LIGHT-deficiency impairs CD8+ T cell expansion, but not effector function

Jinqi Liu1, Clint S. Schmidt2, Feisha Zhao1, Angela J. Okragly1, Andrew Glasebrook2, Niles Fox1, Elizabeth Galbreath1, Qing Zhang1, Ho Yeong Song1, Songqing Na2 and Derek D. Yang1

1 Department of Bio-Research and Technologies and Proteins, and 2 Department of Inflammation and Immunomodulation Research, Eli Lilly & Co., Indianapolis, IN 46285, USA

The first two authors contributed equally to this work
Correspondence to: S. Na; E-mail: na_songqing{at}lilly.com or D. D. Yang; E-mail: yang_derek_di{at}lilly.com
Transmitting editor: R. Medzhitov


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
LIGHT, a newly identified member of the tumor necrosis factor (TNF) family, is expressed on activated T lymphocytes. To evaluate how LIGHT contributes to T cell functions, we generated LIGHT-deficient (LIGHT–/–) mice using gene targeting. Disruption of LIGHT significantly reduced CD8+ T cell-cycle progression, leading to reduced proliferation to anti-CD3, anti-CD3/anti-CD28 or allogeneic stimulation, whereas proliferation of CD4+ T cells remained unchanged. In contrast to the observed proliferative defects, isolated CD8+ T cells from LIGHT–/– mice displayed normal cytotoxic effector function development when compared to wild-type CD8+ T cells. Underlying a potential mechanism of reduced CD8+ T cell proliferation, LIGHT–/– CD8+ T cells displayed reduced surface levels of CD25 and a diminished ability to proliferate in response to exogenous IL-2. Furthermore, addition of IL-12 to LIGHT–/– CD8+ T cell cultures could not ameliorate this proliferative defect. These results reveal a potential mechanism of action for LIGHT as a positive regulator of CD8+ T cell expansion, but not lytic effector function development.

Keywords: CD8+, effector function, LIGHT, T cell expansion


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
LIGHT, a newly identified member of tumor necrosis factor (TNF) family, is expressed on activated T lymphocytes (13) and immature dendritic cells (DC) (4). Herpes virus entry mediator (HVEM), a TNF receptor (TNFR) superfamily member, is expressed on immature DC (5,6), T and B lymphocytes, NK cells, monocytes, and endothelial cells (2,7,8), and was identified as a receptor for LIGHT (1). HVEM also serves as a mediator for herpes simplex virus type 1 entry into activated T lymphocytes, indicating a potential regulatory role of LIGHT in response to viral infection (1,9). LIGHT also binds lymphotoxin ß receptor, which is expressed on epithelial and stromal cells, but is absent on lymphocytes (10). Therefore LIGHT’s activity in T cell immune responses is likely through its interaction with HVEM and LIGHT has been shown to provide a potent co-stimulatory signal in a CD28-independent manner (4,11).

Previous investigations have shown that soluble LIGHT can augment mixed lymphocyte reactions (MLR) and DC maturation (6), along with enhancing tumor immunity by up-regulating the immune response of cytotoxic T lymphocytes (CTL) to tumor antigen (11,12). LIGHT was also shown to be essential to regulate DC-mediated allogeneic T cell responses (4,6), suggesting that LIGHT is involved in regulating T cell activation and plays a critical role in mediating the interaction between T cells and antigen-presenting cells (APC).

Studies in LIGHT transgenic mice demonstrated that constitutive expression of LIGHT on T cells results in sustained T cell activation and inflammation in mucosal tissues (13). LIGHT was also shown to be involved in negative selection of thymocytes (14). However, the physiological functions of LIGHT have not been clearly established and are complicated by the interaction of LIGHT with different receptors, which also bind other ligands.

Recent evidence has shown that disruption of LIGHT in the mouse causes defects in CD8+ T cell activation and selective impairment of CTL functions (15,16). Furthermore, absence of LIGHT prolongs cardiac allograft survival, indicating LIGHT-dependent co-stimulation plays a significant role in T cell-mediated allograft rejection (17). All of these previous studies revealed an important role(s) for LIGHT in T lymphocyte functions, especially for CD8+ T cells. However, the mechanisms of impaired CD8+ T cell activation and function remain unclear. Furthermore, there is a discrepancy among these studies regarding LIGHT-deficient (LIGHT–/–) CD8+ T cell proliferation.

Using a similar approach to further understand the role of LIGHT in T cell activation, we have independently generated LIGHT–/– mice via gene targeting. Our results revealed that absence of LIGHT dramatically reduces CD8+ T cell proliferation, which is due, at least in part, to reduced cell division. The capacity of LIGHT–/– CD8+ T cells to differentiate into CTL, however, was not affected. Additionally LIGHT–/– CD8+ T cells exhibited reduced CD25 surface levels and diminished proliferation in response to exogenous IL-2 or IL-12 upon stimulation. Our data provide further evidence and a possible mechanism by which LIGHT serves as an important regulator in determining the extent of CD8+ T cell expansion without necessarily affecting the development of lytic effector function.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
BALB/c mice (H-2d) were purchased from Harlan (Indianapolis, IN). All animals were kept in American Association for Accreditation of Laboratory Animal Care-accredited pathogen-free facilities, and provided standard laboratory diet and water ad libitum.

Generation of LIGHT–/– mice
Using the sequence of LIGHT exon I (GenBank accession no. AC079564) as a probe, we isolated mouse genomic DNA clones corresponding to the LIGHT locus from mouse strain 129/SvJ (FixII phage library; Stratagene, La Jolla, CA). A targeting vector backbone pGT-N29-tk was constructed as described (18). The targeting vector pKO-LIGHT was constructed by inserting a 1.7-kb HincII fragment obtained from the 5' end of a LIGHT genomic clone into the XhoI and EcoRI sites of pGT-N29-tk using appropriate linkers. A 6.0-kb HindIII–SphI fragment that contained the exon IV of LIGHT gene was inserted into the vector at the BamHI and NotI sites using appropriate linkers. Germline transmission of the LIGHT mutation was confirmed by Southern blot analysis of tail DNA. F2 LIGHT–/– and wild-type littermates were maintained in sterilized microisolator cages and used for further analyses.

Southern blot and Northern blot
Genomic DNAs isolated from murine embryonic stem (ES) cells and tails were examined by Southern blot analysis. The probe was a PCR-amplified 245-bp fragment corresponding to the 3' region of the LIGHT genomic sequence. The forward and reverse primers were 5' -CCAGGTCACAATCCATCACT and 5'-GCCAGATCAGATTAGATCAG respectively. Northern blot analysis was performed using poly(A)+ RNA prepared from mouse spleen using Oligotex (Qiagen, Santa Clarita, CA). Blots were hybridized to a radiolabeled probe corresponding to nucleotides 293–701 (exon IV) of the murine LIGHT cDNA.

Flow cytometric analysis
Lymphocyte lineage development in wild-type and LIGHT–/– mice was determined by flow cytometric analysis. mAb specific for murine cell-surface markers including Thy1.2, CD4, CD8, B220, CD19, IgD, CD69, CD25 and CD44 (BD PharMingen, San Diego, CA) were used for detection. For CD25 staining of activated CD8+ T cells, purified cells (3 x 105 cells/well) in a 96-well tissue culture-treated plate were activated by plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) for 48 h, with or without 10 ng/ml recombinant murine IL-12 (rmIL-12; R & D Systems, Minneapolis, MN). FITC-conjugated anti-mouse CD25 antibody or FITC-conjugated rat IgM isotype control (both from BD PharMingen) was used for staining as described above. All flow cytometric data were collected with a FACSort (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson).

T cell proliferation and MLR assay
Lymph node and splenic cell suspensions were isolated from 8- to 10-week-old wild-type and LIGHT–/– mice by homogenizing tissues between frosted glass slides (Fisher, Pittsburgh, PA) and removing red blood cells with ACK lysing buffer (Biowhittaker, Walkersville, MD). Thy1.2+, CD4+ or CD8+ T cells were enriched by positive selection using magnetic anti-Thy1.2, anti-CD4 or anti-CD8 microbeads respectively, and AutoMACS magnetic cell sorter (Miltenyi Biotec, Auburn, CA). The purity of isolated T cell populations was subsequently analyzed by flow cytometry and found to be 90–95% positive for the indicated population. Purified T cells were cultured in triplicate wells (5 x 105 cells/well) of a Costar 96-well tissue culture plate (Corning, Corning, NY) in complete RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 5.5 x 10–5 M 2-mercaptoethanol and 10% FCS (all supplements from Invitrogen) at 37°C, 5% CO2 in the presence of plate-bound anti-CD3 antibody (0.5 µg/ml) only or a combination of anti-CD3 (0.5 µg/ml) and anti-CD28 (0.5 µg/ml) (BD PharMingen) for 48 h. Proliferation was measured by an uptake of radioactive [3H]thymidine (1 µCi/well; ICN Radiochemicals, Irvine, CA) in the last 15 h of culture using a filtermate harvester (Packard Instrument, Downers Grove, IL) and a 1450 microbeta liquid scintillation counter (Pharmacia Biotech, Uppsala, Sweden).

For MLR assays, wild-type and LIGHT–/– mice were immunized subcutaneously with 107 P815, mouse mastocytoma, cells (ATCC, Manassas, VA) on days –14 and –7. On day 0, spleens from the mice were removed and homogenized into cell suspensions between frosted glass slides (Fisher). CD8+ T cells were enriched by negative selection using a rat anti-mouse antibody cocktail consisting of anti-NK, anti-CD4, anti-B220, anti-CD11b and anti-MHC II mAb (BD PharMingen), and Biomag goat anti-rat IgG magnetic beads (Polysciences, Warrington, PA). CD8+ T cells (8 x 105) were cultured in triplicate wells of a 96-well plate with a titration of {gamma}-irradiated (5000 rad) P815 cells for 5 days. Proliferation was measured by [3H]thymidine uptake the last 15 h of culture.

Cell division and Annexin-V-binding analysis
CD8+ T cell fractions generated from naive LIGHT–/– and wild-type mice as described were labeled with CFSE (Molecular Probes, Eugene, OR) according to established protocols (19). Briefly, suspensions at 107 cells/ml in HBSS were prewarmed to 37°C. CFSE was then added to a final concentration of 1 µM and the cells were incubated for 10 min at 37°C with occasional mixing, followed by addition of 10 volumes of ice-cold RPMI medium containing 10% FCS. After washing 2 times with complete RPMI medium, CFSE-labeled cells were cultured as described above with anti-CD3 (0.5 µg/ml) and anti-CD28 (0.5 µg/ml) antibody for 24, 48 and 72 h prior to flow cytometric analysis.

The binding of Annexin-V to cell-surface phosphatidylserine was assayed on activated (as above) CD8+ cells from wild-type and LIGHT–/– mice using the Annexin-V–FITC apoptosis detection kit (BD PharMingen). CD8+ cell cultures were analyzed by flow cytometry at 24, 48 and 72 h, and histograms depicted represent propidium iodide-negative cells.

IL-2- and IL-12-stimulated CD8+ T cell proliferation
For IL-2-stimulated CD8+ T cell proliferation, purified CD8+ T cells were activated by incubation in pre-coated tissue culture plates with anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) for 48 h. The cells were then washed in complete RPMI medium with 10% FCS and counted. CD8+ T cells (1 x 105) from both wild-type and LIGHT–/– were incubated in 96-well plates in the presence of various concentrations of rmIL-2 (R & D Systems) for 48 h. T cell proliferation was measured by incorporation of radioactive [3H]thymidine as described above for the last 8 h of culture.

For IL-12-stimulated CD8+ T cell proliferation, purified CD8+ T cells were stimulated with plate-bound anti-CD3 (0.5 µg/ml) and anti-CD28 (0.25 µg/ml) in the presence of various concentrations of rmIL-12 (R & D Systems). The cells were then incubated for 48 h and pulsed with [3H]thymidine for the last 8 h of culture.

Cytotoxicity assay
CTL were generated as previously described (20). Briefly, splenocytes from LIGHT–/– mice and their wild-type littermates (H-2b) were cultured with irradiated BALB/c splenocytes (H-2d) in the presence of 10 U/ml rmIL-2 (R & D Systems). After 5 days of culture, live cells were harvested over Ficoll (density 1.077; Sigma, St Louis, MO). Cytotoxic activity of activated splenocyte cultures was assessed by using a Cytotox 96 non-radioactive kit (Promega Biotec, Madison, WI) according to the manufacturer’s instructions. Equal numbers of live cells from wild-type and LIGHT–/– mice were seeded into wells of a 96-well U-bottom plate with P815 mastocytoma (H-2d; ATCC) target cells at different E:T ratios. EL-4 thymoma cells (H-2b; ATCC) were used as a syngeneic control. For CTL assay using purified CD8+ T cells from both wild-type and LIGHT–/– mice, purified cells were cultured with irradiated P815 mastocytoma for 5 days. Live CD8+ T cells were then washed, counted and an equal number of effector cells was then used for CTL assay at different E:T ratios.

Statistical analysis
For each of the experiments, an ANOVA model was used to evaluate the significance of acquired data. The terms used in this model for most of the experiments were ‘Treatment’ (wild-type versus LIGHT–/–), ‘Dilutions’ and ‘Treatment x Dilution’ interaction as effects. The dilution effect refers to the difference between dilution of IL-2, IL-12, effecter cell, stimulating cell or anti-CD3 antibody, as the case may be in a given experiment. Since the distribution of the response levels was not symmetric, the Box–Cox transformation method (21) was used to transform the data to ensure symmetry and homogenous variance that is required for the ANOVA method. These analyses were performed for these transformed data using the JMP software, version 4.0.2 (developed by SAS Institute). Within the framework of this ANOVA model, comparisons between LIGHT–/– and wild-type groups were made for each of the dilutions. Statistical significance was claimed when the two-tailed P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of LIGHT–/– mice
To investigate the physiological role of LIGHT, we generated a LIGHT–/– mouse strain by gene targeting. The targeting construct was designed to replace an internal 3.8-kb HincII–HindIII genomic fragment with the PGK-neo gene cassette. The deleted region encompasses exons 1–3 encoding amino acids 1–98 of murine LIGHT sequence (Fig. 1A). The targeting vector was introduced into R1 ES cells. Two targeted mutant ES clones identified by Southern blotting were injected into C57BL/6 blastocysts and resulted in chimeric mice that transmitted the disrupted LIGHT allele through the germline. Heterozygous mutant mice (+/–) were intercrossed to obtain homozygous mutant mice (–/–). Mouse genotypes were identified by Southern blot analysis of genomic DNA. EcoRI bands, 12 and 17 kb, were detected as mutated and wild-type alleles respectively (Fig. 1B). The null mutation of LIGHT was demonstrated by the absence of LIGHT gene expression as determined by Northern blot analysis of LIGHT mRNA isolated from spleens of wild-type and homozygous mutant mice (Fig. 1C). LIGHT–/– mice were born at the expected Mendelian ratios and were fertile. Necropsy data indicated that there were no remarkable findings on anatomic pathology or clinical pathology in the absence of LIGHT. Histological analysis revealed no significant changes in major organs, including lymphoid tissues. Lymphoid cell populations in bone marrow, lymph node and spleen of LIGHT–/– mice appeared normal by flow cytometric analysis (data not shown). T and B cell numbers from naive wild-type and LIGHT–/– mice also remained at the same level (data not shown). These results indicate that LIGHT alone is not essential for normal mouse development and lymphoid organogenesis.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1. Generation of LIGHT–/– mice by gene targeting. Strategy for targeted disruption of LIGHT gene by homologous recombination was as indicated. pGT-N29-tk vector, targeting vector pKO-LIGHT, the genomic region at the LIGHT locus and the predicted structure of the mutated LIGHT gene are illustrated. The restriction fragments used to construct the targeting vector are indicated (1.7-kb HincII fragment and 6.3-kb BamHI–HindIII fragment). The coding regions of the exons are presented as striped (E1, exon I; E2, exon II; E3, exon III; E4, exon IV). Restriction enzyme sites (B, BamHI; H, HindIII; Hc, HincII; N, NotI; RI, EcoRI; X, XhoI) and the probe for Southern blot analysis are indicated (A). Germline transmission of LIGHT disruption is indicated by Southern analysis of mouse genomic DNA as the presence of all three expected genotypes (+/+, +/– and –/–). The upper band (17 kb) corresponds to the wild-type allele and the lower band (12 kb) corresponds to the mutant allele (B). Absence of LIGHT mRNA expression in LIGHT–/– mice. Poly(A)+ RNAs (2.5 µg/lane) from spleens of mice were analyzed for LIGHT transcripts with a LIGHT cDNA probe derived from exon IV or exon I (C).

 
Impaired LIGHT–/– CD8+ T cell proliferation
To further determine if the absence of LIGHT affects lymphoid cell functions, purified T cells from wild-type and LIGHT–/– mice were cultured in the presence of anti-CD3 antibody or a combination of anti-CD3 and anti-CD28 (Fig. 2A). Under both of these stimulatory conditions, the proliferation of T cells was significantly reduced in the absence of LIGHT, suggesting that LIGHT may serve as a co-stimulator for mediating T cell growth and its regulatory role is CD28 independent. To further dissect which T cell population was impacted by the disruption of LIGHT, CD4+ and CD8+ T cells from wild-type and LIGHT–/– mice were isolated and stimulated with anti-CD3 and anti-CD28. Compared with wild-type CD8+ T cells, the proliferation of LIGHT–/– CD8+ T cells was significantly reduced in response to stimulation, whereas the proliferation of CD4+ T cells was not significantly affected by the absence of LIGHT (Fig. 2B). Furthermore, the impaired CD8+ T cell proliferation upon TCR stimulation was observed at all tested concentrations of anti-CD3 antibody (0.1–1.0 µg/ml, data not shown), indicating that the intrinsic defect of CD8+ T cell proliferation was anti-CD3 antibody dose independent. These results suggest that LIGHT is essential for optimal stimulation of CD8+, but not CD4+, T cell proliferation in the presence of TCR or TCR plus CD28-mediated stimulation.




View larger version (40K):
[in this window]
[in a new window]
 
Fig. 2. Disruption of LIGHT reduces T cell proliferative responses. Purified Thy1.2+ T cells from wild-type and LIGHT–/– mice were cultured for 48 h in 96-well microplates coated with 0.5 µg/ml anti-CD3 mAb (A, left) or 0.5 µg/ml anti-CD3 mAb plus 0.5 µg/ml anti-CD28 mAb (A, right). CD4+ (B, left) and CD8+ (B, right) T cells were purified and stimulated with 0.5 µg/ml of plate-bound anti-CD3 and anti-CD28 mAb for 48 h. The proliferative response of T cells was measured by [3H]thymidine incorporation during the last 15 h of stimulation. (C) For the MLR assay, purified CD8+ T cells from wild-type and LIGHT–/– mice previously immunized with P815 cells were cultured for 5 days in 96-well microplates with a titration of irradiated P815 cells. The proliferative response of CD8+ T cells was measured by [3H]thymidine incorporation during the last 15 h of stimulation. Values shown represent the mean and error bars represent the SD. *P < 0.05; **P < 0.01.

 
To further confirm the results we observed with anti-CD3 stimulation, we next measured CD8+ T cell proliferation from both wild-type and LIGHT–/– mice in a MLR assay using P815 mastocytoma (H-2d) as stimulator cells. Figure 2(C) illustrates that LIGHT–/– CD8+ T cells exhibited significantly reduced proliferation in response to allogeneic stimulator cells. These data are consistent with previous observations that LIGHT is an important regulator for CD8+ T cell proliferation (15,16) and the absence of LIGHT causes impaired CD8+ T cell proliferation in response to mitogenic stimulation through the TCR.

LIGHT–/– CD8+ T cells exhibit defects in cell division progression, but not apoptosis
The impaired CD8+ T cell proliferation in the absence of LIGHT could be explained by either reduced cell division, increased cell death or a combination of both events. To address this question, we compared both cell division and cell death of CD8+ T cells from wild-type and LIGHT–/– mice. Prior to culture with plate-bound anti-CD3 and anti-CD28 antibody, purified CD8+ T cells were labeled with CSFE. The cell division was measured by monitoring loss of CSFE fluorescence after 24, 48 and 72 h of stimulation (Fig. 3A). Compared with wild-type cultures, LIGHT–/– CD8+ T cells clearly exhibited decreased cell division at all the time points examined. Interestingly, we did not observe any gross differences in apoptosis of CD8+ T cells between wild-type and LIGHT–/– mice by Annexin-V-binding analysis (Fig. 3B). These data indicate that LIGHT can influence cell proliferation by regulating cell division.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3. Impaired CD8+ T cell division, but not apoptosis, from LIGHT–/– mice. (A) CD8+ T cell fractions generated from naive wild-type and LIGHT–/– mice were labeled with CFSE prior in vitro stimulation. CFSE-labeled cells were cultured with 0.5 µg/ml plate-bound anti-CD3 and anti-CD28 for 24, 48 and 72 h. Loss of CSFE fluorescence, indicating cell division progression, was measured by flow cytometry. Histogram markers indicate the percentage of CD8+ cells in culture that underwent mitosis. (B) Wild-type (light lines) and LIGHT–/– (dark lines) CD8+ T cells were stimulated as above, and Annexin-V-binding activity was analyzed by flow cytometry at 24, 48 and 72 h. Histograms represent Annexin-V levels on propidium iodide-negative populations.

 
Targeting LIGHT reduced CD8+ T cell-surface CD25 expression, and responses to exogenous IL-2 and IL-12
Signaling through the IL-2 receptor plays a vital role for T cell growth (22). To determine if IL-2 receptor expression was altered in the absence of LIGHT, we examined surface CD25 expression levels on CD8+ T cells upon activation. CD8+ T cells from wild-type and LIGHT–/– mice were activated with anti-CD3 and anti-CD28 for 48 h, and cell-surface expression of CD25 was measured by flow cytometric analysis. Strikingly, CD25 levels were dramatically reduced on LIGHT–/– compared to wild-type CD8+ T cells (Fig. 4A, left), suggesting that the observed reduction in LIGHT–/– CD8+ T cell proliferation may be due to reduced IL-2 signaling. To address this hypothesis, CD8+ T cells were activated by anti-CD3 and anti-CD28 for 48 h, washed, and equal numbers of wild-type and LIGHT–/– CD8+ T cells were stimulated with different concentrations of rmIL-2 for 48 h. As shown in Fig. 4(B), proliferation in response to exogenous IL-2 is significantly reduced in LIGHT–/– compared with wild-type CD8+ T cells, indicating that reduced proliferation of LIGHT–/– CD8+ T cells could potentially be due, at least in part, to the lower level expression of IL-2 receptor.




View larger version (55K):
[in this window]
[in a new window]
 
Fig. 4. Targeting LIGHT reduced CD8+ T cell-surface CD25 expression, and responses to exogenous IL-2 and IL-12. Purified CD8+ T cells from wild-type and LIGHT–/– mice were activated for 48 h in the presence of plate-bound anti-CD3 and anti-CD28 antibody, with (right) or without (left) exogenous rmIL-12. Cells were then washed and analyzed for cell-surface expression of IL-2 receptor, CD25, by flow cytometry (A) or proliferation in the presence exogenous of rmIL-2 (B) or rmIL-12 (C). Cells were labeled with [3H]thymidine for the last 8 h of a 48-h culture. Histogram markers indicate the percentage of cultured CD8+ T cells staining positive for CD25 (shaded histograms) above isotype control (unshaded histograms). Proliferation values shown represent the mean and error bars represent the SD. *P < 0.05; **P < 0.01.

 
In addition to IL-2, IL-12 has been demonstrated to promote CD8+ T cell expansion by providing a third activation signal (in addition to TCR engagement and co-stimulation/IL-2) and up-regulating IL-2 receptor expression (2325). To examine whether exogenous IL-12 could ameliorate the proliferative defect of LIGHT–/– CD8+ T cells, purified wild-type and LIGHT–/– CD8+ T cells were activated by anti-CD3 and anti-CD28 in the presence of various concentrations of rmIL-12 for 48 h. LIGHT–/– CD8+ T cells still exhibited reduced proliferation compared with wild-type CD8+ T cells (Fig. 4C) as measured by [3H]thymidine incorporation. Interestingly, IL-12 did increase the cell-surface expression of CD25 on both LIGHT–/– and wild-type CD8+ T cells (Fig. 4A, right), although CD25 levels on LIGHT–/– CD8+ T cells still remained lower than observed on wild-type CD8+ T cells (wild-type = 90.6 ± 1.6%, LIGHT–/– = 68.9 ± 10.4%). These data suggest that lower CD25 levels may contribute to the proliferative defects of LIGHT–/– CD8+ T cells. However, this phenomenon is not solely responsible since CD25 levels on LIGHT–/– CD8+ T cells are increased upon addition of exogenous IL-12, albeit still to a lesser level than that observed in wild-type CD8+ T cell cultures.

Impaired CTL expansion, but not activity, in LIGHT–/– CD8+ T cells
Since CD8+ T cell proliferation was impaired in the absence of LIGHT, we then examined whether reduced proliferation was accompanied by reduced cytolytic effector function. Effector CTL activity against allogeneic P815 mastocytoma (H-2d) target cells was initially examined using total splenocytes from both wild-type and LIGHT–/– mice. Syngeneic EL-4 thymoma cells (H-2b) were used as a negative control. In these studies, total splenic cytolytic activity against P815 was significantly decreased in the absence of LIGHT, whereas little to no cytolytic activity against syngeneic EL-4 targets was observed in either wild-type or LIGHT–/– cultures (Fig. 5A). The decreased cytolytic activity observed in total splenocyte cultures could be due to either decreased CTL numbers, defective CTL activity or a combination of both events. To further differentiate these possibilities, we performed CTL assays using purified CD8+ T cells from both wild-type and LIGHT–/– mice. When equal numbers of wild-type or LIGHT–/– CD8+ CTL were used, we did not observe any significant difference in CTL activity against P815 cells at all tested E:T ratios (Fig. 5B). These data suggest that LIGHT deficiency results in reduced CD8+ T cell proliferation and subsequent CTL populations, but does not affect cytolytic effector function on a per cell basis.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5. Impaired CTL expansion, but not activity, of LIGHT–/– CD8+ T cells. (A) Total splenocytes from both wild-type and LIGHT–/– mice were cultured with irradiated BALB/c splenocytes (H-2d) in the presence of 10 U/ml rmIL-2. After culturing for 5 days, live effector cells were harvested over Ficoll and cultured with P815 mastocytoma target cells or EL-4 thymoma syngeneic control cells as indicated. (B) CTL assay was performed as described above except for using purified CD8+ T cells from both wild-type and LIGHT–/– mice. Cytotoxic activity was assessed as described in Methods. Values shown represent the mean of triplicate wells and error bars represent the SD. *P < 0.05; **P < 0.01.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TNF/TNFR family members play a critical role in regulating immune responses (26,27). Inducible expression of these family members during lymphocyte activation is essential to control cell proliferation, differentiation and apoptosis (28). CD40 ligand (CD40L), which is of crucial importance in mediating immune responses (29), is highly induced in the activated CD4+ T lymphocytes (30). LIGHT expression is also highly induced upon activation of T lymphocytes (13,31). In contrast to CD40L, LIGHT is preferentially expressed on the CD8+ population (2), indicating that LIGHT may play a regulatory role in the activation of CD8+ T lymphocytes. In vitro studies with soluble LIGHT have shown that LIGHT can serve as a co-stimulatory factor in T cell proliferative responses and transduce CD28-independent co-stimulatory signals (1,4). These data suggest that LIGHT is involved in the induction of cell-mediated immune responses.

In order to further delineate the physiological functions of LIGHT, we have generated LIGHT–/– mice by gene targeting. Disruption of LIGHT resulted in reduced CD8+ T cell proliferation without affecting cell apoptosis. Cell division analysis clearly showed impaired cell division progression in LIGHT–/– CD8+ T cells, which may explain, at least in part, the defects in cell proliferation when compared with wild-type CD8+ T cells. Furthermore, reduced surface expression of CD25 and responses to IL-2 were also observed on activated LIGHT–/– CD8+ T cells, indicating that impaired IL-2 signaling may also contribute to decreased T cell proliferative responses observed. Interestingly, LIGHT deficiency did not appear to impact the cytolytic activity of CD8+ effectors cells on a per cell basis, although the total cytolytic activity of lymphoid tissue cell cultures (i.e. splenocytes) was reduced, presumably through the impaired expansion of CD8+ effectors cells.

Consistent with previous in vitro investigations using soluble LIGHT (11,12), our study demonstrated that disruption of LIGHT negatively affects T cell proliferation and preferentially reduces the CD8+ T cell proliferative responses. However, disruption of LIGHT had no effect on B cell proliferation, Ig isotype switching or antibody production in response to T cell-dependent and -independent antigens (J. Liu, unpublished data). These results provide further evidence that LIGHT plays an important role during CD8+ T lymphocyte activation and expansion.

Recently, three other groups also generated LIGHT–/– mice, and demonstrated important roles of LIGHT in T cell functions, allograft rejection and even cooperative functions with lymphotoxin ß in lymphoid organogenesis (1517). Interest ingly, Tamada et al. (15) observed no differences between wild-type and LIGHT–/– mice in cell proliferation of both purified CD4+ and CD8+ T cells, or total lymph node cells, upon stimulation with anti-CD3 in vitro, although CD8+ T cells showed impaired responses to superantigen in vivo. In contrast, Scheu et al. (16) showed reduced T cell proliferation upon anti-CD3 stimulation and impaired cytolytic MLR responses using total splenocytes from LIGHT–/– mice. The reason for the defective CD8+ T cell proliferation is unknown and the authors suggest the possibility of a difference in cell death. Using purified CD8+ T cells, as opposed to total splenocytes, we clearly show impaired CD8+, but not CD4+, T cell proliferation, which is consistent with results obtained by Scheu et al. (16). Furthermore, for the first time, we provide evidence showing that the impaired LIGHT–/– CD8+ T cell proliferation is mainly due to defects in CD8+ T cell division, not by increasing apoptosis. These data are inconsistent with results reported by Scheu et al. (16) that showed no difference of cell division between wild-type and LIGHT–/– CD4+ or CD8+ T cells. This discrepancy is probably due to the different assay systems performed. Scheu et al. (16) used total splenocytes, and later gated on CD4+ and CD8+ cell populations for flow cytometric cell division analysis, whereas we used purified CD8+ T cells. There may be additional factors expressed by other splenocytes in culture that can provide signals to compensate for the defect of LIGHT–/– CD8+ T cell division. One of these factors could be insufficient IL-2 production, which is vital for stimulating CD8+ T cell proliferation (22). We showed that exogenously provided IL-2 did not recover the proliferation defect of LIGHT–/– CD8+ T cells (Fig. 4B), suggesting that the amount of soluble IL-2 in culture is not the cause responsible for the reduction of cell proliferation. Furthermore IL-2 receptor surface expression was decreased on activated LIGHT–/– CD8+ T cells (Fig. 4A), suggesting that defective IL-2 signaling to the CD8+ T cell itself may contribute, at least in part, to the observed reduction in proliferation. IL-12 is another soluble factor capable of providing a third activation signal, in addition to TCR engagement and co-stimulation/IL-2, that is required for an effective in vitro and in vivo response by naive CD8+ T cells (23,24). Additionally, IL-12 has recently been shown to stimulate both higher and prolonged expression of CD25 on CD8+ T cells stimulated with just TCR engagement and co-stimulation and/or IL-2 (25). Providing exogenous rmIL-12, however, failed to totally ameliorate the proliferation defects of LIGHT–/– CD8+ T cells (Fig. 4C). Addition of rmIL-12 did increase CD25 levels on both wild-type and LIGHT–/– CD8+ T cells (Fig. 4A), although LIGHT–/– cultures still had reduced CD25 levels when compared to wild-type cultures. Thus LIGHT may normally participate in the full necessary contingent of co-stimulatory actions required as a second signal for optimal CD8+ T cell activation and expansion.

Together, we provide evidence here that LIGHT–/– CD8+ T cells have intrinsic defects in TCR-mediated activation, which results in impaired cell division. The exact mechanism(s) of LIGHT that are intrinsic to a complete CD8+ T cell response remain unclear. Recent evidence suggests that LIGHT not only functions as a ligand, but also transduces co-stimulatory signals to stimulated T cells upon cross-linking (32,33). Furthermore, overexpression of LIGHT on T cells in transgenic mice has been shown to stimulate proliferation of autoreactive T cells and tissue destruction (13). In vivo studies have shown that the absence of LIGHT can delay graft injection, suggesting an impaired CTL response in those mice (17). The impaired CTL response in vivo could be a result of either a defect in generating sufficient numbers of effector CTL, defects in the functional activity of CTL or a combination of both events. Consistent with previous results (16), we also observed reduced CTL activity in vitro when total splenocytes were used in the assay culture (Fig. 5A). However, these data could be explained by the fact that LIGHT–/– CD8+ T cells have a proliferation defect and overall reduced CTL numbers in the assay culture. To further address the effect of LIGHT deficiency on CTL generation and function, we used purified wild-type and LIGHT–/– CD8+ T cells that were differentiated in vitro and compared their CTL activity by using equivalent numbers of effector cells. Surprisingly, we did not observe any significant difference between wild-type and LIGHT–/– CTL activity (Fig. 5B), indicating that reported in vivo CTL defects in LIGHT–/– mice probably arise through reduced effector cell expansion and overall numbers, but not necessarily through defective CTL function.

In summary, our findings have revealed the LIGHT plays an important role in the fulfillment of an effective CD8+ T cell response by affecting CD8+ T cell division. Manipulation of the interactions between LIGHT and its receptors may provide therapeutic opportunities in the treatment of various diseases and immune system disorders, including cancer, autoimmunity and allograft rejections.


    Acknowledgements
 
We thank Dr Josef Heuer for critical review of the manuscript, Dr Viswanath Devanarayan for statistical assistance and Teresa Morehead for technical assistance. C. S. S. is a postdoctoral fellow at Eli Lilly & Co.


    Abbreviations
 
APC—antigen-presenting cell

CD40L—CD40 ligand

CTL—cytotoxic T lymphocyte

DC—dendritic cell

ES—embryonic stem

HVEM—herpes virus entry mediator

KLH—keyhole limpet hemocyanin

MLR—mixed lymphocyte reaction

TNF—tumor necrosis factor

TNFR—TNF receptor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Mauri, D. N., Ebner, R., Montgomery, R. I., Kochel, K. D., Cheung, T. C., Yu, G., Ruben, S., Murphy, M., Eisenberg, R. J., Cohen, G. H., Spear, P. G. and Ware, C. F. 1998. LIGHT, a new member of the TNF superfamily, and lymphotoxin {alpha} are ligands for herpesvirus entry mediator. Immunity 8:21.[ISI][Medline]
  2. Morel, Y., Schiano de Colella, J.-M., Harrop, J., Deen, K. C., Holmes, S. D., Wattam, T. A., Khandekar, S. S., Truneh, A., Sweet, R. W., Gastaut, J.-A., Olive, D. and Costello, R. T. 2000. Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor. J. Immunol. 165:4397.[Abstract/Free Full Text]
  3. Granger, S. W. and Ware, C. F. 2001. Turning on LIGHT. J. Clin. Invest. 108:1741.[Free Full Text]
  4. Tamada, K., Shimozaki, K., Chapoval, A. I., Zhai, Y., Su, J., Chen, S., Hsieh, S., Nagata, S., Ni, J. and Chen, L. 2000. LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response. J. Immunol. 164:4105.[Abstract/Free Full Text]
  5. Salio, M., Cella, M., Suter, M. and Lanzavecchia, A. 1999. Inhibition of dendritic cell maturation by herpes simplex virus. Eur. J. Immunol. 29:3245.[CrossRef][ISI][Medline]
  6. Morel, Y., Truneh, A., Sweet, R. W., Olive, D. and Costello, R. T. 2001. The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity. J. Immunol. 167:2479.[Abstract/Free Full Text]
  7. Harrop, J. A., Reddy, M., Dede, K., Brigham-Burke, M., Lyn, S., Tan, K. B., Silverman, C., Eichman, C., DiPrinzio, R., Spampanato, J., Porter, T., Holmes, S.,Young, P. R. and Truneh, A. 1998. Antibodies to TR2 (herpesvirus entry mediator), a new member of the TNF receptor superfamily, block T cell proliferation, expression of activation markers, and production of cytokines. J. Immunol. 161:1786.[Abstract/Free Full Text]
  8. Kwon, B. S., Tan, K. B., Ni, J., Oh, K. O., Lee, Z. H., Kim, K. K., Kim, Y. J., Wang, S., Gentz, R., Yu, G. L., Harrop, J., Lyn, S. D., Silverman, C., Porter, T. G., Truneh, A. and Young, P. R. 1997. A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation. J. Biol. Chem. 272:14272.[Abstract/Free Full Text]
  9. Montgomery, R. I., Warner, M. S., Lum, B. J. and Spear, P. G. 1996. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87:427.[ISI][Medline]
  10. Browning, J. L., Sizing, I. D., Lawton, P., Bourdon, P. R., Rennert, P. D., Majeau, G. R., Ambrose, C. M., Hession, C., Miatkowski, K., Griffiths, D. A., Ngam-ek, A., Meier, W., Benjamin, C. D. and Hochman, P. S. 1997. Characterization of lymphotoxin-alpha beta complexes on the surface of mouse lymphocytes. J. Immunol. 159:3288.[Abstract]
  11. Tamada, K., Shimozaki, K., Chapoval, A. I., Zhu, G., Sica, G., Flies, D., Boone, T., Hsu, H., Fu, Y., Nagata, S., Ni, J. and Chen, L. 2000. Modulation of T cell mediated immunity in tumor and graft versus host disease models through the LIGHT co-stimulatory pathway. Nat. Med. 6:283.[CrossRef][ISI][Medline]
  12. Zhai, Y., Guo, R., Hsu, T. L., Yu, G. L., Ni, J., Kwon, B. S., Jiang, G. W., Lu, J., Ugustus, M. and Carter, K. 1998. LIGHT, a novel ligand for lymphotoxin ß receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer. J. Clin. Invest. 102:1142.[Abstract/Free Full Text]
  13. Shaikh, R. B., Santee, S., Granger, S. W., Butrovich, K., Cheung, T., Kronenberg, M., Cheroutre, H. and Ware, C. F. 2001. Constitutive expression of LIGHT on T cells leads to lymphocyte activation, inflammation, and tissue destruction. J. Immunol. 167:6330.[Abstract/Free Full Text]
  14. Wang, J., Chun, T., Lo, J. C., Wu, Q., Wang, Y., Foster, A., Roca, K., Chen, M., Tamada, K., Chen, L., Wang, C.-R. and Fu, Y.-Y. 2001. The critical role of LIGHT, a TNF family member, in T cell development. J. Immunol. 167:5099.[Abstract/Free Full Text]
  15. Tamada, K., Ni, J., Zhu, G., Fiscella, M., Teng, B., van Deursen, J. M. A. and Chen, L. 2002. Selective impairment of CD8+ T cell function in mice lacking the TNF superfamily member LIGHT. J. Immunol. 168:4832.[Abstract/Free Full Text]
  16. Scheu, S., Alferink, J., Potzel, T., Barchet, W., Kalinke, U. and Pfeffer, K. 2002. Targeted disruption of LIGHT causes defects in costimulatory T cell activation and reveals cooperation with lymphotoxin ß in mesenteric lymph node genesis. J. Exp. Med. 195:1613.[Abstract/Free Full Text]
  17. Ye, Q., Fraser, C. C., Gao, W., Wang, L., Busfield, S. J., Wang, C., Qiu, Y., Coyle, A. J., Gutierrez-Ramos, J.-C. and Hancock, W. W. 2002. Modulation of LIGHT–HVEM costimulation prolongs cardiac allograft survival. J. Exp. Med. 195:795.[Abstract/Free Full Text]
  18. Liu, J., Na, S., Glasebrook, A., Fox, N., Solenberg, P. J., Zhang, Q., Song, H. Y. and Yang, D. D. 2001. Enhanced CD4+ T cell proliferation and Th2 cytokine production in DR6-deficient mice. Immunity. 15:23.[CrossRef][ISI][Medline]
  19. Lyons, A. B. and Parish, C. R. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131.[CrossRef][ISI][Medline]
  20. Delgado, M. and Ganea, D. 2000. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit T cell-mediated cytotoxicity by inhibiting Fas ligand expression. J. Immunol. 165:114.[Abstract/Free Full Text]
  21. Box, G. E. P. and Cox, D. R. 1964. An analysis of transformations. J. R. Stat. Soc. Ser. B 26:211.[ISI]
  22. Mueller, D., Jenkins, M. and Schwartz, R. 1989. Clonal expansion vs functional clonal inactivation. Annu. Rev. Immunol. 7:445.[CrossRef][ISI][Medline]
  23. Curtsinger, J. M., Schmidt, C. S., Mondino, A., Lins, D. C., Kedl, R. M., Jenkins, M. K. and Mescher, M. F. 1999. Inflammatory cytokines provide a third signal for activation of naïve CD4+ and CD8+ T cells. J. Immunol. 162:3256.[Abstract/Free Full Text]
  24. Schmidt, C. S. and Mescher, M. F. 2002. Peptide antigen priming of naïve, but not memory, CD8 T cells requires a third signal that can be provided by IL-12. J. Immunol. 168:5521.[Abstract/Free Full Text]
  25. Valenzuela, J., Schmidt, C. and Mescher, M. F. 2002. The roles of IL-12 in providing a third signal for clonal expansion of naïve CD8 T cells. J. Immunol. 169:6842.[Abstract/Free Full Text]
  26. Watts, T. H. and DeBenedette, M. A. 1999. T cell co-stimulatory molecules other than CD28. Curr. Opin. Immunol. 11:286.[CrossRef][ISI][Medline]
  27. Gravestein, L. A. and Borst, J. 1998. Tumor necrosis factor receptor family members in the immune system. Semin. Immunol. 10:423.[CrossRef][ISI][Medline]
  28. Locksley, R. M., Killeen, N. and Lenardo, M. J. 2001. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104:487.[CrossRef][ISI][Medline]
  29. Xu, J., Foy, T. M., Laman, J. D., Elliott, E. A., Dunn, J. J., Waldschmidt, T. J., Elsemore, J., Noelle, R. J. and Flavell, R. A. 1994. Mice deficient for the CD40 ligand. Immunity 1:423.[ISI][Medline]
  30. Roy, M., Waldschmidt, T., Aruffo, A., Ledbetter, J. A. and Noelle, R. J. 1993. The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells. J. Immunol. 151:2497.[Abstract/Free Full Text]
  31. Yu, K.-Y., Kwon, B., Ni, J., Zhai, Y., Ebners, R. and Kwon, B. S. 1999. The newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis. J. Biol. Chem. 274:13733.[Abstract/Free Full Text]
  32. Shi, G., Luo, H., Wan, X., Salcedo, T. W., Zhang, J. and Wu, J. 2002. Mouse T cells receive costimulatory signals from LIGHT, a TNF family member. Blood 100:3279.[Abstract/Free Full Text]
  33. Wan, X., Zhang, J., Luo, H., Shi, G., Kapnik, E., Kim, S., Kanakaraj, P. and Wu, J. 2002. A TNF family member LIGHT transduces costimulatory signals into human T cells. J. Immunol. 169:6813.[Abstract/Free Full Text]