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International Immunology Advance Access originally published online on March 28, 2006
International Immunology 2006 18(5):797-806; doi:10.1093/intimm/dxl016
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

LIGHT is dispensable for CD4+ and CD8+ T cell and antibody responses to influenza A virus in mice

Bradley J Sedgmen1, Wojceich Dawicki1,3,, Jennifer L Gommerman1, Klaus Pfeffer2 and Tania H Watts1

1 Department of Immunology, Room 5263, Medical Sciences Building, University of Toronto, Toronto, ON M5S 1A8, Canada
2 Institut für Medizinische Mikrobiologie, Universitätsklinikum der Heinrich-Heine-Universität, Düsseldorf, Germany
3 Present address: Department of Microbiology and Immunology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, NS B3H 4H7, Canada

Correspondence to: T. H. Watts; E-mail: tania.watts{at}utoronto.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The tumor necrosis factor family ligands, LIGHT (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), 4-1BBL and CD70, are found in the same gene cluster on mouse chromosome 17. Although the roles of 4-1BB–4-1BBL and CD27–CD70 interactions in anti-viral T cell responses have been well established, the role of LIGHT in T cell activation/expansion in vivo is less clear. Under conditions that were previously employed to demonstrate a role for 4-1BBL in CD8+ T cell memory, wild-type and LIGHT–/– mice were infected with influenza A virus and primary and memory/recall responses were measured at various time points thereafter. Neither primary expansion nor memory/recall CD8+ T cell responses were affected by the absence of LIGHT, as measured up to 2 months post-infection. CD4+ T cell responses were also unaffected by LIGHT deficiency. Furthermore, we found that LIGHT played no role in the induction of influenza-specific IgG1 and IgG2a serum antibodies. Taken together, these data suggest that LIGHT is dispensable for the acquired immune response to influenza virus in mice with no effect on the induction, maintenance or reactivation of CD8+ T cell memory.

Keywords: co-stimulation, influenza, knockout mice, LIGHT, memory T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Activation of T cells requires the simultaneous engagement of the antigen-specific T cell receptor and CD28, which results in T cell expansion and survival through the production of IL-2 and the generation of survival signals (1). In addition to the co-stimulatory properties of CD28, other molecules of the CD28 and tumor necrosis factor receptor (TNFR) superfamilies play instrumental roles in positively and negatively regulating the activation and survival of T cells following antigenic stimulation (1–4).

The TNF superfamily ligand, LIGHT (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), is a type II transmembrane glycoprotein that is expressed on activated T cells, monocytes and granulocytes and immature dendritic cells (DCs) (5–8). It binds three receptors including herpes virus entry mediator (HVEM), lymphotoxin ß receptor (LTßR) and, in humans, the decoy receptor, DcR3/TR6 (5, 9, 10). HVEM is expressed on resting T cells, NK cells, monocytes, immature DCs and endothelial cells (6, 11–13), whereas LTßR is expressed on DCs, endothelial cells and stromal cells but is absent from lymphocytes (14).

LIGHT is found closely linked to 4-1BBL and CD70 on human chromosome 19/mouse chromosome 17 (15, 16) raising the possibility of analogous functions. 4-1BBL (CD137L) controls the magnitude of the CD8+ T cell memory/recall response to viral infection (17–20). 4-1BBL-deficient mice exhibit normal primary expansion and contraction of the CD8+ T cell response to influenza virus, but show decreased T cell numbers 21–38 days after initial infection, and decreased memory/recall CD8+ T cell response to viruses in vivo (19, 20). Recent evidence shows that this is due to a requirement for 4-1BBL in the maintenance of CD8+ T cell survival following antigen clearance (21), although in other models 4-1BBL has also been shown to influence primary responses to viruses (22, 23). Abrogation of CD70–CD27 interaction by genetic manipulation results in decreased numbers of CD4+ and CD8+ T cells in the lung and spleen during the primary and memory/recall immune response to influenza virus (24, 25).

Several lines of evidence suggest that LIGHT enhances T cell-mediated immunity [reviewed in (3, 26)]. Expression of LIGHT on transplanted tumors results in increased anti-tumor responses and tumor clearance (27). Similarly, over-expression of LIGHT in T cells results in a lymphoproliferative disorder, characterized by expanded populations of CD4+ and CD8+ T cells (28–30). Immobilized LIGHT can increase T cell proliferation in vitro, whereas blockade or targeted disruption of LIGHT can ameliorate alloresponses, graft rejection or graft versus host disease (GVHD) (6, 8, 27, 31, 32). Several studies have suggested that CD8+ T cell responses to anti-CD3, anti-CD3/CD28, peptide and superantigen are reduced in LIGHT–/– mice (33–35). However, LIGHT did not appear to influence the primary response to vesicular stomatitis virus and there have been differential reports on whether LIGHT is required for maximal CD4+ T cell responses (33–35). It is of interest that LIGHT and 4-1BBL play a strikingly similar role in skin allograft rejection: deletion of either 4-1BBL or LIGHT alone had no effect on skin allograft rejection, but in the context of CD28 deficiency, 4-1BBL or LIGHT deficiency results in a similar delay in graft rejection (17, 34).

A systematic analysis of the role of LIGHT in T and B cell responses during an infection in vivo has not been reported to date. In view of the extensive evidence that LIGHT can influence T cell responses, together with the findings that adjacent TNF superfamily ligands within the same gene cluster play a positive role in regulating memory anti-viral CD8+ T cell responses, we hypothesized that LIGHT deficiency would result in a similar defect in vivo following influenza A infection. Surprisingly, however, the results reported here suggest that LIGHT is completely dispensable for the acquired immune response to influenza in mice, under conditions where we can show a clear role for 4-1BBL in CD8+ recall responses.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Wild-type (WT) C57BL/6 breeder pairs were obtained from Charles River Laboratories (St-Constant, Quebec, Canada). LIGHT knockout (LIGHT–/–) mice were backcrossed onto the C57BL/6 background at least six times and bred under specific pathogen free conditions at the University of Toronto. All mouse experiments were approved by the University of Toronto animal care committee in accordance with the regulations of the Canadian Council on animal care.

Influenza A virus infection
Sex-matched 6- to 8-week old mice were infected intra-peritoneally (i.p.) with 200 hemagglutinin units (HAU) of influenza A HKx31 (H3N2) or intra-nasally (i.n.) with 5 HAU. At days 21 or 60 post-infection, some mice were challenged with the serologically distinct influenza A PR8/34 (PR8, H1N1), which shares the nucleoprotein peptide (NP) gene with HKx31, but differs in the hemagglutinin (HA) and neuraminidase (NA) surface proteins. The use of the two strains avoids the complication of neutralizing antibodies generated during priming from preventing re-infection and thereby limiting the secondary CTL response. Mice were sacrificed at various time points after infection and their spleens harvested for single-cell suspensions. At later time points, bone marrow cells were flushed from femurs and tibiae of infected mice with ice-cold PBS and tetramer-positive cells measured by flow cytometry. Mediastinal lymph nodes (MLN) and lungs were excised from i.n. infected mice at day 8 post-challenge with 5 HAU of PR8. Lungs were perfused with 5 ml of PBS and lymphocytes from individual mice were then enriched by isolation over an 80/40% percoll gradient.

Flow cytometry
Spleen cell suspensions were prepared in PBS/2% FCS/0.01% sodium azide on ice. For analysis of influenza NP-specific CD8+ T cells, splenocytes were surface stained with FITC-conjugated anti-mouse CD62L (L-selectin), PE- or allophycoerythrin (APC)-conjugated tetramer consisting of the murine class I MHC molecule H-2Db, ß2-microglobulin and influenza nucleoprotein peptide, NP366–374 (36) and APC- or PE-conjugated anti-mouse CD8 (eBioscience, San Diego, CA, USA). Tetramers were a gift from Rafick Sékaly, CANVAC, University of Montreal, or were obtained from the National Institutes of Health (NIH) tetramer facility. For intracellular IFN-{gamma} staining, cell suspensions were re-stimulated in culture medium (RPMI/10% FCS with antibiotics and 2-mercaptoethonol) for 6 h at 37°C with 1µM of NP366–374 peptide and GolgiStop (BD-Pharmingen, San Diego, CA, USA). Cells were then harvested, re-suspended in PBS/2% FCS/sodium azide and surface stained with PE–anti-CD8 and FITC–anti-CD62L as above. Following surface staining, cells were fixed in cytofix/cytoperm solution (BD-Pharmingen) and then stained with APC-conjugated anti-mouse IFN-{gamma} diluted in 1x perm/wash solution (BD-Pharmingen). Samples were analyzed using a FACScalibur (BD Biosciences) and CELLQuest (Becton-Dickinson, Mountain View, CA, USA) or FlowJo (Tree Star Inc., Ashland, OR, USA) software.

Cytotoxicity assay
CTL activity was measured directly ex vivo at 7 days after challenge with influenza A virus. Adherent cells were removed by incubating splenocytes at 5 x 106 cells ml–1 for 1 h at 37°C. At time points when antigen-specific T cell numbers in the spleen were low (i.e. at day 60 post-challenge), splenocytes were re-stimulated in vitro for 5 days with 100 nm of the H-2Db-restricted peptide NP366–374. For both assays, non-adherent effector cells were collected and serially diluted 3-fold and assayed for anti-influenza NP-specific CTL activity against 51Cr-labeled EL4 target cells pulsed with 50 µM NP366–374 peptide. After a 5-h incubation period, 70 µl of supernatant was harvested onto 96-well harvest plates (Canberra Packard, Mississauga, ON, Canada) and counted on a Topcount scintillation counter (Canberra Packard). Maximum and spontaneous release was determined from wells containing 1% SDS or media alone. The percentage of specific lysis was calculated using the following equation: (experimental 51Cr release – spontaneous 51Cr release)/(maximum 51Cr release – spontaneous 51Cr release) x 100% = % specific lysis.

IL-2 bioassay
CD4+ T cell responses were measured by analyzing IL-2 production in response to re-stimulation of T cells with 250 HAU of heat-killed influenza virus as previously described (19). Culture supernatants were harvested after 48 h of stimulation, and the levels of IL-2 were detected using the IL-2-dependent indicator cell line CTLL. [3H]thymidine incorporation (counts per minute) by CTLL was analyzed with a Topcount 96-well scintillation counter.

Detection of influenza A-specific antibodies
Serum was assayed by ELISA for levels of influenza-specific antibodies of the IgG1 and IgG2a isotypes as previously described by Bertram et al. (19). Plates were coated with 500 HAU ml–1 of heat-killed (56°C, 30 min) influenza A HKx31 in 0.1 M NaHCO3 coating buffer. Following blocking with 5% skimmed milk, 5-fold serial dilutions of serum were added and plates were incubated for 2 h at 37°C. HRP-conjugated anti-isotype antibodies (Caltag Laboratories, Burlingame, CA, USA) were then added to each well and OD405 measured 20 min after the addition of ABTS (Sigma-Aldrich, St Louis, MO, USA) and H2O2 (pH 5.0).

Statistical analysis
Where indicated, P-values were obtained using the Student's t-test (unpaired, two tailed, 95% confidence interval).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Primary response of CD8+ T cells to influenza A virus in the presence or absence of LIGHT
To assess primary CD8+ T cell expansion in response to influenza infection, WT and LIGHT–/– mice were infected i.p. with 200 HAU of the influenza A strain HKx31, and the CD8+ T cells specific for the immunodominant epitope, NP366–374 (37), were enumerated at days 5, 7, 9, 14 and 21 post-infection using fluorescent-labeled Db/NP366–374 tetramers. Initial experiments using heterozygous LIGHT+/– mice revealed no difference in T cell expansion compared with the LIGHT+/+ genotype (data not shown) and thus WT and LIGHT–/– mice were used throughout. NP-specific CD8+ T cell expansion in WT and LIGHT-deficient mice were similar in both magnitude and kinetics (Fig. 1A and B). The population of responding Db/NP366–374 tetramer-positive cells in the spleen at day 7 expressed low levels of L-selectin (CD62Llo), indicating an effector/memory phenotype (38), and this phenotype did not differ between WT and LIGHT-deficient mice or change throughout the course of the response (Fig. 1B). Conversion of the percentage of CD8+ Db/NP366–374-positive cells to total numbers of antigen-specific cells based on cell counts also did not reveal differences between the infected WT and LIGHT–/– mice (data not shown).


Figure 1
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  		Fig. 1. Primary CD8+ T cell response to influenza virus in the presence and absence of LIGHT. WT and LIGHT–/– mice were infected i.p. with 200 HAU of influenza A virus (HKx31). (A) Percentages of CD8+ T cells that are Db/NP366–374 positive were determined from splenocytes collected 5, 7, 9, 14 and 21 days after influenza infection using a fluorescent-labeled Db/NP366–374 tetramer. Conversion to total numbers of NP-specific T cells per spleen gives similar results (data not shown). (B) Representative FACS plots of live splenocytes gated on CD8-positive T cells stained for CD62L and Db/NP366–374 tetramer at day 7 post-primary infection. Percentages of CD8+ T cells that are tetramer positive are indicated in upper right quadrants of each plot. (C) Percentages of antigen-specific IFN-{gamma}-producing CD8+ T cells were determined from splenocytes collected 5, 7, 14 and 21 days after influenza infection. At each time point, splenocytes were re-stimulated with NP366–374 peptide for 6 h in the presence of GolgiStop followed by intracellular staining for IFN-{gamma}, as described in the Methods. For both graphs (A and C), data were pooled from three individual experiments containing three to four mice per experiment per time point and presented as mean ± SEM.

 
The effector function of NP366–374-specific CD8+ T cells was assessed by measuring intracellular IFN-{gamma} levels following a 6-h re-stimulation with NP366–374 peptide (Fig. 1C). Influenza-specific CD8+ IFN-{gamma}-producing cells showed indistinguishable kinetics of expansion and contraction as seen by MHC–tetramer analysis, and again there was no difference between WT and LIGHT–/– mice. Thus, LIGHT is not required for the primary expansion or IFN-{gamma} production by CD8+ T cells in response to influenza infection in vivo.

Recall response of CD8+ T cells to influenza A virus in the presence and absence of LIGHT
At day 21 after priming, WT and LIGHT–/– mice were challenged with influenza A PR8 and the re-expansion of NP366–374-specific CD8+ T cells was measured. As discussed in the Methods, PR8 shares the NP gene with X31 but differs in the HA and NA genes. This prevents HA- and NA-specific neutralizing antibodies which develop in the primary infection from limiting secondary infection, which would minimize the secondary CTL response. The secondary expansion of influenza-specific CD8+ T cells in spleens of influenza-infected WT and LIGHT–/– mice was faster and more robust than that of the primary response, peaking at day 5 and subsiding ~1.5-fold by day 14 post-challenge (Fig. 2A and B). Again, there was no significant difference between WT and LIGHT-deficient mice in either the magnitude or the kinetics of recall CD8+ T cell expansion.


Figure 2
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  		Fig. 2. In vivo recall CD8+ T cell responses to influenza A virus in the presence and absence of LIGHT. Memory/recall CD8+ T cells were measured in WT and LIGHT–/– mice following i.p. challenge with 200 HAU of influenza A PR8 at day 21 post-primary infection. (A) Percentages of Db/NP366–374-positive cells in spleen at days 5, 7 and 14 after influenza challenge. Data are pooled from three individual experiments with three to four mice per time point in each experiment and presented as mean ± SEM. (B) Representative FACS plots of live splenocytes gated on CD8-positive T cells stained for CD62L and Db/NP366–374 tetramer. Percentages of tetramer-positive, CD62Llo CD8+ T cells for naive, WT and LIGHT–/– representatives at day 7 of the memory/recall response are indicated in upper right quadrants of each plot. (C) WT and LIGHT–/– mice were immunized i.n. with 5 HAU of influenza A X31 and, 35 days later, challenged with the same dose of influenza A/PR8. Percentages of Db/NP366–374-positive cells were determined from spleen, lung and MLN collected 8 days post-challenge. Data presented are for five to six mice per genotype.

 
Although previous results had shown a defect in influenza-specific recall CD8+ T cell responses in 4-1BBL mice after i.p. injection, the physiological route of infection with influenza virus is the respiratory tract. Therefore, we also analyzed recall CD8+ T cell responses in LIGHT-deficient mice following i.n. priming with influenza A X31 and i.n. challenge with PR8. Again, there was no significant difference in the magnitude of the CD8+ T cell response to influenza in the lung, spleen or MLN of WT and LIGHT-deficient mice (Fig. 2C).

Effector function of CD8+ T cells from WT and LIGHT–/– mice following influenza challenge
To determine the role of LIGHT on the development of T cell effector function, intracellular IFN-{gamma} levels were determined at the peak of the secondary response following peptide re-stimulation of cells collected from WT and LIGHT–/– mice (Fig. 3A). CTL activity of T cells from WT and LIGHT–/– mice was assessed in a direct ex vivo killing assay at day 7 post-i.p. challenge (Fig. 3B). Neither the frequency of IFN-{gamma}-producing cells nor the ability of the CD8+ T cells to kill targets was affected by the absence of LIGHT. Thus, LIGHT is dispensable for effector function of primed CD8+ T cells.


Figure 3
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  		Fig. 3. Effector responses to influenza A virus infection in the presence and absence of LIGHT. (A) Percentages of antigen-specific IFN-{gamma}-producing CD8+ T cells from i.p. influenza-infected mice at the peak of the memory/recall response (day 7). Data presented are the sum of three individual experiments containing three to four mice per group per experiment. (B) Antigen-specific cytotoxicity of adherent cell-depleted splenocytes from influenza-infected WT and LIGHT–/– was measured directly ex vivo using splenocytes collected at day 7 post-secondary influenza challenge. Samples were assayed for release of 51Cr in duplicate and the data are presented as mean ± SEM of three to four mice per group.

 
Role of LIGHT in maintenance and re-expansion of memory CD8+ T cell responses following influenza A virus infection
Previous results have revealed a defect in CD8+ T cell responses to influenza in 4-1BBL–/– mice starting at day 21 post-infection (19). However, it was conceivable that defects in CD8+ T cell memory in LIGHT-deficient mice might take more time to become apparent. Thus, we analyzed influenza-specific CD8+ T cell numbers at 60 days post-priming (Fig. 4A–C). As the bone marrow is a major reservoir and site of homeostatic proliferation of memory CD8+ T cells (39–41), we analyzed both spleen and bone marrow at 2 months post-influenza infection. At this time point, there was a clear defect in influenza-specific CD8+ T cell numbers in 4-1BBL-deficient as compared with WT mice. 4-1BBL-deficient mice had approximately half to one-third the number of antigen-specific CD8+ T cells as WT mice, whereas LIGHT-deficient mice were indistinguishable from WT mice with respect to antigen-specific CD8+ T cell numbers (Fig. 4A and B). Furthermore, upon re-infection of mice at day 60 post-priming, WT and LIGHT-deficient mice showed similar expansion of influenza-specific T cells in the spleen and bone marrow, whereas 4-1BBL-deficient mice showed a marked reduction of the memory response (Fig. 4D and E). The effector function of influenza-specific T cells at this time point, as measured by intracellular IFN-{gamma} staining, was also found to reflect that of the tetramer numbers in the spleen (Fig. 4F). Following a 5-day re-stimulation with peptide in vitro, T cells from LIGHT-deficient mice infected 60 days earlier showed only a marginal and statistically insignificant decrease in CTL activity compared with WT mice. In contrast, there was a marked reduction in the T cell response from 4-1BBL-deficient mice, correlating with decreased antigen-specific memory T cells in the starting culture (Fig. 4C). Similarly, the direct ex vivo CTL assay at day 7 following re-infection of mice 60 days after priming showed a defect in 4-1BBL–/– mice but little or no effect in the LIGHT–/– mice (Fig. 4G). Thus, LIGHT is dispensable for the establishment, maintenance and reactivation of memory CD8+ T cell responses to influenza virus.


Figure 4
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  		Fig. 4. Long-term persistence and recall responses of CD8 influenza-specific memory T cells in WT, LIGHT-deficient and 4-1BBL-deficient mice. Mice were infected i.p. with 200 HAU of influenza virus A X31 and re-challenged with influenza A PR8 at day 60. (A) Percentages of CD62Llo CD8 Db/NP366–374-positive cells in spleen and bone marrow were measured at day 60 post-priming. (B) CTL activity was measured from splenocytes collected at day 60 post-infection with influenza A virus following a 5-day in vitro re-stimulation. Samples were assayed in duplicate and the data presented as mean ± SEM of five to six mice per group. (C) Percentages of CD62Llo CD8 Db/NP366–374 tetramer-positive cells were analyzed in spleen and bone marrow at day 7 post-challenge of mice primed 60 days earlier. It should be noted that in this model the WT influenza-specific CD8+ T cells maintain a CD62Llo (effector memory) phenotype even up to 3 months post-infection and no differences in the CD62L expression between WT and LIGHT–/– mice were detected at 60 days post-infection. (D) Percentages of IFN-{gamma}-producing CD8+ T cells in spleen at day 7 post-challenge of mice primed 60 days earlier, measured following 6-h peptide re-stimulation as in Fig. 1. (E) Direct CTL activity was measured from splenocytes collected at day 7 post-challenge. Samples were assayed in duplicate and the data presented as mean ± SEM of three to five mice per group.

 
CD4+ T cell responses to influenza A virus in the presence and absence of LIGHT
The role of LIGHT on the CD4+ T cell response to influenza infection was assessed in WT and LIGHT–/– mice at the peak of the primary response (day 7), at the end of the primary response (day 21) and the peak of the secondary response (day 7). Splenocytes collected at these time points were cultured in the presence of heat-inactivated influenza A virus, and the levels of IL-2 in supernatant collected 48 h after incubation were quantified using a CTLL bioassay. As shown in Fig. 5, no significant differences were detected at any of the time points analyzed. The finding that the WT and LIGHT–/– CD4+ T cells re-stimulated with influenza produce equivalent levels of IL-2 suggests that the CD4+ T cell response to influenza in LIGHT–/– mice is intact.


Figure 5
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  		Fig. 5. Recall CD4+ T cell responses to influenza A virus in the presence and absence of LIGHT. Mice were infected i.p. with influenza A X31 and challenged with influenza PR8 at day 21 post-infection. Splenocytes isolated from mice sacrificed at 7 days post-challenge were incubated with heat-killed influenza A HKx31 for 48 h to stimulate a recall CD4 T cell response. IL-2 production was measured by a bioassay on CTLL cells, as described in the Methods. Samples were assayed in duplicate and the data presented are mean ± SEM of thymidine incorporation by CTTL responding to culture supernatants from cultures from 9 to 12 mice per group.

 
Serum antibody responses to influenza virus A in the presence and absence of LIGHT
To examine the role of LIGHT on the humoral immune response to influenza A infection, serum antibodies were quantified by ELISA (Fig. 6). At day 7 of the secondary response, high levels of both IgG1 and IgG2a influenza-specific antibodies were observed in the serum of immunized WT and LIGHT–/– mice, as compared with the naive control serum. No significant differences in the influenza-specific titers of either isotype were detected between WT and LIGHT–/– mice at both time points analyzed. Thus, LIGHT deficiency does not influence the quantity or the isotype of the secondary antibody response to influenza virus in mice.


Figure 6
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  		Fig. 6. Antibody responses to influenza A virus infection in the presence and absence of LIGHT. Antibody isotypes (IgG1 and IgG2a) specific for influenza A HKx31 were measured in sera samples by ELISA at day 7 after primary infection and day 7 after influenza A (PR8) challenge, as described in the Methods. Samples were assayed in the duplicate and the data presented are mean ± SEM of 9–12 mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study we show that LIGHT, a TNF superfamily ligand, has no net effect on the immune response to influenza A virus in vivo. Primary and secondary expansion and effector function of memory NP366–374-specific CD8+ T cells were found to be unaffected in mice lacking LIGHT. Similarly, influenza-specific CD4+ T cell and serum antibody responses detected in infected LIGHT–/– mice were comparable to those observed in WT mice. The failure to identify a defect in CD8+ T cell memory in LIGHT–/– mice is unlikely to be due to the sensitivity of the system, given that defects in CD8+ memory in 4-1BBL-deficient mice were found under the same conditions. Taken together, the data presented in this report suggest that LIGHT does not play a major role in the induction or maintenance of T cell immunity to influenza A virus in vivo.

4-1BBL and CD70 are thought to play a role in T cell activation at least in part through binding to their receptors, 4-1BB and CD27, on T cells. HVEM, like CD27 and 4-1BB, signals through TNFR associated factor 2 which in turn can link these receptors to the nuclear factor kappa B pathway and survival signaling [reviewed in (42)]. Thus, the absence of LIGHT might have influenced T cell co-stimulatory signals through HVEM on T cells. However, this did not seem to be the case in the present study. Recently, it was demonstrated that, in addition to binding its TNF family ligands, HVEM also binds to an inhibitory member of the Ig superfamily, B and T lymphocyte attenuator (BTLA) (43, 44). The binding of HVEM to LIGHT or BTLA during cell–cell interaction is thought to be mutually exclusive (45). Thus, the ability of LIGHT to act as a co-stimulatory ligand for HVEM on T cells may well be limited by HVEM binding to BTLA. The finding that HVEM-deficient mice show enhanced T cell responses is consistent with HVEM playing an inhibitory role as a ligand for BTLA and suggests that the more predominant effect of HVEM in systemic T cell activation is as a ligand for BTLA rather than as a receptor for LIGHT (46).

Although two studies have shown that T cells from LIGHT-deficient mice show decreased proliferative responses to anti-CD3 or anti-CD3/CD28 in vitro, these effects may depend on a sufficient culture density to allow HVEM–LIGHT interaction by T–T interaction (33–35). Such an effect might not take place in vivo, at least not in the context of viral infection. The effect of LIGHT deficiency on T cell proliferation was not observed by Tamada et al. (33). However, they did find a defect in CD8+ T cell numbers following in vivo administration of staphylococcal enterotoxin B (SEB). It is possible that the strong antigenic stimulation and high frequency of response afforded by SEB immunization as compared with influenza infection led to more dominant effects of LIGHT on T cell activation. Similarly, an effect of LIGHT deficiency on the recall response to a papilloma peptide may also reflect the recall culture conditions (33). In the present studies, CD8+ T cell responses were quantified immediately ex vivo thus more accurately reflecting the in vivo situation.

In addition to its ability to bind to HVEM on T cells, LIGHT on activated T cells can cooperate with CD40L to contribute to DC conditioning, thereby having an indirect effect on CD8+ T cell activation (47). On DC, this could take place via LIGHT binding to HVEM or LTßR. However, in the present study, LIGHT conditioning of DC clearly could not have played a major role in the influenza response, as CD8+ T cells showed no defects in numbers or function. In contrast, DC conditioning by CD40L is required for maximal CD8+ T cell responses to influenza virus (48). It was recently reported that LIGHT expression is up-regulated on the B cell surface in response to CD40L and LIGHT co-stimulation, and this resulted in enhanced B cell proliferation and antibody production in vitro (49). However, in vivo, the role of LIGHT on humoral immunity is not obvious. In mice lacking LIGHT, no defect in antibody responses was observed following VSV (34) or influenza infection (this report).

Although LIGHT is clearly dispensable for anti-viral antibody and CD4+ and CD8+ T cell responses, there appear to be particularly strong effects of LIGHT in alloresponses, including graft rejection and GVHD (8, 27, 31, 34, 50). Whether this is due to effects of LIGHT on APC via HVEM or LTßR or through effects on endothelial or stromal cells through LTßR stimulation remains to be determined. However, the differences in the effect of LIGHT on graft rejection as compared with anti-viral responses may reflect the prolonged nature of the response, the numbers of T cells activated and the nature of the cell types and receptors involved.

Over-expression of LIGHT in mice has dramatic effects on CD4+ and CD8+ T cell expansion, IgA production in the gut, intestinal inflammation and autoimmunity (28, 30, 51). Although it is difficult to compare effects of over-expression with the effects of removing the ligand by gene targeting, the transgenic over-expression experiments highlight a prominent role for LIGHT in the gut. In an adoptive transfer model of Crohn's disease, LIGHT interaction with both its receptors, HVEM and LTßR, was shown to contribute to disease (51). Since T cells in the human gut express LIGHT constitutively (52), the gut mucosa may represent a niche where LIGHT has a more prominent role, thereby contributing to autoimmunity.

Collectively, the data presented here eliminate LIGHT as an important co-stimulatory molecule for antibody and CD4+ or CD8+ T cell responses to influenza virus. Thus, although similar to 4-1BBL in skin allograft rejection models, LIGHT is clearly very different from 4-1BBL in its role in anti-viral immunity.


   Acknowledgements
 
We thank Edward Bertram for helpful discussion, Laura Snell for critical reading of the manuscript and Kim Ellefsen and Rafick Sékaly as well as the NIH tetramer facility for providing MHC I tetramers. This work was funded by grants from the Canadian Institutes for Health Research to T.H.W. and J.L.G.


   Abbreviations
 
APC, allophycoerythrin
BTLA, B and T lymphocyte attenuator
DC, dendritic cell
GVHD, graft versus host disease
HA, hemagglutinin
HAU, hemagglutinin units
HVEM, herpes virus entry mediator
i.p., intra-peritoneally
LIGHT, lymphotoxin like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes
LTßR, lymphotoxin ß receptor
MLN, mediastinal lymph nodes
NA, neuraminidase
NIH, National Institutes of Health
NP, nucleoprotein peptide
SEB, staphylococcal enterotoxin B
TNF, tumor necrosis factor
WT, wild type

   Notes
 
Transmitting editor: K. Okumura

Received 3 February 2006, accepted 20 February 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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