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Department of Immunology and Bacteriology, University of Glasgow, Western Infirmary, Glasgow, United Kingdom
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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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Th cells can be divided into at least two subsets, Th1 and Th2,
determined by which cytokines they produce upon activation
(8), with Th1 cells producing IL-2, IFN-
, and TNFß,
and Th2 cells producing IL-4, IL-5, IL-6, IL-10, and IL-13. The balance
of cytokines produced by these subsets is a key factor influencing the
character of an immune response (9, 10, 11, 12, 13). Although a number
of studies have demonstrated the ability of both Th1 and Th2 cells to
support B cell responses (14, 15, 16, 17), the latter are widely
believed to be particularly adept in this respect, as the cytokines
they characteristically secrete have all been implicated in various
stages of B cell proliferation and differentiation (11, 18, 19, 20). However, many of the studies noted above were performed
in vitro, employed T cells clones or lines, relied upon different Ags
to induce Th1 or Th2 responses, or could not identify the Ag-specific
cells in question. Recent studies employing the adoptive transfer of
TCR transgenic (tg) T cells have indicated that, in contrast to Th2
cells, CCR7 expression on Th1 cells reduces their migration to
follicles and hence their ability to help B cell responses in vivo
(21). However, Th2 cells may not be essential for B cell
proliferation and differentiation, as IL-4 or IL-5 knockout (KO) mice,
which have deficient Th2 responses, have readily detectable Ab
production and germinal center formation (22, 23, 24).
Furthermore, whereas Th1 cells are associated with cell-mediated
immunity, and generally regarded as less important for B cell
responses, IgG2a isotype switching is promoted by IFN- (20, 25, 26), the prototypical Th1 cytokine. These results suggest
that Th2 cells are not unique in their ability to induce T-dependent B
cell responses and that the mechanisms by which Th1 and Th2 cells
provide B cell help remain to be defined, particularly in vivo.
Understanding the relative roles of Th1 and Th2 cells in providing help
for B cells and the mechanisms they employ to achieve this will be
important for the rational design of vaccines and therapies for
immune-mediated disorders.
To study the role of Th1 and Th2 cells in providing B cell help in vivo, we have modified a recently developed system to investigate the interactions and requirements underlying T cell help for B cell responses. By polarizing Ag-specific TCR tg T cells toward a Th1 or Th2 phenotype in vitro and transferring them into naive syngeneic recipients along with Ag-specific B cell receptor (BcR) tg B cells, we have been able to examine the effect Th1 and Th2 cells have on B cell expansion, differentiation, and Ab production in vivo. Our results indicate that, following immunization, both Th1 and Th2 cells migrate into B cell follicles to support B cell clonal expansion and Ab production in a CD154-dependent manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c (H-2d/d, IgMa), C57BL/6 (H-2b/b, IgMb/b), and BALB/c x C57BL/6 F1 (H-2d/b, IgMa/b) mice were purchased from Harlan Olac (Bichester, U.K.). Mice homozygous for the chicken OVA (cOVA) peptide323–339/I-Ad-specific DO11.10 TCR transgenes (detected using the clonotypic mAb KJ1.26) on the BALB/c background (27) were crossed to C57BL/6 mice to produce animals heterozygous for the DO11.10 transgenes on an F1 background. Similarly, mice heterozygous for the anti-HEL IgMa and IgDa transgenes on the C57BL/6 background (MD4) (28) were crossed to BALB/c mice, and offspring from this cross were screened for the expression of the MD4 transgenes by flow cytometry. Six-week-old, male C57BL/6 x BALB/c F1 mice were used as recipients (Harlan Olac). All animals were maintained under standard conditions at the University of Glasgow Central Research Facility in accordance with Home Office regulations.
Flow cytometry
Cell suspensions were prepared from pooled draining lymph nodes by forcing them through a nylon mesh (Cadisch Precision Meshes, London, U.K.). Aliquots of these cells were stained with PE-labeled anti-CD4 mAb CD4 (L3T4) (RM4-5) (PharMingen, San Diego, CA) and biotinylated KJ1.26 mAb followed by FITC-labeled streptavidin (PharMingen) for the detection of DO11.10 T cells. Tg B cells were detected with PE-labeled anti-B220 mAb (RA3-6B2) (PharMingen) in combination with biotinylated hen egg lysozyme (HEL) (Biozyme, Blaenavon, Gwent, U.K.) or biotinylated anti-IgMa mAb (Igh-6a) (DS-1) (PharMingen) followed by FITC-labeled streptavidin. Stained cells (20,000 per sample) were then analyzed by flow cytometry using CellQuest software (Becton Dickinson, Oxford, U.K.).
Cytokines and Abs for in vitro polarization of Th1 and Th2 cells
Recombinant murine IL-4 was kindly provided by Genzyme
(Cambridge, MA) and recombinant murine IL-12 by Hoffman-LaRoche
(Nutley, NJ). Anti-IL-12 polyclonal rabbit serum and anti-IFN-
polyclonal sheep serum were used at 1/500 dilution. Anti-IL-4 mAb 11B11
was used as described previously (29).
T cell purification and T cell cultures
T cells were maintained in RPMI 1640 supplemented with 10% FCS,
2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin, and 1.25 mg/ml fungizone (complete medium) (all Life
Technologies, Paisley, Scotland, U.K.). Naive
CD4+ T cells were purified from pooled lymph node
and spleen cells from DO11.10 BALB/c x C57BL/6
F1 mice by negative selection. B cells,
monocytes, and CD8+ T cells were removed using
anti-CD19, anti-CD11c, and anti-CD8 mAbs (all Serotec,
Oxford, U.K.), respectively, which were then bound by anti-IgG MACS
beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s
instructions. APCs were obtained by irradiating BALB/c x C57BL/6
spleen cells (2000 rad). T cell differentiation was induced by
culturing 2 x 105/ml
CD4+ T cells with 2 x
106/ml APC and 0.3 µM
OVA323–339 (Genosys, Cambridgeshire, U.K.). In
addition, Th1 cell cultures included 5 ng/ml IL-12 and 10 µg/ml
anti-IL-4 mAb (11B11), whereas Th2 polarizing cultures incorporated
5 ng/ml IL-4, 1/500 anti-IL-12, and 1/500 anti-IFN- serum.
After 3 days of culture, the cells were washed and harvested for
transfer.
Adoptive transfer
Single cell suspensions of naive lymph node and spleen cells were prepared from DO11.10 and MD4 F1 transgenic mice. Th1 and Th2 cells were prepared as described above. The percentage of CD4+, KJ1.26+ DO11.10 T cells or HEL-binding, IgMa+ B cells in these preparations was determined by flow cytometric analysis. Cell suspensions containing 1–6 x 106 tg T and 1–6 x 106 tg B cells were mixed and injected i.v. into unirradiated, age- and sex-matched BALB/c x C57BL/6 F1 recipients at the same time.
Ags and immunizations
cOVA-HEL was prepared by coupling HEL to cOVA using glutaraldehyde as described previously (3). Animals were injected s.c. with 130 µg of coupled Ag in CFA (Sigma, St. Louis, MO).
Ab treatments
Mice were injected with 250 µg anti-CD154 mAb (clone MR-1, kind gift of Dr. R. Noelle, Department of Microbiology, Dartmouth Medical College, Lebanon, NH) or 250 µg hamster IgG (ICN Biochemicals, Aurora, OH) i.p. on the day of immunization and 2 days later.
Cell cultures for cytokine analysis
Seven or fourteen days postimmunization, single cell suspensions were prepared from draining lymph nodes as described above, washed, and resuspended in complete medium at 4 x 106 cells/ml. Cells were added to 24-well plates (Costar, Corning, NY) in 1-ml aliquots and stimulated with 5 µM OVA323–339 peptide. Forty-eight hours later, supernatants were harvested and frozen to be assayed later by ELISA.
ELISAs
Abs. To detect tg B cell-derived anti-HEL IgMa in serum, Immulon 2 plates (Costar) were coated with HEL (20 µg/ml) at 4°C overnight. Plates were then washed at least three times with PBS-0.05% Tween before being blocked with PBS-10% FCS for 1 h at 37°C. Plates were washed and incubated with serially diluted serum samples for 3 h at 37°C before further washing. IgMa levels in serum were determined by incubation with biotinylated IgMa (Igh-6a) (DS-1) (2 µg/ml) (PharMingen) for 1 h at 37°C. Plates were then washed and incubated with Extravidin (1/1000) (Sigma) for 1 h at 37°C. Plates were washed again and TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added. To detect anti-cOVA IgG isotypes from endogenous B cells, a similar protocol as above was followed except that plates were coated with cOVA (2 µg/ml) (Sigma) followed by biotinylated anti-IgG2a (PharMingen) or biotinylated anti-IgG1 (Serotec), Extravidin, and TMB.
Cytokines.
To detect IL-2, IL-5, and IFN- produced from TCR tg T cells
restimulated in vitro, Immulon 4 plates (Costar) were coated with
anti-IL-2, anti-IL-5, or anti-IFN- detection Abs (all
PharMingen) overnight at 4°C. Plates were then washed and blocked as
above. Plates were next washed and incubated with supernatants for
3 h at 37°C before being washed again and incubated with a
biotinylated detection Ab, 0.5 µg/ml anti-IL-2, 2 µg/ml
anti-IL-5, or 4 µg/ml anti-IFN- (all PharMingen) for
1 h at 37°C. Plates were washed and incubated with Extravidin
(1/1000) for 1 h at 37°C before being washed again. TMB
Microwell Peroxidase substrate was then added as above. IL-5 and
IFN- standards were obtained from R&D Systems (Minneapolis, MN).
IL-4 was measured using a kit from Genzyme Diagnostics (Cambridge, MA)
according to the manufacturer’s instructions. All ELISAs were read on
a plate reader at 630 nm.
Immunohistochemistry
Lymph nodes were harvested and frozen in liquid nitrogen in OCT embedding medium (Miles, Diagnostic Division, Elkhart, IN). Tissue sections (6–10 µm) were cut on a cryostat microtome, fixed in acetone, and allowed to dry before being blocked in PBS-2% Goat serum (Vector Laboratories, Peterborough, U.K.). Sections were then stained with KJ1.26 (1/400) for 30 min before being washed in PBS for 15 min. Sections were then incubated with avidin-biotin complex-labeled alkaline phosphatase (Vector) for 30 min before being washed as before. Sections were next incubated with 5-bromo-4-chloro-3-indolyl phosphatase/nitroblue tetrazolium substrate (Vector) for 45 min, and then washed. Sections were next incubated with biotinylated anti-CD45R/B220 (RA3-6B2) (1/500) (PharMingen) for 30 min before washing. Avidin-biotin complex-labeled peroxidase (Vector) was then added for 30 min before washing. Sections were finally incubated in 3,3'-diaminobenzidine substrate for 10 min before a final wash, dehydration in 95% ethanol followed by 100% ethanol and clearing in Histoclear (BS & S, Edinburgh, U.K.). Sections were left to dry before mounting in ImmunoMount (Vector).
Statistics
Results are represented as the mean ± the range where groups contained two animals. If groups contained at least three animals, results are represented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4+ T cells were purified from DO11.10 mice
by negative selection and polarized in vitro to become Th1 or Th2
cells. After 3 days of culture, these cells were harvested for adoptive
transfer. Before transfer, an aliquot of each cell type was cultured in
vitro with OVA323–339 peptide and their cytokine
profile determined by ELISA (Fig. 1
). Th1
cells produced high levels of IFN- and little IL-5, whereas Th2
polarized cells produced significant amounts of IL-5 and low levels of
IFN-. Therefore, after polarization under the appropriate conditions
in vitro, DO11.10 T cells adopted the expected Th1 and Th2
phenotypes.
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Unpolarized, Th1 and Th2 OVA-specific TCR tg T cells and
HEL-specific tg B cells were transferred into naive syngeneic
BALB/c x C57BL/6 F1 mice, which were then
immunized with cOVA-HEL in CFA. The draining lymph nodes were removed
from recipients 3, 5, and 14 days after immunization, and flow
cytometric analysis performed. Th1, Th2, and unpolarized tg T cells
could all be detected in vivo (Fig. 2).
In agreement with our previous studies (3), peak clonal
expansion was observed on day 3 after immunization with unpolarized TCR
tg T cells and this was similar for the Th1 and Th2 groups. Unpolarized
and Th1 cells expanded to similar levels, whereas Th2 cells
proliferated to a lesser extent. All immunized groups displayed
expansion above unimmunized control levels, indicating that in vitro
polarized Th1 and Th2 TCR tg T cells can survive and respond to Ag
in vivo.
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To confirm the phenotype of the polarized tg T cells following
their adoptive transfer, we examined their effect on isotype switching
of the endogenous anti-OVA Ab response of the recipients. The
endogenous anti-OVA Ab responses of animals transferred with naive,
Th1, or Th2 cells was assessed as the HEL-specific tg B cells are
unable to switch their heavy chains. The serum level of anti-OVA
IgG2a was determined 14 days postimmunization (Fig. 3A). OVA-specific IgG2a could
be detected in immunized mice transferred with Th1 or unpolarized T
cells, but not in those transferred with Th2 cells. No OVA-specific
IgG2a could be detected in any unimmunized groups. There was no
difference between immunized mice transferred with Th1 and Th2 cells
when anti-OVA IgG1 levels were examined (data not shown); this may
be explained by the recent report that this isotype is not entirely
Th2-dependent (30).
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To further confirm the functional phenotype of the transferred
polarized TCR tg T cells, lymph nodes from immunized and unimmunized
transferred mice were removed 7 and 14 days postimmunization, and
single cell suspensions were prepared. These cells were then cultured
in vitro with or without OVA323–339 peptide, and
their cytokine production profile was analyzed.
OVA323–339 peptide-specific T cells from animals
receiving Th1 polarized cells and OVA/CFA continue to produce IFN-
(Fig. 3
B) but little IL-4 (Fig. 3C) or IL-5 (Fig. 3D) after peptide stimulation, whereas peptide-specific T
cells from animals transferred with Th2 polarized cells and immunized
with OVA/CFA produce IL-4 (Fig. 3C) and IL-5 (Fig. 3D) but little IFN- (Fig. 3B). Unpolarized
cells produced a mixture of both Th1 and Th2 cytokines when
restimulated in vitro (Fig. 3). Little or no cytokine production was
apparent in the absence of peptide stimulation.
Effect of tg Th1 and Th2 cells on tg B cells in vivo
Clonal expansion.
The relative abilities of Th1 and Th2 cells to provide B cell help
could be reflected in differences in B cell clonal expansion, Ab
production, or both. However, it has previously been difficult to make
such comparisons due to the inability to track Ag-specific T and B
cells in vivo. Therefore, we employed an adaptation of a recently
described adoptive transfer system in which the response of BcR tg B
cells is dependent upon cognate help from TCR tg T cells, as noted
above, to directly assess Ag-specific B cell clonal expansion and Ab
production in vivo in the context of help from Th1 or Th2 cells. The
kinetics and magnitude of the B cell response to immunization with
OVA-HEL/CFA was similar in animals receiving unpolarized, Th1 or Th2
TCR tg cells. In agreement with our previous studies (3),
B cell clonal expansion peaked 5 days postimmunization before declining
toward control levels by day 14 (Fig. 4A). Indeed, even though the
Th2 cells themselves did not expand as well as unpolarized or Th1 cells
(Fig. 2), they displayed a similar capacity to support B cell expansion
(Fig. 4A). Thus, animals adoptively transferred with
unpolarized, Th1, or Th2 cells and immunized with cOVA-HEL/CFA all
appeared capable of supporting similar levels of clonal expansion of
HEL-specific BcR tg B cells. As we have shown previously
(3) and confirmed here (Table I), the BcR tg B cells do not respond in
the absence of cognate help from the TCR tg T cells.
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B). Serum was taken from animals transferred with
unpolarized, Th1, or Th2 tg T cells together with tg B cells at 3, 5,
7, and 14 days postimmunization. All tg T cells could support Ab
production by MD4 BcR tg B cells as measured by anti-HEL
IgMa. Serum levels of anti-HEL
IgMa peaked at 5 days postimmunization in Th1 and
Th2 recipients but not until day 7 in recipients of unpolarized TCR tg
T cells. By day 14 after immunization, HEL-specific
IgMa Ab levels had declined to control levels in
all groups. Thus, when Th1 or Th2 cells were transferred and the
recipients immunized, anti-HEL IgMa was
detected earlier compared with recipients receiving unpolarized tg T
cells. Little or no anti-HEL IgMa was
detected in serum from any unimmunized control animals. Localization of unpolarized, Th1, and Th2 cells in vivo
It has been shown previously that T and B cells alter
their anatomical location within lymph nodes in response to
immunization to facilitate cognate interactions with each other
(3). In the case of the T cells, adoptively transferred,
unpolarized DO11.10 TCR Tg T cells disperse throughout the T cell-rich
paracortex of the lymph node and do not enter B cell follicles
(3) in naive animals. However, following immunization, the
Ag-specific T cells undergo clonal expansion and migrate into B cell
follicles, presumably to provide help to B cells. To determine whether
Th1 and Th2 cells alter their localization within the lymph node in
response to immunization in vivo, and if they do so in a similar
manner, we transferred each cell type into naive mice and immunized
with cOVA-HEL/CFA. In contrast to recent studies (21), we
found that unpolarized, Th1, and Th2 TCR tg T cells all displayed
paracortical localization in naive animals (Fig. 5A) and all displayed a
similar follicular migration following immunization (Fig. 5, B–E). There was no difference in the kinetics of
the migration of unpolarized, Th1, and Th2 TCR tg T cells into B cell
follicles as this occurred within 3 days of immunization and was still
apparent after 5 days in all groups. These results confirmed earlier
findings describing the follicular migration of unpolarized T cells in
response to immunization (3) and significantly extend them
by showing that both Th1 and Th2 T cells undergo similar migration with
similar kinetics. Furthermore, the presence of the BcR tg B cells has
no apparent effect on the response or localization of the TCR Tg T
cells; no differences were detected when similar studies to those
described above were performed using immunization with cOVA-HEL
(targets OVA to BcR tg B cells) vs cOVA + turkey OVA-HEL (no targeting
of OVA), or in µMT KO mice, or intact MD4 BcR tg mice immunized with
cOVA or cOVA-HEL (targeting OVA to a large population of BcR tg B
cells, (P. Garside, K. A. Pape, and M. K. Jenkins,
unpublished observations).
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T cell help for B cells is dependent on interactions between CD40
and CD154 (3, 31). Therefore, we decided to investigate
whether both Th1 and Th2 cells displayed similar requirements to
deliver T cell help to B cells in vivo. As we have reported previously
(3), treatment with anti-CD154 affected the clonal
expansion of T and B cells. The effect of anti-CD154 on T cell
expansion was measured at the peak of T cell expansion 3 days after
immunization. The Th1 and Th2 responses were similarly affected in the
anti-CD154-treated animals as the cells only expanded to 49% and
47% of their respective untreated groups (Th1 clonal expansion =
1.03% vs 0.51% in untreated vs anti-CD154-treated groups; Th2
clonal expansion = 0.68% vs 0.32%) (representative FACS profile,
Fig. 6, A–H; mean values
± range for two to three animals per group, Fig. 7A). We assessed the effect of
anti-CD154 treatment at the peak of B cell clonal expansion on day
5 after immunization. B cells from Th1-transferred
anti-CD154-treated and immunized mice only achieved 26%
(representative FACS profile, Fig. 6, I–P; mean values
± SEM for two to three animals per group, Fig. 7B) of the
clonal expansion of untreated Th1-transferred and immunized mice
(1.69% vs 0.44%), whereas B cells from Th2-transferred mice only
achieved 32% (Fig. 6, I–P) of the clonal expansion
exhibited by untreated Th2-transferred and immunized mice (2.44% vs
0.79%). Ab responses were also decreased in the presence of
anti-CD154. Serum anti-HEL IgMa was
analyzed by ELISA 5 days after immunization. It was found that
anti-CD154-treated mice transferred with Th1 cells only produced
58% of the HEL-specific serum IgMa of untreated
mice and anti-CD154-treated mice transferred with Th2 cells had
only produced 56% of the anti-HEL serum IgMa
of untreated Th2-transferred and immunized mice (Fig. 7C).
These results clearly demonstrate that in response to antigenic
challenge in vivo previously primed Th1 and Th2 cells require
interactions between CD40 and CD154 to expand fully and to mediate B
cell help. Interestingly, treatment with anti-CD154 did not prevent
movement of Ag-specific Th1 or Th2 (Fig. 5) cells into B cell follicles
after immunization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze cognate interactions between Ag-specific Th1, Th2, and B
cells in vivo, it was first necessary to circumvent the obstacles
presented by the extremely low frequency of Ag-specific lymphocytes
present in a normal naive animal. To do this, we modified an adoptive
transfer system in which we have recently shown that BcR tg B cells
require cognate help from TCR tg T cells for clonal expansion and
germinal center formation (3). In our current study, naive
TCR tg T cells were stably polarized toward a Th1 or Th2 phenotype in
vitro, as has been described previously (13, 32, 33, 34, 35, 36, 37),
before adoptive transfer. Our transferred T cells secreted the expected
pattern of cytokines (IFN- for Th1 and IL-4 and IL-5 for Th2) before
adoptive transfer and maintained their phenotypes following
restimulation after recovery from immunized animals. Furthermore, in
contrast to TCR tg Th2 cells, the TCR tg Th1 cells supported the
generation of OVA-specific IgG2a-secreting endogenous B cells,
indicating that the Th1 and Th2 cells maintained their adopted
phenotypes in vivo. Though there was no difference in OVA-specific
serum IgG1 levels between Th1 and Th2 recipients, others
(38) have observed IgG1 production in the absence of IL-4
suggesting that the absence of IgG2a, rather than the presence of IgG1,
may be a better indicator of a Th2 response. Furthermore, a recent
report has indicated that IgG1 may not be entirely Th2-dependent
(30).
Although early in vitro studies indicated that both Th1 and Th2 cells could support B cell responses (15, 16, 17), the capacity of Th1 cells to provide cognate help for B cells is controversial, with some studies reporting that they are relatively deficient in this function (21, 39, 40). However, care must be taken when interpreting such in vitro studies, as examination of several Th1 and Th2 clones revealed considerable variation in their ability to help B cells with some Th2 clones failing to support Ab synthesis at all (15). Recent studies have also attempted to address this issue in vivo (41) and support a role for Th1 cells in helping B cell responses. However, the Th1 and Th2 cells were of differing Ag specificities and induced by different immunizing regimes as this study was more concerned with help for isotype switching than B cell clonal expansion. We have developed a physiologically more relevant system where the TCR tg Th1 and Th2 cells recognize the same Ag and can be tracked in vivo using mAbs, as can the BcR tg B cells, thus allowing analyses of Ag-specific responses.
We found that both Th1 and Th2 cells clonally expanded in response to immunization. Interestingly, whereas the kinetics of T cell clonal expansion were similar, Th2 cells expanded significantly less than Th1 cells. However, both cell types were able to support a similar degree of B cell clonal expansion and Ab production. This might imply that Th2 cells are more adept at supporting B cell responses on a per cell basis, as was suggested by earlier studies (17, 42). Although this issue will require further detailed analysis, it is clear from our studies that Th1 cells are perfectly able to support B cell clonal expansion and Ab production. Alternatively, it may be that Th1 and Th2 cells achieve optimal B cell help in different ways with Th1 cells helping less well on a per cell basis but expanding to provide more cells, whereas Th2 cells provide more help per cell but expand less, thus the net effect is the same.
We have demonstrated previously with this adoptive transfer system that
Ag-specific T cells migrate into B cell follicles to provide B cell
help. Recent studies have emphasized the importance of cellular
localization in the induction of immune responses (43) and
this issue has only begun to be directly addressed for Th1 and Th2
cells in vivo. Therefore, we determined the location of TCR tg Th1 and
Th2 cells following immunization in vivo. We found that, whereas few
Th1 and Th2 cells were present in B cell follicles in naive adoptive
transfer recipients, considerable numbers of both types of cell could
be found in this location following immunization. Our findings contrast
with recent studies (21, 39), which suggested that, unlike
Th2 cells, Th1 cells localize in the paracortex by virtue of their
expression of CCR7 and are unable to support the differentiation of
naive B cells into germinal center cells. These results do not explain
the production of high-affinity IgG2a-secreting B cells that develop
with the help of Th1-derived cytokines such as IFN-. The
discrepancies between the studies may be the result of the in vitro vs
in vivo nature of some of the systems employed, our use of primed T
cells, the use of different adjuvants, or the fact that, in contrast to
the other studies, we have also tracked Ag-specific B cells directly
during detailed kinetic studies in vivo. However, as noted above, it
seems unlikely that the presence of the BcR tg B cells affects the
behavior of the TCR tg B cells, and we have clearly shown that Th1
cells move into the B cell follicle to support B cell clonal expansion,
Ab production, and class-switching to IgG2a.
A considerable number of studies have previously shown that CD40-CD154
interactions are an important component of T cell help for B cells, and
we confirmed this using the adoptive transfer system (3, 31). Therefore, it was important to determine whether both Th1
and Th2 cell help for B cells were CD154-dependent. Both Th1 and Th2
cells can express CD154 (44), and membranes from each cell
type can provide cognate help for B cell responses in vitro, which was
shown to be CD154-dependent (31). However, more recently,
it has become clear that CD154 may be involved in B cell help directly,
or indirectly via effects on the differentiation of T cells or both.
Thus the interaction of CD154 on T cells with CD40 on APC may result in
the production of cytokines, e.g., IL-12, which polarize or activate
the T cell before it interacts with the B cell. Here, we have shown
that the clonal expansion of both Th1 and Th2 cells was reduced by
50% in anti-CD154-treated animals, whereas B cell clonal
expansion and Ab production were almost abolished in both situations.
This is consistent with our previous findings (3) and
suggests that, as reported by others, CD154 is important for the
differentiation of Th1 and Th2 cells and their ability to help B cells.
We cannot distinguish whether the decreased B cell responses in our
anti-CD154-treated animals are a result of blocked interactions
between T cells and APC leading to reduced T cell clonal expansion
(3), altered T cell differentiation (45), or
of a direct blockade of subsequent T cell–B cell interactions.
Nevertheless, our results show clearly that Th1 and Th2 cells require
CD40-CD154 interactions for maximal T and B cell responses.
Interestingly, unpolarized, Th1 and Th2 cells can still enter B cell
follicles after treatment with anti-CD154, suggesting that this
interaction is not required for movement into the follicle and that a
defect in T cell migration does not underlie the failure of T cell help
in this situation, as has been demonstrated in tolerance
(46).
Although our studies have provided the first direct in vivo evidence that both Th1 and Th2 cells support Ag-specific B cell expansion and Ab production in a similar manner and location, all of the molecular interactions involved remain to be elucidated. The most likely candidates would be cytokines, which have been widely implicated in T cell help for B cells. However, as we have demonstrated that Th1 and Th2 cells, which secrete very different patterns of cytokines, are both able to support B cell responses and others have been unable to find a role for a battery of the expected cytokines (39), it is clear that further studies are required to define the molecules involved in T–B cell interactions in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Garside, Department of Immunology and Bacteriology, University of Glasgow, Western Infirmary, Glasgow, G11 6NT, U.K.
3 Abbreviations used in this paper: DC, dendritic cell; tg, transgenic; KO, knockout; BcR, B cell receptor; cOVA, chicken OVA; HEL, hen egg lysozyme.
Received for publication January 18, 2000. Accepted for publication June 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide. J. Immunol. 140:2121.[Abstract]
and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944. receptor. Science 259:1742. production and activates IRAK and NF
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A. P. Matson, L. Zhu, E. G. Lingenheld, C. M. Schramm, R. B. Clark, D. M. Selander, R. S. Thrall, E. Breen, and L. Puddington Maternal Transmission of Resistance to Development of Allergic Airway Disease J. Immunol., July 15, 2007; 179(2): 1282 - 1291. [Abstract] [Full Text] [PDF]
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S. Prickett, P. M. Gray, S. L. Colpitts, P. Scott, P. M. Kaye, and D. F. Smith In vivo recognition of ovalbumin expressed by transgenic leishmania is determined by its subcellular localization. J. Immunol., April 15, 2006; 176(8): 4826 - 4833. [Abstract] [Full Text] [PDF]
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M. L. Fields, S. A. Nish, B. D. Hondowicz, M. H. Metzgar, G. N. Wharton, A. J. Caton, and J. Erikson The Influence of Effector T Cells and Fas Ligand on Lupus-Associated B Cells J. Immunol., July 1, 2005; 175(1): 104 - 111. [Abstract] [Full Text] [PDF]
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S. Matsumoto, M. Matsumoto, K. Umemori, Y. Ozeki, M. Furugen, T. Tatsuo, Y. Hirayama, S. Yamamoto, T. Yamada, and K. Kobayashi DNA Augments Antigenicity of Mycobacterial DNA-Binding Protein 1 and Confers Protection against Mycobacterium tuberculosis Infection in Mice J. Immunol., July 1, 2005; 175(1): 441 - 449. [Abstract] [Full Text] [PDF]
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T. Yasumi, K. Katamura, I. Okafuji, T. Yoshioka, T.-a. Meguro, R. Nishikomori, T. Kusunoki, T. Heike, and T. Nakahata Limited Ability of Antigen-Specific Th1 Responses to Inhibit Th2 Cell Development In Vivo J. Immunol., February 1, 2005; 174(3): 1325 - 1331. [Abstract] [Full Text] [PDF]
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K. M. Smith, J. M. Brewer, C. M. Rush, J. Riley, and P. Garside In Vivo Generated Th1 Cells Can Migrate to B Cell Follicles to Support B Cell Responses J. Immunol., August 1, 2004; 173(3): 1640 - 1646. [Abstract] [Full Text] [PDF]
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P. Maffia, J. M. Brewer, J. A. Gracie, A. Ianaro, B. P. Leung, P. J. Mitchell, K. M. Smith, I. B. McInnes, and P. Garside Inducing Experimental Arthritis and Breaking Self-Tolerance to Joint-Specific Antigens with Trackable, Ovalbumin-Specific T Cells J. Immunol., July 1, 2004; 173(1): 151 - 156. [Abstract] [Full Text] [PDF]
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T. Yasumi, K. Katamura, T. Yoshioka, T.-a. Meguro, R. Nishikomori, T. Heike, M. Inobe, S. Kon, T. Uede, and T. Nakahata Differential Requirement for the CD40-CD154 Costimulatory Pathway during Th Cell Priming by CD8{alpha}+ and CD8{alpha}− Murine Dendritic Cell Subsets J. Immunol., April 15, 2004; 172(8): 4826 - 4833. [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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K. M. Smith, J. M. Brewer, P. Webb, A. J. Coyle, C. Gutierrez-Ramos, and P. Garside Inducible Costimulatory Molecule-B7-Related Protein 1 Interactions Are Important for the Clonal Expansion and B Cell Helper Functions of Naive, Th1, and Th2 T Cells J. Immunol., March 1, 2003; 170(5): 2310 - 2315. [Abstract] [Full Text] [PDF]
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R.-N. E. Dogan, C. Vasu, M. J. Holterman, and B. S. Prabhakar Absence of IL-4, and Not Suppression of the Th2 Response, Prevents Development of Experimental Autoimmune Graves' Disease J. Immunol., February 15, 2003; 170(4): 2195 - 2204. [Abstract] [Full Text] [PDF]
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C. Rush, T. Mitchell, and P. Garside Efficient Priming of CD4+ and CD8+ T Cells by DNA Vaccination Depends on Appropriate Targeting of Sufficient Levels of Immunologically Relevant Antigen to Appropriate Processing Pathways J. Immunol., November 1, 2002; 169(9): 4951 - 4960. [Abstract] [Full Text] [PDF]
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G. Muller, U. E. Hopken, H. Stein, and M. Lipp Systemic immunoregulatory and pathogenic functions of homeostatic chemokine receptors J. Leukoc. Biol., July 1, 2002; 72(1): 1 - 8. [Abstract] [Full Text] [PDF]
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K. M. Smith, F. McAskill, and P. Garside Orally Tolerized T Cells Are Only Able to Enter B Cell Follicles Following Challenge with Antigen in Adjuvant, but They Remain Unable to Provide B Cell Help J. Immunol., May 1, 2002; 168(9): 4318 - 4325. [Abstract] [Full Text] [PDF]
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B. Johansson-Lindbom and C. A. K. Borrebaeck Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development In Vivo J. Immunol., April 1, 2002; 168(7): 3165 - 3172. [Abstract] [Full Text] [PDF]
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J. L. McQualter, R. Darwiche, C. Ewing, M. Onuki, T. W. Kay, J. A. Hamilton, H. H. Reid, and C. C.A. Bernard Granulocyte Macrophage Colony-Stimulating Factor: A New Putative Therapeutic Target in Multiple Sclerosis J. Exp. Med., October 1, 2001; 194(7): 873 - 882. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), Th1 (•), or
Th2 (
) T cells along with 3–6 x 106
B220+ HEL-specific B cells. On day 0, transfer recipients
were injected s.c. with 130 µg cOVA-HEL in CFA. The percentage of
CD4+KJ1.26+ T cells in the draining lymph nodes
of adoptively transferred recipients was assessed by flow cytometry on
days 3, 5, and 14 after immunization. Each time point represents the
mean ± range for at least two mice per group. Unimmunized
controls from each time point were averaged and represented as day 0.
Similar results were obtained in two additional experiments.
