FTY720 Immunosuppression Impairs Effector T Cell Peripheral Homing Without Affecting Induction, Expansion, and Memory

Mice

C57BL/6 mice (H-2^b), 318 TCR transgenic mice, expressing a TCR specific for LCMV-GP33 together with H-2D^b, and RIP-GP transgenic mice expressing the LCMV-GP on pancreatic islet cells were obtained from the breeding colony of the Institut für Labortierkunde, Veterinary Hospital (Zurich, Switzerland). Breeding was performed under specific pathogen-free conditions, and experiments were performed under conventional animal facility conditions.

Viruses, virus detection, and inactivation

Cell analysis and staining

Cell analysis and counting of absolute numbers of transferred cells circulating in the blood were performed by FACS by mixing a defined number of autofluorescent beads with a known volume of blood to permit the calculation of positive cell number per microliter, using Becton Dickinson Trucount Tubes (Mountain View, CA). In addition to the manufacturer’s instructions, the absolute number of autofluorescent beads per sample was re-evaluated by FACS with the instrument settings used for the respective cell analysis. Relative cell numbers varied within a range of ±2%; truly absolute numbers are, however, less precisely measurable.

For counting of transferred specific CTL present in secondary lymphoid organs single-cell suspensions were prepared as described for cytotoxicity assays. The total number of lymphocytes per organ was assessed in a Neubauer chamber (improved bright-line, Brand Wertheim, Germany). The percentage of CD8⁺Vα2⁺Vβ8⁺ triple-positive cells in the lymphocyte population was evaluated by FACS and multiplied by the total number of lymphocytes to obtain the absolute number of CD8⁺Vα2⁺Vβ8⁺ indicator CTLs per organ.

The fluorescent dye CFSE from Molecular Probes (Eugene, OR) was used to label cells before transfer as previously described (19).

TCR transgenic CD8⁺ T cells were detected with anti-mouse CD8α-TRI Ab from Caltag (Burlingame, CA), anti-mouse Vβ8.1/8.2-FITC, and anti-mouse Vα2-PE Abs (PharMingen, San Diego, CA). CFSE-labeled CD4⁺ and CD8⁺ T cells were stained with anti-mouse CD4-TRI and anti-mouse CD8-TRI (both from Caltag), respectively.

Assessment of the primary footpad swelling reaction and of DTH/footpad infiltrating CTL

LCMV-WE (200 PFU) in 30 μl of MEM supplemented with 2% FCS and GP33 (3 μg/ml) or NP52 (1 mg/ml) in 30 μl of balanced salts solution was injected into both hind footpads. The footpad thickness was measured with a spring-loaded caliper (Kroeplein, Schluchtern, Hessen, Germany). Values were taken as the mean of both hind footpads (20, 21).

CTL assay and peptides

Virus-specific cytotoxic T cells were assayed as described previously (22). Briefly, single-cell suspensions were prepared from the spleens and lymph nodes of mice at the indicated time points and used directly in a ⁵¹Cr release assay. Target cells were either GP33-coated EL4 or vaccinia-WR-infected MC57 cells (multiplicity of infection of 3 for 2 h). The LCMV peptide GP33 and the VSV peptide NP52–59 were purchased from Neosystem Laboratoire (Strasbourg, France).

Ab detection

VSV-NJ and VSV-IND neutralizing Abs were determined in a neutralization assay as described previously (23). IgG was determined by its resistance to reduction by β-ME. The difference between total Ig and IgG represents IgM. LCMV-NP-specific Abs were measured by ELISA using the following steps as described previously (24): 1) coating with baculovirus-derived LCMV-NP, 2) blocking with 2% BSA in PBS, 3) 10-fold prediluted mouse serum was titrated in 3-fold dilutions over 12 steps, 4) IgM- or IgG-specific horseradish peroxidase-labeled goat anti-mouse Abs (0.5 μg/ml; Southern Biotechnology Associates, Birmingham, AL), 5) substrate 2,2′-azinobis-3-ethylbenzthiazolazine sulfonic acid (Roche, Mannheim, Germany) and H₂O₂ (Fluka Buchs, Switzerland). Ab titers were determined as the serum dilutions yielding an absorption (OD₄₀₅) of twice background levels.

Diabetes induction and assessment of blood glucose levels

For the induction of an autoimmune diabetes, RIP-GP transgenic mice were infected i.v. with 200 PFU of LCMV-WE. This elicits diabetes in RIP-GP transgenic but not in C57BL/6 mice (25) (see also Results). Glucose levels in the blood were measured using the Haemo-Glucotest color reaction sticks and were quantified with the reflection photometer Reflolux II (Roche) according to the manufacturer’s instructions.

Immunohistology

Freshly removed organs were immersed in HBSS and snap-frozen in liquid nitrogen. Tissue sections of 5-μm thickness were cut in a cryostat, placed on siliconized glass slides, air-dried, fixed with acetone for 10 min, and stored at −70°C. For the staining of lymphocyte differentiation markers, rehydrated sections were incubated with primary rat mAbs against CD4 and CD8 (YTS 191 and YTS 169) (26). Insulin was detected using affinity-purified guinea pig primary Abs (Dako, Glostrup, Denmark). Primary Abs were revealed by sequential incubation with either goat anti-rat Ig Abs (Tago, Burlingame, CA) and alkaline phosphatase-labeled donkey anti-goat Ig Abs or alkaline phosphatase-labeled rabbit anti-guinea pig Ig Abs and alkaline phosphatase-labeled goat anti-rabbit Ig Abs (Jackson ImmunoResearch Laboratories, West Grove, PA). Dilutions of the affinity-purified secondary Abs were made in Tris-buffered saline containing 5% normal mouse serum. Alkaline phosphatase was visualized using naphthol AS-BI (6-bromo-2-hydroxy-3-naphtholic acid-2-methoxy anilide) phosphate and new fuchsin as substrate. Endogenous alkaline phosphatase was blocked by levamisole. Color reactions were performed at room temperatures for 15 min with reagents from Sigma (St. Louis, MO). Sections were counterstained with hemalum, and coverslips were mounted with glycerol and gelatin.

FTY720 administration

FTY720 was obtained from Novartis Pharmaceuticals (Basel, Switzerland) and was dissolved in distilled water. A dose of 0.3 mg/kg was given daily by gavage in a volume of 100 μl/10 g body weight.

Statistical analysis

We performed repeated measures ANOVA with two between factors (group and trial) and one within factor (time point or E:T cell ratio, respectively). In experiments with two groups, post-hoc comparisons at different time points were performed using unpaired t tests with Bonferroni correction. In experiments with three groups, the Bonferroni-Dunn test was used for pairwise between-group comparisons. Between-group comparisons on different days were performed using a two-way ANOVA with post-hoc Bonferroni-Dunn test and an additional Bonferroni correction for tests on various days. Cell counts were log-transformed to achieve an approximate normal distribution and compared using a two-way ANOVA (factors trial and group) with Bonferroni correction for testing of multiple organs. The figures show the data of only one representative experiment, while two or three comparable sets of experiments were used for statistical analysis as stated in the figure legends.

Reduced primary swelling reaction after LCMV infection

Infection of C57BL/6 mice with 200 PFU of LCMV into the hind footpad causes a primary swelling reaction starting on day 6 and peaking around days 7–9. The first peak of the swelling reaction from days 6 to 8 is caused by CD8⁺ cells, whereas the following lesser swelling from days 9 to 14 is mainly CD4⁺ T cell dependent (21). This simple in vivo readout offers a reliable assessment of overall T cell-mediated immune responses. Immunosuppression by any mechanism modulating this response is readily revealed (27). One group of mice was treated daily with 0.3 mg/kg FTY720 orally starting 1 day before infection, and the control group was left untreated. FTY720 treatment significantly reduced the swelling during the entire period (p = 0.001; Fig. 1⇓). This suggested that the early CD8⁺ as well as the CD4⁺ effector T cell activity were impaired at least as assessed locally in the footpad.

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FIGURE 1.

Impairment of the primary footpad swelling reaction by FTY720. C57BL/6 mice were either treated from day −1 until day 15 daily with 0.3 mg/kg FTY720 orally (▪) or left untreated (□; control). On day 0 they were immunized with 200 PFU of LCMV-WE diluted in a volume of 30 μl of MEM/2% FCS into the hind footpad. Footpad swelling was monitored as described in Materials and Methods. Each symbol represents a group of three mice. One representative of two statistically analyzed experiments is shown. Values indicate the mean ± SD. ∗, p < 0.005.

Unimpaired induction and expansion of cytotoxic T cells

To evaluate potential effects of FTY720 on T cell induction and expansion (28, 29) vs T cell effector function in the footpad, the following experiments were performed. Mice were immunized with 200 PFU of LCMV-WE i.v., and CTL activity was measured at its peak on day 8. Because a difference in the distribution of naive T cells had already been shown (9), not only splenocytes but also lymph node cells were tested as effectors in a ⁵¹Cr release assay (Fig. 2⇓). An obvious decrease in cytolytic activity in the spleen (p = 0.004; Fig. 2⇓A) after FTY720 treatment contrasted with an increase in cytotoxic activity in peripheral lymph nodes (p = 0.01; Fig. 2⇓B). This finding rendered a general reduction in the cytotoxic T cell population unlikely. Cells from pooled popliteal, inguinal, mesenteric, para-aortic, axillary, and submandibular lymph nodes and spleen yielded the same cytotoxic activity in treated and untreated mice (p = 0.74; Fig. 2⇓C). The same experiment was repeated with vaccinia-WR with similar results (Fig. 2⇓, D–F). FTY720-treated mice exhibited lower cytotoxic activity in the spleen (p = 0.0001) and moderately more cytotoxicity in lymph nodes (p = 0.01), but activity in pooled secondary lymphoid organs was comparable to that in untreated control mice (p = 0.29). Despite an only moderate difference in lymph nodes (Fig. 2⇓, B and E), statistical analysis of more than one comparable experiment resulted in overall significantly higher activity in lymph nodes of FTY720-treated mice. This finding should be viewed in the context of additional data presented below. Absolute numbers of lymphocytes found in pooled secondary lymphoid organs were about the same as those in controls (not shown). Taken together, these data show that a dose of 0.3 mg/kg FTY720 did not interfere significantly with the induction or expansion of specific cytotoxic T cells, but FTY720 apparently changed the distribution pattern of specific activated CD8⁺ effector T cells in a manner similar to that found for naive lymphocytes (9). To exclude effects of FTY720 on viral replication, mice were infected with 200 PFU of LCMV i.v. Four days later viral titers were measured in the spleen and inguinal lymph nodes and were comparable in treated and untreated mice (data not shown).

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FIGURE 2.

Unimpaired induction and expansion of cytotoxic T cells under FTY720 treatment. A–C, C57BL/6 mice were either treated with 0.3 mg/kg FTY720 orally daily from day −1 until the end of the experiment (▪) or left untreated (□; control). After immunization with 200 PFU of LCMV-WE i.v. on day 0, mice were sacrificed on day 8, and cells from spleen and lymph nodes were tested in a primary in vitro cytotoxicity assay. Specific cytotoxic activity against GP33-labeled EL-4 target cells was measured for spleen cells (A), for inguinal lymph node cells (B), and for pooled cells from popliteal, inguinal, mesenteric, para-aortal, axillary, and submandibular lymph nodes and spleen (C). Each symbol represents a group of three mice. One representative of two statistically analyzed experiments is shown. Spontaneous release was always <20%. Values indicate the mean ± SD. D–F, Mice were treated as described in A–C, but were immunized on day 0 with 2 × 10⁶ PFU of vaccinia-WR i.v. On day 6 mice were sacrificed, and spleen and lymph node cells were tested in a primary in vitro cytotoxicity assay against vaccinia-WR-infected MC57 target cells (D, spleen cells; E, pooled lymph node cells; F, lymph node and spleen cells pooled). One representative of two or three statistically analyzed experiments is shown. Spontaneous release was always <20%. Values indicate the mean ± SD.

Induction of lymphocytopenia and its reversibility

In vitro studies had suggested that specific apoptotic death of T cells was responsible for the immunosuppressive properties of FTY720 (7, 8, 30). Apoptosis might have been expected to reduce the cytotoxic effector population, but the results of overall unimpaired induction of CTL responses did not readily support this idea. In addition, previous studies showed a transient lymphocytopenia after a single dose of FTY720 (3, 9), but did not evaluate recovery due to newly generated lymphocytes after FTY720 treatment was stopped. To address this question, naive spleen cells were labeled with the vital dye CFSE (19) and adoptively transferred i.v. to naive recipients. The recipients were then treated either with a single dose of 0.3 mg/kg FTY720 or with the same dose for 7 consecutive days. The numbers of transferred CD4⁺ (Fig. 3⇓A) and CD8⁺ T cells (Fig. 3⇓B) in the peripheral blood were measured by FACS and compared with those in untreated control mice. A marked drop in both populations was observed a few hours after a single dose of 0.3 mg/kg FTY720; this drop was more severe after repetitive treatments. Within 7 or 21 days after treatment was stopped, respectively, transferred CD4⁺ and CD8⁺ T cells in the blood had recovered levels within 10% of the untreated control values. The same recovery was found for B lymphocytes (data not shown). Additional experiments had shown that the cell numbers slowly but continuously recovered during the time span of 7 and 21 days, respectively (data not shown). Recovery due to proliferation could be excluded, because proliferation and the resulting CFSE dilution would have been easily detected by alteration of the fluorescence intensities (19). These results indicate that the recovery of circulating lymphocytes after transient FTY720 application is not due to newly generated cells, suggesting that FTY720 probably causes sequestration of lymphocytes rather than significantly affecting their viability.

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FIGURE 3.

Induction of lymphocytopenia by FTY720 and its reversibility. On day −1, 3 × 10⁷ naive CFSE-labeled splenocytes were adoptively transferred i.v. into naive syngeneic C57BL/6 recipients. The recipients were then treated with a single dose of 0.3 mg/kg FTY720 orally on day 0 (□), treated with the same dose daily from day 0 until day 6 (▴), or left untreated (control = 100%). The numbers of transferred, CFSE-positive circulating CD4⁺ (A) and CD8⁺ (B) T cells per microliter of blood were measured by FACS analysis; staining of transferred cells was performed as described in Materials and Methods. Gated living white cells were analyzed for CFSE⁺CD4⁺ (A) and CFSE⁺CD8⁺ (B) double-positive cells, respectively. The values are expressed as a percentage of the mean of the control group. Each group consisted of four animals. One representative experiment of two is shown. Values indicate the mean ± SD.

Impaired circulation of specific CD8⁺ cytotoxic effector T cells

The obvious discrepancy between the reduction of the primary swelling reaction and the overall efficient induction and expansion of cytotoxic T cells required analyses of the circulation properties of specific effector T cells (31). To follow specific effector T cells in the blood of mice, 10⁴ indicator spleen cells from mice with a transgenic TCR specific for D^b and LCMV-GP33 (318-mouse) were transferred to naive recipients that were infected i.v. with 200 PFU of LCMV-WE. It has previously been shown that this number of transferred cells replaces about 50% of the endogenous GP33-specific CD8⁺ T cells without changing the overall response against LCMV (32, 33). The number of specific circulating transgenic TCR⁺ CD8⁺ T cells per microliter of blood was measured by FACS analysis. FTY720 treatment reduced the number of specific circulating CD8⁺ T cells by more than a factor of 10 (Fig. 4⇓).

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FIGURE 4.

Impaired circulation of CD8⁺ cytotoxic T cells. C57BL/6 mice were either treated from day −1 until day 15 daily with 0.3 mg/kg FTY720 orally (▪) or left untreated (□; control) and infected with 200 PFU of LCMV-WE i.v. on day 0. The mice had received 10⁴ 318-tgTCR Vα2⁺Vβ8⁺ spleen cells specific for D^b and LCMV-GP33 i.v. on day −2. tgTCR⁺ (Vα2⁺Vβ8⁺) CD8⁺ T cells in the blood were measured at the time points indicated. One representative experiment of three is shown. Values indicate the mean ± SD.

Comparable numbers, but changed distribution pattern, of specific CD8⁺ cytotoxic effector T cells

Comparing the above finding with the data from Fig. 2⇑, it was rather unlikely that a reduced population of specific CD8⁺ cytotoxic effector T cells was the underlying reason for their reduced circulation in the peripheral blood (Fig. 4⇑). To corroborate this conclusion and to further analyze the effect of FTY720 on the distribution pattern of specific CD8⁺ cytotoxic effector T cells, mice were treated as described in Fig. 4⇑ and were sacrificed at the peak of the CTL response on day 8 after infection. Transgenic TCR⁺ CD8⁺ T cells were counted in blood, spleen, inguinal lymph node, and pooled secondary lymphoid organs (spleen pooled with popliteal, inguinal, mesenteric, para-aortal, axillary, and submandibular lymph nodes) as described in Materials and Methods (Fig. 5⇓). Comparable numbers of indicator CTL were present in pooled secondary lymphoid organs of FTY720-treated and untreated mice (p = 0.52), while drug treatment significantly reduced the number of CTL circulating in the blood (p < 0.0001). Similar to the behavior of naive lymphocytes (9) and compatible with the data shown in Fig. 2⇑, A and D, fewer specific CD8⁺ cytotoxic effector T cells could be found in the spleen of FTY720-treated mice compared with untreated control mice (p = 0.0003), while their number was increased in the inguinal lymph node (p = 0.0001). This result taken together with the data from Figs. 1⇑, 2⇑, and 4⇑ suggested that FTY720 impairs the circulation of effector CD8⁺ T cells, apparently by sequestering them in secondary lymphoid organs, mainly peripheral lymph nodes, without impairing their induction or expansion.

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FIGURE 5.

Changed distribution pattern of specific cytotoxic T cells. C57BL/6 mice were either treated from day −1 until day 8 daily with 0.3 mg/kg FTY720 orally (▪) or left untreated (□) and were infected with 200 PFU of LCMV-WE i.v. on day 0. The mice had received 10⁴ 318-tgTCR Vα2⁺Vβ8⁺ spleen cells specific for D^b and LCMV-GP33 i.v. on day −2. Mice were sacrificed at day 8 after infection, and tgTCR⁺ (Vα2⁺Vβ8⁺) CD8⁺ T cells were counted in blood, spleen, inguinal lymph node, and pooled secondary lymphoid organs (pooled spleen and popliteal, inguinal, mesenteric, para-aortal, axillary, and submandibular lymph nodes) as described in Materials and Methods. One representative of two statistically analyzed experiments is shown. Values indicate the mean ± SD.

Reduced LCMV-specific delayed-type hypersensitivity reaction (DTH)

To study the recruitment of effector CD8⁺ T cells to s.c. sites with Ag, mice were immunized with 200 PFU of LCMV-WE i.v. and 13 days later were treated with the immunodominant D^b nonapeptide, gp33–41 of LCMV-glycoprotein (GP33). This peptide elicits an exclusively CD8⁺ T cell-mediated DTH in LCMV-infected, but not in naïve, C57BL/6 mice (20). Footpad injection of the unrelated immunodominant D^b binding octapeptide np52–59 of VSV-nucleoprotein (NP52), which causes a similar reaction in VSV-infected mice (20), did not lead to any footpad swelling (data not shown). Daily treatment with 0.3 mg/kg FTY720 starting 1 day before infection significantly reduced the CD8⁺ T cell-mediated DTH against GP33 (FTY720 day 1 vs control, p = 0.0001; Fig. 6⇓). To avoid an effect of FTY720 on early phases of the anti-LCMV response, a third group of mice was only treated with FTY720 from day 5 after infection onward. Consistent with earlier findings (4, 6), the evoked DTH reaction was reduced (FTY720 day 5 vs control, p = 0.0001) to the same extent as had been found in mice treated from day −1 onward (FTY720 day 1 vs FTY720 day 5, p = 0.51; Fig. 6⇓).

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FIGURE 6.

Reduced DTH reaction under FTY720 treatment. C57BL/6 mice were either treated daily with 0.3 mg/kg FTY720 orally starting on day −1 (▪) and on day 5 after infection (▴), respectively, until the end of the experiment or were left untreated (□ control). Thirteen days after infection with 200 PFU of LCMV-WE i.v. on day 0, 30 μl of GP33 solution in a concentration of 3 μg/ml dissolved in BSS was injected into both hind footpads. Footpad swelling was measured as described in Materials and Methods. Each symbol represents a group of five mice. One representative of two statistically analyzed experiments is shown. Values indicate the mean ± SD. ∗, p < 0.0016 (for ▪ vs □ and for ▴ vs □ only, but not for ▪ vs ▴).

Prevention of a CD8⁺ T cell-mediated autoimmune diabetes

Interference with the homing of effector T cells to peripheral lesions may offer new therapeutic possibilities against immunopathological or autoimmune disease. To examine this, we analyzed a transgenic mouse expressing the glycoprotein of LCMV in the islets of the pancreas (RIP-GP) (25). In this model the transgenic GP is ignored by CD8⁺ T cells in the naive mouse, but after infection with LCMV, CD8⁺ T cell-mediated autoimmune diabetes develops because potentially autoreactive T cells have not been deleted in the thymus or the periphery, and effector CTLs are efficiently induced following LCMV infection of secondary lymphoid organs (25). Consequent perforin-dependent killing by CTLs leads to islet destruction and hyperglycemia (34). In a total of three experiments, daily treatment with 0.3 mg/kg FTY720 starting 1 day before infection with 200 PFU of LCMV-WE i.v. prevented autoimmune diabetes in six and delayed it in three (onset of diabetes >2 days after the last respective control mouse) of 12 mice. All 11 control mice developed diabetes early during the observation period of 20 days (Fig. 7⇓). Similar results were also obtained in RIP-GP mice after LCMV infection into the footpad (data not shown).

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FIGURE 7.

Prevention of CD8⁺ T cell-mediated autoimmune diabetes. RIP-GP transgenic mice were either treated with 0.3 mg/kg FTY720 orally daily from day −1 until day 20 (▪) or left untreated (□). On day 0 they were immunized with 200 PFU of LCMV-WE i.v. Glucose levels in the blood were measured as described in Materials and Methods. Each symbol represents an individual mouse. One representative experiment of three is shown.

Reduced tissue infiltration by CD8⁺ cytotoxic effector T cells

To assess whether the suppressive effects of FTY720 treatment on diabetes development and DTH were due to reduced tissue infiltration, the following experiments were performed. RIP-GP mice were treated as described above (Fig. 7⇑) and sacrificed on days 9 and 20 after infection for histological examination. On day 9 control mice developed diabetes and exhibited considerable CD8⁺ infiltrates (Fig. 8⇓B), while only very few CD8⁺ T cells were visible around the pancreatic islets of FTY720-treated mice (Fig. 8⇓F). Corresponding to the degree of diabetes they had developed (Fig. 7⇑), a similar reduction of CD8⁺ cytotoxic T cells could also be seen 20 days after infection (Fig. 8⇓H, normoglycemic mouse) compared with dense aggregates in untreated control mice (Fig. 8⇓D). Parallel to increased CD8⁺ T cell infiltration staining for insulin decreased (Fig. 8⇓, A, C, E, and G).

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FIGURE 8.

Reduced tissue infiltration under FTY720 treatment. A–H, RIP-GP transgenic mice were treated with 0.3 mg/kg FTY720 orally daily from day −1 until day 9 (E and F) and until day 20, respectively (G and H) or were left untreated (A–D). On day 0 they were immunized with 200 PFU of LCMV-WE i.v. On day 9 (A, B, E, and F) and day 20 (C, D, G, and H) mice were sacrificed, and the pancreas was prepared for histological examination. Sections were stained for insulin (A, E, C, and G) and CD8⁺ T cells (B, F, D, and H) as described in Materials and Methods. One representative sample of four to six per time point and group collected from two different sets of experiments is shown. Original magnification, ×100. I–X, C57BL/6 mice were treated daily with 0.3 mg/kg FTY720 orally starting on day −1 until the end of the experiment (M–P and U–X) or were left untreated (I–L and Q–T). After infection with 200 PFU of LCMV-WE i.v. on day 0, 30 μl of LCMV-GP33 (3 μg/ml; I–R, U, and V) or of VSV-NP52 (1 mg/ml; S, T, W, and X) solution dissolved in BSS was injected into both hind footpads on day 13. Mice were sacrificed 1 h (I and M), 3 h (J, N, Q, and U), 8 h (K, O, R, and V), and 18 h (L, P) later, and feet were prepared for histological analysis. Sections were stained for CD8⁺ (J–P, S, T, W, and X) and CD4⁺ T cells as described in Materials and Methods. One representative sample of five or six per group and time point collected from two different sets of experiments is shown. Original magnification, ×75.

C57BL/6 mice were treated with 0.3 mg/kg FTY720 orally daily starting 1 day before infection with 200 PFU of LCMV-WE i.v. on day 0. On day 13 LCMV-GP33 was injected into the hind footpad, and the mice were sacrificed 1, 3, 8, and 18 h later. Histological sections of the footpad were analyzed for infiltrating CD8⁺ and CD4⁺ T cells. One hour after peptide injection, some perivascular CD8⁺ T cells could be found in the s.c. tissue of untreated control mice (Fig. 8⇑I), while no infiltration was seen in FTY720-treated mice (Fig. 8⇑M). Two hours later massive aggregates of perivascular CD8⁺ T cells and a beginning infiltration of approximate sweat glands (Fig. 8⇑J) were considerably reduced by FTY720 (Fig. 8⇑N). Eight hours after peptide challenge FTY720 treatment reduced the extensive infiltration of sweat glands (Fig. 8⇑, K and O) and muscle (not shown). Another 10 h later, decreasing numbers of CD8⁺ T cells had caused considerable destruction and edema in untreated mice (Fig. 8⇑L), while in the footpad of FTY720-treated mice CD8⁺ T cells had caused correspondingly less damage (Fig. 8⇑P). Only very few to no CD4⁺ T cells were found in the samples above (Fig. 8⇑, Q, R, U, and V, and data not shown) independent of FTY720 treatment. Present mainly at later time points, CD4⁺ T cells were probably attracted by the local inflammatory process initiated by CTLs. Footpad injection of the D^b binding immunodominant VSV peptide NP52 did not cause either swelling (not shown) or considerable infiltration of CD8⁺ (Fig. 8⇑, S, T, W, and X) or CD4⁺ T cells (data not shown) independent of FTY720 treatment. Similarly, GP33 did not cause infiltration in naive C57BL/6 mice (data not shown). This suggests that the infiltrates shown in Fig. 8⇑, I—P, consist in the great majority of specific activated cytotoxic T cells, which are responsible for the swelling reaction (Fig. 6⇑) and tissue damage (Fig. 8⇑L).

These results parallel the data of Yanagawa and collaborators (10) demonstrating reduced lymphocytic allograft infiltration under FTY720 treatment. Together with efficient virus control by CTL in the spleen (data not shown), where LCMV replication initially predominates (35), the present data suggested that FTY720 impaired emigration/homing of effector CTL to the LCMV-GP-expressing pancreatic islets and to the s.c. connective tissue of the footpad and thereby reduced immunopathology.

Unimpaired Ab responses

To study the effects of FTY720 on the humoral immune response, mice were immunized with 200 PFU of LCMV-WE i.v. The anti-LCMV-NP IgM and IgG response is strictly Th cell dependent. Compared with untreated control mice, no reduction in Ab production could be observed in animals treated with 0.3 mg/kg FTY720 daily starting 1 day before infection until the end of the experiment (Fig. 9⇓A). Other mice were infected with 2 × 10⁶ PFU of VSV-IND i.v. The neutralizing IgM response against VSV is Th cell independent (TI-1), whereas the switch to IgG production is largely dependent on T cell help. FTY720 treatment as described above did not measurably change Ab responses (Fig. 9⇓B) or the survival of infected mice (data not shown). This indicated that B cell induction and Ab secretion as well as the capacity of CD4⁺ T cells to deliver help to B cells remained intact under treatment with FTY720.

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FIGURE 9.

Normal humoral immune responses during FTY720 treatment. A, C57BL/6 mice were either treated from day −1 until the end of the experiment daily with 0.3 mg/kg FTY720 orally (▪) or were left untreated (□). On day 0 they were immunized with 200 PFU of LCMV-WE i.v. Serum samples were taken at the indicated time points, and Ab titers against LCMV-NP were measured by ELISA as described in Materials and Methods. Each symbol represents a group of three mice. One representative experiment of two is shown. Values indicate the mean ± SD. B, The experimental groups were set up as described in A. On day 0 the mice were immunized with 2 × 10⁶ PFU of VSV-IND i.v. Serum samples were taken at the indicated time points, and Ab titers were measured in a neutralization assay. One representative experiment of two is shown. Values indicate the mean ± SD.

Unimpaired immunological memory

To address the question of whether the temporarily sequestered lymphocytes remained functional after FTY720 treatment, the following experiments evaluated effects of FTY720 on immunological memory. Mice were immunized with 2 × 10⁶ PFU of VSV-IND i.v. on day 0 and treated with 0.3 mg/kg FTY720 from days 23–48. No reduction of neutralizing IgG Ab titer was observed during the entire observation period up to day 120 (Fig. 10⇓A), indicating that memory B cells and plasma cell function were unimpaired (23, 36). The same results were obtained for memory anti-LCMV-NP Ab titers (data not shown). To evaluate effects of FTY720 on recall responses, mice were immunized with VSV-NJ, transiently treated with FTY720 from day 11 until day 17, and then challenged on day 40 with 2 × 10⁶ PFU of the homologous virus (Fig. 10⇓B). The response was the same as found in immunized but untreated control mice. Mice that had been left unprimed and were now immunized with the booster dose, mounted the expected primary Ab response with IgM (data not shown) and low delayed levels of IgG (Fig. 10⇓B).

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FIGURE 10.

Transitory FTY720 treatment does not affect the immunological memory. A, C57BL/6 mice were immunized with 2 × 10⁶ PFU of VSV-IND i.v. on day 0 and either treated from day 23 until day 48 daily with 0.3 mg/kg FTY720 orally (▴) or left untreated (▵). Serum samples were taken at the indicated time points, and Ab titers were determined in a neutralization assay. Each symbol represents the mean of five (treated) and two (untreated) mice. One of two similar experiments is shown. Values indicate the mean ± SD. B and C, C57BL/6 mice were immunized with 2 × 10⁶ PFU of VSV-NJ i.v. on day 0. They were then either treated from day 11 until day 17 daily with 0.3 mg/kg FTY720 orally (▪ and ▴) or left untreated (□ and ▵). On day 40 the mice were boosted with either 2 × 10⁶ PFU of VSV-NJ (▪ and □) i.v. (B) or 10⁷ PFU of UV inactivated VSV-IND (▴ and ▵) i.v. (C). Additionally, three naive mice were immunized with the same protocols on day 40 (• and ○). Serum samples were taken at the indicated time points and tested in a neutralization assay against VSV-NJ (▪, □, and •) or VSV-IND (▴, ▵, and ○). All mice mounted the expected IgM (data not shown) and IgG (B and C) responses to primary infection with VSV-NJ. In B only IgG responses to booster infection with VSV-NJ are shown, whereas the response to challenge infection with UV-inactivated VSV-IND in C consisted mainly of IgM. The symbols represent the mean of three or four mice. One of two similar experiments is shown. Values indicate the mean ± SD. D, C57BL/6 mice were immunized with 200 PFU of LCMV-WE i.v. They were then either treated with 0.3 mg/kg FTY720 orally daily from day 36 until day 48 (▪) or left untreated (□; control). On day 70 a group of sex- and age-matched naive mice was added to the experiment (▧; naive), and all mice were infected with 2 × 10⁶ PFU of LCMV-WE i.v. Four days later mice were sacrificed, and LCMV titers in the organs were determined. The bars represent the mean of three or four mice. Dotted lines indicate the detection limit. One of two similar experiments is shown. Values indicate the mean ± SD.

To evaluate T-helper cell memory, we profited from the fact that VSV-IND and VSV-NJ induce the same specificity of T-helper cells (37, 38). On the other hand it is well established that Abs raised against VSV-NJ do not neutralize VSV-IND and vice versa (39); by definition they are serologically distinct. Therefore, immunological memory to VSV-NJ at the CD4⁺ T cell level will augment neutralizing IgM responses against a very low level VSV-IND immunization. Because memory B cells and circulating neutralizing Abs induced by VSV-NJ will not neutralize the second virus (VSV-IND), the B cell response against the second infection will not be impaired. Mice were immunized with 2 × 10⁶ PFU VSV-NJ i.v. After treatment with FTY720 from day 11 until day 17 the mice were boosted with 10⁷ PFU of UV-inactivated VSV-IND (Fig. 10⇑C). The anti-VSV-IND IgM response was 20 times stronger in mice with primed cross-reactive T cell help compared with that in naive mice; it was equal in FTY720-treated and untreated preimmunized mice, indicating unimpaired Th cell memory in FTY720-treated mice.

Cytotoxic memory T cell responses were evaluated as follows. LCMV-specific CTL are able to clear LCMV early after challenge infection (40). After infection with 200 PFU of LCMV-WE i.v., mice were treated with 0.3 mg/kg FTY720 from day 36 until day 48 and were challenged with 10⁶ PFU LCMV-WE i.v. on day 70. Four days later mice were sacrificed, and viral titers were determined in blood, spleen, liver, kidney, and lung (Fig. 10⇑D). A temporary 12-day treatment with 0.3 mg/kg FTY720 had no effect on later efficient viral clearance. Nonimmune mice had high viral titers in all organs tested. Taken together, these results show that FTY720 had no significant effect on memory T and B cell responses.