodies, aggregated C3 and normal mouse IgG (nmIgG) for three days. Data show granzyme B expression of CD11a++/CD25+ double positive cells measured by flow cytometry. Gray: isotype control, black line: anti-granzyme B. One representative experiment of three is shown.

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Fig. 5. Inhibition of IFN␥ and enhancement of IL-10 cytokine production upon engagement of CR1 on activated CD4+ T cells. CD4+ T cells were activated for three days in microwells coated with anti-CD3, anti-CD3 or normal mouse IgG (nmIgG) in the presence of excess IL-2 (80 U/ml). The concentration of IFN␥ (panel A) and IL-10 (panel B) cytokines was measured from the supernatant of the activated cells by flow cytomix kits. The amount of cytokines measured in the supernatant of anti-CD3 + IL-2-stimulated cultures was taken 100%. Statistical analysis was done by permutation test. Average values ± SEM obtained in four independent experiments are shown.

Fig. 6. Simultaneous engagement of MCP and CR1 synergistically enhances the anti-CD3 induced proliferation of CD4+ T cells. CD4+ T cells were activated for three days in anti-CD3 coated microwells in the presence of excess IL-2 (80 U/ml). CD3 and CD46 specific antibodies or normal mouse IgG (nmIgG) were also present in the cultures, as indicated in the figure. Proliferation was measured by 3 H-thymidine incorporation. Data were normalized to cpm measured in anti-CD3 + IL-2 cultures, which were taken 100%. Statistical analysis was done by permutation test. Data of five independent experiments + SEM are shown.

CD4+ T cells. To this end isolated CD4+ T cells were cultured in anti-CD3 coated micro wells in the presence of anti-CD3 and/or anti-CD46. To each culture exogenous IL-2 was added (80 U), which is needed for the development of Th cells into the Treg phenotype [22]. We found that the concomitant engagement of CD3 and CD46 significantly enhanced T cell proliferation in a synergistic manner (Fig. 6). 3.5. CD4+CD35+ T cells in human tonsils are predominantly localized in the mantle-marginal zones of the folliculi In our attempt to reveal the in vivo significance of these findings, we looked for CD4+ CD3+ cells in tissue sections of human tonsils. As shown in Fig. 7, CD3+ T helper cells can be identified mainly in the mantle-marginal zones of the folliculi, where T and B cells interact in order to induce the adaptive immune response. This observation is in line with the results of Fuchs et al., who showed that CD46-induced Treg cells stimulate B cell differentiation both through cytokine production and by direct cell–cell contact [44]. To a lesser extent double positive cells can also be found in the GC (Fig. 7B, insert), and the peri-vascular region (Fig. 7C, short arrows).

4. Discussion Complement proteins found in serum are mostly generated by the liver, however several tissue-resident and migratory cells are also able to produce various complement components. In the past years particular attention has been devoted to locally produced, immune-cell derived components – especially C3 and its activation fragments – and their interaction with the corresponding receptors on B cells. Recently the immune regulatory role of complement in T cell biology has been highlighted in several publications [16–18,22–26,28–32,34,36,37,39,40,44–48]. When complement is activated C3, the major component is cleaved into the small C3a and the large C3b fragments. The importance of locally generated anaphylatoxic peptides – such as C3a and C5a – and the up regulation of their receptors in cognate APC–T cell interactions have been demonstrated in the murine system by Straininc et al. [18]. They showed that these cell-derived complement peptides provide both costimulatory and survival signals to naive CD4+ T cells. It has to be mentioned, that so far most studies on the role of locally produced complement have been performed in rodents while studies related to humans are still limited.

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Fig. 7. CR1+ T helper cells localize in folliculi and mantle zone of human tonsil. Double immunofluorescent (IF) labeling for CD4+ T-lymphocytes and CD35+ cells in the follicles and the inter-follicular region of the tonsil (B–D). (A) As reference, hematoxylin–eosin stained cryosection is shown obtained from the lymphoepithelial tonsil tissue that reveals secondary lymphoid follicles composed of germinal centers (GC) surrounded by mantle-marginal zones (darkly stained rim of lymphocytes) and interfollicular mononuclear cells. At upper left corner of the image squamous mucosal epithelial layer is seen (original magnification: 5×). (B) Double IF from serial sections of the above tissue is shown exhibiting CD4+ T-cells (green) mainly located within the inter-follicular regions and to a lesser extent in the follicles. As opposed, CD35+ cells (red) are predominantly situated in the germinal centers of the follicles (GC) in form of a networking pattern with the presence of scattered CD35+/CD4+ double labeled cells (e.g., at short arrow that is magnified in the insert). On the other hand, few CD35+ cells are also found along the mantle-marginal zones with the intimate co-existence of CD4+ T-helper lymphocytes. Note that few of them show co-expression for both markers (long arrows) indicating CD35+ T-cells (original magnification: 20×). (C) and (D) Within the inter-follicular T-cell area, there are scattered T-cells with CD35 co-expression, also (long arrows) and a few double labeled cells located at the peri-vascular region of the medulla (short arrows; v: vessel lumen) (original magnification for C and D: 40× and 50×, respectively). (E) High power view of cytospin sample from the same tonsil showing a double labeled cell for CD4 (green) and CD35 (red) markers which convincingly demonstrates the co-expression, as shown above in the tissues, in situ. Fluorescent antibodies used for the labeling of cytospin samples were the same as in the case of tissue sections (see Section 2). For double IF DAPI nuclear counterstaining was used (blue fluorescence). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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C3b, the larger fragment is a ligand for cell membrane structures, such as MCP and CR1. Kemper et al. showed that coengagement of the TCR and CD46 on human CD4+ T cells induces Treg type lymphocytes, which produce IL-10 as well as IFN␥ [17]. Later it has been proven that activation via CD46 drives the switch from classical IFN␥-producing Th1 cells toward IL-10-producing Th1 cells with immunosuppressive properties in a strictly IL-2 dependent manner [22]. The expression of CR1 (CD35), the other C3b-binding receptor was first described on human T cells more than 30 years ago [10,21], and later its enhanced expression has been shown on mitogenactivated T cells and various T cell lines [11,13,14]. We extend these data by demonstrating the elevated expression of CR1 also on activated tonsil-derived T cells (Figs. 1 and 2). Wagner et al. showed that treatment of CD3-activated, non-separated peripheral human T cells for five days with anti-CR1 antibody induces negative regulatory functions associated with decreased proliferation [16]. Here, we demonstrate that engagement of CR1 on isolated, CD3-activated Th cells either by its natural ligand or by CR1-specific antibodies elevates the levels of CD11a and CD25 as well as granzyme B expression (Figs. 3 and 4). Furthermore, we show that ligation of CR1 enhances IL-10 production and reduces IFN␥ release by CD4+ T cells in the presence of access IL-2 (Fig. 5). It should be noted that we aimed to asses FoxP3 expression by cytofluorimetry as well as immunohistochemistry. We employed several commercially available antibodies, but despite all our efforts we did not get remarkable results. The lack of FoxP3 expression in our experiments however, is in line with the results of Cardone et al. [22], who suggested that complement-induced Treg cells may express FoxP3 unstably as they keep Th1-line expression factors active. Our results prove that engagement of CD35 on activated Th cells contributes to Treg differentiation in a similar fashion as described by Kemper et al. employing anti-CD46 antibody [17]. It is obvious from our data and those of Kemper et al., that culture conditions and kinetics of cellular responses are important in the process of Treg generation. We suppose that the CR1-mediated inhibition of T cell proliferation described by Wagner et al. is mediated by the IL-10 producing Treg cells and the lack of sufficient IL-2 in the cultures. Since C3b is a major ligand for both CD35 and CD46 and under physiological conditions these two receptors are most likely ligated simultaneously, we were curious to see how their co-engagement affects the proliferative capacity of anti-CD3-activated CD4+ T cells. We found that the simultaneous engagement of CD46 and CD35 resulted in a strong synergistic stimulatory effect when proliferation was assessed (Fig. 6). Since CR1 has a short intracellular tale with only one tyrosine phosphorylation site in contrast to MCP which has two different cytoplasmic regions with more activation sites, we assume that CR1 uses the MCP-mediated signaling pathway when co-clustered on CD4+ T cells. Finally, it was important to reveal what the in vivo functional significance of the in vitro findings could be. Investigating the localization of the CR1+CD4+ T cells in human tonsillar sections by double fluorescence labeling we found that these lymphocytes are localized mainly in the interfollicular regions where direct contact of Th cells and B lymphocytes occurs, and partially in the follicles. Since C3b can be generated locally in the secondary lymphoid organs either by the T cells themselves [48] or by macrophages as well as other cell types [49], the effect of ligand binding can be exerted in situ. These findings are consistent with the earlier results of Fuchs et al., who showed that complement induced Treg cells induce B lymphocyte maturation by both cytokine production and direct cell–cell contact [44]. Regarding the in vivo relevance of our finding we propose the following scenario (drafted in the graphical abstract). CD4+ T cells stimulated in the secondary lymphoid organs produce C3, which is cleaved into C3b, iC3b or iC3(H2 O) either through spontaneous

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hydrolysis or by enzymes produced by the same cells. In the beginning of the process these ligands mainly bind to MCP and induce the production of IFN␥ and IL-2 cytokines. When local IL-2 levels reach a relatively high concentration, CR1 expression becomes more pronounced, and upon ligand binding it promotes the switch to the Treg phenotype induced by MCP. Finally, these complementinduced Treg cells may promote B cell responses by direct contact in the mantle zone of the folliculi. Conflict of interest The authors declare no conflict of interest. Acknowledgements The financial support from the Hungarian National Science Fund (OTKA) Grant K 112011, TÁMOP 4.2.1./B-09/1/KMR-2010-0003, and the support from the Hungarian Academy of Sciences are gratefully acknowledged. References [1] Krych-Goldberg M, Atkinson JP. Structure–function relationships of complement receptor type 1. Immunol Rev 2001;180:112–22. [2] Kalli KR, Hsu P, Fearon DT. Therapeutic uses of recombinant complement protein inhibitors. Springer Semin Immunopathol 1994;15:417–31. [3] Dunkelberger JR, Song WC. Complement and its role in innate and adaptive immune responses. Cell Res 2010;20:34–50. [4] Craig ML, Bankovich AJ, Taylor RP. Visualization of the transfer reaction: tracking immune complexes from erythrocyte complement receptor 1 to macrophages. Clin Immunol 2002;105:36–47. [5] Hess C, Schifferli JA. Immune adherence revisited: novel players in an old game. News Physiol Sci 2003;18:104–8. [6] Lloyd B, Klickstein JMM. CR1. In: Walport BJMaMJ, editor. The Complement Facts Book. 2000. p. 136–45. [7] Liu D, Niu ZX. The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35). Immunopharmacol Immunotoxicol 2009;31:524–35. [8] Jozsi M, Prechl J, Bajtay Z, Erdei A. Complement receptor type 1 (CD35) mediates inhibitory signals in human B lymphocytes. J Immunol 2002;168:2782–8. [9] Kremlitzka M, Polgár A, Fülöp L, Kiss E, Poór G, Erdei A. Complement receptor type 1 (CR1, CD35) is a potent inhibitor of B-cell functions in rheumatoid arthritis patients. Int Immunol 2013;25:9. [10] Wilson JG, Tedder TF, Fearon DT. Characterization of human T lymphocytes that express the C3b receptor. J Immunol 1983;131:684–9. [11] Yaskanin DD, Waxman FJ. Expression of the CR1 receptor on human leukemiaderived CD4+ T cell lines. Cell Immunol 1995;163:139–47. [12] Rodgaard A, Christensen LD, Thomsen BS, Wiik A, Bendixen G. Complement receptor type 1 (CR1, CD35) expression on peripheral T lymphocytes: both CD4and CD8-positive cells express CR1. Complement Inflamm 1991;8:303–9. [13] Rodgaard A, Thomsen BS, Bendixen G, Bendtzen K. Increased expression of complement receptor type 1 (CR1, CD35) on human peripheral blood T lymphocytes after polyclonal activation in vitro. Immunol Res 1995;14:69–76. [14] Reilly BD, Pfeiffer A, Petney K, Atkinson JP. Human peripheral CD4+ T-cells expressing complement receptor 1 (CR1, CD35). J Immunol 2010;184:138. [15] Mouhoub A, Delibrias CC, Fischer E, Boyer V, Kazatchkine MD. Ligation of CR1 (C3b receptor, CD35) on CD4+ T lymphocytes enhances viral replication in HIVinfected cells. Clin Exp Immunol 1996;106:297–303. [16] Wagner C, Ochmann C, Schoels M, Giese T, Stegmaier S, Richter R, et al. The complement receptor 1, CR1 (CD35), mediates inhibitory signals in human Tlymphocytes. Mol Immunol 2006;43:643–51. [17] Kemper C, Chan AC, Green JM, Brett KA, Murphy KM, Atkinson JP. Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 2003;421:388–92. [18] Strainic MG, Liu J, Huang D, An F, Lalli PN, Muqim N, et al. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity 2008;28:425–35. [19] Basta M, Hammer CH. A rapid FPLC method for purification of the third component of human and guinea pig complement. J Immunol Methods 1991;142:39–44. [20] Tsakiris I, Torocsik D, Gyongyosi A, Dozsa A, Szatmari I, Szanto A, et al. Carboxypeptidase-M is regulated by lipids and CSFs in macrophages and dendritic cells and expressed selectively in tissue granulomas and foam cells. Lab Invest 2012;92:345–61. [21] Tarshis IA, Meretskov VV, Pinegin BV. Detection of receptors for the IgG Fc fragment as well as for the C3 component of complement in the spleen Tlymphocytes of CBA mice. Zh Mikrobiol Epidemiol Immunobiol 1979:56–9.

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