Complement Activation Regulates the Capacity of Proximal Tubular Epithelial Cell to Stimulate Alloreactive T Cell Response
Ke Li,
Hetal Patel,
Conrad A. Farrar,
Roseanna E.G. Hargreaves,
Steven H. Sacks and
Wuding Zhou
Department of Nephrology and Transplantation, Guy’s Hospital, King’s College London, London, United Kingdom
Correspondence to Dr. Wuding Zhou, Department of Nephrology and Transplantation, 5th Floor, Thomas Guy House, Guy’s Hospital, London SE1 9RT, UK. Phone: 44-207-188-1528; Fax: 44-207-188-5660; E-mail: wuding.zhou{at}kcl.ac.uk
ABSTRACT. Tissue expression of C3 is an unexpected regulatorof the alloimmune response in mouse kidney transplantation.It is unclear, however, whether a direct or an indirect actionof complement on the host immune response is involved. Alsounknown is which of the complement effector products, cleavedC3, cleaved C5, or C5b-9, is responsible. Proximal tubular epithelialcells (PTEC) not only constitute a major target of the alloimmuneresponse but also produce substantial amounts of C3. This studyinvestigated the property of mouse PTEC to stimulate alloreactiveT cells in a complement-dependent manner. The proliferativeand cytokine responses of primed alloreactive T cells were measuredafter exposure to donor-specific PTEC that had been pretreatedwith normal mouse serum, heat-inactivated mouse serum, or complement-deficient (C3, C5, or C6) mouse sera to differentially depositcomplement components. PTEC were able to stimulate alloreactiveT cells in an antigen-specific manner. Complement activationleading to the deposition of cleaved C3 on PTEC enhanced thealloreactive T cell response. This complement-mediated stimulationof the T cell response was dependent on C3 but not on C5 orC6. The primary influence of tissue-bound complement was onCD4+ T cells. Moreover, the effect of complement on alloreactiveT cells was B7 dependent, shown by inhibition studies with CTLA4-Ig.These results suggest that donor epithelium-bound C3 can upregulatethe alloimmune response. It is postulated that surface-boundC3 interacts with complement receptors on alloreactive T cellsor on antigen presenting cells to increase allo-immune stimulation.
Evidence has emerged that the innate immune system regulatesthe development of the adaptive immune response. Complementis one of the major components of the innate immune system andnot only plays an important role in host defense against initialinfection but also modulates both B cell and T cell function.Much of the evidence linking the immunoregulatory propertiesof complement with receptor activation on immune cells is inthe context of infection and autoimmunity. The regulation ofthe alloimmune response by complement is much less clear.
Complement protein C3 is the point of convergence for the threerecognized pathways of complement activation and thus playsa critical role in biologic processes mediated by the complementcascade, which includes anaphylatoxic activity, opsonizationof pathogens and foreign particles, and lysis of cells. In additionto these effector functions, it has become increasingly evidentthat C3 participates in the regulation of the antigen-specificimmune response. The C3 split product C3b or its metaboliteC3d, either conjugated to antigen or bound on antigen-presentingcells (APC), can regulate the specific immune response againstexogenous antigens (1–4). Furthermore, studies in C3-deficient(C3-def) mice have demonstrated that C3 has an ability to augmentthe specific T cell response against viral antigens and autoantibodies(5,6). Recently, two studies in our laboratory provided evidencethat complement also has an important role in the regulationof the alloresponse. In a skin graft model, C3- and C4-def recipientmice but not C5-def mice had a profound defect in the primaryalloantibody response, suggesting that complement participatesin modulating alloantibody production (7). In another study,most allogeneic C3-def mouse kidneys transplanted into complement-sufficientmice survived for at least 100 d, whereas control grafts fromC3-sufficient (C3-suf) donors were rejected within 14 d (8).Both studies strongly suggest that tissue C3 is essential forthe regulation of transplant rejection and that local productionof C3 has a significant effect on the T cell alloresponse.
Alloreactive T cells are critical for mediating acute allograftrejection. Activation of allospecific T cells after transplantationrequires two signals: stimulation by donor antigen (alloantigen)and co-stimulation through antigen-independent pathways (9).Alloantigen is a unique antigen, which could be allo-MHC moleculesor peptide:MHC complexes; the latter could be any peptide:allo-MHCor allo-peptide:self MHC (10–12). Alloreactive T cellsrecognize all of them by either the direct or indirect antigenpresentation pathway. In the kidney, apart from passenger leukocytes,parenchymal cells are also thought to be able to present antigento T cells (13,14). However, the extent, regulation, and significanceof antigen presentation by kidney parenchymal cells are at presentunclear. Renal proximal tubular epithelial cells (PTEC), besidesexpressing MHC class II molecules, are able to produce a numberof inflammatory mediators, including C3, and co-stimulatorymolecules (13,15,16), whose expression is increased by manyinflammatory stimuli. In addition, PTEC reside at the interfacebetween the urinary space, interstitium, and peritubular capillaries.These features allow PTEC to have a potential role in coordinatingproinflammatory and immunoregulatory functions in the renalinterstitium. PTEC functioning as nonprofessional APC thereforecould participate in the alloimmune response during transplantrejection.
Previous work has shown that C3 is markedly upregulated in thetubules of inflamed kidney (i.e., ischemia/reperfusion injuredor rejected) and is deposited on the tubule surface adjacentto the T cell infiltrate (17–19). These data suggest thatcomplement, in addition to regulating the specific immune responsein infection and autoimmunity, participates in the regulationof the alloimmune response in kidney transplantation. In thepresent study, we examined the effect of complement on the capacityof PTEC to stimulate primed alloreactive T cells and investigatedthe precise level of the complement cascade (C3/C5/C6) involved.
Mice
BALB/c (H-2d), C57BL/6 (H-2b), C3H/He (C6-suf), DBA/1 (C5-suf)and DBA/2 (C5-def) were purchased from Harlan UK Ltd (Bicester,UK). Homozygous C3-def mice were derived by homologous recombinationin embryonic stem cells (20) and backcrossed onto the C57BL/6parental strain for 11 generations. C6-def mice, which werederived from a Peruvian strain and backcrossed with C3H/He micefor 10 generations, were provided by Professor P. Lachmann (Cambridge,UK) (21). C3-, C5-, and C6-def mice had no plasma activity oftheir respective deficient complement components. Only malemice were used in the study to avoid gender-related variationsin complement activity that occur in some strains. All procedureswere conducted in accordance with the Home Office Animals (ScientificProcedures) Act of 1986.
PTEC Cultures
Primary PTEC cultures were prepared from kidneys of male C57BL/6(H-2b) or BALB/c (H-2d) mice as described previously (15). Mincedcortex was digested with 0.1% collagenase II and passed througha 40-µm nylon sieve. The cells and tubules were collectedand cultured in a DMEM-12 medium that contained 2% FCS, insulin(5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml),hydrocortisone (40 ng/ml), and tri-iodothyronine (10–2M). All cells used in this study were nonpassaged 5- to 6-dcultures. They displayed epithelial morphology, expressed thebrush border enzyme alkaline phosphatase, and stained positivelyfor cytokeratin. Examination by electron microscopy demonstratedthe presence of numerous apical microvilli of a rudimentarybrush border, with reassembly of tight junctions.
Reverse Transcription of RNA and Subsequent PCR
Total RNA was extracted from PTEC and followed by cDNA synthesisas we described previously (16). PCR was carried out with 2µl of diluted cDNA (reflecting 0.2 µg of total RNA),12.5 pmol of each 3' and 5' primer pair for MHC class II A chain,CD40, intracellular adhesion molecule-1 (ICAM-1), and B7.2 (CD86)(Table 1) in 25 µl of reaction buffer (Promega, Southampton,UK). The PCR cycle consisted of 1 min at 94°C, 1 min at62°C, and 1 min at 72°C. Amplified PCR products werevisualized after electrophoresis on 1.2% agarose gel that containedethidium bromide. Glyceraldehyde-3-phosphate dehydrogenase primers,12.5 pmol each, were also added in every reaction as an internalcontrol.
Serum Preparation
Mouse sera were freshly prepared on the day of use. Normal serum(NS) was obtained from C57BL/6 mice; heat-inactivated serum(HIS) was prepared by incubating this serum at 56°C for30 min. Complement component-sufficient sera (C3, C5, and C6)were obtained from C57BL/6, DBA/1, and C3H/He mice, respectively.Sera deficient in a specific complement component were obtainedfrom C3-, C5-, and C6-def mice.
Preparation of Stimulators (PTEC)
PTEC used in this study were incubated with IFN- for 24 h tomaximize expression of MHC and co-stimulatory molecules. Single-cellsuspensions were prepared by trypsin/EDTA treatment and thenincubated with 10% mouse serum for 30 min, unless otherwisestated. PTEC were then washed three times with endotoxin-freePBS to remove unbound complement proteins and resuspended inT cell culture medium (RPMI-1640 containing 10% heat-inactivatedFCS, 50 µM 2-mecaptoehtonal, 100 U/ml penicillin, and100 µg/ml streptomycin) and irradiated at 1000 RADS. Cellviability was assessed before and after serum treatment, bytrypan blue dye exclusion, which showed no increase in the numberof dead cells after treatment with serum. Viable cells were>90% of the total. Allogeneic H-2b PTEC monolayers were usedin all experiments, except the syngeneic control experimentin which H-2d PTEC were used.
Preparation of Responders (Alloreactive T Cells)
Primed alloreactive T cells were isolated from spleens of BALB/c(H-2d) mice 14 d after they received a C57BL/6 skin graft. Mincedspleen was forced through a 40-µm nylon sieve using a5-ml syringe plunger. The filtrates were collected and washedonce with endotoxin-free PBS. Enriched T cells were preparedusing anti-mouse IgG beads (Dynal, Oslo, Norway) to remove IgG+cells. After enrichment, the T cell preparation routinely consistedof >80% CD3+ T cells. Purified CD4+ and CD8+ T cells wereprepared using Spin-Sep Enrichment Cocktail Kit (StemCell Technologies,London, UK). After the purification, the purity of those T cellpreparations is >90% as determined by flow cytometry.
T Cell Proliferation Assay
After cytokine ± serum treatment, 2 x 105 washed, irradiatedPTEC (H-2b) were added into cultures of 2 x 105 enriched T cellsor purified CD4+ and CD8+ T cells (H-2d) in 96-well plates andcultured for 96 h. 3H-thymidine (1 µCi/well) was addedduring the last 24 h. The amount of [3H] TdR incorporation wascounted. Control cultures included wells of irradiated PTECwith irradiated T cells. Data were expressed as the differencein cpm of experimental and control cultures and referred toas " cpm." T cells alone and PTEC alone controls were also includedin each experiment and gave consistently low backgrounds.
T Cell Function Assay
The amount of IFN- and IL-2 secreted into cell culture supernatantswas measured by ELISA. Sandwich ELISA was performed using ELISAKit for mouse IFN- or IL-2 (R&D Systems, Oxford, UK) accordingto the manufacturer’s instructions.
Flow Cytometry
Single-cell suspensions of PTEC were stained with either FITC-or phycoerythrin (PE)-conjugated antibody or the appropriateisotype control antibody. The stained cells were analyzed byflow cytometry (FACScan; Becton Dickinson, Oxford, UK). Ab reagentsused were FITC-conjugated cross-reactive rabbit anti-human C3dantibody (Dako, High Wycombe, UK), PE-conjugated rat anti-mouseMHC class II (I-A/I-E), PE-conjugated rat anti-mouse CD40, PE-conjugatedArmenian hamster anti-mouse ICAM-1 (CD54), and PE-conjugatedrat anti-mouse B7.2 (CD86; BD Biosciences, Cowley, UK).
Statistical Analyses
All T cell proliferation data shown are representative of atleast three independent experiments. ELISA data shown are representativeof three independent experiments. Statistical analysis was performedon a complete data set of one experiment, with T cell proliferationand ELISA assessed in triplicate. Data are expressed as mean± SEM and subjected to t test.
MHC Class II and Co-stimulatory Molecule Expression
To examine whether primary cultured murine PTEC are able toexpress MHC class II and a number of co-stimulatory moleculesand thereby can potentially function as APC, we performed reversetranscription-PCR (RT-PCR) and flow cytometry on PTEC treatedwith IFN- (1000 U/ml) for 24 h. Gene expression of MHC classII, CD40, ICAM-1, and B7.2 was clearly detected by RT-PCR. Proteinexpression of MHC class II, CD40, and ICAM-1 was correspondinglydetected by flow cytometry, with the exception of B7.2, whichis only weakly detected (Figure 1). In addition, gene expressionof MHC class II, CD40, and ICAM-1 was detected in unstimulatedPTEC by RT-PCR (data not shown).
Figure 1. Detection of MHC class II and co-stimulatory molecules. Monolayers of proximal tubular epithelial cells (PTEC) were stimulated with IFN- (1000 U/ml), followed by reverse transcription-PCR (RT-PCR) and flow cytometry analysis. (A) RT-PCR. An agarose gel showing the size of the detected fragments and the 453-bp glyceraldehyde-3-phosphate dehydrogenase (internal control) band. The 1-kb DNA markers are shown along side the gel. (B) Flow cytometry. In all histogram plots, the control peak (heavy line) corresponds to staining cells with the appropriate isotype control antibody. The detection peak (dotted line) shows surface binding of specific antibody.
C3 Deposition on PTEC
To test whether complement can be activated and deposited onmurine PTEC, the suspension cells were incubated with 10% mousesera (NS, HIS, C3-def, C5-def, or C6-def sera). C3 depositionwas examined by flow cytometry. The result of C3d staining showsthat membrane-bound C3 was clearly detected in NS or C5-defserum-treated cells but barely detected in HIS or C3-def serum-treatedcells (Figure 2). These results indicate that after complementactivation, C3 split product was deposited on PTEC. The veryweak staining of C3d observed in HIS- or C3-def serum-treatedcells could be explained by the C3 produced by PTEC themselves(15,16). The detection of C3d in C5-def (or C6-def; data notshown) serum-treated cells indicates that without C5 or C6,complement activation of C3 occurs despite C5b-9 being unableto form. Our data suggests that mouse PTEC can spontaneouslyactivate complement, confirming observations with human PTEC(22,23).
Figure 2. Detection of complement C3 deposition on PTEC. Single-cell suspension PTEC were treated with 10% various mouse sera (normal serum [NS], heat-inactivated serum [HIS], C3-deficient [C3-def], and C5-def) for 30 min. Washed serum-treated cells were subsequently stained for C3d by FACS. PTEC that were treated with serum are represented by the dotted line, and control cells (no serum added) are represented by the heavy line.
C3 Deposition Enhances the Ability of PTEC to Stimulate Alloreactive T Cell Proliferation
To determine whether PTEC are able to stimulate alloreactiveT cells, nonstimulated or IFN--stimulated PTEC were subsequentlyco-cultured with alloreactive T cells. As shown in Figure 3A,T cell proliferation was clearly detected and was increasedby pretreating the stimulator cells with IFN- compared withuntreated cells. To determine the effect of complement depositionon the activity of PTEC to stimulate T cells, we treated IFN--stimulatedPTEC with NS or HIS before their co-culture with responder Tcells. The results showed that treatment of PTEC with NS significantlyincreased T cell proliferation compared with HIS treatment.Syngeneic control experiments with H-2d stimulator PTEC eliciteda low level of T cell proliferation (Figure 3B). These datademonstrate that PTEC are able to stimulate alloreactive T cellsand that complement activation on PTEC enhances their APC function.
Figure 3. Complement deposition enhances the activity of PTEC to stimulate alloreactive T cell proliferation. (A) PTEC were incubated with IFN- (1000 U/ml) or medium alone for 24 h and then irradiated and assessed as stimulators in a 4-d T cell proliferation assay. (B) PTEC (derived from either H-2b or H-2d mice) were treated with 10% NS or HIS for 30 min, followed by T cell proliferation assay. The incorporation of 3H-thymidine was measured in the last 24 h. P values are for comparisons between IFN- and medium alone in A and for comparisons between NS and HIS treatment in B. ***P < 0.0001; **P < 0.005; ns, no significant difference.
Enhancement of the Alloreactive T Cell Response Is Dependent on PTEC-Bound C3 but not C5 or C6
Complement activation produces a number of biologically activeproducts, including C3b, C5a, and C5b-9, whose presence on thePTEC may participate in the regulation of the immune response.To investigate further which components might contribute toPTEC stimulation of the alloreactive T cell response, we treatedPTEC with different pairs of sera, including C3-suf/C3-def,C5-suf/C5-def, and C6-suft/C6-def sera. The treated PTEC werethen used to stimulate alloreactive T cells. Treatment of PTECwith C3-def serum induced a weaker T cell response comparedwith treatment with C3-suf serum. However, the effect of treatmentof PTEC with either C5-def or C6-def serum on T cell proliferationwas not significantly different from that with their normalwild-type counterparts (Figure 4). These data suggest that theproperty of complement to enhance the alloreactive T cell responseis dependent on C3 but not on C5 or C6.
Figure 4. Enhancement of the alloreactive T cell response is dependent on PTEC-bound C3 but not C5 and C6. IFN--stimulated PTEC were treated with different mouse sera, including C3-suffficient (C3-suf) and C3-def, C5-suf and C5-def, and C6-suf and C6-def sera, and subsequently co-cultured with alloreactive T cells for 96 h. The incorporation of 3H-thymidine was measured in the last 24 h. P value is for comparisons between C3-suf and C3-def serum. **P < 0.005.
Complement C3 Dose-Effect on the Alloreactive T Cell Response
The dose-effect of C3 bound to the stimulator PTEC on the magnitudeof the T cell alloresponse was examined next. These experimentswere performed with C5-def serum to avoid C5a and C5b-9 formationbut to allow deposition of C3. After incubation of PTEC withvarious concentrations of C5-def serum or C5-def HIS, cell-boundC3 was analyzed by FACS and the serum-treated PTEC used in Tcell proliferation assays. The absence of PTEC damage aftertreatment with all serum concentrations was confirmed by theirtrypan blue dye exclusion. FACS analysis showed that the depositionof C3 on the cell surface increased as the concentration ofC5-def serum used (between 2.5 and 50%). This increase was notobserved in cells that were treated with C5-def HIS (Figure 5A).The extent of T cell proliferation was significantly elevatedby treatment of PTEC with freshly prepared C5-def serum butnot the C5-def HIS. There was a significant difference in Tcell proliferation at serum concentrations of 2.5 and 10%, suggestinga dose-dependent effect. However, at higher serum concentrations(25 and 50%, the latter not shown), a plateau was reached, suggestingsaturation of the effect on T cell proliferation (Figure 5B).
Figure 5. Complement dose-effect on the alloreactive T cell response. Single-cell suspensions of IFN--stimulated PTEC were treated with C5-def or HIS C5-def at various concentrations for 30 min. Washed serum-treated cells were subsequently stained for C3d by FACS or co-cultured with alloreactive T cells. (A) FACS analysis. PTEC that were treated with C5-def serum or C5-def HIS are represented by the heavy line, and control cells (no serum added) are represented by the dotted line. (B) T cell proliferation. Serum-treated PTEC were co-cultured with alloreactive T cells for 96 h. The incorporation of 3H-thymidine was measured in the last 24 h. P values are for comparisons between C5-def serum treatment and control (no serum added). *P < 0.05; **P < 0.005; ***P < 0.0003.
IL-2 and IFN- Production by Alloreactive T Cells
Because the T cell proliferation assay detects dividing cellsrather than measures T cell effector function and the majorityof activated T cells produce IL-2 and/or IFN-, we examined IFN-and IL-2 production by ELISA. The co-culture, as before, consistedof PTEC incubated with T cells for up to 96 h. AlloreactiveT cells produced both IL-2 and IFN-, but with different kinetics.The production of IL-2 peaked at 48 h, whereas IFN- peaked at72 h. The effect of NS-treated PTEC was significant at 24 and48 h for IL-2 and at 48, 72, and 96 h for IFN- compared withHIS-treated PTEC (Figure 6). These data correspond with theT cell proliferation results and confirm that T cell respondersare functionally activated as well as being stimulated to dividein a complement-dependent manner, after exposure to allogeneicPTEC.
Figure 6. Detection of IL-2 and IFN- by ELISA. IFN--stimulated PTEC were treated with NS or HIS for 30 min and subsequently co-cultured with T cells. (A and B) Supernatants were collected from co-culture of 2 x 105 PTEC with 2 x 105 T cells at various time points and assayed for IL-2 and IFN- by sandwich ELISA. P values are for comparisons between NS and HIS treatment. *P < 0.05; **P < 0.005; ***P < 0.0003.
CD4+ T Cells Are Responsive to Complement-Mediated PTEC Stimulation of Alloreactive T Cell Response
To evaluate the relative involvement of CD4+ and CD8+ T cellsin the complement-enhanced alloreactive T cell response, weprepared purified alloreactive CD4+ and CD8+ T cells and co-culturedthese cells with NS- or HIS-treated PTEC. Supernatants werecollected at 72 h for ELISA analysis of IFN- (Figure 7). Theproduction of IFN- was much higher in CD4+ T cell preparationcompared with CD8+ T cell preparation, regardless of the serumtreatment of PTEC. For CD4+ T cell preparation, the productionof IFN- was significantly higher in NS-treated PTEC comparedwith either untreated or HIS-treated PTEC. However, for CD8+T cell preparation, we did not observe a difference of IFN-production between NS-treated PTEC and HIS-treated PTEC. Thesedata indicate that the primary influence of tissue-bound complementwas on CD4+ T cells.
Figure 7. Relative involvement of CD4+ and CD8+ T cells in complement-enhanced alloreactive T cell response. IFN--stimulated and NS- or HIS-treated PTEC were co-cultured with purified alloreactive CD4+ or CD8+ T cells. Supernatants were collected from co-culture of PTEC with T cells after 72 h of co-culturing and assayed for IFN- by sandwich ELISA. P values are for comparisons between NS and HIS treatment. **P < 0.005.
Complement-Mediated PTEC Stimulation of Alloreactive T Cell Response Can Be Inhibited with CTLA4-Ig
To investigate further how complement activation and depositionon PTEC influence T cell co-stimulation, we performed a CD80and CD86 blockade experiment. We co-cultured purified alloreactiveCD4+ T cells with NS- or HIS-treated PTEC in the presence ofCTLA4-Ig (5 µg/ml) or control Ig (human IgG1, 5 µg/ml)in the culture medium. Supernatants were collected at 72 h forELISA analysis of IFN- (Figure 8). The production of IFN- wassignificantly decreased in the presence of CTLA4-Ig comparedwith either medium alone or control Ig (human IgG1), where 70%reduction in NS-treated PTEC and 57% reduction in HIS-treatedPTEC were observed. These data indicate that the co-stimulationof B7 plays an important role in PTEC stimulation of alloreactiveT cell response and that the effect of complement on this responseis dependent on the B7 co-stimulation.
Figure 8. Complement-mediated PTEC stimulation of alloreactive T cell response can be inhibited with CTLA4-Ig. IFN--stimulated and NS- or HIS-treated PTEC were co-cultured with purified alloreactive CD4+ T cells in medium alone or in the presence of CTLA4-Ig (5 µg/ml) or control Ig (human IgG1, 5 µg/ml). Supernatants were collected from co-culture of PTEC with T cells after 72 h of co-culturing and assayed for IFN- by sandwich ELISA. P values are for comparisons between CTLA4-Ig treatment and medium alone or control Ig treatment. **P < 0.005.
The present investigation is a sequel to mouse kidney transplantationstudies, in which local tissue expression of C3 was found toincrease the antidonor T cell response and lead to more rapidallograft rejection (8). There are several possible explanationsfor the effect of immunoregulatory C3 in this animal model,which include the nonspecific effects of complement-mediatedinflammation on antigen presentation and a direct effect ofcomplement on T cell stimulation. The present results providea strong case for a direct action of complement on the antigen-presentingproperty of PTEC in vitro. Moreover, the results suggest thatbecause the effect of complement on the tubular cells is independentof C5 and C6, this effect is unrelated to C5b-9 but rather isdue to C3 fragment covalently attached to the epithelial cellsurface. Our results suggest that direct interaction betweentissue-bound C3 fragment and recipient immune cells providesat least one mechanism by which local complement augments theallospecific immune response.
T lymphocytes express receptors that detect a variety of complementactivation products, resulting in T cell activation. For example,CR1/2 expression on primed CD4+ T cells potentially allows Tcells to detect bound C3b or its proteolytic cleavage productsiC3b and C3d, which remain attached to the activating surface(24). Mouse lymphocytes also express complement control proteinssuch as CD46, CD55, and Crry that bind C3 and participate insignal transduction, as well as act as co-factors for the proteolyticcleavage of C3 (25–27). Thus, C3 receptor ligand engagementleading to improved contact with tissue APC, with or withoutensuing signal transduction, might explain the effect of complementin our T cell stimulation assays. The close physical relationshipbetween the tubular deposition of C3 and infiltrating leukocyteswith complement receptor expression, seen in stained histologicsections of rejecting mouse grafts, provides important circumstantialevidence for the interaction suggested by our experiments (8).Moreover, the same tubule cells are a prominent site of C3 synthesisin rejecting grafts (28). Taken together, these data provideevidence for a cooperative effect of C3 on the interaction betweendonor PTEC and recipient T cells, which may in part explainthe effect of complement on the immune response.
Complement receptors that are capable of interacting with attachedC3 fragments have also been reported to occur on professionalAPC, such as macrophages and dendritic cells (29). Previousstudies have shown that the immunostimulatory capacity of APCprimed by tetanus toxoid antigen is increased with C3-opsonizedantigen, thought to be due to more efficient antigen uptakeand processing in the presence of C3 "chaperone" activity (30).Possibly, therefore, an alternative or additional explanationfor the effect of C3 observed in our T cell stimulation studiesis that C3b-opsonized alloantigen, either stripped from or maintainedon the cell surface, is taken up or processed more effectivelyby APC, increasing their capacity as stimulators of T cells.
Indeed, in our B7-CD28 co-stimulation pathway blockade study,CTLA4-Ig was effective at inhibiting the stimulatory effectof complement. Thus, allogeneic T cell stimulation was dependenton B7 pathway signaling as well as complement activation. Becausethe expression of B7 on PTEC is weak, this suggests that themain effect of complement is mediated via another cell type.We suggest that this cell type could be a professional APC inthe co-culture, because such cells normally express higher levelsof B7 and stimulate T cells in a B7-dependent manner. Possibilitiesinclude contaminating donor passenger cells in the PTEC preparationor recipient APC (e.g., B cells or macrophages) in the T cellpreparation. Given that our purified T cell preparation respondsto mitogen stimulation (data not shown), it seems likely thatthe T cell preparation contained recipient APC. Therefore, themechanism of action of complement on T cell stimulation couldinvolve indirect antigen presentation in a manner already describedfor exogenous antigen (30).
In addition to the ability to detect surface bound C3, leukocytesexpress receptors that are capable of interacting with the solublefragments C3a and C5a, leading to cell activation (31,32). Ourexperimental protocol to activate complement on PTEC includedthe removal of unbound soluble complement fragments in the wash,making it unlikely for T cells to encounter C3a and C5a. Therefore,C3aR- and C5aR-mediated stimulation of the T cell response isunlikely to have played a significant role in our study. Itshould be noted, however, that the absence of an effect of C3a-or C5a-mediated T cell activation in our co-culture studiesdoes not preclude an effect in vivo.
Complement activation through C3 leads to cleavage of C5 andsubsequently to the insertion of C5b-9 in the cell membrane.C5b-9-induced cell activation can lead to upregulation of moleculesconcerned with immunologic function and therefore potentiallyenhance the immune response (22,23). In our study, however,we found no evidence of increased expression of MHC, co-stimulatory,or adhesion molecules on PTEC after serum treatment. Moreover,in the absence of C5 or C6, complement had an equal effect onthe T cell stimulatory function of PTEC, indicating that theaction of complement in vitro was independent of C5b-9-mediatedmembrane injury.
Overall, our data provide evidence for complement-mediated regulationof the alloresponse at the level of graft tissue-bound C3 interactingrecipient immune cells. Although the precise details of thisputative mechanism remain to be worked out, our experimentsshow an effect independent of C5a and C5b-9 that is likely toinvolve direct interaction of tissue-bound C3 with complementreceptor-bearing alloreactive T cells that lead to CD4+ T cellstimulation.
Acknowledgments
This work was supported by the Wellcome Trust and the MedicalResearch Council of the United Kingdom.
We thank Dr. Stipo Jurvevic for scientific discussions.
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Received for publication February 24, 2004.
Accepted for publication May 27, 2004.
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354(19):
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Abstract
Introduction
chain, CD40, intracellular adhesion molecule-1 (ICAM-1), and B7.2 (CD86) (Table 1) in 25 µl of reaction buffer (Promega, Southampton, UK). The PCR cycle consisted of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C. Amplified PCR products were visualized after electrophoresis on 1.2% agarose gel that contained ethidium bromide. Glyceraldehyde-3-phosphate dehydrogenase primers, 12.5 pmol each, were also added in every reaction as an internal control. 
for 24 h to maximize expression of MHC and co-stimulatory molecules. Single-cell suspensions were prepared by trypsin/EDTA treatment and then incubated with 10% mouse serum for 30 min, unless otherwise stated. PTEC were then washed three times with endotoxin-free PBS to remove unbound complement proteins and resuspended in T cell culture medium (RPMI-1640 containing 10% heat-inactivated FCS, 50 µM 2-mecaptoehtonal, 100 U/ml penicillin, and 100 µg/ml streptomycin) and irradiated at 1000 RADS. Cell viability was assessed before and after serum treatment, by trypan blue dye exclusion, which showed no increase in the number of dead cells after treatment with serum. Viable cells were >90% of the total. Allogeneic H-2b PTEC monolayers were used in all experiments, except the syngeneic control experiment in which H-2d PTEC were used. 
cpm." T cells alone and PTEC alone controls were also included in each experiment and gave consistently low backgrounds. 
