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Human T cell derived, cell-bound complement iC3b is integrally involved in T cell activation
Immunology Letters 143 (2012) 131–136
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Human T cell derived, cell-bound complement iC3b is integrally involved in T cell activation Katalin Török a , Mariann Kremlitzka a , Noémi Sándor a,b , Eszter Angéla Tóth a , Zsuzsa Bajtay a,b , Anna Erdei a,b,∗ a b
Department of Immunology, Eötvös Loránd University, Budapest, Hungary Immunology Research Group of the Hungarian Academy of Sciences at Eötvös Loránd University, Budapest, Hungary
a r t i c l e
i n f o
Article history: Available online 22 February 2012 Keywords: Human T cell Complement C3 production Cell-bound iC3b T cell proliferation
a b s t r a c t Although the complement system is thought to be mainly involved in innate immunity and in the humoral arm of adaptive responses, evidence implicating that complement impacts T cell responses are accumulating recently. The role of the various activation products of the major complement component C3 were mainly studied so far in animal systems, and investigations regarding the effect of different C3-fragments on human T cells are sparse. Here we show that anti-CD3 activated human T lymphocytes derived from the blood and tonsil of healthy individuals produce C3, and the major cleavage fragment that appears on the T cell surface is iC3b. Based on studies carried out in allogenic system we demonstrate that the T cell membrane bound iC3b binds to the CR3 and probably to CR4 receptors expressed on monocyte-derived dendritic cells, and this interaction leads to significantly enhanced T-cell proliferation. Since neither C3aR and nor C3a binding could be detected on the membrane of anti-CD3 activated T cells, our findings indicate that in humans – in contrast to mice – the C3a peptide is most probably not involved directly in the T cell activation process. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The complement system is an integral part of our immune defense. Its components participate both in the innate and adaptive responses to various pathogens and antigens. The major source of the circulating components is the liver, however the importance of locally produced complement proteins is also well accepted by now, particularly due to their participation in the regulation of adaptive immune processes in peripheral lymphoid organs [1–5]. It is well known that several cell types are able to produce various complement proteins, including C3, the central component of the activation cascade [6,7]. C3 production by mouse T cells and the role of C3a, its small cleavage product along with the other anaphylatoxic complement peptide C5a have been extensively studied recently during the interaction with dendritic cells (DC), using a mouse model systems [8]. The role of C5a in driving Th17 differentiation and triggering arthritis has also been described [9]. Moreover, recent findings in experimental models of allergic rhinitis and allergic asthma suggest that C3a, C5a and the activation of antigen-presenting cells via their corresponding receptors regulate
the development of maladaptive Th2 and Th17 immunity [10]. Our knowledge regarding complement synthesis by human T cells however is sparce, and thorough studies on the interaction of C3 activation fragments and T lymphocytes are not available. Employing a sensitive, radio-immunoprecipitation method Pantazis et al. demonstrated earlier that PHA-activated and HTLV infected T cell lines produce C3 [11], and recently Cardone et al. showed that under certain circumstances C3b appears on the surface of activated human T cells [12], but no further analysis was carried out. Here we studied C3 production by primary human T lymphocytes in detail. We show that anti-CD3 activated T cells produce C3, and demonstrate that the major C3-derived protein found on the surface of CD3-activated T cells is the iC3b fragment. We show that these fragments – by their capacity to bind to CR3 and possible also to CR4 expressed by DCs – enhance the activation of alloreactive T cells. Based on our data the possible involvement of C3a in the human system can be excluded, since we could not detect C3aR expression and C3a binding neither on non-activated nor on activated T cells. 2. Materials and methods
∗ Corresponding author at: Department of Immunology, Eötvös Loránd University, Budapest, Hungary, Pázmány Péter s. 1/C, 1117 Budapest, Hungary. Tel.: +36 1 3812 175; fax: +36 1 3812 176. E-mail address: [email protected] (A. Erdei). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2012.02.003
2.1. Cells T cells were isolated from tonsils of children aged 2–8, and from buffy coats of healthy donors. Tonsils were provided by the
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St Imre Hospital, Budapest, and buffy coats were obtained from the Hungarian National Blood Transfusion Service based on an ethical permission. Informed consent was provided for the use of tonsils and blood samples according to the Declaration of Helsinki, and the use of tonsils was implemented by the acquiescent declaration of the donor’s parents. Mononuclear cells were isolated by sedimentation on FicollPaqueTM Plus (GE Healthcare Bio-Sciences AB). T cells from blood and tonsil were negatively isolated by Vario MACS Separator using anti-CD19- and anti-CD14-microbeads on LS Columns (Miltényi) or by sorting using anti-CD19-APC, anti-CD16-FITC and anti-CD14PE (BD-Pharmingen) antibodies and FACSAria III instrument. The percentage of T cells in the final suspensions of tonsils was always greater than 96% when applying MACS, of blood it was greater than 90%, and by the FACSAria III it was always more than 99.5%. Cells were characterised by flow cytometric analysis (FACSCalibur) using the following antibodies: anti-CD3-FITC, anti-CD3-PE, anti-CD19-APC, m-IgG1-FITC, m-IgG1-PE (Immunotools); polyclonal goat anti-human-C3-FITC F(ab) 2 (Cappel); anti-C3aR-Al647 (AbD Serotec); anti-CR3-RPE (Dako); m-IgG1-Al647 (BioLegend); anti-m-IgG (H+L), anti-m-IgG-Al488, anti-rabbit-IgG-Al488 (Invitrogen); anti-iC3b (Quidel); goat serum (control), rabbit serum (control); rabbit antiserum to human C3a (Behring Institute). 2.2. T cell activation T cells (105 cells/well) were activated for three days by culturing in microwells coated with 1 g/ml anti-CD3 (BD Pharmingen) or pooled mIgG, for control. 2.3. C3 PCR Total RNA from MACS purified non-activated and activated T cells and from the U937 human monocytic cell line were isolated using Rnaesy Mini Kit (Qiagen). The isolated RNA was converted into cDNA by reverse transcription (ThermoHybaid PCR Express) using 500 ng of each RNA sample and a mixture of 0.5 g OligoDT (synthesized by BioBasic Inc.), 200 unit/l RevertAid H Minus M-MuLV Reverse transcriptase (Fermentas), DEPC treated water, 5x reaction buffer (Fermentas), 40 unit/l RiboLock RNase inhibitor (Fermentas), 10 mM dNTPs (Fermentas) in the concentration providing by the manufacturer. The reaction profile was as follows: 70 ◦ C 5 , 37 5 , 42 ◦ C 1 h, 70 ◦ C 10 , followed by final cooling to 4 ◦ C. Samples were frozen until further studies. For the C3 PCR a mixture was made up from the following ingredients in the concentration providing by the manufacturer: 10 M forward (5 -GTGGCCAKGCRGCAGAAGGC-3 ) and reverse (5 -TCACCACSTGGGAGATTCTG-3 ) primers (IDT), 10 mM dNTPs (Fermentas), 2.5 l cDNS, RNase free water, 25 mM MgCl2 (Fermentas), 10x Taq puffer +KCl, −MgCl2 (Fermentas), 1 unit/l Taq polymerase (Fermentas). The reaction profile consisted of 94 ◦ C 5 ; 60 ◦ C 2 ; 72 ◦ C 22 , 94 ◦ C 22 , 60 ◦ C 22 through 40 cycles; 72 ◦ C 10 ; followed by final cooling to 4 ◦ C. Twenty l of the PCR reaction was applied to a 2% agarose gel (Serva) and the PCR products were detected by an UV-transilluminator. As control -actin primers reverse: 5 (forward: 5 -GGCTACAGCTTCACCACCAC-3 , GCGCTCAGGAGGAGCAATG-3 ) PCR were applied. The length of the C3 PCR product is 233 bp, while that of the -actin is 411 bp. 2.4. Biotinylation of cell surface proteins and immunoprecipitation 107 freshly isolated and 3-day activated peripheral T cells were surface biotinylated using the Pierce cell surface protein isolation kit (Pierce Biotechnology). After biotinylation, cells were
solubilized in 0.5 ml lysis buffer containing 50 mM HEPES, (pH 7.4), 1% Triton X-100, 100 mM NaF, 10 mM EDTA, 2 mM sodium orthovanadate, 10% glycerol, 2 g/ml aprotinin, 2 g/ml pepstatin, 5 g/ml leupeptin, and 1 mM PMSF at 4 ◦ C for 30 min (the enzyme inhibitors were all purchased from Sigma Aldrich). The cell extracts were centrifuged at 15,000 × g for 10 min, and pre-cleared with Protein-G Sepharose beads (Pierce Biotechnology) at 4 ◦ C for 3 h. Then the samples were immunoprecipitated with anti-human C3c (Dako) using Protein-G Sepharose beads (10 g rabbit IgG/sample). After rotating the samples overnight at 4 ◦ C beads were washed, boiled in reducing sample buffer and run on 10% SDS–PAGE. Separated proteins were transferred to nitrocellulose membrane. After blocking with 5% BSA for 1 h, the blots were developed by incubation with streptavidin–HRP (1:500, R&D Systems). Bands were visualized by the ECL method (Millipore). 2.5. Conjugate formation between CR3-bearing U937 cells and activated T lymphocytes 105 U937 cells labeled with 0.5 M CFSE (Molecular Probes) were co-incubated with 4 × 105 T cells for 2 h at 37 ◦ C. For the inhibition studies cells were incubated in the presence of TMG6-5, the iC3b ligand-binding site specific anti-CD11b antibody, kindly provided by Dr. Istvan Ando (BRC, Szeged, Hungary), the anti-iC3b antibody recognizing a neo-epitope on iC3b (Quidel), or an isotype control (mouse IgG1, Sigma) at 10 g/ml concentration. The number of interactions was counted and normalized to 100 cells using an Olympus IX81 confocal laser scanning microscope with further digital magnification. 2.6. Monocyte-derived dendritic cells (MDCs) Monocytes were isolated from buffy coat using CD14 microbeads (Miltenyi). Cells were cultivated in CellGro serum free medium (Cell Genix) supplemented with 100 ng/ml rHu GMCSF (R&D) and 1500 unit/ml rHu IL-4 (Promokine), and cytokines were added every other day. On day four CD14− immature MDCs were resuspended in serum-free medium containing 100 ng/ml LPS (Sigma). On day five mature MDCs were washed and used for further studies. 2.7. Allogenic T cell activation Mature MDCs were washed and transferred to 96 well plates and further cultured with allogenic T cells at a DC:T cell ratio of 1:5 for 16 h. T cells, obtained from a different donor had been preactivated as described above. Cell proliferation was measured by 3 H-thymidine incorporation in the presence or in the absence of polyclonal goat anti-human-C3 (Fab )2 (Cappel), anti-CR3 (TMG65) and anti-iC3b (Quidel), all in the concentration of 25 g/ml. Either T cells or DCs, or both cell types were preincubated with the antibodies for half an hour, then washed and put to co-culture. 2.8. 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 was 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. 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.
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Fig. 2. T cell derived C3 associated with the cell membrane. Flow separated human T cells were surface biotinylated either without activation (lane 1) or after stimulation by anti-CD3 for three days (lane 2). Cell extracts were immunoprecipitated with anti-C3c antibody and separated on 10% SDS–PAGE under reducing conditions. Biotinylated C3, which has been degraded to some extent, was also run for comparison (lane 3). The following protein-bands are indicated by arrow on the figure: 110 kDa: ␣-chain of C3; 75 kDa: intact -chain of C3; 67 and 45 kDa: cleavage fragments derived from the ␣-chain of C3.
Fig. 1. Demonstration of C3 expression by human T lymphocytes. (a) Expression of C3 mRNA. Isolated non-activated and activated human T lymphocytes and the human monocytic U937 cell line were subjected to mRNA preparation followed by PCR, as described in Section 2. Lane 1: non-activated blood-derived T cells; lane 2: CD3activated blood-derived T cells; lane 3: non-activated tonsil-derived T cells; lane 4: CD3-activated tonsil-derived T cells; lane 5: U937 cells (control). The figure shows data obtained with MACS-purified cells. Similar data were obtained when flow sorted cells were used. The results of one representative experiment of three are shown. (b and c) Demonstration of T cell derived iC3b on the surface of activated human T cells by cytofluorimetry. Non-activated and activated tonsil-derived T cells were labelled with FITC-labelled polyclonal anti-C3 F(ab )2 antibody. One representative histogram of three with similar results is shown. (b). For the analysis of blood-derived T cells a monoclonal anti-iC3b antibody recognizing a neoepitope on cell-bound iC3b was used. One representative experiment of two with similar results is shown (c). Both the anti-C3 F(ab )2 and the anti-iC3b antibodies reacted with tonsil- and blood-derived T-cells to a similar extent.
3. Results 3.1. C3 synthesis by activated human T lymphocytes To study whether human T cells are able to synthesize complement C3, first we looked for the C3-message at the mRNA-level. We used T cells from tonsils and blood isolated either by MACS or by sorting as described in Section 2. Cells were activated by anti-CD3 for three days, then used for mRNA preparation. As shown in Fig. 1a, in the case of purified, non-activated blood-derived T cells only a small amount of C3 mRNA can be detected, while upon activation of the cells via the TCR, a strong C3-message appears. In the case of tonsil-derived T cells we found no major difference between the in vitro stimulated and non activated cells; both expressed mRNA for C3 to the same extent, most probably due to in vivo activation of the cells. As positive control the C3-producing human monocytic U937 cell line was used. Next we attempted to detect C3 in the supernatant of nonactivated and activated T cells, employing a sensitive indirect ELISA system which detects C3 concentration as low as 2 ng/ml. However no C3 protein could be demonstrated in these samples, while the
culture supernatant of U937 cells, used as positive control, contained a substantial amount of C3 (data not shown). 3.2. Demonstration of cell-derived, surface-bound iC3b-fragments on activated human T cells by cytofluorimetry Since Cardone et al. demonstrated C3b deposition on the surface of purified, activated human CD4+ T cells [12], in the next experiments we set out to examine whether the larger C3-fragments can be detected also in our experimental system. We found that both the F(ab )2 fragment of the goat polyclonal anti-C3 IgG and a monoclonal antibody specifically recognizing a neoepitope on iC3b strongly react with the flow separated activated T cells (Fig. 1b and c). Since the iC3b neoepitope is hidden in the intact C3, these results suggest that the larger complement fragment most probably becomes surface bound after activation. Since only flow separated T lymphocytes were present in the cultures investigated in these experiments, and the FCS supplementing the cell culture medium does not contain C3-protein [13], our data provide evidence that C3-fragments found on the cellsurface were generated by the T lymphocytes. 3.3. Demonstration of cell-derived surface-bound iC3b-fragments on activated human T cells by immunoprecipitation To confirm the cytofluorimetric data and also in our attempt to elucidate the nature of the interaction between C3 and the cell membrane of human T cells, we performed immunoprecipitation. For the experiments we used the extract of surface biotinylated non activated and activated T cells and employed C3c-specific antibody. As demonstrated in Fig. 2, iC3b was present mainly on the surface of activated T lymphocytes (lane 2), however a very small amount of the C3-fragments could also be found in the detergent extract of the cell membrane of non activated cells (lane 1). Comparing the surface-bound complement protein (lane 2) with the purified, biotinylated C3 (lane 3), it can be seen that the intact, 110 kDa ␣-chain of the complement protein is almost completely
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Fig. 3. Lack of C3aR expression and C3a binding by human T cells. Non-activated and anti-CD3 activated T cells were subjected to FACS analysis using human C3a and C3aRspecific antibodies. As control C3a-binding and C3aR expression of monocytes derived from the same healthy donor is shown. We gated on monocytes on the basis of forward and side scatter. Solid lines show the binding of C3aR- and C3a-specific antibodies, respectively, dotted line shows the binding of the control antibody and the grey background indicates autofluorescence in each histogram. Results of one representative experiment out of three with similar results are shown.
missing, proving that activation/cleavage of C3 took place. Under the reducing conditions employed, the 67 and 45 kDa fragments of the ␣ -chain of iC3b appear, while the -chain (75 kDa) remains intact. We could not identify any larger protein complexes containing C3-fragments in these cell membrane extracts, suggesting that most probably the interaction between T cells and C3 is of non covalent nature.
3.4. T cells of healthy individuals do not express C3aR As shown in Figs. 1b, c and 2 the larger fragments of C3 are present on the surface of activated human T cells. Since these products are generated by the proteolytic cleavage of C3, next we studied the possibility, whether the small peptide, C3a also interacts with the T cells. Earlier it had been demonstrated that T lymphocytes of patients suffering from severe inflammatory skin diseases express C3aR, while unstimulated T cells of healthy individuals do not [14]. The same authors also demonstrated that type I IFNs added to freshly isolated or cloned T lymphocytes induce C3aR expression. Based on these data and the results of Strainic et al., who had shown in a mouse system that T cell derived C3a (and also C5a) interact with their respective receptors and influence T cell
proliferation [8], we aimed to clarify whether anti-CD3 activated human T cells also express this anaphylatoxin receptor. As shown in Fig. 3 however, no C3aR could be detected on the T lymphocytes activated in our experimental system, suggesting that inflammatory conditions are needed for its expression. The possibility that cell-bound C3a might sterically inhibit the binding of the C3aR specific antibody could be excluded, since we did not find any C3a peptide bound to the surface of the activated T cells (Fig. 3). Used as positive control, human monocytes were found to possess C3aR and also to bind C3a.
3.5. iC3b-mediated adherence of activated T lymphocytes to CR3 expressing cells As demonstrated in Figs. 1c and 2, activated T cells bear substantial amount of iC3b on their surface, which might serve as ligand for CR3 and CR4 expressed by various cell types. In order to find out whether these surface-bound C3-fragments are involved in cellular adherence, we investigated conjugate-formation between activated T cells and the CR3-expressing U937 cells, as described in Section 2. As seen in Fig. 4 activated T cells adhered to the CFSE-labelled U937 cells, and this interaction could be inhibited
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Fig. 4. Conjugate formation between activated human T cells and U937 cells. CFSE labeled CR3-expressing U937 cells were coincubated with activated human T lymphocytes in a 1:5 ratio for 2 h. Conjugate formation was analyzed by Olympus Fluoview 500 confocal laser scanning microscope at 60× magnification with further digital enlargement (b). Average number of interactions is shown (a).
by preincubation of the activated T cells with anti-iC3b and antiCD11b.
3.6. T cell-bound C3 fragments enhance proliferation induced by allogenic DCs Next we set out to study whether the iC3b-mediated adherence may lead to changes in the proliferative capacity of T cells. To this end we generated DCs from monocytes and after activation for one day by LPS they were cocultured for 16 h with pre-activated alloreactive T cells in a 1:5 ratio. Cells were harvested after pulsing with H3 -thymidine overnight. As seen in Fig. 5, under these conditions T cell proliferation was significantly enhanced compared to the activity of non activated T cells. When activated T cells were preincubated with a mixture of antibodies reacting with C3, iC3b and CR3, this enhancement was significantly diminished. Interestingly, when the DCs were pretreated with the same antibodies, the T cells’ proliferative capacity did not change compared to the untreated control (Fig. 5). There might be two explanations for this phenomenon. One possibility is that CR4 – which also binds iC3b – is also involved in the interaction between T cells and DCs. Another explanation might be that newly expressed CR3 molecules appear on the surface of DCs.
Fig. 5. Allogenic T cell activation is mediated by T cell-derived and T cell-bound iC3b. Human monocyte-derived DCs after stimulation with LPS for 1 day and T cells activated by anti-CD3 for 3 days were cocultured in a 1:5 ratio for 16 h. Cells pretreated with a mixture of anti-iC3b, anti-C3 (Fab )2 and anti-CR3 at 25 g/ml are indicated by a frame around the abbreviation (DC: dendritic cell; aT: activated T cell; naT: non-activated T cell). Cells treated with the antibody-cocktail were washed and cocultured for one day. Results of one representative experiment out of three with similar results are shown.
4. Discussion The involvement of complement in the instruction of adaptive immunity has been investigated for a long time, beginning in 1974 by Pepys [15]. Whereas in the past decades the role of complement in various B-cell functions has been clarified to a great extent [16–19] its influence on various T cell functions has been studied in detail only recently. Most of the available data have been obtained in various mouse systems – particularly those describing the role of C3a and C5a providing costimulatory signals in cognate interactions between antigen-presenting cells and T lymphocytes [5] In human systems however, the local production of complement proteins and the possible contribution of the larger fragments (C3b, iC3b) to various T cell functions have not been investigated thoroughly so far. By radioimmune precipitation followed by SDS–PAGE Pantazis et al. demonstrated earlier that mitogen activated human T cells and HTLV infected T cell lines produce C3 [11]. Recently Cardone et al. showed C3b deposition onto activated CD4+ T cells by flow cytometry, however no further details were provided regarding the origin and mode of attachment of the complement fragment [12]. Here we show both at the mRNA and the protein level that blood and tonsil derived human T cells produce C3 when activated via CD3. While secreted C3 could not be detected in the supernatant of the cell cultures even by using a very sensitive sandwich ELISA system, C3 bound to the cell surface of activated T cells was easily demonstrated. Using a neoepitope specific antibody we could identify iC3b as the major cleavage product of C3 on T cells. Since iC3b is the natural ligand of both CR3 and CR4, it could be assumed that iC3b bearing T lymphocytes might interact with cells expressing these complement receptors. Indeed, we found that this interaction occurs in allogenic systems and leads to enhanced T cell proliferation. This most probably results in changes in the production of various cytokines, thus may participate in the regulation of various immune functions. These data complement earlier results obtained in animal experiments, where the role of macrophage-derived C3 in enhancing antigen presentation has been clearly shown [20,21]. Using mice for their experiments Strainic et al. demonstrated that complement-derived anaphylatoxic peptides (C3a, C5a) produced locally by APCs and T cells during cognate interactions, are integrally involved in the activation of T lymphocytes [8]. The possible involvement of the larger fragments of C3 however, which are generated simultaneously, has not been investigated in this model system so far. In contrast to the studies carried out in mice, we could not detect C3 released by human DCs [22], implying that in our system the enhanced proliferation of alloreactive T lymphocytes is caused by T cell derived C3-fragments. This has been clearly proven by the inhibition of the MLR by specific antibodies. Our data demonstrate the bridge-forming role of iC3b, which most probably plays an important role under physiologic conditions in lymph nodes. Certainly the deposition of various C3-fragments may
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influence other T cell functions as well, as shown by Cardone et al. [12]. While the synthesis and production of C3 by human T cells is clearly shown in this paper, the exact nature of the cell membrane bound C3-fragments could not be revealed so far. Our results obtained by immunoprecipitation of surface labelled T cells do not provide evidence of covalent fixation, since we could not detect complexes of C3 and cell membrane constituents by Western blotting. Another possibility could be that the produced C3 molecule is nonproteolytically activated, thus C3(H2 O) is generated which is processed to iC3(H2 O). This iC3b-like molecule serves then as a ligand for complement receptors expressed by different cell types. This reaction has been described earlier in the case of activated thrombocytes [23]. Regarding the molecular mechanism of the “iC3b-bridge” formation between T cells and APCs it can also be assumed that iC3(H2 O) forms dimers (or oligomers), enabling the interaction with various complement receptors expressed on both cell types. Finally, it may further be hypothesized that the C3 molecules synthesized by the T cells – or its various products – are retained in the membrane of T lymphocytes via a so far unknown mechanism. Acknowledgements The financial support from the Hungarian National Science Fund (OTKA) grant K72026, from TÁMOP – 4.2.1/B-09/1/KMR-20100003, and the support from the Hungarian Academy of Sciences are gratefully acknowledged. References [1] Erdei A, Fust G, Gergely J. The role of C3 in the immune response. Immunol Today 1991;12:332–7. [2] Sacks S, Zhou W. The effect of locally synthesised complement on acute renal allograft rejection. J Mol Med (Berl) 2003;81:404–10. [3] Sewell DL, Nacewicz B, Liu F, Macvilay S, Erdei A, Lambris JD, et al. Complement C3 and C5 play critical roles in traumatic brain cryoinjury: blocking effects on neutrophil extravasation by C5a receptor antagonist. J Neuroimmunol 2004;155:55–63. [4] Lalli PN, Strainic MG, Yang M, Lin F, Medof ME, Heeger PS. Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis. Blood 2008;112:1759–66. [5] Heeger PS, Kemper C. Novel roles of complement in T effector cell regulation. Immunobiology 2011.
[6] van Kooten C, Fiore N, Trouw LA, Csomor E, Xu W, Castellano G, et al. Complement production and regulation by dendritic cells: molecular switches between tolerance and immunity. Mol Immunol 2008;45:4064–72. [7] Veerhuis R, Nielsen HM, Tenner AJ. Complement in the brain. Mol Immunol 2011;48:1592–603. [8] 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. [9] Hashimoto M, Hirota K, Yoshitomi H, Maeda S, Teradaira S, Akizuki S, et al. Complement drives Th17 cell differentiation and triggers autoimmune arthritis. J Exp Med 2010;207:1135–43. [10] Laumonnier Y, Schmudde I, Kohl J. The role of complement in the diagnosis and management of allergic rhinitis and allergic asthma. Curr Allergy Asthma Rep 2011;11:122–30. [11] Pantazis P, Kalyanaraman VS, Bing DH. Synthesis of the third component of complement (C3) by lectin-activated and HTLV-infected human T-cells. Mol Immunol 1990;27:283–9. [12] Cardone J, Le Friec G, Vantourout P, Roberts A, Fuchs A, Jackson I, et al. Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells. Nat Immunol 2010;11:862–71. [13] Triglia RP, Linscott WD. Titers of nine complement components, conglutinin and C3b-inactivator in adult and fetal bovine sera. Mol Immunol 1980;17:741–8. [14] Werfel T, Kirchhoff K, Wittmann M, Begemann G, Kapp A, Heidenreich F, et al. Activated human T lymphocytes express a functional C3a receptor. J Immunol 2000;165:6599–605. [15] Pepys MB. Role of complement in induction of antibody production in vivo. Effect of cobra factor and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J Exp Med 1974;140:126–45. [16] Tsokos GC, Berger M, Balow JE. Modulation of human B cell immunoglobulin secretion by the C3b component of complement. J Immunol 1984;132:622–6. [17] Daha MR, Bloem AC, Ballieux RE. Immunoglobulin production by human peripheral lymphocytes induced by anti-C3 receptor antibodies. J Immunol 1984;132:1197–201. [18] Melchers F, Erdei A, Schulz T, Dierich MP. Growth control of activated, synchronized murine B cells by the C3d fragment of human complement. Nature 1985;317:264–7. [19] Nemerow GR, McNaughton ME, Cooper NR. Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes. J Immunol 1985;135:3068–73. [20] Kerekes K, Prechl J, Bajtay Z, Jozsi M, Erdei A. A further link between innate and adaptive immunity: C3 deposition on antigen-presenting cells enhances the proliferation of antigen-specific T cells. Int Immunol 1998;10:1923–30. [21] Erdei A, Kohler V, Schafer H, Burger R. Macrophage-bound C3 fragments as adhesion molecules modulate presentation of exogenous antigens. Immunobiology 1992;185:314–26. [22] Sandor N, Pap D, Prechl J, Erdei A, Bajtay Z. A novel, complement-mediated way to enhance the interplay between macrophages, dendritic cells and T lymphocytes. Mol Immunol 2009;47:438–48. [23] Hamad OA, Nilsson PH, Wouters D, Lambris JD, Ekdahl KN, Nilsson B. Complement component C3 binds to activated normal platelets without preceding proteolytic activation and promotes binding to complement receptor 1. J Immunol 2010;184:2686–92.
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Human T cell derived, cell-bound complement iC3b is integrally involved in T cell activation Katalin Török a , Mariann Kremlitzka a , Noémi Sándor a,b , Eszter Angéla Tóth a , Zsuzsa Bajtay a,b , Anna Erdei a,b,∗ a b
Department of Immunology, Eötvös Loránd University, Budapest, Hungary Immunology Research Group of the Hungarian Academy of Sciences at Eötvös Loránd University, Budapest, Hungary
a r t i c l e
i n f o
Article history: Available online 22 February 2012 Keywords: Human T cell Complement C3 production Cell-bound iC3b T cell proliferation
a b s t r a c t Although the complement system is thought to be mainly involved in innate immunity and in the humoral arm of adaptive responses, evidence implicating that complement impacts T cell responses are accumulating recently. The role of the various activation products of the major complement component C3 were mainly studied so far in animal systems, and investigations regarding the effect of different C3-fragments on human T cells are sparse. Here we show that anti-CD3 activated human T lymphocytes derived from the blood and tonsil of healthy individuals produce C3, and the major cleavage fragment that appears on the T cell surface is iC3b. Based on studies carried out in allogenic system we demonstrate that the T cell membrane bound iC3b binds to the CR3 and probably to CR4 receptors expressed on monocyte-derived dendritic cells, and this interaction leads to significantly enhanced T-cell proliferation. Since neither C3aR and nor C3a binding could be detected on the membrane of anti-CD3 activated T cells, our findings indicate that in humans – in contrast to mice – the C3a peptide is most probably not involved directly in the T cell activation process. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The complement system is an integral part of our immune defense. Its components participate both in the innate and adaptive responses to various pathogens and antigens. The major source of the circulating components is the liver, however the importance of locally produced complement proteins is also well accepted by now, particularly due to their participation in the regulation of adaptive immune processes in peripheral lymphoid organs [1–5]. It is well known that several cell types are able to produce various complement proteins, including C3, the central component of the activation cascade [6,7]. C3 production by mouse T cells and the role of C3a, its small cleavage product along with the other anaphylatoxic complement peptide C5a have been extensively studied recently during the interaction with dendritic cells (DC), using a mouse model systems [8]. The role of C5a in driving Th17 differentiation and triggering arthritis has also been described [9]. Moreover, recent findings in experimental models of allergic rhinitis and allergic asthma suggest that C3a, C5a and the activation of antigen-presenting cells via their corresponding receptors regulate
the development of maladaptive Th2 and Th17 immunity [10]. Our knowledge regarding complement synthesis by human T cells however is sparce, and thorough studies on the interaction of C3 activation fragments and T lymphocytes are not available. Employing a sensitive, radio-immunoprecipitation method Pantazis et al. demonstrated earlier that PHA-activated and HTLV infected T cell lines produce C3 [11], and recently Cardone et al. showed that under certain circumstances C3b appears on the surface of activated human T cells [12], but no further analysis was carried out. Here we studied C3 production by primary human T lymphocytes in detail. We show that anti-CD3 activated T cells produce C3, and demonstrate that the major C3-derived protein found on the surface of CD3-activated T cells is the iC3b fragment. We show that these fragments – by their capacity to bind to CR3 and possible also to CR4 expressed by DCs – enhance the activation of alloreactive T cells. Based on our data the possible involvement of C3a in the human system can be excluded, since we could not detect C3aR expression and C3a binding neither on non-activated nor on activated T cells. 2. Materials and methods
∗ Corresponding author at: Department of Immunology, Eötvös Loránd University, Budapest, Hungary, Pázmány Péter s. 1/C, 1117 Budapest, Hungary. Tel.: +36 1 3812 175; fax: +36 1 3812 176. E-mail address: [email protected] (A. Erdei). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2012.02.003
2.1. Cells T cells were isolated from tonsils of children aged 2–8, and from buffy coats of healthy donors. Tonsils were provided by the
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St Imre Hospital, Budapest, and buffy coats were obtained from the Hungarian National Blood Transfusion Service based on an ethical permission. Informed consent was provided for the use of tonsils and blood samples according to the Declaration of Helsinki, and the use of tonsils was implemented by the acquiescent declaration of the donor’s parents. Mononuclear cells were isolated by sedimentation on FicollPaqueTM Plus (GE Healthcare Bio-Sciences AB). T cells from blood and tonsil were negatively isolated by Vario MACS Separator using anti-CD19- and anti-CD14-microbeads on LS Columns (Miltényi) or by sorting using anti-CD19-APC, anti-CD16-FITC and anti-CD14PE (BD-Pharmingen) antibodies and FACSAria III instrument. The percentage of T cells in the final suspensions of tonsils was always greater than 96% when applying MACS, of blood it was greater than 90%, and by the FACSAria III it was always more than 99.5%. Cells were characterised by flow cytometric analysis (FACSCalibur) using the following antibodies: anti-CD3-FITC, anti-CD3-PE, anti-CD19-APC, m-IgG1-FITC, m-IgG1-PE (Immunotools); polyclonal goat anti-human-C3-FITC F(ab) 2 (Cappel); anti-C3aR-Al647 (AbD Serotec); anti-CR3-RPE (Dako); m-IgG1-Al647 (BioLegend); anti-m-IgG (H+L), anti-m-IgG-Al488, anti-rabbit-IgG-Al488 (Invitrogen); anti-iC3b (Quidel); goat serum (control), rabbit serum (control); rabbit antiserum to human C3a (Behring Institute). 2.2. T cell activation T cells (105 cells/well) were activated for three days by culturing in microwells coated with 1 g/ml anti-CD3 (BD Pharmingen) or pooled mIgG, for control. 2.3. C3 PCR Total RNA from MACS purified non-activated and activated T cells and from the U937 human monocytic cell line were isolated using Rnaesy Mini Kit (Qiagen). The isolated RNA was converted into cDNA by reverse transcription (ThermoHybaid PCR Express) using 500 ng of each RNA sample and a mixture of 0.5 g OligoDT (synthesized by BioBasic Inc.), 200 unit/l RevertAid H Minus M-MuLV Reverse transcriptase (Fermentas), DEPC treated water, 5x reaction buffer (Fermentas), 40 unit/l RiboLock RNase inhibitor (Fermentas), 10 mM dNTPs (Fermentas) in the concentration providing by the manufacturer. The reaction profile was as follows: 70 ◦ C 5 , 37 5 , 42 ◦ C 1 h, 70 ◦ C 10 , followed by final cooling to 4 ◦ C. Samples were frozen until further studies. For the C3 PCR a mixture was made up from the following ingredients in the concentration providing by the manufacturer: 10 M forward (5 -GTGGCCAKGCRGCAGAAGGC-3 ) and reverse (5 -TCACCACSTGGGAGATTCTG-3 ) primers (IDT), 10 mM dNTPs (Fermentas), 2.5 l cDNS, RNase free water, 25 mM MgCl2 (Fermentas), 10x Taq puffer +KCl, −MgCl2 (Fermentas), 1 unit/l Taq polymerase (Fermentas). The reaction profile consisted of 94 ◦ C 5 ; 60 ◦ C 2 ; 72 ◦ C 22 , 94 ◦ C 22 , 60 ◦ C 22 through 40 cycles; 72 ◦ C 10 ; followed by final cooling to 4 ◦ C. Twenty l of the PCR reaction was applied to a 2% agarose gel (Serva) and the PCR products were detected by an UV-transilluminator. As control -actin primers reverse: 5 (forward: 5 -GGCTACAGCTTCACCACCAC-3 , GCGCTCAGGAGGAGCAATG-3 ) PCR were applied. The length of the C3 PCR product is 233 bp, while that of the -actin is 411 bp. 2.4. Biotinylation of cell surface proteins and immunoprecipitation 107 freshly isolated and 3-day activated peripheral T cells were surface biotinylated using the Pierce cell surface protein isolation kit (Pierce Biotechnology). After biotinylation, cells were
solubilized in 0.5 ml lysis buffer containing 50 mM HEPES, (pH 7.4), 1% Triton X-100, 100 mM NaF, 10 mM EDTA, 2 mM sodium orthovanadate, 10% glycerol, 2 g/ml aprotinin, 2 g/ml pepstatin, 5 g/ml leupeptin, and 1 mM PMSF at 4 ◦ C for 30 min (the enzyme inhibitors were all purchased from Sigma Aldrich). The cell extracts were centrifuged at 15,000 × g for 10 min, and pre-cleared with Protein-G Sepharose beads (Pierce Biotechnology) at 4 ◦ C for 3 h. Then the samples were immunoprecipitated with anti-human C3c (Dako) using Protein-G Sepharose beads (10 g rabbit IgG/sample). After rotating the samples overnight at 4 ◦ C beads were washed, boiled in reducing sample buffer and run on 10% SDS–PAGE. Separated proteins were transferred to nitrocellulose membrane. After blocking with 5% BSA for 1 h, the blots were developed by incubation with streptavidin–HRP (1:500, R&D Systems). Bands were visualized by the ECL method (Millipore). 2.5. Conjugate formation between CR3-bearing U937 cells and activated T lymphocytes 105 U937 cells labeled with 0.5 M CFSE (Molecular Probes) were co-incubated with 4 × 105 T cells for 2 h at 37 ◦ C. For the inhibition studies cells were incubated in the presence of TMG6-5, the iC3b ligand-binding site specific anti-CD11b antibody, kindly provided by Dr. Istvan Ando (BRC, Szeged, Hungary), the anti-iC3b antibody recognizing a neo-epitope on iC3b (Quidel), or an isotype control (mouse IgG1, Sigma) at 10 g/ml concentration. The number of interactions was counted and normalized to 100 cells using an Olympus IX81 confocal laser scanning microscope with further digital magnification. 2.6. Monocyte-derived dendritic cells (MDCs) Monocytes were isolated from buffy coat using CD14 microbeads (Miltenyi). Cells were cultivated in CellGro serum free medium (Cell Genix) supplemented with 100 ng/ml rHu GMCSF (R&D) and 1500 unit/ml rHu IL-4 (Promokine), and cytokines were added every other day. On day four CD14− immature MDCs were resuspended in serum-free medium containing 100 ng/ml LPS (Sigma). On day five mature MDCs were washed and used for further studies. 2.7. Allogenic T cell activation Mature MDCs were washed and transferred to 96 well plates and further cultured with allogenic T cells at a DC:T cell ratio of 1:5 for 16 h. T cells, obtained from a different donor had been preactivated as described above. Cell proliferation was measured by 3 H-thymidine incorporation in the presence or in the absence of polyclonal goat anti-human-C3 (Fab )2 (Cappel), anti-CR3 (TMG65) and anti-iC3b (Quidel), all in the concentration of 25 g/ml. Either T cells or DCs, or both cell types were preincubated with the antibodies for half an hour, then washed and put to co-culture. 2.8. 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 was 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. 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.
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Fig. 2. T cell derived C3 associated with the cell membrane. Flow separated human T cells were surface biotinylated either without activation (lane 1) or after stimulation by anti-CD3 for three days (lane 2). Cell extracts were immunoprecipitated with anti-C3c antibody and separated on 10% SDS–PAGE under reducing conditions. Biotinylated C3, which has been degraded to some extent, was also run for comparison (lane 3). The following protein-bands are indicated by arrow on the figure: 110 kDa: ␣-chain of C3; 75 kDa: intact -chain of C3; 67 and 45 kDa: cleavage fragments derived from the ␣-chain of C3.
Fig. 1. Demonstration of C3 expression by human T lymphocytes. (a) Expression of C3 mRNA. Isolated non-activated and activated human T lymphocytes and the human monocytic U937 cell line were subjected to mRNA preparation followed by PCR, as described in Section 2. Lane 1: non-activated blood-derived T cells; lane 2: CD3activated blood-derived T cells; lane 3: non-activated tonsil-derived T cells; lane 4: CD3-activated tonsil-derived T cells; lane 5: U937 cells (control). The figure shows data obtained with MACS-purified cells. Similar data were obtained when flow sorted cells were used. The results of one representative experiment of three are shown. (b and c) Demonstration of T cell derived iC3b on the surface of activated human T cells by cytofluorimetry. Non-activated and activated tonsil-derived T cells were labelled with FITC-labelled polyclonal anti-C3 F(ab )2 antibody. One representative histogram of three with similar results is shown. (b). For the analysis of blood-derived T cells a monoclonal anti-iC3b antibody recognizing a neoepitope on cell-bound iC3b was used. One representative experiment of two with similar results is shown (c). Both the anti-C3 F(ab )2 and the anti-iC3b antibodies reacted with tonsil- and blood-derived T-cells to a similar extent.
3. Results 3.1. C3 synthesis by activated human T lymphocytes To study whether human T cells are able to synthesize complement C3, first we looked for the C3-message at the mRNA-level. We used T cells from tonsils and blood isolated either by MACS or by sorting as described in Section 2. Cells were activated by anti-CD3 for three days, then used for mRNA preparation. As shown in Fig. 1a, in the case of purified, non-activated blood-derived T cells only a small amount of C3 mRNA can be detected, while upon activation of the cells via the TCR, a strong C3-message appears. In the case of tonsil-derived T cells we found no major difference between the in vitro stimulated and non activated cells; both expressed mRNA for C3 to the same extent, most probably due to in vivo activation of the cells. As positive control the C3-producing human monocytic U937 cell line was used. Next we attempted to detect C3 in the supernatant of nonactivated and activated T cells, employing a sensitive indirect ELISA system which detects C3 concentration as low as 2 ng/ml. However no C3 protein could be demonstrated in these samples, while the
culture supernatant of U937 cells, used as positive control, contained a substantial amount of C3 (data not shown). 3.2. Demonstration of cell-derived, surface-bound iC3b-fragments on activated human T cells by cytofluorimetry Since Cardone et al. demonstrated C3b deposition on the surface of purified, activated human CD4+ T cells [12], in the next experiments we set out to examine whether the larger C3-fragments can be detected also in our experimental system. We found that both the F(ab )2 fragment of the goat polyclonal anti-C3 IgG and a monoclonal antibody specifically recognizing a neoepitope on iC3b strongly react with the flow separated activated T cells (Fig. 1b and c). Since the iC3b neoepitope is hidden in the intact C3, these results suggest that the larger complement fragment most probably becomes surface bound after activation. Since only flow separated T lymphocytes were present in the cultures investigated in these experiments, and the FCS supplementing the cell culture medium does not contain C3-protein [13], our data provide evidence that C3-fragments found on the cellsurface were generated by the T lymphocytes. 3.3. Demonstration of cell-derived surface-bound iC3b-fragments on activated human T cells by immunoprecipitation To confirm the cytofluorimetric data and also in our attempt to elucidate the nature of the interaction between C3 and the cell membrane of human T cells, we performed immunoprecipitation. For the experiments we used the extract of surface biotinylated non activated and activated T cells and employed C3c-specific antibody. As demonstrated in Fig. 2, iC3b was present mainly on the surface of activated T lymphocytes (lane 2), however a very small amount of the C3-fragments could also be found in the detergent extract of the cell membrane of non activated cells (lane 1). Comparing the surface-bound complement protein (lane 2) with the purified, biotinylated C3 (lane 3), it can be seen that the intact, 110 kDa ␣-chain of the complement protein is almost completely
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Fig. 3. Lack of C3aR expression and C3a binding by human T cells. Non-activated and anti-CD3 activated T cells were subjected to FACS analysis using human C3a and C3aRspecific antibodies. As control C3a-binding and C3aR expression of monocytes derived from the same healthy donor is shown. We gated on monocytes on the basis of forward and side scatter. Solid lines show the binding of C3aR- and C3a-specific antibodies, respectively, dotted line shows the binding of the control antibody and the grey background indicates autofluorescence in each histogram. Results of one representative experiment out of three with similar results are shown.
missing, proving that activation/cleavage of C3 took place. Under the reducing conditions employed, the 67 and 45 kDa fragments of the ␣ -chain of iC3b appear, while the -chain (75 kDa) remains intact. We could not identify any larger protein complexes containing C3-fragments in these cell membrane extracts, suggesting that most probably the interaction between T cells and C3 is of non covalent nature.
3.4. T cells of healthy individuals do not express C3aR As shown in Figs. 1b, c and 2 the larger fragments of C3 are present on the surface of activated human T cells. Since these products are generated by the proteolytic cleavage of C3, next we studied the possibility, whether the small peptide, C3a also interacts with the T cells. Earlier it had been demonstrated that T lymphocytes of patients suffering from severe inflammatory skin diseases express C3aR, while unstimulated T cells of healthy individuals do not [14]. The same authors also demonstrated that type I IFNs added to freshly isolated or cloned T lymphocytes induce C3aR expression. Based on these data and the results of Strainic et al., who had shown in a mouse system that T cell derived C3a (and also C5a) interact with their respective receptors and influence T cell
proliferation [8], we aimed to clarify whether anti-CD3 activated human T cells also express this anaphylatoxin receptor. As shown in Fig. 3 however, no C3aR could be detected on the T lymphocytes activated in our experimental system, suggesting that inflammatory conditions are needed for its expression. The possibility that cell-bound C3a might sterically inhibit the binding of the C3aR specific antibody could be excluded, since we did not find any C3a peptide bound to the surface of the activated T cells (Fig. 3). Used as positive control, human monocytes were found to possess C3aR and also to bind C3a.
3.5. iC3b-mediated adherence of activated T lymphocytes to CR3 expressing cells As demonstrated in Figs. 1c and 2, activated T cells bear substantial amount of iC3b on their surface, which might serve as ligand for CR3 and CR4 expressed by various cell types. In order to find out whether these surface-bound C3-fragments are involved in cellular adherence, we investigated conjugate-formation between activated T cells and the CR3-expressing U937 cells, as described in Section 2. As seen in Fig. 4 activated T cells adhered to the CFSE-labelled U937 cells, and this interaction could be inhibited
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Fig. 4. Conjugate formation between activated human T cells and U937 cells. CFSE labeled CR3-expressing U937 cells were coincubated with activated human T lymphocytes in a 1:5 ratio for 2 h. Conjugate formation was analyzed by Olympus Fluoview 500 confocal laser scanning microscope at 60× magnification with further digital enlargement (b). Average number of interactions is shown (a).
by preincubation of the activated T cells with anti-iC3b and antiCD11b.
3.6. T cell-bound C3 fragments enhance proliferation induced by allogenic DCs Next we set out to study whether the iC3b-mediated adherence may lead to changes in the proliferative capacity of T cells. To this end we generated DCs from monocytes and after activation for one day by LPS they were cocultured for 16 h with pre-activated alloreactive T cells in a 1:5 ratio. Cells were harvested after pulsing with H3 -thymidine overnight. As seen in Fig. 5, under these conditions T cell proliferation was significantly enhanced compared to the activity of non activated T cells. When activated T cells were preincubated with a mixture of antibodies reacting with C3, iC3b and CR3, this enhancement was significantly diminished. Interestingly, when the DCs were pretreated with the same antibodies, the T cells’ proliferative capacity did not change compared to the untreated control (Fig. 5). There might be two explanations for this phenomenon. One possibility is that CR4 – which also binds iC3b – is also involved in the interaction between T cells and DCs. Another explanation might be that newly expressed CR3 molecules appear on the surface of DCs.
Fig. 5. Allogenic T cell activation is mediated by T cell-derived and T cell-bound iC3b. Human monocyte-derived DCs after stimulation with LPS for 1 day and T cells activated by anti-CD3 for 3 days were cocultured in a 1:5 ratio for 16 h. Cells pretreated with a mixture of anti-iC3b, anti-C3 (Fab )2 and anti-CR3 at 25 g/ml are indicated by a frame around the abbreviation (DC: dendritic cell; aT: activated T cell; naT: non-activated T cell). Cells treated with the antibody-cocktail were washed and cocultured for one day. Results of one representative experiment out of three with similar results are shown.
4. Discussion The involvement of complement in the instruction of adaptive immunity has been investigated for a long time, beginning in 1974 by Pepys [15]. Whereas in the past decades the role of complement in various B-cell functions has been clarified to a great extent [16–19] its influence on various T cell functions has been studied in detail only recently. Most of the available data have been obtained in various mouse systems – particularly those describing the role of C3a and C5a providing costimulatory signals in cognate interactions between antigen-presenting cells and T lymphocytes [5] In human systems however, the local production of complement proteins and the possible contribution of the larger fragments (C3b, iC3b) to various T cell functions have not been investigated thoroughly so far. By radioimmune precipitation followed by SDS–PAGE Pantazis et al. demonstrated earlier that mitogen activated human T cells and HTLV infected T cell lines produce C3 [11]. Recently Cardone et al. showed C3b deposition onto activated CD4+ T cells by flow cytometry, however no further details were provided regarding the origin and mode of attachment of the complement fragment [12]. Here we show both at the mRNA and the protein level that blood and tonsil derived human T cells produce C3 when activated via CD3. While secreted C3 could not be detected in the supernatant of the cell cultures even by using a very sensitive sandwich ELISA system, C3 bound to the cell surface of activated T cells was easily demonstrated. Using a neoepitope specific antibody we could identify iC3b as the major cleavage product of C3 on T cells. Since iC3b is the natural ligand of both CR3 and CR4, it could be assumed that iC3b bearing T lymphocytes might interact with cells expressing these complement receptors. Indeed, we found that this interaction occurs in allogenic systems and leads to enhanced T cell proliferation. This most probably results in changes in the production of various cytokines, thus may participate in the regulation of various immune functions. These data complement earlier results obtained in animal experiments, where the role of macrophage-derived C3 in enhancing antigen presentation has been clearly shown [20,21]. Using mice for their experiments Strainic et al. demonstrated that complement-derived anaphylatoxic peptides (C3a, C5a) produced locally by APCs and T cells during cognate interactions, are integrally involved in the activation of T lymphocytes [8]. The possible involvement of the larger fragments of C3 however, which are generated simultaneously, has not been investigated in this model system so far. In contrast to the studies carried out in mice, we could not detect C3 released by human DCs [22], implying that in our system the enhanced proliferation of alloreactive T lymphocytes is caused by T cell derived C3-fragments. This has been clearly proven by the inhibition of the MLR by specific antibodies. Our data demonstrate the bridge-forming role of iC3b, which most probably plays an important role under physiologic conditions in lymph nodes. Certainly the deposition of various C3-fragments may
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influence other T cell functions as well, as shown by Cardone et al. [12]. While the synthesis and production of C3 by human T cells is clearly shown in this paper, the exact nature of the cell membrane bound C3-fragments could not be revealed so far. Our results obtained by immunoprecipitation of surface labelled T cells do not provide evidence of covalent fixation, since we could not detect complexes of C3 and cell membrane constituents by Western blotting. Another possibility could be that the produced C3 molecule is nonproteolytically activated, thus C3(H2 O) is generated which is processed to iC3(H2 O). This iC3b-like molecule serves then as a ligand for complement receptors expressed by different cell types. This reaction has been described earlier in the case of activated thrombocytes [23]. Regarding the molecular mechanism of the “iC3b-bridge” formation between T cells and APCs it can also be assumed that iC3(H2 O) forms dimers (or oligomers), enabling the interaction with various complement receptors expressed on both cell types. Finally, it may further be hypothesized that the C3 molecules synthesized by the T cells – or its various products – are retained in the membrane of T lymphocytes via a so far unknown mechanism. Acknowledgements The financial support from the Hungarian National Science Fund (OTKA) grant K72026, from TÁMOP – 4.2.1/B-09/1/KMR-20100003, and the support from the Hungarian Academy of Sciences are gratefully acknowledged. References [1] Erdei A, Fust G, Gergely J. The role of C3 in the immune response. Immunol Today 1991;12:332–7. [2] Sacks S, Zhou W. The effect of locally synthesised complement on acute renal allograft rejection. J Mol Med (Berl) 2003;81:404–10. [3] Sewell DL, Nacewicz B, Liu F, Macvilay S, Erdei A, Lambris JD, et al. Complement C3 and C5 play critical roles in traumatic brain cryoinjury: blocking effects on neutrophil extravasation by C5a receptor antagonist. J Neuroimmunol 2004;155:55–63. [4] Lalli PN, Strainic MG, Yang M, Lin F, Medof ME, Heeger PS. Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis. Blood 2008;112:1759–66. [5] Heeger PS, Kemper C. Novel roles of complement in T effector cell regulation. Immunobiology 2011.
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