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CUTTING EDGE |
Signaling Directly Enhances Primary Antiviral CD4+ T Cell Responses1
Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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drives CD4+ T cell differentiation toward the Th1 phenotype (Th1) and suppresses Th2 development. Current evidence indicates that IFN- inhibits T cell proliferation and decreases T cell survival. In contrast to the above, we show here that antiviral CD4+ T cell generation after infection is reduced in the absence of IFN- signals. The deficient expansion of cells was not due to perturbations in T cell sensitivity to peptide or to altered migratory patterns through nonlymphoid tissues. Instead, IFN- enhanced early antiviral CD4 responses largely through direct signals into these cells. Our data challenge prevailing dogma and have implications for how the sizes of the CD8+ and CD4+ T cell responses are established. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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is a key antiviral effector molecule, acting on target cells to directly inhibit virus replication. The importance of IFN- in antiviral immunity is underlined by the numerous viral strategies that have evolved to escape the effects of this cytokine: many viruses block IFN- synthesis, IFN- signaling processes, or the effects of IFN-induced proteins (1). In addition to antiviral activity, IFN- also has several immunoregulatory functions. For CD8+ T cells, rapid IFN- production correlates with cell abundance during acute infection and with their maturation into the memory pool (2). For CD4+ T cells, IFN- directs Th1 differentiation, and blocks Th2 differentiation, by modulating the expression of transcription factors and cytokine receptors (3, 4, 5); epigenetic changes include histone shifting in the promoter region of IFN- as well as methylation of the IL-4 (6, 7). However, beyond these differentiation effects, which are transmitted from effector cells into the memory phase (8), IFN- is widely thought to suppress T cell responses (9, 10). T cell proliferative responses in vitro are reduced in the presence of IFN- (10). In vivo T cell responses to mycobacterial infection (9) or mutant Listeria monocytogenes infection (11) are enhanced in the absence of IFN-, suggesting that the cytokine reduces these responses, possibly by inducing apoptosis via a caspase-8-dependent mechanism (10). In addition, T cell hyperproliferation has been reported in the absence of IFN- signaling (12). Furthermore, IFN-R is down-regulated on differentiated Th1 cells but not on Th2 cells when the cells are cultured in Th1-inducing conditions (13). These findings are consistent with the hypothesis that IFN-R-derived signals are suppressive: during in vitro culture conditions that favor Th1 differentiation, IFN--mediated suppression is relieved by reducing surface expression of the IFN-R, whereas IFN-R expression and suppression are maintained during Th2 differentiation.
Contrary to these suppressive effects, recent data have shown that IFN- signaling increases the abundance of virus-specific CD8+ T cells during acute lymphocytic choriomeningitis virus (LCMV)3 infection (14). However, many of the studies showing suppressive effects of IFN- were conducted with CD4+ T cells (9, 10), suggesting that these cells may differ from their CD8+ counterparts in their response to this critical immunoregulatory cytokine. In this study, we investigate this issue by evaluating the effects of IFN- on virus-specific CD4+ T cells over the course of infection. We show that IFN-R is increased on effector CD4+ T cells and that CD4+ T cell abundance is reduced in the absence of IFN- signals. Moreover, we report that IFN- acts directly on CD4+ T cells, because cells that express IFN-R are preferentially increased in number. Our data suggest that current models embracing the inherently suppressive effects of IFN- should be reconsidered.
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Materials and Methods |
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C57BL/6 mice were purchased from The Scripps Research Institute (TSRI) breeding facility. IFN-R1KO (B6.129S7-Ifngr1tm1Agt/J) and IFN-KO mice (B6.129S7-Ifngtm1Ts/J), both strains backcrossed 10 generations to C57BL/6), and C57BL/6 mice congenic for Thy1.1 (B6.PL-Thy1a/CyJ) were purchased from Jackson ImmunoResearch Laboratories. C57BL/6.Ly5a mice (B6.SJL-Ptprca Pep3b/BoyJ) were provided by Dr. C. Surh (TSRI). SMARTA TCR-transgenic mice, which express a TCR specific for the LCMV epitope GP61–80 (15) were crossed to C57BL/6.Ly5a mice to generate SMARTA.Ly5a mice and to IFN-R1KO mice to generate SMARTA/IFN-RKO mice. Mice were infected by i.p. administration of 2 x 105 PFU of LCMV, Armstrong strain. All experiments were approved by TSRI Animal Care and Use Committee.
Adoptive transfers
The frequency of V
2, V
8.3+-transgenic CD4+ T cells among all spleen cells was determined by flow cytometry. Defined numbers of the transgenic cells were given i.v. to mice in 0.9 ml of 0% RPMI 1640. In some experiments, the donor cells from wild-type (wt) and IFN-R–/– SMARTA mice were mixed so that equal numbers of SMARTA cells (3 x 104 each) were given to recipient mice, which were infected 1–4 days after transfer. As a control, some mice were given a mix containing a 
10-fold higher number of SMARTA cells and were left uninfected. The larger number of cells in these uninfected controls allowed us to show that 1) the "take" of transferred cells was 10% of the input dose and 2) the 1:1 ratio of IFN-RKO and wt SMARTA cells remained extremely stable over time, in the absence of infection.
Lymphocyte isolation from tissues
Spleen cells and lymph node cells (mix of inguinal, brachial, and axillary nodes) were prepared with RBC lysis using standard protocols. Peripheral blood lymphocytes were suspended in 4% sodium citrate, floated onto a Histopaque cushion, centrifuged, and isolated from the interface using a standard protocol. Lymphocyte isolation from other tissues was done using methods previously described (16). Mice were first perfused with PBS through the heart. The liver was additionally perfused directly by injecting PBS through the hepatic artery. The lungs and small intestine (with the Peyer’s patches removed) were minced and digested with collagenase. The liver and brain were ground over a mesh to make a cell suspension. Lymphocytes were separated from the rest of the tissue cells by resuspending them in 44% Percoll and floating them onto a 56% Percoll cushion, followed by centrifugation. Lymphocytes were isolated at the interface of the two layers.
Flow cytometry
Cells were stained directly ex vivo with anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-Thy1.2 (CD90.2 clone 53-2.1), anti-CD44 (clone IM7), anti-Ly5a (Ly5.1, clone A20), anti-Ly5b (Ly5.2 clone 104) all purchased from eBioscience.com. Anti-IFN-R1 (rat, clone GR20) and the corresponding isotype control Ab were purchased from BD Pharmingen. The intracellular staining assay was performed using anti-IFN- Ab from eBioscience. Cells were acquired on a BD Biosciences FACSCalibur and were analyzed using FlowJo software (Tree Star).
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Results |
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R1
We have recently demonstrated that IFN- acts on CD8+ T cells, contributing to their increased abundance (14). To evaluate the importance of IFN- signaling in determining the abundance of virus-specific CD4+ T cells, we first evaluated the expression pattern of IFN-R1 before and during virus infection. Naive CD4+ T cells express IFN-R1 (dashed line, Fig. 1A) and, following infection, the expression of IFN-R was modulated compared with the naive levels; it was reduced 1 day after infection (light gray) but increased, on a substantial proportion of CD4+ T cells, by day 8 (dark gray). IFN-R1 levels on tissue culture cells can be reduced by ligand-dependent internalization (17), and the Ab that we used to detect the receptor recognizes an epitope within the ligand-binding domain and consequently is unable to detect ligand-receptor complexes (18); either of these mechanisms would represent a potentially trivial explanation for the down-regulation that we observed at 1 day postinfection. However, a proportion of CD4+ T cells in mice lacking IFN- show a reduction in IFN-R1 expression following LCMV infection (Fig. 1B), demonstrating that the transient down-regulation cannot be fully attributed to ligand-mediated internalization or to receptor blockade by the cytokine. We have reported similar kinetics of altered IFN-R1 expression on virus-specific CD8+ T cells (14), and others have confirmed this observation, also showing that IFN-R1 mRNA levels in responding CD8+ T cells declined immediately after infection (19). Although a direct comparison between CD4+ and CD8+ T cells (Fig. 1C) shows that the changes are more pronounced in the latter population, these data, taken together, are consistent with the hypothesis that IFN-R1 expression levels may be one factor regulating the response of both types of T cells. To determine the possible effects of IFN- signaling on the abundance of virus-specific CD4+ T cells, we enumerated epitope-specific CD4+ T cells 8 days after LCMV infection of wt mice and of mice lacking IFN-R1 (IFN-RKO). The percentage of CD4+ T cells specific for the GP61–80 epitope was 2- to 6-fold higher in wt mice than in IFN-RKO mice (example in Fig. 1D); the difference between wt and IFN-RKO mice was magnified when the absolute numbers of epitope-specific cells were calculated (5- to 10-fold difference, Fig. 1E).
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R become more abundant than cells that lack the receptor
For several reasons, studies of antiviral T cell responses in mice lacking IFN-R (or IFN-) are difficult to interpret: for example, APC activation, viral load, replication kinetics, and distribution differ dramatically from those observed in wt mice, and the altered T cell responses may merely reflect these differences. To definitively resolve whether IFN-R expression directly modifies CD4+ T cell responses, a mixed cotransfer experiment was set up in which equal numbers of CD4+ T cells from wt and IFN-RKO mice were mixed and adoptively transferred into B6.PL mice, some of which were infected with LCMV. The recipient mice will mount normal T cell responses and will clear the infection, thus avoiding the potentially confounding effects of persistent virus, and the responding T cells from all three cell populations (two donor, one host) can be independently identified, allowing us to establish the effects of IFN-R expression on CD4+ T cell responses. Without infection, the relative proportion of wt and IFN-RKO donor CD4+ T cells remained at 1:1 for at least 8 days posttransfer (Fig. 2A, cf left and center panels). However, after infection, the wt cells were preferentially expanded (Fig. 2A, right panel). The difference between wt and IFN-RKO cells was more striking when virus-specific cells were enumerated; GP61–80-specific cells were identified by IFN- production and cells that expressed the IFN-R outnumbered their receptor-deficient counterparts by 3.5:1 (Fig. 2B, upper right panel). A comparable ratio was found for CD8+ T cells (Fig. 2B, lower panels), indicating that the benefits of IFN-R expression are similar for CD4+ and CD8+ T cells.
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signals for optimal expansion, but not for functional maturation or spread to nonlymphoid tissues
The above experiments indicate that IFN- signals delivered directly into CD4+ T cells augment their primary responses to LCMV. However, although the same total number of wt and IFN-RKO CD4+ donor T cells were transferred into recipient mice (Fig. 2A), the formal possibility exists that the IFN-RKO donor mice might have had some deficiency in T cell development that resulted in fewer GP61–80-specific precursor cells being present in the transferred population. To allow us to ensure that the same numbers of naive GP61–80-specific cells were transferred, we used as donors SMARTA mice, which express a transgenic TCR specific for this epitope (15). These mice were bred to be IFN-RKO/Ly5a–/Thy1.2 and wt/Ly5a+/Thy1.2; the relationship between Ly5a and IFN-R expression is confirmed in Fig. 3A. Equal numbers of naive Ly5a+ and Ly5a– SMARTA cells were mixed and transferred into normal (Thy1.1) recipients, and the donor cells were enumerated 8 days after LCMV infection (Fig. 3B). The cells (Fig. 3, circled) had undergone a massive expansion and constituted 13% of total splenocytes; within the donor cells, wt SMARTA cells outnumbered IFN-RKO cells by at least 4-fold (shown) and, in some mice, by 20-fold. On average, wt cells were approximately seven times more numerous than IFN-RKO cells (Fig. 3C), demonstrating that IFN-R expression by CD4+ T cells profoundly increases their abundance. Moreover, following peptide stimulation a similar proportion of each donor population produced IFN- (Fig. 3D), TNF, and IL-2 (data not shown), indicating that the absence of direct IFN- signals did not detrimentally affect these functions. The functional integrity of the IFN-RKO CD4+ T cells was further confirmed by measurements of the amount of IFN- that they produced after peptide contact and of their functional avidity (ability to respond to differing doses of peptide (20)). By both of these criteria, wt and IFN-RKO cells were indistinguishable (data not shown). Thus, direct IFN- signaling contributes to the abundance of CD4+ T cells, but is unnecessary for their functional development.
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RKO SMARTA in the spleen might reflect altered migratory patterns for these cells compared with wt cells; perhaps cells lacking the IFN-R preferentially exit the spleen. To examine whether IFN- signals affect the movement of CD4+ T cells, the two populations of donor SMARTA cells were enumerated in lymphoid and nonlymphoid tissues at 8 days after infection (Fig. 3E). In all tissues analyzed, donor cells (Thy1.2+) were abundant (Fig. 3E, top row) and the numbers of wt cells far exceeded those of IFN-RKO cells (Fig. 3E, bottom row; in the mouse shown, by >10-fold in most tissues), except the intraepithelial lymphocytes, in which the ratio was consistently somewhat lower. Furthermore, almost all of the cells were activated (CD44high; Fig. 3E, bottom row), again indicating that the absence of the IFN-R does not inhibit CD4+ T cell maturation. These results indicate that IFN- signals are not required for CD4 localization into peripheral sites and allow us to conclude that IFN- acts directly on CD4+ T cells to increase the size of the primary response. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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acts directly on CD4+ T cells, increasing their abundance during virus infection. The stimulatory effect of IFN- may constitute a central control mechanism for T cell abundance, because we have recently reported that IFN- also confers a survival advantage on CD8+ T cells (2) and that this effect appears to be direct (14). The positive influence of IFN- upon T cells must be exerted early in infection, since the results can be discerned within days thereafter. One possibility is that these stimulatory signals may be delivered within minutes or hours of infection. Such early effects on naive T cells are consistent with several observations: 1) very brief Ag contact is sufficient to initiate the entire program of expansion and contraction in naive CD8+ T cells (21, 22); 2) naive CD8+ T cells synthesize IFN- within 20 h of their initial encounter with appropriate dendritic cells (23); and 3) IFN- signaling acts directly on naive CD4+ T cells, determining their ultimate differentiation pathway (24). Alternatively, IFN- may provide continual stimulation throughout the T cell expansion phase, although a recent publication indicates that CD8+ T cells become unresponsive to this cytokine within 1–2 days of infection (19). Our proposal controverts most published data, which indicate that IFN- is suppressive; for example, IFN-–/– T cells proliferate or survive better in vitro and increased T cell responses are observed in IFN-–/– mice. How might the differences between our results and most published data be explained? A key distinction is that we examine the consequences of defective IFN- signaling in a wt host. In this setting, APC activation and viral elimination are normal; this is not necessarily true of studies conducted in immunodeficient mice, and this may have complicated earlier interpretations. However, some of the apparently discrepant findings still could be reconciled if, as we have previously proposed, the effects of IFN- on T cell abundance change over the course of infection (14); direct IFN- signals delivered early in infection act to boost CD4+ and CD8+ T cell responses and later effects of IFN- may be proapoptotic (12). This hypothesis is contrary to the concept that early IFN- signaling has negative effects on the T cell response, by programming the contraction phase (25); this paradox awaits resolution. After LCMV infection, CD8+ T cell responses are at least 10-fold greater than CD4 responses. This is not due to a difference in precursor frequency (Ref.26 , and J. K. Whitmire, N. Benning, and J. L. Whitton, submitted for publication) and appears unlikely to be due to a difference in resting levels of IFN-R1 expression, which are similar for both types of naive T cells (Fig. 1C, day 0). Nevertheless, it is possible that IFN- signaling plays a part in the differing abundances of CD4+ and CD8+ T cells during virus infection. In conclusion, our data suggest that IFN- simultaneously suppresses microbial infection and enhances CD4+ T cell responses and have implications for both the treatment and prophylaxis of infectious disease. |
Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grant AI 52351. This is manuscript number 17652-NP from The Scripps Research Institute. 
2 Address correspondence and reprint requests to Dr. J. Lindsay Whitton, Molecular and Integrative Neurosciences Department, CVN-9, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: lwhitton{at}scripps.edu
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; wt, wild type.
Received for publication August 12, 2005. Accepted for publication September 2, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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synthesis increases markedly over the course of infection, and correlates with immunodominance. J. Immunol. 173:456.-462. promoter is hypomethylated from early T cell development through mature naive and Th1 cell differentiation, but is hypermethylated in Th2 cells. J. Immunol. 173:7377.-7384. eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J. Exp. Med. 192:117.-122. is required for activation-induced death of T lymphocytes. J. Exp. Med. 196:999.-1005. . Science 290:1354.-1358. interferon receptor knockout mice. J. Virol. 72:7815.-7821. receptor chain and the prevention of interferon signaling in TH1 cells. Science 269:245.-247. acts directly on CD8+ T cells to increase their abundance during virus infection. J. Exp. Med. 201:1053.-1059. on the human histiocytic lymphoma cell line U937. Biochem. J. 274:775.-780.
receptor. J Leukocyte Biol. 51:507.-516. [Abstract]
signaling in antigen-specific CD8+ T cells responding to infection. J. Immunol. 174:6791.-6802. This article has been cited by other articles:
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  J. K. Whitmire, N. Benning, B. Eam, and J. L. Whitton Increasing the CD4+ T Cell Precursor Frequency Leads to Competition for IFN-{gamma} Thereby Degrading Memory Cell Quantity and Quality J. Immunol., May 15, 2008; 180(10): 6777 - 6785. [Abstract] [Full Text] [PDF]
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M. Quigley, X. Huang, and Y. Yang STAT1 Signaling in CD8 T Cells Is Required for Their Clonal Expansion and Memory Formation Following Viral Infection In Vivo J. Immunol., February 15, 2008; 180(4): 2158 - 2164. [Abstract] [Full Text] [PDF]
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K. E. Foulds, M. J. Rotte, M. A. Paley, B. Singh, D. C. Douek, B. J. Hill, J. J. O'Shea, W. T. Watford, R. A. Seder, and C.-Y. Wu IFN-{gamma} Mediates the Death of Th1 Cells in a Paracrine Manner J. Immunol., January 15, 2008; 180(2): 842 - 849. [Abstract] [Full Text] [PDF]
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P. S. Biswas, V. Pedicord, A. Ploss, E. Menet, I. Leiner, and E. G. Pamer Pathogen-Specific CD8 T Cell Responses Are Directly Inhibited by IL-10 J. Immunol., October 1, 2007; 179(7): 4520 - 4528. [Abstract] [Full Text] [PDF]
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B. R. Mothe, B. S. Stewart, C. Oseroff, H.-H. Bui, S. Stogiera, Z. Garcia, C. Dow, M. P. Rodriguez-Carreno, M. Kotturi, V. Pasquetto, et al. Chronic Lymphocytic Choriomeningitis Virus Infection Actively Down-Regulates CD4+ T Cell Responses Directed against a Broad Range of Epitopes J. Immunol., July 15, 2007; 179(2): 1058 - 1067. [Abstract] [Full Text] [PDF]
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J. K. Whitmire, B. Eam, N. Benning, and J. L. Whitton Direct Interferon-{gamma} Signaling Dramatically Enhances CD4+ and CD8+ T Cell Memory J. Immunol., July 15, 2007; 179(2): 1190 - 1197. [Abstract] [Full Text] [PDF]
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S. J. Turner, E. Olivas, A. Gutierrez, G. Diaz, and P. C. Doherty Disregulated Influenza A Virus-Specific CD8+ T Cell Homeostasis in the Absence of IFN-{gamma} Signaling J. Immunol., June 15, 2007; 178(12): 7616 - 7622. [Abstract] [Full Text] [PDF]
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 S. J. Crocker, J. K. Whitmire, R. F. Frausto, P. Chertboonmuang, P. D. Soloway, J. L. Whitton, and I. L. Campbell Persistent Macrophage/Microglial Activation and Myelin Disruption after Experimental Autoimmune Encephalomyelitis in Tissue Inhibitor of Metalloproteinase-1-Deficient Mice Am. J. Pathol., December 1, 2006; 169(6): 2104 - 2116. [Abstract] [Full Text] [PDF]
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 J. S. Haring and J. T. Harty Aberrant Contraction of Antigen-Specific CD4 T Cells after Infection in the Absence of Gamma Interferon or Its Receptor Infect. Immun., November 1, 2006; 74(11): 6252 - 6263. [Abstract] [Full Text] [PDF]
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S. Dhanji, M. T. Chow, and H.-S. Teh Self-Antigen Maintains the Innate Antibacterial Function of Self-Specific CD8 T Cells In Vivo J. Immunol., July 1, 2006; 177(1): 138 - 146. [Abstract] [Full Text] [PDF]
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C. Havenar-Daughton, G. A. Kolumam, and K. Murali-Krishna Cutting Edge: The Direct Action of Type I IFN on CD4 T Cells Is Critical for Sustaining Clonal Expansion in Response to a Viral but Not a Bacterial Infection J. Immunol., March 15, 2006; 176(6): 3315 - 3319. [Abstract] [Full Text] [PDF]
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