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Originally published online as doi:10.1189/jlb.0407240 on August 17, 2007

Published online before print August 17, 2007
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(Journal of Leukocyte Biology. 2007;82:1201-1211.)
© 2007 by Society for Leukocyte Biology

A role for TNF in limiting the duration of CTL effector phase and magnitude of CD8 T cell memory

Anju Singh and M. Suresh1

Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA

1 Correspondence: Department of Pathobiological Sciences, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA. E-mail: sureshm{at}svm.vetmed.wisc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is known that TNF-{alpha} (TNF) exerts distinct tissue-protective or -destructive effects in the pathogenesis of T cell-dependent immunopathology, depending on the context and amount of cytokine produced. To better understand the cellular mechanisms underlying the regulation of T cells by TNF, we have analyzed the role of TNF in regulating various facets of the antigen-specific CD8 T cell response to lymphocytic choriomeningitis virus (LCMV) in mice. We show that expansion and differentiation of virus-specific effector CD8 T cells and LCMV clearance are not dependent on TNF. Instead, we demonstrate that TNF limits the duration of the effector phase of the CD8 T cell response by regulating apoptosis and not proliferation of effector cells in vivo. We further show that attenuation of effector cell apoptosis induced by TNF deficiency led to a substantial increase in the number of virus-specific memory CD8 T cells without affecting their function. The enhancement in the number of memory CD8 T cells in TNF-deficient (TNF–/–) mice was not associated with up-regulation of IL-7R{alpha} or Bcl-2 in effector cells, which indicated that TNF might limit differentiation of memory cells from IL-7Rlo effector cells. Collectively, these data are strongly suggestive of a role for TNF in down-regulating CD8 T cell responses and the establishment of CD8 T cell memory during an acute viral infection. These findings further our understanding of the regulation of CD8 T cell homeostasis and have implications in vaccine development and clinical use of anti-TNF therapies to treat T cell-dependent, inflammatory disorders.

Key Words: homeostasis • virus • apoptosis • immunity • proliferation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TNF-{alpha} (TNF) is a pleiotropic cytokine produced by immune cells, including neutrophils, macrophages, NK cells, and T cells [1 ]. The immune-regulatory effects of TNF in host-protective immune reactions versus the pathogenesis of T cell-dependent autoimmune diseases could be considered as a "double-edged sword", depending on the quantity, duration, and timing of cellular TNF production [2 ]. When produced at physiological levels, TNF plays an important role in the establishment and maintenance of cellular architecture in secondary lymphoid organs [3 , 4 ] and host defense against certain viruses and intracellular bacteria [5 6 7 8 9 10 11 12 ]. However, over-expression or under-expression of TNF could have deleterious consequences: Although hyperinduction of TNF causes severe tissue damage, which is associated with peptide immunization of immune mice [13 ] and chronic inflammatory disorders, such as rheumatoid arthritis (RA) [14 , 15 ] and Crohn’s inflammatory bowel disease (IBD) [16 , 17 ], under-expression of TNF might be the underlying defect in autoimmune diseases such as multiple sclerosis (MS) [16 ], Type 1 diabetes [18 , 19 ], and systemic lupus erythematosus [20 ]. Therefore, systemic blockade of TNF activity ameliorates inflammation and clinical symptoms in RA and IBD patients but not in MS patients [2 , 14 , 15 , 17 , 21 ]. Moreover, therapeutic inhibition of TNF has been reported to induce active disease in MS patients [22 , 23 ]. Thus, host-protective, beneficial effects are elicited when TNF is produced at a certain threshold level; production of TNF at supra- or subthreshold levels enhances inflammation and T cell-dependent autoimmunity, respectively. The cellular and molecular mechanisms underlying the regulation of T cell responses by TNF are unclear.

Studies using the mouse model of lymphocytic choriomeningitis virus (LCMV) infection have provided seminal insights into the mechanisms of CD8 T cell immunity. Like other acute viral infections, LCMV induces a potent CD8 T cell response, which clears infectious virus by Days 8–10 postinfection (PI) [24 , 25 ]. At the peak of the primary response (Day 8 PI), LCMV-specific CD8 T cells constitute up to 90% of the activated CD8 T cells in the spleen [26 27 28 ]. Between Days 8 and 30 PI, 90–95% of the expanded LCMV-effector CD8 T cells are eliminated by apoptosis [27 , 29 ]; the remainder of virus-specific, effector CD8 T cells differentiates gradually into long-lived, memory T cells, which provide lifelong immunity to reinfection [24 , 25 ]. The number of memory T cells generated at the conclusion of a T cell response is a function of the extent of clonal expansion (clonal burst size) minus the apoptosis (clonal downsizing) [24 , 27 , 30 ]. The mechanism(s), which regulate the apoptosis of effector CD8 T cells, have been a topic of intense investigation by several groups. There is increasing evidence that in addition to their well-recognized function as effector molecules of CD8 T cells [31 ], perforin, IFN-{gamma}, and TNF also might down-regulate CD8 T cell responses by inducing apoptosis [32 33 34 ]. TNF and lymphotoxin (LT), the cytokine ligands for TNFRs, are known to induce apoptosis of activated T cells [32 , 35 , 36 ]. However, the relative importance of TNF versus LT in down-regulating CD8 T cell responses is yet to be determined.

To better define how TNF regulates CD8 T cell responses, we have compared CD8 T cell responses to LCMV between wild-type (+/+) and TNF-deficient (TNF–/–) mice. We show that TNF deficiency did not affect the primary expansion of LCMV-specific CD8 T cells significantly but prolonged the effector phase of the CD8 T cell response and enhanced the number of memory CD8 T cells. Further, we demonstrate that the net enhancement in the number of LCMV-specific memory CD8 T cells was associated with reduced apoptosis and not increased proliferation of effector CD8 T cells during the contraction phase. These findings should have implications in vaccine development and might explain the disease-enhancing effect of anti-TNF therapies in patients with T cell-dependent immunopathology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Wild-type C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). TNF–/– mice on the C57BL/6 background, kindly provided by Dr. Lloyd Old (Memorial-Sloan Kettering Cancer Centre, NY, NY, USA) [37 ], were bred at the University of Wisconsin-Madison (WI, USA). All mice were used at 6–8 weeks of age, and experiments were conducted in accordance with the approved institutional animal welfare guidelines.

Virus
The Armstrong strain of LCMV (LCMV-Arm) was used in this study. Mice were infected with 2 x 105 PFU LCMV by i.p. injection, and virus titers in the tissues were quantitated by a plaque assay using Vero cell monolayers [38 ].

CTL assay
The MHC I-restricted, LCMV-specific CTL activity in spleens was assessed directly ex vivo by a standard 51Cr-release assay using virus-infected and uninfected MC57 target cells [38 ].

Quantitation of LCMV-specific CD8 T cells by flow cytometry
MHC I (Db) tetramers, which are specific to the LCMV CTL epitopes NP396-404, GP33-41, and GP276-285, were prepared as described elsewhere [27 ]. All antibodies were purchased from BD PharMingen (San Diego, CA, USA) unless mentioned otherwise. Single-cell suspensions of splenocytes were prepared by standard procedures. Mononuclear cells from liver and lungs were isolated as described elsewhere [39 ]. Erythrocyte-depleted, single-cell suspensions of splenocytes or mononuclear cells from liver and lungs were stained with anti-CD8, anti-CD44, and MHC I tetramers at 4ºC for 1 h. In some experiments, cells were costained with anti-L selectin (CD62L), anti-CD127, anti-LFA-1, and anti-CD122, along with anti-CD8 and MHC I tetramers. After staining, cells were fixed in 2% paraformaldehyde and acquired on a FACSCalibur flow cytometer (Becton Dickinson, San Franscisco, CA, USA). Flow cytometry data were analyzed using the FlowJo (Tree Star, Ashland, OR, USA) or CellQuest (Becton Dickinson) software.

Intracellular staining for cytokines and Bcl-2
To assess cytokine production by LCMV-specific CD8 T cells, splenocytes were stimulated with LCMV epitope peptides in the presence of brefeldin A for 5 h. After culture, cells were stained for cell surface CD8 and intracellular IFN-{gamma} and IL-2 using the Cytofix/cytoperm intracellular staining kit (BD PharMingen). The number of epitope-specific IFN-{gamma}- and IL-2-producing CD8 T cells was quantitated by flow cytometry. To quantitate Bcl-2 levels in LCMV-specific CD8 T cells, splenocytes were stained with anti-CD8, MHC I tetramers, and anti-Bcl-2 using the Bcl-2 staining kit (BD PharMingen).

Quantitation of apoptotic cells directly ex vivo by Annexin V staining
Splenocytes were stained with ant-CD8, MHC I tetramers, and Annexin V (BD PharMingen) directly ex vivo [29 , 40 ]. The percentages of Annexin Vhi LCMV-specific CD8 T cells were quantitated by flow cytometry.

Intracellular staining to detect BrdU incorporation by antigen-specific CD8 T cells
Mice were administered BrdU in drinking water (0.8 mg/ml) for 8 days at different intervals during the expansion and contraction phases of the CD8 T cell response to LCMV. On the 8th day, after the initiation of BrdU treatment, splenocytes were stained with anti-CD8, MHC I tetramers, and anti-BrdU using the BrdU staining kit (BD PharMingen). The percentages of BrdU+ve cells were quantitated by flow cytometry [40 ].

Statistical analysis
Experimental data were analyzed using the commercially available statistical software (SYSTAT, Version 10.2, Chicago, IL, USA). Groups were compared by Student’s t-test, and significance was defined at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Primary expansion of CD8 T cells in TNF–/– mice
Adult, immunocompetent mice mount a potent CD8 T cell response and clear infectious LCMV within 8–10 days PI [24 , 27 ]. The peak of CD8 T cell expansion occurs on Day 8 PI when LCMV-specific CD8 T cells constitute ~90% of activated CD8 T cells in the spleen [26 27 28 ]. To investigate the effect of TNF deficiency on activation and expansion of antigen-specific CD8 T cells, we infected groups of +/+ and TNF–/– mice with LCMV-Arm. On the 8th day after infection, the number of CD8 T cells in the spleen, which are specific to the three Db-restricted epitopes NP396, GP33, and GP276, was quantitated by staining with MHC I tetramers. As shown in Figure 1A , the absolute numbers of NP396-, GP33-, and GP276-specific CD8 T cells in spleens of TNF–/– mice were ~1.5-fold higher (P<0.05 for NP396 and P<0.08 for GP33) than in +/+ mice. Thus, the primary CD8 T cell response to LCMV-Arm is not dependent on TNF.


Figure 1
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  		Figure 1. Primary expansion of CD8 T cells in TNF–/– mice. Groups of wild-type (+/+) and TNF–/– mice were infected with LCMV-Arm, and on the 8th day PI, the CD8 T cell response to LCMV was quantitated in the spleen. (A) Splenocytes were stained with anti-CD8{alpha}, anti-CD44, and Db NP396-404, Db GP33-41, or Db GP276-286 MHC I tetramers. (B and C) Splenocytes were stimulated with the indicated LCMV epitope peptides for 5 h ex vivo and stained for cell surface CD8 and intracellular IFN-{gamma}. (A and B) The total number of LCMV-specific CD8 T cells in spleens of +/+ and TNF–/– mice based on MHC I tetramer staining (A) or IFN-{gamma} production (B). Each symbol represents an individual mouse, and data are from two independent experiments. (C) The immunodominance hierarchy of the primary CD8 T cell response in +/+ and TNF–/– mice. Data are the calculated percentages of CD8 T cells specific to individual epitopes amongst total LCMV-specific CD8 T cells (combined all epitopes); data are the mean of three mice/group from one of two independent experiments.

 
Next, we determined whether TNF deficiency affected the cytokine-producing ability of CD8 T cells specific to the immunodominant (NP396 and GP33) and subdominant (GP276, GP118, and NP205) epitopes by intracellular cytokine staining for IFN-{gamma}. CD8 T cells from +/+ and TNF–/– mice produced comparable levels of IFN-{gamma} upon antigenic stimulation ex vivo; the mean fluorescence intensities (MFI) of staining for intracellular IFN-{gamma} were comparable between +/+ and TNF–/– LCMV-specific CD8 T cells (data not shown). The total numbers of epitope-specific, IFN-{gamma}-producing CD8 T cells in spleens of TNF–/– mice were slightly higher than in +/+ mice (Fig. 1B) . In addition, to determine whether TNF deficiency affected the immunodominance hierarchy of the CD8 T cell response to LCMV, we calculated the relative proportions of CD8 T cells specific to each epitope amongst total LCMV-specific CD8 T cells (Fig. 1C) . Data in Figure 1C clearly show that the immunodominance hierarchy of the anti-LCMV CD8 T cell response in TNF–/– mice was similar to +/+ mice. Taken together, these data suggested that TNF deficiency did not affect the IFN-{gamma}-producing ability or the immunodominance hierarchy of LCMV-specific CD8 T cells. To determine whether TNF deficiency affected LCMV clearance, we quantified infectious LCMV in liver and lungs of +/+ and TNF–/– mice on Day 8 PI. Infectious LCMV was undetectable (data not shown) in +/+ and TNF–/– mice (n=6), which was consistent with the potent CD8 T cell response in both groups of mice.

Persistence of CD8 T cell effector function in TNF–/– mice
The clearance of LCMV is dependent on MHC I-restricted, perforin-dependent, cell-mediated cytotoxicity [41 ]. Here, we have studied the kinetics of direct ex vivo CTL activity of LCMV-specific effector CD8 T cells following an acute LCMV infection. As illustrated in Figure 2 , the CTL activity in the spleen of TNF–/– mice was comparable with +/+ mice on Day 8 PI. However, on Days 15 and 30 PI, the CTL activity in the spleen of TNF–/– mice was approximately threefold higher than in +/+ mice; the specific lysis for +/+ CD8 T cells from mice at a 50:1 E:T ratio was equivalent to specific lysis of CD8 T cells from TNF–/– mice at a 16:1 E:T ratio. These data indicated that effector function of CD8 T cells persists longer in TNF–/– mice than in +/+ mice, suggesting a role for TNF in limiting the duration of the effector phase of the CD8 T cell response to an acute viral infection.


Figure 2
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  		Figure 2. Persistence of CTL activity in TNF–/– mice. Groups of +/+ and TNF–/– mice were infected with LCMV-Arm, and on indicated days PI, the MHC class I-restricted CTL activity in spleens was measured directly ex vivo by a 51 Cr-release assay using LCMV-infected and uninfected MC57G (H-2b) cells as target cells. Data are the means of three mice per group ± SD and representative of two independent experiments (Days 8 and 15 PI). Three to five mice per group were analyzed for the CTL assay on Day 30 PI, and the data represent the mean ± SD. There was no significant lysis of control, uninfected MC57G target cells by splenocytes from +/+ and TNF–/– mice (data not shown). It should be noted that the E:T ratios for Days 15 and 30 PI are different from those of Day 8 PI.

 
Contraction of LCMV-specific CD8 T cells in TNF–/– mice
Previous work has shown that ensuing the expansion phase (after Day 8 PI), 90–95% of the LCMV-specific CD8 T cells are eliminated by apoptosis, and the remaining 5–10% persist as memory cells indefinitely [27 ]. As both LT{alpha} and TNF induce apoptosis of activated T cells in vitro [32 , 36 ], we investigated whether TNF deficiency alone would affect the contraction of virus-specific effector CD8 T cells by staining splenocytes from +/+ and TNF–/– mice with anti-CD8 antibody and MHC I tetramers on Days 8, 15, and 30 PI. As shown in Figure 3A , the magnitude of CD8 T cell contraction differed substantially between +/+ and TNF–/– mice. The frequencies of NP396- or GP33-specific CD8 T cells in +/+ and TNF–/– mice dropped by approximately seven- and 3.5-fold, respectively, between Days 8 and 30 PI. Similarly, the contraction of GP276-specific CD8 T cells in TNF–/– mice was approximately twofold lower than in +/+ mice. To compare the magnitude of contraction more accurately, we calculated percent difference in the absolute numbers of epitope-specific CD8 T cells/spleen between +/+ and TNF–/– mice on Days 8, 15, and 30 PI (Fig. 3B) . Data in Figure 3B show that the absolute number of LCMV-specific CD8 T cells in spleens of +/+ and TNF–/– mice was comparable on Day 8 PI. It is striking that on Day 15 PI, spleens of TNF–/– mice contained up to fivefold more LCMV-specific CD8 T cells than in +/+ mice. On Day 30 PI, spleens of TNF–/– mice still contained two- to 2.6-fold more LCMV-specific CD8 T cells compared with +/+ mice. Based on total number of epitope-specific CD8 T cells in the spleen, the overall decline in the number of LCMV-specific CD8 T cells between Days 8 and 30 PI was calculated to be ~14- and sevenfold, respectively, in +/+ and TNF–/– mice. Taken together, these data suggested a role for TNF in regulating the contraction phase of a LCMV-specific CD8 T cell response.


Figure 3
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  		Figure 3. Contraction of LCMV-specific CD8 T cells in TNF–/– mice. Groups of +/+ and TNF–/– mice were infected with LCMV-Arm. (A) At indicated days PI, CD8 T cells, which are specific to the indicated LCMV epitopes, were quantitated by flow cytometry after staining splenocytes with anti-CD8 antibodies and Db MHC I tetramers. The zebra plots are gated on total viable splenocytes, and the numbers are the percentages of epitope-specific CD8 T cells among total splenocytes. (B) Total number of LCMV-specific CD8 T cells in the spleen of +/+ and TNF–/– mice was determined. The average number of epitope-specific CD8 T cells in the spleen of +/+ mice was compared with individual TNF–/– mice to calculate percent difference between +/+ and TNF–/– mice. Data are the means of three to five mice at each time-point and representative of two independent experiments; data represent the mean ± SD.

 
Effect of TNF deficiency on the apoptosis and proliferation of LCMV-specific effector CD8 T cells
Data in Figure 3 showed that TNF deficiency attenuated the contraction of LCMV-specific, effector CD8 T cells after Day 8 PI. Here, we investigated whether TNF down-regulates CD8 T cell responses during the contraction phase by affecting T cell apoptosis and/or proliferation. We [40 ] and others [29 ] have reported previously that proapoptotic, LCMV-specific effector CD8 T cells could be visualized directly ex vivo by staining with Annexin V. On Days 8 and 9 PI, we quantitated the number of proapoptotic (Annexin Vhi), LCMV-specific CD8 T cells in spleens of LCMV-infected +/+ and TNF–/– mice directly ex vivo. As shown in Figure 4A and 4B , on Days 8 and 9 PI, the relative proportions of Annexin Vhi cells amongst LCMV-specific CD8 T cells were significantly lower in TNF–/– mice compared with +/+ mice. These data show that TNF might down-regulate CD8 T cell responses by inducing apoptosis. Previous work has shown that LCMV-specific effector CD8 T cells, which survive the contraction phase and differentiate into long-lived memory cells, preferentially express IL-7R{alpha} and high levels of Bcl-2 [42 ]. Therefore, we examined whether reduced apoptosis of effector CD8 T cells in TNF–/– mice was associated with alterations in the expression of IL-7R{alpha} and Bcl-2. As shown in Figure 4C , percentages of IL-7R{alpha}-expressing cells amongst LCMV-specific effector CD8 T cells were comparable between +/+ and TNF–/– mice. In addition, the level of Bcl-2 expression in LCMV-specific effector CD8 T cells in TNF–/– mice was similar to +/+ mice (Fig. 4D) . These data implied that reduced apoptosis of LCMV-specific effector CD8 T cells in TNF–/– mice was independent of IL-7R{alpha} or Bcl-2 induction. Therefore, it is possible that IL-7Rlo effector CD8 T cells, which are highly susceptible to TNF-induced apoptosis, will survive in TNF–/– mice but not in +/+ mice.


Figure 4
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  		Figure 4. Effect of TNF deficiency on the apoptosis and proliferation of LCMV-specific, effector CD8 T cells. +/+ or TNF–/– mice were infected with LCMV-Arm. (A and B) On Days 8 and 9 PI, splenocytes were stained with anti-CD8, Db NP396 MHC I tetramer, and Annexin V to assess apoptosis directly ex vivo. The percentages of Annexin Vhi apoptotic NP396-specific T cells were determined by flow cytometry. (A) Dot-plots are gated on tetramer-binding CD8 T cells, and the numbers are the percentages of Annexin Vhi cells of tetramer-binding cells. (C) Eight days after LCMV infection, splenocytes were stained with anti-CD8, anti-CD127 (IL-7R{alpha}), and Db/NP396 tetramers. The dot-plots are gated on total CD8 T cells, and the numbers are the percentages of IL-7R{alpha}hi cells among NP396-specific CD8 T cells. (D) Bcl-2 expression in LCMV-specific CD8 T cells. On Day 8 PI, splenocytes were stained for intracellular Bcl-2 after surface staining with anti-CD8 and Db/NP396 tetramers. The histograms are gated on Db/NP396 tetramer-binding CD8 T cells, and the numbers are the MFI for Bcl-2 staining ± SD. The dotted and bold lines represent staining with isotype control and anti-Bcl-2 antibodies, respectively. (B and D) Data are the mean ± SD of three mice/group and representative of two independent experiments. (C) Data are the mean ± SD of three mice/group.

 
Next, we examined whether TNF deficiency affected the proliferation of LCMV-specific CD8 T cells by measuring BrdU incorporation in vivo. Groups of +/+ and TNF–/– mice were infected with LCMV and exposed to BrdU at the following intervals: Days 0–8, 8–15, and 15–21 PI. At the end of each pulse, we assessed BrdU incorporation by LCMV-specific CD8 T cells by flow cytometry (Fig. 5 ). As illustrated in Figure 5 , the percentages of LCMV-specific CD8 T cells, which incorporated BrdU in TNF–/– mice, were similar to +/+ mice at all time-points examined. These data show that TNF deficiency had a minimal effect on the proliferation of LCMV-specific CD8 T cells during the contraction phase of the CD8 T cell response. Taken together, results presented in Figures 4 and 5 suggested that TNF down-regulates the CD8 T cell response by inducing apoptosis of CD127lo/Bcl-2lo LCMV-specific, effector CD8 T cells.


Figure 5
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  		Figure 5. Effect of TNF deficiency on proliferation of LCMV-specific CD8 T cells. Groups of +/+ or TNF–/– mice were infected with LCMV, and BrdU was administered in drinking water between days 0–8, 8–15, or 15–21 after infection. At the end of each BrdU pulse (i.e., on Days 8, 15, and 21), splenocytes were stained with MHC I tetramers, anti-CD8, and anti-BrdU antibodies. (A) Dot-plots are gated on total CD8 T cells, and numbers are the percentages of BrdU+ve cells among epitope-specific, tetramer-binding cells. (B) Percentages of BrdU+ve cells, and data are the mean ± SD of three mice/group.

 
CD8 T cell memory in TNF–/– mice
According to the current axiom, mechanisms, which regulate the initial clonal burst size (expansion) and the ensuing clonal deletion (contraction) of antigen-specific effector CD8 T cells, are key determinants of the magnitude of CD8 T cell memory [24 , 27 , 43 , 44 ]. Here, we investigated whether loss of TNF-induced effects during the contraction phase affected the magnitude of CD8 T cell memory. The number of memory CD8 T cells specific to the three LCMV epitopes was quantitated in spleens of +/+ and TNF–/– mice on Days 200–250 PI. As shown in Figure 6A , the absolute numbers of LCMV-specific memory CD8 T cells in the spleen of TNF–/– mice were significantly (three- to fourfold; P<0.05) higher than in +/+ mice. These data suggested that TNF might play a role in limiting the number of memory CD8 T cells by inducing apoptosis of effector CD8 T cells. Memory CD8 T cells are found in the secondary lymphoid organs and peripheral tissues [39 ]. Therefore, it is possible that increased numbers of LCMV-specific memory CD8 T cells in the spleen of TNF–/– mice could be a result of anatomic relocalization of memory CD8 T cells from nonlymphoid tissues into the spleen. To address this issue, we quantitated LCMV-specific memory CD8 T cells in liver and lungs of +/+ and TNF–/– mice on Day 110 PI. As shown in Figure 6B , the frequencies of LCMV-specific memory CD8 T cells in liver and lungs of TNF–/– mice were similar to those in +/+ mice. These data suggested that TNF–/–-induced enhancement in the number of memory CD8 T cells in the spleen was not a result of anatomical redistribution of nonlymphoid memory cells.


Figure 6
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  		Figure 6. CD8 T cell memory in TNF–/– mice. (A) Between Days 200 and 250 after infection with LCMV, the number of memory CD8 T cells, which are specific to the indicated LCMV epitopes, was quantitated by intracellular staining for IFN-{gamma}. Data show the total number of epitope-specific memory CD8 T cells in spleens of +/+ and TNF–/– mice. Data are the mean ± SD of seven mice/group and are pooled from two individual experiments. (B) On Day 110 after LCMV infection, mononuclear cells (MNC) from liver and lungs were stained with anti-CD8 and MHC I tetramers. The dot-plots are gated on total MNC, and the numbers are the mean percentages of tetramer-binding CD8 T cells amongst total MNC (n=3).

 
The development of protective immunity depends on "quantity" and "quality" of memory CD8 T cells. Therefore, we asked whether differentiation of effectors into memory cells under conditions of TNF deficiency altered qualitative attributes of memory CD8 T cells. First, we examined the expression of selected cytokine receptors and adhesion molecules on LCMV-specific memory CD8 T cells in +/+ and TNF–/– mice. As illustrated in Figure 7 , the relative proportions of CD62Lhi memory CD8 T cells in TNF–/– mice were lower than in +/+ mice. The expression levels of CD127 (IL-7R{alpha}) and CD122 (IL-15Rβ) on LCMV-specific memory CD8 T cells were largely unaffected by TNF deficiency. Although not striking, the levels of CD44 and LFA-1 on LCMV-specific memory CD8 T cells in TNF–/– mice appeared to be lower compared with +/+ mice. Second, we assessed the kinetics of cytokine production by LCMV-specific memory CD8 T cells upon antigenic stimulation ex vivo. As expected, a large proportion of LCMV-specific memory CD8 T cells from +/+ mice produced IFN-{gamma} and IL-2 within 2 h after antigenic stimulation (Fig. 8 ). The kinetics of cytokine production by LCMV-specific memory CD8 T cells from TNF–/– mice was similar to +/+ mice. Thus, TNF deficiency did not significantly affect the cell surface phenotype or cytokine-producing ability of LCMV-specific memory CD8 T cells.


Figure 7
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  		Figure 7. Surface phenotype of memory CD8 T cells in TNF–/– mice. On Day 200 after LCMV infection, cell-surface phenotype of LCMV-specific memory T cells in spleens of +/+ and TNF–/– mice was determined after staining with MHC I tetramers, anti-CD62L, anti-CD127, anti-CD44, anti-LFA-1, and anti-CD122 antibodies. The histograms are gated on NP396 tetramer-binding CD8 T cells, and the numbers in histograms represent the MFI of staining for the respective molecule ± SD. The numbers in the histogram for CD62L represent percent of CD62L-high cells among NP396-specific CD8 T cells. Data are the mean ± SD of three mice/group.

 

Figure 8
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  		Figure 8. Kinetics of cytokine production by LCMV-specific memory CD8 T cells in TNF–/– mice. On Day 200 after LCMV infection, splenocytes from +/+ and TNF–/– mice were stimulated with the indicated LCMV epitope peptides directly ex vivo for the indicated time intervals and stained for cell-surface CD8 and intracellular IFN-{gamma} and IL-2. The number of IFN-{gamma}- and IL-2-producing CD8 T cells at each time-point was quantitated by flow cytometry. The results are expressed as percentage of maximum response, which was attained at 5 h after stimulation. The results are the mean of three mice per group ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The pathophysiology of TNF actions is highly complex, and the immune effects elicited by this cytokine depend on: cellular source and location; duration of production; quantity produced; and context in which it is produced and expressed in relationship to the afferent or the efferent phase of the immune response [2 , 15 , 45 , 46 ]. Therefore, it is not surprising that depending on the above-mentioned factors, the spectrum of effects induced by TNF could include immune activation and immune suppression. In this manuscript, we have focused our studies to systematically dissect the role of TNF in the regulation of antigen-specific CD8 T cell response to an acute LCMV infection. Here, we have documented that TNF plays a nonredundant role in down-regulating the effector phase of the CD8 T cell response by inducing apoptosis of virus-specific effector cells. In addition, we show that TNF is a factor, which limits the magnitude of long-term CD8 T cell memory. These findings have implications in understanding viral pathogenesis in the context of development of protective immunity and design of effective vaccines. Moreover, these results might also provide insight into the mechanism(s) underlying the down-regulation of T cell-dependent, autoimmune diseases by TNF.

It has been reported recently that TNF–/– CD8 T cells exhibit poor proliferative responses to lymphopenia in vivo and antigenic stimulation in vitro, which suggested that TNF might promote activation and expansion of CD8 T cells [47 ]. In striking contrast to this report, our previous studies in TNFR–/–mice and data presented in this manuscript clearly show that activation and expansion of virus-specific CD8 T cells can occur in the apparent absence of TNF/TNFR interactions [9 , 40 ]. The discrepancy in the results could be related to differences in the experimental approaches. First, the activation requirements for CD8 T cells in vitro or during lymphopenia are likely to differ from an acute viral infection. Second, the potent stimulation of innate immunity/inflammation, including induction of Type I IFNs [48 49 50 ] by the replicating virus, could compensate for TNF deficiency in promoting CD8 T cell activation during an acute LCMV infection.

Acute viral infections often induce massive activation and expansion of CD8 T cells during the primary response [27 ]. After viral clearance, to preserve sufficient lymphoid space for immune responses to other pathogens, it is necessary to eliminate most of the expanded, virus-specific CD8 T cells [44 ]. In addition, timely elimination of effector CD8 T cells might prevent inadvertent CD8 T cell-mediated immunopathology [51 , 52 ]. Not only is this event important to re-establish immune homeostasis, but also, the magnitude of contraction is a critical determinant of the "size" of CD8 T cell memory. Our studies show that the effector phase of the CD8 T cell response is prolonged in the absence of TNF. The persistence of higher levels of CTL activity in TNF–/– mice could result from an increased number of LCMV-specific effector T cells or increased cellular effector function. Our studies suggested that delayed contraction of effector CD8 T cells contributed, at least in part, to extend the effector phase of the anti-LCMV CTL response in TNF–/– mice.

It has been reported that contraction of effector CD8 T cells is programmed during the expansion phase of the CD8 T cell response [53 , 54 ]. It remains to be determined whether this programming is intrinsic to the effector CD8 T cell and/or influenced by cell extrinsic factors. Our studies show that TNF plays a role in inducing apoptosis of LCMV-specific effector CD8 T cells and establishment of immune homeostasis. Partial impairment of the contraction phase, induced by TNF deficiency, not only prolonged the effector phase of the CD8 T cell response but also led to a substantial increase in the number of LCMV-specific memory CD8 T cells. These findings suggest that execution of the apoptotic program in effector CD8 T cells, at least in part, is dependent on TNF. These data are in agreement with previous studies [55 , 56 ], which showed that TNF plays a role in peptide-induced deletion of TCR-transgenic, CD8 T cells. However, studies by Pircher’s group [57 ] showed that TNF deficiency did not affect contraction of monoclonal TCR-transgenic, CD8 T cells. These studies, along with our data, suggest that sensitivity of CD8 T cells to TNF-induced apoptosis depends on the clonotype of CD8 T cells, and studies using monoclonal TCR-transgenic CD8 T cells might not always be representative of a polyclonal CD8 T cell response. It is noteworthy that in our studies, TNF deficiency only impaired but did not abrogate the contraction of LCMV-specific effector CD8 T cells, which is suggestive of the existence of other mechanisms. Indeed, IFN-{gamma} deficiency has been shown to impair the contraction phase of the CD8 T cell response to LCMV [34 ].

Although our studies suggested a role for TNF in regulating the contraction of LCMV-specific, effector CD8 T cells, it will be of interest to determine whether TNF regulates CD8 T cells via direct or indirect effects. We have shown recently that TNFRs down-regulate CD4 T cell expansion via indirect effects on T cells [58 ], and there is evidence that apoptosis of activated CD8 T cells induced by tumor-infiltrating macrophages can be mediated by any of the following: TNFRs, NO, reactive oxygen species (ROS), or IFN-{gamma} [59 ]. Although the relationship among IFN-{gamma}, TNF, ROS, and NO in regulating CD8 T cell homeostasis is not known, it is possible that TNF might regulate CD8 T cell homeostasis by indirect effects, which need further investigation. We have reported previously that deficiency in both TNFR I and TNFR II attenuated contraction of effector CD8 T cells and enhanced the magnitude of CD8 T cell memory [40 ]. However, in this study, the ligand responsible for inducing TNFR signaling was not determined. Not only do they use the same receptors (TNFRs I and II), but also, TNF and LT are known to induce apoptosis of activated T cells [32 , 36 ]. In the present study, TNF deficiency alone recapitulated the "phenotype" of TNFR–/– mice, which indicated that TNF and not LT down-regulates CD8 T cell responses during an acute LCMV infection.

In the immune system, two signaling pathways initiate T cell apoptosis. One pathway is triggered by signaling via the members of the TNFR family, which have the intracellular death domain [60 ]. The second pathway is initiated by events such as withdrawal of trophic cytokines or exposure to cytotoxic drugs. This pathway of cell death, known as Bcl-2-regulated pathway or mitochondrial pathway, is regulated by the interplay of the pro- and antiapoptotic members of the Bcl-2 family [61 , 62 ]. In the Bcl-2-regulated pathway, death signals triggered by cytokine deprivation activate the proapoptotic members of the Bcl-2 family such as BIM. Activated BIM in turn activates the proapoptotic Bcl-2 members BAX and BAK, which disrupt the outer mitochondrial membrane directly or indirectly, causing the release of cytochrome c and other apoptogenic proteins. The death of ~90% of the activated CD8 T cells during the contraction phase of the T cell response has been a topic of intense investigation. It has been reported that superantigen-induced T cell deletion of activated T cells and early contraction of virus-specific CD8 T cells only in the spleen (not lymph nodes) during a HSV infection are dependent on BIM [63 , 64 ]. However, the effect of BIM deficiency on long-term CD8 T cell memory to HSV has not been studied. Although collectively, these studies might indicate that cytokine/antigen withdrawal-induced, BIM-dependent apoptosis contributes to loss of activated T cells in vivo, data presented in this manuscript along with published work [34 , 40 , 65 ] argue that TNF/IFN-{gamma}-dependent and mitochondrial pathways of cellular apoptosis contribute to the contraction of effector CD8 T cells following an acute LCMV infection. In addition, the relative importance of these two mechanisms in contraction of CD8 T cells in different models might be dictated by several factors, including early inflammation, dose of infection, virus strain, and duration/intensity of antigenic stimulation [49 , 53 ]. Nonetheless, our studies suggest that abrogating TNF activity might be a fruitful strategy to enhance long-term, CD8 T cell memory to noncytopathic viruses such as LCMV and other arenaviruses. It was reported recently that TNF produced by T cells versus macrophages/neutrophils has distinct and nonredundant functions in immune regulation [45 ]. It would be informative to determine the cellular source of TNF, which down-regulates CD8 T cell response during LCMV infection.

CD8 T cell memory-dependent, protective immunity depends on quantity and quality of memory CD8 T cells. Our studies indicated that TNF deficiency enhanced the quantity of CD8 T cell memory without affecting their quality significantly. Except for LFA-1, memory CD8 T cells in TNF–/– mice expressed normal levels of cell surface molecules, which mediate adhesion (CD44) and proliferative renewal (CD127 and CD122). Although this is suggestive of a role for TNF in up-regulating expression of LFA-1 on memory CD8 T cells, the biological significance of lower LFA-1 expression on the protective ability of the cell remains to be determined. Functionally, LCMV-specific, memory CD8 T cells in TNF–/– mice were similar to those in +/+ mice; the amount and kinetics of IFN-{gamma}/IL-2 production by memory CD8 T cells from +/+ and TNF–/– mice were similar. It is noteworthy that the rapidity with which memory CD8 T cells produce IFN-{gamma} upon antigenic stimulation ex vivo correlated with immunodominance, which is in agreement with published findings [66 ]. Based on these findings, it had been speculated that rapid production of IFN-{gamma} might regulate immunodominance [66 ]. However, here, we show that the rapidity of IL-2 production by LCMV-specific CD8 T cells also correlates with immunodominance; although 75–100% of NP396- and GP33-specific CD8 T cells produced IL-2 within 2 h, it required up to 4 h for all GP276-specific CD8 T cells to produce IL-2. Thus, memory CD8 T cells, which are specific to immunodominant epitopes, are poised to produce cytokines rapidly, compared with subdominant, epitope-specific CD8 T cells. The epitope-specific differences in the kinetics of cytokine production of memory CD8 T cells are likely a result of signaling alterations and/or epigenetic changes in the cytokine genes, which are reflective of their differentiation status, proliferative history, or exposure to different duration/intensity of antigenic stimulation [44 , 67 68 69 ].

Elegant studies by Homann et al. [70 ] have shown that LCMV-specific, memory CD8 T cells and CD4 T cells exhibit differential stability in their number in immune mice; the number of virus-specific memory CD4 T cells but not memory CD8 T cells undergoes slow attrition over time. In addition, previous work by Zheng et al. [32 ] has shown that activated CD8 T cells are more sensitive to TNF-induced apoptosis as compared with activated CD4 T cells. Therefore, we examined whether TNF differentially regulates CD8 and CD4 T cell memory. Our studies showed that TNF–/– enhanced the number of LCMV-specific memory CD4 T cells (data not shown). However, increased CD4 T cell memory in TNF–/– mice correlated with enhanced, primary expansion and not reduced contraction, which is consistent with our studies published recently with TNFR–/– mice [58 ]. Nonetheless, collectively, these data suggest that TNF limits CD8 and CD4 T cell memory, albeit by different mechanisms.

In summary, in this manuscript, we have provided strong evidence supporting a role for TNF in the establishment of immune homeostasis after a potent CD8 T cell response to an acute viral infection. What are the implications of these findings? From the standpoint of vaccine-induced, protective immunity, our findings suggest that blocking TNF activity might enhance CD4 and CD8 T cell memory. In the context of T cell-dependent autoimmunity, along with published findings, our results indicate that localized induction of TNF might be a strategy to induce apoptosis of autoaggressive CD8 T cells and ameliorate autoimmunity such as Type I diabetes [18 ]. Therefore, research findings reported in this manuscript are expected to have implications in the treatment of T cell-dependent autoimmunity and development of effective vaccines, which can engender potent, CD8 T cell-mediated immunity.


   ACKNOWLEDGEMENTS
 
This work was supported by Public Health Service grant AI48785 from National Institutes of Health to M. S. We thank Yumi Nakayama, Nicole Miller, Katie Skell, and Erin Hemmila Plisch for excellent technical assistance.

Received April 23, 2007; revised July 24, 2007; accepted July 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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