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The Journal of Immunology, 2003, 171: 4089-4095.
Copyright © 2003 by The American Association of Immunologists

Functional Segregation of the TCR and Antigen-MHC Complexes on the Surface of CTL 1

Divya J. Mekala* and Terrence L. Geiger2,*,{dagger}

* Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and {dagger} Department of Pathology, University of Tennessee School of Medicine, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As CTL adhere to and lyze their targets, they extract cognate Ag-MHC and represent this on their own cell surface. Whether such self-presented cognate Ag stimulate the TCR of a CTL is uncertain. To analyze this, we examined TCR capping in response to self-presented Ag. We found that OVA peptide-specific OT-1 CTL that were pulsed with cognate peptide Ag did not cap their TCR, implying that the autologously presented MHC-Ag complex does not normally stimulate the TCR. However, this functional separation of the TCR and its ligand on the cell surface was not absolute. Treatment of Ag-pulsed OT-1 CTL with agents that alter cell surface charge, including trypsin, papain, tunicamycin, neuraminidase, and polybrene, allowed Ag-specific TCR capping. The TCR capped together with the restricting MHC molecule on the surface of the cell, implying an interaction between the TCR and cell-associated Ag. Further, the treated CTL underwent a time- and dose-dependent suicidal death that was both Fas- and perforin-dependent. Therefore, our results indicate that the association of the TCR with its MHC-peptide ligand on the surface of a CTL is normally proscribed by biophysical properties of the plasma membrane. Overcoming this restriction allows TCR stimulation and induces CTL effector functions and cell suicide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytotoxic T lymphocytes are potent killers, lyzing target cells expressing cognate Ag-MHC through the release of perforin and granzyme stored in cytoplasmic granules and through the expression of Fas ligand (FasL) 3 (1). Nevertheless, CTL themselves are largely resistant to lysis by other CTL (2, 3, 4, 5). Several mechanisms for the resistance of CTL to its own effector mechanisms have been proposed. Early studies demonstrated that CTL cell membranes only poorly bind perforin (2, 6), a pore-forming protein necessary for target cell lysis. More recently, cell surface cathepsins have been found to prevent CTL suicide after degranulation, possibly by degrading perforin (7). Whether other potential mechanisms for resistance to CTL lysis, including expression of granzyme-inhibitory serpins (8) and altered expression of bcl-2 family proteins (9), also play a role in lysis resistance has not been resolved.

The issue of CTL lysis resistance has recently taken on added interest with the findings of Huang et al. (10) and Hudrisier et al. (11), showing that after CTL kill and disengage from target cells, they incorporate into their membrane cognate Ag-MHC molecules. Therefore, after target engagement, a CTL naturally possesses on its cell surface Ag that may be capable of stimulating its own TCR. The TCR of the CTL would be expected to bind to this autologously presented cognate Ag, thereby stimulating the CTL and promoting the expression of FasL and the release of toxic perforin- and granzyme-containing granules. Such self-induced activation may even be expected to kill the CTL. However, despite recent evidence that perforin/granzyme released by CTL during lytic encounters can promote low levels of CTL suicide (12), CTL can kill multiple target cells without exhaustion (13). Therefore, the acquisition of cognate Ag typically not only does not kill CTL, but it also seems not to alter their function.

There are two possible explanations for the seeming lack of effect of cognate Ag presented on the surface of the CTL. The TCR of a CTL may engage and be stimulated by this Ag, however, the intrinsic resistance of the cell to lysis may protect it from suicidal death. Alternatively, the TCR of a CTL may not be able to engage the MHC-Ag complex presented on the same plasma membrane. To examine these possibilities, we analyzed the association of TCR and cognate Ag-MHC presented on CTL. We demonstrate that the TCR of a CTL does not normally associate with cognate Ag-MHC present on its plasma membrane. However, we also demonstrate that this segregation can be overcome by treatments that disrupt the surface charge of a cell (zeta potential). Breakdown of this charge barrier permits association of the TCR and MHC-peptide on a single cell and induces cell suicide by a Fas- and perforin-dependent mechanism. Therefore, these results provide evidence for a functional separation of the TCR and its ligand on the surface of a CTL, and further suggest that this separation is maintained by biophysical properties of the membrane itself.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of primary CTLs

Lymph node cells isolated from 6- to 8-wk-old mice (C57BL/6J or OT-1 transgenic; The Jackson Laboratory, Bar Harbor, ME) were isolated and suspended in enriched Eagle’s medium (EHAA; BioSource International, Camarillo, CA) containing glutamine, 10% FCS, antibiotics, and 1 ng/ml rIL-2 (BD PharMingen, San Diego, CA). OT-1 cells were activated with anti-CD3 and CD28 Abs for 4–5 days and expanded in rIL-2 containing medium for 2 more days before their experimental use, or restimulated in an identical fashion to maintain short-term cultured lines. Nontransgenic CD8+ T cells were flow cytometrically isolated from the C57BL/6J lymph node cells and stimulated in the presence of irradiated syngeneic feeder cells as above.

Peptide pulsing of CTLs

CTL were incubated for 2 h in EHAA medium with 50 µg/ml of the 257–264 peptide fragment of OVA at 37°C. Cells were then washed three times with PBS. Control unpulsed cells were mock-treated in EHAA medium with peptide diluent (PBS).

CTL cell death

OT-1 CTLs either pulsed or unpulsed with cognate peptide were incubated with different concentrations of trypsin (Sigma-Aldrich, St. Louis, MO), papain (Sigma-Aldrich), tunicamycin (Sigma-Aldrich), neuraminidase (Calbiochem, La Jolla, CA), heparin (Sigma-Aldrich), or polybrene (Sigma-Aldrich) for different time periods in PBS in a 96-well plate, unless otherwise described. After treatment, cells were placed on ice, supernatant was removed, and viability was immediately measured microscopically by trypan blue exclusion. To analyze fratricidal cell death, peptide-pulsed or unpulsed CTL lines were labeled with 5 µM CFSE (Molecular Probes, Eugene, OR). They were then incubated at a 2:1 E:T ratio with OT-1 CTLs in the presence of 0.1 mg/ml trypsin for 45 min. Ethidium bromide was added to the medium at the end of incubation period and death was determined by the incorporation of ethidium bromide in CFSE-labeled cells. All samples were assayed in triplicate. Mean ± 1 SD is plotted.

Perforin/FasL-mediated cell death

Concanamycin A (CMA; Sigma-Aldrich), an inhibitor of perforin maturation was used to analyze perforin-mediated cell death. OT-1 CTLs were preincubated with the indicated concentration of CMA for 16 h before and during treatment with trypsin for 45 min at 37°C. Cell death was analyzed by trypan blue incorporation. Similarly, rhFas:Fc (human recombinant; Alexis, Carlsbad, CA) fusion protein was used to determine the role of Fas-FasL-mediated cell death. Cells were incubated for 45 min with the indicated concentration of rhFas:Fc along with 1 µg/ml enhancer (Alexis) and trypsin and then analyzed for cell death.

Confocal microscopy

Cocapping of MHC-H-2Kb with TCR after treatment of peptide-pulsed or unpulsed OT-1 CTLs with trypsin or anti-CD3{epsilon} (clone 145-2C11; BD PharMingen) was analyzed by confocal microscopy. Briefly, treated CTLs were cytospun onto glass slides and fixed with 2% paraformaldehyde for 20 min. Slides were then washed with PBS and blocked with 10% FCS for 1 h. Cells were stained with FITC- or PE-conjugated anti-TCR (clone H57-597; BD PharMingen), CD8 (clone 53-6.7; BD PharMingen), H-2Db (clone K495; BD PharMingen) and H-2Kb (clone AF6-88.5; BD PharMingen) Abs as indicated for 1 h each with intermediate PBS washings (three times). After staining, the slides were again washed with PBS three times, mounted with p-phenylenediamine (Sigma-Aldrich) and viewed and photographed under a Leica TCS NT SP laser scanning confocal microscope (Deerfield, IL). Brightness and contrast of captured images were adjusted using Adobe Photoshop software (Adobe Systems, Mountain View, CA).

Electron microscopy

Peptide-pulsed or unpulsed OT-1 CTLs were treated with or without 0.1 mg/ml trypsin for 45 min, then fixed with 2.5% glutaraldehyde (Tousimis, Rockville, MD) overnight. Eighty-nanometer sections were cut and analyzed using a JEOL 1200 EXII TEMSCAN electron microscope (Peabody, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TCR capping after treatment with antigenic peptide

Functional engagement of TCR with its Ag ligand in metabolically active T cells leads to cytoskeletal rearrangement and the migration and accumulation of TCR into a cap structure (14, 15). To determine whether cognate Ag presented on the surface of a CTL binds to and stimulates the TCR of the cell, we therefore analyzed TCR capping. We incubated an H-2b CTL line derived from OT-1 TCR transgenic mice with its cognate peptide, the H-2Kb-restricted 257–264 fragment of OVA, at 37°C for intervals from 30 min to 2.5 h. Alternatively, we pulsed the OT-1 CTL with peptide, washed the cells, and then incubated them at 37°C. The cells were then fixed, stained with Abs specific for TCR and H-2Kb, and analyzed by confocal microscopy.

As expected, control untreated cells displayed no cap formation (Fig. 1, A–C) while cells treated with anti-CD3{epsilon} Ab showed good cap formation (Fig. 1, D–F). In contrast to anti-CD3{epsilon} treatment, when the OT-1 cells were pulsed with antigenic peptide, TCR capping was not observed regardless of the incubation time after pulsing (Fig. 1, G–I and data not shown). This implies that the TCR of a CTL does not functionally engage cognate Ag presented on its own surface.



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  		FIGURE 1. Capping of TCR and Kb. Except as indicated below, peptide-pulsed or unpulsed OT-1 CTL were incubated for 30–45 min at 37°C with the indicated treatment, formalin-fixed, stained with PE-TCR- and FITC-Kb-specific Abs, and analyzed for receptor capping by confocal microscopy. Images from identical fields showing TCR, H-2Kb, and an overlay of these stains are shown. A–C, Untreated OT-1 CTL. D–F, OT-1 CTL were stained with biotinylated CD3{epsilon}-specific Ab and then cross-linked by incubation at 37°C in the presence of FITC-streptavidin. After fixation, cells were stained with a PE-labeled anti-Kb. G–I, OVA peptide-pulsed OT-1 CTL. J–L, OT-1 CTL treated with 100 µg/ml trypsin during the 37°C incubation. M–O, Peptide-pulsed OT-1 CTL treated with 100 µg/ml trypsin. P–R, OT-1 CTL treated with 2.5 mU/ml neuraminidase during the 37°C incubation. S–U, Peptide-pulsed OT-1 CTL treated with neuraminidase. AA–FF, Effect of trypsin and peptide treatment on the nonrestricting class I Db molecule. GG–LL, CD8 cocapping with TCR after trypsin treatment. Results are representative of three experiments.

 
We hypothesized that cell surface proteins play a role in preventing the association of the TCR with the cognate Ag-MHC complex. To test for this, we treated either unpulsed or peptide-pulsed OT-1 CTL with trypsin protease to determine whether alteration of the cell surface protein content would influence TCR association with Ag. Trypsin treatment of control CTL not pulsed with peptide did not induce TCR capping (Fig. 1, J–L). However, by 30 min of trypsin treatment, significant TCR capping was observed on peptide-pulsed cells (Fig. 1, M–O). This occurred even using concentrations of trypsin >10-fold lower than those commonly used for dislodging adherent tissue culture cells. Interestingly, in contrast to TCR capping after anti-CD3{epsilon} treatment in which H-2Kb did not cocap with the TCR, a fraction of the restricting cell surface H-2Kb molecules capped together with the TCR after peptide and trypsin treatment. This likely reflected the colocalization of cognate Ag-Kb complex with the TCR in cap structures, a possibility supported by the failure of the nonrestricting class I Db molecule to cocap with TCR after trypsin and peptide treatment (Fig. 1, AA–FF).

To provide further evidence for TCR association with restricting MHC in cap structures, we analyzed colocalization of the CD8 coreceptor for the TCR. CD8 would be expected to bind to the class I MHC-Ag complex during TCR engagement. CD8 was observed to cap together with the TCR in trypsin- and peptide-treated CTLs, but not in cells treated with peptide alone (Fig. 1, GG–LL). These results show that mild protease treatment releases the inhibition of TCR engagement with peptide-MHC on the cell surface. TCR engages MHC-peptide complexes in an Ag-specific manner, and this engagement leads to capping of the TCR and CD8 coreceptor on the CTL surface.

Induction of cell death by Ag and protease treatment

TCR stimulation promotes the release of CTL granules and the expression of FasL at the site of CTL/target cell engagement, thereby inducing target cell death. To determine whether trypsin treatment of Ag-pulsed OT-1 T cells also induced cell death, we measured the viability of treated cells. Ag-pulsed CTL treated with trypsin died in both a time- and trypsin-dose-dependent manner (Fig. 2, A and B). Controls, including CTL that were pulsed with Ag but not trypsin-treated, and unpulsed and trypsin-treated CTL, showed only limited loss of viability. The induction of cell death by protease and Ag was not specific to trypsin as similar results for both TCR capping and cell death were observed when the OT-1 CTL were treated with an alternative protease, papain (Fig. 2, C and D, and data not shown). This demonstrates that protease treatment releases the block in TCR engagement with cognate Ag, ultimately leading to their death.



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  		FIGURE 2. Impact of peptide and protease treatment on OT-1 viability. A, Trypsin dose dependence of cell death. Peptide-pulsed or unpulsed OT-1 cells were treated for 60 min with the indicated concentration of trypsin before viability analysis by trypan blue exclusion. B, Time dependence of cell death with trypsin treatment. Peptide-pulsed or unpulsed OT-1 CTL were treated with 100 µg/ml trypsin or no trypsin for the indicated time. C and D, Analyses of the effect of papain on OT-1 CTL were performed as in A and B. D, One-hundred micrograms per milliliter or no papain were used. Results are representative of four experiments.

 
The most likely explanation for this protease-induced, Ag-dependent CTL death is the induction of the endogenous Fas and/or perforin-dependent effector responses of the CTL after TCR stimulation by Ag. To test for this, we treated OT-1 CTL with CMA (16), an inhibitor of lytic granule maturation that blocks perforin mediated cytotoxicity, or with Fas-Fc, an inhibitor of Fas-induced cell death. Either CMA or Fas-Fc inhibited cell death by ~40–50% in peptide-pulsed trypsin treated cells, implicating both mechanisms in OT-1 cell death (Fig. 3, A and B). Treatment with both agents further diminished cell death, demonstrating complementarity in the Fas- and perforin-dependent mechanisms (Fig. 3C). These results show that protease treatment relieves a block in the induction of CTL effector pathways by self-expressed cognate Ag, and that this induces cell death through both perforin/granzyme- and Fas-dependent mechanisms.



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  		FIGURE 3. Role of perforin and Fas pathways in trypsin/Ag-mediated OT-1 cell death. Peptide-pulsed or unpulsed OT-1 CTL were treated with 100 µg/ml trypsin for 45 min before determining cell viability by trypan blue exclusion. Cells were either treated for 16 h preceding and during trypsin treatment with the indicated concentration of CMA, an inhibitor of perforin maturation and lysosomal acidification (A), or at the time of trypsin treatment cultured with the indicated amount of an inhibitor of FasL-mediated apoptosis, FasFc, in a volume of 200 µl (B). C, Peptide-pulsed OT-1 CTL were treated with either CMA, FasFc, or both agents. Results are representative of four experiments (A and B) and two experiments (C).

 
Cell death is suicidal and not fratricidal

Because the restricting MHC, H-2Kb, was incorporated in the TCR cap after treatment with peptide and trypsin, we suspected that T cell death resulted from engagement of cognate Ag by TCR on the membrane of the T cell. However, either intercellular (fratricidal) or intracellular (suicidal) engagement may have caused the death of the Ag-pulsed, protease-treated CTL. To distinguish between these possibilities, we performed two analyses. First, we plated peptide-pulsed OT-1 CTL at extremely low density, 2 x 106 µm2/cell, to prevent cell contact and therefore fratricidal lysis during trypsin treatment. Physical separation of the T cells was readily apparent microscopically during incubation. Nevertheless, similar levels of cell death were apparent in these highly diluted cultures as in cultures incubated at higher density, implicating suicidal death as the mechanism of lysis (Fig. 4A).



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  		FIGURE 4. Protease- and Ag-induced OT-1 CTL death results from cell suicide. A, Analysis of the effect of cell contact on trypsin-dependent cell death. Ten-thousand peptide-pulsed or unpulsed OT-1 CTL were incubated with 100 µg/ml trypsin in a 15-cm circular tissue culture dish for 45 min before placement on ice, the gentle removal of supernatant, and the addition of trypan blue for cell viability analysis. Physical separation of cells was readily apparent microscopically during the time of incubation. B, Fratricidal lysis of C57BL/6 CTL pulsed with OVA peptide. CFSE-labeled C57BL/6 CTL, pulsed or unpulsed with antigenic peptide, were incubated with or without OT-1 CTL and trypsin in U-shaped wells. OT-1 CTL were added at a 2:1 E:T ratio to the C57BL/6 CTL. After 45 min, supernatant was removed, the cells were transferred to an ice-cold flat-bottom plate, ethidium bromide was added, and viability was determined by the percentage of CFSE+ cells staining with ethidium. C, Control analysis performed identically to B, except a 2:1 ratio of unlabeled and CFSE-labeled OT-1 CTL were added to each well.

 
Second, we directly assayed fratricidal lysis by measuring the targeted killing of CTL lacking an OVA-specific TCR. We created a polyclonal C57BL/6 CTL line by activating flow cytometrically purified CD8+ T cells with CD3- and CD28-specific Abs. This line, which did not itself recognize OVA, was lytically active in redirected lysis assays (data not shown). It was pulsed with OVA peptide, labeled with the fluorescent dye CFSE, extensively washed, and coincubated at a 1:2 ratio with unlabeled OT-1 CTL effectors. If the death of OT-1 CTL in the experiments above was fratricidal, the added OT-1 cells should kill the peptide-pulsed C57BL/6 CTL after trypsin treatment. Death of C57BL/6 CTL was measured by the addition of ethidium bromide and fluorescent microscopic counting of CFSE/ethidium double-positive vs CFSE single-positive cells. No increased cell death of the trypsin-treated, peptide-pulsed C57BL/6 CTL was observed compared with control wells containing unpulsed C57BL/6 CTL or to which trypsin was not added (Fig. 4B). In contrast, when peptide-pulsed OT-1 CTL were similarly CFSE-labeled and incubated in an identical manner with unlabeled OT-1 CTL, the labeled cells showed significant cell death with trypsin treatment (Fig. 4C). Therefore, CTL death requires coexpression of cognate Ag and TCR on the same cell, demonstrating that protease treatment induces suicidal activation of cognate-Ag-pulsed CTL.

Role of glycosylation in preventing autoactivation-mediated cell death

Both trypsin and papain are promiscuous in their targets and will cleave large numbers of cell surface proteins. The proteases may have either disrupted specific proteins important in segregating TCR and MHC. Alternatively, they may have more globally altered the chemical nature of the cell surface. In the latter case, surface glycosylation would be a likely candidate as protein-linked glycans form a coat on the cell surface that critically influence protein-protein interactions (17). To test for the role of glycosylation, we pulsed OT-1 CTL with antigenic peptide and treated them with tunicamycin, an inhibitor of N-linked glycosylation. Whereas limited cell death occurred after tunicamycin treatment or peptide treatment alone, tunicamycin treatment of peptide-pulsed cells led to significant cell death (Fig. 5A and data not shown). This cell death was first apparent ~24 h after initiating tunicamycin treatment, suggesting that this period of time was required to reduce pre-existing glycosylation to levels permissive for cell suicide. These results show that surface glycosylation is critical in preventing Ag-mediated autolysis of CTL.



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  		FIGURE 5. Glycosylation inhibition and sialic acid removal promotes Ag-induced CTL death. A, OVA peptide-pulsed or unpulsed OT-1 CTL were cultured in the presence of 0.1 µg/ml tunicamycin for the indicated time intervals before assessment of cell viability by trypan blue exclusion. B, Peptide-pulsed or unpulsed OT-1 CTL were treated with 2.5 mU/ml neuraminidase for the indicated times before viability analysis. Results are representative of two experiments.

 
Glycosylation forms a complex web of polysaccharides on the cell surface. Prominent among these sugars is sialic acid. Sialic acid commonly forms the terminal residue of the N-linked oligosaccharides, the formation of which was inhibited by tunicamycin in the experiment above (18). Sialic acid generates a strong negative charge on the cell surface, and is a significant contributor to the zeta potential of a cell. This charge potential provides an electrostatic barrier to cell-cell association. It could similarly prevent association of TCR and peptide-MHC present within a the membrane of a cell. To test the role of sialylation in cell death and TCR association with cognate Ag, we treated OT-1 cells with neuraminidase, an enzyme specific for sialic acid. This treatment mimicked treatment with either protease or tunicamycin. It both induced the death of peptide-pulsed OT-1 CTL and promoted TCR and MHC cocapping (Figs. 1, S–U, and 5B). This demonstrates that sialylation is required to maintain the segregation of TCR and MHC on the CTL surface.

The activity of neuraminidase may have been specifically due to removal of sialic acid or to the removal of the cell surface negative charge, carried by these sugars. In the latter case, other methods that neutralize surface charge should likewise promote TCR association with Ag-MHC and cell death. To test this, we treated OT-1 CTL with either a polycation, hexadimethrine bromide (polybrene), or with a control polyanion, heparin, a sulfated polysaccharide. Polybrene is known to promote RBC agglutination, an effect dependent on the neutralization of sialic acid residues and on the consequent restructuring of water molecules around the membrane (19, 20). Polybrene does not affect the agglutination of cells deficient in sialic acid, demonstrating that this sugar residue is the main target of this agent. Whereas treatment with polybrene promoted peptide-dependent cell death and TCR-MHC cocapping, similar concentrations of heparin had no effect (Fig. 6 and data not shown). These results confirm our data with neuraminidase treatment and further imply that cell surface charge is critical in maintaining the isolation of TCR and MHC-peptide on the cell surface.



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  		FIGURE 6. Effect of polybrene and heparin on CTL death. The indicated concentration of polybrene (A) or heparin (B) was added to cultured antigenic peptide-pulsed or unpulsed OT-1 CTL for 2 h. Cell viability was assessed by trypan blue exclusion. Time course analysis showed progressively increasing cell death of polybrene-treated peptide-pulsed OT-1 CTL during the course of incubation (data not shown). Results are representative of two experiments.

 
Ultrastructure of treated cells

To provide a structural correlate for the functional effects we observed, we performed electron microscopy of OT-1 CTL treated with trypsin and/or antigenic peptide. Peptide-pulsed OT-1 CTL displayed no detectable differences compared with control untreated cells (Fig. 7, A–D). In contrast, trypsin treatment in the absence of peptide visibly altered the CTL surface, despite its inability to induce TCR capping or significant CTL death (Fig. 7, E and F). Microvilli, prominent in control cells, were noticeably reduced or absent. Consistent with these findings, studies by others have likewise correlated loss of cell surface charge with diminished numbers of microvilli, and biochemical measurements have shown that a loss of cell surface charge can impact both membrane fluidity and curvature (21, 22, 23). These results demonstrate that trypsin, but not peptide Ag, alters the macromolecular structure of the cell membrane, and support the notion that the trypsin-dependent interaction of TCR with cognate Ag-MHC on the surface of a cell is associated with membrane reorganization.



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  		FIGURE 7. Ultrastructure of OT-1 CTL treated with antigenic peptide and/or trypsin. Transmission electron micrographs of OT-1 CTL (A and B), peptide-pulsed OT-1 CTL (C and D), trypsin-treated (45 min) OT-1 CTL (E and F), and peptide-pulsed and trypsin-treated CTL (G and H) are shown at low and high magnification; mv, microvilli.

 
Electron microscopic analysis of peptide-pulsed, trypsin-treated OT-1 CTL demonstrated the same microvillar loss apparent with trypsin alone (Fig. 7, G and H). It also showed typical apoptotic changes in treated cells, including chromatin condensation and nuclear disintegration. This histologically verifies the cell death observed in Figs. 2–4 and further identifies apoptosis as the mechanism of death.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrate that CTL pulsed with cognate antigenic peptide are resistant to TCR activation and self-lysis through a cell membrane-dependent mechanism. This resistance may be overcome by treatment of CTL with agents that disrupt cell surface charge, including trypsin, papain, tunicamycin, neuraminidase, and polybrene. These treatments permit TCR and CD8 cocapping with MHC, and promote cell suicide through a Fas- and perforin-dependent process.

Although CTL are highly resistant to lysis, they can undergo an Ag-dependent fratricidal death at the hands of other CTL. For example, Su et. al. (24) observed an ~10% per hour rate of CTL attrition due to fratricide after pulsing with high concentrations of peptide Ag. We did not observe any influence of trypsin treatment on fratricidal lysis, as indicated both in assays in which cells were tested after extreme dilution and assays measuring the lysis of CFSE-labeled, polyclonal, peptide-pulsed target CTL. This may imply that fratricidal lysis was not promoted by the treatments that we administered. Alternatively, the low efficiency of fratricidal lysis, background cell death due to incubation with trypsin, and the short time course of our assays (generally 45 min-1 h) may have prevented us from detecting enhanced fratricidal killing. In contrast to fratricidal lysis, which requires engagement of two cells and association of receptor and ligand across them, suicidal activation only requires ligand receptor association within a single membrane and therefore has a lower entropic barrier. Therefore, it would be expected that suicidal mechanisms would be more significantly enhanced than fratricidal ones by treatments that disrupt a membrane impediment to TCR engagement with its ligand.

Glycosylation, and specifically sialylation of cell membrane proteins, appears primarily responsible for protecting the CTL from self-activation caused by coexpression of the TCR and its Ag-MHC ligand. The importance of surface glycosylation in TCR activity has been demonstrated in other recent studies (25, 26). Galectin 1, a galactose-binding protein, has been found to be important in setting the TCR signaling threshold, and deficiency in its binding partner leads to autoimmune manifestations (27). Induction of deficiency of the ST3Gal-I sialyltransferase on naive CD8+ T cells using a loxP-flanked targeting vector induces their apoptosis, presumably through CD43 engagement or aggregation (28). Likewise, sialic acids present on the CD8 molecule have recently been found to be important in regulating its coreceptor activity and possibly TCR thresholds. Increased sialylation with T cell development decreases the affinity of CD8 for MHC (29, 30).

Our data do not support these documented roles for sialylation of specific cell surface proteins in preventing CTL self-activation and suicide. For CD8 and CD43, removal of sialic acid enhances protein function, and thereby decreases the triggering threshold. In contrast, in our studies, trypsin and papain were as effective in promoting CTL death as treatments that eliminated or neutralized sialic acid. Trypsin and papain would be expected to degrade specific proteins, inhibiting their function rather than increasing their activity. The effectiveness of these proteases implies that the treatments studied here allowing TCR-MHC association do not act to reduce the charge effects and thereby enhance the function of a specific protein involved in T cell activation, but have a more global effect. As all treatments are able to reduce total cell surface charge, or zeta potential, it seems likely that this charge, or physical properties of the membrane that it maintains, is responsible for preventing TCR/MHC association on the CTL surface.

The role of cell surface charge in maintaining cell surface integrity has not been well-studied. Cell surface charge may either act directly, forming a potential barrier that must be breeched. Alternatively, the charge barrier itself may be less important than the resulting structuring of H20 molecules. This may form an insulating barrier preventing membrane-membrane association. Sialic acid is an important contributor to net cell surface charge, and therefore would be expected to have a significant role in any such process.

Therefore, our results support a model in which sialic acid residues form a barrier that prevents TCR and MHC from interacting on a single cell. Considering our data (Fig. 7) and that of others showing that loss of this barrier alters membrane structure, we suspect that this potential barrier acts in part by preventing the cell membrane from folding upon itself in a manner that would permit such a ligand pair association. This barrier normally protects T cells that have captured cognate Ag from self-destruction and its disruption permits self-activation and CTL death. Additional studies will be required to better define how such a barrier forms, its biophysical properties, and the impact it has on T cell physiology and the association of other ligand-receptor pairs on the cell surface.


   Acknowledgments
 
We thank Dr. K. Gopal Murti for assistance with confocal and electron microscopy.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R21 AI49872 and P30 CA21765 (to T.L.G.) and by American Lebanese Syrian Associated Charities/St. Jude Children’s Research Hospital (to T.L.G. and D.J.M.). Back

2 Address correspondence and reprint requests to Dr. Terrence L. Geiger, Department of Pathology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, D-4047, Memphis, TN 38105. E-mail address: terrence.geiger{at}stjude.org Back

3 Abbreviations used in this paper: FasL, Fas ligand; EHAA, enhanced Eagle’s medium; CMA, concanamycin A. Back

Received for publication February 13, 2003. Accepted for publication July 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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