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Departments of
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Pathology and of
Medicine, Case Western Reserve University School of Medicine, Cleveland, OH 44106
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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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Purified protein Ags and peptides, the currently favored subunit vaccine candidates, are poor immunogens. Injection of these substances usually induces immunological tolerance unless administered with an adjuvant (6, 7, 8, 9), traditionally CFA. This generally known yet poorly understood need to contaminate pure Ags with mycobacteria to induce immunity has been called the "immunologist’s dirty little secret" (10). To explain this and similar observations made over the decades, models that tie the induction of either an immune response or tolerance to the activation state of the innate immune system have been proposed (11, 12). According to these models, microbial constituents and other signals recognized as "infectious nonself" or as "danger" activate professional APCs, which causes them to acquire costimulatory properties. The "second signal" required for T cells to be primed (13, 14) is delivered by activated APC; Ag encountered on resting APC inactivates T cells, thus accounting for naturally developing as well as experimentally induced tolerance. Infectious agents are thought to be the prime candidates causing activation of APC (reviewed in Refs. 15 and 16). The classic Ag injection protocols with CFA or IFA seemed to support this hypothesis. Inoculations with Ag in CFA (oil containing inactivated mycobacteria) has been thought to be the most forceful way of inducing an immune response to protein Ags (6), while the injection of Ag in IFA (oil alone) has been thought to induce tolerance, particularly when done i.p. (17, 18, 19).
While revisiting neonatal Ag:IFA injections, we observed that this method of administering Ags does not induce immunological tolerance but triggers a vigorous type-2 immune response (20). These data have since been confirmed by several groups (21, 22, 23). Because neonates are thought to be biased toward type-2 immunity (24, 25), we performed the experiments presented here to test stringently the hypothesis that the supposedly tolerance-inducing Ag:IFA injections would also trigger type-2 immunity in adult mice. Having found this to be the case and because of the rigor that would be required if a classic paradigm were to be redefined, we decided to characterize closely the immune response induced. We showed that hen eggwhite lysozyme (HEL)4 is equally immunogenic when injected in CFA and IFA in that it induces T cell responses of comparable clonal size, fine specificity, and avidity but differing in cytokine profile, being type-1 and type-2, respectively. Moreover, we show that these results may be generalizable as they were seen with seven protein Ags and in a total of six mouse strains. We therefore conclude that it is not the "infectious/noninfectious" or "dangerous/nondangerous" character of the immune challenge that provides the "second signal" determining whether T cell immunity or T cell tolerance is induced. Instead, this dichotomy seems to determine whether T cells differentiate along the type-1 or type-2 pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and bred at Case Western Research University under specific pathogen-free conditions. Female mice were injected at 6–10 wk of age. HEL, OVA, and BSA were purchased from Sigma (St. Louis, MO). Guinea pig myelin basic protein (MBP), rabbit renal tubular Ag (RTA), Schistosoma egg Ag (provided by C. King), and Leishmania Ags were prepared as described (26, 27, 28). The peptides OVA:323–339 (KISQAVHAAHAEINEAG), HEL:106–116 (NAWVAWRNRCK), and proteolipid protein peptide (PLP) PLP:140–152 (HCLGKWLGHPDKF) were purchased from Princeton Biomolecules (Columbus, OH). IFA was purchased from Life Technologies (Grand Island, NY), and CFA was made by mixing M. tuberculosis H37RA (Difco Laboratories, Detroit, MI) at 2.5 mg/ml into IFA. Alum (Imject Alum) was purchased from Pierce (Rockford, IL). Individually, Ags or peptides were mixed with the adjuvant to yield a 2-mg/ml emulsion, of which 50 µl (100 µg/mouse) was injected once, i.p. or s.c., as specified.
Proliferation assays
Spleen cell proliferation assays were performed as previously described (29). Briefly, single-cell suspensions were prepared, and 1 x 106 spleen cells (single-cell suspensions that had not been depleted of erythrocytes) were plated per well in flat-bottom, 96-well microtiter plates in serum-free HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with L-glutamine at 1 mM. Ags were added at a final concentration of 100 µg/ml. During the last 18 h of a 4-day culture, [3H]thymidine was added (1 µCi/well); incorporation of label was measured by liquid scintillation counting. The lymph node (LN) assays followed the same protocol except that 5 x 105 cells were plated per well.
Cytokine ELISPOT assays
The use of plates and of an image analyzer customized for
ELISPOT assays greatly improved the resolution and sensitivity of this
assay to the extent that it has single-cell resolution and is suited to
measure cytokine production of low-frequency Ag-specific memory T cells
in freshly isolated cellular material (20). Briefly, plates
(ImmunoSpot, Resolution Technology, Columbus, OH) were coated overnight
at 4°C with the cytokine-specific capture Abs specified below. The
plates were then blocked with 1% BSA in PBS for 1 h at room
temperature and washed four times with PBS. Subsequently, the Ags,
peptides, and freshly isolated single-cell suspensions of spleen or LN
cells were added under the conditions specified for proliferation
assays. After 24–48 h of cell culture in the incubator, the cells were
removed by washing three times with PBS and four times with PBS
containing 0.05% Tween (PBST). Then, the detection Abs were added and
incubated at 4°C overnight (the detection Abs were either horse
radish peroxidase (HRP)-labeled or biotinylated). The plates were
washed three times with PBST. If biotinylated detection mAbs were used,
streptavidin-HRP conjugate (Dako, Carpenteria, CA) was added at 1:3000
dilution, incubated for 2 h at room temperature, and removed by
washing twice with PBST and twice with PBS. The spots were visualized
by adding HRP substrate 3-amino-9-ethylcarbozole (Pierce, Rockford,
IL). We used the following combinations of capture and detection mAbs
for IL-2, IL-4, IL-5, and IFN-
assays respectively: JES6-1A12 (5
µg/ml) and JES6-5H4-biotin (2 µg/ml), BVD4-1D11 (2 µg/ml) and
BVD4-24G2-biotin (2.5 µg/ml), TRFK5 (5 µg/ml) and TRFK4-HRP (2
µg/ml), and R46A2 (5 µg/ml) and XMG1.2-HRP (2 µg/ml). Image
analysis of ELISA spot assays was performed on a Series 1 ImmunoSpot
Image Analyzer (Resolution Technology, Columbus, OH) customized for
analyzing ELISA spots to meet objective criteria for size, chromatic
density, shape, and color. To evaluate the results in the HEL system,
we used image analysis; some of the results summarized in Table I
were obtained before we had access to an image
analyzer and so were counted visually.
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Plates (Nunc Immunoplate, Fisher Scientific, Pittsburgh, PA) were coated with HEL (10 µg/ml) overnight at 4°C, then blocked for 1–2 h with 0.1% gelatin, both in PBST. The test serum was added and incubated overnight at 4°C. Plate-bound Ab was detected by alkaline-phosphatase-coupled anti-mouse Ig. Affinity-purified goat anti-mouse IgG(H+L) from Southern Biotechnology (Birmingham, AL) was used to detect total Ig; the isotype-specific Abs used to detect IgG1 and IgG2a were also from Southern Biotechnology. P-Nitrophenyl phosphate disodium salt (Research Organics, Cleveland, OH) was used for the development of the colorimetric reaction.
Determinant mapping with overlapping peptides
The 118 HEL 12-mer peptides that covered the entire sequence of the HEL molecule in single steps of amino acids (30) were purchased from Chiron Mimotopes (Raleigh, NC). Peptides were dissolved at 560 µM in pH-optimized buffer (either 0.2 M HEPES or 0.2 M acetic acid), aliquoted into 96-well microtiter plates, and stored frozen at -85°C. Freshly thawed peptides were used in the cytokine ELISPOT assay at 7 µM.
Cell separations
Animals were injected with Ag in adjuvant, and 3–4 wk later single-cell suspensions were prepared from their spleens. Erythrocytes were depleted by Ficoll density gradient separation. CD4 cells were obtained by passing erythrocyte-depleted spleen cells through a murine CD4+ T cell enrichment column (R & D Systems, Minneapolis, MN) and further separated using Dynabeads (Dynal, Lake Success, NY) to obtain L-selectin+ and L-selectin- cells. FACS analysis was performed after each fractionation step and showed >90% enrichment for the desired phenotype (as specified in the figure legends for the experiments shown). All cell fractions were plated at 2 x 105 cells/well and tested in proliferation and ELISPOT assays with irradiated BALB/c SCID APCs in the presence or absence of HEL at 100 µg/ml or anti-CD3 mAb 2C11 (American Type Culture Collection, Manassas, VA) at 1 µg/ml in HL-1 medium. The bioassays were performed as specified above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Spleen cells of mice injected i.p. with HEL:IFA showed a vigorous
Ag-specific proliferative recall response of a magnitude comparable to
that induced by s.c. HEL:CFA immunization (Fig. 1).
Specific proliferative memory responses were also detected after i.p.
IFA injections of other protein Ags in several mouse strains (data not
shown, summarized for cytokine recall in Table I
), indicating that Ag
i.p. injected in IFA induces an immune response rather than immunologic
tolerance.
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A). This lack of response in
nondraining LN was seen after all i.p. injections, irrespective of the
adjuvants used, mouse strains tested, or harvest times after injection
(1–8 wk). Similarly, mice injected s.c. with HEL:CFA yielded recall
responses in the draining popliteal LN but not in the nondraining
brachial LN (Fig. 2B), the latter of which may have been
caused by the down-modulation of the LN homing receptor (L-selectin) on
memory cells (31, 32). To test this hypothesis, we separated
subpopulations of primed spleen cells and showed that the responding
cells were L-selectin-, CD4+ cells (Fig. 2, C and D). The lack of LN-homing-receptor
expression may explain why few memory cells migrated to nondraining
LN and, thus, why the previous studies, which focused on measuring T
cell responses in LN, missed the fact that immunity rather than
tolerance had been induced after the i.p. injections.
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The fact that memory cells occur at low frequencies (19, 33) has made it challenging to obtain accurate measurements of the cytokine recall responses of freshly isolated T cells to single nominal Ags such as HEL. To overcome this limitation, we developed a modification of the cytokine ELISPOT assay that can detect and accurately measure the frequency of single cytokine-producing cells.5 Using this assay, we have characterized the cytokine signatures of the T cell responses induced by Ag injections in IFA and CFA.
Freshly isolated spleen cells from i.p. HEL:IFA-injected mice produced
20–100 IL-5 spots per million spleen cells when cultured with HEL for
48 h; fewer than three spots (usually none at all) were seen in
cultures containing medium alone (Fig. 3), which yields
a stimulation index (SI) ranging from 7 to 100. This IL-5 was
produced by CD4+ T cells, and when challenged with HEL
these CD4+ cells also produced comparable numbers of
specific IL-4 and IL-2 spots (data not shown), but no HEL-specific
IFN- production was seen (Fig. 3, <8 spots, SI = 1). In
contrast, in mice injected s.c. with HEL:CFA, a specific IFN-
response of comparable magnitude was recalled (SI = 40–90) with
no IL-5 production in the spleen (Fig. 3). No IL-5 or IL-4 spots were
induced by HEL in the draining LN of such mice, but apparently
Ag-specific IL-4 production was seen in the spleen (however, cell
separation experiments showed that this IL-4 was not derived from T
cells, but was induced as a cytokine-mediated bystander reaction in
APC; A.Y.K, manuscript in preparation). In both the type-1-biased
B10.D2 and the type-2-biased BALB/c strains (34), injection of HEL in
either IFA or CFA induced virtually unipolar cytokine recall responses
(Fig. 3). Similar results were obtained with six mouse strains of mixed
type-1 and type-2 biases and six protein Ags other than HEL (Table I).
Even proteins with well-defined intrinsic type-1/type-2-polarizing
properties such as Leishmania and Schistosoma egg
Ags (35, 36) followed this pattern. These data suggest that, for
protein Ags, the choice of adjuvant (facilitated by the route of
injection, see below) might suffice to override both the type-1 and
type-2 biases of the hosts and Ags. This observation, which we
first reported for a prototypic "foreign" and "self"
Ag (HEL and MBP) in two strains of mice (BALB/c and B10.PL) at
neonatal and adult ages (20), seems, therefore, to be generalizable to
adult mice (and neonatal mice as well; T.F., manuscript in
preparation).
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). PLP peptide 140–152 in SJL mice and HEL peptide 106–116 in
DBA/2 mice behaved as did the protein Ags. Although the injection of
the third peptide, OVA:323–339, in CFA, into BALB/c mice also induced
a unipolar IFN- response, when injected in IFA it triggered mixed
IFN-:IL-5 memory (Table I). Why the response to this OVA peptide was
type-1 biased is unclear. Unlike proteins, peptides can directly bind
to MHC molecules on the cell surface, reaching MHC/peptide densities
that would not occur after intracellular processing of the native
protein. Such a high ligand density might have favored type-1
differentiation (37, 38). Intraperitoneal HEL:IFA injections induce specific IgG1 but not IgG2a production
We chose to use HEL, one of the best-characterized protein Ags, to
define the adjuvant-induced immune responses more closely. Mice
injected i.p. with HEL:IFA produced specific IgG1 but did not produce
detectable levels of IgG2a Abs (Fig. 4). Because
switching of Ig isotype to IgG2a is IFN- dependent (39), this
finding supports the notion that IFA induces Th cells that do not
produce IFN-. In CFA-immunized mice, IgG2a was produced, though IgG1
was also. This coexpression of IgG1 and IgG2a is not surprising because
switching to IgG1 is upstream of IgG2a and is not strictly
IL-4-dependent, but can also be promoted by IL-2 (40, 41). Therefore,
specific IgG1 Abs may be a less reliable surrogate marker for type-2
immunity than generally anticipated. We found that the sera of
HEL:CFA-immunized BALB/c mice tested negative for IgE in specific,
passive cutaneous anaphylaxis assays, confirming previous findings
(42), while sera of HEL:IFA-injected mice tested positive (data not
shown). Because IgE Abs are IL-4-dependent (41, 43, 44), their
absence provided further evidence that the memory T cells
induced by the CFA immunization did not produce IL-4 (and that the IL-4
bystander reaction detected in the spleens of such mice might have
limited functional relevance; see Footnote 3).
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Having observed unexpected type-1 and type-2 polarization when
using Freund’s classic adjuvants, we decided to include alum in our
studies as it is the only adjuvant presently in general use for human
vaccinations (9). Injection of HEL in alum, both s.c. and i.p.,
resulted in the production of IgG1 but not IgG2a Abs comparable in
magnitude to the IFA-induced response (Fig. 4); however, significant
cytokine recall responses were seen only after i.p. alum injections
(Fig. 3). These were unipolarly IL-5+:IFN--
but involved an overall frequency of IL-5-producing cells fivefold
lower than that after i.p. IFA injections. These data suggest that,
although both alum and IFA promote type-2 responses, they differ in
their propensity to engage the cellular, as opposed to the humoral,
effector arm of type-2 immunity; IFA is more proficient at driving the
clonal expansion of IL-5-producing memory cells.
With IFA and alum, the i.p. route facilitates generation of a type-2 cellular response
The clonal size of Ag-specific IL-5-producing cells induced by
i.p. HEL:IFA injection was 
10-fold greater than that seen after s.c.
administration of HEL:IFA (Fig. 3). Moreover, the weak cytokine recall
response engaged using the s.c. route included some IFN--producing
cells in addition to the IL-5 producers (Fig. 3). With alum, the i.p.
route also facilitated the generation of IL-5-producing memory cells as
with IFA. In contrast to the cytokine response, the Ab levels induced
by i.p. and s.c. injections with either IFA or alum were of a
comparable titer and had a pure IgG1+ and
IgG2a- profile (Fig. 4). Apparently, the relatively few
type-2 memory cells primed by s.c. injection were sufficient to provide
maximum help toward IgG1 Ab production even though the i.p. route
facilitated the generation of 10 times more type-2 memory cells.
Because the immune response occurs in LN associated with either
systemic or mucosal immunity when the Ag is injected s.c. (popliteal
LN) or i.p. (mediastinal postthymic LN), respectively, different
lymphoid tissues are involved when the Ag is injected s.c. and i.p.,
and the differences associated with the route of immunization might be
the result of differences in the influence of local cytokines (45) or
hormones (46).
Anti-HEL T cell responses induced by i.p. IFA and CFA injections have identical fine specificities
Our finding, that i.p. injection of Ag induces type-2 immunity may
explain why some earlier reports implied that this protocol induces
tolerance by activating suppressor cells. Because fine specificities
that differ from those of CD4+ cells have been attributed
to suppressor cells (47), we mapped the anti-HEL response after
i.p. IFA and CFA immunization using a complete HEL peptide series that
covered every possible determinant on the molecule in steps of single
amino acids. The data showed that identical T cell specificities were
engaged in both instances (Fig. 5, A and
B), leading to the recognition of region amino acid 105–119
(core: 108–116), located within the well-defined,
I-Ed-restricted determinant, 106–116 (30). The recognition
of this MHC class II-restricted determinant suggested that
CD4+ cells had been induced, confirming the results of the
cell separation experiments shown in Fig. 2.
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One possible interpretation of our data was that i.p. HEL:IFA
injections deleted T cell clones with high avidity for the Ag (48) and
that the type-2 response measured after this treatment represented
residual activity from low-avidity T cells. To address this
possibility, spleen cells from BALB/c mice were injected i.p. with
either HEL:IFA or HEL:CFA, and the responses were recalled in
ELISPOT assays in which the immunodominant peptide
HEL:106–116 was titrated. Following the classic definition of
functional avidity for polyvalent ligand-receptor interactions,
functional T cell avidity can be defined as the concentration of
peptide that leads to the activation of 50% of T cells in a given
population for a constant number of APCs (49). T cells become activated
(a process that includes the induction of cytokine production) when a
critical number of MHC-peptide ligands are bound by their TCR (50).
This number depends on the intrinsic affinity of the TCR for the
particular MHC-peptide complex (51): high-avidity T cell clones will be
activated at low peptide concentrations and those of low avidity at
high concentrations. Because the frequencies of cytokine-producing T
cells measured by ELISPOT in the cells primed with CFA and IFA had
similar dose-response characteristics (Fig. 5, C and
D), we can conclude that the T cells primed with CFA and IFA
had comparable functional avidities. These data suggest that similar
precursor-cell repertoires, including the high-affinity end of the
spectrum, were primed by both immunizations.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), memory T cells primed
after i.p. immunization are LN-homing-receptor-negative and do not
migrate efficiently to LN, though they are present in the spleen. This
compartmentalization of memory T cells is consistent with the present
understanding of T cell migration patterns (56, 57, 58, 59). Fourth,
proliferative recall assays that detect CD4+ memory in the
spleen became feasible only with the advent of serum-free assay
conditions, at which time attention was drawn to the detection of
CD4+ memory cells in the spleen (29). Finally, measurements
of the cytokines produced by Ag-specific cells have been challenging,
particularly for type-2 cytokines. Performing ELISAs on culture
supernatants and ELISPOT assays in parallel, we found that between 200
to 400 T cells had to produce IL-4 and IL-5 before these cytokines
could be detected by ELISA in the culture supernatants. This relatively
low sensitivity might be why the immunogenicity of Ag:IFA injections
was not detected, even in TCR transgenic adoptive transfer models;
however, there are several earlier reports according to which s.c. IFA
injections induce "humoral" but not "cell-mediated" (type-1)
immunity (7, 8, 60).
Our finding that i.p. IFA injections are strongly immunogenic might
provide new insights into what is required to induce T cell responses
in general and type-1 and type-2 immunity in particular. It has
generally been held that to promote a strong immune response an
adjuvant must simultaneously perform two critical functions (reviewed
in Refs. 7 and 8). First, the adjuvant must function as an Ag depot.
The oil-based Freund’s adjuvants excel at retaining and slowly
releasing Ag; the half-life of OVA in IFA is 90 days (60). Second,
it is now widely accepted that adjuvants must locally activate APC to
engage the "second signal," a reaction that is thought to be
primarily induced by microbial products (reviewed in Refs. 15 and 16).
Because we show that IFA is as strong an adjuvant for inducing type-2
immunity as CFA is for type-1 responses, the "infectious
nonself" hypotheses might have to be revised in favor of one
of the following models.
Model 1 states that the activation of the innate immune system by "infectious nonself" signals is required for the induction of type-1 but not type-2 immunity. In this model, "infectious nonself" might correspond to the up-regulation of B7-1 molecules and/or the induction of IL-12. When cells of the innate immune system, which include dendritic cells, are exposed to certain microbial products that these cells have evolved to recognize as common features of "foreign," such as carbohydrates (reviewed in 16), they up-regulate B7-1 and secrete IL-12, resulting in a microenvironment that favors T cell differentiation along the type-1 pathway (61, 62). In contrast, type-2 immunity may develop by default when T cells encounter Ag on "unstimulated" APC (which in a normal microbial environment may constitutively express enough costimulatory molecules, including B7-2, to activate T cells). This view might be substantiated by an increasing number of recent reports of soluble protein Ags inducing type-2 immunity when administered epicutaneously (63), orally (64), nasally (65), and i.v. as aggregated proteins (66). The fact that HEL:IFA promotes a stronger T cell response than does HEL:alum while the same dose of soluble HEL is barely immunogenic might merely reflect the unique Ag-retaining properties of IFA. In this scenario, the role of IFA as an adjuvant is passive, limited to serving as an Ag depot (60).
Model 2 predicts that there are two fundamentally different "infectious nonself"/"danger" reactions of the innate immune system that direct T cell maturation along either the type-1 or type-2 pathway. Whereas both induce the up-regulation of costimulatory molecules on APC, one type of stimulus would induce IL-12 production, e.g., in dendritic cells (67), while the other would stimulate IL-4 and/or IL-6 production, e.g., in mast cells or eosinophils (68), the latter creating a cytokine microenvironment that favors type-2 T cell differentiation (69, 70, 71). The oils that IFA consists of might directly induce IL-4 or IL-6 production or might cause a sterile inflammation associated with prevalently type-2 cytokine microenvironment.
Model 3 predicts that there are two thresholds in the magnitude of the "infectious nonself" or "danger" reaction that define whether an Ag encounter results in tolerance, in a type-2 response, or in type-1 immunity. The recognition of Ag on resting APC by T cells, which would be below the first threshold (no or minimal "danger"), results in clonal inactivation and deletion in the absence of the induction of the "second signal," as originally postulated and as it seems to occur after i.v. injection of deaggregated protein (72) and during naturally developing self-tolerance. Beyond a certain level of stimulation of the innate immune system, between the first and second thresholds, the "second signal" becomes engaged on APC, but IL-12 is not induced: moderate "danger" results in type-2 immunity. Once the magnitude of stimulation of the innate immune system exceeds the second threshold, the production of IL-12 is also induced: great "danger" causes type-1 immunity. In this model gradations of the same inflammatory reactions of the innate immune system therefore determine the type-1 or type-2 environment for T cell differentiation. In a modification of this model, it is conceivable that the strength of signal 1 and signal 2 synergistically define the class of the response (strong 1st x weak 2nd = weak 1st x strong 2nd). Our finding that OVA peptide 323–339 induced a mixed response when injected in IFA can be interpreted along these lines.
While it remains unclear why IFA acts as a strong type-2 adjuvant, our
data provide clues about what it takes to generate a type-1 adjuvant:
the presence of microbial products that signal "infectious
nonself." Confirming this notion, we found that supplementing IFA
with pertussis toxin, LPS, or with oligonucleotides containing the CpG
motif characteristic of bacterial DNA (substances that induce IL-12
production) all yield adjuvants that show the type-1-polarizing
properties of CFA (Ref. 73 , and T.F., unpublished observations). This
information might be of particular relevance for human adjuvant design
because CFA is not suited to human use due to the induction of severe
granulomatous reactions and because aluminum, the only adjuvant
recommended for human use, has type-2-inducing properties (Figs. 3 and 4).
We wish to point out that the polarizing effects of adjuvants reported
here apply only to the priming of naive T cells. In ongoing
experiments, we find that the response induced by the primary CFA or
IFA immunization largely maintains its type-1 or type-2 cytokine recall
and Ab isotype profile after the mice are reinjected with the opposite
type of adjuvant (data not shown). This finding is consistent with the
notion that, unlike naive T cells, memory cells are committed to their
type-1 or type-2 lineages (74). The data suggest that even after a
primary injection adjuvants will not polarize a response toward the
type-1 or type-2 pathway if the Ag-specific T cells are not naive but
have been preprimed to type-1 or type-2 memory cells by cross-reactive
environmental stimulation or by homologous self-proteins (22, 75).
Furthermore, we found that IFA/Ag preparations needed to be injected
i.p. (Fig. 3) and to be free of contaminating LPS or CpG-containing-DNA
to induce pure type-2 responses, otherwise the IFA-induced response
will be switched to type-1 (73). The same applies if the mouse colony
is infected.
As with mice of different genetic backgrounds, humans also show considerable heterogeneity in their type-1/type-2 bias (76); this is a factor that must be overridden by vaccination protocols to ensure uniform success. Once type-1/type-2-polarizing adjuvants suitable for human use have been identified, exploiting the intrinsic properties of these adjuvants should be the simplest, cheapest, and most reliable subunit vaccination strategy for selectively engaging the desired class of immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul V. Lehmann, Case Western Reserve University, Department of Pathology, BRB 929, 10900 Euclid Avenue, Cleveland, OH 44106-4943. E-mail address:
3 Note to type-1/type-2 classification: Presently, it is controversial whether the T cells that mediate type-1 and type-2 responses in vivo secrete mutually exclusive sets of type-1 and type-2 cytokines (Th1/Th2 model) (1), whether they randomly coexpress these cytokines in a continuous range of concentrations (stochastic model) (2), or whether each T cell produces only a single cytokine (3). To avoid these uncertainties of cytokine coexpression in individual memory T cells in vivo, we purposefully chose the type-1/type-2 terminology, which describes types of in vivo responses (4, 5), instead of the Th1/Th2 nomenclature (1), which implies cytokine expression patterns in the individual memory T cells that mediate these responses. We used the term type-1 to describe the production of IFN-+, IL-5- by memory T cells, which mediate delayed-type hypersensitivity and promote specific IgG2a but not IgE production. Type 2 responses were defined as IFN--, IL-5+ production in memory T cells, which do not mediate delayed-type hypersensitivity but promote IgE and IgG1 production in the absence of IgG2a.
4 Abbreviations used in this paper: HEL, hen eggwhite lysozyme; MBP, myelin basic protein; RTA, renal tubular Ag; PLP, proteolipid protein; LN, lymph node; PBST, PBS with Tween; HRP, horseradish peroxidase; SI, stimulation index.
5 A. Y. Karulin, M. D. Hesse, M. Tary-Lehmann, and P. V. Lehmann. Direct visualization of cytokine coexpression by individual in vivo differentiated CD4 cells. Submitted for publication.
Received for publication August 13, 1998. Accepted for publication January 6, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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K. Hagenow, K. A. Gelderman, M. Hultqvist, P. Merky, J. Backlund, O. Frey, T. Kamradt, and R. Holmdahl Ncf1-Associated Reduced Oxidative Burst Promotes IL-33R+ T Cell-Mediated Adjuvant-Free Arthritis in Mice J. Immunol., July 15, 2009; 183(2): 874 - 881. [Abstract] [Full Text] [PDF]
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 W. Kim, Y.-Y. Fan, R. Smith, B. Patil, G. K. Jayaprakasha, D. N. McMurray, and R. S. Chapkin Dietary Curcumin and Limonin Suppress CD4+ T-Cell Proliferation and Interleukin-2 Production in Mice J. Nutr., May 1, 2009; 139(5): 1042 - 1048. [Abstract] [Full Text] [PDF]
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 L.-Q. Qiu, P. Cresswell, and K.-C. Chin Viperin is required for optimal Th2 responses and T-cell receptor-mediated activation of NF-{kappa}B and AP-1 Blood, April 9, 2009; 113(15): 3520 - 3529. [Abstract] [Full Text] [PDF]
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C. Richter, M. H. S. Juan, J. Will, R. P. Brandes, U. Kalinke, S. Akira, J. M. Pfeilschifter, M. Hultqvist, R. Holmdahl, and H. H. Radeke Ncf1 Provides a Reactive Oxygen Species-Independent Negative Feedback Regulation of TLR9-Induced IL-12p70 in Murine Dendritic Cells J. Immunol., April 1, 2009; 182(7): 4183 - 4191. [Abstract] [Full Text] [PDF]
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 J. Sun, X. Song, and S. Hu Ginsenoside Rg1 and Aluminum Hydroxide Synergistically Promote Immune Responses to Ovalbumin in BALB/c Mice Clin. Vaccine Immunol., February 1, 2008; 15(2): 303 - 307. [Abstract] [Full Text] [PDF]
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 A. Pellegrini, N. Guinazu, M. P. Aoki, I. C. Calero, E. A. Carrera-Silva, N. Girones, M. Fresno, and S. Gea Spleen B cells from BALB/c are more prone to activation than spleen B cells from C57BL/6 mice during a secondary immune response to cruzipain Int. Immunol., December 1, 2007; 19(12): 1395 - 1402. [Abstract] [Full Text] [PDF]
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Z. Miao, B. A. Premack, Z. Wei, Y. Wang, C. Gerard, H. Showell, M. Howard, T. J. Schall, and R. Berahovich Proinflammatory Proteases Liberate a Discrete High-Affinity Functional FPRL1 (CCR12) Ligand from CCL23 J. Immunol., June 1, 2007; 178(11): 7395 - 7404. [Abstract] [Full Text] [PDF]
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F. S. Lichtenegger, S. Kuerten, S. Faas, B. O. Boehm, M. Tary-Lehmann, and P. V. Lehmann Dissociation of Experimental Allergic Encephalomyelitis Protective Effect and Allergic Side Reactions in Tolerization with Neuroantigen J. Immunol., April 15, 2007; 178(8): 4749 - 4756. [Abstract] [Full Text] [PDF]
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 B. Mahesh, H.-S. Leong, A. McCormack, P. Sarathchandra, A. Holder, and M. L. Rose Autoantibodies to Vimentin Cause Accelerated Rejection of Cardiac Allografts Am. J. Pathol., April 1, 2007; 170(4): 1415 - 1427. [Abstract] [Full Text] [PDF]
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B. A. Maletto, A. S. Ropolo, D. O. Alignani, M. V. Liscovsky, R. P. Ranocchia, V. G. Moron, and M. C. Pistoresi-Palencia Presence of neutrophil-bearing antigen in lymphoid organs of immune mice Blood, November 1, 2006; 108(9): 3094 - 3102. [Abstract] [Full Text] [PDF]
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A. Valujskikh, Q. Zhang, and P. S. Heeger CD8 T Cells Specific for a Donor-Derived, Self-Restricted Transplant Antigen Are Nonpathogenic Bystanders after Vascularized Heart Transplantation in Mice J. Immunol., February 15, 2006; 176(4): 2190 - 2196. [Abstract] [Full Text] [PDF]
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J. Tian, D. Zekzer, Y. Lu, H. Dang, and D. L. Kaufman B Cells Are Crucial for Determinant Spreading of T Cell Autoimmunity among beta Cell Antigens in Diabetes-Prone Nonobese Diabetic Mice J. Immunol., February 15, 2006; 176(4): 2654 - 2661. [Abstract] [Full Text] [PDF]
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C. Nicolo, G. Di Sante, M. Orsini, S. Rolla, S. Columba-Cabezas, V. R. Spica, G. Ricciardi, B. M. C. Chan, and F. Ria Mycobacterium tuberculosis in the adjuvant modulates the balance of Th immune response to self-antigen of the CNS without influencing a "core" repertoire of specific T cells Int. Immunol., February 1, 2006; 18(2): 363 - 374. [Abstract] [Full Text] [PDF]
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S. Gregoire, A. S. Bergot, C. Feraudet, C. Carnaud, P. Aucouturier, and M. B. Rosset The Murine B Cell Repertoire Is Severely Selected against Endogenous Cellular Prion Protein J. Immunol., November 15, 2005; 175(10): 6443 - 6449. [Abstract] [Full Text] [PDF]
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Y. Tanaka, T. So, S. Lebedeva, M. Croft, and A. Altman Impaired IL-4 and c-Maf expression and enhanced Th1-cell development in Vav1-deficient mice Blood, August 15, 2005; 106(4): 1286 - 1295. [Abstract] [Full Text] [PDF]
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A. P. Olcott, J. Tian, V. Walker, H. Dang, B. Middleton, L. Adorini, L. Washburn, and D. L. Kaufman Antigen-Based Therapies Using Ignored Determinants of {beta} Cell Antigens Can More Effectively Inhibit Late-Stage Autoimmune Disease in Diabetes-Prone Mice J. Immunol., August 1, 2005; 175(3): 1991 - 1999. [Abstract] [Full Text] [PDF]
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C. B. Stephensen, X. Jiang, and T. Freytag Vitamin A Deficiency Increases the In Vivo Development of IL-10-Positive Th2 Cells and Decreases Development of Th1 Cells in Mice J. Nutr., October 1, 2004; 134(10): 2660 - 2666. [Abstract] [Full Text] [PDF]
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W. C. Bartholomae, F. H. Rininsland, J. C. Eisenberg, B. O. Boehm, P. V. Lehmann, and M. Tary-Lehmann T Cell Immunity Induced by Live, Necrotic, and Apoptotic Tumor Cells J. Immunol., July 15, 2004; 173(2): 1012 - 1022. [Abstract] [Full Text] [PDF]
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 M. Le Hir Histopathology of humorally mediated anti-glomerular basement membrane (GBM) glomerulonephritis in mice Nephrol. Dial. Transplant., July 1, 2004; 19(7): 1875 - 1880. [Abstract] [Full Text] [PDF]
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K. Darabi, A. Y. Karulin, B. O. Boehm, H. H. Hofstetter, Z. Fabry, J. C. LaManna, J. C. Chavez, M. Tary-Lehmann, and P. V. Lehmann The Third Signal in T Cell-Mediated Autoimmune Disease? J. Immunol., July 1, 2004; 173(1): 92 - 99. [Abstract] [Full Text] [PDF]
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F. Ria, A. Gallard, C. R. Gabaglia, J.-C. Guery, E. E. Sercarz, and L. Adorini Selection of Similar Naive T Cell Repertoires but Induction of Distinct T Cell Responses by Native and Modified Antigen J. Immunol., March 15, 2004; 172(6): 3447 - 3453. [Abstract] [Full Text] [PDF]
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 R. Hakamada-Taguchi, Y. Uehara, K. Kuribayashi, A. Numabe, K. Saito, H. Negoro, T. Fujita, T. Toyo-oka, and T. Kato Inhibition of Hydroxymethylglutaryl-Coenzyme A Reductase Reduces Th1 Development and Promotes Th2 Development Circ. Res., November 14, 2003; 93(10): 948 - 956. [Abstract] [Full Text] [PDF]
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A. S. Lonsdorf, H. Kuekrek, B. V. Stern, B. O. Boehm, P. V. Lehmann, and M. Tary-Lehmann Intratumor CpG-Oligodeoxynucleotide Injection Induces Protective Antitumor T Cell Immunity J. Immunol., October 15, 2003; 171(8): 3941 - 3946. [Abstract] [Full Text] [PDF]
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C. A. Velikovsky, F. A. Goldbaum, J. Cassataro, S. Estein, R. A. Bowden, L. Bruno, C. A. Fossati, and G. H. Giambartolomei Brucella Lumazine Synthase Elicits a Mixed Th1-Th2 Immune Response and Reduces Infection in Mice Challenged with Brucella abortus 544 Independently of the Adjuvant Formulation Used Infect. Immun., October 1, 2003; 71(10): 5750 - 5755. [Abstract] [Full Text] [PDF]
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S.-C. Wong, E. Oh, C.-H. Ng, and K.-P. Lam Impaired germinal center formation and recall T-cell-dependent immune responses in mice lacking the costimulatory ligand B7-H2 Blood, August 15, 2003; 102(4): 1381 - 1388. [Abstract] [Full Text] [PDF]
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C. Bourquin, A. Schubart, S. Tobollik, I. Mather, S. Ogg, R. Liblau, and C. Linington Selective Unresponsiveness to Conformational B Cell Epitopes of the Myelin Oligodendrocyte Glycoprotein in H-2b Mice J. Immunol., July 1, 2003; 171(1): 455 - 461. [Abstract] [Full Text] [PDF]
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J. C. Eisenberg, S. J. Czinn, C. A. Garhart, R. W. Redline, W. C. Bartholomae, J. M. Gottwein, J. G. Nedrud, S. E. Emancipator, B. B. Boehm, P. V. Lehmann, et al. Protective Efficacy of Anti-Helicobacterpylori Immunity following Systemic Immunization of Neonatal Mice Infect. Immun., April 1, 2003; 71(4): 1820 - 1827. [Abstract] [Full Text]
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C. M. Denkinger, M. Denkinger, J. J. Kort, C. Metz, and T. G. Forsthuber In Vivo Blockade of Macrophage Migration Inhibitory Factor Ameliorates Acute Experimental Autoimmune Encephalomyelitis by Impairing the Homing of Encephalitogenic T Cells to the Central Nervous System J. Immunol., February 1, 2003; 170(3): 1274 - 1282. [Abstract] [Full Text] [PDF]
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J. Tian, A. P. Olcott, and D. L. Kaufman Antigen-Based Immunotherapy Drives the Precocious Development of Autoimmunity J. Immunol., December 1, 2002; 169(11): 6564 - 6569. [Abstract] [Full Text] [PDF]
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D. O. Gor, X. Ding, Q. Li, J. R. Schreiber, M. Dubinsky, and N. S. Greenspan Enhanced Immunogenicity of Pneumococcal Surface Adhesin A by Genetic Fusion to Cytokines and Evaluation of Protective Immunity in Mice Infect. Immun., October 1, 2002; 70(10): 5589 - 5595. [Abstract] [Full Text] [PDF]
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 R. Maron, G. Sukhova, A.-M. Faria, E. Hoffmann, F. Mach, P. Libby, and H. L. Weiner Mucosal Administration of Heat Shock Protein-65 Decreases Atherosclerosis and Inflammation in Aortic Arch of Low-Density Lipoprotein Receptor-Deficient Mice Circulation, September 24, 2002; 106(13): 1708 - 1715. [Abstract] [Full Text] [PDF]
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 B. Maletto, A. Ropolo, V. Moron, and M. C. Pistoresi-Palencia CpG-DNA stimulates cellular and humoral immunity and promotes Th1 differentiation in aged BALB/c mice J. Leukoc. Biol., September 1, 2002; 72(3): 447 - 454. [Abstract] [Full Text] [PDF]
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K. Boztug, M. J. Carson, N. Pham-Mitchell, V. C. Asensio, J. DeMartino, and I. L. Campbell Leukocyte Infiltration, But Not Neurodegeneration, in the CNS of Transgenic Mice with Astrocyte Production of the CXC Chemokine Ligand 10 J. Immunol., August 1, 2002; 169(3): 1505 - 1515. [Abstract] [Full Text] [PDF]
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H. H. Hofstetter, C. L. Shive, and T. G. Forsthuber Pertussis Toxin Modulates the Immune Response to Neuroantigens Injected in Incomplete Freund's Adjuvant: Induction of Th1 Cells and Experimental Autoimmune Encephalomyelitis in the Presence of High Frequencies of Th2 Cells J. Immunol., July 1, 2002; 169(1): 117 - 125. [Abstract] [Full Text] [PDF]
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B. V. Stern, B. O. Boehm, and M. Tary-Lehmann Vaccination with Tumor Peptide in CpG Adjuvant Protects Via IFN-{gamma}-Dependent CD4 Cell Immunity J. Immunol., June 15, 2002; 168(12): 6099 - 6105. [Abstract] [Full Text] [PDF]
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S. Saarelainen, T. Zeiler, J. Rautiainen, A. Narvanen, M. Rytkonen-Nissinen, R. Mantyjarvi, P. Vilja, and T. Virtanen Lipocalin allergen Bos d 2 is a weak immunogen Int. Immunol., April 1, 2002; 14(4): 401 - 409. [Abstract] [Full Text] [PDF]
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A. Y. Karulin, M. D. Hesse, H. C. Yip, and P. V. Lehmann Indirect IL-4 Pathway in Type 1 Immunity J. Immunol., January 15, 2002; 168(2): 545 - 553. [Abstract] [Full Text] [PDF]
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B. M. Naiman, D. Alt, C. A. Bolin, R. Zuerner, and C. L. Baldwin Protective Killed Leptospira borgpetersenii Vaccine Induces Potent Th1 Immunity Comprising Responses by CD4 and gamma delta T Lymphocytes Infect. Immun., December 1, 2001; 69(12): 7550 - 7558. [Abstract] [Full Text] [PDF]
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A. Billiau and P. Matthys Modes of action of Freund's adjuvants in experimental models of autoimmune diseases J. Leukoc. Biol., December 1, 2001; 70(6): 849 - 860. [Abstract] [Full Text] [PDF]
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 S. Mirshahidi, C.-T. Huang, and S. Sadegh-Nasseri Anergy in Peripheral Memory Cd4+ T Cells Induced by Low Avidity Engagement of T Cell Receptor J. Exp. Med., September 17, 2001; 194(6): 719 - 732. [Abstract] [Full Text] [PDF]
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M. D. Hesse, A. Y. Karulin, B. O. Boehm, P. V. Lehmann, and M. Tary-Lehmann A T Cell Clone's Avidity Is a Function of Its Activation State J. Immunol., August 1, 2001; 167(3): 1353 - 1361. [Abstract] [Full Text] [PDF]
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J. D. Ahlers, I. M. Belyakov, S. Matsui, and J. A. Berzofsky Mechanisms of cytokine synergy essential for vaccine protection against viral challenge Int. Immunol., July 1, 2001; 13(7): 897 - 908. [Abstract] [Full Text] [PDF]
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K. G. Tournoy, J. C. Kips, and R. A. Pauwels The Allergen-Induced Airway Hyperresponsiveness in a Human-Mouse Chimera Model of Asthma Is T Cell and IL-4 and IL-5 Dependent J. Immunol., June 1, 2001; 166(11): 6982 - 6991. [Abstract] [Full Text] [PDF]
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O. S. Targoni, J. Baus, H. H. Hofstetter, M. D. Hesse, A. Y. Karulin, B. O. Boehm, T. G. Forsthuber, and P. V. Lehmann Frequencies of Neuroantigen-Specific T Cells in the Central Nervous System Versus the Immune Periphery During the Course of Experimental Allergic Encephalomyelitis J. Immunol., April 1, 2001; 166(7): 4757 - 4764. [Abstract] [Full Text] [PDF]
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H. Jyonouchi, S. Sun, T. Winship, and M. J. Kuchan Dietary Ribonucleotides Modulate Type 1 and Type 2 T-Helper Cell Responses against Ovalbumin in Young BALB/cJ Mice J. Nutr., April 1, 2001; 131(4): 1165 - 1170. [Abstract] [Full Text]
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D. Abraham, O. Leon, S. Leon, and S. Lustigman Development of a Recombinant Antigen Vaccine against Infection with the Filarial Worm Onchocerca volvulus Infect. Immun., January 1, 2001; 69(1): 262 - 270. [Abstract] [Full Text] [PDF]
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P. Matthys, K. Vermeire, H. Heremans, and A. Billiau The protective effect of IFN-{gamma} in experimental autoimmune diseases: a central role of mycobacterial adjuvant-induced myelopoiesis J. Leukoc. Biol., October 1, 2000; 68(4): 447 - 454. [Abstract] [Full Text]
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G. B. Lipford, T. Sparwasser, S. Zimmermann, K. Heeg, and H. Wagner CpG-DNA-Mediated Transient Lymphadenopathy Is Associated with a State of Th1 Predisposition to Antigen-Driven Responses J. Immunol., August 1, 2000; 165(3): 1228 - 1235. [Abstract] [Full Text] [PDF]
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B. M. Segal, J. T. Chang, and E. M. Shevach CpG Oligonucleotides Are Potent Adjuvants for the Activation of Autoreactive Encephalitogenic T Cells In Vivo J. Immunol., June 1, 2000; 164(11): 5683 - 5688. [Abstract] [Full Text] [PDF]
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P. S. Heeger, T. Forsthuber, C. Shive, E. Biekert, C. Genain, H. H. Hofstetter, A. Karulin, and P. V. Lehmann Revisiting Tolerance Induced by Autoantigen in Incomplete Freund's Adjuvant J. Immunol., June 1, 2000; 164(11): 5771 - 5781. [Abstract] [Full Text] [PDF]
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G. Foucras, L. Gapin, C. Coureau, J. M. Kanellopoulos, and J.-C. Guery Interleukin 4-Producing Cd4 T Cells Arise from Different Precursors Depending on the Conditions of Antigen Exposure in Vivo J. Exp. Med., February 21, 2000; 191(4): 683 - 694. [Abstract] [Full Text] [PDF]
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A. Y. Karulin, M. D. Hesse, M. Tary-Lehmann, and P. V. Lehmann Single-Cytokine-Producing CD4 Memory Cells Predominate in Type 1 and Type 2 Immunity J. Immunol., February 15, 2000; 164(4): 1862 - 1872. [Abstract] [Full Text] [PDF]
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D. C. Lenz, N. A. Wolf, and R. H. Swanborg Strain Variation in Autoimmunity: Attempted Tolerization of DA Rats Results in the Induction of Experimental Autoimmune Encephalomyelitis J. Immunol., August 15, 1999; 163(4): 1763 - 1768. [Abstract] [Full Text] [PDF]
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