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*
Institute of Experimental Immunology and
Laboratory for Special Techniques, Department of Pathology, University Hospital, Zurich, Switzerland; and
Novartis Pharma AG, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro studies on FTY720 suggested that selective apoptotic cell death of lymphocytes, mainly of the T cell subset, was a possible mechanism of action (7, 8). However, the concentrations needed for lymphocyte apoptosis in vitro were several orders of magnitude greater than the blood concentrations in rats given FTY720 at the therapeutic dose of 1 mg/kg (9). In vivo FTY720 has been reported to shift the distribution of lymphocytes from spleen to Peyer’s patches and peripheral lymph nodes and accelerate homing of naive lymphocytes to these organs (9). Another study suggested that low blood lymphocyte counts may reflect reduced emigration of effector cells to the periphery (10), but they could not distinguish effects on the circulation of naive mature lymphocytes from specific effector cells. Whether FTY720 interferes with the generation of specific effector T cells has not been shown. Therefore, the mechanisms responsible for the reported reduced graft infiltration under FTY720 treatment (10) remain unclear. In the present study we analyzed the immunosuppressive mechanisms of 0.3 mg/kg FTY720 daily as monotherapy on T cell responses to lymphocytic choriomeningitis virus (LCMV) and B cell responses to vesicular stomatitis virus (VSV) in the mouse. This dose reveals biological effects in vivo and may be given to transplantation patients in combination therapy with other immunosuppressants. An in vivo half-life around or below 24 h (data not shown) excludes the possibility of major drug accumulation.
Primary infection with LCMV, a noncytopathic RNA virus, is controlled
almost exclusively by CTL (11, 12). Immunopathology in
this infection is also T cell-mediated (11, 13, 14), and
ßTCR transgenic CD8+ T cells are available
against a major CTL target LCMV glycoprotein peptide 33–41 (GP33); so
LCMV infection offers a good model to assess beneficial or detrimental
effects of immunomodulation. On the other hand, VSV, a highly
cytopathogenic RNA virus, was used to study the effects of FTY720 on B
cell responses. An early neutralizing Ab response against VSV is
required to prevent lethal encephalitis, and VSV infection induces
lifelong protective B cell memory (15, 16).
We now show that FTY720 does not impair efficient priming of T and B cell/Ab responses, but inhibits immune responses by preventing the homing of effector T cells to lesions in peripheral organs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice (H-2b), 318 TCR transgenic mice, expressing a TCR specific for LCMV-GP33 together with H-2Db, 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
Viruses. LCMV-WE was originally obtained from Dr. F. Lehmann-Grube (Heinrich Pette Institute, Hamburg, Germany) and was grown on L929 cells. VSV Indiana (VSV-IND; Mudd-Summers isolate) and VSV New Jersey (VSV-NJ; Pringle isolate) were obtained from Dr. D. Kolakovsky (University of Geneva, Geneva, Switzerland) and were grown on BHK21 cells. Vaccinia-WR was generously provided by Dr. B. Moss (National Institutes of Health, Bethesda, MD) and was propagated on BSC40 cells.
Infectious LCMV in the organs was detected by focus-forming assays performed as previously described (17).
For UV inactivation a small volume of high titer VSV preparation was exposed as a thin layer in a petri dish to a UV lamp (Philips, 15W) for 3 min at a distance of 8 centimeters (18).
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+V2+Vß8+
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+V2+Vß8+
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 CD8-TRI Ab from Caltag (Burlingame, CA),
anti-mouse Vß8.1/8.2-FITC, and anti-mouse V2-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 ß-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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. 2A) after FTY720 treatment contrasted with an increase in
cytotoxic activity in peripheral lymph nodes (p
= 0.01; Fig. 2B). 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. 2C). 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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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. 3A) and
CD8+ T cells (Fig. 3B) 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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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
Db 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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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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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
Db 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 Db 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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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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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. 8B), while only very few
CD8+ T cells were visible around the pancreatic
islets of FTY720-treated mice (Fig. 8F). 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. 8H, normoglycemic mouse) compared
with dense aggregates in untreated control mice (Fig. 8D).
Parallel to increased CD8+ T cell infiltration
staining for insulin decreased (Fig. 8, A, C,
E, and G).
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I), while
no infiltration was seen in FTY720-treated mice (Fig. 8M).
Two hours later massive aggregates of perivascular
CD8+ T cells and a beginning infiltration of
approximate sweat glands (Fig. 8J) were considerably reduced
by FTY720 (Fig. 8N). 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. 8L), while in the footpad of FTY720-treated mice
CD8+ T cells had caused correspondingly less
damage (Fig. 8P). 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 Db 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. 8L). 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. 9A). Other mice were infected
with 2 x 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. 9B) 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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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 x 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. 10A), 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 x
106 PFU of the homologous virus (Fig. 10B). 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. 10B).
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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. 10D). 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of immunopathological conditions, including allograft rejection, chronic hepatitis caused by hepatitis B and C virus, and some autoimmune diseases are at least partially T cell mediated and directed against peripherally located tissues. Because these disease processes require emigration of effector T cells, they may be efficiently treated with FTY720. The findings here, that FTY720 impairs DTH or induction of a T cell-mediated autoimmune diabetes, support this idea. As revealed by results from several experimental allotransplantation model situations (5, 6, 10), FTY720 synergizes efficiently with other immunomodulating agents that directly suppress T cell activation; this indicates that blockade at two different steps of the T cell response, i.e., induction-proliferation and emigration-homing to the periphery, results in very potent immunosuppression.
A risk of general immunosuppression may be increased susceptibility to infection and lymphomas. FTY720 impaired neither humoral immune responses nor immunological memory at both the T and B cell levels and did not suppress the generation of primary virus-specific CTL in lymph nodes. Again, this may offer new possibilities of combining immunomodulators with distinct mechanisms of action that overall interfere less with immunity to infection. In addition, these data suggest that lymphocyte effector functions in lymphoid organs are largely unimpaired under FTY720 treatment and that susceptibility to lymphomas should not increase.
Our findings here are compatible and extend recent results (9) demonstrating accelerated homing of naive lymphocytes to secondary lymphoid organs under FTY720 treatment. In addition, they also fit findings on reduced allograft infiltration by T cells and delayed allograft rejection in FTY720-treated rats (10).
Of course, other possibilities to interfere with the circulation-emigration step of effector T cells are being evaluated, e.g. with Abs against adhesion molecules or homing receptors including ICAM-1, LFA-1, or selectins. The mechanism by which FTY720 modulates lymphocyte emigration and/or homing to peripheral lesions remains to be investigated. Modification of cell adhesion molecules, such as selectins and integrins, chemokines, and other attractants, could be envisaged. A combination of Abs to the classical adhesion molecules L-selectin and CD11a and CD49d integrins has been shown to effectively inhibit accelerated trafficking of adoptively transferred naive lymphocytes to lymph nodes of FTY720-treated animals (9). Convincing evidence for FTY720-mediated modulation of the adhesion process or particularly for modification of the above molecules is however missing. Selectins and integrins are responsible for the homing of naive lymphocytes to secondary lymphoid organs as well as for the delivery of effector T cells to peripheral sites of infection. Therefore, modification of these molecules as a single mechanism could hardly explain the one-sided sequestration of effector T cells to secondary lymphoid organs and reduced infiltration of peripheral tissue at the same time. Preliminary findings suggest that FTY720 may function through G protein-coupled receptors on lymphocytes, possibly chemokine receptors (41) binding to ligands that are expressed in secondary lymphoid organs and normally attract preferentially naive lymphocytes. FTY720 treatment may thereby result in a changed homing pattern of naive as well as activated lymphocytes.
Taken together the present data show that FTY720 impairs circulation and homing of effector T cells to peripheral lesions without affecting the induction and expansion of immune responses in secondary lymphoid organs. Such a mechanism of action has not been observed with any other immunosuppressive drug. Therefore, FTY720 may offer new intervention possibilities to treat organ graft rejection and ongoing autoimmune and immunopathological diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rolf M. Zinkernagel, Institute of Experimental Immunology Schmelzbergstrasse 12, 8091 Zurich, Switzerland.
3 Abbreviations used in this paper: FTY720, 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride; LCMV, lymphocytic choriomeningitis virus; CFSE, 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester; LCMV-WE, LCMV strain WE; LCMV-NP, nucleoprotein of LCMV; LCMV-GP33/GP33, peptide 33–41 of the LCMV glycoprotein; vaccinia-WR, vaccinia virus strain WR; VSV, vesicular stomatitis virus; VSV-IND, VSV strain Indiana; VSV-NJ, VSV strain New Jersey; VSV-NP52/NP52, peptide 52–59 of the VSV nucleoprotein; tg, transgenic; DTH, delayed-type hypersensitivity reaction.
Received for publication May 7, 1999. Accepted for publication March 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß interferon receptor-deficient mice. J. Virol. 69:2153.[Abstract]
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 M. Mehling, V. Brinkmann, J. Antel, A. Bar-Or, N. Goebels, C. Vedrine, C. Kristofic, J. Kuhle, R.L.P. Lindberg, and L. Kappos FTY720 therapy exerts differential effects on T cell subsets in multiple sclerosis Neurology, October 14, 2008; 71(16): 1261 - 1267. [Abstract] [Full Text] [PDF]
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 D. Marsolais, B. Hahm, K. H. Edelmann, K. B. Walsh, M. Guerrero, Y. Hatta, Y. Kawaoka, E. Roberts, M. B. A. Oldstone, and H. Rosen Local Not Systemic Modulation of Dendritic Cell S1P Receptors in Lung Blunts Virus-Specific Immune Responses to Influenza Mol. Pharmacol., September 1, 2008; 74(3): 896 - 903. [Abstract] [Full Text] [PDF]
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 C. Daniel, N. Sartory, N. Zahn, G. Geisslinger, H. H. Radeke, and J. M. Stein FTY720 Ameliorates Th1-Mediated Colitis in Mice by Directly Affecting the Functional Activity of CD4+CD25+ Regulatory T Cells J. Immunol., February 15, 2007; 178(4): 2458 - 2468. [Abstract] [Full Text] [PDF]
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 K. Kabashima, N. M. Haynes, Y. Xu, S. L. Nutt, M. L. Allende, R. L. Proia, and J. G. Cyster Plasma cell S1P1 expression determines secondary lymphoid organ retention versus bone marrow tropism J. Exp. Med., November 27, 2006; 203(12): 2683 - 2690. [Abstract] [Full Text] [PDF]
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 L. Kappos, J. Antel, G. Comi, X. Montalban, P. O'Connor, C. H. Polman, T. Haas, A. A. Korn, G. Karlsson, E. W. Radue, et al. Oral Fingolimod (FTY720) for Relapsing Multiple Sclerosis N. Engl. J. Med., September 14, 2006; 355(11): 1124 - 1140. [Abstract] [Full Text] [PDF]
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 R. Schmouder, D. Serra, Y. Wang, J. M. Kovarik, J. DiMarco, T. L. Hunt, and M.-C. Bastien FTY720: Placebo-Controlled Study of the Effect on Cardiac Rate and Rhythm in Healthy Subjects. J. Clin. Pharmacol., August 1, 2006; 46(8): 895 - 904. [Abstract] [Full Text] [PDF]
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 R. Fujii, T. Kanai, Y. Nemoto, S. Makita, S. Oshima, R. Okamoto, K. Tsuchiya, T. Totsuka, and M. Watanabe FTY720 suppresses CD4+CD44highCD62L- effector memory T cell-mediated colitis Am J Physiol Gastrointest Liver Physiol, August 1, 2006; 291(2): G267 - G274. [Abstract] [Full Text] [PDF]
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 A. S. Awad, H. Ye, L. Huang, L. Li, F. W. Foss Jr., T. L. Macdonald, K. R. Lynch, and M. D. Okusa Selective sphingosine 1-phosphate 1 receptor activation reduces ischemia-reperfusion injury in mouse kidney Am J Physiol Renal Physiol, June 1, 2006; 290(6): F1516 - F1524. [Abstract] [Full Text] [PDF]
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Q. Zhang, Y. Chen, R. L. Fairchild, P. S. Heeger, and A. Valujskikh Lymphoid Sequestration of Alloreactive Memory CD4 T Cells Promotes Cardiac Allograft Survival J. Immunol., January 15, 2006; 176(2): 770 - 777. [Abstract] [Full Text] [PDF]
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A. Habicht, M. R. Clarkson, J. Yang, J. Henderson, V. Brinkmann, S. Fernandes, M. Jurewicz, X. Yuan, and M. H. Sayegh Novel Insights into the Mechanism of Action of FTY720 in a Transgenic Model of Allograft Rejection: Implications for Therapy of Chronic Rejection J. Immunol., January 1, 2006; 176(1): 36 - 42. [Abstract] [Full Text] [PDF]
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E. Sawicka, G. Dubois, G. Jarai, M. Edwards, M. Thomas, A. Nicholls, R. Albert, C. Newson, V. Brinkmann, and C. Walker The Sphingosine 1-Phosphate Receptor Agonist FTY720 Differentially Affects the Sequestration of CD4+/CD25+ T-Regulatory Cells and Enhances Their Functional Activity J. Immunol., December 15, 2005; 175(12): 7973 - 7980. [Abstract] [Full Text] [PDF]
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I. I. Singer, M. Tian, L. A. Wickham, J. Lin, S. S. Matheravidathu, M. J. Forrest, S. Mandala, and E. J. Quackenbush Sphingosine-1-Phosphate Agonists Increase Macrophage Homing, Lymphocyte Contacts, and Endothelial Junctional Complex Formation in Murine Lymph Nodes J. Immunol., December 1, 2005; 175(11): 7151 - 7161. [Abstract] [Full Text] [PDF]
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P. Bandhuvula, Y. Y. Tam, B. Oskouian, and J. D. Saba The Immune Modulator FTY720 Inhibits Sphingosine-1-phosphate Lyase Activity J. Biol. Chem., October 7, 2005; 280(40): 33697 - 33700. [Abstract] [Full Text] [PDF]
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N. Czeloth, G. Bernhardt, F. Hofmann, H. Genth, and R. Forster Sphingosine-1-Phosphate Mediates Migration of Mature Dendritic Cells J. Immunol., September 1, 2005; 175(5): 2960 - 2967. [Abstract] [Full Text] [PDF]
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 C. Halin, M. L. Scimone, R. Bonasio, J.-M. Gauguet, T. R. Mempel, E. Quackenbush, R. L. Proia, S. Mandala, and U. H. von Andrian The S1P-analog FTY720 differentially modulates T-cell homing via HEV: T-cell-expressed S1P1 amplifies integrin activation in peripheral lymph nodes but not in Peyer patches Blood, August 15, 2005; 106(4): 1314 - 1322. [Abstract] [Full Text] [PDF]
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 K. A. Vora, E. Nichols, G. Porter, Y. Cui, C. A. Keohane, R. Hajdu, J. Hale, W. Neway, D. Zaller, and S. Mandala Sphingosine 1-phosphate receptor agonist FTY720-phosphate causes marginal zone B cell displacement J. Leukoc. Biol., August 1, 2005; 78(2): 471 - 480. [Abstract] [Full Text] [PDF]
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X. Martinez, H. T. C. Kreuwel, W. L. Redmond, R. Trenney, K. Hunter, H. Rosen, N. Sarvetnick, L. S. Wicker, and L. A. Sherman CD8+ T Cell Tolerance in Nonobese Diabetic Mice Is Restored by Insulin-Dependent Diabetes Resistance Alleles J. Immunol., August 1, 2005; 175(3): 1677 - 1685. [Abstract] [Full Text] [PDF]
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C. G. Lo, Y. Xu, R. L. Proia, and J. G. Cyster Cyclical modulation of sphingosine-1-phosphate receptor 1 surface expression during lymphocyte recirculation and relationship to lymphoid organ transit J. Exp. Med., January 18, 2005; 201(2): 291 - 301. [Abstract] [Full Text] [PDF]
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S. Han, X. Zhang, G. Wang, H. Guan, G. Garcia, P. Li, L. Feng, and B. Zheng FTY720 suppresses humoral immunity by inhibiting germinal center reaction Blood, December 15, 2004; 104(13): 4129 - 4133. [Abstract] [Full Text] [PDF]
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C. A. Welsch, L. W. A. Roth, J. F. Goetschy, and N. R. Movva Genetic, Biochemical, and Transcriptional Responses of Saccharomyces cerevisiae to the Novel Immunomodulator FTY720 Largely Mimic Those of the Natural Sphingolipid Phytosphingosine J. Biol. Chem., August 27, 2004; 279(35): 36720 - 36731. [Abstract] [Full Text] [PDF]
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A. C. Yopp, S. Fu, S. M. Honig, G. J. Randolph, Y. Ding, N. R. Krieger, and J. S. Bromberg FTY720-Enhanced T Cell Homing Is Dependent on CCR2, CCR5, CCR7, and CXCR4: Evidence for Distinct Chemokine Compartments J. Immunol., July 15, 2004; 173(2): 855 - 865. [Abstract] [Full Text] [PDF]
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 U. Kunzendorf, E. Ziegler, and D. Kabelitz FTY720--the first compound of a new promising class of immunosuppressive drugs Nephrol. Dial. Transplant., July 1, 2004; 19(7): 1677 - 1681. [Full Text] [PDF]
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 K Mayer, F Birnbaum, T Reinhard, A Reis, S Braunstein, F Claas, and R Sundmacher FTY720 prolongs clear corneal allograft survival with a differential effect on different lymphocyte populations Br. J. Ophthalmol., July 1, 2004; 88(7): 915 - 919. [Abstract] [Full Text] [PDF]
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 J. M Kovarik, R. L Schmouder, D. Barilla, M. Buche, M. Rouilly, S. Berthier, Y. Wang, C. Van Saders, T. Mayer, and A. B Gottlieb FTY720 and Cyclosporine: Evaluation for a Pharmacokinetic Interaction Ann. Pharmacother., July 1, 2004; 38(7): 1153 - 1158. [Abstract] [Full Text] [PDF]
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T. Kimura, A. M. Boehmler, G. Seitz, S. Kuci, T. Wiesner, V. Brinkmann, L. Kanz, and R. Mohle The sphingosine 1-phosphate receptor agonist FTY720 supports CXCR4-dependent migration and bone marrow homing of human CD34+ progenitor cells Blood, June 15, 2004; 103(12): 4478 - 4486. [Abstract] [Full Text] [PDF]
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 M. Forrest, S.-Y. Sun, R. Hajdu, J. Bergstrom, D. Card, G. Doherty, J. Hale, C. Keohane, C. Meyers, J. Milligan, et al. Immune Cell Regulation and Cardiovascular Effects of Sphingosine 1-Phosphate Receptor Agonists in Rodents Are Mediated via Distinct Receptor Subtypes J. Pharmacol. Exp. Ther., May 1, 2004; 309(2): 758 - 768. [Abstract] [Full Text] [PDF]
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J. M. Kovarik, R. Schmouder, D. Barilla, G.-J. Riviere, Y. Wang, and T. Hunt Multiple-Dose FTY720: Tolerability, Pharmacokinetics, and Lymphocyte Responses in Healthy Subjects J. Clin. Pharmacol., May 1, 2004; 44(5): 532 - 537. [Abstract] [Full Text]
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E. Sawicka, C. Zuany-Amorim, C. Manlius, A. Trifilieff, V. Brinkmann, D. M. Kemeny, and C. Walker Inhibition of Th1- and Th2-Mediated Airway Inflammation by the Sphingosine 1-Phosphate Receptor Agonist FTY720 J. Immunol., December 1, 2003; 171(11): 6206 - 6214. [Abstract] [Full Text] [PDF]
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A. Billich, F. Bornancin, P. Devay, D. Mechtcheriakova, N. Urtz, and T. Baumruker Phosphorylation of the Immunomodulatory Drug FTY720 by Sphingosine Kinases J. Biol. Chem., November 28, 2003; 278(48): 47408 - 47415. [Abstract] [Full Text] [PDF]
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H. Rosen, C. Alfonso, C. D. Surh, and M. G. McHeyzer-Williams Rapid induction of medullary thymocyte phenotypic maturation and egress inhibition by nanomolar sphingosine 1-phosphate receptor agonist PNAS, September 16, 2003; 100(19): 10907 - 10912. [Abstract] [Full Text] [PDF]
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A. C. Yopp, G. J. Randolph, and J. S. Bromberg Leukotrienes, Sphingolipids, and Leukocyte Trafficking J. Immunol., July 1, 2003; 171(1): 5 - 10. [Full Text] [PDF]
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A. M. Woltman and C. van Kooten Functional modulation of dendritic cells to suppress adaptive immune responses J. Leukoc. Biol., April 1, 2003; 73(4): 428 - 441. [Abstract] [Full Text] [PDF]
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M. Fujino, N. Funeshima, Y. Kitazawa, H. Kimura, H. Amemiya, S. Suzuki, and X.-K. Li Amelioration of Experimental Autoimmune Encephalomyelitis in Lewis Rats by FTY720 Treatment J. Pharmacol. Exp. Ther., April 1, 2003; 305(1): 70 - 77. [Abstract] [Full Text]
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J. H. Xie, N. Nomura, S. L. Koprak, E. J. Quackenbush, M. J. Forrest, and H. Rosen Sphingosine-1-Phosphate Receptor Agonism Impairs the Efficiency of the Local Immune Response by Altering Trafficking of Naive and Antigen-Activated CD4+ T Cells J. Immunol., April 1, 2003; 170(7): 3662 - 3670. [Abstract] [Full Text] [PDF]
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V. Brinkmann, M. D. Davis, C. E. Heise, R. Albert, S. Cottens, R. Hof, C. Bruns, E. Prieschl, T. Baumruker, P. Hiestand, et al. The Immune Modulator FTY720 Targets Sphingosine 1-Phosphate Receptors J. Biol. Chem., June 7, 2002; 277(24): 21453 - 21457. [Abstract] [Full Text] [PDF]
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H. Li, G. M. L. Meno-Tetang, K. Chiba, N. Arima, P. Heining, and W. J. Jusko Pharmacokinetics and Cell Trafficking Dynamics of 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol Hydrochloride (FTY720) in Cynomolgus Monkeys after Single Oral and Intravenous Doses J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 519 - 526. [Abstract] [Full Text] [PDF]
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 K. Budde, R. L. Schmouder, R. Brunkhorst, B. Nashan, P. W. Lucker, T. Mayer, S. Choudhury, A. Skerjanec, G. Kraus, and H. H. Neumayer First Human Trial of FTY720, a Novel Immunomodulator, in Stable Renal Transplant Patients J. Am. Soc. Nephrol., April 1, 2002; 13(4): 1073 - 1083. [Abstract] [Full Text] [PDF]
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Y. Bai, J. Liu, Y. Wang, S. Honig, L. Qin, P. Boros, and J. S. Bromberg L-Selectin-Dependent Lymphoid Occupancy Is Required to Induce Alloantigen-Specific Tolerance J. Immunol., February 15, 2002; 168(4): 1579 - 1589. [Abstract] [Full Text] [PDF]
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G. Henning, L. Ohl, T. Junt, P. Reiterer, V. Brinkmann, H. Nakano, W. Hohenberger, M. Lipp, and R. Forster CC Chemokine Receptor 7-dependent and -independent Pathways for Lymphocyte Homing: Modulation by FTY720 J. Exp. Med., December 17, 2001; 194(12): 1875 - 1881. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) 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.
), 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.
). 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 x 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 (
). Serum samples were taken at the indicated time
points and tested in a neutralization assay against VSV-NJ (
; naive),
and all mice were infected with 2 x 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.