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* Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101; and Departments of
Pathology and
Neurology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
IL-2R
are each characterized by a rapid lethal autoimmune
lymphoproliferative disorder that complicates their use in studies
aimed at investigating the role of these cytokines and receptors for
immune responses in vivo. We have previously characterized a novel
transgenic (Tg) mouse on the IL-2R-/- genetic
background (Tg-/- mice) that lacks autoimmune disease but
still contains peripheral T cells that are nonresponsive to IL-2 and
IL-15. In the present study, these mice were used to investigate the
extent by which IL-2 and IL-15 are essential for T cell immunity in
vivo. Tg-/- mice generated near normal primary and
secondary Ab responses to OVA, readily mounted first and second set
allogeneic skin graft rejection responses, and developed primary and
recall CD8 T cell responses to vaccinia virus. However,
Tg-/- mice generated a slightly lower level of IgG2a Abs
to OVA, exhibited a somewhat delayed first set skin graft rejection
response with lower allo-specific CTL, and developed a significantly
lower number of IFN-
-producing vaccinia-specific CD8+ T
cells. Thus, although T effector function is somewhat impaired, T cell
immunity is largely functional in the absence of IL-2- and
IL-15-induced signaling through IL-2R. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunits that impose
ligand binding specificity, and shared and (common -chain;
c)3
subunits that contribute to ligand binding and deliver intracellular
signals (1, 2). As a consequence, IL-2R and IL-15R share
common signaling elements and functional activities, including
induction of T cell proliferation, stimulation of lymphokine-activated
killer cells, and activation of NK cells. However, distinct functional
activities have been attributed to each of these cytokine receptors.
For example, in vitro IL-2 is most efficient in programming activated T
cells toward apoptosis or activation-induced cell death, which is
thought to contribute to restoring T cell homeostasis after an immune
response (3, 4, 5). In vivo, the absence of IL-2 function
leads to extensive T cell lymphoproliferation and autoimmunity
(6, 7, 8, 9, 10). In contrast, IL-15 functions as a survival factor
for T cells and inhibits IL-2-dependent apoptosis (11, 12). In contrast to IL-2/IL-2R deficiency, the absence of IL-15
in vivo does not result in autoimmunity, but reduced numbers of
memory-phenotypic CD8+ T cells as well as a
failure to produce NK and NK T cells (13, 14).
Although IL-2 is the dominant cytokine for T cell responses in vitro,
the requirement for IL-2 during in vivo immune responses is still not
precisely defined. A direct assessment of the necessity for IL-2 might
be expected to emerge by evaluating T cell-mediated immunity in
IL-2-deficient mice. In this regard, IL-2-/-
mice often developed immune responses in vivo, but sometimes disparate
results were noted even when the same agent was used to elicit the
immune response (4, 6, 15, 16, 17, 18, 19). One serious complication
in evaluating immunocompetency in IL-2-, IL-2R-, and
IL-2R-deficient mice is that their rapid and extreme autoimmunity
provides an abnormal environment that might potentially augment or
suppress a specific antigenic challenge. Furthermore, recent studies
have raised the possibility that the initial T cell proliferation to Ag
challenge in vivo is dependent upon IL-15 (20). These
experiments also showed that expression of IL-2 and IL-2R was
detected from activated T cells only after 5–7 cell divisions,
suggesting that IL-2 may function primarily during a later phase of the
T cell response. A direct evaluation of immune responses by IL-15- and
IL-15R-deficient mice showed that these mice mounted primary and memory
virus-specific CD8-dependent T cell responses, albeit at somewhat lower
levels than generated in wild-type mice (21, 22). Whether
this response was due to IL-2 was not evaluated. Therefore, regardless
of some of their nonoverlapping activities, it is still unclear whether
IL-2 and IL-15 are mandatory cytokines for T cell-dependent immune
responses in vivo.
An ideal scenario to investigate whether IL-2 and IL-15 are essential,
but function redundantly, for T cell immunity is to immunologically
challenge IL-2R-/- mice that are deficient
in responding to both cytokines without complications associated with
severe systemic autoimmunity. Our laboratory has developed such a
mouse model by expressing IL-2R as a transgene in the thymus of
IL-2R-/- mice (23, 24).
Peripheral T cells in these mice (designated transgenic
(Tg)-/-) remained nonresponsive to IL-2 and
IL-15, yet Tg-/- mice did not exhibit
autoimmunity and uncontrolled expansion of activated peripheral T cells
(23, 24). More recent studies demonstrated that
autoimmunity was prevented in Tg-/- mice by
restoring production of
CD4+CD25+ T regulatory
cells (25). Thus, the essential function of IL-2 in vivo
encompasses its role in the production of this suppressor cell rather
than an intrinsic T cell defect in IL-2-dependent contraction of
autoreactive peripheral T cells.
Previous in vitro studies demonstrated that T cells from
Tg-/- mice readily proliferated (
50% of
normal control mice) after stimulation by anti-CD3
(23, 24). This proliferation was substantially inhibited
by blocking costimulation through CD40 ligand and CD28, but
unaffected by the addition of mAbs to IL-2 and c
(24). Both CD4 and CD8 Tg-/- T
cells were induced by anti-CD3 to undergo 3–4 divisions, but
thereafter, these T cells became hyporesponsive to IL-4 and IL-7, in
addition to their nonresponsiveness to IL-2 and IL-15, preventing
further growth. Anti-CD3-activated Tg-/- T
cells were also markedly deficient in their capacity to secrete IFN-
and produce CTL (24). Thus, in vitro IL-2R is essential
for long-term T cell growth and programming of some T cell
effector activities. In some instances, these in vitro defects were
partially overcome by culturing the anti-CD3-stimulated
Tg-/- T cells with exogenous IL-4,
demonstrating the existence of a partial compensatory pathway in the
absence of IL-2R function (24). In the current study,
we have used Tg-/- mice to revisit the issue of
whether typical CD4 and CD8 T cell-dependent primary and secondary
immune responses in vivo require signaling through IL-2R. The
results indicate that IL-2R, and hence IL-2 and IL-15, are
dispensable for T cell immune responses, although the magnitude and
effector activity of the response is sometimes impaired.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thymic targeted Tg wild-type IL-2R expressed in
IL-2R-/- mice on the C57BL/6 genetic
background (designated Tg-/- in this report)
have been previously described (23, 24). C57BL/6 and
BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME).
In most experiments, IL-2R+/- littermate mice
were used as controls, but occasionally
IL-2R+/+ C57BL/6 mice were the controls. Where
indicated, mice were injected i.p. with 100 µg of anti-CD3
(145-2C11) in PBS.
Induction and assay of Ab responses
Mice were immunized with 100 µg OVA i.p. in CFA. Six weeks
later the primed mice received a second injection consisting of 10 µg
OVA in IFA. Resulting antisera were tested for anti-OVA Ab by
ELISA. Wells were coated with OVA (2 µg/ml in 0.1 M carbonate, pH
9.5), blocked with 0.25% gelatin in PBS, incubated with a serial
dilution of antiserum for 2 h, washed with PBS containing 0.05%
Tween 20, and then incubated with HRP-conjugated mAbs (BD PharMingen,
San Diego, CA) to mouse-IgG1, IgG2a, or 
L chains. After washing,
color was developed by incubation with tetramethylbenzidine
substrate reagent set (BD PharMingen), the OD was read at 450 minus 570
nm correction, and Ab titers were determined by the reciprocal of the
dilution that yielded 50% maximal response.
Allogeneic skin grafts
Grafts from the tail skin of donor BALB/c mice were prepared after mice were sacrificed and tails swabbed with 70% ethanol. Briefly, skin was incised on the dorsal side, down the length of the tail, and skin was peeled and removed in one piece. The internal surface was sterilely placed on PBS-moistened filter paper, carefully flattened and spread, and then cut in horizontal pieces (10–12/tail). Recipient mice were anesthetized using Avertin and the left thorax shaved and swabbed with 70% ethanol. Graft beds were prepared and tail skin was orthotopically placed on the lateral thorax. Mice were plaster cast and observed daily. After 7 days, casts were removed and 6–8 h later, all grafts were scored. Only grafts that appeared vascularized and healthy were followed and scored daily. Grafts were scored as rejected following complete epithelial breakdown (100% necrotic).
Virus infection
To generate primary responses to viral infection, mice were challenged i.p. with 5 x 107 PFU of recombinant vaccinia virus (vJS510) expressing the immunodominant H-2b-restricted CD8+ T cell epitope from the spike protein (S510) of the JHM strain of mouse hepatitis virus (JHMV) (26). Secondary responses were induced by immunizing mice i.p. with 5 x 107 PFU of recombinant Sindbis virus (SINJS510) expressing the same JHMV S510 epitope and challenging 4 wk later with 5 x 107 PFU of vJS510. SINJS510 was generated by inserting hybridized oligonucleotides encoding the S510 epitope (5'-CTAGATGTGTTCTCTTTGGAATGGGCCCCATTTGTGA-3' and 5'-CTAGTCACAAATGGGGCCCATTCCAAAGAGAACACAT-3') into the XbaI site of the parent SIN vector pTE3'2J (27). Stocks of SINJS510 virus were generated via transfection of BHK-21 cells with infectious in vitro transcribed mRNA from XhoI linearized pSINJS510 and titered using BHK-21 cells (27). Mice were sacrificed at indicated time points postchallenge and single-cell suspensions were prepared from the inguinal lymph nodes and spleen.
In vitro T cell functional assays
Splenocytes from virus-challenged mice were assayed for cytolytic activity either directly ex vivo or following 6 days of culture. For in vitro cultures, 1 x 108 splenocytes were placed in a T75 tissue culture flask with 40 ml of RPMI 1640 medium supplemented with 2 mM glutamine, 25 µg/ml gentamicin, 1 mM sodium pyruvate, 5 x 10-5 M 2-ME, nonessential amino acids, 10% FCS, and 1 µM S510 peptide. CTL assays were performed as described previously (28). Briefly, EL-4 (H-2b) and P815 (H-2d) target cells were labeled with 100 µCi Na51CrO4 (NEN, Boston, MA) at 37°C for 1 h and washed three times before use. For virus-specific CTL, EL-4 target (1 x 104/well) and effector cells were transferred to 96-well plates in the presence or absence of 1 µM S510 peptide. For ex vivo allospecific or redirected CTL, lymph node effector cells were directly incubated with P815 targets (1 x 104/well). After 4 h of incubation, supernatant (100 µl) was removed and specific 51Cr release was determined. Specific lysis was defined as 100 x (experimental release - spontaneous release)/(detergent release - spontaneous release). Maximum spontaneous release values were <10% of the total detergent release values in all experiments.
S510 peptide (CSLWNGPHL) used in functional and proliferative assays is derived from the immundominant JHMV S510 CD8+ T cell epitope in H-2b mice (26, 29).
In vitro T cell proliferative responses were preformed as previously described (23). In brief, unfractionated lymph node cells (1 x 105/well) or spleen cells (2 x 105/well) from skin-grafted or virus-infected mice were cultured with PMA (10 ng/ml), IL-2 (50 U/ml), IL-4 (10 ng/ml), IL-7 (10 ng/ml), or anti-CD3 (1 µg/ml) for 48 h. [3H]Thymidine was added during the last 6 h of culture.
ELISPOT assays were performed as described previously
(28). Briefly, 3.3-fold dilutions of spleen cells from
virus-challenged mice were plated in triplicate and stimulated with
irradiated (25 Gy) splenocytes from naive wild-type mice (4 x
105/well) in the presence or absence of 1 µM
S510 peptide. Cells were incubated for 40 h at 37°C in the
presence of plate-bound anti-IFN- mAb (10 µg/ml, R4.6A2; BD
PharMingen). Captured IFN- was detected by an 8-h incubation at
4°C with biotinylated anti-IFN- mAb (5 µg/ml, XMG1.2; BD
PharMingen), followed by consecutive incubations with
streptavidin/peroxidase and diaminobenzidine as a substrate
(Sigma-Aldrich, St. Louis, MO). Spots from two mononuclear cell
dilutions (n = 6) were counted for each sample.
FACS analysis
CyChrome-anti-CD8 and FITC-IFN- were obtained from BD
PharMingen. The Db/510 tetramer has been
described previously (30). For detection of intracellular
IFN-, spleen cells were stimulated for 6 h in RPMI supplemented
with 10% FCS and Goglistop (BD PharMingen) in the presence or absence
of 1 µM S510 peptide. Cells were first stained with anti-CD8 mAb,
permeabilized with Cytofix/Cytoperm reagents (BD PharMingen) as per the
manufacturer’s recommendations, and then stained with anti-IFN-
mAb. FACS analysis was performed using a BD Biosciences FACScan and
CellQuest software (BD Biosciences, Mountain View, CA). Typically
100,000 viable cells were analyzed per sample based on forward vs side
scatter gating.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several distinct agents were used to challenge the
Tg-/- mice to evaluate their capacity to mount
T cell-dependent immune responses. Initially, the induction of primary
and secondary Ab responses to OVA was assessed. When compared with
control mice, Tg-/- mice readily elicited
primary and secondary responses to OVA (Fig. 1
). Because induction of IFN- by
Tg-/- T cells is impaired in vitro, we also
examined the level of OVA-specific IgG1 and IgG2a in the serum after a
secondary challenge (Fig. 2). Although
there was some individual variation, most Tg-/-
mice produced lower levels of IgG2a, but this difference was not
statistically significant (p = 0.09) when
compared with titers from control mice.
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Control and Tg-/- mice received a fully
allogeneic BALB/c skin graft. Both groups of mice readily rejected the
skin graft, although the rejection by the Tg-/-
mice was significantly (p = 0.008) delayed
(Fig. 3A). Mean rejection time
was 9.8 ± 0.6 days for control littermate mice and 12.2 ±
0.4 days for Tg-/- mice. Ex vivo analysis of
CTL activity to H-2d alloantigens by T cells from
lymph nodes of mice that received BALB/c skin graft 7 days previously
indicated that alloantigen-specific CTL were detected from control, but
not Tg-/-, mice (Fig. 3B). No CTL
activity was detected against either H-2b EL4 or
H-2d P815 targets when the T cells were obtained
from untreated normal mice (data not shown). By 14 days after receiving
the skin graft, no ex vivo CTL activity was observed by lymph node T
cells from either control or Tg-/- mice (data
not shown). It is also important to point out that lymph node T cells
from the grafted control mice, but not untreated normal or grafted
Tg-/- mice, readily proliferated when cultured
in exogenous IL-2, further confirming the absence of IL-2R function
by the Tg-/- T cells (Fig. 3C).
Furthermore, when these mice received a second graft, they exhibit no
difficulty in mounting a second set rejection response, which was
slightly more rapid in the Tg-/- mice (Fig. 3A). Histological assessment indicated that extensive and
comparable infiltrates were observed just before rejection in control
and Tg-/- transplant recipients (data not
shown). Therefore, despite the absence of detectable cytolytic activity
and proliferation to IL-2, Tg-/- mice still
successfully rejected the allogeneic skin graft, perhaps by CD4
effector cells, as reported by others (31, 32, 33, 34).
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The failure to induce detectable allospecific CTL from
Tg-/- T cells in vivo and in vitro
(23) pointed to the importance of IL-2R in the
generation of cytotoxic effector cells. However, in vitro IL-4
redundantly functioned to promote CTL (23), indicating
that there is not a strict requirement for IL-2R to generate CTL in
culture. To test whether a redundant pathway might function in vivo,
control and Tg-/- mice were injected with
anti-CD3. Two days later, lymph nodes were examined ex vivo for
their ability to lyse P815 targets due to redirected lysis (Fig. 4). We reasoned that the use of
anti-CD3 would afford the greatest chance to induce a potential
redundant activity for CTL in the Tg-/- mice.
Under these conditions, a similar level of CTL activity was observed
for control and Tg-/- T cells. No CTL activity
was detected when normal untreated T cells were used as the effector
cells (data not shown). Again, exogenous IL-2 induced proliferation of
lymph node cells from control, but not Tg-/-
mice (23) (data not shown). Thus, this experiment
demonstrates that Tg-/- T cells indeed have the
capacity to develop into CTL in vivo.
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To investigate primary antiviral responses, control and
Tg-/- mice were infected with a recombinant
vaccinia virus (vJS510) expressing the immunodominant JHMV S510 CTL
epitope, thereby permitting a direct assessment of virus-specific
CD8+ T cell expansion via staining with the
DbS510 tetramer. To assess secondary responses,
mice were primed with a recombinant Sindbis virus expressing the JMHV
S510 epitope (SINJS510) and then challenged 4 wk later with vJS510.
Following both primary and secondary viral challenges,
Db S510
tetramer+CD8+ T cells were
detected in both spleen and inguinal nodes of control and
Tg-/- mice (Fig. 5, A and C).
Primary responses in Tg-/- mice were comparable
to those seen in control littermate mice. Frequencies of virus-specific
CD8+ T cells during secondary responses were
somewhat lower in Tg-/- mice (Fig. 5, B and C); however, this difference was not
statistically significant (p = 0.21).
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).
IL-2-dependent responses were not generated by
Tg-/- T cells in the presence of PMA,
confirming the absence of signaling through the IL-2R by
Tg-/- peripheral T cells. By contrast, T cells
from Tg-/- mice generated substantial, but
somewhat reduced, responses to both PMA/IL-4 and anti-CD3 when
compared with T cells from littermate controls, confirming that T cells
from Tg-/- mice were not anergic. Furthermore,
nearly comparable CD8+ T cell responses to the
S510 peptide as well as CD4+ T cell responses to
UV-inactivated vJS510 indicated that virus-specific T cells from
Tg-/- mice and control mice display similar
capacities for expansion in direct response to specific Ags in vitro.
However, exogenous IL-2 enhanced these proliferative responses to S510
peptide and UV-inactivated vJS510 by control, but not
Tg-/- T cells, when assayed days 5 and 7 post
primary infection and day 5 post secondary infection (data not shown).
These data provide additional evidence for the IL-2 nonresponsiveness
of Tg-/- T cells when assayed directly ex
vivo.
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. Direct ex vivo
cytolytic activity was not detected using splenocytes from either
control or Tg-/- mice during either primary or
secondary responses to vJS510 (data not shown). By contrast, cytolytic
activity was readily detected from both Tg-/-
and control splenocytes after culture of unfractionated spleen cells
for 6 days in the presence of the S510 peptide (Fig. 7A). Although 60% fewer
viable cells were recovered from the cultures of
Tg-/- splenocytes, cytolytic activity from both
groups of cells was similar based on comparable numbers of live
effector cells in the CTL assay.
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ELISPOT 7 days post
primary infection with vJS510 revealed a striking difference in the
number of IFN- secreting cells between Tg-/-
mice and littermate controls, i.e., 36 vs 241 per
106 splenocytes, respectively. As the frequencies
of
DbS510+CD8+
T cells were almost identical during primary responses, this result
suggested that Tg-/- T cells are impaired in
generating IFN--secreting CD8+ effector cells.
Similar results were obtained 5 days post secondary vJS510 infection. A
total of 3- to 4-fold fewer
IFN-+CD8+ T cells were
detected via intracellular IFN- staining in splenocytes from
Tg-/- mice compared with control mice (Fig. 7
, B and C). Even though spleens of
Tg-/- mice contain 20% fewer
CD8+ T cells during the secondary response
compared with control mice (Fig. 5A), the differences in the
frequency of IFN--producing cells was not accounted for by the
slightly lower fraction of DbS510
tetramer+/CD8+ T cells in
the spleen. Collectively, these data indicate that
Tg-/- mice developed primary and secondary T
cell responses to vaccinia virus, but with an impaired capacity of
these T cells to secrete IFN-. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signaling for extended T proliferation and differentiation into
effector T cells (23, 24). Therefore, we were somewhat
surprised at the extent of immunocompetence of
Tg-/- mice when challenged in vivo to develop T
cell-dependent immune responses. This included induction of Ab
responses to OVA that depend on CD4 T cell help, responses to vaccinia
virus that depend on CD8 T cells, and rejection of allogeneic skin that
uses both CD4 and CD8 T effector cells. The magnitude of both the
primary and secondary responses to each of these in vivo immune
responses by Tg-/- mice was largely comparable
to that generated by control mice. Although the responses by the
Tg-/- mice were often slightly lower, these
differences were usually not statistically significant. Therefore,
these findings demonstrate that IL-2R signaling, and hence IL-2 and
IL-15, are largely dispensable to mount primary and secondary T
cell-dependent immune responses in vivo.
It is extremely unlikely that the immune responses developed by the
Tg-/- mice were due to some residual IL-2R
activity by peripheral T cells. We have extensively documented that
responses by Tg-/- T cells in the presence of
IL-2 and IL-15 are exceptionally impaired in vitro (23, 24). Confirming these results, T cells from
Tg-/- mice that were first stimulated in vivo
remained essentially nonresponsive to IL-2 when assayed directly ex
vivo. Furthermore, as discussed more fully below, some T effector cell
functions that were impaired in vitro were also diminished in vivo
following immunological challenge of Tg-/-
mice, directly demonstrating impaired IL-2R function in
vivo.
Past studies have shown that immune responses were generated in
IL-2-deficient mice (4, 6, 15, 16, 17, 18, 19, 35). Ag-specific T cell
proliferation by TCR Tg
IL-2-/-CD8+ T cells was
induced in vivo by influenza nucleoprotein peptide (19).
IL-2-/- mice were shown to be competent to
reject allogeneic islet allografts (16). Furthermore,
primary and secondary CTL were generated to lymphocytic
choriomeningitis (LCMV) and vaccinia virus infections as well as Ab
responses to vesicular stomatitis virus (VSV) infection in
IL-2-/- mice (15). However, other
studies have reported impaired immune responses in
IL-2-/- and IL-2R-/-
mice (7, 17, 35, 36). For example, the magnitude of the
primary response to LCMV was dramatically reduced in
IL-2-/- mice although these T cells
developed cytolytic activity (17).
IL-2R-/- mice failed to mount immune
responses to VSV and LCMV (7). More recently,
IL-2-/- mice were shown to be resistant to
infection with avirulent Salmonella serovar
Choleraesuis while IL-2R-/- mice were
susceptible to infection (35). The immunocompetence of
Tg-/- mice strongly suggests that failed
responses in IL-2 or IL-2R nonresponsive animals results from
complications of autoimmunity, which become superimposed on evaluations
of specific immune responses. Nevertheless, the development of primary
and secondary immune responses to a variety of stimuli in
autoimmune-free Tg-/- mice rules out the
possibility that immune responses were induced in
IL-2-/- mice solely as a consequence of
autoimmunity. In any case, the severe autoimmunity associated with
IL-2/IL-2R deficiency represents a serious limitation in most studies
that use these mice to evaluate T cell immunity.
Two recent studies have investigated antiviral CD8-dependent T cell responses in IL-15- and IL-15R-deficient mice (21, 22). Both primary and secondary responses to VSV and LCMV were readily generated, although the responses to VSV were suboptimal especially after 5–6 days when compared with control mice. Thus, IL-15 is also not mandatory for an immune response to virus. However, these and other studies revealed that the key role for IL-15 resides in the long-term maintenance of the memory pool (21, 22, 37, 38, 39, 40, 41). Although we have not investigated the Ag-specific memory pool, our findings are largely in agreement with these reports. In contrast to the studies described above, an obvious difference in studying T cell immunity in Tg-/- mice is that the contribution of IL-2 and IL-15 to a particular response is assessed simultaneously. Our study indicates that neither cytokine is essential for diverse T cell responses in vivo in the time frame of 1–6 wk for primary and recall responses. This conclusion seems at odds with recent work that suggests that IL-15 may be a key cytokine that promotes the initial growth of Ag-activated T cells in vivo, especially CD8+ T cells (20). In these experiments, T cell proliferation was assessed by adoptively transferred T cells into lethally irradiated allogeneic recipients early after transfer. Lethal irradiation induces host cytokine production and a lymphopenic environment, two conditions that favor IL-15-induced CD8 T cell growth in vivo. This setting is much different from both our and other studies (21, 22) where immune responses were investigated in unirradiated nonlymphopenic mice. We contend that these distinct experimental conditions represent a plausible explanation for markedly different requirements for IL-15 in the initial response to Ag.
T cell responses in vitro are highly dependent upon IL-2R signaling,
including the induction of IFN- secretion (24).
Although IL-2R was largely dispensable for T cell immunity in vivo,
optimal IFN- secretion still required IL-2R. Slightly diminished
class switching to IgG2a, which in part is dependent upon IFN-, and
a substantially lower frequency of IFN--producing virus-specific
CD8+ T cells was observed for immunized
Tg-/- mice. The impairment in IFN-
production to vaccinia virus was not accounted for by a lower precursor
frequency of S510-specific CD8+ T cells. Other
studies have noted impaired IFN- production in the absence of IL-2R
signaling in vitro and in vivo (6, 17, 42). Thus, IL-2
appears to be an important cytokine in vivo for efficient production of
IFN- by T effector cells.
Primary stimulation of Tg-/- T cells in vitro
resulted in markedly impaired production of CTL due to reduced
induction of perforin and nearly absent induction of granzyme B
(24). By contrast, induction of CTL activity in vivo was
only sometimes impaired in Tg-/- mice when
compared with control mice, notably to alloantigens after priming with
skin grafts. In contrast, when Tg-/- mice were
challenged with anti-CD3, they readily produced CTL when assayed ex
vivo. Other studies point to a variable requirement for IL-2 in vivo
for induction of CTL (15, 17, 19). These findings suggest
that other cytokines or cell interactions may bypass the requirement
for IL-2R signaling, and such a signal may be limiting in some
immune responses in vivo. In this regard, it is pertinent that
exogenous IL-4 in vitro effectively overcame the impairment of CTL
development by Tg-/- T cells (24).
However, in very limited experiments to date, we have been unsuccessful
in implicating IL-4 as a redundant cytokine for production of CTL by
Tg-/- mice in vivo (data not shown).
Furthermore, LCMV-specific CTL and protective viral responses were
induced in vivo in IL-2/IL-4 double knockout mice (18).
Thus, it is likely that IL-4-independent signals may substitute for the
absence of IL-2R for production of CTL. It is also interesting to
note that although no direct ex vivo cytolytic activity was detected
after primary or secondary challenge with S510-modified vaccinia virus
from either control or Tg-/- mice, upon in
vitro culture, cytolytic activity was readily induced from both type of
T cells, although many fewer cells were recovered from the cultures
containing Tg-/- T cells. We believe that the
signals necessary for S510-specific CTL were received during the in
vivo priming, perhaps due to an innate immune response, because direct
in vitro stimulation of Tg-/- T cells by
anti-CD3 or alloantigen resulted in negligible induction of CTL
(24).
The immunocompetence of Tg-/- mice demonstrates
that there must be compensatory pathways that permit relatively
efficient T cell immunity in the absence of IL-2 and IL-15. However,
the magnitude of some of the responses in Tg-/-
mice was sometimes slightly diminished and effector activity, such as
IFN- production, was impaired, indicating that such compensatory
mechanisms do not fully substitute for failed IL-2 and IL-15 signaling
in vivo. Therefore, there are likely some situations where effective
immunity may still depend on IL-2 and/or IL-15. In this regard, it is
important to point out that when IL-15-/- mice
were infected with the highly virulent neurotropic WR vaccinia virus
strain, all mice died 4–9 days after infection (14). This
result markedly contrasts with the nonlethal infection by attenuated
recombinant vJS510 vaccinia virus that nevertheless stimulated strong T
cell responses in both control and Tg-/-
mice.
The main unanswered question and the subject for future investigation
from this work is what accounts for the capacity to induce T cell
responses in vivo in the absence of IL-2R-dependent IL-2 and IL-15
signaling. With respect to c-dependent cytokines, it is
unlikely, as discussed above, that IL-4 is solely responsible for these
responses. Studies of alloantigen-induced T cell proliferation in
adoptively transferred lethally irradiated mice demonstrated a role for
IL-15 in driving the initial proliferative response (20).
The one very attractive feature of this result is that IL-15 is
produced by nonlymphoid cells and is readily available to stimulate T
cells when present at a low frequency, as expected at the initiation of
the response. Perhaps nonlymphoid-derived IL-7, whose signaling is very
similar to that induced by IL-2 and IL-15, might substitute for lack of
IL-2R in a nonmanipulated host in a manner analogous to that
recently reported for IL-7 and IL-15 in the regulation of
CD8+ memory T cells (40, 41, 43).
Another candidate might be IL-21, which is secreted by activated
CD4+ T cells and uses a receptor related to
IL-2R and IL-4R (44). Alternatively, there are many
surface molecules on T lymphocytes whose function in T cell
immunobiology are very poorly understood and may serve to promote
immune responses in an IL-2/IL-15-independent fashion. Thus, although
the IL-2R is essential for in vitro T cell responses, there are
undoubtedly alternative pathways to induce T cell-dependent immune
responses in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas R. Malek, Department of Microbiology and Immunology, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136. E-mail address: tmalek{at}med.miami.edu
3 Abbreviations used in this paper: c, common -chain; LCMV, lymphocytic choriomeningitis virus; VSV, vesicular stomatitis virus; Tg, transgenic.
Received for publication August 23, 2002. Accepted for publication October 28, 2002.
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