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The Journal of Immunology, 2005, 175: 5043-5049.
Copyright © 2005 by The American Association of Immunologists

Rapid Production of TNF-{alpha} following TCR Engagement of Naive CD8 T Cells1

Michael A. Brehm, Keith A. Daniels and Raymond M. Welsh2

Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The acquisition of effector functions by naive CD8 T cells following TCR engagement is thought to occur sequentially with full functionality being gained only after the initiation of division. We show that naive CD8 T cells are capable of immediate effector function following TCR engagement, which stimulates the rapid production of TNF-{alpha}. Stimulation of splenocytes from naive mice of differing genetic backgrounds with anti-CD3{epsilon} mAb resulted in significant production of TNF-{alpha} by naive CD8 T cells within 5 h. Moreover, naive lymphocytic choriomeningitis virus-specific TCR-transgenic CD8 T cells stimulated with either their cognate peptide ligand or virus-infected cells produced TNF-{alpha} as early as 2 h poststimulation, with production peaking by 4 h. Naive CD8 T cells produced both membrane-bound and soluble TNF-{alpha}. Interfering with TNF-{alpha} activity during the initial encounter between naive CD8 T cells and Ag loaded dendritic cells altered the maturation profile of the APC and diminished the overall viability of the APC population. These findings suggest that production of TNF-{alpha} by naive CD8 T cells immediately after TCR engagement may have an unappreciated impact within the local environment where Ag presentation is occurring and potentially influence the development of immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many CD8 T cells are important components of the acquired immune system for the control of intracellular pathogens (1, 2, 3, 4). Effector CD8 T cells have numerous mechanisms at their disposal to control invading pathogens, including perforin- and Fas ligand-dependent cytotoxic activity and cytokine production (5). The transition of naive CD8 T cells to effector cells is thought to be a sequential process, with activation-specific functions occurring in a designated order, and with the acquisition of full effector function only occurring after cell division commences (6, 7, 8, 9, 10, 11). The activation of naive CD8 T cells is initiated when an epitope-specific T cell engages its peptide ligand presented on an APC. This T cell interaction with the APC stimulates a programmed pathway of differentiation for the CD8 T cells, resulting in cell division and the acquisition of effector functions (12, 13, 14, 15, 16). Commitment of naive CD8 T cells to this programmed differentiation pathway requires only a brief TCR engagement with the peptide-MHC complex and can be as short as 2 h. Because CD8 T cells are committed to differentiation so early, environmental conditions at the site where the initial interaction between T cells and APC occurs could impact the generation of the T cell response.

The early phase of an immune response against infection is mediated predominantly by the innate arm of the immune system, which may initially control the infection and then influence the generation of the acquired immune response (17, 18, 19). The effector T cell response contributes later during infection, after naive CD8 T cells have proliferated and acquired full functionality. However, previous studies have suggested that mRNA encoding TNF-{alpha} rapidly accumulates within naive T cells from both mice and humans after their initial TCR engagement (20, 21, 22). T cell production of TNF-{alpha} early during the generation of an immune response could have important implications, as TNF-{alpha} is a potent inflammatory cytokine that can stimulate differentiation and proliferation but also induce cell death (23, 24, 25). Moreover, recent studies have demonstrated that CD8 T cell-produced TNF-{alpha} can mediate severe immunopathology associated with infection (26, 27).

In this study we have examined the ability of naive CD8 T cells to produce TNF-{alpha} within the first few hours of TCR engagement and evaluated the impact of TNF-{alpha} on the initial interaction between a T cell and an APC. We show that naive (CD44low or CD11alow) bulk or transgenic CD8 T cells from C57BL/6, BALB/c, and CBA mice produce TNF-{alpha} within 5 h of TCR engagement and that this TNF-{alpha} both altered the maturation of the dendritic cells and decreased their overall viability. These findings suggest that the production of TNF-{alpha} during the initial encounter between a naive CD8 T cell and an APC can alter the differentiation of the APC and potentially influence the nature of the T cell response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Male B6 mice (H2b), BALB/cJ (H2d), and CBA/J (H2k) were purchased from The Jackson Laboratory and used at 6–10 wk of age. TCR-LCMV-P14/Rag2 mice harboring transgenic CD8 T cells specific for the lymphocytic choriomeningitis virus (LCMV)3 epitope GP33-41 were purchased from Taconic Farms. B6.TNF-{alpha}–/– mice (B6;129S6-TNFtm1gk1) were originally purchased from The Jackson Laboratory and were then backcrossed to B6 mice for three generations before use. All mice were maintained under specific-pathogen free conditions within the Department of Animal Medicine at the University of Massachusetts Medical School (Worcester, MA). All experiments were done in compliance with institutional guidelines as approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.

Viruses and cells lines

LCMV, strain Armstrong, stocks were prepared in baby hamster kidney cells (BHK21) as previously described (28). Peritoneal exudate cells (PEC) were isolated by peritoneal lavage from mice that had been injected i.p. with thioglycolate medium (1 ml) 3 days previously. LCMV-infected PEC were generated by infecting mice with 5 x 104 PFU 1 day after injection of thioglycolate. Recovered PEC were gamma irradiated (2000 rad) and then stored frozen at –70°C. DC2.4 provided by K. Rock (Department of Pathology, University of Massachusetts Medical School, Worcester, MA) is an immortalized murine bone marrow-derived dendritic cell line and was maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 2 mM L-glutamine, 1% (v/v) nonessential amino acid solution (Sigma-Aldrich), and 50 µM 2-ME.

Synthetic peptides

LCMV-GP33-41 (KAVYNFATC) and LCMV-NP396-404 (FQPQNGQFI) peptides were generated by BioSource International. The peptide was purified with RP-HPLC to 90% purity.

Intracellular cytokine staining

Cytokine-producing CD8 T cells were detected using the Cytofix/Cytoperm Plus kit (with GolgiPlug; BD Pharmingen), as previously described (29). Splenocytes (2 x 106 cells) were incubated with the indicated concentration of anti-mouse CD3{epsilon} mAb (145-2C11; BD Pharmingen), 1 µM synthetic peptide or LCMV-infected PEC (5 x 105 stimulator cells per sample) in the presence of 10 U/ml human rIL-2 (BD Pharmingen), and 1 µl/ml GolgiPlug at 37°C/5% CO2 for either 5 h or the indicated time. Following the incubation splenocytes were stained with mAb specific for CD8 (53-6.7) and CD44 (IM7). Samples were then fixed and permeabilized with Cytofix/Cytoperm solution and stained with mAb specific for IFN-{gamma} (XMG1.2; BD Pharmingen) or TNF-{alpha} (MP6-XT22; BD Pharmingen), or with an IgG1 isotype control (R3-34; BD Pharmingen). P14-CD8 T cells were treated with actinomycin-D (Sigma-Aldrich), 20 µg/ml, or cycloheximide (Sigma-Aldrich), 5 µg/ml, as indicated. The samples were analyzed using a BD Biosciences FACSCalibur and CellQuest software (BD Biosciences). When shown, error bars are representative of SD.

Activation of P14-CD8 T cells

Single cell suspensions were prepared from the spleens of naive TCR-LCMV-P14 mice, and splenocytes were then stimulated for the indicated time with 1 µM GP33 peptide in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 2 mM L-glutamine, and 10 U/ml human rIL-2. Following the incubation, splenocytes were stained with mAb specific for CD8, CD44, CD69 (H1.2F3), and CD62 ligand (CD62L, MEL-14), and were then treated with Cytofix (BD Pharmingen). Samples were also stained for cell surface expression of TNF-{alpha} and IFN-{gamma}. The samples were analyzed as described.

Cytokine ELISA

Single cell suspensions were prepared from the spleens of naive P14 mice, and splenocytes were stimulated for 4 h with 1 µM GP33 peptide in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 2 mM L-glutamine, and 10 U/ml human rIL-2. Following the incubation, TNF-{alpha} and IFN-{gamma} levels were evaluated in the cell culture supernatants with a sandwich ELISA using a commercially available immunoassay kit (BioSource International).

DC2.4 maturation assays

For coculture experiments, DC2.4 cells were incubated with media containing 1 µM GP33 or no peptide for 1.5 h and were then washed to remove excess peptide. Splenocytes from naive P14 mice were labeled with 2 µM Cell Trace Far Red DDAO-SE (Molecular Probes), to allow discrimination from DC2.4 cells. Labeled P14 splenocytes were then added to the DC2.4 cells to achieve a CD8 T cell to DC2.4 ratio of 1:20, and cultures were incubated for 20 h at 37°C/5% CO2. Following the incubation, DC2.4 cells were first incubated with Fc block (2.4G2; BD Pharmingen) and were then stained with mAbs specific for the MHC class II molecule IAb (AF6-120.1; BD Pharmingen) and for CD80 (16-10A1; BD Pharmingen) for 30 min or stained with 7-aminoactinomycin D (7-AAD) and annexin V (both BD Pharmingen) to evaluate cell viability. Samples were then washed and treated with Cytofix. In some cases the cultures contained 1 x 10–7 M ENBREL (Immunex), which is a human TNFR2 (CD120b)-IgG1 fusion protein that has been demonstrated to inhibit murine TNF-{alpha} activity, or 12.5 µg/ml of a mAb specific for IFN-{gamma} (R4-6A2; eBioscience). For experiments in which supernatants from stimulated T cells were transferred to DC2.4 cultures, splenocytes (5 x 105 cells) from naive P14 mice were stimulated with 1 µM GP33 peptide or left unstimulated for 4 h in a total volume of 200 µl supplemented RPMI 1640. Following the incubation, the T cell-conditioned supernatants were added to 1 x 105 DC2.4 cells, and the cells were incubated for 12 h. DC2.4 cells were then harvested and stained as described for IAb or 7-AAD. In some cases the cultures contained 1 x 10–7 M ENBREL. The samples were analyzed as described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TCR engagement stimulates the rapid production of TNF-{alpha}

The capacity of naive CD8 T cells to rapidly synthesize cytokines after TCR engagement was examined using a mAb specific for CD3{epsilon}. Splenocytes from B6 mice were stimulated with varying concentrations of anti-CD3 mAb for 5 h, and then CD8 T cells were stained for intracellular TNF-{alpha} and IFN-{gamma} or the appropriate isotype controls, as described in Materials and Methods (Fig. 1A). Exposure to 250 ng/ml anti-CD3{epsilon} stimulated a substantial proportion of CD8 T cells to produce TNF-{alpha} but only a small population to produce IFN-{gamma}. These percentages decreased with the dilution of the anti-CD3{epsilon} mAb. We next examined the phenotype of CD8 T cells stimulated to produce TNF-{alpha} (Fig. 1B). Following stimulation of splenocytes from naive B6 mice with mAb specific for CD3{epsilon}, CD44low or naive phenotype CD8 T cells were stimulated to produce TNF-{alpha} but not IFN-{gamma}. CD44high or memory phenotype CD8 T cells were able to produce TNF-{alpha} and IFN-{gamma}. Culturing of splenocytes from naive mice of varying genetic backgrounds, including BALB/c and CBA mice, with mAb specific for CD3{epsilon} also stimulated the synthesis of TNF-{alpha} by a substantial proportion of naive phenotype CD8 T cells (Fig. 1C). TNF-{alpha} production was not detectable following TCR engagement of CD8 T cells from B6.TNF-{alpha} knockout mice, confirming the specificity of the anti-TNF Ab used for these studies. These results show that CD3 engagement on naive CD8 T cells is sufficient to stimulate the rapid production of TNF-{alpha}.



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  		FIGURE 1. Naive CD8 T cells rapidly produce TNF-{alpha} but not IFN-{gamma} after TCR engagement. A and B, Splenocytes from naive B6 mice were stimulated for 5 h with the indicated concentrations of mAb specific for CD3{epsilon}, as described in Materials and Methods, and then stained for cell surface markers and for intracellular cytokines. For analysis, samples were gated on CD8-positive cells, and values (inset) represent either the percentage of CD8 T cells (A) staining positive for TNF-{alpha} and/or IFN-{gamma} or the percentage of CD44low or CD44high cells (B) staining positive for either cytokine. C, Splenocytes from naive B6, BALB/c, CBA, or B6.TNF-{alpha}–/– mice were stimulated for 5 h with 250 ng/ml mAb specific for CD3{epsilon} and then stained for cell surface markers and for intracellular cytokines. For analysis, cells were gated on naive CD8 T cells (CD44low for B6 and CBA, and CD11alow for BALB/c). Graphs are representative of an average of three mice and the error bars represent ±SD. The data are representative of three experiments.

 
Naive virus-specific CD8 T cells rapidly produce TNF-{alpha} after recognition of cognate ligand

We next evaluated whether naive CD8 T cells would be stimulated to rapidly produce TNF-{alpha} by their cognate peptide ligand. Splenocytes from naive TCR-LCMV-P14 mice were incubated with either no peptide or 1 µM GP33 for 5 h, and then CD8 T cell cytokine expression was evaluated (Fig. 2A). Incubation with the GP33 peptide stimulated a large proportion of P14-CD8 T cells to synthesize TNF-{alpha} with low levels of IFN-{gamma} within the 5-h period. As observed with anti-CD3{epsilon} stimulation (Fig. 1), naive phenotype, CD44low P14-CD8 T cells produced TNF-{alpha} but only had a limited capacity to produce IFN-{gamma} following stimulation with GP33 (Fig. 2B). In contrast, memory phenotype CD44high P14-CD8 T cells were able to produce both TNF-{alpha} and IFN-{gamma}. An irrelevant peptide, LCMV-NP396, did not stimulate cytokine production (0.6% P14-CD8 T cells producing TNF-{alpha}). To confirm that a physiologically relevant level of Ag that would be presented by a virus-infected APC is sufficient to stimulate TNF-{alpha} expression by P14-CD8 T cells, splenocytes from naive TCR-LCMV-P14 mice were incubated with either uninfected or LCMV-infected syngeneic PEC for 5 h (Fig. 2C). A large proportion of P14-CD8 T cells produced TNF-{alpha} following stimulation with LCMV-infected PEC but not with uninfected PEC. Thus, naive virus-specific CD8 T cells can rapidly produce TNF-{alpha} following stimulation with a physiologically relevant level of Ag.



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  		FIGURE 2. Naive virus-specific CD8 T cells rapidly produce TNF-{alpha} but not IFN-{gamma} after TCR engagement. A and B, Splenocytes from naive TCR-LCMV-P14 mice were stimulated for 5 h with either GP33 or no peptide, as described in Materials and Methods, and then stained for cell surface markers and for intracellular cytokines. For analysis, samples were gated on CD8-positive cells, and values (inset) represent either the percentage of CD8 T cells (A) staining positive for TNF-{alpha} and/or IFN-{gamma} or the percentage of CD44low or CD44high cells (B and C) staining positive for either cytokine. C, Splenocytes from naive TCR-LCMV-P14 mice were stimulated for 5 h with either uninfected PEC or LCMV-infected PEC, as described in Materials and Methods, and then stained for cell surface markers and for intracellular cytokines. The data are representative of three experiments.

 
We next evaluated the kinetics for TNF-{alpha} production by P14-CD8 T cells following stimulation with the GP33 peptide. Splenocytes from naive TCR-LCMV-P14 mice were incubated with 1 µM GP33 for the indicated times, and then CD8 T cell cytokine expression was evaluated (Fig. 3A). TNF-{alpha} production was first detectable at low levels as early as 1 h after stimulation. The number of P14-CD8 T cells synthesizing TNF-{alpha} continued to increase thereafter and peaked after 4 h of stimulation. In separate experiments, splenocytes from TCR-LCMV-P14 mice were stimulated with 1 µM GP33 without brefeldin A. At the indicated time points the cell surface expression of the early activation makers CD69 and CD62L were evaluated on P14-CD8 T cells (Fig. 3B). CD69 was up-regulated on a majority of P14-CD8 T cells by 2 h following GP33 stimulation. CD62L down-regulation was initially detectable by 1 h after stimulation, and a majority of P14-CD8 T cells have lost expression by 2 h. To examine the requirement of transcription for TNF-{alpha} production, P14-CD8 T cells were treated with actinomycin D. Actinomycin D-treated P14-CD8 T cells did not produce TNF-{alpha} following stimulation with GP33 peptide (Fig. 4). Similarly, P14-CD8 T cells treated with cycloheximide to block protein synthesis did not produce TNF-{alpha}. Thus, the peak of TNF-{alpha} production by CD8 T cells occurs shortly after changes in the surface expression of early markers of activation, but transcription is required for synthesis.



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  		FIGURE 3. Kinetics of TNF-{alpha} production by P14-CD8 T cells. A, Splenocytes from naive TCR-LCMV-P14 mice were stimulated for the indicated times with GP33 in the presence of brefeldin A, as described in Materials and Methods, and then stained for cell surface markers and for intracellular cytokines. For analysis, samples were gated on CD8-positive cells and plotted as the percentage of CD44low CD8 T cells staining positive for TNF-{alpha} or as the MFI of the TNF-{alpha} stain. Each point is an average of four mice. B, Splenocytes from naive TCR-LCMV-P14 mice were stimulated for the indicated times with GP33 in the absence of brefeldin A, as described in Materials and Methods, and then stained for the CD69 or CD62L. Each point is an average of three mice. The data are representative of three experiments.

 


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  		FIGURE 4. Inhibitors of RNA or protein synthesis block TNF-{alpha} production by naive P14-CD8 T cells. Splenocytes from naive TCR-LCMV-P14 mice were pretreated with actinomycin D (20 µg/ml) or cycloheximide (5 µg/ml) for 30 min, and GP33 peptide was then added to a concentration of 1 µM. Samples were incubated for 4 h in the presence of brefeldin A and IL-2 and then stained for cell surface markers and for intracellular cytokines. For analysis, samples were gated on CD8-positive cells, and the values represent the percentage of CD8 T cells staining positive for TNF-{alpha} and/or IFN-{gamma}. The data are representative of two experiments.

 
Naive CD8 T cells produce both membrane-bound and soluble TNF-{alpha}

TNF-{alpha} exists in two active forms, a membrane-bound form and a soluble form (30). We examined the ability of naive CD8 T cells to produce both forms of TNF-{alpha} during the first 4 h of TCR engagement. Splenocytes from naive TCR-LCMV-P14 mice were stimulated with 1 µM GP33 without brefeldin A for 4 h. Supernatants were then harvested, and the cells were stained for surface expression of TNF-{alpha} and IFN-{gamma}. TNF-{alpha} but not IFN-{gamma} was detectable on the cell surface of P14-CD8 T cells that were stimulated with GP33 peptide (Fig. 5A). This is in contrast to the detection of intracellular IFN-{gamma} in stimulated P14-CD8 T cells (Fig. 2). TNF-{alpha} and IFN-{gamma} were not detectable on the surface of unstimulated cells. TNF-{alpha} was also detectable in the culture supernatants of GP33-stimulated P14-CD8 T cells, but not in supernatants from unstimulated T cells (Fig. 5B). IFN-{gamma} was not detectable in the supernatants from peptide stimulated P14-CD8 T cells at levels above the limit of detection (1 pg/ml). These findings indicated that TNF-{alpha} produced by naive CD8 T cells can be both on the surface of the T cells and secreted.



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  		FIGURE 5. P14-CD8 T cells produce both membrane-bound and soluble TNF-{alpha}. Splenocytes (5 x 105) from naive TCR-LCMV-P14 mice were stimulated for 4 h with GP33, as described in Materials and Methods. A, Following the incubation, the unpermeabilized cells were stained for cell surface expression of TNF-{alpha} or IFN-{gamma}. Samples were gated on CD8-positive cells, and the values (inset) represent the percentage of CD8 T cells staining positive for TNF-{alpha} or IFN-{gamma}. B, Soluble TNF-{alpha} levels were determined in the culture supernatants by ELISA, and the results from three individual mice are shown for each stimulation. The limit of detection for TNF-{alpha} is 3 pg/ml. The data are representative of three experiments.

 
TNF-{alpha} interferes with T cell-induced maturation of APC

TNF-{alpha} is a pleiotropic cytokine that has been demonstrated to stimulate cell death as well as cell differentiation (25). The rapid production of TNF-{alpha} by naive CD8 T cells during their initial encounter with an APC may have an important effect in the local environment by altering the maturation of the APC. To examine the impact of TNF-{alpha} during the interaction between a naive CD8 T cell and an APC, splenocytes from naive TCR-LCMV-P14 mice were cocultured with DC2.4 cells, an immortalized dendritic cell line that expresses low levels of MHC class II molecules, which had been either pulsed with 1 µM GP33 or left unpulsed. To assess the impact of TNF-{alpha}, ENBREL was added to some cultures. The DC2.4 cells were cocultured with the P14-CD8 T cells for 20 h and were then evaluated for expression of the class II MHC molecule IAb. Unpulsed DC2.4 cells that were cultured with P14-CD8 T cells did not up-regulate IAb (Fig. 6A). In contrast, coculturing of P14-CD8 T cells with GP33-coated DC2.4 cells resulted in increased expression of IAb on the surface of the DC2.4 cells. The increase in IAb expression was attributed to IFN-{gamma} produced during the coculture, as adding a neutralizing mAb specific for IFN-{gamma} substantially blocked IAb up-regulation (Fig. 6B). Blocking TNF-{alpha} activity with ENBREL during the coculture of P14-CD8 T cells with GP33-coated DC2.4 cells enhanced the expression of IAb on the DC2.4 cells (Fig. 6A) relative to expression levels in the absence of ENBREL. However when IFN-{gamma} was neutralized, the addition of ENBREL had no effect on class II expression (Fig. 6B). Coculturing of GP33-coated DC2.4 cells with P14-CD8 T cells resulted in increased expression of CD80 (B7-1) on the surface of the dendritic cell line, and ENBREL did not affect this up-regulation (mean fluorescence intensity (MFI): No peptide = 113 ± 0.6, GP33 = 322 ± 12, GP33 and ENBREL = 319 ± 14). These results suggest that TNF-{alpha} produced during the early phase of the interaction between a T cell and an APC can alter the maturation of the APC by interfering with IFN-{gamma}-dependent up-regulation of class II MHC.



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  		FIGURE 6. TNF-{alpha} can impede the T cell-induced maturation of dendritic cells. DC2.4 dendritic cells were either untreated or pulsed with 1 µM GP33 peptide for 1.5 h and then washed to remove excess peptide. Splenocytes from naive TCR-LCMV-P14 mice were labeled with Cell Trace Far Red DDAO-SE, to allow discrimination from DC2.4 cells. The labeled splenocytes were added to the DC2.4 cells at a ratio of 1:20 P14-CD8 T cell to DC2.4 cells and then incubated for 20 h. In some samples 1 x 10–7 M ENBREL (A and C) and/or a mAb specific for IFN-{gamma} (B) were added for the length of the coculture. After the incubation DC2.4 cells were stained with mAb to MHC class II, IAb (A and B) or with 7-AAD (C). Values (inset) represent the percentage of DC2.4 cells expressing IAb, and the MFI for each population is at the bottom of the histogram (A and B). The data are representative of three experiments.

 
TNF-{alpha} production reduces viability of APC

TNF-{alpha} can induce cell death under certain conditions, and therefore we examined the viability of DC2.4 cells that had been cocultured with P14-CD8 T cells as earlier described using 7-AAD, which can bind cellular DNA in dying cells with permeable membranes. Unpulsed DC2.4 cells that were cultured with P14-CD8 T cells for 20 h had a low level of 7-AAD staining (Fig. 6C). In contrast, DC2.4 cells that had been pulsed with GP33 and cocultured with P14-CD8 T cells had the highest uptake of 7-AAD, and this was reduced when ENBREL was added to the cultures to block TNF-{alpha} activity (Fig. 6C). GP33-coated DC2.4 cells that were cultured with P14-CD8 T cells in the absence or presence of ENBREL showed no difference in annexin V binding (annexin V-positive cells: No peptide = 5.1 ± 1.1, GP33 = 8.3 ± 0.6, GP33 and ENBREL = 8.5 ± 0.9). TNF-{alpha} has been previously demonstrated to decrease the cell membrane integrity of cell lines, resulting in increased uptake of 7-AAD without increasing annexin V binding (31), and this phenotype would be consistent with the DC2.4 cells being in an early phase of necrosis (32, 33). Overall these results suggest that TNF-{alpha} signaling during interactions between CD8 T cells and APC can reduce the viability of the APC.

The findings presented in Fig. 6 demonstrate an important role for TNF-{alpha} during the encounter of a naive CD8 T cell with an APC, but do not address whether soluble TNF-{alpha} or membrane bound TNF-{alpha} is driving the changes in DC2.4 cells. To determine whether soluble TNF-{alpha} produced by CD8 T cells is sufficient to stimulate the effects on DC2.4 cells, CD8 T cells from TCR-LCMV-P14 mice were stimulated with GP33 peptide for 4 h, and then the T cell-conditioned supernatants were transferred onto DC2.4 cultures. The DC2.4 cells were cultured in the supernatants for 12 h and were then evaluated for expression of IAb and for 7-AAD staining. Supernatants from unstimulated P14-CD8 T cells did not increase the expression of IAb on the DC2.4 cells, but supernatants from GP33-stimulated P14-CD8 T cells did increase expression (Fig. 7A). As with the coculture experiments, blocking TNF-{alpha} activity by adding ENBREL to the supernatants from GP33-stimulated P14-CD8 T cells further enhanced IAb expression. Moreover, blocking TNF-{alpha} increased the viability of the DC2.4 cells when supernatants from activated P14-CD8 T cells were added to the cultures, as indicated by the decrease in 7-AAD staining (Fig. 7B). No differences in annexin V binding were observed between samples that were treated with ENBREL and samples that were not (annexin V-positive cells: No peptide = 6.6 ± 0.6, GP33 = 10.4 ± 0.5, GP33 and ENBREL = 11.6 ± 0.2). These results suggest that soluble TNF-{alpha} can interfere with APC maturation during T cell activation and reduce the viability of the APC.



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  		FIGURE 7. TNF-{alpha} from T cell-conditioned supernatants impedes maturation of dendritic cells. Splenocytes (5 x 105 cells) from naive TCR-LCMV-P14 mice were either unstimulated or stimulated for 4 h with 1 µM GP33, as described in Materials and Methods. ENBREL was included in some cultures at 1 x 10–7 M. After the incubation, supernatants were transferred to DC2.4 cultures (1 x 105 cells), and the cultures were then incubated for 12 h. Following the incubation, DC2.4 cells were stained with mAb to MHC class II, IAb (A) or with 7-AAD (B). The values (inset) represent the percentage of DC2.4 cells expressing IAb, and the MFI for each population is at the bottom of the histogram (A). The data are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The contribution of CD8 T cells to immunity against pathogens is expected to be mediated during the adaptive phase of the immune response. It is at this point that the T cells have increased in number and have a full repertoire of effector functions that can be used to aid in the resolution of the infection. However, we show that naive CD8 T cells can rapidly synthesize TNF-{alpha} within a few hours of TCR engagement. The production of TNF-{alpha} by CD8 T cells was stimulated by engagement of the CD3 complex by Ab or by peptide-MHC complexes, and was observed in three different mouse strains. TNF-{alpha} production by CD8 T cells early during the interaction between T cells and APC would introduce a powerful inflammatory cytokine into the local environment during the initial generation of the immune response. Our results show that the presence of TNF-{alpha} during the first few hours of T cell engagement can alter APC maturation and decrease the overall viability of the APC.

The unexpected production of TNF-{alpha} by naive phenotype CD8 T cells suggests that T cells are capable of immediate effector function after recognition of their cognate ligand. This production of TNF-{alpha} by naive CD8 T cells was first detectable as early as 1 h after TCR engagement. Effector and memory CD8 T cells also rapidly produce cytokines such as TNF-{alpha} and IFN-{gamma}, and this cytokine production is tightly regulated by the presence of Ag (34, 35, 36, 37). Prior studies have suggested that TCR engagement on naive CD4 T cells stimulates splicing of pre-mRNA complexes that encode TNF-{alpha} to a mature form of mRNA, and this activity was independent of transcription (22). Thus the ability of naive CD8 T cells to rapidly produce TNF-{alpha} may be attributed to a splicing event that would be independent of transcription. However our data suggest that blocking transcription in naive CD8 T cells abrogates the production of TNF-{alpha} following Ag stimulation.

The ability of naive CD8 T cells to rapidly produce TNF-{alpha} following TCR engagement may have important implications during the initial phase of the immune response, as T cells encounter their cognate ligands for the first time. However, because the frequency of CD8 T cells specific for any individual epitope is extremely low in a naive animal (38), T cell-produced TNF-{alpha}, both membrane-bound and soluble forms, may have a greater effect within the local environment rather than a global impact. The early signals that a naive T cell receives during an encounter with Ag have been demonstrated to initiate a programmed pathway of T cell activation (13, 14, 15), and the presence of TNF-{alpha} within the local environment could influence this programming. Moreover, recent studies have demonstrated that CD8 T cells and Ag-presenting dendritic cells form contacts that can persist for several hours in vivo (7, 39), and TNF-{alpha} produced during this long-lived interaction would be optimally localized to mediate an effect on the APC.

TNF-{alpha} is a pleiotropic cytokine that can induce a range of effects, including cellular proliferation, differentiation, and death, and it can also contribute to the control of pathogens (40). Studies with TNF-{alpha} knockout mice and mice lacking TNFR have shown that TNF-{alpha} is important for the control of some bacterial and viral infections (41, 42, 43, 44, 45, 46, 47). Many types of cells can produce TNF-{alpha} including macrophages, NK cells, T cells, and B cells (23). Interestingly, TNF-{alpha} produced by T cells can have dramatic effects on a host. Pretreatment of transplant recipients with anti-CD3 Abs to delete T cells and prolong graft survival has resulted in severe TNF-mediated reactions in the host (48, 49, 50). Moreover, recent studies have shown that TNF-{alpha} made by CD8 T cells can contribute to immunopathology in the lung following infection with influenza (27) and to the induction of autoimmune hepatitis by Con A (26).

Our results suggest that TNF-{alpha} produced during the initial stages of an interaction between a CD8 T cell and an APC can affect the differentiation of the APC. TNF-{alpha} diminished the IFN-{gamma}-induced up-regulation of MHC class II molecules on a peptide-coated dendritic cell line that was cocultured with naive P14-CD8 T cells. GP33-coated DC2.4 cells that were cocultured with P14-CD8 T cells, in the presence or absence of ENBREL, up-regulated CD80 to a similar extent, suggesting that class II expression was being specifically affected by TNF-{alpha}. Prior studies have shown that TNF-{alpha} can interfere with IFN-{gamma} up-regulation of MHC class II in a number of cell lines (51, 52). One mechanism by which TNF-{alpha} can abrogate the IFN-{gamma} enhancement of MHC class II expression is by suppressing the transcription of a trans-activator protein (or CIITA) that is critical for the expression of class II molecules (53). Further study will be necessary to evaluate the impact of TNF-{alpha} produced by naive CD8 T cells on APC in vivo during the initial phase of Ag recognition.

A recent study has proposed that the initial signals that an APC receives from the environment will start in motion a molecular "timer" that will limit the lifespan of the APC through a balance of proapoptotic and antiapoptotic proteins (54). In addition, during the first few days of an immune response against intracellular pathogens, APC are lost in vivo after interactions with naive T cells (55, 56, 57, 58). Together these findings indicate that the initial signals that an APC receives from the surrounding environment will influence their maturation and survival. A recent study found that mice deficient in TNFRI and TNFRII (p55R and p75R, respectively) were able to control an infection with LCMV but generated significantly higher frequencies of virus-specific CD8 T cells compared with wild-type mice during the acute phase of infection and in memory (45). These findings, along with our data would suggest that TNF-{alpha} may play a role in limiting the magnitude of T cell responses to infection. Our data suggest that TNF-{alpha} present during the encounter between a CD8 T cell and an APC would decrease the viability of the APC, contributing to their loss in vivo and would have important implications for the generation of adaptive immune responses.


   Acknowledgments
 
We thank Dr. Liisa K. Selin and Lee Wilkinson for providing backcrossed B6.TNF-{alpha}–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health research Grants AI-17672, AR-35506, AI-46629, and AI-42669, by a Charles A. King Trust Fellowship from the Medical Foundation (to M.A.B.), and an institutional Diabetes Endocrinology Research Center Grant DK52530. The contents of this publication are solely the responsibility of the authors and do not represent the official views of the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Raymond M. Welsh, Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: raymond.welsh{at}umassmed.edu Back

3 Abbreviations used in this paper: LCMV; lymphocytic choriomeningitis virus; PEC, peritoneal exudate cell; MFI, mean fluorescence intensity; 7-AAD, 7-aminoactinomycin D; CD62L, CD62 ligand. Back

Received for publication March 21, 2005. Accepted for publication August 3, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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