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Adjuvant Effect of IL-12: Conversion of
Peptide Antigen Administration from
Tolerizing to Immunizing for CD8+ T Cells
In Vivo
Clint S. Schmidt and Matthew F. Mescher
J Immunol 1999; 163:2561-2567; ;
http://www.jimmunol.org/content/163/5/2561
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Copyright © 1999 by The American Association of
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References
Adjuvant Effect of IL-12: Conversion of Peptide Antigen
Administration from Tolerizing to Immunizing for CD81
T Cells In Vivo1
Clint S. Schmidt and Matthew F. Mescher2
I
n vivo presentation of soluble peptide Ag to naive T cells can
result in either full activation and development of effector
function, or, conversely, may induce tolerance in an Ag-specific manner. Many factors influence the two outcomes, such as the
physical state of the Ag along with dose and route of immunization
(1–3). It has been argued that the in vivo difference between a
tolerogenic and an immunogenic T cell response depends on
whether the cell senses “danger” in the environment (4, 5). Indeed,
full effector function in vivo is only achieved when soluble peptide
Ag is administered with adjuvant or as part of an intact organism
(6 – 8). Thus the adjuvant, whether in the form of CFA, LPS, or
other bacterial cell wall components, or aggregate protein, must
incite an environment that is interpreted as “dangerous” by the host
immune system. This awareness could be attributed to many
causes, but a prevalent view is that inflammatory cytokines, produced by cells of the innate immune system and/or APC, provide
the signals necessary for the induction of an effector response (5).
Previous CD41 T cell studies have confirmed the importance of
adjuvant to prevent tolerance in vivo, and have even begun to
define molecular characteristics involved in full T cell activation
(9, 10). In addition to costimulation and the resulting production of
IL-2, IL-1 has been identified as a cytokine capable of inducing a
Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota, Minneapolis, MN 55455
Received for publication April 9, 1999. Accepted for publication June 21, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants AI34824 and
AI35296. C.S. was supported in part by National Institutes of Health Grant
T32-AI07313.
2
Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for
Immunology, University of Minnesota, Box 334 Mayo, 420 Delaware Street S.E.,
Minneapolis, MN 55455. E-mail address: [email protected]
Copyright © 1999 by The American Association of Immunologists
strong in vivo peptide response for CD41 T cells (11). In vitro
studies have also identified some inflammatory mediators produced by professional APCs, including IL-1, IL-6, IL-12, and
IFN-g, that can positively influence a naive CD81 T cell’s proliferation and/or differentiation to effector CTL (12–15). Recent in
vitro studies using latex microspheres coated with various combinations of class I MHC/peptide complexes and costimulatory molecules have shown that IL-12, but not IL-1, acts directly on the
naive CD81 T cell to promote clonal expansion and differentiation
(16). The in vitro results demonstrating a direct effect of IL-12 on
CD81 T cells suggested that this cytokine might prevent Ag-specific tolerance upon peptide administration, and support an effective in vivo CD81 T cell response.
The heterodimeric cytokine IL-12 was originally identified as
NK cell stimulating factor, and is known to stimulate CD41 T cell
differentiation to Th1 effectors (14, 17). IL-12 has also been shown
to have anti-tumor effects in vivo, effects that at least in some cases
can be attributed to production of IFN-g (18 –20). Clinical trials
have also shown IL-12 to induce a higher CTL precursor frequency in humans, although this clinical effect was accompanied
by significant side effects (21, 22). Aside from, but not excluding,
the past studies, little is known about the effects of IL-12 on the in
vivo etiology, kinetics, anatomy, and relative stability of a primary
CD81 T cell response. To examine this, we have used a system in
which a small number of naive Ag-specific CD81 T cells are adoptively transferred into naive, syngeneic recipients (23) to monitor
their response to peptide immunization in the presence or absence
of adjuvant and inflammatory cytokines. IL-12 was found to mimic
the effects of CFA in preventing tolerance, and to be effective in
supporting clonal activation, migration, development of effector
function, and establishment of immunologic memory. While IL-12
administration is sufficient to support a strong response, it is not an
absolute requirement, since a strong response can still be obtained
using peptide and CFA in IL-12 (p40)-deficient mice.
0022-1767/99/$02.00
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CD81 T cells from TCR transgenic 2C mice, specific for SIYRYYGL peptide bound to H-2Kb, were adoptively transferred into
C57BL/6 recipients to allow monitoring of their location, numbers, and phenotype upon peptide challenge. Recipients were primed
by s.c. injection of SIYRYYGL alone or with CFA or IL-12, and the transferred cells then tracked by flow cytometry using the
1B2 mAb specific for the 2C TCR. Peptide alone induced a transient and weak expansion of 1B21 cells in the draining lymph nodes
(DLN) by day 3, but these cells were tolerant to secondary peptide challenge. In contrast, priming with CFA/peptide resulted in
a large clonal expansion of 1B21 cells in DLN by day 3, and the cells exhibited a CD25highCD44high phenotype, blast transformation, and lytic effector function. By day 5, 1B21 cell numbers decreased in the DLN and increased in the spleen and blood. 1B21
cells with a memory phenotype persisted through day 60 in the DLN, spleen, and blood and responded to secondary peptide
challenge. Immunization with peptide, along with IL-12, mimicked the adjuvant effects of CFA with respect to phenotype, clonal
expansion, effector function, and establishment of memory. IL-12 was not unique in providing this adjuvant effect however, since
CFA/peptide immunization of IL-12-deficient recipient mice also resulted in 1B21 T cell activation and clonal expansion. Thus,
CFA or IL-12 can enhance Ag-specific CD81 T cell responses to peptide, demonstrating that an inflammatory cytokine(s) can
support activation and prevent tolerance induction. The Journal of Immunology, 1999, 163: 2561–2567.
IL-12 AS ADJUVANT FOR NAIVE CD81 T CELLS
2562
Materials and Methods
Mice
2C TCR transgenic mice (24) were obtained as a kind gift from Dr. Dennis
Loh (Washington University, St. Louis, MO) and were bred to wild-type
C57BL/6 mice to generate mice heterozygous for the 2C TCR transgene.
CD81 T cells from these mice were used as donors in all adoptive transfer
experiments, and C57BL/6 mice (Charles River Laboratories, Wilmington,
MA) or IL-12 knockout (KO)3 mice on a C57BL/6 background (25) (The
Jackson Laboratory, Bar Harbor, ME) were used as recipients. All mice
were housed in a specific pathogen-free environment at all times.
Antibodies
Cell lines
The thymoma EL4 (H-2b) was grown in vitro in complete RPMI medium
(see above). Cultures were always 90 –100% viable, as measured by trypan
blue exclusion, and cells were washed with PBS before use in in vitro
chromium release assays (see below).
Adoptive transfer of 2C transgenic cells
Lymph node cells (axillary, brachial, cervical, inguinal, periaortic, and
mesenteric) from heterozygous 2C transgenic mice were removed, homogenized, ammonium chloride-treated to remove RBC, and adherence-depleted for 90 min. The nonadherent cells were washed in PBS and enriched
for CD81 cells using the CD81 Cellect column purification kit (Biotex
Laboratories, Edmonton, Canada). Before transfer, the purified population
was analyzed by flow cytometry to determine the percentage of 1B21
CD81 cells and their phenotype with respect to CD25, CD44, and forward
scatter (FSC) to insure that the transferred population was phenotypically
naive. A total of 3–5 3 106 1B21 CD81 cells in 500 ml PBS was transferred via tail vein injection into age- and sex-matched naive 6- to 8-wk-old
recipients. Recipient mice were then rested for 24 h before immunizations.
Immunizations
The synthetic peptide SIYRYYGL (27) (Chiron Mimotopes, Clayton, Victoria, Australia) was prepared in PBS and injected alone (peptide only) or
emulsified in CFA (CFA/peptide) (Sigma, St. Louis, MO). All peptide
immunizations involved the s.c. injection of 50 mg peptide in 300 ml per
mouse distributed between two sites on the back. Recombinant murine
IL-12 (Genetics Institute, Cambridge, MA) was administered i.p. at 1 mg in
100 ml PBS with 0.1% sterile mouse serum on days 0, 1, and 2. As controls,
transferred animals were also immunized with PBS alone (Transfer Only).
Flow cytometric analysis of in vivo populations
Transferred and immunized mice were sacrificed at varying times after
priming or rechallenge, and the lymph nodes and spleen were removed,
homogenized, and ammonium chloride-treated to lyse RBC. PBL was
drawn from the heart using a heparin-loaded syringe, and the RBC were
lysed by ammonium chloride treatment. Brachial, axillary, and inguinal
lymph nodes (LN) were pooled as draining lymph nodes (DLN). Each cell
population (DLN, spleen, and PBL) was counted for total cell population
using trypan blue to exclude dead cells. Isolated cells (1–2 3 106) from
each site were stained with 1B2-biotin mAb, anti-CD8a-CyChrome, and
either anti-CD44-FITC (Pgp1) or anti-CD25-FITC. After 20 –30 min of
incubation, cells were washed and streptavidin-APC was added for detection of 1B2-biotin. Stained cells were fixed with 1% formaldehyde and
analyzed by flow cytometry using the CellQuest software package (Becton
3
Abbreviations used in this paper: KO, knockout; FSC, forward scatter; LN, lymph
node; DLN, draining LN; MLN, mesenteric LN.
Chromium release assay
Following immunizations, DLN cells of duplicate mice were harvested,
pooled, homogenized, and enriched for CD81 cells using the CD81 Cellect
column purification kit (Biotex Laboratories). These cells (of which 0.12–
1.12% are 1B21 CD81) were then assayed using a standard 4-h 51Cr release assay with SIYRYYGL-pulsed EL4 target cells.
Results
Primary in vivo CD81 T cell response to soluble peptide Ag in
the presence or absence of CFA
The primary response of naive CD81 T cells to soluble peptide Ag
was monitored in vivo through the use of an adoptive transfer
system utilizing 2C TCR transgenic CD81 T cells that recognize
the synthetic peptide SIYRYYGL in the context of H-2Kb (23, 24,
27). Phenotypically naive CD81 2C T cells (3–5 3 106) were
injected i.v. into naive age/sex-matched C57BL/6 recipients, and
the resulting 2C populations were monitored throughout the course
of an immune response by the use of the anticlonotypic mAb 1B2
(26) and flow cytometric analysis. The 1B21 CD81 population
comprises 0.2– 0.4% of total LN cells 1 day after transfer and
before immunization (23). Transferred mice were rested for 1 day
and then immunized with SIYRYYGL peptide (s.c. 50 mg/mouse,
distributed between two sites on the back) in the presence or absence of CFA. The amount of SIYRYYGL peptide used was based
on previous in vivo dose/response titrations that resulted in optimal
1B21CD81 responses (data not shown).
Immunization with SIYRYYGL emulsified in CFA resulted in
massive 1B21 CD81 clonal expansion in the DLN (inguinal, brachial, axillary) by day 3 (Fig. 1A). The cells had increased levels
of CD44 (Fig. 1C), CD25, and blast transformation (49.7%
CD25high, 74.8% FSChigh; Table I), indicating that they were proliferating. To confirm that the increased 1B21 numbers reflected
clonal expansion and not simply increased migration to the site,
donor cells were labeled with PKH26 lipophilic dye before adoptive transfer, and the fluorescence intensity was found to be highly
diluted by day 3 as cells expanded (data not shown, and Ref. 16).
1B21 CD81 cell numbers peak at day 3, and the cells then migrate
out of the DLN, and, by day 5, large numbers could be detected in
the spleen and PBL. The numbers at these sites gradually decline
as the primary response subsides, and, by day 20, only a small
population of 1B21CD81 cells (4 – 6 3 104 in DLN) with a memory phenotype (54.6% CD44high) is still present (data not shown,
and see below). The 2C cells detected in nondraining lymph nodes,
such as mesenteric lymph nodes (MLN), did not proliferate in
response to peptide Ag and retained a CD252CD44low phenotype
through day 20.
In contrast to immunization with adjuvant, 2C cells in mice
immunized with SIYRYYGL alone in PBS showed only a low
level of clonal expansion at all sites by day 3 (Fig. 1B, note change
of scale). By day 1, significant numbers of these cells in the DLN
had acquired high levels of CD25 (34.7%) and CD44 (39.8%) and
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The 1B2 mAb, specific for the transgenic 2C TCR, was from the 1B2
hybridoma (26) (a gift from Dr. Herman Eisen, Massachusetts Institute of
Technology, Boston, MA) grown in vitro in complete RPMI media (RPMI
1640; Cellgro, Herndon, VA) with 10% FCS (Tissue Culture Biologicals,
Tulare, CA), 0.2% L-glutamine, 0.1% penicillin/streptomycin, 0.1%
HEPES (BioWhittaker, Walkersville, MD), 0.1% nonessential amino acids, 0.1% sodium pyruvate (Cellgro), and 0.05% 2-ME. The 1B2 mAb was
purified from supernatants using a protein A-Sepharose column with elution by citric acid at pH 3.5. Fractions containing Ab were pooled, dialyzed
into PBS, and biotinylated for 4 h at room temperature with 0.15 mg/ml
biotin in DMSO and 0.15 mg/ml NaHCO3 per 1 mg/ml mAb. Other
Abs used were anti-CD44-FITC (Pgp1), anti-CD25-FITC, anti-CD8aCyChrome, streptavidin-APC, and rat IgG2a k-FITC isotype control (all
from PharMingen, San Diego, CA).
Dickinson, San Jose, CA), as described (23). All cytometer settings were
identical for all time points within a given experiment. A total of 35– 40 3
103 lymphocyte-gated events were collected and analyzed, and the percentage of 1B21 CD81 cells was multiplied by the total number of cells
recovered from the site to determine the total number of 1B21 CD81 cells.
All determinations were done in duplicate mice for each condition and time
point. Values shown are averages, and error bars represent the range of the
duplicates. Phenotypes of the 2C cells from various sites were determined
by gating on the 1B21CD81 cells and collecting 200 events examining the
FITC fluorescence of the various phenotype marker mAb. Gates denoting
high and low expression of each surface marker were set based on the
phenotype of naive T cells. Replacing the specific mAbs with isotype control mAbs resulted in almost no events (,1%) falling into the high gate for
each marker.
The Journal of Immunology
2563
had undergone blast transformation (52.7% FSChigh) (Fig. 1C, bottom, and Table I), indicating that the 2C cells had indeed recognized and responded to the peptide challenge. This low-level response, however, was very transient, since 2C cell numbers in all
sampled sites dropped to a point where accurate phenotypic analysis could not be made by day 8, and, by day 20, these cells were
only detectable close to background (0.01% of total cells). To determine whether this response was specific, adoptively transferred
mice were also immunized with an equal amount of SIINFEKL, an
OVA peptide also bound by H-Kb. The 1B21CD81 cells in these
mice remained essentially the same in both absolute numbers and
phenotype as those found in transferred mice immunized with PBS
(data not shown).
The reduced and transient CD81 T cell response to soluble antigenic peptide immunization (as compared with CFA and peptide)
is consistent with the adjuvant effect noted in previous studies
examining CD41 T cells in an adoptive transfer system. Immunization with peptide alone caused some CD41 T cell clonal expansion, but this response was short-lived and resulted in long-term
Ag-specific nonresponsiveness. In contrast, peptide immunization
with adjuvant caused much larger clonal expansion (up to 3-fold
higher than with peptide alone) along with development of effector
function (9). The adjuvant effect is also present for CD81 T cells,
but the level of proliferation obtained with adjuvant in comparison
to peptide alone is much greater (up to 25-fold greater with adjuvant; Fig. 1, A and B). The fact that transferred 2C cells appeared
to proliferate simultaneously in all sampled sites with peptide
alone, as opposed to proliferating first in the DLN and subsequent
migration to the spleen and PBL, as seen with CFA and peptide,
may suggest that the adjuvant and peptide emulsification serves as
Table I. Phenotype of 1B21CD81 cellsa
Day 1
Day 3
Phenotype
DLN (%)
PBL (%)
CD25high
CD44high
FSChigh
4.5
14.1
13.7
4.8
14.1
13.7
CD25high
CD44high
FSChigh
34.7
39.8
52.7
12.5
38.8
33.6
DLN (%)
Day 5
PBL (%)
DLN (%)
After CFA/peptide challenge
49.7
26.4
70.9
84.5
74.8
47.5
After peptide only challenge
7.2
3.8
27.4
26.1
17.0
16.2
Day 8
PBL (%)
DLN (%)
PBL (%)
12.4
64.0
7.4
8.4
87.0
3.6
16.0
69.9
12.8
3.7
85.1
13.8
9.3
24.0
1.3
ND
ND
ND
ND
ND
ND
ND
ND
ND
a
Phenotype percentages of transferred 1B21CD81 cells in the DLN and PBL from day 1 to day 8 after SIYRYYGL peptide immunization (50 mg), either in the presence
or absence of CFA. All mice were adoptively transferred with 3–5 3 106 1B21CD81 T cells 24 h prior to immunization. FSC high values are indicative of blast transformation.
Phenotypes were based on 200 1B21CD81 gated events, and gates denoting high and low expression of each surface marker were set based on the phenotype of naive CD81
T cells. Percentages shown are representative of 10 independent experiments in which duplicate mice were used for each time point and immunization. ND, accurate phenotype
analysis was unavailable due to low 1B21CD81 cell numbers.
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FIGURE 1. 1B21CD81 cell numbers, location, and CD44 phenotype during the primary
response to SIYRYYGL. Transferred 2C cells
(3–5 3 106) were detected during the course of
the primary response by flow cytometry using
the 1B2 mAb specific for the 2C TCR and flow
cytometry. A, 1B21CD81 cell numbers in the
DLN, MLN, spleen, and PBL from days 1– 8
after challenge with SIYRYYGL emulsified in
CFA (50 mg peptide in 300 ml delivered s.c. to
two sites on the back of the mouse). B, As in A,
except after challenge with SIYRYYGL (50 mg)
in PBS. Inset graph shows the same data plotted
using a different scale on the y-axis. C, CD44
levels of 1B21CD81 populations from recipient
mice after indicated immunizations. Data is representative of 10 independent experiments. Each
time point and condition was performed in duplicate mice. Values shown represent the average of the duplicate mice and error bars represent the range.
2564
an Ag depot to prevent the peptide from being distributed systemically in the animal. Comparable results to those shown in Fig. 1
and Table I were obtained in 10 independent experiments. Additional experiments demonstrated that IFA had similar effects to
CFA in this system (data not shown).
IL-12 mimics the CFA adjuvant effect during an in vivo peptide
response
Based on its proinflammatory properties and previous in vitro results (14, 16), IL-12 was examined to determine whether it could
replace CFA in the context of clonal expansion and development
of effector function. Cells from the DLN, spleen, and PBL were
examined on day 3 after immunization to correspond to the time of
maximum clonal expansion seen in the CFA/peptide experiments
described above. IL-12 (1 mg/day, 2.7 3 103 6 1.2 3 103 U/mg)
was administered i.p. on days 0, 1, and 2 in 100 ml PBS with 0.1%
sterile mouse serum in addition to s.c. peptide immunization. IL12/peptide supported nearly the same extent of clonal expansion as
CFA/peptide in the DLN on day 3 (Fig. 2). IL-12 also caused
substantial clonal expansion in the spleen, most likely a consequence of both peptide and IL-12 being systemic, as opposed to the
Ag remaining localized in CFA (Fig. 2). The phenotypes of
1B21CD81 cells responding to IL-12/peptide were also nearly
identical to those primed with CFA/peptide, with high levels of
CD25, CD44, and blast transformation by day 3, and also showed
decreased PKH26 fluorescence as described above (data not
shown, and Ref. 16). To address whether this IL-12 effect was
CD41 T cell-dependent, CD42/2 mice were also used as naive
adoptive transfer recipients. Proliferation and phenotype changes
of transferred cells in response to CFA/peptide and IL-12/peptide
were essentially the same as those observed in normal C57BL/6
recipients (data not shown), demonstrating that CD41 T cells do
not contribute in a detectable way to these responses.
FIGURE 3. CFA and/or IL-12 support development of lytic effector
function of transferred 1B21CD81 cells primed with SIYRYYGL. Adoptively transferred mice were immunized with the indicated combinations of
Ag on day 0, and lytic effector function was measured on day 3 by direct
ex vivo 51Cr-release assay against SIYRYYGL-pulsed EL4 target cells.
CD81 cells were first purified from transferred animals by negative selection columns. Transferred 1B21CD81 cells represent a small percentage of
the purified CD81 cells; thus, at the 100:1 E:T data points, the actual
1B21CD81:target ratios are: transfer only, 0.12:1; peptide only, 0.32:1;
IL-12/peptide, 1.33:1; CFA/peptide, 0.52:1; no transfer, CFA/peptide ,
0.01:1. The “no transfer:CFA/peptide” group represents mice that did not
receive an adoptive transfer of 1B21CD81 cells, but were immunized with
CFA/peptide. Transfer only, peptide only, and no transfer:CFA/peptide
samples all had specific lysis of ,1.6%. Data is representative of four
independent experiments. Duplicate mice were used for each immunization, and DLN cells were pooled before CD81 column purification to increase the number of 1B21CD81 T cells in the assay culture.
Along with causing increased clonal expansion, IL-12 was also
effective in supporting effector function development. CTL activity
of 1B21CD81 cells was directly measured ex vivo using
SIYRYYGL-pulsed target cells in a standard 4-h 51Cr release assay. DLNs of transferred and immunized animals were harvested
and pooled for two mice on day 3, and total CD81 cells were
column-purified by negative selection (see Materials and Methods). The purified CD81 T cells were then assayed for lytic activity at varying E:T ratios (Fig. 3) and analyzed through flow
cytometry to determine the percentages of 1B21CD81 T cells in
this population. To establish that target cell lysis was indeed due to
the transferred 1B21 population, naive C57BL/6 mice that were
not adoptively transferred were also immunized with CFA/peptide
and assayed. IL-12/peptide and CFA/peptide did induce lytic effector function of transferred cells (30.6% and 19.2% specific lysis, respectfully) as measured at a 100:1 E:T ratio using the total
CD81 population. When corrected for the number of 1B21CD81
cells in the purifed CD81 populations, the actual E:T ratios at
100:1 are as follows: transfer only, 0.12:1; peptide only, 0.32:1;
IL-12/peptide, 1.33:1; CFA/peptide, 0.52:1; no transfer, CFA/peptide , 0.01:1. The CFA/peptide-primed, nontransfer control samples had little detectable killing above background, nor did the
transfer only and peptide only samples show significant activity
(,1.6% specific lysis; Fig. 3). These data suggest that both IL-12
and CFA support development of effector function during peptide
immunization and prevent tolerance induction.
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FIGURE 2. 1B21CD81 cell numbers in the DLN, spleen, and PBL on
day 3 after immunizing with SIYRYYGL in the presence or absence of
adjuvant or IL-12. Immunizations on day 0 include PBS alone (transfer
only), SIYRYYGL in PBS (peptide only), SIYRYYGL in CFA (CFA/
peptide), and SIYRYYGL 1 IL-12 (IL-12/peptide). All immunizations
included 50 mg peptide in 300 ml CFA or PBS distributed between two
sites on the back. Recombinant murine IL-12 (1 mg/mouse in 100 ml PBS
with 0.1% sterile mouse serum) was injected i.p. on days 0, 1, and 2. Data
is representative of seven independent experiments. Each time point and
condition was performed in duplicate mice. Values shown represent the
average of the duplicate mice and error bars represent the range.
IL-12 AS ADJUVANT FOR NAIVE CD81 T CELLS
The Journal of Immunology
2565
adoptively transferred into either IL-12 KO mice or naive
C57BL/6 mice, to evaluate the effects of both CFA and IL-12
during peptide immunization. Fig. 4 shows the absolute numbers
of 1B21CD81 cells in the DLN on days 3 and 5 after immunization. The CFA/peptide response was still intact in the IL-12 KO
mice, indicating that IL-12 is not unique in providing the adjuvant
effect. IL-12 was also still able to support a strong proliferative
response in these mice.
IL-12 acts as an adjuvant to prevent tolerance, while supporting
memory establishment
Although immunization with peptide in the absence of adjuvant
(peptide only) does stimulate some limited clonal expansion (Fig.
1B), this does not appear sufficient to induce lytic function in these
cells (Fig. 3). No killing activity was detectable in cells from the
mice that received just peptide (peptide only) when lysis was examined at a 1B21CD81:target ratio, where killing was readily
apparent by cells from mice immunized with peptide along with
CFA or IL-12.
IL-12 is not required for the CFA adjuvant effect
While IL-12 can be necessary and sufficient to support a response
to peptide Ag, it may not be the only means of supporting this
response. To examine this, IL-12 (p40)-deficient mice (25) were
used as naive recipients for the 2C adoptive transfer to determine
whether a response could still be obtained to CFA/peptide immunization. Column-purified 1B21CD81 T cells from 2C mice were
FIGURE 5. CFA 1 SIYRYYGL and
IL-12 1 SIYRYYGL induce a memory
population of 1B21 cells capable of responding to secondary SIYRYYGL
challenge. Naive C57BL/6 mice were
adoptively transferred with 1B21CD81
cells on day 21, and immunized with
the Ag combinations listed first on day
0. Sixty days later, the mice were rechallenged with the Ag combinations listed
second, and 1B21CD81 numbers were
measured on day 63 by flow cytometry
using the 1B2 mAb. Results shown are
averages of duplicate mice analyzed for
each condition, and error bars indicate
the range of values obtained. Essentially,
identical results were obtained in two independent experiments.
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FIGURE 4. CFA 1 SIYRYYGL and IL-12 1 SIYRYYGL administration to 2C transferred, IL-12 KO recipient mice results in 1B21CD81 T
cell clonal expansion. IL-12 KO mice (25) and naive C57BL/6 mice were
first transferred with 5 3 106 column-purified 1B21CD81 cells on day 21
and then immunized on day 0 with the indicated combinations of Ag. The
resulting 1B21CD81 clonal expansion was measured on day 3 and day 5.
Results are the averages of duplicate mice for each condition, and error
bars indicate the range of values obtained. Essentially, identical results
were obtained in two independent experiments.
To directly analyze the effects of IL-12 and CFA on tolerance vs
establishment of a memory population, transferred and immunized
animals were given a secondary immunization 60 days after the
primary challenge. The DLN, spleen, and PBL of the animals were
then analyzed on day 63 (day 3 of secondary immunization) for
1B21CD81 clonal expansion. Animals that were transferred but
only given PBS for both immunizations (PBS/PBS: primary immunizations for mice groups are listed first, followed by the secondary challenge) had a low but detectable level of 1B21CD81
cells in the DLN (3.1 3 103 cells/mouse) and spleen (7.0 3 103
cells/mouse) on day 63 (Fig. 5). These cells, being naive, were
capable of responding to CFA/peptide challenge (PBS/CFA 1
peptide). Animals that were immunized first with peptide alone
and then received just PBS (peptide only/PBS) also had small populations of 1B21CD81 cells in the DLN (3.4 3 103 cells/mouse)
and spleen (7.0 3 103 cells/mouse) (Fig. 5), and these had a
CD44high phenotype (39%), indicating that they had made some
response to the initial immunization (data not shown). These cells,
however, were incapable of responding to a secondary challenge of
peptide even in the presence of CFA (peptide only/CFA 1 peptide). This lack of proliferation to secondary challenge with peptide was prevented by initially using CFA or IL-12 during the
primary peptide immunization. This resulted in a larger
1B21CD81 population in the DLN by day 63 (6.5 3 104 for CFA
1 peptide/PBS and 3.6 3 104 for IL-12 1 peptide/PBS), which is
11- to 19-fold larger than the population observed with peptide
alone (peptide only/PBS). In addition, these cells had a phenotype
characteristic of memory cells (.75% CD44 high; data not shown).
Secondary challenge of these cells with peptide and CFA or IL-12
on day 60 resulted in significant proliferation within 3 days. If both
2566
peptide immunizations were accompanied by CFA (CFA 1 peptide/CFA 1 peptide), a 3-fold increase in 1B21CD81 cell numbers (over CFA 1 peptide/PBS) was observed in the DLN. If
IL-12 was administered with peptide on both days 0 and 60 (IL-12
1 peptide/IL-12 1 peptide), the subsequent memory response
dwarfed that seen with CFA, and even approached the levels seen
in a primary response (nearly 22-fold higher than IL-12 1 peptide/
PBS; Fig. 5). To insure that this 1B21CD81 response to secondary
challenge is indeed a memory response, donor cells were labeled
with PKH26 lipophilic dye before adoptive transfer and primary
immunization, as mentioned above. The fluorescence intensity of
1B21CD81 cells was completely lost by day 8 as the primary
response occurred, demonstrating that all detectable cells had responded to the initial Ag encounter (data not shown, and Ref. 16).
Discussion
responded to secondary challenge with IL-12 and peptide (up to
22-fold expansion; Fig. 5)
Although these results suggest that IL-12 is necessary and sufficient to prevent Ag-specific tolerance, the exact mechanisms of
how this occurs in vivo are not known. Previous in vitro models
have demonstrated that IL-12 acts directly on the CD81 T cell,
along with TCR ligation and costimulation, to induce proliferation
and development of lytic effector function (16). Although the in
vivo proliferation and CTL activity shown above are consistent
with these previous studies, it is not known whether administered
IL-12 acts directly on the 1B21CD81 T cell in the recipient animal. Given the broad immunological effects of IL-12, including
Th1 differentiation and NK cell stimulation, this proinflammatory
cytokine may serve to systematically increase levels of IFN-g
and/or effect the expression of costimulatory molecules present on
the surface of APC. Recent evidence is suggesting that CD41 T
cells actually provide help to CD81 T cells by conditioning APC,
via CD40 ligation, to more effectively present Ag to CD81 T cells
and stimulate their response (29 –31). It is possible that this involves induction of IL-12 production by the APC, known to be
stimulated by CD40 ligation (32, 33), and that the IL-12 then supports CD81 T cell proliferation and differentiation in response to
Ag and costimulation on the APC.
Even though the effects of IL-12 during a peptide response appear to mimic those observed when CFA is used, IL-12 may only
provide part of the overall adjuvant effect. Our data suggest that
IL-12 does not fully account for the adjuvant effect of CFA, as the
response is still intact in IL-12 KO mice (Fig. 4). The exact mechanisms of how CFA prevents tolerance are not known, although
increased costimulation or combinations of other cytokines may be
responsible for the observed effects. IL-12 also had a strong effect
(well above peptide alone) in these same IL-12 KO animals, further confirming that IL-12 prevents Ag-specific tolerance. The in
vivo CD81 T cell effects of IL-12 directly contrast with previous
in vivo studies of CD41 T cells, which indicate that IL-1, and not
IL-12, is sufficient to promote clonal expansion and differentiation
(11). Conversely, IL-1 was previously found to not promote these
same events in CD81 T cells (16). Thus, it appears that these two
distinct populations of T cells are influenced differently with respect to inflammatory mediators.
The antitumor effects of IL-12 have been well-characterized;
murine tumor models have focused heavily on delivery of this
cytokine, ranging from locally with transduction and Ab-fusion
protein experiments to systemic i.p. injections, all with promising
results (34 –37). Other in vivo murine tumor models indicate that
IL-12 may even contribute to CTL migration to the site of tumor
load (38). Clinical trials have implicated IL-12 in promoting a
higher CTL precursor frequency, although initial IL-12 protocols
resulted in severe human toxicity (21, 22). In addition to the potential therapeutic value of IL-12 for established pathological conditions, the results reported here suggest that it may provide a very
effective adjuvant for immunizing for protective CD81 T cell responses. T cell activation, proliferation, lytic effector function, and
memory establishment are all comparable to those induced by traditional adjuvants, and toxic effects may be minimal or absent with
the limited amounts of IL-12 required for the adjuvant effect.
Acknowledgments
We thank Debra Lins for excellent technical assistance, and Drs. Marc
Jenkins and Daniel Mueller for critical reading of the manuscript.
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