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Departments of
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Neurology and
Microbiology and Molecular Immunology, University of Southern California School of Medicine, Los Angeles, CA 90033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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by CD8+ T cells from IL-4-deficient mice was also
elevated, reflecting their enhanced activation. Thus, IL-4 appears to
limit the activation, expansion, and differentiation of
CD8+ T cells with high cytolytic
potential. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF-
, whereas Th2
cells characteristically secrete IL-4, IL-5, IL-6, IL-10, and IL-13.
Immune responses may be dominated by either a Th1- or Th2-type response
as cytokine secretion from one Th subset negatively regulates the other
1 .
The pathological significance of differential type 1 or type 2
activation has been demonstrated in various bacterial, fungal,
helminthic, and protozoan parasitic infections 2, 3, 4, 5 . On these models,
susceptibility studies combined with the depletion of cytokine subsets
suggested that Th1 responses protect the host from intracellular
pathogens, whereas Th2 responses provide protection against
extracellular microbes. The balance between type 1 and type 2 responses
also plays a role in protection against viral infections. Studies with
HIV 6 - and hepatitis B virus 7 -infected patients suggested that
predominant Th1 responses promote cell-mediated immunity and may be
associated with better control of viral infection and delayed onset of
symptomatic chronic disease. Diminished IFN- and IL-2 production
concomitant with increased IL-4, IL-10, and Ab production during HIV
infection in a subset of patients indicated that progression to AIDS
may be associated with a Th1 to Th2 switch. Cytokine production by
CD8+ T cells in HIV-infected patients with low CD4 T cell
counts supports a role for type 2 T cells in pathogenesis 8 . Further
evidence for detrimental Th2 responses is found in murine
retrovirus-induced immunodeficiency 9 . Therefore, an early Th1
response comprising antiviral CD8+ T cells may not only be
a critical component during the natural immune response to viral
infection, but also may prove to be an essential component of
prophylactic antiviral vaccines.
CD8+ T cells also have the potential to develop into functionally distinct type 1 or type 2 subsets with cytokine secretion patterns similar to CD4+ Th1 and Th2 subsets 10, 11, 12, 13 . However, the relative importance of cytokines during CD8+ T cell activation and regulation is not as well defined as for the CD4+ T cell compartment. IL-4 plays a key role in suppressing type 1 cytokines and CTL function of activated CD8+ T cells both in vivo and in vitro 12, 13, 14, 15, 16, 17 . This is evidenced by IL-4 treatment during infection, either by direct administration or via recombinant vaccinia viruses (VV),3 which delays viral clearance and inhibits both antiviral cytokines and CTL responsiveness 16, 17 . An inhibitory effect of IL-4 on CTL-mediated cytolysis is also implied by the detection of type 2 CD8+ cells with reduced cytolytic capacity in certain HIV-1-infected patients 18 . Furthermore, mice treated with anti-IL-4 Ab concomitant with immunization responded with augmented CTL activity following challenge with respiratory syncytial virus (RSV) 19 . However, neither CD8+ T cell activation nor viral clearance is significantly affected in IL-4-deficient (IL-4-/-) mice 20, 21, 22 . In vitro, IL-4 may function as a growth factor for CD8+ T cells and CTL generation during primary stimulation 12, 23, 24, 25 , indicating multiple roles in CD8+ T cell differentiation and function. As both type 1 and type 2 CD8+ T cell subsets exert cytolytic activity 11, 15 , the conditions under which IL-4 affects cytolysis are not clear.
The apparent multiple effects of IL-4 on CD8+ T cell
function are important to dissect as CD8+ T cells are
critical for the control of viral infections via direct cytolysis as
well as secretion of the antiviral cytokines IFN- and TNF- 26, 27 . An inhibitory influence of IL-4 on CD8+
effector function implies that induction of antiviral CD8+
T cells in a Th1-biased cytokine milieu may be beneficial in priming
CD8+ CTL with a more responsive effector phenotype
following Ag challenge. This report tests this hypothesis by analyzing
the role of IL-4 in CD8+ T cell activation during the acute
and memory phase using the HIV-1IIIB gp160-derived p18 CTL
epitope 28 . Similar frequencies of Ag-specific CTL precursors (CTLp)
in IL-4-deficient (IL-4-/-) vs wild-type
(IL-4+/+) mice indicated that IL-4 is not essential for the
induction phase of CTL generation. A comparison of Ag-specific
IFN--producing cells, IL-2 secretion, and proliferation from both
groups of immunized mice revealed that IL-4 influenced the activation,
expansion, and differentiation of primed CD8+ T cells. On a
per cell basis, CTL from IL-4-/- mice exhibited enhanced
cytolytic activity at a temporal as well as quantitative level
following in vitro restimulation of both primary and memory T cells.
These results suggest that induction of CTL with enhanced cytolytic
function, either during infection or following vaccination, may be more
efficient in an IL-4-deficient environment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/cAnN mice homozygous for an IL-4 gene disruption 29 were supplied by Dr. R. Coffman (DNAX, Palo Alto, CA). BALB/cAnN IL-4+/+ mice were purchased from Charles River Laboratories (Frederick, MD) at 6 wk of age. Mice were housed in microisolator cages in an accredited animal facility at the University of Southern California. For immunizations, groups of 3–4 IL-4-/- or IL-4+/+ control mice received 5 x 107 plaque-forming units (pfu) of rVV i.p. Immunized groups of mice were both sex- and age-matched, and results were independent of these parameters. Mice were sacrificed and spleens were harvested at the indicated time points after immunization.
Viruses, virus titration, and cell lines
The derivation of rVV vSC8 (encoding the Escherichia coli lacZ gene) and vpN-38-p18 (encoding the dominant p18 CTL epitope as a fusion peptide comprising the V3 loop of HIV-1 gp160 linked to a second CTL epitope derived from mouse hepatitis virus) have been described 30, 31 . Replication of rVV in vivo was titrated in ovaries as described elsewhere 32 . Briefly, pairs of ovaries from individual mice were homogenized in 1 ml HBSS buffer and virus titers in supernatants determined by plaque assay on BSC-1 monolayers 33 . The J774.1 (H-2d) macrophage cell line was propagated as described 28 .
Synthetic peptides
Peptide p18 (RGPGRAFVTI) was synthesized on an automated model 430A synthesizer (Applied Biosystems, Foster City, CA) by the University of Southern California Norris Cancer Center Microchemistry Core Laboratory and purified as described 30 .
Generation of CTL, CTL assays, and CTLp frequency analysis
Following immunization of age- and sex-matched IL-4-/- or IL-4+/+ mice, spleen cells were obtained at the indicated time points and stimulated in Iscove’s modified Dulbecco’s medium supplemented with 2 mM L-glutamine, 25 µg/ml gentamicin, 1 mM sodium pyruvate, 5 x 10-5 M 2-ME, nonessential amino acids, and 10% FCS (Gemini Bioproducts, Calabasas, CA) in the presence of 1 µM p18 peptide as previously described 30, 31 . Cultures (10 ml) contained 2.5 x 106 splenocytes per ml, and CD8+ T cell percentages in IL-4-/- or IL-4+/+ immune splenocytes were similar. Unless otherwise indicated, polyclonal p18-specific CTL cultures were not supplemented with an exogenous source of IL-2. Selected cultures received daily doses of 10 ng/ml mouse rIL-4 (kindly provided by Dr. R. Coffman). Cytolytic activity was measured in a 4-h 51Cr release assay in the presence of 2–5% FCS as described 30, 31 . Target cells (104 J774.1 cells per well) were used either untreated or coated with 1 µM p18 peptide. Effector cells were added at the indicated numbers or E:T ratios. Data are expressed as percentage specific lysis defined as 100 x [(experimental lysis) - (spontaneous lysis)]/[total (detergent lysis) - (spontaneous lysis)]. Maximum spontaneous release values were always <20% of total release values. For quantitative comparison of individual cultures, data are presented in lytic units (LU). One LU was arbitrarily defined as the number of lymphocytes required to yield 20% and 30% lysis of 104 target cells for cultures set up 7 days and over 4 wk postimmunization, respectively; values were obtained from the linear region of T cell titration curves depicting the values of specific lysis vs the log of effector cell numbers.
CTLp frequency analysis by limiting dilution analysis (LDA) was conducted using splenocytes from mice immunized i.p. with 5 x 107 pfu rVV as previously described 31 . Briefly, pooled spleen cells from 2–3 mice per group primed 8 or 53 days previously were plated into 96-well plates at decreasing densities starting at 5 x 105/well at 24 replicates per cell density. Effectors were stimulated using irradiated, syngeneic spleen cells (4 x 105/well) coated with 1 µM p18 peptide. Following 6 days of stimulation, cytotoxicity of individual wells was measured on labeled J774.1 target cells coated with p18 peptide in a 4- to 5-h 51Cr release assay. Wells were scored positive if cytotoxicity exceeded spontaneous release plus 3 SD. CTLp frequencies were obtained from the inverse of the unfractionated responder cell concentration at which 37% of the wells scored as negative 31 .
CD4+ T cell depletion and proliferation assay
Splenocyte suspensions were depleted of CD4+ T cells by immunomagnetic separation. Magnetic beads (Dynal, Lake Success, NY) coated with mAb GK1.5 (American Type Culture Collection, Manassas, VA) were used to remove CD4+ cells. CD4-depleted preparations routinely contained <1% CD4+ splenocytes and 19–20% CD8+ cells. CD4-depleted splenocytes (4 x 105 cells/well) were incubated in medium only or with 1 µM p18 peptide for up to 4 days. The culture medium was Iscove’s modified Dulbecco’s medium supplemented with 2 mM L-glutamine, 25 µg/ml gentamicin, 1 mM sodium pyruvate, 50 µM 2-ME, nonessential amino acids, and 2% FCS. The cultures received 2 µCi/well [3H]TdR (5 Ci/mmol; ICN, Irvine, CA) for the last 16 h of incubation.
Detection of IFN--producing cells
For intracellular staining of IFN-, splenocytes from immune
or naive mice were stimulated in vitro with or without p18 peptide for
20–22 h. Monensin (1 µM; PharMingen, San Diego, CA) was added during
the last 4 h incubation. Fc receptors were blocked by adding
normal mouse serum. Cells were incubated with phycoerythrin-conjugated
anti-CD8 mAb (PharMingen). After fixation and permeabilization,
cells were incubated with FITC-conjugated anti-IFN- mAb XMG1.2
in the presence of permeabilization buffer according to the
manufacturer’s instructions (PharMingen). Double-stained
preparations were analyzed by flow cytometry in a FACScan flow
cytometer (Becton Dickinson, Mountain View, CA). Fifty thousand events
were scored for each sample.
IFN--secreting cells were detected using Ag-specific ELISA spot
(ELISPOT) assays as described 34 . Briefly, 96-well plates supporting
cellulose ester membranes (MultiScreen HA, Millipore, Bedford, MA) were
coated with 2 µg/ml R4-6A2 mAb (PharMingen) in sterile PBS by
overnight incubation at 4°C. Empty binding sites were blocked by
incubation with Iscove’s modified Dulbecco’s medium containing 10%
FCS. Serial dilutions of immune splenocytes were added to mAb-coated
wells in the presence of p18 peptide-pulsed splenocytes from naive mice
(2.5 x 105/well). Nontreated APC were included as
controls in each assay. Rat Con A supernatant was added as a source of
IL-2 to a final 2% concentration, and cultures were incubated for
18 h at 37°C. Bound IFN- was detected by overnight incubation
at 4°C with biotinylated anti-IFN- mAb (0.5 µg/ml; XMG1.2,
PharMingen) followed by consecutive incubation with
streptavidin/peroxidase (Sigma, St. Louis, MO) and aminoethyl carbazole
as substrate (Zymed, San Francisco, CA). Each sample was tested in
triplicate.
Detection of secreted IL-2, IL-4, and IFN- by ELISA
Secreted IL-2, IL-4, and IFN- were measured in supernatants
of unfractionated or CD4-depleted splenocyte cultures. mAb JES6-1A12
for IL-2, BVD4-1D11 for IL-4, and R4-6A2 for IFN- were used as
capture reagents. Detection Ab were biotinylated mAb JES6-5H4 for IL-2,
BVD6-24G2 for IL-4, and XMG1.2 for IFN- (PharMingen). Ag/Ab
complexes were detected by incubation with streptavidin/peroxidase
conjugate and reaction with O-phenylenediamine
dihydrochloride (both from Sigma). Recombinant IL-2, IL-4, and IFN-
(PharMingen) were used for the preparation of standard curves. Each
sample was tested in duplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Observations that the induction of type 2-like CD8+ T
cells with reduced cytolytic activity and IFN- secretion in the
presence of IL-4 may result in limited control of viral infections
14, 15, 16, 17, 18 suggest that priming of CD8+ T cells in the
absence of IL-4 may promote type 1 effector function. However, this
possibility appears to contradict the potential role of IL-4 as a
growth factor for CD8+ T cells 23, 24, 25 . To evaluate the
role of IL-4 in the activation of Ag-specific CD8+ T cells
into cytolytic effectors, rVV was used as a prototype vaccine to
generate HIV-1IIIB gp160-specific CTL in
IL-4-/- and IL4+/+ mice. Mice were immunized
with a rVV expressing a cytoplasmic form of gp160 containing the
dominant p18 epitope in the context of the V3 loop, designated
vpN-38-p18 31 . This construct was chosen due to its relative enhanced
immunogenicity compared with rVV-expressing gp160 31 . To determine
whether the absence of IL-4 affects CTL effector function during a
primary response, splenocytes were recovered 7 days postinfection
(p.i.) and restimulated in vitro with p18 peptide to monitor CTL
activity, as cytolysis was not detected from splenocytes ex vivo (data
not shown). Ag-specific CTL activity was measured following 1, 2, 4, or
6 days of restimulation (Fig. 1
A). Cytolysis was not
detected in either culture at day 1. At day 2, specific lysis was
approximately fourfold higher in splenocytes from IL-4-/-
mice compared with IL-4+/+ mice. Enhanced cytolysis in
IL-4-/- cultures was maintained at day 4 and 6 of in
vitro restimulation. To allow direct comparison of the cytolytic
potential on a per cell basis, Fig. 1B displays the data
shown in Fig. 1A as LU/106 effector cells from
cultures at each time point. The difference in cytolytic activity
between IL-4-/- and IL-4+/+ cultures was the
greatest at day 4, when CTL derived from IL-4-/- mice
exhibited 10-fold higher cytolysis. Furthermore, whereas the cytolytic
activity per IL-4-/- effector did not increase
significantly after day 4, cytolysis by IL-4+/+ cells was
increased 3-fold at day 6. This suggests that CD8+ T cell
differentiation in the absence of IL-4 not only gives rise to cell
populations with higher cytolytic potential, but may also occur more
rapidly. When adjusted for total number of viable cells per culture,
cytolytic activity was also 6- to 10-fold higher at day 4 and day 6 in
cultures from IL-4-/- mice and peaked at day 6 (Fig. 1C). Despite exhibiting a minimal increase from day 4 to day
6 at a single-cell level, cytolysis in total cultures from
IL-4-/- mice was still enhanced 3-fold during this time
interval. These results suggested that increased cytolytic activity was
associated with a higher differentiation state of activated effectors
in IL-4-/- cultures. Although the CD8+
population in day 6 cultures from IL-4-/- mice was
approximately twofold higher than in cultures from IL-4+/+
mice (28.4% vs 12.1%, compared with 9–10% for either culture at day
0), cytolytic activity adjusted to CD8+ cells was still 3-
to 4-fold higher in effectors from IL-4-/- mice.
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D).
Despite a 2.5- to 3-fold increase in cytolysis by IL-4+/+
cultures at day 4 and 6, cytolysis was not restored to the level
observed in splenocytes from IL-4-/- mice not
supplemented with IL-2 (Fig. 1D). Although no influence of
exogenous IL-2 was detected at day 2 and 4 in IL-4-/-
splenocytes, IL-2 induced a fourfold increase in cytolysis at day 6,
presumably by preventing apoptosis of activated effectors. The fact
that exogenous IL-2 was not sufficient to overcome lower cytolytic
activity in IL-4+/+ splenocytes indicates involvement of a
mechanism other than down-regulation of IL-2 production by IL-4.
To confirm that the enhanced cytolysis exerted by IL-4-/-
splenocytes was due to the absence of IL-4, splenocytes from
IL-4-/- or IL-4+/+ mice recovered 7 days p.i.
were cultured with Ag in the presence or absence of rIL-4. The addition
of rIL-4 reduced the cytolytic activity of IL-4-/-
splenocytes by 10-fold at day 4 and by 40-fold at day 6 of in vitro
restimulation (Fig. 2). An inhibitory
effect of IL-4 on cytolysis was also observed in restimulated
IL-4+/+ splenocytes, although the reduction was less
dramatic compared with splenocytes from IL-4-deficient mice. The
results confirmed the notion that IL-4 limits the expansion and/or
cytolytic activity of CD8+ effectors 14, 17 . However, the
addition of anti-IL-4 Ab to IL-4+/+ splenocyte cultures
did not enhance cytolytic activity (data not shown), suggesting that
enhanced IL-4-/- CD8+ T cell responsiveness
may be due to an indirect effect in the absence of IL-4, possibly
already primed in vivo.
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To test whether the enhanced cytolysis exerted by
IL-4-/- splenocytes was due to a higher frequency of
Ag-specific effectors, the number of Ag-specific IFN- secreting
splenocytes was determined by ELISPOT assay 34 . At day 7 p.i.,
the initial percentage of CD8+ splenocytes (9–10%) and
the frequencies of IFN--secreting splenocytes were similar in
both groups (Fig. 3). After 6 days in
culture, the number of Ag-specific IFN--secreting cells was 2- to
3-fold higher in IL-4-/- splenocytes, correlating with a
2-fold higher percentage of CD8+ T cells in cultures from
IL-4-/- mice (28.4% for IL-4-/- and 12.1%
for IL-4+/+ splenocytes). Although the increase in
Ag-specific IFN--secreting cells after 6 days of restimulation
indicated enhanced expansion of IL-4-/- CD8+
T cells, the increase was not sufficient to account for the
sixfold difference in cytolytic activity. These data suggest that
increased cytolytic activity in IL-4-/- cultures is due
to enhanced responsiveness to Ag both at the level of expansion and
differentiation.
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-producing cells was confirmed
by FACS analysis following intracellular staining of IFN- in cells
from IL-4-/- or IL-4+/+ mice explanted 7 days
p.i. and restimulated with p18 peptide for 24 h in the absence of
exogenous IL-2 (Table I). All
IFN-+ cells in cultures stimulated with peptide
coexpressed CD8, excluding a contribution by NK cells. Overall, the
frequencies of IFN--producing cells were at least 2-fold higher
compared with the ELISPOT assay. In addition, the frequency of
IFN-+ CD8+ splenocytes was 40% higher in
IL-4-/- mice despite similar percentages of
CD8+ splenocytes in both groups. IFN- production was
strictly specific for the p18 peptide because signals elicited by media
only or a heterologous H-2Dd binding peptide were
negligible. No IFN-+ cells were detected in splenocytes
from naive mice cultured in medium alone or with Ag. These data
contrasted results obtained from ELISPOT assays, where differences in
IFN--secreting cell numbers were evident only after prolonged in
vitro restimulation. These results indicate that the activation of
IL-4-/- splenocytes leads to enhanced IFN- synthesis,
albeit not secretion, as early as 24 h after restimulation. Thus,
higher frequencies observed by staining in the absence of exogenous
IL-2 reflect an earlier parameter of CD8+ T cell
activation. Overall, these data indicate that CD8+
splenocytes from IL-4-/- mice are more responsive to Ag
stimulation than IL-4+/+ splenocytes.
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).
Similar viral titers in both IL-4-/- and
IL-4+/+ mice indicated that differences in CD8+
T cell responsiveness were not due to varying viral load.
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The enhanced CD8+ T cell effector function exhibited
by splenocytes from IL-4-/- mice suggested that priming
in the absence of IL-4 may be beneficial for vaccine strategies.
Efficient vaccines should prime for a strong memory response upon
encounter with their specific Ag, which is dependent on both a high
frequency of responder cells and their rapid activation. Enhanced
activation and expansion of primary CD8+ splenocytes from
IL-4-/- mice following Ag stimulation in vitro may
reflect a higher frequency of Ag-specific CD8+ T cells
during memory in vivo. Therefore, LDA was used to compare the
frequencies of p18 specific CTLp 30 in splenocytes during memory and
primary responses in rVV-immunized mice (Table III). CTLp frequencies were similar at 8
days p.i., correlating with overall higher but similar frequencies of
IFN--secreting splenocytes ex vivo (Table III and Fig. 3). At 8 wk
p.i., the frequencies of CTL were reduced to the same extent in
splenocytes from IL-4+/+ and IL-4-/- mice
compared with day 8. These data indicated that the absence of IL-4 does
not significantly affect CTLp priming, expansion, or transition to
memory in vivo.
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production. Cytolytic activity by memory T cells from
IL-4-/- mice was indeed invariably higher compared with
IL-4+/+ cells at all time points tested, with the greatest
difference (
5-fold) detectable at day 4 (Fig. 4). Further resembling the primary
cultures was the higher percentage of CD8+ T cells in
cultures from IL-4-/- vs IL-4+/+ splenocytes
at day 6 (60.7% vs 43.6%, respectively), despite similar ex vivo
CD8+ percentages (14%). These results show a 40%
enhanced expansion of T cells from immune IL-4-/- mice in
response to specific Ag.
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production following short-term (24 h) and several
days in vitro stimulation. The number of
IFN-+CD8+ cells 24 h after explant was
50% higher in IL-4-/- splenocytes with similar
percentages of CD8+ cells in both groups (Table IV), confirming enhanced activation of
these cells. A maximal increase of 70% in absolute numbers of
IFN-+CD8+ cells was observed at day 4. When
adjusting for increased percentage of CD8+ cells at this
time point, the ratios of IFN-+ cells within the
CD8+ population remained similar. At day 6, the number of
IFN--producing CD8+ declined markedly in both cultures,
while the CD8+ percentages remained constant relative to
day 4, possibly reflecting a decline of IFN- secretion in highly
activated T cells 35 . These data indicate that enhanced cytolytic
activity does not correlate in a linear fashion with the modest
increase in CD8+ memory IL-4-/- T cells
observed 24 h poststimulation. A potential mechanism may reside in
a lower threshold of activation, as already suggested by the results
from primary CTL.
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secretion in supernatants from CD4-depleted splenocytes cultured with
or without Ag were monitored by ELISA (Fig. 5). IFN- levels in culture
supernatants from IL-4-/- mice peaked 2 days earlier
compared with IL-4+/+ cultures, namely between day 2 and 3.
Furthermore, IFN- levels were enhanced by 2.5-fold at day 2,
indicating both more rapid and increased responses to Ag. At early time
points, the kinetics of secretion correlate with the data obtained by
intracellular staining, indicating that the enhanced numbers of
activated IL-4-/- splenocytes observed by intracellular
ex vivo staining results in enhanced IFN- secretion by day 2. A 68%
higher number of IFN-+ cells in IL-4-/-
CD8+ T cells at day 4 was not paralleled by secreted
cytokine (compare Table IV and Fig. 5). This data may reflect the
earlier differentiation of IL-4-/- CD8+ T
cells, which at this time point are already highly cytolytic. An
assessment of IL-4 production by ELISA under the conditions described
above revealed no IL-4 secretion in either unfractionated or
CD4-depleted splenocytes above detection thresholds (50 pg/ml).
Overall, these data show that activated memory CD8+ T cells
from IL-4-/- mice are characterized by both augmented
cytolytic and noncytolytic effector functions, thus retaining the
effector activity displayed during the primary response.
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A). IL-2 concentrations in
supernatants of IL-4-/- CD8+ T cell cultures
were at least 2-fold higher than in supernatants from
IL-4+/+ mice at all time points, reaching a maximum 3-fold
difference at day 2, which coincided with peak levels of IL-2 in both
cultures. Importantly, the marked secretion of IL-2 observed at day 1
of in vitro stimulation, when proliferation was similar for both
IL4-/- and IL-4+/+ splenocytes, indicates
that Ag-driven activation is higher and precedes the expansion of
IL-4-/- CD8+ T cells (Fig. 6B).
Proliferation of IL-4-/- CD8+ splenocytes was
maximal at day 2 and at most 50% higher than their IL-4+/+
counterpart. Therefore, increased IL-2 secretion contributes to the
expansion of CD8+ T cells following in vitro stimulation
for 4 or 6 days of IL-4-deficient splenocytes. The level of expansion
correlates with the twofold increase in CD8+ percentages
observed in splenocytes from IL-4-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF. To
understand the regulation of antiviral CD8+ T cells by IL-4
following immunization, our studies focused on CD8+ T cell
responses to the HIV gp120-derived p18 epitope 28 . The results show
enhanced CTL-mediated cytolytic activity on a per cell basis in
splenocytes from IL-4-/- mice compared with
IL-4+/+ mice in response to rVV immunization, both during
the primary and the memory response. No significant differences in
viral titers in the ovaries between both groups of mice indicated that
enhanced responsiveness was not simply attributed to higher viral load
in IL-4-/- mice. The fact that the addition of rIL-4 to T
cell cultures resulted in markedly reduced cytolysis not only by
splenocytes from IL-4-/- mice but also by
IL-4+/+ splenocytes indicated that enhanced responsiveness
in IL-4-/- splenocytes was directly attributed to the
lack of IL-4. However, cytolytic activity was not affected by the
addition of anti-IL-4 Ab, consistent with the inability to detect
IL-4 in culture supernatants. These findings may reflect low levels
and/or rapid consumption of IL-4. An alternative explanation is that
enhanced responder function is already primed in vivo and amplified in
vitro, especially in light of the culture conditions to specifically
stimulate Ag-specific CD8+ T cells. Therefore, our data
demonstrating IL-4 sensitivity in Ag-specific CD8+ T cells
primed in vivo confirm a direct role for IL-4 in limiting
CD8+ CTL effector function 14, 36 . By contrast, in vitro
studies of allogeneic CTL suggested that IL-4 induces secretion of type
2 cytokines by CD8+ T cells 10, 11, 15 , without
compromising their short-term cytolytic potential in 4-h CTL assays
15 . This may reflect a delayed effect of IL-4, because the killing of
tumor cells cocultured with allogeneic CTL was only detectable at early
but not late time points 36 . This observation may reflect the
quantitative differences in the CD8+ T cell repertoire
capable of responding to allogeneic Ag as compared with a single viral
epitope. Excess IL-4 in vivo may limit CTL priming, because the CTLp frequency was reduced in splenocytes from mice infected with a rVV-expressing IL-4 17 . Conversely, enhanced cytolysis in IL-4-/- splenocyte cultures may be attributed to more efficient priming of CTLp. In our system, this latter possibility seems unlikely because of similar frequencies of p18-specific CTLp in spleen cells derived from either IL-4-/- or IL-4+/+ mice. Contrasting our results, intranasal infection of IL-4-/- mice with Sendai virus resulted in reduced CTLp frequencies in the spleen and draining lymph nodes 20 ; however, bronchoalveolar T cells exhibited no differences in ex vivo cytolysis, suggesting that enhanced activation of CTL effectors may have compensated for lower CTLp frequency. Similarly, CTL function in IL-4-/- mice infected with lymphocytic choriomeningitis virus or VV did not differ from their IL-4+/+ counterpart 21 . Variable virulence and sites of infection may account for these differing observations. To our knowledge there is no evidence for enhanced IL-4 induction by VV as opposed to lymphocytic choriomeningitis virus, Sendai, or influenza virus infections 16, 17 . Thus, effects on CD8+ T cell function exerted by the absence of IL-4 may be more evident in self-limiting, asymptomatic infections or may be more likely to emerge when the T cell response to a single epitope is studied.
Enhanced activation and expansion in response to Ag-stimulation could
account for the increased cytolytic activity of splenocytes from
IL-4-/- mice. Enhanced temporal as well as quantitative
responses were indicated by higher levels of IFN- in supernatants of
memory CD8+ T cells from IL-4-/- mice,
preceding those in IL-4+/+ splenocyte cultures.
Intracellular staining of splenocytes from immunized mice revealed
consistently higher numbers of Ag-specific IFN- producing
CD8+ T cells ex vivo in the IL-4-deficient population both
7 days and 8 wk p.i., suggesting that IFN--producing
CD8+ T lymphocytes differentiate into secreting cells upon
further stimulation. This was also supported by higher numbers of
IFN--secreting cells in IL-4-deficient cultures detected by ELISPOT
at day 6. However, the numbers of IFN- secreting cells in
IL-4-deficient cultures compared with IL-4+/+ cultures
never exceeded a 2-fold difference, even after prolonged in vitro
stimulation. Therefore, we favor the notion that enhanced cytolytic
potential of CD8+ T cells is primarily due to increased
responsiveness to Ag in primed precursors from IL-4-/-
mice.
The higher number of IFN--producing cells in IL-4-/-
splenocytes resulted from an overall increased expansion of
CD8+ T cells in these cultures, as detected by
intracellular staining. Augmented expansion of IL-4-/-
CD8+ T cells was also indicated by increased proliferation
in response to Ag. High levels of IL-2 in the supernatants of
IL-4-/- splenocytes suggested that increased activation
further potentiates expansion of CD8+ T cells, resulting in
a larger number of highly activated CTL. Therefore, Ag-specific IL-2
secretion by CD8+ T cells in IL-4-/- mice may
function in an autocrine fashion. Supporting this observation, the
addition of anti-IL-2 mAb, but not of anti-IL-4 mAb, abrogated
the development of cytolytic function (data not shown). Thus,
exogenously added IL-2 may skew parameters affecting CD8+ T
cell regulation via endogenous IL-2. Indeed, whereas assays requiring
exogenous IL-2, i.e., ELISPOT and LDA, failed to reveal differences in
the numbers of Ag-specific CD8+ T cells from
IL-4-/- and IL-4+/+ mice, they were readily
detected in assays without IL-2 addition. This may be explained by
receptor competition, as IL-2 and IL-4 as well as other cytokines share
the common -chain of their receptors 37, 38 . Thus, the lack
of IL-4 may provide additional receptor engagement by IL-2, explaining
the enhanced proliferation of IL-4-/- CD8+ T
cells. Supporting this notion, the signal mediated by IL-4 binding to
its receptor may be less efficient at inducing proliferation than the
signal delivered by IL-2 39 .
Our results using virally primed T cells support the concept that
CD8+ populations with a type 1 cytokine profile and
effector function are inhibited by IL-4 and promoted by IFN- 11, 12 . Fundamental to the dichotomy of cytokine production, IL-4 inhibits
IFN- secretion by both CD4+ and CD8+ T cells
1, 11, 12, 13, 14 . Our data further suggest that in the absence of IL-4 to
counterbalance IFN- production, IFN- may contribute to enhanced
CD8+ T cell activation and expansion via its stimulating
role in processing and presentation of viral peptides in vitro and in
vivo 40, 41 . The enhanced potential for cytolytic and noncytolytic
CD8+ T cell effector function in the absence of IL-4 is not
only crucial for limiting viral infections but also for vaccine design.
Mechanisms responsible for inducing polarization of type 1 or type 2
responses appear to be established rapidly following exposure to Ag and
to be sustained during memory. Therefore, the cytokine environment
during T cell activation and expansion is a critical parameter for the
outcome of infections and protective immunity. In vivo, abundant
production of IL-4 is detrimental for clearance of influenza virus or
VV 16, 17 . Equally important, the nature of the cytokine profile
dominating during one infection may bias responses to subsequent
infections 42 . Although a polarized response may not always be
evident immediately after immunization, it may become potentiated upon
challenge, as shown for the envelope proteins of RSV 43 . In our
model, enhanced effector functions of memory CD8+ T cells
suggests that CTLp priming in the absence of IL-4 may give rise to a
more potent response following challenge. In viral infections and
cancers, where CTL responses are protective, therapy modulating the
IL-4 response by administration of antagonists 44 , soluble receptors
45 , or Ab-mediated neutralization would be beneficial, as
demonstrated for RSV 19, 46 and renal cell carcinoma 47 .
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cornelia Bergmann, 1333 San Pablo Street, MCH142, University of Southern California School of Medicine, Los Angeles, CA 90033. E-mail address:
3 Abbreviations used in this paper: VV, vaccinia virus; ELISPOT, ELISA spot; LDA, limiting dilution analysis; p.i., post infection; RSV, respiratory syncytial virus; CTLp, CTL precursor; LU, lytic unit; pfu, plaque-forming unit.
Received for publication March 5, 1998. Accepted for publication November 20, 1998.
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and IL-4 regulate the growth and differentiation of CD8+ T cells into subpopulations with distinct cytokine profiles. J. Immunol. 155:2928.[Abstract]
. Annu. Rev. Immunol. 15:749.[Medline]
is a prerequisite for optimal antigen processing of viral peptides in vivo. J. Immunol. 158:3303.[Abstract]
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