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The Journal of Immunology, 2007, 179, 2115-2125
Copyright © 2007 by The American Association of Immunologists, Inc.
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Role of Direct Effects of IFN-{gamma} on T Cells in the Regulation of CD8 T Cell Homeostasis1

Kavita Tewari, Yumi Nakayama and M. Suresh2

Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI 53706


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is well recognized that IFN-{gamma} plays a critical role in the control of CD8 T cell expansion and contraction during immune responses to several intracellular pathogens. However, our understanding of the mechanisms underlying the regulation of T cell fate by IFN-{gamma} is sorely incomplete. Specifically, it is unclear whether regulation of CD8 T cell homeostasis occurs by a T cell intrinsic IFN-{gamma} pathway. In this study, we have determined the role of the direct effects of IFN-{gamma} on T cells in regulating the expansion, contraction, and memory phases of the polyclonal CD8 T cell response to an acute viral infection. Using two complementary approaches we demonstrate that the direct effects of IFN-{gamma} suppress IL-7R expression on Ag-specific effector CD8 T cells, but clonal expansion or deletion of activated CD8 T cells in vivo can occur in the apparent absence of IFN-{gamma}R signaling in T cells. These findings have clarified fundamental features of control of T cell homeostasis by IFN-{gamma} in the context of CD8 T cell memory and protective immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The pleiotropic cytokine IFN-{gamma} is produced primarily by activated T cells and NK cells. The cellular effects of IFN-{gamma} are mediated by its heterodimeric cell surface receptor IFN-{gamma}R. The IFN-{gamma}R is comprised of {alpha}- and beta-chains, both of which belong to the class II family of cytokine receptors (1, 2). IFN-{gamma}R is expressed ubiquitously on all nucleated cells, including T cells, and therefore IFN-{gamma} exerts distinct effects in a wide range of target cells (1, 3). The broad effects of IFN-{gamma} include activation of macrophages and antiviral immunity, enhancement of Ag presentation and induction of MHC-peptide complexes, orchestration of lymphocyte-endothelial interactions, regulation of T cell polarization toward TH1/TH2, cellular proliferation, and stimulation of apoptosis (3). The obvious importance in cellular immunity of these diverse IFN-{gamma}-mediated actions is highlighted in the observation that humans and mice with IFN-{gamma}R deficiency show a marked susceptibility to intracellular bacterial and several viral infections (2, 4).

In addition to its well-established role in cellular immunity, evidence from diverse studies has been accumulating that IFN-{gamma} plays a key role in regulating T cell homeostasis during the immune response. Infection of IFN-{gamma}-deficient (IFN-{gamma}–/–) mice with Mycobacterium is associated with accumulation of a large number of activated CD4+ T cells (5), which is linked to lack of macrophage-dependent apoptotic signals (5). Another study has shown that IFN-{gamma} deficiency exacerbates experimental autoimmune encephalomyelitis in mice again due to uncontrolled expansion of activated CD4+ T cells in the CNS (6). The deletion of parasite-specific CD4+ T cells in malaria infection has also been shown to depend on IFN-{gamma} action (7). After infection with lymphocytic choriomeningitis virus (LCMV)3 or an attenuated strain of Listeria monocytogenes, IFN-{gamma}–/– mice exhibited impaired contraction of effector CD8 T cells (8, 9). Similarly, IFN-{gamma}-dependent early inflammation appears to control the contraction of CD8 T cells following L. monocytogenes infection in mice (10). Thus, IFN-{gamma} appears to control the cell-fate decision of effector CD8 T cells: apoptosis vs differentiation into memory cells. However, unraveling the multitude of IFN-{gamma}-dependent cell type-specific actions that could be involved in determining the fate of effector CD8 T cells has been accomplished only in part.

It has been shown that purified T cells from IFN-{gamma}–/– and IFN-{gamma}R-deficient (IFN-{gamma}R–/–) mice both appear refractory to apoptosis in vitro (9, 11). Moreover, in transgenic mice engineered for T cell-specific unresponsiveness to IFN-{gamma} stimulation, the magnitude of proliferative responses to allogeneic stimulation is also affected (12). At the same time, IFN-{gamma} signaling in Ag-specific CD8 T cells in vivo, during an acute L. monocytogenes infection has been demonstrated to change in a highly dynamic fashion (13). Although these studies collectively might suggest that IFN-{gamma} regulates CD8 T cell homeostasis by direct effects on CD8 T cells, it has not been formally demonstrated in comprehensive studies in vivo that IFN-{gamma} regulation is CD8 T cell autonomous. Additionally, lack of this information has imposed a constraint on studies to explore the molecular mechanisms underlying the direct effects of IFN-{gamma} on CD8 T cells. In light of these issues, we investigated whether IFN-{gamma} regulates the expansion, contraction, and memory phases of the polyclonal CD8 T cell response by direct effects on T cells during an acute LCMV infection in mice. Using two complementary experimental approaches, mixed bone marrow chimeras and transgenic mice in which T cells specifically are refractory to stimulation by IFN-{gamma}, we show that regulation of CD8 T cell homeostasis by IFN-{gamma} is essentially independent of direct effects on T cells in vivo. These findings are important for design of future studies to elucidate the mechanisms underlying the regulation of CD8 T cell memory.


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

C57BL/6/Ly-5.2 mice were purchased from the National Cancer Institute (Bethesda, MD). BALB/c, C57BL/6/Ly-5.1, IFN-{gamma}R–/– (14), and C57BL/6/Rag1–/– mice were purchased from The Jackson Laboratory. BALB/c mice expressing a dominant negative (DN) form of IFN-{gamma}R {alpha}-chain (12) were provided by Dr. R. Schreiber (Washington University, St. Louis, MO). All animal experiments were performed in strict accordance with the approved institutional animal welfare guidelines.

Generation of mixed bone marrow chimeras

Bone marrow cells were collected from the femur and tibia of C57BL/6/Ly-5.1, C57BL/6/Ly-5.2, or IFN-{gamma}R–/–/Ly-5.2 mice and depleted of T cells using magnetically labeled anti-CD5 (Ly-1) microbeads (Miltenyi Biotec). A total of 7 x 106 C57BL/6/Ly-5.1 bone marrow cells were mixed with an equal number of C57BL/6/Ly-5.2 or IFN-{gamma}R–/–/Ly-5.2 cells and injected i.v. into irradiated C57BL/6/Rag1–/– mice (950 rad). The engrafted C57BL/6/Rag1–/– mice were maintained on antibiotic water for 6 wk postirradiation.

Virus

Mice were infected with 2 x 105 PFU of the Armstrong CA 1371 strain of LCMV by i.p. injection (15). All LCMV stocks used in this study were triple plaque purified on Vero cells, and stocks were grown in BHK-21 cells. Infectious LCMV in the tissues of infected mice was quantitated by a plaque assay on Vero cells as previously described (16).

Quantitation of LCMV-specific CD8 T cells using MHC class I tetramers

Single-cell suspensions of splenocytes and PBMC were obtained by standard procedures. Mononuclear cells were isolated from the livers as previously described (17). The MHC class I tetramers specific to the Db-restricted LCMV CTL epitopes NP396–404, GP33–41, GP276–286, and the Ld-restricted CTL epitope NP118 were prepared as described elsewhere (15). Single-cell suspensions of splenocytes, hepatic mononuclear cells, or PBMC were stained with anti-CD8, anti-Ly-5.1, anti-Ly-5.2, and MHC class I tetramers for 1 h at 4°C (15). Following staining, cells were fixed in 2% paraformaldehyde and acquired on a FACSCalibur flow cytometer (BD Biosciences). All Abs were purchased from BD Pharmingen.

Intracellular staining for cytokines, granzyme B, and Bcl-2

Intracellular staining for IFN-{gamma}, TNF, and IL-2 was performed as described (15). Briefly, freshly explanted splenocytes (106 cells/well) with or without the LCMV CTL epitope peptides (0.1 µg/ml) in the presence of brefeldin A (Golgiplug; BD Pharmingen) and human recombinant IL-2 (10 U/well; BD Pharmingen) were cultured in 96-well flat-bottom plates. After 5 h of culture, cells were stained for surface CD8 and intracellular IFN-{gamma}, TNF, and IL-2 with the Cytofix/Cytoperm kit (BD Pharmingen), according to the manufacturer’s instructions. Direct ex vivo staining of LCMV-specific CD8 T cells for intracellular granzyme B and Bcl-2 was performed using the Cytofix/Cytoperm kit as described (18, 19).

Assessment of in vivo proliferation of LCMV-specific CD8 T cells with BrdU

Proliferation of virus-specific CD8 T cells in LCMV-infected mice was assessed by administering BrdU (0.8 mg/ml) in drinking water for 8 days. After 8 days of BrdU treatment, splenocytes were stained with anti-CD8 and anti-CD44 Abs and MHC class I tetramers. Following surface staining, cells were permeabilized and stained with anti-BrdU Abs (B44; BD Biosciences) as previously described (15).

Western blot analysis

Single-cell suspensions of splenocytes were separated into T cells and non-T cells using magnetically labeled anti-CD90 (Thy-1.2) microbeads (Miltenyi Biotec). Total cell lysates were prepared from T cells and non-T cells in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM NaF, 0.4 mM EDTA, 10 mM Tris (pH 7.5), and protease inhibitors). Protein derived from 2.5 x 105 cells was separated by SDS-PAGE under reducing conditions and immobilized onto polyvinylidene difluoride Immobilon membranes (Millipore). The membranes were probed with rabbit anti-phospho-STAT1 and anti-STAT1 Abs from Cell Signaling Technology. The binding of Abs was visualized using ECL Advance Western Blotting Detection kit (Amersham Biosciences).

Statistical analysis

Experimental data were analyzed using commercially available statistical software (SYSTAT v.8.0). 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
 Disclosures
 References
 
Kinetics of virus-specific CD8 T cell responses in IFN-{gamma}R–/– mice

Previous work have shown that IFN-{gamma} might play a role in regulating the expansion and contraction phases of the CD8 T cell response to LCMV (8, 9). However, the effect of IFN-{gamma}R deficiency on the "quality" and "quantity" of long-term CD8 T cell memory has not been studied. In this study, we compared the kinetics of the CD8 T cell response and generation of CD8 T cell memory between wild-type C57BL/6 (+/+) and IFN-{gamma}R–/– mice (14) following infection with the Armstrong strain of LCMV. On various days postinfection (PI), we quantitated LCMV-specific CD8 T cells in the spleen by intracellular cytokine staining (15). As illustrated in Fig. 1A, on day 8 PI, expansion of CD8 T cells specific to the three LCMV epitopes NP396–404, GP33–41, and GP276–286 was substantially reduced (~10-fold) in the spleen of IFN-{gamma}R–/– mice, compared with wild-type C57BL/6 mice. Similarly, the expansion of LCMV-specific CD8 T cells in the nonlymphoid organs liver and lung of IFN-{gamma}R–/– mice was lower than in wild-type C57BL/6 mice (data not shown). Thus, IFN-{gamma}-IFN-{gamma}R interactions are essential for optimal expansion of CD8 T cells during a primary LCMV infection. Ensuing the expansion phase, in the wild-type C57BL/6 mice, between days 8 and 54 PI, there was a ~60-fold reduction in the number of LCMV-specific CD8 T cells (Fig. 1A), but that stayed relatively stable thereafter until day 500 PI. Strikingly, in the IFN-{gamma}R–/– mice, there was no detectable reduction in the number of LCMV-specific CD8 T cells between days 8 and 500 PI (Fig. 1A). Thus, despite lower expansion, a lack of contraction resulted in a substantial enhancement (~6-fold) in the number of memory CD8 T cells in the spleen of IFN-{gamma}R–/– mice compared with wild-type C57BL/6 mice. The nonlymphoid organs of IFN-{gamma}R–/– mice also contained a higher number of Ag-specific memory CD8 T cells than found in wild-type C57BL/6 mice (data not shown). We confirmed the data from intracellular cytokine staining by performing parallel analyses using MHC class I tetramers (data not shown). These data showed that IFN-{gamma}-IFN-{gamma}R interactions are causally involved in the contraction of LCMV-specific CD8 T cells, thereby limiting the size of CD8 T cell memory. Coupled with published findings (8), these data also demonstrated that, as in BALB/c mice, IFN-{gamma} regulates CD8 T cell homeostasis during an acute LCMV infection in C57BL/6 mice. Although infectious LCMV in the tissues of wild-type C57BL/6 mice was below the level of detection by day 8 PI, IFN-{gamma}R–/– mice harbored LCMV up to day 15 PI (data not shown).


Figure 1
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  		FIGURE 1. Phenotype and functional attributes of effector and memory CD8 T cells in wild-type C57BL/6 (+/+) and IFN-{gamma}R–/– mice. A, On the indicated days after LCMV infection, LCMV-specific CD8 T cells were quantitated in the spleen by intracellular staining for IFN-{gamma}. B, On days 8 (effector) and 250 (memory) PI, the surface phenotype of effector and memory CD8 T cells was analyzed using flow cytometry. FACS plots are gated on Db/NP396-specific CD8 T cells. C, Intracellular granzyme B expression in Db/NP396-specific CD8 T cells on days 8 (effector) and 120 (memory) PI. Staining with isotype control (open histogram) and anti-granzyme Abs (gray-filled histogram) are depicted. D–F, Kinetics of IFN-{gamma} production by Db/NP396-specific memory CD8 T cells from LCMV immune (day 500 PI) wild-type C57BL/6 and IFN-{gamma}R–/– mice; the percentage of IFN-{gamma}-producing CD8 T cells was quantitated by intracellular cytokine staining. Data in D are expressed as a percentage of maximum response attained at 5 h for each mouse. Data in E are the MFI of staining for IFN-{gamma}. F, On day 500 PI, IFN-{gamma} production by LCMV-specific CD8 T cells was determined by intracellular cytokine staining. Dot plots are gated on total splenocytes, and the percentage shown represents high and low IFN-{gamma}-producing cells of total epitope-specific CD8 T cells. Value in parentheses is the MFI of IFN-{gamma} staining for high and low IFN-{gamma}-producing CD8 T cells. G, Homeostatic proliferation of memory CD8 T cells. LCMV-immune mice were administered BrdU for 1 wk. Splenocytes were stained with anti-CD8, anti-CD44, MHC class I tetramers, and anti-BrdU Abs. The histograms are gated on the indicated populations of CD8 T cells, and the percentage of BrdU+ cells among the gated cell population is shown. All experiments were conducted with three to four mice per group and repeated two to three times.

 
Data in Fig. 1A clearly showed the importance of IFN-{gamma}-IFN-{gamma}R interactions in regulating the expansion and contraction phases of the CD8 T cell response to LCMV. We investigated whether IFN-{gamma}R deficiency affected phenotypic attributes of effector and memory CD8 T cells. As shown in Fig. 1B, LCMV-specific effector CD8 T cells (day 8 PI) in both wild-type C57BL/6 and IFN-{gamma}R–/– mice were L-selectinlow (CD62L)low, CD44high, CD27high, and CD43high 1B11high. However, notably a larger proportion of LCMV-specific effector CD8 T cells from IFN-{gamma}R–/– mice were IL-7Rhigh (CD127)high compared with CD8 T cells from wild-type C57BL/6 mice (Fig. 1B). Except for CD62L expression, cell surface phenotype of LCMV-specific memory CD8 T cells in IFN-{gamma}R–/– mice was similar to those in wild-type C57BL/6 mice; a larger proportion of NP396-specific memory CD8 T cells exhibited the CD62Llow effector memory phenotype in the IFN-{gamma}R–/– mice compared with wild-type C57BL/6 mice (Fig. 1B). Moreover, as in wild-type C57BL/6 mice, only LCMV-specific effector CD8 T cells (day 8 PI), and not memory CD8 T cells expressed intracellular granzyme B in IFN-{gamma}R–/– mice (Fig. 1C).

It is known that memory CD8 T cells are hyperreactive to antigenic stimulation and produce cytokines more rapidly than naive T cells (20, 21). We determined whether generation of memory CD8 T cells under conditions of IFN-{gamma}R deficiency affected their cytokine producing abilities ex vivo. On day 500 PI, splenocytes from wild-type C57BL/6 and IFN-{gamma}R–/– mice were stimulated with LCMV CTL epitope peptides, and the kinetics of IFN-{gamma}, TNF, and IL-2 production by LCMV-specific CD8 T cells was assessed from 1 to 5 h after stimulation. LCMV-specific memory CD8 T cells from wild-type C57BL/6 mice produced cytokines rapidly, whereas IFN-{gamma}R–/– mice exhibited a slight delay in producing IFN-{gamma}; at 1-h poststimulation ~50% and 25% of LCMV-specific CD8 T cells produced IFN-{gamma} in wild-type C57BL/6 and IFN-{gamma}R–/– mice, respectively. However, after 1 h, the kinetics of IFN-{gamma} production by LCMV-specific CD8 T cells was similar between wild-type C57BL/6 and IFN-{gamma}R–/– mice (Fig. 1D). As an index of cytokine-producing ability, we compared the mean fluorescence intensity (MFI) of staining for intracellular IFN-{gamma} between wild-type C57BL/6 and IFN-{gamma}R–/– memory CD8 T cells at various times after antigenic stimulation ex vivo. Fig. 1E shows that the MFI for IFN-{gamma} staining was lower in memory CD8 T cells from IFN-{gamma}R–/– mice than in wild-type C57BL/6 mice. Dot plots in Fig. 1F clearly illustrate the presence of subpopulations of high IFN-{gamma} producers and low IFN-{gamma} producers among LCMV-specific memory CD8 T cells in both wild-type C57BL/6 and IFN-{gamma}R–/– mice. Notably, 19.1 ± 1.8% and 40.1 ± 2.4% of memory CD8 T cells produce low levels of IFN-{gamma} in wild-type C57BL/6 and IFN-{gamma}R–/– mice, respectively. The MFI of IFN-{gamma} staining was comparable between wild-type C57BL/6 (69 ± 5) and IFN-{gamma}R–/– (59 ± 5) low IFN-{gamma}-producing CD8 T cells. However, the average MFI of IFN-{gamma} staining of wild-type C57BL/6 high IFN-{gamma}-producing CD8 T cells (633 ± 29) was higher than in IFN-{gamma}R–/– high IFN-{gamma}-producing CD8 T cells (466 ± 46). Collectively, data presented in Fig. 1, D–F, strongly suggested that LCMV-specific memory CD8 T cells in IFN-{gamma}R–/– mice produced lower levels of IFN-{gamma} compared with cells produced in wild-type C57BL/6 mice.

The ability to undergo cytokine-driven proliferative renewal is considered as one of the hallmark features of memory CD8 T cells (22). Between days 180 and 188 PI, we assessed whether differentiation of memory CD8 T cells without IFN-{gamma}R signaling affected the proliferative renewal of LCMV-specific memory CD8 T cells by quantitating BrdU incorporation in vivo (15). Our results show that 19–20% of CD44high and LCMV-specific memory CD8 T cells incorporated BrdU during a span of 1 wk in wild-type C57BL/6 mice (Fig. 1G). In IFN-{gamma}R–/– mice, BrdU incorporation by CD44low and CD44high CD8 T cells was comparable to incorporation in wild-type C57BL/6 mice. However, the percentage of BrdU+ LCMV-specific CD8 T cells (NP396-specific CD8 T cells in particular) in IFN-{gamma}R–/– mice was lower than in wild-type C57BL/6 mice. The lower proliferation of LCMV-specific memory CD8 T cells in IFN-{gamma}R–/– mice is consistent with their CD62Llow effector memory phenotype (Fig. 1B); data are in agreement with a study that compared proliferative renewal of central with effector memory CD8 T cells (19). In summary, these data show that: 1) proliferative renewal of LCMV-specific memory CD8 T cells is reduced in IFN-{gamma}R–/– mice, and 2) enhanced number of LCMV-specific memory CD8 T cells in IFN-{gamma}R–/– mice was not due to an increased rate of proliferation.

Expansion of IFN-{gamma}R-sufficient and IFN-{gamma}R-deficient CD8 T cells in bone marrow chimeric mice

Because IFN-{gamma}R is expressed ubiquitously, regulation of CD8 T cell expansion and contraction by IFN-{gamma} could be mediated by direct effects on CD8 T cells per se and/or by indirect effects through non-CD8 T cells. It was recently reported that the expansion of monoclonal TCR transgenic CD8 T cells during an acute LCMV infection was compromised without the direct effects of IFN-{gamma} (23). In these studies, IFN-{gamma}R–/– TCR transgenic CD8 T cells exhibited poor expansion compared with their IFN-{gamma}R-expressing counterparts in LCMV-infected mice. Monoclonal TCR transgenic mice do not necessarily mimic a physiological polyclonal response, and it remains to be determined whether IFN-{gamma} regulates CD8 T cell contraction and memory cell survival by direct effect on CD8 T cells. To address these issues, we generated bone marrow chimeric mice, which will allow us to compare the responses of IFN-{gamma}R-deficient and IFN-{gamma}R-sufficient polyclonal CD8 T cells in the same mouse. Lethally irradiated Rag1–/– mice were reconstituted with a 1:1 mixture of T cell-depleted bone marrow cells from wild-type C57BL/6/Ly-5.1 and IFN-{gamma}R–/–/Ly-5.2 mice (Fig. 2A). At 8 wk posttransfer of cells, we assessed the reconstitution of the hemopoietic system by the donor bone marrow cells, by staining PBMC with anti-CD8, anti-CD4, anti-Ly-5.1, and anti-Ly-5.2 Abs (Fig. 2B). Dot plots in Fig. 2B show that donor bone marrow cells from wild-type C57BL/6 and IFN-{gamma}R–/– mice successfully reconstituted the lymphoid system of Rag1–/– mice. The observed ~1:1 ratio of wild-type C57BL/6/Ly-5.1 to IFN-{gamma}R–/–/Ly-5.2 CD8 T cells in Rag1–/– mice showed that the reconstitution abilities of wild-type C57BL/6 and IFN-{gamma}R–/– bone marrow cells were similar. To examine whether expansion of polyclonal CD8 T cells during an acute viral infection is dependant on direct effects of IFN-{gamma}, we infected mixed bone marrow chimeric mice with LCMV. On day 8 PI, we quantitated LCMV-specific CD8 T cells in the spleen and liver by using MHC class I tetramers. As shown in Fig. 2C, there was a strong activation of LCMV-specific CD8 T cells in mixed bone marrow chimeric mice. Next, we determined the relative proportions of C57BL/6/Ly-5.1 vs IFN-{gamma}R–/–/Ly-5.2 cells among LCMV-specific CD8 T cells in spleen, liver, and blood of mixed bone marrow chimeric mice. As shown in Fig. 2D, the ratio of C57BL/6/Ly-5.1 to IFN-{gamma}R–/–/Ly-5.2 LCMV-specific effector CD8 T cells was close to 1:1 in the spleen and liver of all bone marrow chimeric mice examined. Data shown in Fig. 2 are representative of three independent experiments. These studies show that lack of IFN-{gamma}R on CD8 T cells did not adversely affect expansion of polyclonal LCMV-specific CD8 T cells in vivo. Thus, direct effect of IFN-{gamma} on CD8 T cells does not appear to be required for expansion of CD8 T cells during an acute LCMV infection.


Figure 2
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  		FIGURE 2. Expansion of wild-type C57BL/6 (+/+) and IFN-{gamma}R-deficient CD8 T cells in bone marrow chimeric mice. A, Generation of mixed bone marrow chimeras (BMC). Lethally irradiated C57BL/6/Rag1–/– mice were reconstituted with 1:1 mixture of bone marrow cells from C57BL/6/Ly-5.1 and C57BL/6/Ly-5.2 or IFN-{gamma}R–/–/Ly-5.2 mice. B, The percentage of wild-type C57BL/6/Ly-5.1 and IFN-{gamma}R–/–/Ly-5.2 CD8 T cells in the peripheral blood of bone marrow chimeras was determined 6 wk after cell transfer; dot plots of individual bone marrow chimeras are gated on total CD8 T cells, and the percentage of Ly-5.1 vs Ly-5.2 CD8 T cells in the CD8 T cell compartment is shown. C, The bone marrow chimeric mice (C57BL/6/Ly-5.1:IFN-{gamma}R–/–/Ly-5.2) were infected with LCMV, and the number of LCMV epitope-specific CD8 T cells in the spleen were determined by staining with MHC class I tetramers on day 8 PI. Dot plots of individual bone marrow chimeric mice are gated on total viable splenocytes, and the percentage of tetramer-binding CD8 T cells among splenocytes is shown. D, On day 8 PI, the percentage of wild-type C57BL/6 (Ly-5.1) and IFN-{gamma}R-deficient (Ly-5.2) cells of total LCMV-specific CD8 T cells was determined in the spleen and liver of bone marrow chimeric mice by staining with MHC class I tetramers, anti-CD8, anti-Ly-5.1, and anti-Ly-5.2 Abs. Dot plots are gated on the indicated tetramer-positive CD8 T cells; percentage of wild-type C57BL/6 or IFN-{gamma}R-deficient CD8 T cells of total epitope-specific CD8 T cells is indicated. E, Granzyme B and IFN-{gamma} expression by Db/NP396-specific effector CD8 T cells in the spleen of bone marrow chimeric mice. On day 8 PI, splenocytes were stained with anti-CD8, MHC class I tetramer, anti-Ly-5.1, anti-Ly-5.2, and anti-granzyme B Abs. Staining with isotype control (open histogram) and anti-granzyme B Abs (gray-filled histogram) are depicted at top. The histograms at the bottom are gated on Ly-5.1+ or Ly-5.2+ IFN-{gamma}-producing CD8 T cells and show staining for IFN-{gamma} in NP396–404 peptide stimulated CD8 T cells from the spleens of bone marrow chimeric mice at day 8 PI. The MFI ± SD for staining for granzyme B or IFN-{gamma} is shown. F, Bcl-2 expression in wild-type C57BL/6 and IFN-{gamma}R-deficient CD8 T cells. On day 8 PI, splenocytes were stained with anti-CD8, MHC class I tetramer, anti-Ly-5.1, anti-Ly-5.2, and anti-Bcl-2 Abs. Specific staining for Bcl-2 in Db/NP396-specific CD8 T cells at day 8 PI in bone marrow chimeric mice (gray-filled histogram) is depicted. Staining with an isotype control Ab (open histogram) is also indicated. Data are from three to four mice per group and are representative of three independent experiments.

 
Expression of granzyme B is a reliable surrogate marker of effector CD8 T cells (19). Data in Fig. 2E show that the levels of granzyme in IFN-{gamma}R–/– LCMV-specific effector CD8 T cells were comparable to those in wild-type C57BL/6 CD8 T cells. Thus, lack of direct effects of IFN-{gamma} on CD8 T cells did not affect differentiation of effector cells from naive CD8 T cells. Next, we determined whether loss of IFN-{gamma} signaling on CD8 T cells affected the ability of effector CD8 T cells to secrete cytokines directly ex vivo. The MFI of staining for intracellular IFN-{gamma} in wild-type C57BL/6 and IFN-{gamma}R–/– effector CD8 T cells were comparable. Therefore, IFN-{gamma}R deficiency had no detectable effects on the production of IFN-{gamma} upon peptide stimulation ex vivo by effector CD8 T cells (Fig. 2E). Nor did the absence of IFN-{gamma}R signaling affect the level of antiapoptotic molecule Bcl-2; both the C57BL/6/Ly-5.1 and IFN-{gamma}R–/–/Ly-5.2 effector CD8 T cells expressed similar levels of Bcl-2 in the mixed bone marrow chimeric mice (Fig. 2F). In summary, lack of IFN-{gamma}R signaling in CD8 T cells did not affect clonal expansion and differentiation of effector CD8 T cells from naive T cells.

Contraction and homeostasis of IFN-{gamma}R-sufficient and IFN-{gamma}R-deficient CD8 T cells in bone marrow chimeric mice

Fig. 1A showed that the contraction of LCMV-specific effector CD8 T cells was impaired in IFN-{gamma}R–/– mice. In this model, using bone marrow chimeric mice, we examined whether direct effects of IFN-{gamma} on T cells are essential to effect the contraction phase of CD8 T cell response. As described, we generated bone marrow chimeric mice by reconstituting lethally irradiated Rag1–/– mice with a 1:1 mixture of bone marrow cells from wild-type C57BL/6/Ly-5.1 and wild-type C57BL/6/Ly-5.2 or IFN-{gamma}R–/–/Ly-5.2 mice. FACS plots in Fig. 3B, show the percentages of Ly-5.1 vs Ly-5.2 CD8 T cells among total circulating CD8 T cells in C57BL/6/Ly-5.1 to C57BL/6/Ly-5.2 (control) bone marrow chimeric mice and wild-type C57BL/6/Ly-5.1 to IFN-{gamma}R–/–/Ly-5.2 (experimental) bone marrow chimeric mice 8 wk after immune reconstitution (before LCMV infection). Control and experimental bone marrow chimeric mice were infected with LCMV, and longitudinal analysis of CD8 T cell responses in the blood was performed in individual mice at different days after infection. Upon LCMV infection, both control and experimental groups of bone marrow chimeric mice exhibited strong activation of virus-specific CD8 T cells. Longitudinal analysis of circulating LCMV-specific CD8 T cells in individual mice (Fig. 3A) showed that the overall kinetics of the CD8 T cell response in control and experimental bone marrow chimeras were comparable; the kinetics of LCMV-specific CD8 T cell response in blood of control and experimental chimeras were similar to those in unmanipulated wild-type C57BL/6 mice (data not shown). Next, we quantitated the percentage of Ly-5.1 vs Ly-5.2 CD8 T cells among LCMV-specific CD8 T cells in the blood on various days after infection. To reiterate, the preinfection ratio of circulating Ly-5.1+ and Ly-5.2+ CD8 T cells was close to 1:1 in both control and experimental bone marrow chimeric mice (Fig. 3B, top). On day 8 PI, the relative proportions of Ly-5.1+ and Ly-5.2+ effector CD8 T cells among LCMV-specific CD8 T cells were similar to preinfection values for total CD8 T cells (Fig. 3B). These data confirm our findings shown in Fig. 2D that the expansion of LCMV-specific CD8 T cells was not dependent on the direct effect of IFN-{gamma}. Importantly, during the contraction phase, (between days 8 and 40 PI) there was little change in the ratio of wild-type C57BL/6/Ly-5.1 to IFN-{gamma}R–/–/Ly-5.2 LCMV-specific CD8 T cells (Fig. 3B). These findings showed that lack of IFN-{gamma}R signaling did not significantly affect the clonal depletion of LCMV-specific CD8 T cells in vivo. Moreover, the generation and maintenance of long-term CD8 T cell memory (day 250 PI) was normal in the absence of direct effects of IFN-{gamma} on CD8 T cells. Data in Fig. 3 are representative of two independent experiments. Taken together, these data show that the regulation of CD8 T cell homeostasis including the expansion phase, contraction phase, and memory phase is largely independent of the direct effects of IFN-{gamma} on CD8 T cells.


Figure 3
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  		FIGURE 3. Contraction of wild-type C57BL/6 (+/+) and IFN-{gamma}R-deficient LCMV-specific CD8 T cells in bone marrow chimeric mice. Longitudinal analyses of LCMV-specific CD8 T cell responses in bone marrow chimeric (BMC) mice. Groups of C57BL/6/Ly-5.1:C57BL/6/Ly-5.2 (control) or C57BL/6/Ly-5.1:IFN-{gamma}R–/–/Ly-5.2 (experimental) bone marrow chimeric mice were infected with LCMV. A, Normal kinetics of CD8 T cell response to LCMV in bone marrow chimeric mice. On the indicated days after LCMV infection, the percentage of NP396- and GP33-specific CD8 T cells in the peripheral blood was quantitated by staining with MHC class I tetramers. B, Relative proportions of wild-type C57BL/6 and IFN-{gamma}R-deficient CD8 T cells among LCMV-specific CD8 T cells in bone marrow chimeric mice. LCMV-infected bone marrow chimeric mice were bled on the indicated days after infection, and PBMC were stained with anti-CD8, Db/NP396 tetramer, anti-Ly-5.1, and anti-Ly-5.2 Abs. Dot plots for preinfection are gated on total CD8 T cells, but other dot plots are gated on tetramer-binding CD8 T cells. All experiments were conducted with six to eight mice per group.

 
Effect of T cell-specific deficiency of IFN-{gamma}R signaling on primary expansion of CD8 T cells during an acute LCMV infection

Studies in bone marrow chimeric mice showed that the responses of IFN-{gamma}R–/– CD8 T cells were similar to wild-type C57BL/6 CD8 T cells during an acute LCMV infection (Fig. 2). These data strongly suggested that CD8 T cell homeostasis might occur independently of the direct effect of IFN-{gamma} on CD8 T cells. However, it is possible that IFN-{gamma}R-expressing wild-type C57BL/6 CD8 T cells could have indirectly promoted the expansion and contraction of IFN-{gamma}R–/– CD8 T cells in the bone marrow chimeric mice. To address this issue, we investigated CD8 T cell responses to LCMV in transgenic mice that express a DN form of IFN-{gamma}R selectively in T cells (12), (referred to in this study as DN mice). In the DN mice, Lck promoter-driven transgenic expression of a truncated form of IFN-{gamma}R {alpha}-chain acts as a "dominant negative" in the T cell compartment; as a result, in these mice, only T cells are refractory to direct effects of IFN-{gamma}. Previous characterization of these transgenic mice has shown that IFN-{gamma}-induced up-regulation of MHC class I is selectively impaired in T cells (12). To confirm that only T cells in DN mice are refractory to IFN-{gamma}-mediated effects, we examined IFN-{gamma}-mediated phosphorylation of STAT1 in T cells and non-T cells in vitro. As shown in Fig. 4A, IFN-{gamma}-induced phosphorylation of STAT1 in both T and non-T cells from nontransgenic BALB/c mice. However in the DN mice, IFN-{gamma} induced phosphorylation of STAT1 only in non-T cells, but not in T cells (Fig. 4A). Therefore, it appears that T cells are selectively refractory to IFN-{gamma} in the DN mice.


Figure 4
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  		FIGURE 4. Primary expansion of CD8 T cells in the absence of direct effects of IFN-{gamma} on T cells. A, T cells from DN mice are refractory to IFN-{gamma}-induced phosphorylation of STAT1. Splenocytes from BALB/c (+/+) and DN mice were separated into T cells and non-T cells using magnetically labeled anti-Thy1.2 microbeads, and exposed to IFN-{gamma} for 30 min. After in vitro stimulation with IFN-{gamma}, cell lysates were analyzed for phosphorylation of STAT1 by Western blotting. B, Expansion of LCMV-specific CD8 T cells in DN mice. On the eighth day after LCMV infection, Ld/NP118-specific CD8 T cells were quantitated in the spleen of BALB/c and DN mice by staining spleen cells with anti-CD8 and Ld/NP118 MHC class I tetramer. Data are the mean of eight mice per group. C and D, Cell surface phenotype of NP118-specific effector CD8 T cells in DN mice on day 8 PI. Splenocytes were stained for the indicated cell surface molecules or intracellular Bcl-2 in conjunction with anti-CD8 and Ld/NP118 tetramer. The FACS plots are gated on Ld/NP118-specific CD8 T cells, and the percentage of cell surface molecules or intracellular Bcl-2 is indicated. E, IFN-{gamma} production by NP118-specific CD8 T cells in DN mice. On day 8 PI, splenocytes from BALB/c and DN mice were stimulated with the NP118 peptide ex vivo, and the number of IFN-{gamma}-producing CD8 T cells was quantitated by intracellular cytokine staining. The histograms are gated on CD8 T cells secreting IFN-{gamma} in response to ex vivo stimulation with NP118–126 peptide, and MFI ± SD of staining for IFN-{gamma} is shown. All the experiments were conducted with four mice per group and repeated three times.

 
Please note that the effects of IFN-{gamma} deficiency on CD8 T cell homeostasis during LCMV infection in BALB/c mice (8) is similar to those in C57BL/6 mice (Fig. 1A). Hence, we believe that our studies of LCMV-specific CD8 T cell homeostasis using DN mice on the BALB/c background will validate experiments done in C57BL/6 mice (Fig. 3). In this model, we determined the role of IFN-{gamma}-mediated direct effects on T cells during the activation and expansion of virus specific CD8 T cells following an acute LCMV infection. Groups of nontransgenic BALB/c (+/+) and DN mice were infected with LCMV, and the number of CD8 T cells specific to the immunodominant epitope Ld/NP118 was quantitated by using MHC class I tetramers. Data in Fig. 4B show that the expansion of Ld-restricted NP118-specific CD8 T cells in DN mice was similar to BALB/c mice. Thus, similar to studies using bone marrow chimeric mice (Fig. 2D), the expansion of CD8 T cells during an acute LCMV infection can occur independent of direct effects of IFN-{gamma}. Furthermore, the expression of LFA-1, CD62L, and CD43 on LCMV-specific effector CD8 T cells in DN mice was similar to BALB/c mice (Fig. 4C). Previous studies have suggested that effector CD8 T cells that express higher levels of inhibitory killer cell lectin-like receptor G-1 (KLRG-1) and low levels of IL-7R represent apoptosis-prone terminally differentiated or senescent effector cells (18). We next determined whether absence of signaling through IFN-{gamma}R affected the expression of KLRG-1 and IL-7R on LCMV-specific CD8 T cells. FACS plots in Fig. 4D show that ~56% of the Ag-specific CD8 T cells were KLRG-1high in both BALB/c and DN mice. However, the proportion of IL-7Rhigh LCMV-specific effector CD8 T cells was significantly higher in the spleen of DN mice as compared with the BALB/c mice (Fig. 4D). Additionally, purified naive T cells from BALB/c mice but not DN mice down-regulated IL-7R expression in response to IFN-{gamma} in vitro (data not shown). Thus, direct signaling through IFN-{gamma}R on T cells appears to regulate IL-7R expression on Ag-specific effector CD8 T cells.

Next, we examined the effect of T cell-specific IFN-{gamma}R deficiency on the expression of cytokines IFN-{gamma}, TNF, and IL-2 in LCMV-specific CD8 T cells. The Ag-induced production of IFN-{gamma} (Fig. 4E), TNF, and IL-2 (data not shown) by LCMV-specific CD8 T cells was similar in BALB/c and DN mice. Thus, deficiency in IFN-{gamma}R signaling did not significantly affect the expression of effector cytokines by LCMV-specific CD8 T cells.

Contraction and CD8 T cell memory in the absence of IFN-{gamma}R signaling in T cells

To determine the role of direct effects of IFN-{gamma} on T cells in regulating CD8 T cell homeostasis during an acute LCMV infection, we performed longitudinal analysis of virus-specific CD8 T cells in the blood of individual LCMV-infected BALB/c and DN mice. The frequency of LCMV-specific CD8 T cells in BALB/c and DN mice were comparable on day 8 PI (Fig. 5A). After day 8 PI, during the contraction phase, the frequencies of Ld/NP118-specific CD8 T cells declined in both BALB/c and DN mice. Although not dramatic, the contraction phase of the CD8 T cell response was slightly attenuated in DN mice compared with BALB/c mice. Between day 8 and 60 PI, there was a ~17- and ~8-fold reduction in the frequencies of LCMV-specific CD8 T cells in BALB/c and DN mice, respectively. Reproducibly, the frequencies of Ld/NP118-specific memory CD8 T cells (day 60 PI) in DN mice were ~2-fold more than in BALB/c mice. Consistent with this conclusion, on day 250 PI, spleens of DN mice contained 2- to 3-fold more LCMV-specific memory CD8 T cells than found in BALB/c mice (Fig. 5B). The modest effect of IFN-{gamma}R signaling deficiency on the contraction of LCMV-specific CD8 T cells was not linked to increased proliferation between days 8 and 15 PI (Fig. 5C), but correlated with reduced susceptibility to apoptosis in vitro (Fig. 5D). Additionally, the cell surface phenotype and cytokine producing ability of LCMV-specific memory CD8 T cells in DN mice were comparable to BALB/c mice (data not shown). Taken together, data in Figs. 4 and 5 showed that direct effects of IFN-{gamma} on T cells might not be required to regulate expansion and contraction of Ag-specific CD8 T cells during an acute LCMV infection.


Figure 5
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  		FIGURE 5. Contraction of LCMV-specific CD8 T cells is independent of direct effects of IFN-{gamma} on T cells. A and B, Longitudinal analyses of LCMV-specific CD8 T cells in BALB/c (+/+) and DN mice. On the indicated days after infection with LCMV, the percentage of Ld/NP118-specific CD8 T cells was quantitated in the peripheral blood of individual BALB/c and DN mice by staining with anti-CD8 and Ld/NP118 tetramers. The dot plots are gated on total viable PBMC, and the percentage of Ld/NP118-specific cells of total mononuclear cells is shown. B, The total number of Ld/NP118-specific CD8 T cells in the spleen of LCMV immune (day 270 PI) BALB/c and DN mice. C, In vivo proliferation of LCMV-specific CD8 T cells in BALB/c and DN mice. Groups of BALB/c and DN mice were infected with LCMV and administered BrdU in drinking water between days 8 and 15 PI. On day 15 PI, splenocytes and liver mononuclear cells were stained with anti-CD8, anti-CD44, Ld/NP118 tetramers, and anti-BrdU Abs. The histograms are gated on Ld/NP118-specific CD8 T cells, and the percentage ± SD of BrdU+ cells of Ld/NP118-binding CD8 T cells is shown. D, In vitro apoptosis of LCMV-specific CD8 T cells from BALB/c and DN mice. On days 8 and 15 PI, the percentage of annexin V-binding Ld/NP118-specific CD8 T cells was determined after overnight culture in medium. Dot plots are gated on Ld/NP118 tetramer-binding CD8 T cells, and the percentage ± SD of annexin Vhigh cells of total Ld/NP118-specific CD8 T cells is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well established that contraction of activated CD8 T cells following L. monocytogenes and LCMV infections is dependent upon IFN-{gamma}-IFN-{gamma}R interactions (8). Because the expression of IFN-{gamma}R is ubiquitous in nucleated cells of all tissues, the immunoregulatory activity of IFN-{gamma} could act through any one of a vast number of mechanisms intrinsically feasible for IFN-{gamma}-dependent cell-mediated action. Recent studies, in vitro (11) and in vivo (13), have indicated that IFN-{gamma} might regulate CD8 T cell responses by direct action on the T cells themselves. Yet, it is still unknown whether expression of IFN-{gamma}R on CD8 T cells is required for IFN-{gamma} to regulate CD8 T cell homeostasis in vivo during an acute viral infection. The thrust of our experiments addresses this basic uncertainty; we report that, during an acute LCMV infection, the presence of functional IFN-{gamma}R on CD8 T cells is not necessary for normal expansion and contraction of polyclonal Ag-specific CD8 T cells or the generation of CD8 T cell memory. These findings directly address the cellular and molecular bases of CD8 T cell memory.

Although it has been reported that the contraction phase of the CD8 T cell response to LCMV was impaired in IFN-{gamma}–/– mice (8), the phenotype and functional attributes of memory CD8 T cells generated under conditions of IFN-{gamma} deficiency have not been examined. Our studies have confirmed previous reports that primary expansion of CD8 T cells is substantially reduced in IFN-{gamma}R–/– mice compared with wild-type C57BL/6 mice (8, 24). Following expansion, unlike in the wild-type C57BL/6 mice, LCMV-specific CD8 T cells in IFN-{gamma}R–/– mice showed no detectable contraction up to day 500 PI; the number of LCMV-specific memory CD8 T cells in the spleen of IFN-{gamma}R–/– mice was ~5-fold higher than in the spleen of wild-type C57BL/6 mice. With the one exception that a larger fraction of LCMV-specific memory CD8 T cells in IFN-{gamma}R–/– mice was of the CD62Llow effector memory phenotype, and accordingly exhibited slightly lower rate of proliferative renewal (19), the expression of other cell surface molecules was not significantly affected by IFN-{gamma}R deficiency. Notably, however, a larger fraction of LCMV-specific memory CD8 T cells from IFN-{gamma}R–/– mice produced lower amounts of IFN-{gamma} upon Ag stimulation ex vivo, compared with those from wild-type C57BL/6 mice. The overall lower cytokine production by IFN-{gamma}R-deficient memory CD8 T cells could be effected by a slightly protracted LCMV infection that has been observed in IFN-{gamma}R–/– mice (24); and/or, by the effects of IFN-{gamma}R deficiency on the activities of non-T cells in IFN-{gamma}R–/– mice. Studies in bone marrow chimeric mice suggested that stimulation of wild-type C57BL/6 CD8 T cells by less effective IFN-{gamma}R-deficient APCs may have adversely affected cytokine production of memory CD8 T cells. In any event, these findings show that IFN-{gamma}-IFN-{gamma}R interactions regulate both quantity and quality of CD8 T cell memory.

We and others (8, 23, 24) have shown that loss of IFN-{gamma}-IFN-{gamma}R interactions effected a substantial reduction in the primary activation and expansion of CD8 T cells during an acute LCMV infection. In this study, using two different approaches, we show that direct effects of IFN-{gamma} on T cells are not obligatory for CD8 T cell expansion and differentiation into effector cells. Our findings contradict experiments by Whitmire et al. (23), who reported that direct effects of IFN-{gamma} are required for optimal primary expansion of LCMV-specific CD8 T cells. Differences in the experimental approaches used by Whitmire et al. (23) and by us might explain the discrepancies. First, in the design of adoptive transfer, used in one of the experiments of Whitmire et al. (23), an equal number of total splenocytes from wild-type C57BL/6 mice and IFN-{gamma}R–/– mice were mixed and adoptively transferred into congenic (+/+) C57BL/6 mice. A potential problem with this approach is that the number of CD8 T cell precursors in the T cell repertoire of C57BL/6 and IFN-{gamma}R–/– mice were not shown to be the same; additionally, inadvertent introduction of unrecognized artifacts associated with adoptive transfer of CD8 T cells cannot be excluded. Second, in studies using adoptive transfer of monoclonal P14 TCR transgenic CD8 T cells, the ratio of wild-type C57BL/6 to IFN-{gamma}R-deficient donor CD8 T cells in ~50% of the recipient LCMV-infected mice was close to 1, which suggested that in these mice, the expansion of IFN-{gamma}R-deficient CD8 T cells was indeed comparable to their wild-type counterparts. At the same time, it is possible that the responses of monoclonal TCR transgenic CD8 T cells will not mimic a physiological polyclonal CD8 T cell response. In our studies, we have used mixed bone marrow chimeras in which wild-type C57BL/6 and IFN-{gamma}R-deficient CD8 T cells both develop within an identical milieu in vivo throughout thymic development, in addition to coexisting during the full duration of an LCMV-specific immune response. Thymic selection and maturation of T cells does not appear to require IFN-{gamma}R signaling in T cells (12). Therefore, in the mixed bone marrow chimeras, any alteration in the responses of IFN-{gamma}R-deficient CD8 T cells during LCMV infection are directly linked to lack of CD8 T cell-specific IFN-{gamma} action. Again, in these mixed bone marrow chimeras, we have compared LCMV-specific responses of polyclonal wild-type C57BL/6 and IFN-{gamma}R-deficient CD8 T cells within the same mouse. Our results strongly indicate that IFN-{gamma} stimulation of non-CD8 T cells rather than the LCMV-activated CD8 T cells themselves is required to promote exponential activation and expansion of Ag-specific CD8 T cells during an acute LCMV infection. As we have mentioned, there is a pitfall in the mixed bone marrow chimera model that cannot be excluded or controlled: IFN-{gamma}R-mediated effects induced directly in wild-type CD8 T cells could indirectly promote IFN-{gamma}-dependent expansion of IFN-{gamma}R-deficient CD8 T cells. We addressed this caveat by studying CD8 T cell responses to LCMV using transgenic DN mice, in which only T cells are refractory to direct stimulation by IFN-{gamma}. In the DN mice, similar to mixed bone marrow chimeras, there was normal expansion of LCMV-specific CD8 T cells. Because the direct action of IFN-{gamma} on CD8 T cells is minimized in DN mice, indirect effects resulting from IFN-{gamma} stimulation of non-CD8 T cells appear to be predominantly responsible for normal expansion of CD8 T cells during an acute LCMV infection. The study by Sercan et al. (25) indicates that at least some indirect effects of IFN-{gamma} on CD8 T cells might occur through CD11b+ cells, like macrophages. It is possible that activation of CD8 T cells by LCMV-infected macrophages is potentiated in particular by IFN-{gamma}-stimulated enhancement of Ag processing and presentation in these APCs (26, 27). Priming of CD8 T cell responses appear to be dependent upon Ag presentation by CD8{alpha}+ dendritic cells in vivo (28), and IFN-{gamma} might promote the activation and expansion of LCMV-specific CD8 T cells by enhancing Ag processing, expression of MHC class I, and up-regulating expression of costimulatory molecules like 4-1BB ligand and B7 on these APCs (27, 29, 30, 31). In contrast to infection with LCMV, both direct and indirect effects of IFN-{gamma} are dispensable for activation and expansion of CD8 T cells during infection of mice with L. monocytogenes (32, 33) or vaccinia virus (Y. Nakayama and M. Suresh, manuscript in preparation). Differences in cell tropism and pathogenesis between infectious agents are likely factors that determine the requirement for IFN-{gamma} to elicit potent CD8 T cell responses.

In the 2- to 3-wk phase of clonal contraction (days 8–30 PI) that follows the massive expansion (days 0–8 PI) of Ag-specific CD8 T cells, ~90% of the newly formed effector CD8 T cells are eliminated by apoptosis. An important unresolved question in T cell immunology is: what determines the fate of effector CD8 T cells between apoptosis vs survival with differentiation into memory CD8 T cells? One of the dramatic effects of either IFN-{gamma} or IFN-{gamma}R deficiency is the abrogation of CD8 T cell contraction following LCMV and L. monocytogenes infections in mice (8), possibly by inducing apoptosis of effector T cells (5, 6, 9, 11). Using mixed bone marrow chimeras, we compared contraction of wild-type C57BL/6 and IFN-{gamma}R-deficient LCMV-specific effector CD8 T cells within the same mouse. A larger fraction of IFN-{gamma}R-deficient LCMV-specific effector CD8 T cells in mixed bone marrow chimeras and DN mice expressed IL-7R, compared with wild-type C57BL/6 CD8 T cells. However, the magnitude and kinetics of the contraction of LCMV-specific IFN-{gamma}R-deficient and IFN-{gamma}R-sufficient effector CD8 T cells were similar in the bone marrow chimeric mice. Moreover, in the DN mice, T cell-specific deficiency of IFN-{gamma}R signaling had a minimal effect on both the contraction of LCMV-specific effector CD8 T cells and survival of memory CD8 T cells. These data provided strong evidence that contraction of LCMV-specific effector CD8 T cells is essentially independent of IFN-{gamma} activity in T cells themselves. Studies of CD8 T cell responses to experimental L. monocytogenes infection in C57BL/6 and BALB/c mice have shown that IFN-{gamma} regulates CD8 T cell homeostasis only in BALB/c mice but not in C57BL/6 mice (33). Therefore, it could be argued that experiments with bone marrow chimeric mice and DN mice are not directly comparable due to differences in the genetic background of mice: C57BL/6 vs BALB/c. The present study along with published work (8, 24) have clearly demonstrated that IFN-{gamma} regulates CD8 T cell homeostasis in both C57BL/6 and BALB/c mice during an acute LCMV infection, unlike during L. monocytogenes infection. Therefore, at least for the LCMV infection, studies of IFN-{gamma}-mediated CD8 T cell homeostasis in C57BL/6 and BALB/c mice provide valid comparisons.

How does IFN-{gamma} regulate contraction of activated CD8 T cells in vivo? The present study shows that expansion and contraction of LCMV-specific CD8 T cells are minimally affected in the absence of direct effects of IFN-{gamma} on T cells. Indirect effects of IFN-{gamma} on LCMV-specific CD8 T cells could be mediated via accessory cells, like macrophages, because: 1) CD11b+ splenocytes have been shown to effect IFN-{gamma}-dependent contraction of CD8 T cells in vivo (25) and 2) in a mycobacterial infection, elimination of activated CD4 T cells by IFN-{gamma} appears to require NO production by Mac-1+ macrophages (5). However, our own studies have shown that the overall kinetics of the anti-LCMV CD8 T cell response in the inducible NO synthase-deficient mouse model were similar to those in wild-type C57B46 mice, which indicated that NO activity alone is not sufficient to induce contraction of LCMV-specific effector CD8 T cells (data not shown). We have reported that TNFR deficiency attenuated the contraction phase of the CD8 T cell response, and enhanced the number of LCMV-specific memory CD8 T cells (34). Others have shown that apoptosis of activated CD8 T cells induced by tumor-infiltrating macrophages can be inhibited by blocking any of the following: TNFR, NO, reactive oxygen species (ROS), or IFN-{gamma} (35). Additionally, it has been reported that inhibition of ROS reduced both expansion and contraction of LCMV-specific CD8 T cells (36). Nevertheless, the actual interplay between IFN-{gamma}, TNF, ROS, and NO in regulating CD8 T cell homeostasis has not been systematically examined. Further studies are required to determine whether CD8 T cell responses to LCMV in IFN-{gamma}R–/– mice might be recapitulated by treating mice deficient for both TNF and NO with ROS inhibitors. Studies to evaluate the effects of conditional ablation of IFN-{gamma}R in CD11b+ cells on CD8 T cell homeostasis would also be informative. At the molecular level, BIM, a proapoptotic member of the Bcl-2 family appears to be required for the early acute contraction of LCMV-specific CD8 T cells in vivo (37). It remains to be determined whether IFN-{gamma} induces contraction of LCMV-specific CD8 T cells by activating cellular BIM by indirect effects. Data presented in this study conclusively demonstrate that regulation of CD8 T cell expansion, contraction, and memory by IFN-{gamma} occurs by mechanisms essentially independent of any direct action by IFN-{gamma} on T cells. Our findings have clarified fundamental features of control by IFN-{gamma}-IFN-{gamma}R of T cell homeostasis during the immune response; as a result, directions for future study of mechanisms have been greatly simplified toward developing models that focus on non-T cell targets of IFN-{gamma}.


   Acknowledgments
 
We thank Dr. J. Walent and N. Miller for preparing the MHC class I tetramers. We appreciate technical help by Erin Hemmila Plisch and Katie Skell.


   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 Grant AI059804 from the Public Health Service, National Institutes of Health (to M.S.). Back

2 Address correspondence and reprint requests to Dr. M. Suresh, Department of Pathobiological Sciences, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706. E-mail address: sureshm{at}svm.vetmed.wisc.edu Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; DN, dominant negative; PI, postinfection; CD62L, CD62 ligand; MFI, mean fluorescence intensity; ROS, reactive oxygen species. Back

Received for publication April 16, 2007. Accepted for publication May 26, 2007.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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M. K. L. MacLeod, A. McKee, F. Crawford, J. White, J. Kappler, and P. Marrack
CD4 memory T cells divide poorly in response to antigen because of their cytokine profile
PNAS, September 23, 2008; 105(38): 14521 - 14526.
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