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Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous CD4+ 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 strong in vivo peptide response for
CD4+ 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-
, that can
positively influence a naive CD8+ T cell’s
proliferation and/or differentiation to effector CTL
(12, 13, 14, 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 CD8+ T cell to promote
clonal expansion and differentiation (16). The in vitro
results demonstrating a direct effect of IL-12 on
CD8+ T cells suggested that this cytokine might
prevent Ag-specific tolerance upon peptide administration, and support
an effective in vivo CD8+ T cell response.
The heterodimeric cytokine IL-12 was originally identified as NK cell
stimulating factor, and is known to stimulate
CD4+ 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- (18, 19, 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
CD8+ T cell response. To examine this, we have
used a system in which a small number of naive Ag-specific
CD8+ 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. CD8+ 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
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-CD8
- CyChrome, streptavidin-APC, and
rat IgG2a 
-FITC isotype control (all from PharMingen, San
Diego, CA).
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 CD8+ cells using the CD8+ Cellect column purification kit (Biotex Laboratories, Edmonton, Canada). Before transfer, the purified population was analyzed by flow cytometry to determine the percentage of 1B2+ CD8+ 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 x 106 1B2+ CD8+ cells in 500 µl 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 µg peptide in 300 µl per mouse distributed between two sites on the back. Recombinant murine IL-12 (Genetics Institute, Cambridge, MA) was administered i.p. at 1 µg in 100 µl 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 x 106) from each site were stained
with 1B2-biotin mAb, anti-CD8-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 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 x 103
lymphocyte-gated events were collected and analyzed, and the percentage
of 1B2+ CD8+ cells was
multiplied by the total number of cells recovered from the site to
determine the total number of 1B2+
CD8+ 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 1B2+CD8+ 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.
Chromium release assay
Following immunizations, DLN cells of duplicate mice were harvested, pooled, homogenized, and enriched for CD8+ cells using the CD8+ Cellect column purification kit (Biotex Laboratories). These cells (of which 0.12–1.12% are 1B2+ CD8+) were then assayed using a standard 4-h 51Cr release assay with SIYRYYGL-pulsed EL4 target cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary response of naive CD8+ T cells to soluble peptide Ag was monitored in vivo through the use of an adoptive transfer system utilizing 2C TCR transgenic CD8+ T cells that recognize the synthetic peptide SIYRYYGL in the context of H-2Kb (23, 24, 27). Phenotypically naive CD8+ 2C T cells (3–5 x 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 1B2+ CD8+ 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 µg/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 1B2+CD8+ responses (data not shown).
Immunization with SIYRYYGL emulsified in CFA resulted in massive
1B2+ CD8+ clonal expansion
in the DLN (inguinal, brachial, axillary) by day 3 (Fig. 1
A). 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
1B2+ 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). 1B2+
CD8+ 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 1B2+CD8+
cells (4–6 x 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
CD25-CD44low phenotype
through day 20.
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B, 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 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
1B2+CD8+ 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 CD8+ 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 CD4+ T cells in an adoptive
transfer system. Immunization with peptide alone caused some
CD4+ 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
CD8+ 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 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
µg/day, 2.7 x 103 ± 1.2 x
103 U/µg) was administered i.p. on days 0, 1,
and 2 in 100 µl PBS with 0.1% sterile mouse serum in addition to
s.c. peptide immunization. IL-12/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
1B2+CD8+ 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 CD4+ T cell-dependent,
CD4-/- 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 CD4+ T cells do not contribute
in a detectable way to these responses.
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) and
analyzed through flow cytometry to determine the percentages of
1B2+CD8+ T cells in this
population. To establish that target cell lysis was indeed due to the
transferred 1B2+ 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
CD8+ population. When corrected for the number of
1B2+CD8+ cells in the
purifed CD8+ 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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B), 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
1B2+CD8+: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 1B2+CD8+ T
cells from 2C mice were 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
1B2+CD8+ 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.
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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
1B2+CD8+ 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
1B2+CD8+ cells in the DLN
(3.1 x 103 cells/mouse) and spleen
(7.0 x 103 cells/mouse) on day 63 (Fig. 5). These cells, being naive, were
capable of responding to CFA/peptide challenge (PBS/CFA + peptide).
Animals that were immunized first with peptide alone and then received
just PBS (peptide only/PBS) also had small populations of
1B2+CD8+ cells in the DLN
(3.4 x 103 cells/mouse) and spleen
(7.0 x 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 + 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
1B2+CD8+ population in the
DLN by day 63 (6.5 x 104 for CFA +
peptide/PBS and 3.6 x 104 for IL-12 +
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 peptide immunizations
were accompanied by CFA (CFA + peptide/CFA + peptide), a 3-fold
increase in 1B2+CD8+ cell
numbers (over CFA + peptide/PBS) was observed in the DLN. If IL-12 was
administered with peptide on both days 0 and 60 (IL-12 + peptide/IL-12
+ 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 + peptide/PBS; Fig. 5). To insure that this
1B2+CD8+ 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
1B2+CD8+ 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B). This disseminated response could possibly be due to the
peptide being systemic after s.c. injection, as opposed to localized
when administered with CFA. The initial response, however, is very
transient, and few 1B2+CD8+
cells can be detected by day 20. Ex vivo effector CTL activity was not
detected in these mice, but this could be a consequence of the small
number of 1B2+CD8+ cells
present in the entire CD8+ population. These
cells were present in the DLN and spleen up to day 63, but appeared
tolerized since they were incapable of responding to secondary peptide
challenge even in the presence of adjuvant (<1.2-fold expansion;
Fig. 5).
In contrast, when peptide is administered with adjuvant (CFA), a large
1B2+CD8+ proliferative
response is detected first in the DLN (Fig. 1A), followed by
migration to the spleen and peripheral blood. The initial proliferation
is very localized to the DLN (axillary, brachial, and inguinal), while
1B2+CD8+ cells detected in
nondraining (mesenteric) LN remain at a constant low level throughout
the response. As the initial proliferative response subsides, the cell
numbers decline to leave a small, stable population of cells with a
memory phenotype (54.6% CD44high). In addition
to proliferating, these
1B2+CD8+ cells also develop
effector CTL activity by day 3, as measured by a direct ex vivo
51Cr-release assay (Fig. 3). CFA/peptide
immunization results in immunologic memory by day 60, as these same
cells were capable of responding to secondary challenge with peptide
and adjuvant by expanding up to 3-fold (Fig. 5).
Administration of IL-12 during peptide immunization resulted in massive
clonal expansion similar to that seen with CFA/peptide (Fig. 2). This
proliferation was not limited to the DLN, as large numbers of
1B2+CD8+ cells could also
be detected in the spleen. IL-12/peptide immunization also induced
lytic effector development of
1B2+CD8+ cells, since cells
isolated directly ex vivo were capable of killing peptide-pulsed EL4
target cells (Fig. 3). In addition to preventing Ag-specific tolerance
during a primary response (as measured by proliferation and effector
function), IL-12 was also capable of generating a stable memory
population that 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 CD8+
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
1B2+CD8+ 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- and/or effect the expression of costimulatory molecules present
on the surface of APC. Recent evidence is suggesting that
CD4+ T cells actually provide help to
CD8+ T cells by conditioning APC, via CD40
ligation, to more effectively present Ag to CD8+
T cells and stimulate their response (29, 30, 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 CD8+ 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 CD8+ T cell
effects of IL-12 directly contrast with previous in vivo studies of
CD4+ 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 CD8+ 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, 35, 36, 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 CD8+ 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.
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Acknowledgments |
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Footnotes |
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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:
3 Abbreviations used in this paper: KO, knockout; FSC, forward scatter; LN, lymph node; DLN, draining LN; MLN, mesenteric LN.
Received for publication April 9, 1999. Accepted for publication June 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) interferon for their growth and differentiation into effector cells. Curr. Top. Microbiol. Immunol. 126:173.[Medline]
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M. J. Anderson, K. Shafer-Weaver, N. M. Greenberg, and A. A. Hurwitz Tolerization of Tumor-Specific T Cells Despite Efficient Initial Priming in a Primary Murine Model of Prostate Cancer J. Immunol., February 1, 2007; 178(3): 1268 - 1276. [Abstract] [Full Text] [PDF]
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S. Koido, E. Hara, S. Homma, A. Torii, M. Mitsunaga, S. Yanagisawa, Y. Toyama, H. Kawahara, M. Watanabe, S. Yoshida, et al. Streptococcal Preparation OK-432 Promotes Fusion Efficiency and Enhances Induction of Antigen-Specific CTL by Fusions of Dendritic Cells and Colorectal Cancer Cells J. Immunol., January 1, 2007; 178(1): 613 - 622. [Abstract] [Full Text] [PDF]
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G. Murugaiyan, R. Agrawal, G. C. Mishra, D. Mitra, and B. Saha Functional Dichotomy in CD40 Reciprocally Regulates Effector T Cell Functions J. Immunol., November 15, 2006; 177(10): 6642 - 6649. [Abstract] [Full Text] [PDF]
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 N. Sanarico, A. Ciaramella, A. Sacchi, D. Bernasconi, P. Bossu, F. Mariani, V. Colizzi, and S. Vendetti Human monocyte-derived dendritic cells differentiated in the presence of IL-2 produce proinflammatory cytokines and prime Th1 immune response J. Leukoc. Biol., September 1, 2006; 80(3): 555 - 562. [Abstract] [Full Text] [PDF]
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J. M. Curtsinger, D. C. Lins, C. M. Johnson, and M. F. Mescher Signal 3 Tolerant CD8 T Cells Degranulate in Response to Antigen but Lack Granzyme B to Mediate Cytolysis J. Immunol., October 1, 2005; 175(7): 4392 - 4399. [Abstract] [Full Text] [PDF]
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M. A. Markiewicz, L. N. Carayannopoulos, O. V. Naidenko, K. Matsui, W. R. Burack, E. L. Wise, D. H. Fremont, P. M. Allen, W. M. Yokoyama, M. Colonna, et al. Costimulation through NKG2D Enhances Murine CD8+ CTL Function: Similarities and Differences between NKG2D and CD28 Costimulation J. Immunol., September 1, 2005; 175(5): 2825 - 2833. [Abstract] [Full Text] [PDF]
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 Y. Li, J. S. Heuser, S. D. Kosanke, M. Hemric, and M. W. Cunningham Protection against Experimental Autoimmune Myocarditis Is Mediated by Interleukin-10-Producing T Cells that Are Controlled by Dendritic Cells Am. J. Pathol., July 1, 2005; 167(1): 5 - 15. [Abstract] [Full Text] [PDF]
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A. A. Filatenkov, E. L. Jacovetty, U. B. Fischer, J. M. Curtsinger, M. F. Mescher, and E. Ingulli CD4 T Cell-Dependent Conditioning of Dendritic Cells to Produce IL-12 Results in CD8-Mediated Graft Rejection and Avoidance of Tolerance J. Immunol., June 1, 2005; 174(11): 6909 - 6917. [Abstract] [Full Text] [PDF]
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J. M. Curtsinger, J. O. Valenzuela, P. Agarwal, D. Lins, and M. F. Mescher Cutting Edge: Type I IFNs Provide a Third Signal to CD8 T Cells to Stimulate Clonal Expansion and Differentiation J. Immunol., April 15, 2005; 174(8): 4465 - 4469. [Abstract] [Full Text] [PDF]
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J. O. Valenzuela, C. D. Hammerbeck, and M. F. Mescher Cutting Edge: Bcl-3 Up-Regulation by Signal 3 Cytokine (IL-12) Prolongs Survival of Antigen-Activated CD8 T Cells J. Immunol., January 15, 2005; 174(2): 600 - 604. [Abstract] [Full Text] [PDF]
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S. Reich-Zeliger, J. Gan, E. Bachar-Lustig, and Y. Reisner Tolerance Induction by Veto CTLs in the TCR Transgenic 2C Mouse Model. II. Deletion of Effector Cells by Fas-Fas Ligand Apoptosis J. Immunol., December 1, 2004; 173(11): 6660 - 6666. [Abstract] [Full Text] [PDF]
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A. T. Stock, S. N. Mueller, A. L. van Lint, W. R. Heath, and F. R. Carbone Cutting Edge: Prolonged Antigen Presentation after Herpes Simplex Virus-1 Skin Infection J. Immunol., August 15, 2004; 173(4): 2241 - 2244. [Abstract] [Full Text] [PDF]
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A. Moroz, C. Eppolito, Q. Li, J. Tao, C. H. Clegg, and P. A. Shrikant IL-21 Enhances and Sustains CD8+ T Cell Responses to Achieve Durable Tumor Immunity: Comparative Evaluation of IL-2, IL-15, and IL-21 J. Immunol., July 15, 2004; 173(2): 900 - 909. [Abstract] [Full Text] [PDF]
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M. L. Salem, A. N. Kadima, Y. Zhou, C. L. Nguyen, M. P. Rubinstein, M. Demcheva, J. N. Vournakis, D. J. Cole, and W. E. Gillanders Paracrine Release of IL-12 Stimulates IFN-{gamma} Production and Dramatically Enhances the Antigen-Specific T Cell Response after Vaccination with a Novel Peptide-Based Cancer Vaccine J. Immunol., May 1, 2004; 172(9): 5159 - 5167. [Abstract] [Full Text] [PDF]
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 C. L. Ahonen, C. L. Doxsee, S. M. McGurran, T. R. Riter, W. F. Wade, R. J. Barth, J. P. Vasilakos, R. J. Noelle, and R. M. Kedl Combined TLR and CD40 Triggering Induces Potent CD8+ T Cell Expansion with Variable Dependence on Type I IFN J. Exp. Med., March 15, 2004; 199(6): 775 - 784. [Abstract] [Full Text] [PDF]
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J. Chang, J.-H. Cho, S.-W. Lee, S.-Y. Choi, S.-J. Ha, and Y.-C. Sung IL-12 Priming during In Vitro Antigenic Stimulation Changes Properties of CD8 T Cells and Increases Generation of Effector and Memory Cells J. Immunol., March 1, 2004; 172(5): 2818 - 2826. [Abstract] [Full Text] [PDF]
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J. M. Curtsinger, C. M. Johnson, and M. F. Mescher CD8 T Cell Clonal Expansion and Development of Effector Function Require Prolonged Exposure to Antigen, Costimulation, and Signal 3 Cytokine J. Immunol., November 15, 2003; 171(10): 5165 - 5171. [Abstract] [Full Text] [PDF]
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H. Beacock-Sharp, A. M. Donachie, N. C. Robson, and A. M. Mowat A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo Int. Immunol., June 1, 2003; 15(6): 711 - 720. [Abstract] [Full Text] [PDF]
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 L. Krishnan, S. Sad, G. B. Patel, and G. D. Sprott Archaeosomes Induce Enhanced Cytotoxic T Lymphocyte Responses to Entrapped Soluble Protein in the Absence of Interleukin 12 and Protect against Tumor Challenge Cancer Res., May 15, 2003; 63(10): 2526 - 2534. [Abstract] [Full Text] [PDF]
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J. M. Curtsinger, D. C. Lins, and M. F. Mescher Signal 3 Determines Tolerance versus Full Activation of Naive CD8 T Cells: Dissociating Proliferation and Development of Effector Function J. Exp. Med., May 5, 2003; 197(9): 1141 - 1151. [Abstract] [Full Text] [PDF]
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S. W. Lee, Y. Park, J. K. Yoo, S. Y. Choi, and Y. C. Sung Inhibition of TCR-Induced CD8 T Cell Death by IL-12: Regulation of Fas Ligand and Cellular FLIP Expression and Caspase Activation by IL-12 J. Immunol., March 1, 2003; 170(5): 2456 - 2460. [Abstract] [Full Text] [PDF]
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J. Goldberg, P. Shrikant, and M. F. Mescher In Vivo Augmentation of Tumor-Specific CTL Responses by Class I/Peptide Antigen Complexes on Microspheres (Large Multivalent Immunogen) J. Immunol., January 1, 2003; 170(1): 228 - 235. [Abstract] [Full Text] [PDF]
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J. Valenzuela, C. Schmidt, and M. Mescher The Roles of IL-12 in Providing a Third Signal for Clonal Expansion of Naive CD8 T Cells J. Immunol., December 15, 2002; 169(12): 6842 - 6849. [Abstract] [Full Text] [PDF]
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L. A. E. Harmala, E. G. Ingulli, J. M. Curtsinger, M. M. Lucido, C. S. Schmidt, B. J. Weigel, B. R. Blazar, M. F. Mescher, and C. A. Pennell The Adjuvant Effects of Mycobacterium tuberculosis Heat Shock Protein 70 Result from the Rapid and Prolonged Activation of Antigen-Specific CD8+ T Cells In Vivo J. Immunol., November 15, 2002; 169(10): 5622 - 5629. [Abstract] [Full Text] [PDF]
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J. Hernandez, S. Aung, K. Marquardt, and L. A. Sherman Uncoupling of Proliferative Potential and Gain of Effector Function by CD8+ T Cells Responding to Self-Antigens J. Exp. Med., August 5, 2002; 196(3): 323 - 333. [Abstract] [Full Text] [PDF]
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C. S. Schmidt and M. F. Mescher Peptide Antigen Priming of Naive, But Not Memory, CD8 T Cells Requires a Third Signal That Can Be Provided by IL-12 J. Immunol., June 1, 2002; 168(11): 5521 - 5529. [Abstract] [Full Text] [PDF]
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A. D. Higgins, M. A. Mihalyo, P. W. McGary, and A. J. Adler CD4 Cell Priming and Tolerization Are Differentially Programmed by APCs upon Initial Engagement J. Immunol., June 1, 2002; 168(11): 5573 - 5581. [Abstract] [Full Text] [PDF]
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J. Gong, S. Koido, D. Chen, Y. Tanaka, L. Huang, D. Avigan, K. Anderson, T. Ohno, and D. Kufe Immunization against murine multiple myeloma with fusions of dendritic and plasmacytoma cells is potentiated by interleukin 12 Blood, April 1, 2002; 99(7): 2512 - 2517. [Abstract] [Full Text] [PDF]
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G.-X. Zhang, H. Xu, M. Kishi, D. Calida, and A. Rostami The Role of IL-12 in the Induction of Intravenous Tolerance in Experimental Autoimmune Encephalomyelitis J. Immunol., March 1, 2002; 168(5): 2501 - 2507. [Abstract] [Full Text] [PDF]
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 P. Lee, F. Wang, J. Kuniyoshi, V. Rubio, T. Stuges, S. Groshen, C. Gee, R. Lau, G. Jeffery, K. Margolin, et al. Effects of Interleukin-12 on the Immune Response to a Multipeptide Vaccine for Resected Metastatic Melanoma J. Clin. Oncol., September 15, 2001; 19(18): 3836 - 3847. [Abstract] [Full Text] [PDF]
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 R. M. Kedl, M. Jordan, T. Potter, J. Kappler, P. Marrack, and S. Dow CD40 stimulation accelerates deletion of tumor-specific CD8+ T cells in the absence of tumor-antigen vaccination PNAS, August 23, 2001; (2001) 191371898. [Abstract] [Full Text] [PDF]
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D. Stober, R. Schirmbeck, and J. Reimann IL-12/IL-18-Dependent IFN-{{gamma}} Release by Murine Dendritic Cells J. Immunol., July 15, 2001; 167(2): 957 - 965. [Abstract] [Full Text] [PDF]
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W. C. Kieper, M. Prlic, C. S. Schmidt, M. F. Mescher, and S. C. Jameson IL-12 Enhances CD8 T Cell Homeostatic Expansion J. Immunol., May 1, 2001; 166(9): 5515 - 5521. [Abstract] [Full Text] [PDF]
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E. J. Roy, U. Gawlick, B. A. Orr, L. A. Rund, A. G. Webb, and D. M. Kranz IL-12 Treatment of Endogenously Arising Murine Brain Tumors J. Immunol., December 15, 2000; 165(12): 7293 - 7299. [Abstract] [Full Text] [PDF]
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S. Koido, M. Kashiwaba, D. Chen, S. Gendler, D. Kufe, and J. Gong Induction of Antitumor Immunity by Vaccination of Dendritic Cells Transfected with MUC1 RNA J. Immunol., November 15, 2000; 165(10): 5713 - 5719. [Abstract] [Full Text] [PDF]
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P. J. Mosca, A. C. Hobeika, T. M. Clay, S. K. Nair, E. K. Thomas, M. A. Morse, and H. K. Lyerly A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment Blood, November 15, 2000; 96(10): 3499 - 3504. [Abstract] [Full Text] [PDF]
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P. J. Skinner, M. A. Daniels, C. S. Schmidt, S. C. Jameson, and A. T. Haase Cutting Edge: In Situ Tetramer Staining of Antigen-Specific T Cells in Tissues J. Immunol., July 15, 2000; 165(2): 613 - 617. [Abstract] [Full Text] [PDF]
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R. M. Kedl, M. Jordan, T. Potter, J. Kappler, P. Marrack, and S. Dow CD40 stimulation accelerates deletion of tumor-specific CD8+ T cells in the absence of tumor-antigen vaccination PNAS, September 11, 2001; 98(19): 10811 - 10816. [Abstract] [Full Text] [PDF]
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