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The Journal of Immunology, 2000, 165: 734-742.
Copyright © 2000 by The American Association of Immunologists

An Epithelial Cell Line That Can Stimulate Alloproliferation of Resting CD4+ T Cells, But Not After IFN-{gamma} Stimulation1

Charlotte Lawson*, Ann M. McCormack*, David Moyes*, Sheng Yun{dagger}, John W. Fabre{dagger}, Magdi Yacoub* and Marlene L. Rose2,*

* Transplant Immunology Group, Imperial College School of Medicine, National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Harefield, Middlesex, United Kingdom; and {dagger} Department of Clinical Sciences, Institute of Liver Studies, Kings College Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has previously been shown that IFN-{gamma}-induced up-regulation of HLA class II on the surface of epithelial cells is not sufficient to induce proliferation of allospecific CD4+ T cells in vitro. To further investigate this phenomenon, a human epithelial bladder carcinoma, T24, was induced to constitutively express HLA class II without IFN-{gamma} stimulation, by permanent transfection with the full-length class II transactivator (CIITA) gene. Proliferation of allospecific T cells to transfected and wild-type cells with and without prior activation with saturating levels of IFN-{gamma} for 4 days was examined. IFN-{gamma}-activated T24 did not induce any response from CD4+ T cells. However, T24.CIITA induced significant levels of alloproliferation, which could be abrogated by pretreatment of T24.CIITA with a mAb to LFA-3. Prestimulation of T24.CIITA with saturating levels of IFN-{gamma} for 4 days also prevented allospecific CD4+ T cell proliferation. These findings suggest that epithelial cells may be intrinsically able to process and present alloantigen and provide adequate costimulation. We propose that IFN-{gamma} has a secondary, as yet unidentified, effect that acts to negatively regulate this response, at least in some epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The role of MHC class II-expressing parenchymal cells as stimulators or suppressers of the alloimmune response remains controversial in the field of allotransplantation. It is known that MHC class II is widely expressed within the vasculature in human, but not rodent, organs, particularly on microvascular, although also to a lesser extent, on large vessel endothelial cells (1, 2, 3). A number of groups have shown that IFN-{gamma}-treated large vessel endothelial cells cause alloproliferation of resting CD4+ T cells in vitro, allowing them to postulate that constitutively HLA class II-positive endothelial cells cause allostimulation of resting T cells in vivo (4, 5, 6). Similarly, human epithelial cells associated with allografts have been shown to be constitutively positive for HLA-DR in vivo (7, 8, 9, 10) and to up-regulate HLA-DR after IFN-{gamma} stimulation in vitro (11, 12, 13). However, IFN-{gamma}-stimulated epithelial cells do not induce proliferation of allospecific CD4+ T lymphocytes, and may even induce a state of anergy or nonresponsiveness in vitro (14). This suggests that epithelial cells may act to suppress the immune response after IFN-{gamma} stimulation, while endothelial cells seem to have an opposite function.

It is known that T cells require two signals to become activated: one is the occupancy of the TCR; the second is activation of accessory molecules on T cells (15). Although allostimulation by dendritic cells depends on B7.1/B7.2 (CD80/CD86) interacting with CD28 on the responding T cells, endothelial cells lack B7.1/B7.2 expression (16). Thus, in contrast to professional APC, allostimulation of resting T cells by endothelial cells depends on interactions between LFA-3 on the endothelial cells and TCR CD2 (17). However, in view of the fact that epithelial cells also express LFA-3 (7, 11), this does not explain the contrasting abilities of endothelial cells and epithelial cells to cause T cell activation.

To further dissect the responses of allospecific resting peripheral CD4+ T cells to nonprofessional APC, we have permanently transfected two cell lines with a construct containing the full-length cDNA for class II transactivator (CIITA),3 previously described as a regulator of the expression of HLA class II and related molecules, both in vivo and in vitro (18, 19). In this way we have induced expression of HLA class II without the pleiotropic effects of IFN-{gamma}. Two cell lines have been used, which differ in their ability to up-regulate HLA-DR and support allospecific proliferation of CD4+ T cells in vitro. EaHy.926, a hybridoma of primary HUVEC and a human airway epithelial cell line (20), has been previously shown to behave similarly to primary endothelial cells in vitro, expressing high levels of the endothelial specific marker, CD31, as well as retaining the ability to up-regulate HLA-DR and cause proliferation of allospecific T cells in vitro (16, 21). In contrast, the second cell line we have transfected is a bladder epithelial carcinoma T24 (previously misidentified as a spontaneously transformed endothelial cell line ECV.304) (22). This cell line does not express HLA-class II determinants after IFN-{gamma} stimulation for several days, while responding with up-regulation of other molecules influenced by IFN-{gamma} treatment (21). It is CD31 negative and does not cause allospecific T cell proliferation in vitro. We have examined the effect of these cell lines on alloproliferation of resting CD4+ T cells both before and after IFN-{gamma} stimulation of transfected and wild-type cells. Our results suggest that the inability of epithelial cells to stimulate proliferation of allospecific T cells may be due to negative signals induced by IFN-{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

EaHy.926 (20) were maintained in DMEM (Sigma, Poole, U.K.) with 2 mM L-glutamine (Life Technologies, Paisley, U.K.), 150 U/ml penicillin/streptomycin (Life Technologies), 10% FCS (Sigma), and hypoxanthine/aminopterin/thymidine (Flow Laboratories, McLean, VA). T24 (American Type Culture Collection, Manassas, VA) were maintained in DMEM with 2 mM L-glutamine, 150 U/ml penicillin/streptomycin, and 10% FCS. Transfected cells were maintained in full growth medium with the addition of 100 µg/ml G418 (Life Technologies).

Transfection

Cells were seeded at 50% confluency on six-well plates and allowed to adhere. The following day, the medium was replaced, and after 4 h cells were transfected with a construct containing the full-length CIITA cDNA (a kind gift of Dr. V. Steimle, University of Geneva Medical School, Geneva, Switzerland) cloned into pcDNA3 (23). Briefly, 1 µg plasmid DNA was added to 16 µg polybrene in 2 ml full medium, and the mix was allowed to stand at room temperature for 5 min. Medium was removed from the cells and replaced with the polybrene:DNA mix, and cells were incubated for 48 h at 37°C. The medium was removed and replaced with selection medium. After ~2 wk, growing colonies of cells were trypsinized from dishes and pooled before two rounds of magnetic separation using Dynabeads coated with mouse anti-human DR Ab (Dynal, Wirral, U.K.). Cells that bound to the Dynabeads were replated and allowed to recover. Once confluent, they were reselected as above and maintained in G418 selection medium.

Quantitative flow cytometry

Transfected and wild-type T24 and EaHy.926 cells were plated onto 25-cm2 tissue culture flasks and allowed to become confluent. The medium was changed and replaced with fresh full medium without G418, with or without the addition of 500 U/ml IFN-{gamma} (R&D Systems, Abingdon, U.K.). Cells were incubated for 4 days at 37°C. They were trypsinized and stained for the presence of surface markers including HLA-DR, HLA-DP, and HLA-DQ (clone L243, B7/21, SK10, respectively; all from Becton Dickinson, Oxford, U.K.); HLA class I (clone W6/32; ATCC); CD40 (R&D Systems); ICAM-1 (24); LFA-3 (HB205; ATCC); and for positive staining with EN-4 (endothelial specific Ab; Monosan, Bradsure Biologicals, Loughborough, U.K.) and binding of CTLA-4-Ig (25), followed by goat anti-mouse Ig F(ab')2 FITC (F0479; Dako, Cambridge, U.K.). Cells were analyzed using a Coulter (Palo Alto, CA) EPICS flow cytometer. In conjunction with staining of cells, QIFI beads (Dako) were stained with goat anti-mouse Ig F(ab')2 FITC and then analyzed, to accurately quantitate the levels of binding of Abs to the cell surface. Relative binding was calculated using an algorithm, based on the fluorescence intensity of the FITC-labeled beads, according to the manufacturer’s instructions. Relative binding was expressed as surface Ag-binding capacity (SABC).

Reverse-transcription PCR

Transfected and wild-type T24 and EaHy.926 cells were plated onto 24-well plates and allowed to become confluent. The medium was changed and replaced with fresh full medium without G418, with or without the addition of 500 U/ml IFN-{gamma}. Cells were incubated for 4 days at 37°C. The medium was removed from each well, and total RNA was extracted from the cells using RNeasy extraction columns (Qiagen, Crawley, U.K.) according to the manufacturer’s instructions, or Purescript total RNA isolation kits (Flowgen, Stafford, U.K.) according to the manufacturer’s instructions.

cDNA synthesis was conducted using 10 U AMV reverse transcriptase (Promega, Southampton, U.K.) and 200 ng oligo(dT) primer together with the manufacturer’s buffer, 20 nmol dNTP, and 12.5 nmol MgSO4 for 1 h at 48°C. cDNA was stored at -20°C. The PCR was conducted using 10 U Taq DNA polymerase (Promega). A total of 1 µl of each cDNA was used per PCR reaction. Oligodeoxynucleotide primers (25 nmol) to HLA class II determinants were synthesized by MWG Biotech (Milton Keynes, U.K.); primers for CIITA and ß-actin were used as previously described (26). For sequences of primers, see Table IGo.


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  		Table I. Sequences of primers used for RT-PCR

 
T cell purification

Allogeneic CD4+ T cells were isolated from human blood, as previously described (27). Briefly, PBMC were separated by centrifugation on a Lymphoprep gradient (Life Technologies), followed by removal of the adherent population by plating onto tissue culture plastic for 1 h at 37°C. The CD4+ population was then isolated by negative selection using a Minimacs CD4 isolation kit (Miltenyi Biotech, Surrey, U.K.). The negatively selected cells were incubated with tissue culture supernatant from an anti-human DR{alpha}-specific mAb-secreting hybridoma (clone L243; ATCC), followed by rabbit complement to lyse all HLA-DR-expressing cells. The purity of isolated T cells was established by PHA assay. Briefly, purified CD4+ T cells were incubated with PHA (2 µg/ml) in the presence or absence of {gamma}-irradiated (30 Gy) monocytes recovered after the adherence step, and proliferation was measured by addition of 1 µCi [3H]TdR for the last 18 h of culture. Plates were incubated for an additional 20 h before freeze-thawing, followed by harvesting on a Tomtec harvester (Tomtec, Orange, CT) and analysis on a Wallac micro beta counter (Wallac, Milton Keynes, U.K.). Purified CD4+ T cells did not proliferate to PHA in the absence of accessory cells (data not shown).

T cell proliferation assays

Transfected and wild-type T24 and EaHy.926 cells were plated onto 25-cm2 tissue culture flasks and allowed to become confluent. The medium was changed and replaced with fresh full medium without G418, with or without the addition of 500 U/ml IFN-{gamma} (R&D Systems). Cells were incubated for 4 days at 37°C, before treatment with mitomycin C (Sigma), at a final concentration of 60 µg/ml for 25 min at 37°C, to prevent further proliferation. Cells were trypsinized and replated onto flat-bottom 96-well plates at 2.5 x 104 cells/well, in triplicate. They were allowed to adhere overnight; the medium was removed and replaced with AIMV (Life Technologies). A total of 1 x 105 CD4+ T cells was added per well in a final volume of 200 µl. Plates were incubated for 3–6 days at 37°C. A total of 1 µCi [3H]TdR was added, and plates were incubated for an additional 20 h before freeze-thawing, followed by harvesting on a Tomtec harvester and analysis on a Wallac micro beta counter. For Ab blocking, saturating amounts of either anti-LFA-3 or anti-HLA-DR{alpha} were added to mitomycin C-treated epithelial or endothelial cells before addition of CD4+ T cells and remained present for the duration of the experiment.

Two-step T cell coculture assays

IFN-{gamma}-treated and resting T24.CIITA were mitomycin C treated, as described above, and seeded onto six-well plates at a density of 5 x 105 cells/well. They were allowed to adhere overnight and then washed with serum-free RPMI. Allogeneic CD4+ T cells were purified as described above and resuspended in RPMI containing 10% human male AB serum (Sigma). They were cocultured with the adherent cells at a density of 2 x 106 CD4+ T cells/well, or they were cultured on tissue culture plastic at a density of 10 x 106 cells/well, for 5 days at 37°C. CD4+ T cells were then recovered and washed twice in RPMI containing 10% FCS. They were cocultured with T24.CIITA or EaHy.CIITA up to 7 days on 96-well plates, and 1 µCi [3H]TdR was added 24 h before harvesting, as described above. Alternatively, T cells recovered after the first coculture step were incubated with 10 µg/ml IL-2 (Roche Diagnostics, Lewes, U.K.) in the presence of 1 µCi [3H]TdR for 24 or 48 h at 37°C before harvesting.

Statistical analysis

SigmaStat (SPSS, San Rafael, CA) was used to perform Mann-Whitney-U nonparametric tests for all statistics presented in this study.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of MHC class II

EaHy.926 endothelial cells and T24 epithelial cells were permanently transfected with a construct containing the full-length human CIITA gene, as described in Materials and Methods. Cells that were able to grow in G418 selection medium were examined for surface expression of HLA class II determinants before and after stimulation with saturating levels of IFN-{gamma} for 4 days, by flow cytometry and quantitation with QIFI beads (Dako) (Fig. 1Go). Neither cell type expressed HLA class II constitutively. Eahy.926 expressed high levels of HLA-DR and HLA-DP with moderate levels of surface DQ expression after 4 days of IFN-{gamma} treatment (Fig. 1GoA). In contrast, there was no HLA class II expression on the surface of T24 after IFN-{gamma} treatment (Fig. 1GoB). CIITA transfection of both cell types led to surface expression of HLA-DR, HLA-DP, and HLA-DQ. HLA class I expression was up-regulated to a similar level in all cell lines after IFN-{gamma} treatment (Fig. 1Go). This suggests that the inability of T24 to express HLA class II after IFN-{gamma} treatment is due to a deficiency in the arm of the signaling pathway leading from the IFN-{gamma} receptor to CIITA expression rather than a lack of functional receptors for IFN-{gamma}.



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  		FIGURE 1. Analysis by flow cytometry of expression of HLA determinants after CIITA transfection of EaHy.926 or T24. CIITA-transfected or wild-type cells were cultured for 4 days in the presence or absence of 500 U/ml IFN-{gamma}. Cells were harvested and immunostained for HLA determinants, and analyzed by quantitative flow cytometry. A and B, Quantitation of flow cytometry data; bar chart, percentage of stained cells; open bars, untreated, untransfected cells; stippled bars, IFN-{gamma}-treated, untransfected cells; striped bars; untreated CIITA-transfected cells; dashed bars, IFN-{gamma}-treated, CIITA-transfected cells. Line graphs, quantitation of binding capacity (SABC). A, EaHy.926. B, T24. C, Flow cytometry histograms.

 
Expression of mRNA for HLA determinants was also examined by RT-PCR, and expression mirrored that observed at the cell surface (Fig. 2GoA). Expression of HLA-DM{alpha} and HLA-DMß, and invariant chain (Ii) was examined. As expected, HLA-DM and Ii were expressed in EaHy.926 after IFN-{gamma} treatment or after CIITA transfection. mRNA for HLA-DM and Ii was expressed in T24 only after CIITA transfection. Resting, untransfected cells of both types did not express mRNA for CIITA, but message could be detected after transfection. IFN-{gamma}-treated, untransfected EaHy.926 expressed CIITA, but IFN-{gamma}-treated T24 did not (Fig. 2GoB).



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  		FIGURE 2. mRNA analysis of HLA determinants after CIITA transfection of EaHy.926 or T24. CIITA-transfected or wild-type cells were cultured for 4 days in the presence or absence of 500 U/ml IFN-{gamma}. Cells were harvested and total RNA was isolated, as described in Materials and Methods. After cDNA synthesis, PCR was conducted for {alpha}- and ß-chains of HLA-DM, HLA-DP, HLA-DQ, HLA-DR, Ii, and CIITA. Lane 1, Untreated, untransfected cells; lane 2, untransfected cells + 500 U/ml IFN-{gamma}; lane 3, CIITA-transfected cells; lane 4, CIITA-transfected cells + IFN-{gamma}; lane 5, Raji-positive control; lane 6, -RT negative control. A, EaHy.926. B, T24.

 
T cell proliferation

The ability of EaHy.926 and T24 to present alloantigen to resting peripheral CD4+ T cells was examined. As expected from previous results (6, 16), IFN-{gamma}-treated EaHy.926 were able to induce proliferation of allospecific T cells, while there was minimal alloproliferation to IFN-{gamma}-treated T24 (Fig. 3Go). CIITA-transfected cells of both cell types could induce a T cell response, although the magnitude of the response to EaHy.CIITA was up to 10–15-fold higher than that to T24.CIITA (Fig. 3Go). Interestingly, IFN-{gamma} treatment of EaHy.CIITA or T24.CIITA reduced the T cell response after 6 days of coculture. Although this did not reach significance in the case of EaHy.CIITA, T cell responses to IFN-{gamma}-treated T24.CIITA were reduced to almost background levels (p = 0.008 T24.CIITA vs IFN-{gamma} T24.CIITA).



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  		FIGURE 3. Proliferation of allospecific T cells to CIITA-transfected EaHy.926 or T24. CIITA-transfected or wild-type cells were cultured for 4 days in the presence or absence of 500 U/ml IFN-{gamma}. Cells were treated with mitomycin C and seeded onto flat-bottom 96-well plates at a density of 5 x 104 per well. Peripheral CD4+ T cells were purified, as described in Materials and Methods, and cocultured with mitomycin C-treated adherent cells for 6 days at a starting density of 1 x 105 per well. [3H]TdR was added for the last 18 h of culture. Results are expressed as mean cpm for triplicate cultures and are representative of at least two different allogeneic individuals. A, CD4+ T cell response to EaHy.926. B, CD4+ T cell response to T24.

 
We next examined whether the time course of this allospecific proliferation was altered in responses to CIITA-transfected cells compared with IFN-{gamma}-treated wild-type cells. Proliferation of resting peripheral CD4+ T cells was measured after 4–7 days of incubation with mitomycin C-treated cells (Fig. 4Go). As shown previously (6), the response to IFN-{gamma}-treated EaHy.926 peaked at day 6. This pattern of response was similar to IFN-{gamma}-treated EaHy. CIITA (Fig. 4GoA). There was no response to IFN-{gamma}-treated T24 at any time point. The response to unstimulated T24.CIITA continued to increase with time up to 7 days of coculture (Fig. 4GoB), but there was no response to IFN-{gamma}-treated T24.CIITA at any time point.



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  		FIGURE 4. Time course of proliferation of allospecific T cells to CIITA-transfected EaHy.926 or T24. CIITA-transfected or wild-type cells were cultured for 4 days in the presence or absence of 500 U/ml IFN-{gamma}. Cells were treated with mitomycin C and seeded onto flat-bottom 96-well plates at a density of 5 x 104 per well. Peripheral CD4+ T cells were purified, as described in Materials and Methods, and cocultured with mitomycin C-treated adherent cells for 4–7 days at a starting density of 1 x 105 per well. [3H]TdR was added for the last 18 h of culture. Results are expressed as mean cpm for triplicate cultures and are representative of at least two different allogeneic individuals. A, CD4+ T cell response to EaHy.926. B, CD4+ T cell response to T24.

 
Expression of costimulatory molecules on transfected and wild-type cells

Because the kinetics and magnitude of stimulation of resting allospecific CD4+ T cells were altered after transfection of CIITA into either T24 epithelial cells or EaHy.926 endothelial, epithelial hybridoma cells, surface expression of potentially costimulatory molecules was examined in transfected and wild-type cells before and after stimulation with saturating doses of IFN-{gamma} for 4 days. All cell types expressed LFA-3, and levels were not affected by IFN-{gamma} treatment or CIITA transfection. ICAM-1 and CD40 were also expressed on all cells, and levels were increased to similar levels after IFN-{gamma} treatment in transfected and untransfected cells. None of the cell lines expressed B7.1 or B7.2 (as assessed by their ability to bind CTLA-4-Ig), and this was not altered after IFN-{gamma} treatment (Fig. 5Go).



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  		FIGURE 5. Analysis by flow cytometry of expression of costimulatory molecules after CIITA transfection of EaHy.926 or T24. CIITA-transfected or wild-type cells were cultured for 4 days in the presence or absence of 500 U/ml IFN-{gamma}. Cells were harvested and stained by indirect immunofluorescence for the presence of CD40, ICAM-1, LFA-3, and binding to CTLA-4-Ig and EN-4, and analyzed by quantitative flow cytometry. A and B, Quantitation of flow cytometry data; bar chart, percentage of stained cells; open bars, untreated, untransfected cells; stippled bars, IFN-{gamma}-treated, untransfected cells; striped bars, untreated CIITA-transfected cells; dashed bars, IFN-{gamma}-treated, CIITA-transfected cells. Line graphs, SABC. A, EaHy.926. B, T24.

 
T cell proliferation to T24.CIITA can be blocked with Abs to LFA-3 and HLA-DR

It has previously been shown that alloproliferation of CD4+ T cells to endothelial cells is inhibited by blocking cognate interactions (with Abs against HLA-DR (16, 28)) or by blocking the second signal (using Abs directed against LFA-3 (17)). As shown in Fig. 6Go, proliferation of resting CD4+ T cells to both T24.CIITA and EaHy.CIITA was inhibited by greater than 90% in the presence of Abs to either HLA-DR or LFA-3. The response to IFN-{gamma}-treated EaHy.926 and IFN-{gamma}-treated EaHy.CIITA was also blocked by either one of these Abs.



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  		FIGURE 6. Blocking of proliferation of allospecific T cells to CIITA-transfected EaHy.926 or T24, using Abs to LFA-3 and HLA-DR{alpha}. CIITA-transfected or wild-type cells were cultured for 4 days in the presence or absence of 500 U/ml IFN-{gamma}. Cells were treated with mitomycin C and seeded onto flat-bottom 96-well plates at a density of 5 x 104 per well. Peripheral CD4+ T cells were purified, as described in Materials and Methods, and cocultured with mitomycin C-treated adherent cells for 6 days at a starting density of 1 x 105 per well with or without prior incubation with saturating doses of anti-LFA-3 or anti-DR{alpha}, and continued incubation with Ab for the duration of the experiment. [3H]TdR was added for the last 18 h of culture. Results are expressed as mean cpm for triplicate cultures and are representative of at least two different allogeneic individuals. A, CD4+ T cell response to EaHy.926. B, CD4+ T cell response to T24.

 
After coculture with IFN-{gamma}-treated T24.CIITA, allogeneic CD4+ T cells respond to IL-2 stimulation, but are not anergized

Since we observed that T24 were able to induce allospecific CD4+ T cell proliferation after transfection with CIITA, but not after prior treatment with maximal doses of IFN-{gamma}, we next investigated whether coculture of allogeneic T cells with IFN-{gamma}-treated T24.CIITA could elicit nonresponsiveness to further coculture with untreated T24.CIITA. As shown in Fig. 7GoA, CD4+ T cells that had been cocultured with IFN-{gamma}-treated T24.CIITA for 5 days were able to respond to untreated T24.CIITA, less well than CD4+ T cells that had been cultured on untreated T24.CIITA. However, they also gave less response to EaHy.CIITA than T cells cultured on T24.CIITA or EaHy.CIITA, suggesting a nonspecific down-regulation of allospecific T cell proliferation, rather than induction of anergy. T cells recovered after coculture with IFN-{gamma}-treated T24.CIITA were able to proliferate in response to exogenous IL-2, indicating that they had interacted with the epithelial cells in an Ag-dependent manner. Those T cells that were precultured on tissue culture plastic were not (Fig. 7Gob).



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  		FIGURE 7. Two-step coculture of allospecific T cells with CIITA-transfected EaHy.926 or T24. T24.CIITA were cultured for 4 days in the presence or absence of 500 U/ml IFN-{gamma}. Cells were treated with mitomycin C and seeded onto six-well plates at a density of 5 x 105 per well. Peripheral CD4+ T cells were purified, as described in Materials and Methods, and cocultured either with mitomycin C-treated adherent cells or on tissue culture plastic at a starting density of 2 x 106 per well. After 5 days, T cells were recovered and A, restimulated for 6 days with fresh mitomycin C-treated T24.CIITA or EaHy.CIITA, which had previously been seeded onto 96-well flat-bottom plates ([3H]TdR was added for the last 18 h of culture), or B, cultured in the presence of 10 µg/ml rIL-2 and [3H]TdR for 48 h. Results are expressed as mean cpm for triplicate cultures and are representative of at least two different allogeneic individuals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of this study was to use CIITA transfectants to investigate the apparent discrepancy in the ability of different parenchymal cells to cause alloproliferation of resting CD4+ T cells. There is a broad agreement that HLA class II-positive endothelial cells (5, 6, 16, 28, 29), but not epithelial cells (7, 11, 12, 13, 14, 30), fibroblasts (31, 32), or smooth muscle cells (28, 33), cause alloproliferation of resting CD4+ T cells. The advantage of CIITA transfection is that it can cause up-regulation of HLA-class II molecules without the pleiotropic effects of IFN-{gamma}. We showed first that transfecting CIITA into either EaHy.926 or T24 up-regulated HLA-DR, HLA-DP, and HLA-DQ to the same or greater extent as saturating doses of IFN-{gamma} on untransfected EaHy.926. HLA-DM and Ii were also up-regulated on both cell lines. It was then shown that CIITA transfection did not alter cell surface expression of LFA-3, known to be a second signal for endothelial cell-mediated stimulation of CD4+ T cells (17, 28, 34). Expression of ICAM-1, CD40, and HLA class I also remained unaltered after CIITA transfection. This, however, does not rule out the possibility that CIITA transfection does not up-regulate other genes, which might be involved in T cell activation. We are currently taking a proteomic approach to investigate whether the expression of any other proteins is modified after CIITA transfection.

Recent studies have shown that utilizing CIITA to up-regulate HLA class II in porcine endothelial cells is sufficient to induce them to present Ag to specific T cells without prior incubation with IFN-{gamma} (2, 43). Together with the results presented in this work, this shows that endothelial cells constitutively express the costimulatory molecules required to induce allospecific T cell proliferation in vitro.

As expected, T24 epithelial carcinoma cells were not able to stimulate alloproliferation even after several days of treatment with maximal levels of IFN-{gamma} (Fig. 3Go), due to lack of expression of HLA class II Ags (Fig. 1Go). However, increased expression of CD40 and HLA class I was observed (Figs. 1Go and 5Go), suggesting that the IFN-{gamma} receptor was functional in these cells. To our surprise, CIITA-transfected T24 were able to cause allostimulation of resting CD4+ peripheral T cells (Fig. 3Go), and the response was not reduced after 7 days of coculture, but appeared to be continuing to rise (Fig. 4Go). Thus, these epithelial cells were able to induce alloproliferation of resting CD4+ T cells with the same molecular requirements (i.e., dependence on HLA-DR and LFA-3) as endothelial cells. This is the first demonstration of HLA class II-positive epithelial cells causing alloproliferation of resting peripheral blood CD4+ T cells. In other studies, CIITA has been transfected into fibroblasts (35) and hepatocytes (36), leading to cell surface expression of HLA-class II determinants and efficient presentation of peptide epitopes to HLA-DR-restricted peptide-specific CD4+ T cell clones. It is, however, not unusual to show proliferation of T cell clones and lines to HLA-DR-expressing epithelial cells (11, 12, 14). It is likely that cloned T cells acquire different costimulatory requirements after prolonged culture, including independence from B7-mediated signals (13). The novel observation in this study leads us to suggest that B7-independent alloreactive T cells exist in PBL, which are able to respond to HLA class II expressed on certain epithelial cells, but only in the absence of IFN-{gamma}.

Pretreatment of T24.CIITA with IFN-{gamma} for 4 days completely inhibited the alloproliferation observed with untreated T24.CIITA (Fig. 3Go). We believe this correlates with previous studies using epithelial cells treated with maximal doses of IFN-{gamma} to increase HLA-class II expression at the cell surface (7, 11, 12, 13, 30, 37, 38, 39). In these studies, there was no proliferative response of alloreactive T cells to HLA-DR-expressing IFN-{gamma}-treated epithelial cells. Indeed, in several studies, T cells exposed to IFN-{gamma}-treated epithelial cells became unresponsive to the same HLA Ags presented by professional APC. For example, Frasca et al. (12) and Singer et al. (39) showed that kidney epithelial cells could induce nonresponsiveness in peripheral CD4+ T cells, while Cunningham et al. (11) described a similar phenomenon using lung epithelial cells and Marelli-Berg et al. (13) reported similar findings with a thyroid epithelial cell line. This group also showed that at least one of the mechanisms by which epithelial cells are able to induce nonresponsiveness is via Fas (CD95)-mediated apoptosis. Addition of an Ab directed against CD95 could protect the CD45RA+ naive T cell subset, but not the CD45RO+ memory subset, from the induction of anergy (13). In our study, anergy was not observed. We have to conclude that in our study, failure to activate allospecific CD4+ T lymphocytes by IFN-{gamma}-treated HLA class II-positive T24 epithelial cells is not due to lack of functional costimulatory molecules, but by a negative regulatory mechanism induced by IFN-{gamma}.

The proposed negative regulation may serve to prevent activation of self-reactive T cells, which have not been deleted in the thymus due to lack of expression of nonthymic Ags. This would be of particular importance in the microenvironment of a vigorous inflammatory response, in the presence of IFN-{gamma}, which will up-regulate MHC class II expression and the presentation of self Ags. Whether anergy or nonresponsiveness occurs will depend on many other factors (T cell maturation, presence of apoptotic signals). It is possible that the breakdown of self tolerance observed during autoimmunity may be, at least in part, due to a defect in this mechanism, so that peripheral epithelial cells do not cause anergy of potentially self-reactive T cells in the presence of IFN-{gamma}. One can only speculate why endothelial cell stimulation of CD4+ T cells is less regulated by IFN-{gamma}. The response does decrease with time, but regulation only becomes evident at higher levels of proliferation. It may be that, in vivo, the activation of CD4+ T cells by endothelial cells results in transendothelial migration and subsequent removal of the T cells from the activated endothelial cells (14). In the transplant setting, donor epithelial cells could contribute to the initiation and maintenance of T cell tolerance observed in several allograft models after prolonged graft presence. This is not the first report that IFN-{gamma} can act as a negative regulator of T cell proliferation and graft rejection. In two models of tolerance induction (40, 41), tolerance could not be induced in IFN-{gamma}-/- mice, or mice treated with neutralizing Abs to IFN-{gamma}. It was also shown that IFN-{gamma} limited the expansion of activated T cells, in vitro (40). IFN-{gamma} has also been shown to suppress TNF-{alpha}- and IL-1ß-induced up-regulation of the adhesion molecules E- and P-selectin on endothelial cells, suggesting that it can act as an anti-inflammatory cytokine (42).

In conclusion, this study confirms the negative effect class II-expressing epithelial cells have on CD4+ T cells, but suggests a novel mechanism at least operating within T24, namely via IFN-{gamma}. Whether this mechanism applies to other epithelial cell types (or is unique to the T24 bladder carcinoma line) is currently under investigation.


   Footnotes
 
1 This work was supported by the British Heart Foundation. Back

2 Address correspondence and reprint requests to Dr. Marlene Rose, Transplant Immunology Group, Imperial College School of Medicine, National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Harefield, Middlesex UB9 6JH, U.K. Back

3 Abbreviations used in this paper: CIITA, class II transactivator; Ii, invariant chain; SABC, surface Ag-binding capacity. Back

Received for publication February 16, 2000. Accepted for publication April 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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