CD8 T Cells Erosion in the Quality of ''Helpless'' Memory TRAIL ...

The Journal of Immunology

TRAIL Deficiency Delays, but Does Not Prevent, Erosion in the Quality of “Helpless” Memory CD8 T Cells1 Vladimir P. Badovinac,* Kelly A. Nordyke Messingham,* Thomas S. Griffith,† and John T. Harty2*‡ In this study, we investigated the role of TRAIL in Ag-specific CD8 T cell homeostasis after viral infection. TRAIL deficiency does not influence the kinetics of the Ag-specific CD8 T cell responses, and CD8 T cells in TRAIL-deficient mice were able to expand, contract, and generate functional memory cell numbers that were indistinguishable from TRAIL-sufficient wild-type CD8 T cells after acute lymphocytic choriomeningitis virus infection. Interestingly, the ability of “helpless” CD8 T cells to retain their memory phenotypic and functional (i.e., secondary expansion) characteristics was prolonged in TRAIL-deficient mice compared with wild-type CD4-depleted controls. However, TRAIL deficiency only delayed, but did not prevent, the eventual erosion in the quality of helpless memory CD8 T cells, and that correlated with their inability to respond to a second round of Ag-driven proliferation. These data, which suggest that CD4 help consists of both TRAIL-dependent and -independent components, may help to resolve the current controversy between the early programming and maintenance models that were put forward to explain the role of CD4 T cell help in Ag-specific CD8 T cell homeostasis. The Journal of Immunology, 2006, 177: 999 –1006.

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he Ag-specific CD8 T cell response is an important arm of adaptive immunity and is required for protection and clearance of many viral and bacterial infections (1). CD8 T cell responses to infection initiate when relatively rare naive pathogen-specific CD8 T cells enter secondary lymphoid organs and encounter Ag-expressing mature dendritic cells that express high levels of costimulatory molecules. Upon activation, naive CD8 T cells undergo a period of substantial proliferative expansion in number that coincides with their differentiation into effector cells, which participate in clearance of infection (2). The expansion phase is followed by an equally rapid contraction, where 90 – 95% of the effector CD8 T cells die by apoptosis. The remaining Ag-specific CD8 T cells form the initial memory pool and their numbers can remain stable for the life of the host. Despite their relatively stable numbers, the maintenance of memory CD8 T cell pool is a dynamic process consisting of cytokine-driven homeostatic proliferation that is balanced by equivalent death (1). Recent evidence suggests that the expansion and the contraction phases of the CD8 T cell response to infection are programmed and that a relatively brief contact with Ag (hours to days) is sufficient to drive conversion of naive CD8 T cell precursors into long-lived memory CD8 T cells (3–7). Although initial recognition of Ag-expressing mature dendritic cells is critical for induction and generation of memory CD8 T cell responses after infection, cooperation with other cell types, including CD4 T cells, might influence this process.

*Department of Microbiology, †Department of Urology, and ‡Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242 Received for publication March 29, 2006. Accepted for publication April 26, 2006. 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 Grants ROIAI42767, ROIAI46653, ROIAI50073, ROIAI059752, POIAI60699 (to J.T.H.), and ROICAI09446 (to T.S.G.); by an American Cancer Society (ACS) grant administered through The Holden Comprehensive Cancer Center at the University of Iowa (IRG77-004-28) (to V.P.B.); and by ACS Grant PF-06-048-01-LIB (to K.A.N.M.). 2 Address correspondence and reprint requests to Dr. John T. Harty, Department of Microbiology, University of Iowa, 3-512 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242. E-mail address: [email protected]

Copyright © 2006 by The American Association of Immunologists, Inc.

CD8 T cell responses against pathogens have been classified as mainly independent from CD4 T cell help (reviewed in Ref. 8). The inflammatory stimuli provided by replicating pathogens are thought to provide signals (i.e., TLR triggering) required for full activation (maturation) of APCs and therefore the need for CD4 T cell help is diminished (9 –11). Conversely, in immunizations with Ag from a noninflammatory source, CD4 T cells might be required for full activation of APC (i.e., by CD40 signaling) to stimulate maximal CD8 T cell responses (12–15). However, recent studies have revisited the need for CD4 T cell help in CD8 T cell responses to noninflammatory Ags and suggested that, whereas primary CD8 T cell responses were CD4 T cell independent, secondary responses were dependent on CD4 T cell help that occurred during initial priming (programming model) (16, 17). In contrast to the programming model, other studies with murine models of infection showed that CD4 T cells were required for maintaining fully functional memory CD8 T cells (i.e., ability to respond to secondary Ag challenge) (18 –20). Therefore, CD4 T cell help might not be required for the programmed conversion of naive to effector to memory CD8 T cells after infection, but instead was needed for maintaining the memory CD8 T cell pool (maintenance model) (21). Finally, other evidence suggests that the requirement for CD4 T cell help in memory CD8 T cell maintenance and function might be pathogen-specific (17, 22–25), adding an additional layer of complexity to understanding the role of CD4 T cell help for CD8 T cell memory. In experiments designed to address the role of CD4 T cell help in programming CD8 T cell memory, Schoenberger and colleagues (26) recently showed that mRNA for TRAIL was selectively upregulated in “helpless” CD8 T cells (CD8 T cells primed in the absence of CD4 T cell help) compared with the CD8 T cells initially primed in the presence of CD4 T cells (“helped”). Using cross-priming against cell-associated Ag as well as lymphocytic choriomeningitis virus (LCMV)3 infection models, they showed that, despite similar expression of TRAIL receptor death receptor

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; LCMV-Arm, Armstrong strain of LCMV; WT, wild type; p.i., postinfection; DR5, death receptor 5.

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CD8 T CELLS IN THE ABSENCE OF TRAIL AND/OR CD4 T CELLS

5 (DR5) (26) on helped and helpless CD8 T cells, the increased ability of helpless CD8 T cells to produce TRAIL correlated with the increased activation-induced cell death upon secondary Ag challenge (26). In addition, treatment with recombinant TRAIL of the helped CD8 T cells inhibited their secondary expansion, suggesting that CD8 T cells primed in the presence of CD4 T cells are also susceptible to TRAIL-mediated death (26). These data suggested that CD4 T cell help might be imprinted early after naive CD8 T cell activation and that signals delivered by CD4 T cells will be remembered (i.e., modulation of TRAIL expression) as CD8 T cells progress to memory. In this study, we report that TRAIL deficiency does not influence the normal kinetics of the Ag-specific CD8 T cell responses after acute LCMV infection. CD8 T cells in TRAIL-deficient mice were able to expand, contract, and generate memory cell numbers that were indistinguishable from TRAIL-sufficient wild-type (WT) CD8 T cells. In the absence of CD4 T cell help, WT CD8 T cells were maintained at similar numbers as helped counterparts but quickly lost their ability to respond to secondary bacterial (Listeria monocytogenes) or viral (LCMV) challenge, confirming the importance of CD4 T cells in memory CD8 T cell generation after primary viral infection. Consistent with previous data (26), the ability of helpless CD8 T cells to retain their memory phenotypic and functional (i.e., secondary expansion) characteristics was prolonged in TRAIL-deficient mice. However, TRAIL deficiency only delayed, but did not prevent, the eventual erosion in the quality of helpless memory CD8 T cells, suggesting that the protective CD4 T cell help for memory CD8 T cell responses has both TRAIL-dependent and -independent components.

Materials and Methods Mice, LCMV, and L. monocytogenes C57BL/6 (H-2b) mice were obtained from the National Cancer Institute (Frederick, MD). TRAIL-deficient mice (H-2b) had been backcrossed 10 generations onto a B6 background (27, 28). Pathogen-infected mice were housed in the appropriate biosafety conditions. All mice were used at 8 –16 wk of age. All animal experiments followed approved Institutional Animal Care and Use Committee protocols. The Armstrong strain of LCMV (2 ⫻ 105 PFU/mouse for primary, and 2 ⫻ 106 PFU/mouse for secondary immunizations, given i.p.) was used as described (29). The virulent L. monocytogenes strain XFL204 that expresses LCMV-derived NP396 – 404 H-2Db MHC class I-restricted epitope (provided by Dr. H. Shen, University of Pennsylvania, Philadelphia, PA) (30) was resistant to streptomycin and was grown, injected i.v., and quantified as described (31).

from the peptide-stimulated value to determine the percentage of Ag-specific CD8 T cells. The total number of epitope-specific CD8 T cells per spleen was determined from the percentage of IFN-␥⫹CD8⫹ T cells, the percentage of CD8 T cells in each sample, and the total number of cells per spleen.

Results LCMV-specific CD8 T cell responses in the absence of TRAIL To investigate the role for TRAIL in regulating Ag-specific CD8 T cell responses after viral infection, naive WT (C57BL/6) and TRAIL-deficient mice were infected i.p. with 2 ⫻ 105 PFU of the Armstrong strain of LCMV (LCMV-Arm). As previously described (34), WT CD8 T cells recognizing the dominant LCMVderived GP33 and NP396 epitopes undergo vigorous expansion after LCMV infection (Fig. 1, A and B). After the completion of the expansion phase, both CD8 T cell responses went through contraction (death) phase and cells that survived (5–10% of numbers detected at day 8 postinfection (p.i.)) initiated the stable memory CD8 T cell pool (Fig. 1B). TRAIL deficiency did not influence the kinetics and magnitude of GP33- and NP396-specific CD8 T cell responses to LCMV infection. In addition, similar magnitudes of CD8 T cell expansion were observed for both TRAIL-deficient subdominant GP276 and NP205 epitopes when compared with WT counterparts (data not shown). Furthermore, in the absence of TRAIL, the extent of contraction of effector CD8 T cells was indistinguishable from WT CD8 T cell responses suggesting that TRAIL does not play a substantial role in regulation of contraction. Importantly, overlapping memory CD8 T cell numbers were achieved in both mice for multiple CD8 T cell epitopes analyzed (Fig. 1, A and B). Thus, Ag-specific CD8 T cell homeostasis, including the contraction phase of the CD8 T cell responses, is not influenced by TRAIL deficiency in vivo after viral infection. Functional and phenotypic characteristics of TRAIL-deficient CD8 T cells after LCMV infection Similar stable numbers of Ag-specific memory CD8 T cells were present in WT and TRAIL-deficient mice after LCMV infection.

Abs and peptides The following mAbs were used: PE- or FITC-conjugated Ab to IFN-␥ (anti-IFN-␥; clone XMG1.2; eBioscience), FITC- or CyChrome-conjugated anti-CD8 (clone 53-6.7; BD Pharmingen), FITC-anti-Thy1.2 (clone 53-2.1; BD Pharmingen), PE-anti-TNF (clone MP6-XT22; eBioscience), PE-anti-CD127 (clone A7R34; eBioscience), PE-anti-CD27 (clone LG.7F9; eBioscience), PE-anti-IL-2 (clone JES6-5H4; BD Pharmingen), and PE-rat IgG2a, IgG2b, and IgG1 isotype controls (clones eBR2a, KLH/ G2b-1-2, and eBRG1, respectively; eBioscience). Defined LCMV GP33– 41 and NP396 – 404 H-2b MHC class I-restricted peptides were described previously (32).

In vivo CD4 depletion At 5 and 2 days before challenging with LCMV, mice were treated with 0.4 mg of purified anti-CD4 (GK1.5) Ab i.p. After the infection, anti-CD4 (0.4 mg/mouse) treatment was continued weekly until the end of the experiment. Noncompeting FITC-conjugated anti-CD4 mAb (clone RM4-4; BD Pharmingen) was used for CD4 depletion assessment in vivo.

Quantification of Ag-specific CD8 T cell response The magnitude of the epitope-specific CD8 T cell response was determined by peptide-stimulated intracellular staining for IFN-␥, IFN-␥ and TNF, or IFN-␥ and IL-2 as previously described (33). The percentage of IFN␥⫹CD8⫹ T cells in unstimulated samples from each mouse was subtracted

FIGURE 1. TRAIL-deficient CD8 T cell responses after LCMV infection. C57BL/6 (WT, H-2b) and TRAIL-deficient (H-2b) mice were infected with LCMV-Arm (2 ⫻ 105 PFU/mouse). A, GP33- and NP396-specific CD8 T cells detected by intracellular IFN-␥ staining in representative mice on day 8 and day 120 p.i. Numbers represent frequencies of Ag-specific CD8 T cells in the spleen. B, Total number of GP33- and NP396-specific CD8 T cells in the spleen at indicated days after LCMV infection. Data are mean ⫾ SD of three to six mice per time point.

The Journal of Immunology To further characterize the fitness of TRAIL-deficient memory CD8 T cells, we analyzed the expression of phenotypic and functional markers at the effector (day 8) and memory (day 90 p.i.) phases of the Ag-specific CD8 T cell response in both groups of mice (Fig. 2). As previously observed in multiple infection models, including LCMV infection of WT mice (35), IL-7R␣ (CD127) expression was down-regulated at day 8 on GP33-specific IFN-␥producing effector CD8 T cells in both groups of mice. Similarly, Ag-specific effector CD8 T cells in both groups of mice showed up-regulation of the epitope recognized by the 1B11 mAb (CD43) (36), and down-regulation of CD27 at day 8 p.i., and 75% of IFN-␥ producing cells produced TNF after in vitro peptide stimulation (37). Finally, low frequencies of Ag-specific effector CD8 T cells from both groups scored positive for IL-2 production after Ag stimulation (Fig. 2). Thus, TRAIL deficiency does not alter the phenotype or cytokine production properties of Ag-specific effector CD8 T cells after LCMV infection. WT CD8 T cells showed phenotypic and functional traits of memory at 3 mo after LCMV infection (37). Most of the memory GP33-specific CD8 T cells showed high levels of CD127 expression, a modest increase in CD27 when compared with effector cells and low levels of 1B11 epitope on CD43 (Fig. 2). All of the IFN␥-producing CD8 T cells produce TNF, and 35– 40% of those cells produced IL-2 upon peptide stimulation (37). Importantly, the expression of phenotypic and functional markers on TRAIL-deficient memory CD8 T cells was essentially identical when compared with WT memory cells (Fig. 2). Similar data were obtained when NP396-specific CD8 T cells were evaluated (data not shown). Taken together, true memory CD8 T cells (38) were observed after viral infection of TRAIL-deficient mice, suggesting that effector to memory CD8 T cell progression (numbers, phenotype, and function) in vivo is TRAIL independent.

1001 inflammation (present early after infection) is thought to dictate the extent and timing of CD4 T cell influence on CD8 T cell homeostasis. In some, but not all models of infection (and CD4 depletion), CD4 T cell help is not required for primary CD8 T cell expansion but is required for memory maintenance and ability of those (helpless) CD8 T cells to respond after secondary Ag encounter (8, 17, 22, 24, 25). In addition, TRAIL expression, which differs among helped and helpless CD8 T cells (26), has been described as a potential mechanism that controls the ability of helpless memory cells to respond after secondary infection (26). To explore the requirement for TRAIL in helpless CD8 T cell homeostasis after viral infection, WT and TRAIL-deficient mice were treated on days 5 and 2 before and day 2 after LCMV-Arm challenge with 0.4 mg/day CD4-depleting Ab (GK1.5) (Fig. 3A). The control groups of mice initially received the same concentration of control Ab (rat IgG). Efficient and long-lasting depletion of CD4 T cell compartment was achieved in vivo with weekly GK1.5 treatments (Fig. 3B). In our hands, CD4 T cell depletion did not influence the overall kinetics and memory CD8 T cell numbers in WT and TRAILdeficient mice after LCMV infection (Fig. 3C). Helpless CD8 T cells specific for GP33 and NP396 expanded to similar numbers as helped counterparts, and identical numbers of stable memory WT and TRAIL-deficient CD8 T cells were achieved despite the absence of CD4 T cells throughout the experiment (Fig. 3C). Thus, in contrast to previous studies based on MHC class II-deficient mice (20, 21), CD4 T cell help in the presence or absence of

Helped and helpless CD8 T cell responses in the absence of TRAIL The requirement for CD4 T cell help for optimal CD8 T cell responses differs in various experimental models (8). In this regard,

FIGURE 2. Phenotypic and functional characteristics of LCMV-specific effector (d8) and memory (d90) CD8 T cells in WT and TRAILdeficient mice. WT and TRAIL-deficient mice were injected with LCMVArm and on day 8 or day 90 p.i., GP33-specific CD8 T cells in the spleen were detected with intracellular IFN-␥ staining in the presence of GP33 peptide stimulation. Shaded histogram represents the isotype control staining, and the thick line represents staining with mAbs of the indicated specificity of CD8⫹IFN-␥⫹ cells from representative mice. Numbers represent the percentage of cells positive for the indicated molecules.

FIGURE 3. WT and TRAIL-deficient LCMV-specific CD8 T cell responses in the presence or absence of CD4 T cells. A, WT and TRAILdeficient mice were treated with anti-CD4 T cell depleting (GK1.5) or control (rat IgG) Abs (0.4 mg/mouse) 5 and 2 days before the LCMV-Arm infection and continued weekly after the infection. B, Frequencies of CD8 and CD4 T cells in the spleens of CD4 T cell nondepleted (Ig group) and CD4 T cell depleted (␣CD4 group) 90 days after LCMV infection. C, Total number of GP33- and NP396-specific CD8 T cells in the spleen at indicated days after LCMV infection. Data are mean ⫾ SD of three to six mice per time point.

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TRAIL synthesis was not required for maintenance of endogenous memory CD8 T cell numbers after LCMV infection. We next sought to investigate the fitness of memory CD8 T cells in the CD4-depleted WT and TRAIL-deficient mice. The expression of IL-7R␣ as well as the ability of memory CD8 T cells to produce TNF and IL-2 was diminished for both GP33 and NP396specific helpless WT CD8 T cells, at all memory time points (60 and 90 days p.i.) analyzed (Fig. 4). Interestingly and consistent with previous reports (26), TRAIL-deficient CD8 T cells in CD4 T cell-depleted mice showed similar effector to memory phenotypic and functional transition as their helped controls at 60 days after infection. Similar frequencies of TRAIL-deficient GP33- and NP396-specific CD8 T cells were positive for CD127 expression, most of the IFN-␥-producing cells produced TNF, and 30% were IL-2 positive after peptide stimulation of helped and helpless TRAIL-deficient CD8 T cells (Fig. 4). However, equal expression of memory CD8 T cell characteristics by TRAIL-deficient CD8 T cells was not sustained in the absence of CD4 T cell help, and 3 mo after infection helpless CD8 T cells showed diminished CD127 expression, and decreased TNF and IL-2 production (Fig. 4). Therefore, TRAIL deficiency delays, but does not prevent the eventual erosion in the quality of helpless memory CD8 T cells. These data suggest that CD4 T cell help for memory CD8 T cell maintenance consists of more than TRAIL-dependent regulation. Secondary expansion of helpless CD8 T cells in the absence of TRAIL In the absence of CD4 T cell help, primary memory CD8 T cells showed diminished expansion potential after secondary stimulation (infection) (18 –20). In the model used here (in vivo CD4 depletion), similar numbers of helped and helpless Ag-specific CD8 T cells were maintained in vivo, but the fitness of helpless WT memory CD8 T cells was impaired, suggesting that their ability to expand after rechallenge might be compromised as well. In turn, the prolonged fitness of helpless TRAIL-deficient CD8 T cells suggests that those cells might be able to respond to secondary Ag encounter at the time when their phenotypic (i.e., CD127)

and functional (i.e., IL-2) status was similar to CD8 T cells primed in the presence of CD4 T cell help. To address this notion, additional experiments were performed where naive WT and TRAILdeficient mice (in the presence or absence of CD4 T cells) were infected with LCMV and their CD8 T cells were analyzed 60 days p.i. TRAIL-deficient NP396-specific CD8 T cells showed similar levels of expression of CD127 and IL-2 regardless of the presence or absence of CD4 T cells (Fig. 5, A and B). At the same time, CD127 and IL-2 expression was diminished in helpless WT cells (Fig. 5, A and B). All four groups of mice were infected with 2 ⫻ 106 PFU of LCMV (10-fold higher dose than used for primary immunizations), and 5 days later the expansion of NP396-specific CD8 T cells was determined in the spleens of those mice (Fig. 5C). As expected, helpless WT CD8 T cells were unable to respond and similar frequencies as well as total numbers of NP396-specific CD8 T cells were present before and 5 days after the LCMV rechallenge (Fig. 5, D and E). Importantly, TRAIL-deficient NP396-specific CD8 T cells from CD4-depleted and nondepleted groups responded to secondary stimulation similarly as helped WT cells, showing that memory CD8 T cell phenotype correlates with the ability of those cells to enter the second round of Ag-specific proliferation. A similar analysis was performed at the time (day 90 p.i.) when helpless TRAIL-deficient memory CD8 T cells showed decreased IL-7R␣ expression and IL-2 production compared with helped TRAIL-deficient CD8 T cells (Fig. 6, A and B). In contrast to differences observed between helpless WT and TRAIL-deficient memory CD8 T cell responses analyzed 60 days p.i. (Fig. 5, A and B), both CD8 T cell responses showed a similar decrease in fitness 90 days after infection (Fig. 6, A and B). In addition to decreased expression of CD127 and IL-2, WT and TRAIL-deficient helpless CD8 T cells produced less IFN-␥ and TNF upon NP396 – 404 peptide stimulation ex vivo when compared with helped NP396-specific CD8 T cells (Fig. 6, C and D). Consistent with their phenotype, WT and TRAIL-deficient memory NP396-specific CD8 T cells that were primed and maintained for 90 days in the absence of CD4 T cell help failed to expand after secondary LCMV challenge (Fig. 6, F and G). As a consequence of the large memory CD8 T cell pool after primary LCMV infection, secondary responses to LCMV-Arm are modest even by helped WT CD8 T cells. To determine whether helpless CD8 T cell memory in TRAIL-deficient mice were compromised in response to strong stimulation, control and helpless LCMV immune WT and TRAIL-deficient mice (90 days p.i.) were challenged with recombinant L. monocytogenes that expresses LCMV-derived NP396 epitope (Fig. 7A) (30). Similar to helpless WT CD8 T cells (Fig. 7B), helpless TRAIL-deficient NP396-specific CD8 T cells at day 90 p.i. were unable to respond to L. monocytogenes infection (Fig. 7, C and D) and the total numbers of helpless NP396-specific CD8 T cells remained similar to numbers of GP33-specific CD8 T cells, which are not reactive to L. monocytogenes-NP396 infection. Therefore, the absence of TRAIL is not sufficient for the in vivo maintenance of long-term memory CD8 T cells in the absence of CD4 T cells.

Discussion FIGURE 4. Acquisition of memory CD8 T cell characteristics in the absence of CD4 T cells in WT and TRAIL-deficient mice after LCMV infection. WT and TRAIL-deficient nondepleted (Ig) and CD4-depleted mice (␣CD4) were infected with LCMV-Arm on day 0, and phenotypic (CD127) and functional (TNF, IL-2) status of GP33 and NP396-specific CD8 T cells in the spleen was determined on indicated days p.i. The results are presented as frequencies of CD8⫹IFN-␥⫹ that were positive for CD127, TNF, or IL-2. Data are mean ⫾ SD of three to six mice per time point.

The results presented in this study demonstrate that 1) endogenous CD8 T cell homeostasis after acute viral infection, including the contraction phase of the Ag-specific CD8 T cell responses is not influenced by the absence of TRAIL, and 2) TRAIL deficiency might have an initial protective role but is not sufficient for the in vivo maintenance of long-term memory CD8 T cells in the absence of CD4 T help. These data, which suggest that CD4 help consists of both TRAIL-dependent and -independent components, may aid


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FIGURE 5. Expansion potential of helped and helpless LCMV-specific CD8 T cells after LCMV reinfection of WT and TRAIL-deficient mice. Two months after primary LCMV infection (2 ⫻ 105 PFU/mouse; 1⫻), NP396-specific CD8 T cells from CD4 nondepleted (Ig) and depleted (␣CD4) WT and TRAIL-deficient mice were analyzed for expression of CD127 (A) and IL-2 (B). The shaded histogram represents the isotype control staining, and the thick line represents staining with mAbs of the indicated specificity of CD8⫹IFN-␥⫹ cells from representative mice. Numbers represent the percentage of cells positive for the indicated molecules. C, All four groups of mice were reinfected with LCMV-Arm (2 ⫻ 106 PFU/mouse; 10⫻) on day 60 after initial LCMV challenge. D, Frequency of NP396-specific CD8 T cells in the spleen from representative mice before (day 60) and 5 days after LCMV rechallenge (day 60 ⫹ 5). Data are presented on a gated CD8 T cells as the percentage of Thy1.2⫹IFN-␥⫹ cells in the presence (upper number) or absence (lower number) of NP396 peptide stimulation. E, Total number per spleen of NP396-specific CD8 T cells before and after the secondary LCMV challenge. Data are presented as mean ⫾ SD for three mice per group. Numbers inside panels indicate the fold increase in total numbers of NP396-specific CD8 T cells 5 days after LCMV challenge.

in resolving the current controversy between the early programming and maintenance models (8, 26) that were put forward to explain the role of CD4 T cell help in Ag-specific CD8 T cell homeostasis. Since its description, TRAIL has been implicated in apoptosis of tumor cells (39, 40). Recent studies showed that TRAIL is also involved in the death of other nontransformed cell types (41, 42). Less is known about its role(s) during infections in vivo, although it has been shown that TRAIL might influence the course of L. monocytogenes infection (43). In vivo blocking of TRAIL by soluble DR5 ameliorated the disease in WT mice and due to increased apoptosis of neutrophils and macrophages, 10 –100 times greater numbers of L. monocytogenes were detected in WT compared with TRAIL-deficient mice (43). To address TRAIL deficiency after acute viral infection in vivo, WT and TRAIL-deficient mice were challenged with the Armstrong strain of LCMV and their Ag-specific CD8 T cell response was followed throughout the expansion, contraction, and memory phases of CD8 T cell homeostasis. Similar numbers and indistinguishable kinetics were observed in the WT and TRAIL-deficient Ag-specific CD8 T cell responses, suggesting that TRAIL synthesis is not required for expansion and death phases of Ag-specific CD8 T cells. Importantly, memory CD8 T cells generated in the TRAIL-deficient mice showed phenotypic and functional traits of WT memory CD8 T cells (1, 37,

38). In addition, both TRAIL-sufficient and TRAIL-deficient memory CD8 T cells were able to proliferate upon secondary bacterial (L. monocytogenes) or viral (LCMV) infections. These results showed that, in the presence of CD4 T cells, TRAIL is dispensable for Ag-specific CD8 T cell homeostasis after LCMV infection in vivo. Mouse Ag-specific CD8 T cells express the functional TRAIL receptor DR5 and are susceptible to TRAIL-mediated apoptosis (26). In the absence of CD4 T cell help, memory CD8 T cells produced TRAIL upon secondary Ag encounter and were unable to proliferate due to the TRAIL-mediated activation-induced cell death. Conversely, memory CD8 T cells primed and maintained in the presence of CD4 T cells respond vigorously to secondary stimulation and did not produce TRAIL (26). Importantly, in those experiments, no defects in primary CD8 T cell responses in the absence of CD4 T cells were described. These results led to the proposal of the programming model in which CD4 T cell help might be transmitted during the initial priming of naive CD8 T cells and imprinted in their clonal progeny (17). Schoenberger and colleagues (26) showed that helpless TCR-transgenic CD8 T cells a month after LCMV infection were unable to undergo a second round of proliferation upon peptide restimulation in vitro unless those cells were initially primed in the absence of TRAIL. Consistent with these data, we show that phenotypic and functional

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FIGURE 6. Erosion in the quality of helpless TRAIL-deficient memory CD8 T cells 90 days after LCMV infection. Ninety days after primary LCMV infection (2 ⫻ 105 PFU/mouse; 1⫻), NP396-specific CD8 T cells from CD4 nondepleted (Ig) and depleted (␣CD4) WT and TRAIL-deficient mice were analyzed for expression of CD127 (A) and IL-2 (B). Shaded histogram represents the isotype control staining, and the thick line represents staining with mAbs of the indicated specificity of CD8⫹IFN-␥⫹ cells from representative mice. Numbers represent the percentage of cells positive for the indicated molecules. C, Spleen cells from mice 90 days after LCMV infection were stimulated with NP396 peptide for 5 h ex vivo. The percentage of TNF-producing cells is determined on gated CD8⫹IFN-␥⫹ T cells. D, The levels of intracellular IFN-␥ and TNF were determined by measuring the mean fluorescence index using flow cytometry. E, All four groups of mice were reinfected with LCMV-Arm (2 ⫻ 106 PFU/mouse; 10⫻) on day 90 after initial LCMV challenge. F, Frequency of NP396-specific CD8 T cells in the spleen from representative mice before (day 90) and 4 days after LCMV rechallenge (day 90 ⫹ 4). Data are presented on a gated CD8 T cells as the percentage of Thy1.2⫹IFN-␥⫹ cells in the presence (upper number) or absence (lower number) of NP396 peptide stimulation. G, Total number per spleen of NP396-specific CD8 T cells before and after the secondary LCMV challenge. Data are presented as mean ⫾ SD for three mice per group. Numbers inside panels indicate the fold increase in total numbers of NP396-specific CD8 T cells 4 days after LCMV challenge.

characteristics of helpless TRAIL-deficient CD8 T cells were similar to helped LCMV-specific CD8 T cells in WT and TRAILdeficient mice at 60 days after LCMV infection. At the same time, WT CD8 T cell primed and maintained in the absence of CD4 T cell help lost their ability to respond to secondary stimulation,

consistent with the notion that TRAIL might regulate the fitness of helpless memory CD8 T cells. Using the LCMV model of infection and MHC class II-deficient mice, Bevan and colleague (20) showed that, in the absence of CD4 T cells, maintenance of CD8 T cell memory was defective


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FIGURE 7. Impaired secondary expansion potential of helpless TRAIL-deficient memory CD8 T cells after L. monocytogenes challenge. A, WT and TRAIL-deficient mice were infected with LCMV on day 0 and helped (Ig) or helpless (␣CD4) NP396- and GP33-specific memory CD8 T cells were analyzed 3 mo later, at the time of challenge with virulent recombinant L. monocytogenes (LM-NP396; 1 ⫻ 105; 10 LD50) that expresses NP396 LCMV-derived epitope. B, Total number per spleen of WT NP396- and GP33-specific CD8 T cells before (memory) and after (d5) the LM-NP396 challenge. Data are presented as mean ⫾ SD for three mice per group. C, Percentage of NP396- and GP33-specific CD8 T cells in the spleen from representative TRAIL-deficient mice before (memory) and 6 days after LM-NP396 infection. Data are presented on a gated CD8 T cells as the percentage of Thy1.2⫹IFN-␥⫹ cells in the presence (upper number) or absence (lower number) of NP396 or GP33 peptide stimulation. D, Total number per spleen of TRAIL-deficient NP396- and GP33-specific CD8 T cells before (memory) and after (d5 or d6) the LM-NP396 challenge. Two independent experiments are shown. Data are presented as mean ⫾ SD for three mice per group. Numbers inside panels indicate the fold increase in total numbers of NP396-specific CD8 T cells 5 or 6 days after LM-NP396 infection.

and that those helpless CD8 T cells were unable to respond to secondary Ag challenge or provide the same degree of protection when compared with helped CD8 T cells. Adoptive transfer of effector and memory CD8 T cells into WT or MHC class II-deficient mice showed that the presence of CD4 T cells was required late and not during the early CD8 T cell programming phase (21). Although the mechanism(s) of CD4 T cell help in maintenance of CD8 T cell memory remain unknown, these and other studies (8, 18 –20) suggested that the effector to memory CD8 T cell transition is independent of CD4 T cells whereas maintenance of long-lived memory CD8 T cells is dependent on CD4 T cell help after infections. Interestingly, here we show that TRAIL deficiency delays

but does not prevent the erosion in the quality of helpless LCMVspecific memory CD8 T cells. These data suggest that CD4 T cell help likely consisted of TRAIL-dependent and -independent activity and that TRAIL may influence early imprinting. In this scenario, unknown TRAIL-independent activities of CD4 T cells may be required for long-term maintenance of CD8 T cell memory. In conclusion, the data presented here showed that CD4 T cells might exert their influence on CD8 T cell responses at the time of the priming as well as at the time when memory CD8 T cells are maintained. Elucidating the mechanisms that govern the generation and maintenance of memory CD8 T cells, including the need for CD4 T cells, remains an important goal that will facilitate our

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ability to manipulate and improve CD8 T cell responses after vaccination.

Acknowledgments The expert technical assistance of Rebecca Podyminogin is greatly appreciated.

Disclosures The authors have no financial conflict of interest.

References 1. Kaech, S. M., E. J. Wherry, and R. Ahmed. 2002. Effector and memory T-cell differentiation: implications for vaccine development. Nat. Rev. Immunol. 2: 251–262. 2. Harty, J. T., and V. P. Badovinac. 2002. Influence of effector molecules on the CD8⫹ T cell response to infection. Curr. Opin. Immunol. 14: 360 –365. 3. Badovinac, V. P., B. B. Porter, and J. T. Harty. 2002. Programmed contraction of CD8⫹ T cells after infection. Nat. Immunol. 3: 619 – 626. 4. Kaech, S. M., and R. Ahmed. 2001. Memory CD8⫹ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat. Immunol. 2: 415– 422. 5. Mercado, R., S. Vijh, S. E. Allen, K. Kerksiek, I. M. Pilip, and E. G. Pamer. 2000. Early programming of T cell populations responding to bacterial infection. J. Immunol. 165: 6833– 6839. 6. van Stipdonk, M. J., E. E. Lemmens, and S. P. Schoenberger. 2001. Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation. Nat. Immunol. 2: 423– 429. 7. Wong, P., and E. G. Pamer. 2001. Cutting edge: antigen-independent CD8 T cell proliferation. J. Immunol. 166: 5864 –5868. 8. Bevan, M. J. 2004. Helping the CD8⫹ T-cell response. Nat. Rev. Immunol. 4: 595– 602. 9. Buller, R. M., K. L. Holmes, A. Hugin, T. N. Frederickson, and H. C. Morse III. 1987. Induction of cytotoxic T-cell responses in vivo in the absence of CD4 helper cells. Nature 328: 77–79. 10. Rahemtulla, A., W. P. Fung-Leung, M. W. Schilham, T. M. Kundig, S. R. Sambhara, A. Narendran, A. Arabian, A. Wakeham, C. J. Paige, R. M. Zinkernagel, et al. 1991. Normal development and function of CD8⫹ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353: 180 –184. 11. Wu, Y., and Y. Liu. 1994. Viral induction of co-stimulatory activity on antigenpresenting cells bypasses the need for CD4⫹ T-cell help in CD8⫹ T-cell responses. Curr. Biol. 4: 499 –505. 12. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, and W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393: 478 – 480. 13. Bennett, S. R., F. R. Carbone, F. Karamalis, J. F. Miller, and W. R. Heath. 1997. Induction of a CD8⫹ cytotoxic T lymphocyte response by cross-priming requires cognate CD4⫹ T cell help. J. Exp. Med. 186: 65–70. 14. Ridge, J. P., F. Di Rosa, and P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4⫹ T-helper and a T-killer cell. Nature 393: 474 – 478. 15. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, and C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393: 480 – 483. 16. Bourgeois, C., B. Rocha, and C. Tanchot. 2002. A role for CD40 expression on CD8⫹ T cells in the generation of CD8⫹ T cell memory. Science 297: 2060 –2063. 17. Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, and S. P. Schoenberger. 2003. CD4⫹ T cells are required for secondary expansion and memory in CD8⫹ T lymphocytes. Nature 421: 852– 856. 18. Khanolkar, A., M. J. Fuller, and A. J. Zajac. 2004. CD4 T cell-dependent CD8 T cell maturation. J. Immunol. 172: 2834 –2844. 19. Shedlock, D. J., and H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300: 337–339. 20. Sun, J. C., and M. J. Bevan. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300: 339 –342. 21. Sun, J. C., M. A. Williams, and M. J. Bevan. 2004. CD4⫹ T cells are required for the maintenance, not programming, of memory CD8⫹ T cells after acute infection. Nat. Immunol. 5: 927–933.

22. Estcourt, M. J., A. J. McMichael, and T. Hanke. 2005. Altered primary CD8⫹ T cell response to a modified virus Ankara(MVA)-vectored vaccine in the absence of CD4⫹ T cell help. Eur. J. Immunol. 35: 3460 –3467. 23. Jennings, S. R., R. H. Bonneau, P. M. Smith, R. M. Wolcott, and R. Chervenak. 1991. CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/6 mice. Cell. Immunol. 133: 234 –252. 24. Marzo, A. L., V. Vezys, K. D. Klonowski, S. J. Lee, G. Muralimohan, M. Moore, D. F. Tough, and L. Lefrancois. 2004. Fully functional memory CD8 T cells in the absence of CD4 T cells. J. Immunol. 173: 969 –975. 25. Riberdy, J. M., J. P. Christensen, K. Branum, and P. C. Doherty. 2000. Diminished primary and secondary influenza virus-specific CD8⫹ T-cell responses in CD4-depleted Ig⫺/⫺ mice. J. Virol. 74: 9762–9765. 26. Janssen, E. M., N. M. Droin, E. E. Lemmens, M. J. Pinkoski, S. J. Bensinger, B. D. Ehst, T. S. Griffith, D. R. Green, and S. P. Schoenberger. 2005. CD4⫹ T-cell help controls CD8⫹ T-cell memory via TRAIL-mediated activation-induced cell death. Nature 434: 88 –93. 27. Schmaltz, C., O. Alpdogan, B. J. Kappel, S. J. Muriglan, J. A. Rotolo, J. Ongchin, L. M. Willis, A. S. Greenberg, J. M. Eng, J. M. Crawford, et al. 2002. T cells require TRAIL for optimal graft-versus-tumor activity. Nat. Med. 8: 1433–1437. 28. Sedger, L. M., M. B. Glaccum, J. C. Schuh, S. T. Kanaly, E. Williamson, N. Kayagaki, T. Yun, P. Smolak, T. Le, R. Goodwin, and B. Gliniak. 2002. Characterization of the in vivo function of TNF-␣-related apoptosis-inducing ligand, TRAIL/Apo2L, using TRAIL/Apo2L gene-deficient mice. Eur. J. Immunol. 32: 2246 –2254. 29. Badovinac, V. P., S. E. Hamilton, and J. T. Harty. 2003. Viral infection results in massive CD8⫹ T cell expansion and mortality in vaccinated perforin-deficient mice. Immunity 18: 463– 474. 30. White, D. W., V. P. Badovinac, G. Kollias, and J. T. Harty. 2000. Cutting edge: antilisterial activity of CD8⫹ T cells derived from TNF-deficient and TNF/perforin double-deficient mice. J. Immunol. 165: 5–9. 31. Harty, J. T., and M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of IFN-␥. Immunity 3: 109 –117. 32. van der Most, R. G., K. Murali-Krishna, J. L. Whitton, C. Oseroff, J. Alexander, S. Southwood, J. Sidney, R. W. Chesnut, A. Sette, and R. Ahmed. 1998. Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice. Virology 240: 158 –167. 33. Badovinac, V. P., and J. T. Harty. 2000. Intracellular staining for TNF and IFN-␥ detects different frequencies of antigen-specific CD8⫹ T cells. J. Immunol. Methods 238: 107–117. 34. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8: 177–187. 35. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, and R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191–1198. 36. Harrington, L. E., M. Galvan, L. G. Baum, J. D. Altman, and R. Ahmed. 2000. Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans. J. Exp. Med. 191: 1241–1246. 37. Wherry, E. J., and R. Ahmed. 2004. Memory CD8 T-cell differentiation during viral infection. J. Virol. 78: 5535–5545. 38. Badovinac, V. P., and J. T. Harty. 2006. Programming, demarcating, and manipulating CD8 T cell memory. Immunol. Rev. In press. 39. Pan, G., K. O’Rourke, A. M. Chinnaiyan, R. Gentz, R. Ebner, J. Ni, and V. M. Dixit. 1997. The receptor for the cytotoxic ligand TRAIL. Science 276: 111–113. 40. Wiley, S. R., K. Schooley, P. J. Smolak, W. S. Din, C. P. Huang, J. K. Nicholl, G. R. Sutherland, T. D. Smith, C. Rauch, C. A. Smith, et al. 1995. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3: 673– 682. 41. Lamhamedi-Cherradi, S. E., S. J. Zheng, K. A. Maguschak, J. Peschon, and Y. H. Chen. 2003. Defective thymocyte apoptosis and accelerated autoimmune diseases in TRAIL⫺/⫺ mice. Nat. Immunol. 4: 255–260. 42. Mundt, B., F. Kuhnel, L. Zender, Y. Paul, H. Tillmann, C. Trautwein, M. P. Manns, and S. Kubicka. 2003. Involvement of TRAIL and its receptors in viral hepatitis. FASEB J. 17: 94 –96. 43. Zheng, S. J., J. Jiang, H. Shen, and Y. H. Chen. 2004. Reduced apoptosis and ameliorated listeriosis in TRAIL-null mice. J. Immunol. 173: 5652–5658.