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The Journal of Immunology, 2003, 170: 3315-3322.
Copyright © 2003 by The American Association of Immunologists

IFN-{gamma} Reverses the Stop Signal Allowing Migration of Antigen-Specific T Cells into Inflammatory Sites1

Szun S. Tay, Ann McCormack, Charlotte Lawson and Marlene L. Rose2

National Heart and Lung Institute, Imperial College School of Medicine, Harefield Hospital, Harefield, Middlesex, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In humans the majority of endothelial cells (EC) constitutively express MHC class II Ags. We know that in vitro ECs can activate CD45RO+ B7-independent CD4+ T cells to proliferate and produce IL-2. The in vivo correlate of this T cell response is not known, and here we have explored whether endothelial expression of MHC class II Ags affects the transendothelial migration of alloreactive CD4+ CD45RO+ B7-independent T cells. Alloreactive CD4+ T cell clones and lines were generated against HLA-DR11, DR13, DR4, and DR1 MHC Ags, and their rates of migration across untreated EC line Eahy.926 (MHC class II negative) or Eahy.926 transfected with CIITA (EahyCIITA) to express DR11 and DR13 were investigated. The migrations of EahyCIITA-specific T cell clones and lines were retarded in a DR-specific manner, and retardation was reversed in the presence of mAb to DR Ag. When investigating the ability of T cells to proliferate in response to EahyCIITA before and after transmigration, migrated cells were still able to proliferate, but the frequency of EahyCIITA-specific cells was much reduced compared with that of nonmigrated cells. The use of fluorescently labeled T cells revealed that specific cells become trapped within the endothelial monolayer. Pretreatment of EahyCIITA with IFN-{gamma} restored the ability of DR11- or DR13-specific T cells to transmigrate and proliferate, thus abrogating DR-specific retardation. We conclude that cognate interaction between T cells and endothelial MHC class II initiates a stop signal possibly similar to an immunological synapse, but this is overcome in an inflammatory milieu.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukocyte recruitment to sites of inflammation is known to involve a number of distinct steps (1). The initial contact of the endothelium by leukocytes, a process known as tethering, is mediated via selectins and causes leukocytes to roll over the endothelial surface (2, 3, 4). These initial contacts allow integrin activation, via chemokines (5) or contact-dependent mechanisms (6, 7). Subsequent firm adhesion and transmigration are mediated via integrin binding to Ig-like molecules, ICAM-1, ICAM-2, or VCAM-1 (8, 9, 10, 11, 12). An additional set of molecular interactions controls the migration of cells through the endothelial layer and basement membrane (13, 14). Most of what we know about these processes relates to the migration of neutrophils, with far less known about T cells.

In contrast, molecules regulating the distinct circulatory patterns of T cells from blood to lymph nodes are well established. Naive T cells migrate from blood to lymph via high endothelial venules within lymph nodes. In contrast, memory and effector T cells are able to migrate into peripheral tissues (15, 16). The differences between these migratory patterns are determined by the different array of cell surface adhesion molecules (17, 18) and chemokine receptors (19, 20) displayed by naive and memory T cells as well as their specific ligands displayed on the endothelium from different sites (21, 22).

This paper addresses the issue of T cell migration into inflammatory sites. In particular, it asks whether the expression of MHC class II Ags on human endothelial cells (EC)3 influences transendothelial migration of T cells that recognize such Ags. Human EC constitutively express MHC class II Ags (23, 24); thus, the immunocytochemistry of human organs reveals all microvascular EC, including venules where egress of lymphocytes occurs, to be MHC class II positive. In vitro experiments have shown that human EC can effectively present Ag to CD4+ CD45RO+ T cells, resulting in T cell activation, as measured by proliferation or cytokine release. Such studies have been performed using autoantigens (25), nominal Ag (26, 27, 28), and alloantigens (29, 30, 31, 32, 33). The costimulatory molecule provided by EC is LFA-3 (34, 35, 36), and such responses are mediated by B7-independent T cells. The in vivo correlates of this response are not known, and it may well be that that the majority of Ag presentation is performed by professional APC in lymphoid tissues (37). It has previously been reported that cognate interaction between CD4+ T cells and endothelial MHC class II stops migration of B7-independent T cells (38). This possibility is counterintuitive, since we would expect effector T cells to be able to enter the inflamed tissue to carry out effector functions. Indeed, in the context of transplantation, we know that allospecific T cells selectively accumulate within the graft (39, 40), and similarly, Ag-specific T cells accumulate in Ag-rich sites in nontransplant diseases (41, 42). Here we have specifically investigated whether presentation of alloantigen on endothelial cells influences the transmigration of B7-independent allospecific CD4+ T cells.

Alloreactive CD4+ T cell clones and lines were raised against HLA-DR11, DR13, DR4, and DR1 MHC class II Ags, and their migrations were tested across the EC line Eahy.926 (43). This cell line has been shown to respond to cytokines and support transendothelial migration of leukocytes in a similar manner as primary endothelial cells (44). Untreated Eahy.926 do not express MHC class II (44), but MHC class II expression can be induced by permanent transfection with the MHC class II trans-activator CIITA (EahyCIITA) or by 4-day treatment of Eahy.926 with IFN-{gamma} (45). We have previously shown that transfection of Eahy.926 with CIITA induced MHC class II expression (DR13, DR11) whereas other molecules, including ICAM-1 and VCAM-1, which may be involved in transmigration, are not up-regulated (45). In this way we have been able to dissect the effects of MHC class II expression on T cell migration from other proinflammatory effects that might be mediated by IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
EC isolation and culture

The EC hybridoma Eahy.926 is a fusion product between HUVEC and the epithelial cell line A549 (43). Eahy.926 cells were serially cultured in DMEM (Sigma-Aldrich, Dorset, U.K.) containing 10% heat-inactivated FCS, 150 U/ml penicillin, 150 U/ml streptomycin, 2 mM L-glutamine, and 0.04% HAT (hypoxanthine, aminopterin, thymine) supplement (all from Invitrogen, Paisley, U.K.) and passaged weekly. Eahy.926 that was permanently transfected with CIITA (EahyCIITA) (45) were cultured as described above, with the addition of 0.1–0.2 mg/ml geneticin (G418 sulfate; Invitrogen). Aortic EC was isolated from donor aorta (with permission from the local ethics committee) and cultured in medium 199 supplemented with 10% heat-inactivated human AB serum (Sigma-Aldrich), 10% FCS, and 10 ng/ml EC growth factor (Roche, Sussex, U.K.) in 1% gelatin (Sigma-Aldrich)-coated flasks. Confluent cells were used between passages 5–12. EC cultures showed typical cobblestone morphology, and purity was further assessed by staining with mAbs against CD31 (EN4; Monosan, Uden, The Netherlands). Where appropriate, EC were treated with 500 U/ml recombinant human IFN-{gamma} (ImmunoKontact, Oxon, U.K.) for 3–4 days to up-regulate MHC class II expression.

PBMC isolation

Peripheral blood was obtained from healthy adult volunteers by venipuncture, and 0.5% (w/v) EDTA was added as an anticoagulant. Blood was diluted 1/1 in PBS and layered over Lymphoprep (Nycomed, West Midlands, U.K.) at a ratio of 1/1 before density centrifugation at 2000 rpm for 20 min at room temperature. PBMC collected from the buffy coat were washed twice and resuspended in RPMI 1640 medium (Invitrogen) supplemented with 10% AB serum.

Purification of CD4+ T cells

Resting CD4+ T cells were purified by negative selection using the RosetteSep Ab cocktail following the manufacturer's instructions (StemCell Technologies, Vancouver, Canada). Briefly, donor peripheral blood was obtained by venipuncture, and 0.5% (w/v) EDTA was added as an anticoagulant. The blood was incubated for 20 min at room temperature with the cocktail of bispecific Ab complexes directed against CD8, CD16, CD19, CD36, CD56, and glyphorin A on RBC. It was then diluted 1/1 in PBS and layered over Lymphoprep for density centrifugation. CD4+ T cells collected from the buffy coat were washed twice and resuspended in RPMI 1640 medium supplemented with 10% AB serum. The purity of the CD4+ T cells was assessed by flow cytometry.

T cell lines and T cell clones

HLA-DR-restricted T cell lines were generated by stimulating purified CD4+ T cells with selected allogeneic PBMC. Stimulator PBMC were gamma-irradiated (30 Gy) before use. The T cell culture and assay medium was RPMI 1640 supplemented with 10% human AB serum. Purified CD4+ T cells were cocultured at a ratio of 1/1 with stimulator PBMC for 7 days, and purified T cells were then separated by density centrifugation in Lymphoprep and restimulated with fresh PBMC. On day 10, 20 U/ml recombinant human IL-2 (Roche) was added. Subsequently, T cell lines were restimulated weekly with fresh PBMC, medium, and 20 U/ml rIL-2. This process was repeated (usually three or four times) until T cell lines were obtained that showed at least a 5 times greater proliferative response to stimulator PBMC than third-party PBMC. The T cells were then ready for use. T cell clones were generated by limiting dilution of CD4+ T cell lines that were at least 2 wk old. T cells were dispensed at 20 µl/well into Terasaki plates (Greiner Labortechnik, Tyne and Wear, U.K.), such that each well contained <1.0 T cell, 1 x 104 stimulator PBMC, and 20 U/ml rIL-2 in RPMI 1640 supplemented with 10% AB serum. After incubation for 10 days at 37°C in 5% CO2, plates were screened for clonally expanded T cells. Established clones were expanded by weekly restimulation with PBMC and 20 U/ml IL-2, transferring from 96-well plates to 48-well plates (Nunc, Kamstrup, Denmark) when appropriate. For use in experiments, T cells were purified by density centrifugation on a Lymphoprep gradient and washed three times before use to exclude any contamination from accessory cells. Where appropriate, T cells were labeled with the intracellular dye CFSE at a final concentration of 0.05 µM (Molecular Probes, Eugene, OR). Cells were labeled in the dark in 2 ml of PBS for 5 min at room temperature, and the reaction was quenched with 2 ml of FCS for 10 min at room temperature. Excess CFSE dye was removed by three washes in RPMI 1640 supplemented with 2% FCS.

HLA typing of PBMC or EC

HLA typing of donors of PBMC, Eahy.926, and EahyCIITA were performed by the Tissue Typing Department, Harefield Hospital, using the PCR-single-strand polymorphism method (46). DNA was phenol-extracted from blood collected from donors or from EC before use in PCR.

T cell proliferation assays.

Responder T cells (1 x 104) were cocultured in 96-well U-bottom (96U) or 96-well flat-bottom (96F) plates (Nunc) with 1 x 105 PBMC or 2.5 x 104 EC stimulators in a final volume of 200 µl. Allogeneic PBMCs were gamma-irradiated (30 Gy) before use. Aortic EC were gamma-irradiated (30 Gy) and seeded onto gelatin-coated 96F plates 24 h before use. Eahy.926 or EahyCIITA were mitomycin C-treated (60 µg/ml; Sigma-Aldrich) for 25 min at 37°C and seeded onto 96F plates 24 h before use. Coculture plates were incubated in at 37°C in 5% CO2 for 2–7 days, and proliferation was assessed at by addition of 1 µCi of [3H]TdR (5 Ci/mmol; Amersham Pharmacia Biotech, Little Chalfont, U.K.) 18 h before harvesting onto glass-fiber filters (Wallac; EG&G, Salem, MA). [3H]TdR incorporation assessed by liquid scintillation counting (Wallac 1450 Microbeta counter; EG&G). All experiments were performed in triplicate, and results were expressed as cpm ± SD. Wells containing responder or stimulator cell populations alone were included in all experiments. Wells containing stimulator cells alone gave <200 cpm, which was subtracted from all experimental values. Cell concentrations and mAbs or chemicals added are detailed in appropriate figure legends.

Flow cytometry and Abs

Cells (1 x 105) were resuspended in 100 µl of PBS and stained with unconjugated primary Ab for 20 min on ice. mAbs used were either purified from hybridomas (anti-DR{alpha} (L243), anti-DR{beta} (DA6.231), anti-ICAM-1 (6.5B5), anti-VCAM-1 (1.4C3), anti-LFA-1 (TS1/22), anti-LFA-3 (TS2/9)) or were commercially available (EN4 (Monosan, The Netherlands); CTLA4-Ig (Repligen, Waltham, MA); anti-CD25 (Coulter, Luton, U.K.)). Excess primary Ab was removed in two washes before addition of FITC-conjugated rabbit anti-mouse IgG secondary Ab at 1/40 (F0313; Dakopatts, High Wycombe, U.K.) for 20 min on ice. Unbound Ab was removed in two washes, and cells were fixed in 400 µl of 0.5% formaldehyde (BDH, Essex, U.K.) before 5000 events were acquired on an EPICS XL flow cytometer (Coulter). Direct staining of T cells were performed using mAb directly conjugated to FITC against the CD3, CD4, and CD8 Ags (BD Biosciences, Mountain View, CA) with an appropriate isotype control. Results were expressed as the percentage of cells staining positively above the control level, which was set at 2% using cells with no primary Ab staining or stained with isotype control.

In vitro migration assay

The in vitro transmigration assay has been previously described (47). EC (1 x 105) were grown on polycarbonate filters of 3 µm pore size (Falcon; Marathon Laboratories, London, U.K.) precoated with 100 µl of recombinant human fibronectin (50 µg/ml; Sigma-Aldrich) and left overnight to form a monolayer. Monolayers were rinsed once in assay medium and transferred to wells of 24-well plates containing 600 µl of fresh prewarmed RPMI 1640 medium supplemented with 10% AB serum. T cells (1 x 105) in 100 µl of RPMI 1640 medium supplemented with 10% AB serum were added to the upper chamber and allowed to migrate to the lower chamber for selected times. Control wells contained an equivalent number of lymphocytes in 600 µl of medium. The numbers of migrated cells in the lower chamber were counted using the EPICS XL flow cytometer (Coulter) set to acquire data for 1 min at 60 µl/min. All wells were set up in triplicate. The percentage of cells migrated was calculated by dividing the number of migrated cells by the total number of cells added to control wells, and results were expressed as the percent migration ± SD.

Limiting dilution analysis

Responder T cells were diluted into four to seven serial 2-fold dilutions, and 24 replicate wells of each dilution were plated into 96U plates containing 2.5 x 104 mitomycin C-treated stimulator EahyCIITA. After 24 h of incubation, supernatant was removed and frozen once at -40°C to kill carryover cells. CTLL-2 (5 x 103/well) in 10 µl of medium were added to thawed supernatants. Eight hours later 1 µCi of [3H]TdR was added to each well, and following a further 18-h incubation, cells were harvested onto glass-fiber filters. [3H]TdR incorporation into CTLL-2 was assessed by liquid scintillation counting (Wallac 1450 Microbeta counter). Background control wells contained supernatants from EahyCIITA stimulator cells pulsed with CTLL-2.

Statistics

Data are presented as the mean ± SD unless otherwise stated. Statistical significance was assessed using unpaired Student's t test. A value of p < 0.05 was considered significant. Analysis was performed using SPSS software (SPSS, Chicago, IL). For limiting dilution analysis, the frequencies of responding cells were determined using maximum likelihood estimation (GLIM software; NAG, Oxford, U.K.). The proportion of negative wells in each sample of responder cells is linearly related to the frequency of responder cells according to the Poisson distribution, as -loge X Pneg = fX, where Pneg is the proportion of negative wells, f is the frequency of responder cells, and X is the number of responders per well. The 95% confidence limits of the frequencies and {chi}2 estimates of probability were calculated. Frequencies were regarded as significantly different if their 95% confidence limits did not overlap.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Surface and functional phenotypes of CD4+ T cell lines and clones

Alloreactive T cell lines and clones against specific HLA-DR haplotypes assessed by flow cytometry revealed a purity of >98.5% for CD3+ CD4+ and CD45RO+ T cells. All T cell lines were >99.8% positive for LFA-1. Different T cell lines expressed CD25 to different degrees (ranging from 68–95%). HLA-DR was similarly expressed to various degrees (ranging from 77–97% positive). Taken together these results represent the phenotype of activated memory T cells. The proliferative response of T cells to PBMC or EC stimulators of defined HLA-DR haplotypes was determined. T cells raised against DR11 PBMC proliferated in response to DR11-expressing PBMC or EahyCIITA (DR11, DR13), but not third-party stimulators (Table I). The DR-dependent nature was confirmed by inhibition of the response to both PBMC and EC by anti-DR{beta} mAb (DA6.231). CTLA4Ig inhibited the PBMC stimulated response, but had no inhibitory effect on the EC-stimulated response, while Abs against LFA-3 inhibited the EC-stimulated response, confirming the EC response to be mediated by B7-independent T cells. The T cell clones R3 and A1 are B7-independent and restricted by DR13 and DR0404, respectively; they proliferate upon stimulation by EC expressing appropriate HLA-DR molecules, and this response is inhibited by anti-DR{beta} mAb (DA6.231; Table I).


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  		Table I. Proliferative response of T cell lines and T cell clones to PBMC and EahyCIITAa

 
Cognate recognition retards transmigration of T cell clones

Migration of the T cell clones R3 (EahyCIITA-specific) and A1 (third-party) across EahyCIITA was determined over 8 h. The number of migrated R3 clones was significantly less (p < 0.01) compared with A1 at all time points measured (Fig. 1, top panel). Clone A1 was also migrated across aortic EC, untreated or pretreated with IFN-{gamma} to up-regulate MHC class II expression. A1 is specific for DR0404 expressing aortic EC, but not for DR0401 expressing aortic EC (Table I). Migration of A1 across IFN-{gamma}-treated allogeneic DR0404 EC was significantly reduced compared with that across untreated EC (Fig. 1, middle panel) at 4 h (p = 0.036) and 8 h (p = 0.0024). In contrast, there was no significant difference in the rate of migration across untreated or IFN-{gamma}-treated DR0401 (third-party) EC expressing DR0401 (Fig. 1, bottom panel). At 4 h, retardation of A1 migration on DR0404-expressing EC was completely reversed in the presence of DA6.231, while DA6.231 had no significant effect on A1 migrating across DR0401-expressing EC (Table II). These results demonstrate that cognate recognition of allogeneic MHC class II on EC retarded transmigration of CD4+ T cell clones.



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  		FIGURE 1. Migration of T cell clones. Top panel, Migration of A1 (DR4 restricted) or R3 (DR13 restricted) T cell clones across DR13 expressing EahyCIITA (top panel). Middle and bottom panels, Migration of A1 across DR0404 aortic EC (middle panel) or across DR0401 aortic EC (bottom panel) with or without pretreatment with IFN-{gamma}. Confluent EC monolayers were grown on tissue culture inserts and transferred to 600 µl of fresh medium, forming upper and lower chambers. T cells (1 x 105) were seeded into the upper chamber or into medium-only controls. At selected time points, migrated cells were harvested and counted on a flow cytometer; the stop volume was set at 60 µl. During data acquisition, lymphocyte gates were constructed using side and forward scatter profiles. The results are expressed as the mean percentage of migrated cells ± SD and are representative of three experiments.

 

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  		Table II. Migration of DR0404-specific T cell clone (A1) across aortic ECa

 
Cognate recognition retarded transendothelial migration of T cell lines

T cell clones are unphysiological; therefore, these results were confirmed using T cell lines in which the frequencies of alloreactive cells more resembles those found in vivo. T cell lines that proliferated to DR11 or DR13 on EahyCIITA or third-party DR4-restricted T cell lines were used (Table I). The frequency of EahyCIITA-specific T cells in DR11- or DR13-restricted T cell lines ranged from 1/500 to 1/3000. As shown in Fig. 2, top panel, cognate recognition of MHC class II on EahyCIITA resulted in retardation of migration across EahyCIITA compared with Eahy.926 at 12 h (p = 0.03) and 24 h (p = 0.001). In contrast, migration of a DR1-restricted (third-party) T cell line remained unchanged across Eahy.926 or EahyCIITA at both time points (Fig. 2, bottom panel). Interestingly, retardation of migration due to cognate recognition by T cell lines was not observed at earlier (<8 h) time points.



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  		FIGURE 2. The migration of DR13-restricted (top panel) or DR1-restricted (bottom panel) T cell line across untreated MHC class II-negative Eahy.926 or MHC class II-expressing (DR11, DR13) EahyCIITA is shown. Confluent EC monolayers were grown on tissue culture inserts and transferred to 600 µl of fresh medium, forming upper and lower chambers. T cells (1 x 105) were seeded onto the upper chamber or into medium-only controls. At selected time points, migrated cells were harvested and counted by flow cytometry, with the stop volume set at 60 µl. During data acquisition, lymphocyte gates were constructed using side and forward scatter profiles. The results are expressed as the mean percentage of migrated cells ± SD and are representative of four experiments.

 
Reduced proliferation of migrated cells was due to lower frequencies of EahyCIITA-specific cells

It has been suggested that migration and proliferation are mutually exclusive properties (38). Therefore, in addition to measuring the rate of migration, we measured the ability of migrated cells to proliferate to EahyCIITA. T cell lines that had migrated through EahyCIITA showed a significantly reduced proliferation response (<50%) compared with control T cell lines that had migrated through Eahy.926 (p < 0.01 in 16 experiments; Fig. 3). There was no reduction in the proliferative responses of T cells that had been cocultured with EahyCIITA (T cells retrieved from upper chambers) compared with control T cells cocultured with Eahy.926 (Fig. 3). Reduced proliferation is therefore not a consequence of contact with EahyCIITA, but is specific to transmigrated T cell populations.



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  		FIGURE 3. Proliferation of migrated and nonmigrated DR13-restricted T cells to EahyCIITA. Twenty-four hours after migrating through Eahy.926 (A) or EahyCIITA (B) T cells were recovered from the lower chamber and restimulated with EahyCIITA. At the same time, T cells from the upper chambers that had been in coculture with Eahy.926 (C) or EahyCIITA (D) were recovered for restimulation with EahyCIITA. For restimulation, 1 x 104 T cells were cocultured with EahyCIITA for 24 h before 1 µCi [3H]TdR was added, and 18 h later cells were harvested for liquid scintillation counting. All experiments were performed in triplicate, and the results are expressed as the mean cpm ± SD. Wells containing EahyCIITA stimulators alone produced <600 cpm, which is subtracted from all experimental values. *, Significant reduction of cpm in cells that had migrated across EahyCIITA (B) compared with cells that had migrated across Eahy.926 (A; p < 0.01). The data are representative of 16 experiments.

 
We confirmed that the reduced proliferation response was due to a reduced frequency of EahyCIITA-specific T cells within the migrated population using limiting dilution analysis, where a CTLL-2 bioassay was used to measure IL-2 secretion by responding T cells. The frequency of EahyCIITA-specific T cells (1/983, before transmigration) was reduced 7-fold (1/7011) after transmigration though EahyCIITA, but not through Eahy.926 (1/857). However, this was not accompanied by a reciprocal increase in the frequency of EahyCIITA-specific T cells in the upper chamber (Table III), which remained at 1/1114.


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  		Table III. Frequency of EahyCIITA-specific T cells before and after transmigrationa

 
Retardation in migration was due to retention of T cells on or within the EC monolayer

The fact that EahyCIITA-specific T cells are not increased in number within the upper chamber suggests that such specific cells have either died or remain trapped on or within the endothelial layer. To distinguish between these possibilities, DR11- or DR13-restricted T cell lines were fluorescently labeled with CFSE before migration across Eahy.926 or EahyCIITA for 24 h. Migrated cells from the lower chamber as well as nonadherent cells from the upper chambers were harvested by gentle washing. Adherent T cells on the insert were released by trypsin digestion. Fluorescent T cells were gated and counted on a flow cytometer. The distribution of T cells after 24 h was calculated. The decrease in the percentage of migrated cells through EahyCIITA (27.2 ± 0.9%) compared with Eahy.926 (39.2 ± 1.8%) was accompanied by a concurrent increase in the adherent population within EahyCIITA (12.3 ± 1.0%) compared with Eahy.926 (4.4 ± 0.2%). There were no significant differences in the percentage of nonadherent cells on Eahy.926 or EahyCIITA (n = 3). This demonstrates that the nontransmigrated cells have not died and disappeared from the system; they simply cannot negotiate their way through the endothelial layer.

Molecules involved in tight adhesion of EahyCIITA-specific T cells to the EC monolayer

We used the proliferation assay, in which migrated T cells were restimulated with EahyCIITA, to assess the effect of blocking Abs on the transmigration of DR11- or DR13-restricted T cell lines. mAbs to HLA-DR{beta}, LFA-3, ICAM-1, and CD14 (the isotype control) were added to EC for 30 min before the transmigration assay (Fig. 4). The results show that the reduced proliferation after migration across EahyCIITA (p < 0.05) was completely abrogated by the presence of anti-HLA-DR{beta} (p < 0.05), while the isotype control has no effect. This confirmed that the reduced proliferation was due to cognate recognition of HLA-DR molecules expressed by EahyCIITA. Abs against LFA-3 did not reverse the reduction in proliferation of migrated cells, although it could directly inhibit T cell activation by EahyCIITA (Table I), suggesting that the stop signal was not a consequence of T cell activation. Abs against ICAM-1 did not reverse the stop signal either, suggesting that the stop signal was not mediated by increased avidity of LFA-1 interacting with its EC ligand, ICAM-1.



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  		FIGURE 4. Effect of blocking Abs on transmigration of DR13-restricted T cells. Twenty-four hours after migrating through Eahy.926 (A) or EahyCIITA (B) in the presence of saturating concentrations of DA6.231 (C), anti-LFA-3 (D), anti-ICAM-1 (E), or anti-CD14 (F) Abs, migrated T cells were recovered from the lower chamber and restimulated with EahyCIITA. For restimulation, 1 x 104 T cells were cocultured with EahyCIITA for 24 h, 1 µCi of [3H]TdR was added, and 18 h later cells were harvested for liquid scintillation counting. All experiments were performed in triplicate, and the results are expressed as the mean cpm ± SD. Wells containing EahyCIITA stimulators alone produced <600 cpm, which was subtracted from all experimental values. *, Significant reduction of proliferation of T cells that migrated through EahyCIITA (B) compared with Eahy.926 (A; p < 0.01); **, significant increase in cpm after migration through EaHyCIITA in the presence of DA6.231 (C) compared with EahyCIITA (B; p < 0.01). The data are representative of three experiments.

 
Effect of IFN-{gamma} pretreatment of EahyCIITA on transmigration

In view of the fact that T cell migration into nonlymphoid tissue is likely to be low in the absence of an inflammatory response, we investigated the effect of cytokine pretreatment of EahyCIITA on the transmigration of DR11- or DR13-restricted T cell lines. EahyCIITA were pretreated with 500 U/ml IFN-{gamma} for 3 or 4 days before use in the migration assay. Pretreatment of EahyCIITA with IFN-{gamma} restored the migration of EahyCIITA-specific T cells; the reduction in proliferative response that was observed after migration through EahyCIITA compared with Eahy.926 (p = 0.01) was abrogated, and the migrated T cells could subsequently proliferate to in response toEahyCIITA (p = 0.01; Fig. 5).



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  		FIGURE 5. Effect of IFN-{gamma} pretreatment on transmigration of DR13-restricted T cells. Twenty-four hours after migrating through Eahy (A), EahyCIITA (B), or EahyCIITA pretreated with IFN-{gamma} (C) cells were recovered from the lower chamber and restimulated with EahyCIITA. For restimulation, 1 x 104 T cells were cocultured with EahyCIITA for 24 h, 1 µCi of [3H]TdR was added, and at 18 h cells were harvested for liquid scintillation counting. All experiments were performed in triplicate, and the results are expressed as the mean cpm ± SD. Wells containing EahyCIITA stimulators alone produced <600 cpm, which was subtracted from all experimental values. *, Significant reduction of proliferation of T cells that migrated through EahyCIITA (B) compared with Eahy.926 (A; p < 0.01); **, significant increase in cpm after migration through IFN-{gamma}-treated EahyCIITA (C) compared with EahyCIITA (B; p < 0.01). The data are representative of nine experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This paper has addressed the issue of whether endothelial expression of MHC class II molecules influenced the transmigration of allospecific CD4+ CD45RO+ B7-independent T cells. The question is particularly pertinent in the context of human allotransplantation, where there is a large endothelial mass (all EC in a grafted organ) expressing alloantigens of donor origin. In our hands and those of the majority of workers, CD4+ CD45RO+ T cells respond to MHC class II-positive allogeneic endothelial cells in vitro by proliferation and IL-2 release in a B7-independent manner (31, 35). The CD4+ CD45RO+ phenotype broadly corresponds to effector memory T cells, cells that migrate into inflammatory tissues in vivo (48); hence, the question we asked was how the ability to become activated by EC affected their transmigration capabilities.

The first experiments investigating the migration of HLA-DR-restricted, B7-independent alloreactive T cell clones demonstrated that cognate interaction retarded transmigration of T cell clones up to 12 h. We believed that it was important to reproduce the data in T cell lines, arguing that T cell clones might form homotypic aggregates upon TCR stimulation, preventing their migration through the EC. Furthermore, in T cell lines, the frequencies of EahyCIITA-specific clones encountered (1/500–1/3000) would be more like those found during an immune response in vivo. A similar phenomenon was observed using T cell lines, i.e., migration was retarded by cognate interaction with endothelial MHC class II.

The disadvantages of using T cell lines is that they are a mixed population of B7-dependent and B7-independent T cells. To focus on the migratory properties of the B7-independent population (which proliferate in response to EahyCIITA stimulation), we measured the ability of migrated T cells to proliferate to EahyCIITA. The reduced ability of migrated T cells to proliferate to EahyCIITA could reflect the relative infrequency of EahyCIITA-specific T cells in the migrated population (as shown in Figs. 1–3). On the other hand, it might suggest that T cells have become anergized by transmigration through MHC class II-expressing EC, as has recently been suggested (49). The present study argues against anergy induction; we have shown that functional T cells with IL-2Rs were present in the migrated population, as measured by flow cytometry and their ability to proliferate to rIL-2 (data not shown). In addition, conducting the 24-h migration assay in the presence of rIL-2 (20 U/ml) (50) does not alter the original observations (reduced proliferative responses of migrated T cells; data not shown). In addition, the fact that cells from the upper chamber that had been in contact with EC for 24 h could still proliferate suggests that anergy as a result of EC contact had not occurred. This confirms a previous study showing that contact of CD45RO+ T cells with EC does not induce anergy (51). The frequency experiments (Table III) confirmed that the reduction in T cell proliferation was due to a reduced frequency of EahyCIITA-specific T cells within the migrated population. We have to date confirmed the observations by Marelli-Berg et al. (38), who used T cell clones to show that B7-independent specific T cells failed to migrate across Ag pulsed endothelial cells expressing the same peptides.

We next went on to ask what happened to the cells that do not transmigrate. The 7-fold decrease in the frequency of alloreactive T cells that transmigrated was not reflected by a reciprocal increase in such cells in the upper chamber. This raises the possibility that cognate recognition caused activation-induced cell death of EahyCIITA-specific T cells that have been serially cultured in vitro for 3–4 wk. However, there was no significant difference in the number of dead T cells retrieved from upper chambers of Eahy.926 compared with EahyCIITA, as determined by acridine-orange/ethidium bromide staining (data not shown).

Using fluorescently labeled T cells we showed that reduction of transmigration was in fact due to retention of allospecific T cells within the endothelial monolayer. At this point we have not determined whether T cells are firmly adhered onto the apical surface of the endothelial layer or trapped between the endothelial monolayer and the basement membrane, but we will use electron microscopy to resolve this issue. There are several explanations for retention of T cells. Trapping of EahyCIITA-specific T cells could be a direct consequence of TCR-MHC interactions, as described in the formation of an immunological synapse with professional APC, whereby EC is behaving like an APC (52). However, it is surprising that synapse formation could be so long-lived (>24 h); synapses between dendritic cells and Ag-specific T cells have been analyzed over minutes to 4 h. As a consequence of cognate recognition, T cell activation and adhesion strengthening could be delivered via other molecules, leading to retention of allospecific T cells. For instance, triggering of LFA-1 to high avidity conformation by TCR signaling has been described (53). We investigated these possibilities by including blocking mAbs in the 24-h migration assay. Abs to LFA-3 and ICAM-1 had no effect on the stop signal generated, arguing against activation-induced integrin avidity changes due to cognate recognition and also against adhesion strengthening via LFA-1/ICAM-1 interactions. However, this does not rule out the involvement of other integrin-mediated strengthening signals, such as interactions between LFA-1 and junctional adhesion moelcule-1 (54, 55) or between very late Ag-4 and fibronectin (12).

We reasoned that in vivo, especially during an inflammatory response, ECs will be bathed in inflammatory cytokines and chemokines. We therefore pretreated ECs with IFN-{gamma} to determine whether this would alter the migration of EahyCIITA-specific T cells. It was clear that in nine of 12 experiments, IFN-{gamma} pretreatment restored the migration of EahyCIITA-specific T cells that were trapped within the EC monolayer. Currently it is not known how IFN-{gamma} works. One possibility is that it up-regulates chemokine production by EahyCIITA (56, 57). Bromley et al. (58) suggested that some chemokines can act to overcome the stop signal mediated by TCR recognition of MHC Ags. It may be that IFN-{gamma} up-regulated the expression of dominant chemokines, thus allowing the migration of Ag-specific T cells. Preliminary experiments using RT-PCR have shown lower levels of MCP-2 in EahyCIITA than Eahy.926 (data not shown). Other possibilities are that IFN-{gamma} could induce reorganization or redistribution of EC adhesion molecules (59) and junctional adhesion molecules (60). These possibilities are currently being explored. It is also possible that other inflammatory cytokines would allow migration of such "stopped" EahyCIITA-specific T cells.

This study therefore extends that of Marelli-Berg (38) in several aspects; it confirms that migration of B7-independent T cells is retarded by cognate recognition, but suggest that the phenomenon is temporary and can be overcome by an inflammatory milieu. Moreover, migration and proliferation are not mutually exclusive, suggesting that once effector cells have reached an inflammatory site, they can continue to expand in situ and carry out effector functions. The mechanisms that inhibit the migration of EahyCIITA-specific T cells across MHC class II-expressing EC are not akin to T cell proliferation, since the stop signal was abrogated by mAb to HLA-DR, but not by mAb to LFA-3. Instead, it could involve intracellular signaling from the TCR, resulting in lack of T cell motility. Alternatively, it could be mediated by adhesive interactions of the TCR and/or CD4 to the MHC class II molecules. The in vivo relevance of Ag-specific retardation of T cell transmigration is unclear at present. Time-lapse intravital videomicroscopy of murine leukocytes migrating into cremaster muscle has demonstrated that transmigration can occur rapidly (within 1 h) (61), whereas the time course of the present experiments was up to 24 h. Looking at the transmigration of Ag-specific T cells under flow conditions would be more physiological, but ultimately it will be necessary to use intravital microscopy and transgenic mice to investigate the migration of Ag-specific T cells in response to relevant Ag. A thorough understanding of the mechanisms that regulate Ag-specific T cell migration may lead to therapeutic strategies for preventing the migration of T cells into chronic inflammatory lesions.


   Acknowledgments
 
We thank the Tissue Typing Department of Harefield Hospital for their assistance, and the people who kindly donated their blood.


   Footnotes
 
1 This work was supported by Studentship FS/99035 from the British Heart Foundation (to S.S.T.). Back

2 Address correspondence and reprint requests to Prof. Marlene L. Rose, National Heart and Lung Institute, Heart Science Center, Harefield Hospital, Harefield, Middlesex, U.K. UB9 6JH. E-mail address: marlene.rose{at}ic.ac.uk Back

3 Abbreviation used in this paper: EC, endothelial cell. Back

Received for publication October 23, 2002. Accepted for publication January 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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