CD8+ T Cells Secreting Type 2 Lymphokines Are Defective in Protection against Viral Infection

Cellular Immunology 202, 13–22 (2000) doi:10.1006/cimm.2000.1639, available online at http://www.idealibrary.com on

CD8 ⫹ T Cells Secreting Type 2 Lymphokines Are Defective in Protection against Viral Infection Susanne Wirth,* ,1 Maries van den Broek,† Christophe P. Frossard,* Ambros W. Hu¨gin,‡ Isabelle Leblond,* Hanspeter Pircher,§ and Conrad Hauser* ,‡ ,2 *Allergy Unit, Division of Immunology and Allergy, and ‡Department of Dermatology, Hoˆpital Cantonal Universitaire, Geneva, Switzerland; †Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland; and §Institute for Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, Germany Received November 29, 1999; accepted March 16, 2000

pathological situations in humans, such as AIDS (9, 10). Elimination of virus is considered to be one of the principal effector functions of CD8 ⫹ T cells. To date, there is little information on whether the lk profile of CD8 ⫹ T cells affects this effector function in vivo. Therefore, we wished to compare the elimination of virus by CD8 ⫹ effector T cells secreting different lk profiles (Tc1, Tc2, Tc0). To this end, we chose two murine models of viral infection: lymphocytic choriomeningitis virus (LCMV) infection, in which viral elimination has previously been shown to be carried out mainly by CD8 ⫹ T cells and a perforin-dependent mechanism (11, 12); and vaccinia virus infection, in which IFN-␥ has been shown to be critical (11, 13–16). We used CD8 ⫹ T cells originating from transgenic (tg) mice that express the P14 TCR that recognizes LCMV glycoprotein (gp) 33– 41 in the context of H-2D b and generated gp 33– 41-specific Tc1, Tc2, or Tc0 in vitro. These three CD8 ⫹ T cell subsets were compared with respect to perforin- and Fas-dependent cytolysis in vitro, as well as to the capacity to control virus in vivo. Interestingly, neither the lk profile nor the cytotoxicity provided a direct explanation for the differential antiviral protection by the CD8 ⫹ effector T cell subsets in vivo. Because adhesion molecules like VLA-4 and LFA-1 have been shown to play a role in antiviral defense in vivo (17, 18), we compared the expression of VLA-4, LFA-1, and ICAM-1 integrins by Tc1, Tc2, and Tc0.

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8 ⴙ T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-␥ secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules. © 2000 Academic Press

INTRODUCTION CD4 ⫹ effector T cell subsets can secrete distinct lymphokines (lk), of which the most polarized profiles are termed type 1 (IL-2, IFN-␥) and type 2 (IL-4, IL-5). In CD4 ⫹ T cells, there is abundant evidence that the lk profile produced by these cells profoundly influences their effector function in vitro and in vivo (reviewed in Ref. 1). Interleukin-4-secreting T cells have also been derived in vitro from CD8 ⫹ T cells by polyclonal or allogeneic stimulation (2–5). In addition, CD8 ⫹ T cells secreting a type 2 lk profile (Tc2) were observed following immunization of mice (6 – 8) or identified in certain

MATERIALS AND METHODS Mice Transgenic mice (line 327 and line 318) expressing the tg P14 T cell receptor (C57BL/6, V␣ 2V␤ 8) specific for the H-2D b-restricted lymphocytic choriomeningitis virus glycoprotein (LCMV gp 33– 41) have been previously described. Mice of lines 327 and 318 express the tg receptor on approximately 90 and 50%, respectively,

1

Present address: Institute for Microbiology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne-CHUV, Switzerland. 2 To whom correspondence should be addressed at Allergy Unit, Hoˆpital Cantonal Universitaire, 24, Rue Micheli-du-Crest, CH 1211 Geneva 14, Switzerland. Fax: (41) 22-372 94 16. E-mail: [email protected]. 13

0008-8749/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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of their CD8 ⫹ T cells (19, 20). P14 TCR transgenic mice with deficient perforin expression were obtained by breeding transgenic line 318 into perforin knockout mice (11). Transgenic and mutant mice were maintained under SPF conditions. Eight- to sixteen-weekold animals were used. C57BL/6 mice were purchased from IFFA–Credo (L’Arbresle, France) and were used between 8 and 12 weeks of age. Culture Media and Reagents CD8 ⫹ lymph node cells were cultured in Dulbecco’s modified Eagle’s medium with L-glutamine, supplemented with 1% MEM nonessential amino acids, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM L-glutamine (all from Life Technologies, Paisley, Scotland), 10% heat-inactivated FCS (Life Technologies, Munich, Germany), 1 mM sodium pyruvate (Fluka, Buchs, Switzerland), and 2 ⫻ 10 ⫺5 M 2-ME (Sigma, St. Louis, MO). CTLL were maintained in RPMI supplemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin, 2 mM L-glutamine (all from Life Technologies), and 5 ⫻ 10 ⫺5 M 2-ME. Monoclonal antibodies to CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.72), CD44 (I42/5), Thy 1.2 (HO 13.49), VLA-4 (R1-2 and PS/2.3.2), ICAM-1 (YN1.7.4), LFA-1 (H35-89.9), rat Ig␬ (MAR 18.5), Fc␥RII (2.4G2), and MHC class II (M5/114.15.2) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Purified anti-VLA-4 mAb PS/2.3.2 was generously provided to us by Dr. Linda Burkly (Biogen Inc., Cambridge, MA). Control rat IgG was from ICN ImmunoBiologicals (Costa Mesa, CA). For phenotyping by flow cytometry, the following biotin and fluorochrome conjugates were purchased from PharMingen (San Diego, CA): anti-CD4 –phycoerythrin (PE) (Rm45), anti-CD8 –FITC (53-6.72), anti-CD3⑀–FITC or –PE (145-2C11), anti-V␣2–PE (B20.1), and anti-V␤8.1,8.2– biotin (MR5-2). As secondary reagents for the detection of human IgG1 fusion proteins and unlabeled rat antibodies, biotinylated goat anti-human IgG (Fc fragment specific, Jackson ImmunoResearch Laboratories, West Grove, PA) and goat anti-rat IgG–FITC (mouse adsorbed, Caltag, San Francisco, CA), respectively, were used. Biotinylated antibodies were revealed by streptavidin–PE (Southern Biotechnology Associates, Inc., Birmingham, AL), avidin–FITC (Molecular Probes, Eugene, OR), or streptavidin–Red 670 (Life Technologies). Synthetic LCMV glycopeptide 33– 41 (LCMV gp 33– 41, KAVYNFATM) was purchased from Neosystem (Strasbourg, France). Purity was 65% for the LCMV gp peptide, as assessed by high-performance liquid chromatography. The peptide was solubilized in balanced salt solution at 1 mg/ml, sterile filtered, and stored frozen. The VCAM–Ig fusion protein containing the two N-terminal domains of the human VCAM-1 gene

fused to human IgG1 sequences has been previously reported (21) and was a kind gift of Dr. Linda Burkly. Virus LCMV strain WE was originally obtained as tripleplaque-purified stock from Dr. F. Lehmann-Grube (Heinrich–Pette Institut, Hamburg, Germany) (22). Recombinant vaccinia virus expressing the LCMV glycoprotein (vacc LCMV gp) was grown and used as described (23). Generation of CD8 ⫹ T Cell Subtypes To generate CD8 ⫹ T cell subtypes, culture conditions similar to those described by Erard et al. (3) were established. CD8 ⫹ T cells were purified from lymph nodes of naive tg mice by passage over nylon wool columns followed by depletion of CD4 ⫹ and MHC class II ⫹ cells by treatments with antibodies GK1.5 and M5, with MAR 18.5, and with rabbit low-tox M complement (Cedarlane, Hornby, Ontario, Canada). These more than 95% CD8 ⫹ T cells (0.5–1 ⫻ 10 4 cells/well) were submitted to primary stimulation in 96-well roundbottom plates (Nunc, Roskilde, Denmark) in the presence of 50 U/ml rhuIL-2 (Eurocetus, Amsterdam, The Netherlands) or 50 U/ml rhuIL-2 and 1000 U/ml rmIL-4 [supernatant of X63/O transfectant (24)] for the generation of effector cells with a type 1, type 0, or type 2 lk pattern. Either plate-bound anti-CD3 antibody (0.3 ␮g/well) or T-cell-depleted, irradiated splenocytes (SC-T, 3000 rad, 10 5 cells/well) with 5 ⫻ 10 ⫺8 M LCMV gp peptide were used as stimulators. After 5 days of culture, cells were expanded in 6-well plates at 1.5 ⫻ 10 6 cells/well without further stimulus in the presence of either 50 U/ml rhuIL-2 or 50 U/ml rhuIL-2 and 1000 U/ml rmIL-4. Thirty-six hours later, cultures were harvested and viable cells were recovered by centrifugation over a lympholyte M gradient (Cederlane). The cells were washed four times before further use. Cell Surface Phenotype of CD8 ⫹ T Cell Subsets Before and after culture, the surface phenotype was assessed by immunolabeling and flow-cytometric analysis on a FACScan (Becton–Dickinson, Mountain View, CA). Expression of CD8 and of the tg TCR was assessed simultaneously by three-color immunostaining with anti-CD8 –FITC, anti-V␣ 2–PE, and biotinylated anti-V ␤8 followed by streptavidin–Red 670. CD44 expression was determined by incubation with unlabeled I42/5 or control rat isotype mAb, followed by goat anti-rat IgG–FITC with or without mouse anti-V␣ 2– PE. FasL expression was assessed by FACScan analysis using three-step immunolabeling with huFas.Fc or control huIL-4R.Fc fusion proteins, biotinylated goat anti-huIgG, and streptavidin–PE. Cells activated or not for 3 h at 37°C with PMA (10 ng/ml) and ionomycin

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(3 ␮g/ml) were analyzed. VCAM–Ig binding of CD8 ⫹ T cells was assessed either by flow cytometry as reported (21) or by an adhesion assay in vitro. VCAM Adhesion Assay To determine adhesion of CD8 ⫹ T cell subtypes to VCAM–Ig, 96-well plates (Maxisorp, Nunc) were coated with 50 ␮g/well of VCAM–Ig at 20 ␮g/ml PBS for 3 h at 37°C. Free binding sites were blocked by incubation with Tris-buffered saline (24 mM Tris–HCl and 150 mM NaCl, pH 7.4) supplemented with 1% BSA, 2 mM glucose, and 10 mM Hepes (TBS) at 37°C for 1 h. After the plate was washed twice with TBS, 10 5 cells in 100 ␮l TBS ⫾ 1 mM MnCl 2, previously incubated on ice for 1 h with either 2.4G2 or R1-2 (1 ml of hybridoma supernatant/10 6 cells), was added to the plate. Adhesion of cells was allowed for 30 min at room temperature under gentle shaking. The plate was washed five times with the respective TBS buffer (⫾MnCl 2) and the number of cells adhering to the plate was counted under a light microscope by two different persons in a blinded way. Results are expressed as the numbers of cells/field with all conditions counted at the same magnification. CTL Assay, Proliferation, and Lymphokine Secretion in Vitro The cytolytic activity of in vitro-generated T cell lines was assessed in a standard 4-h 51Cr-release assay using RMA (H-2 b (25)), MC57G (fibrosarcoma, H-2 b), or YAC-1 as target cells. Calcium-independent cytotoxicity against RMA target cells was determined in the presence of 4 mM EGTA and 3 mM MgCl 2 (26). All experiments were performed in duplicate. Percent specific 51Cr release was calculated as follows: (exp. cpm ⫺ spontaneous cpm)/(total cpm ⫺ spontaneous cpm) ⫻ 100. Spontaneous release (medium alone) was always less than 15% of total release (1 N HCl). For the measurement of proliferation and lymphokine secretion, 10 5 CD8 ⫹-primed T cells were incubated in flat-bottom 96-well plates (Nunc) with 10 5 SC-T that were previously incubated at 37°C for 90 min with the indicated concentration of added LCMV gp peptide. Parallel control cultures consisted of T cells that were polyclonally activated by plate-bound antiCD3 antibody (0.3 ␮g/well). After 24, 48, or 72 h of culture, 100 ␮l of supernatant was harvested and cell proliferation was measured by [methyl- 3H]thymidine incorporation after pulsing the cultures (0.5 ␮Ci/well) for the final 6 to 8 h of the culture period. Supernatants were stored at ⫺20°C until lk analysis. The presence of IL-2 and IL-4 in supernatants was determined with a specific bioassay using CTLL cells and by neutralizing mAbs against IL-2 and IL-4 (27). Internal standards in the form of recombinant murine IL-2 and recombinant murine IL-4 (obtained from Dr.

W. E. Paul) were included in each assay. Detection levels for IL-2 and IL-4 were 0.03 and 3 U/ml, respectively. TNF-␣/␤ production was measured by its cytotoxic effect on the TNF-sensitive mouse fibrosarcoma cell line WEHI 164.13 (28), which could be completely blocked by a TNF-specific rabbit antiserum, kindly provided by Dr. Georges Grau, Centre Me´dical Universitaire (Geneva, Switzerland). The detection level was 0.06 U/ml. The presence of IFN-␥ in culture supernatants was determined by a monospecific ELISA as described (29). The sensitivity of the assay was 1 U/ml. IL-5 was measured by ELISA using mAbs TRFK-4 and TRFK-5 (from Dr. T. Mosmann, DNAX, Palo Alto, CA), as described (30). The detection level was 4 U/ml. The commercially available recombinant mouse cytokines TNF-␣, IFN-␥, and IL-5 (all from Genzyme, Cambridge, MA) served as internal standards for the lk assays. Assessment of Antiviral Protective Effect by CD8 ⫹ T Cells in Vivo C57BL/6 recipient mice were intravenously infected with LCMV-WE 24 h prior to adoptive transfer of CD8 ⫹ T cells. An infecting dose of 200 plaque (focus)-forming units (PFU) was used to determine the protective effect in spleen, whereas a dose of 2 ⫻ 10 5 PFU was necessary for measurement of protection in liver and kidney. Four days after adoptive transfer of different numbers of CD8 ⫹ T cells by an intravenous route, mice were sacrificed and the virus titer was determined in spleen, liver, or kidney with the immunological focus-forming assay described (31). Briefly, organs were homogenized in glass tubes and spun at 1700g for 10 min at 4°C. Serial 10-fold dilutions of homogenized supernatants were incubated with the adherent fibrosarcoma cell line MC57G in 24-well plates. After adsorption of virus by cells and 48 h culture under a methylcellulose overlay, monolayers were fixed, permeabilized, and stained with a rat monoclonal rat anti-LCMV antibody and a peroxidase-labeled second-step antibody. Virus titers are expressed as PFU/g tissue. The antiviral protection against recombinant vaccinia virus gp was assessed as follows: C57BL/6 mice were first infected with 2 ⫻ 10 6 PFU vacc LCMV gp by intravenous route and 4 h later injected intravenously with the indicated number of CD8 ⫹ T cells. Alternatively, mice first received the CD8 ⫹ T cells and, 4 h later, 2 ⫻ 10 6 PFU vacc LCMV gp. Virus titers in ovaries were determined 4 days after cell transfer (32). RESULTS In Vitro Priming of LCMV-Specific CD8 ⫹ T Cells with IL-4 Induces Type 2 Lymphokines and CTL Function To generate in vitro CD8 ⫹ effector T cells with defined antigen specificity and distinct lk patterns, we isolated CD8 ⫹ lymph node cells from a mouse line

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TABLE 1 Lymphokine Production by CD8 ⴙ T Cell Subsets Generated in Vitro Lymphokine produced (U/10 6 cells) during restimulation b Primary stimulation in vitro a

Type obtained

IL-2

IFN-␥

IL-4

IL-5

TNF

Anti-CD3 IL-2 Anti-CD3⫹IL-2⫹IL-4 APC⫹Ag⫹IL-2⫹IL-4

Tc1 Tc2 Tc0

104 4 300

22,000 7,400 30,000

<100 33,400 4,400

<120 2,400 280

1,200 2,000 ⬎22,000

a Tc1, Tc0, or Tc2 was generated from CD8 ⫹ lymph node cells of naive TCR tg mice by stimulation in vitro with plate-bound anti-CD3 mAb (Tc1, Tc2) or with syngeneic T-depleted spleen cells as APC and LCMV gp peptide (Tc0) in the absence or presence of IL-4 as described under Materials and Methods. The resulting effector cells were washed and restimulated with plate-bound anti-CD3, and lymphokines were measured in 24-h supernatants. b No lk secretion was detectable without restimulation. Values with ⬎ indicate above detection limits and values with ⬍ indicate below detection limits of the lymphokine assays at the cell densities and supernatant dilutions used.

expressing a tg TCR specific for LCMV gp 33– 41 on 95% of CD8 ⫹ T cells (19, 20) and activated them under conditions described to induce a distinct lk pattern. The lk profile of effector cells was determined after restimulation with plate-bound anti-CD3 mAb (Table 1). The same lk pattern, but at lower levels, was observed after restimulation with APC plus peptide (data not shown). The highest type 2 lk (IL-4, IL-5) levels were obtained from cells primed with anti-CD3 and IL-2 plus IL-4 (Tc2). The IFN-␥ production in this subset was four- to eightfold lower than that of cells primed with anti-CD3 and IL-2 (Tc1, n ⫽ 8 experiments). Lower levels of type 2 lk were released by cells primed with APC, peptide, IL-2, and IL-4 (Tc0, Table 1); however, these cells also released IFN-␥ at levels similar to those of Tc1 cells. After culture, the cells

from all conditions were at least 95% CD8 ⫹, and 95% of the CD8 ⫹ cells expressed the V ␣2V ␤8 TCR and high levels of CD44. The mean fluorescence intensity of V ␣2 and V ␤8 did not differ among cells from the different culture conditions. We next analyzed the CTL activity of the different effector subsets induced in vitro. When tested against the lymphoid RMA cells (Fig. 1A) or the MC57G fibrosarcoma cells (Fig. 1B), all subsets displayed similar levels of Ag-dependent CTL activity. No significant lytic activity was detected in the absence of Ag or against YAC-1 cells (data no shown). Because Fasmediated cytotoxicity is in part responsible for the lysis of RMA cells and because Fas-dependent cytotoxicity has been reported to be decreased in CD8 ⫹ T cells primed in the presence of IL-4 (26), we assessed the

FIG. 1. Cytolytic activity of CD8 ⫹ T cell subsets. In vitro generated Tc1, Tc2, and Tc0 cells were assayed for cytolytic activity in a standard 4-h 51Cr-release assay on LCMV gp peptide-pulsed target cells (solid symbols). (A) Cytolytic activity of TCR tg CD8 ⫹ T cell subtypes against LCMV gp-pulsed RMA cells. The data shown are representative of 10 experiments (Tc1 and Tc2) and of 6 experiments (all subtypes). (B) Cytolytic activity of TCR tg CD8 ⫹ T effectors against LCMV gp-pulsed MC57G fibroblasts. Data representative of 6 experiments (Tc1 and Tc2) and 2 experiments (Tc1, Tc2, and Tc0) are shown. (C) Cytolytic activity of perforin-deficient, TCR tg CD8 ⫹ Tc1 and Tc0 cells against LCMV gp-pulsed RMA cells. One of two similar experiments is shown. Target cell lysis in the absence of LCMV gp peptide was always less than 5% (open symbols), and spontaneous 51Cr release by targets cells was below 15% in all experiments.

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FIG. 2. Antiviral protective effect of CD8 ⫹ T cell subsets in vivo. (A) C57BL/6 recipient mice were infected with 200 PFU LCMV-WE 24 h prior to adoptive transfer by iv injection of the indicated number of CD8 ⫹ T cell effectors. LCMV-WE titers in spleens were determined 4 days after cell transfer. One of two similar effector cell titration experiments is shown. (B) C57BL/6 recipient mice were infected with 2 ⫻ 10 6 PFU recombinant vaccinia virus (vacc LCMV gp). Four hours later mice received various numbers of CD8 ⫹ T cell effectors by iv route. Vacc LCMV gp titers in ovaries were measured 4 days after cell transfer. One of two similar experiments is shown.

cytotoxicity of CD8 ⫹ T cells from mice with the same tg TCR bred into a perforin-deficient mouse line (11) using RMA cells as target. Tc1 and Tc0 TCR tg cells with a deleted perforin gene exhibited identical CTL activity against RMA target cells (Fig. 1C). Furthermore, no differences in FasL expression were found in Tc1 and Tc2 from TCR tg mice using immunolabeling with a Fas–Ig fusion protein and flow cytometry (not shown). In addition, the CD8 ⫹ T cell subsets showed the same responsiveness with regard to cytotoxicity, proliferation, and lk secretion in response to stimulation with different doses of LCMV gp peptide and different numbers of APC (not shown). Antiviral Activity of CD8 ⫹ T Cell Subsets with Type 2 Lymphokines Is Decreased in Vivo The in vitro generated effector subsets were tested for antiviral activity in vivo. C57BL/6 mice were first infected either with LCMV-WE or with a recombinant LCMV gp-expressing vaccinia virus (vacc LCMV gp). Graded numbers of Tc1 cells and Tc2 cells were adoptively transferred and the virus titer in the target organs was determined 4 days later. Transferred Tc2 cells were markedly less efficient in eliminating LCMV-WE (Fig. 2A) and vacc LCMV gp virus (Fig. 2B) from the spleen and the ovaries, respectively, than Tc1 cells. Although some variations were noted between individual experiments, effectors secreting type 2 lk in vitro (Tc2, Tc0) were always less protective; the mean difference in LCMV-WE load was 150-fold (range 20 – 500, n ⫽ 6 experiments, P ⫽ 0.0313, Wilcoxon twotailed test). At least 10 times as many Tc2 cells were required to obtain the same level of protection mediated by Tc1 cells (Fig. 2). Deficient antiviral protection by Tc2 cells relative to T1 cells was confirmed in kidney

and liver following infection with high doses of LCMV (data not shown). Tc0 cells, like Tc2 cells, exhibited defective protection not only against LCMV (Fig. 3A), but also against vacc LCMV gp (Fig. 3B). Elimination of this latter virus requires IFN-␥ (11, 13–16). A transfer of 1:1 mixed Tc1 and Tc2 cells mediated the same level of protection against LCMV as Tc1 cells alone did (data not shown), indicating that Tc2 cells did not suppress the function of simultaneously transferred Tc1 cells in vivo. These results also show that Tc0 cells behave like a homogenous lk-secreting population, suggesting that most of the Tc0 cells secrete both type 1 and type 2 lk. To ascertain whether protection against LCMV depended on perforin in this transfer setting, as in standard viral infection of immune mice (11), Tc1 and Tc0 cells were generated from a TCR tg mouse carrying a deficient perforin gene and tested in vivo for protection against LCMV. Deletion of the perforin gene almost completely abolished the antiviral protection mediated by tg Tc1 cells and reduced the already diminished protection conferred by tg Tc0 cells (Fig. 4), confirming that elimination of LCMV occurs mainly via a perforin-dependent mechanism in the adoptive transfer setting. Together, these experiments show that CTL function in Tc2 and Tc0 or type 1 lk secreted by Tc0 cells (IFN-␥) is not sufficient for protection against LCMV or vacc LCMV gp, respectively. CD8 ⫹ Effector T Cells with Type 2 Lymphokines Have Decreased Integrin Expression and Function We consistently observed a marked difference between Tc1 and Tc2 cells with respect to homotypic aggregation following secondary stimulation by platebound anti-CD3 mAb. As presented in Fig. 5, Tc1 cells

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We further investigated adhesion molecules in Tc1 and Tc2 cells by flow cytometry. Whereas comparable densities of ICAM-1 and LFA-1 were found on Tc1 and Tc2 cells, VLA-4 expression was decreased or undetectable on Tc2 cells, the mean fluorescence intensity being three times less than that of Tc1 cells (n ⫽ 16 experiments, range 2–5 times, Fig. 6). The expression of VLA-4 remained lower in Tc2 cells than in Tc1 cells, even after reactivation with plate-bound anti-CD3 mAb or APC and LCMV gp peptide. Tc0 cells were also deficient in VLA-4 expression (data not shown). To confirm VLA-4 deficiency in Tc2 cells on a functional level, Tc1 and Tc2 cells were tested for their ability to adhere to immobilized VCAM-1–Ig fusion protein with and without pretreatment with MnCl 2 (Fig. 7). Tc2 cells were nearly incapable of adhering to plasticbound VCAM–Ig, in sharp contrast to Tc1 cells. The addition of MnCl 2, which induces the activation of VLA-4, further increased binding of Tc1 cells but only of very few Tc2 cells. Anti-VLA-4, but not an irrelevant isotype control antibody, completely abolished the adhesion of Tc1 cells, confirming the interaction of VLA-4 with VCAM–Ig fusion protein in this assay. Together these data demonstrate decreased VLA-4 density on the surface of CD8 ⫹ effector cells secreting type 2 lk (Tc2 and Tc0 cells) and decreased VLA-4 and LFA-1/ ICAM-1-dependent adhesive function of these cells. FIG. 3. Antiviral protection by Tc1, Tc2, and Tc0 CD8 ⫹ effector subsets. (A) C57BL/6 recipient mice were infected with 200 PFU LCMV-WE 24 h prior to adoptive transfer by iv injection of 1 ⫻ 10 6 Tc1, Tc2, or Tc0 CD8 ⫹ T cell effectors. Control mice received 2 ⫻ 10 6 naive TCR tg lymph node cells that were previously shown to mediate protection of C57BL/6 mice against LCMV infection. LCMV-WE titers were assessed 4 days after cell transfer. One of six similar experiments is shown. (B) C57BL/6 recipient mice were infected with vacc LCMV gp as described in Materials and Methods. The indicated numbers of CD8 ⫹ T cell effectors of type Tc1, Tc2, and Tc0 were transferred 4 h later and vacc LCMV gp titers in ovaries were determined 4 days after cell transfer.

form discrete aggregates in response to reactivation, in contrast to Tc2 cells, which remain dispersed in the culture well. Because the adhesion assay readout was performed 20 h after reactivation of Tc1 and Tc2 cells and because Tc1 and Tc2 cells did not proliferate differently after reactivation with anti-CD3 (see above), the difference in aggregation was not explained by preferential proliferation of Tc1. Incubation of Tc1 cells with mAb directed to ICAM-1 (Fig. 5) and LFA-1 (not shown), but not to VLA-4 (Fig. 5), completely inhibited activation-induced homotypic aggregation. Activationinduced homotypic aggregation was also absent in Tc2 and Tc0 cells derived from nontransgenic C57/BL6 mice (data not shown). These results indicate that activation-induced aggregation of Tc1 cells is dependent on ICAM-1 and LFA-1 and suggest that Tc2 and Tc0 cells may present a defect in the expression or function of these adhesion molecules.

DISCUSSION We generated in vitro CD8 ⫹ effector T cell populations that did or did not secrete type 2 lk (Tc1, Tc2, Tc0) using CD8 ⫹ T cells from a well-characterized TCR tg mouse line that expresses the transgene on the major-

FIG. 4. The role of perforin in protection against LCMV mediated by adoptively transferred CD8 ⫹ T cell subtypes. CD8 ⫹ Tc1 and Tc0 cells were generated in vitro from TCR tg mice carrying a deficient perforin gene (“ko”) and from TCR tg control mice (“wt”) as described under Materials and Methods. A total of 5 ⫻ 10 5 Tc1 or Tc0 cells (all about 95% CD8 ⫹) were transferred by iv injection into C57BL/6 recipient mice that had been infected with 200 PFU LCMV-WE 24 h before. Virus titers in spleens were determined 4 days after cell transfer.

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FIG. 5. Differential homotypic aggregation of CD8 ⫹ Tc1 and Tc2 cells following activation with plate-bound anti-CD3 antibody. Tc1 and Tc2 cells were incubated in 24-well plates, previously coated with anti-CD3 mAb, in the presence of mAbs anti-ICAM-1, anti-VLA-4, or control rat IgG2b. Twenty hours later, the cells were fixed in the plate with 2% glutaraldehyde and photographed. Original magnification, ⫻10. Results are representative of three similar experiments.

ity of CD8 ⫹ T cells. Tc2 cells were derived by stimulation in vitro with anti-CD3 mAb in the presence of IL-4, whereas Tc0 cells secreting at the same time type 1 and type 2 lk were generated by stimulation with APC, LCMV peptide, and IL-4. In the absence of IL-4, Tc1 cells were obtained independently of the stimulus used. Similar observations regarding the influence of culture conditions/APC on the lk secretion profile induced with IL-4 have been made by other groups (4, 5). The cytolytic functions against Fas ⫹ and Fas ⫺ target cells in vitro were similar in all CD8 ⫹ effector populations tested. Decreased CTL activity of Tc2 was previously observed in a similar study (26) and when cells were primed with PMA (3), which was not used here. Sad et al. also reported no difference in the cytolytic activity of Tc1 and Tc2 (5). Furthermore, we found no difference in FasL expression in Tc1 and Tc2 cells. This contrasts with the situation in CD4 ⫹ Th cells, where Th2 have reduced FasL expression compared to that of Th1 cells (data not shown (33)). With respect to cytolytic activity, lk production and proliferation in all CD8 ⫹ T cell subsets showed similar responsiveness to different doses of the LCMV peptide gp 33– 41. Tc1, Tc2, and Tc0, however, greatly differed in antiviral protection in vivo. The reduction of LCMV-WE and of vaccinia virus carrying the LCMV gp gene by Tc2 and Tc0 cells was significantly lower than that by the corresponding Tc1 cells. Previous studies have established that elimination of LCMV requires CD8 ⫹ T cells and mainly perforin-dependent CTL activity in vivo (11, 12) and to a lesser extent IFN-␥ secretion (16, 34 –36). Despite similar CTL activity in vitro, Tc2 and Tc0 cells were deficient in controlling LCMV compared to Tc1 cells. We ascertained, however, that pro-

tection against LCMV was mediated by perforin in the present adoptive transfer setting (Fig. 4). Despite the fact that recent work has shown that IFN-␥ can play a role in protection against LCMV (37), our LCMV setup was not sensitive to the blocking of IFN-␥ in recipient mice (data not shown). In contrast, IFN-␥ has been shown to be required for the control of vaccinia virus infection (11, 13–16). Therefore, the result with vacc LCMV gp was, on the one hand, in apparent agreement with the somewhat lower (four- to eightfold) secretion of IFN-␥ in Tc2 cells, although these cells still produced substantial amounts of IFN-␥. On the other hand, Tc0 cells that produced IFN-␥ levels comparable to those of Tc1 cells did not protect against vacc LCMV gp. Thus, the secretion of type 2 lk but not in vitro CTL activity and IFN-␥ secretion correlated with the failure to protect against LCMV and vacc LCMV gp, respectively. It can be argued that type 2 lk from Tc2 and Tc0 may have inhibited the function of both donor and recipient cells involved in protection. Administration of mAb to IL-4 into LCMV-infected mice receiving Tc2 cells did not reverse the failure in virus control, excluding a possible inhibitory effect of IL-4 (data not shown). In addition, Tc2 cells did not suppress the function of simultaneously transferred Tc1 cells in vivo because the transfer of 1:1 mixed Tc1 and Tc2 cells mediated the same level of protection against LCMV as Tc1 cells alone did (data not shown). From our results, it was conceivable that a process preceding target cell lysis or IFN-␥ production was deficient in virus-infected mice that received Tc2 and Tc0 cells. Adhesion of CD8 ⫹ T cells is required for migration into target tissue and may be required for efficient interaction with target cells. We thus ana-

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FIG. 6. Flow-cytometric analysis of adhesion molecule expression by CD8 ⫹ Tc1 and Tc2 cells. Tc1 and Tc2 cells were stained with anti-VLA-4 (R1-2, rat IgG2b), anti-LFA-1 (H35-89.9, rat IgG2b), anti-ICAM-1 (YN1.7.4, rat IgG2a), or irrelevant rat IgG2b (2.4G2), followed by goat anti-rat IgG–FITC, and were analyzed by flow cytometry. The thick line shows staining of the indicated adhesion molecule, and the thin line shows background staining by the irrelevant rat IgG2b. The experiment was repeated 16 times with the same results.

lyzed adhesion molecules on the surface of the CD8 ⫹ T cell subsets with different lk profiles. We consistently observed deficient homotypic adhesion of reactivated Tc2 and Tc0 cells. We confirmed that activation-induced homotypic adhesion of Tc1 cells was dependent on LFA-1 and ICAM-1. Activation-induced increase in LFA-1 function on T cell blasts (38) and deficient activation-induced homotypic T cell adhesion in LFA-1 knockout mice (39) have been reported previously. Our in vitro findings point to a defect in the activationinduced LFA-1/ICAM-1 interaction in Tc2 and Tc0 cells because the adhesion defect was not associated with a decrease in surface density of LFA-1 or ICAM-1 on reactivated Tc2 or Tc0 cells. In addition to deficient activation-induced homotypic adhesion of T2 and T0 cells, we found decreased VLA-4 density on Tc2 and Tc0 compared to that on Tc1. This was corroborated with decreased adhesion of Tc2 and Tc0 to immobilized VCAM–Ig fusion protein. Even MnCl 2-induced activation of VLA-4 did not induce adhesion of Tc2 and Tc0 to VCAM–Ig fusion protein. It has been reported that mice genetically deficient in LFA-1 generated normal CTL responses in the spleen after infection with LCMV (39). Furthermore, the same TCR tg mouse as that used here, when ge-

netically deficient in LFA-1, also raised normal CTL responses in the spleen (40). In both reports, however, analysis of antiviral protection in vivo was not presented. A third study revealed that ICAM-1- and LFA1-deficient mice show a reduced ability to eliminate LCMV (Traub strain) (18). When the results of these studies are taken together, an LFA-1/ICAM-1 interaction appears to be important for protection against virus once efficient LFA-1/ICAM-1-independent CTL induction has taken place in the spleen. This is conceivable with our results and points to an important role for LFA-1/ICAM-1 interaction in migration and/or target cell interaction of CD8 ⫹ T cells in this viral model. The role of VLA-4 has also been investigated in vivo using the LCMV model (17). These authors showed that LCMV-immune CD8 ⫹ effector T cells expressed increased VLA-4 density and that these cells transferred delayed-type hypersensitivity to naive infected mice when given intravenously but not when injected into the footpad. The intravenous transfer of delayed type-hypersensitivity was blocked by anti-VLA mAb, suggesting that the migration of LCMV-immune CD8 ⫹ T cells into the site of delayed-type hypersensitivity was VLA-4 dependent. Thus, decreased VLA-4 density

DEFICIENT ANTIVIRAL EFFECT OF Tc2 IN VIVO

21

ACKNOWLEDGMENTS We thank Laurence Tropia for excellent technical assistance and Dr. J. Soriano (Centre Me´dical Universitaire, Geneva, Switzerland) for taking microphotographs. We gratefully acknowledge Dr. L. C. Burkly (Biogen Inc., Cambridge, MA) for the generous gift of VCAM–Ig and Drs. H. Hengartner and R. Zinkernagel (Institute of Experimental Immunology, Zurich, Switzerland), F. Erard (Novartis, Basel, Switzerland), and Dr. B. Imhof (Centre Me´dical Universitaire, Geneva, Switzerland) for helpful discussions. We are grateful to D. Wohlwend for help with flow cytometry, to B. Mermillod for advice on statistical analysis, and to Glaxo Welcome Geneva Biomedical Research Institute (Plan-les-Ouates, Switzerland) for breeding TCR transgenic mice. This work was supported in part by the Swiss National Foundation for Scientific Research (31-40466.94 to C.H.). FIG. 7. Adhesion of CD8 ⫹ Tc1 and Tc2 cells to immobilized VCAM–Ig in vitro. Tc1 and Tc2 cells were preincubated with either control rat IgG2b mAb (2.4G2) or anti-VLA-4 mAb (R1-2, rat IgG2b), washed, and added to a VCAM–Ig-coated 96-well plate in the presence (solid bars) or absence (hatched bars) of MnCl 2. After 30 min, the plate was washed five times with the respective buffer (⫾MnCl 2) and the number of cells adhering to the plate was counted under a light microscope. Results are expressed as numbers of cells/field with all conditions counted at the same magnification. One of three experiments is shown.

on Tc2 and Tc0 cells may have contributed to decreased tissue homing and consequently decreased clearance of virus from tissue. Effector cells primed with IL-4 can have reduced expression of determinants other than integrins important in cell adhesion and migration. E- and P-selectin ligands were reported to be reduced in Th2. Reduced expression of these ligands in Th2 may be responsible for deficient antigen-nonspecific homing of these cells to skin (41, 42). Very recently, similar differential antiviral protection by Tc1 and Tc2 was reported by Cerwenka et al. using an influenza model (43). They found a correlation between the level of antiviral protection and the anatomic localization of the Tc1 and Tc2 subsets in the lung. These findings support the significance of differential LFA-1 and VLA-4 function/expression in Tc subsets, as we have shown here, and the importance of these adhesion molecules in tissue migration and resulting virus elimination. In conclusion, by using two independent models of virus infection that look at different, unrelated effector functions of CD8 ⫹ T cells, we provide evidence that deficient homing to the target tissue may be responsible for the failing antiviral activity of Tc2 and Tc0 cells. Decreased antiviral protection by T cells secreting type 2 lk may also be relevant in human pathology. Acute worsening in patients with asthma is frequently associated with respiratory viral infections and patients with atopic eczema have an apparently increased susceptibility to certain viral skin infections. In both disorders, a predominance of type 2 lk-secreting T cells has been identified.

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