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Department of Immunology & 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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Recently, the adoptive transfer of TCR transgenic (tg)3 lymphocytes has improved our ability to assess the effect Ag has on a single Ag-specific population of T cells in vivo (22). However, functional analysis of these T cells has usually required additional in vitro restimulation. Although these in vitro techniques have proved useful in the past, they remain unphysiological and recent studies have questioned their relevance (23). Furthermore, in vivo functional studies of Ag-specific populations are technically challenging and often rely upon indirect assays of T cell function. One method of assessing T cell functional capacity in vivo is to analyze Ag-specific Ab production to demonstrate the capacity of T cells to provide B cell help (24). However, the potential for fed Ag to directly inactivate B cell responses may confound the interpretation of the effect feeding has on an Ag-specific T cell’s ability to provide cognate help. Using a double tg T and B cell adoptive transfer system, we have previously shown that expansion of Ag-specific B cells is dependent on cognate interactions with Ag-specific T cells (24, 25). In this study, we have modified this system to assess the ability of orally primed or tolerized T cells to provide B cell help. DO11.10 OVA-specific T cells were transferred into naive recipients which were subsequently primed by feeding a high dose of OVA in the presence of the mucosal adjuvant cholera toxin (CT), or tolerized by feeding a high dose of OVA alone. Subsequently, the animals received MD4 hen egg lysozyme (HEL)-specific B cells before being challenged in the periphery with conjugated OVA-HEL in adjuvant. Thus, we have an in vivo system to assess the functional ability of primed or tolerized T cells to provide cognate help for B cell responses. Furthermore, because B cells were not present at the time of primary Ag exposure and because they are specific for a different Ag (HEL vs OVA) the priming or tolerizing treatments should have no direct affect on the tg B cells. Using this approach, we have been able to show that, in contrast to orally primed T cells, orally tolerized T cells display an initial inability to enter B cell follicles and support primary Ab responses. Following challenge in the periphery, primed and tolerized T cells are both able to enter B cell follicles but tolerized T cells remain unable to provide adequate B cell help. However, this failure of B cell help is not a result of reduced T cell clonal expansion or migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c (H-2d/d, IgMa) and C57BL/6 (H-2b/b, IgMb/b) mice were purchased from Harlan-Olac (Bicester, U.K). Congenic IgHb BALB/c (H-2d/d, IgMb) and (C57BL/6 x IgHb BALB/c)F1 (H-2b/d, IgMb) recipient mice were bred in the Central Research Facility of the University of Glasgow. Mice homozygous for the chicken OVA (cOVA) peptide323–339/I-Ad-specific DO11.10 TCR transgenes on the BALB/c background (26) were crossed to C57BL/6 mice to produce animals on an F1 background, which were used as TCR tg T cell donors. Similarly, mice heterozygous for the anti-HEL IgMa and IgDa transgenes on the C57BL/6 background (MD4) (27) were crossed to congenic IgHb BALB/c to produce animals on an F1 background. Offspring were screened for the expression of the MD4 transgenes by flow cytometry and tgs were used as BcR tg B cell donors. Six-week-old male (C57BL/6 x IgHb BALB/c) F1 mice were used as recipients. All animals were specified pathogen free and were maintained under standard animal house conditions in accordance with Home Office regulations.
Preparation of cell suspensions for adoptive transfer
Peripheral lymph nodes (PLN: axillary, inguinal, and cervical), mesenteric lymph nodes (MLN), and spleens from DO11.10 F1 and MD4 F1 mice were pooled and forced through Nitex mesh (Cadisch Precision Meshes, London, U.K.) using a syringe plunger. Suspensions were washed in RPMI 1640 (Life Technologies, Paisley, U.K.). The percentage of CD4+KJ1.26+ T cells and IgMa+B220+ B cells were determined by flow cytometric analysis as described below. TCR tg T cells (1–6 x 106) were injected i.v. into age- and sex- matched IgHb (BALB/c x C57BL/6)F1 recipients as described previously (25). Ten days later, 1–6 x 106 IgMa+B220+ B cells were similarly transferred.
Ag Administration
cOVA (fraction V) and CT were obtained from Sigma-Aldrich and HEL from Biozyme (Gwent, U.K.). Following adoptive transfer, recipient mice were exposed to Ag by feeding PBS, 100 mg OVA, 100 mg OVA with 20 µg CT (OVA/CT; Sigma-Aldrich, Poole, U.K.) or by s.c. injection with 100 µg OVA in 100 µl saline/50% CFA (OVA/CFA; Sigma-Aldrich). cOVA-HEL was prepared by coupling HEL to cOVA using glutaraldehyde as described previously (24). Twenty-four hours after transfer of tg B cells, secondary immunizations were performed by injecting 130 µg OVA-HEL in 100 µl saline/50% IFA (Sigma-Aldrich) s.c.
Flow cytometry
PLN were harvested between days 1 and 10 after Ag challenge. Cell suspensions were prepared as described above. Aliquots of cells were incubated with FcR blocking buffer (anti-CD16/32 hybridoma supernatant, 10% mouse serum (Diagnostic Scotland, Edinburgh, U.K.) and 0.1% sodium azide (Sigma-Aldrich) for 10 min at 4°C to prevent binding of Ab to cells via Fc regions.
For two-color analysis of CD4+ tg T cells, cells were incubated with PE-conjugated anti-CD4 (BD PharMingen, Oxford, U.K.) and biotinylated clonotypic anti-TCR Ab, KJ1.26 (28), for 40 min at 4°C. Cells were washed in FACS buffer (PBS, 2% FCS, and 0.05% sodium azide) and then incubated with FITC-conjugated streptavidin (BD PharMingen) for 40 min at 4°C. tg B cells were detected with PE-labeled anti-B220 (BD PharMingen) in combination with biotinylated anti-IgMa (BD PharMingen) followed by FITC-labeled streptavidin. Following a wash in FACS buffer, cells were resuspended in FACS flow (BD PharMingen) for analysis with a FACScan and CellQuest software (BD PharMingen). Two-color analysis was performed on 20,000 events.
ELISAs
To detect anti-HEL Abs in serum, Immulon 2 plates (Costar; Corning Glass, Corning, NY) were coated with HEL and then blocked with PBS-10% FCS for 1 h at 37°C. Serum samples were added for 3 h at 37°C before incubation with biotinylated anti-IgMa (BD PharMingen) for 1 h at 37°C. Plates were then incubated with extravidin-peroxidase (1/1000; Sigma-Aldrich) for 1 h at 37°C. TMB Microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to detect enzymatic activity as described previously (25). Anti-OVA serum Abs were detected similarly but plates were coated with OVA and Abs were detected with biotinylated anti-IgG1 or biotinylated anti-IgG2a (both BD PharMingen).
Immunohistochemistry
PLN and MLN were frozen in liquid nitrogen in OCT embedding medium (Miles Diagnostic Division, Elkart, IN) and stored at -70°C. Six- to 10- µm sections were cut then stored at -20°C. For staining, sections were incubated in acetone for 10 min, air dried, and rehydrated with PBS before incubation in 0.1% azide/3% H2O2 for 45 min. Avidin solution (Vector Laboratories, Burlingame, CA) was added for 15 min and then biotin solution (Vector Laboratories) was added and finally Fc block for 30 min. Sections were washed in PBS after each treatment.
Single staining for the DO11.10 TCR
To detect tg T cells, sections were stained with 1/1600 KJ1.26
in TNB (0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.5% blocking
reagent (NEN Life Science, Boston, MA)) for 30 min, before being washed
twice in TNT (0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05% Tween
20). Subsequently, sections were incubated with streptavidin-HRP
(1/100 in TNB block; NEN Life Science) for 30 min before washing as
before. Biotinyl-tyramide (1/50 in TNB; NEN Life Science) was then
added for 10 min, followed by two washes in TNT. Streptavidin-HRP was
added again for 30 min before washing twice in TNT. Enzymatic activity
was detected with 3,3'-diaminobenzidine substrate (Vector Laboratories)
before washing in H2O, followed by incubation
with diaminobenzidine-enhancing solution (Vector Laboratories)
for 
10 s and a wash in H2O. Harris hematoxylin
(Vector Laboratories) was used to counterstain before rinsing in
H2O and dipping in acid alcohol, tap water,
bicarbonate, then tap water. Sections were subsequently exposed to 70%
ethanol, 95% ethanol twice, then 100% ethanol for dehydration before
clearing in Histoclear (BS & S, Edinburgh, U.K.) and immediate mounting
in Histomount (BS & S).
Double staining tg T cells and B cell areas or tg B cells and T cell areas
To detect tg T or B cells, sections were stained with KJ1.26 (1/400) or bio-IgMa, respectively, for 30 min before being washed in PBS for 15 min. Sections were then incubated with avidin-biotin complex-labeled alkaline phosphatase (Vector Laboratories) for 30 min before being washed as before. Sections were next incubated with 5-bromo-4-chloro-3-indolyl phosphatase/nitroblue tetrazolium substrate (Vector Laboratories) for 45 min and then washed. To detect T or B cell areas (paracortex vs follicle), sections were next incubated with biotinylated anti-Thy1.2 or anti-CD45R/B220 (1/500; BD PharMingen), respectively, for 30 min before washing. Avidin-biotin complex-labeled peroxidase (Vector Laboratories) 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) before mounting in Histomount (BS & S).
Statistics
Results are expressed as mean ± SEM or mean + range. To
test significance, Student’s unpaired t test was performed;
a p value of 
0.05 was regarded as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To confirm the ability of orally delivered Ag to prime or tolerize
systemic Ab responses, animals were fed OVA/CT or OVA and serum was
collected and analyzed for OVA-specific Abs by ELISA. Serum was
assessed from adoptively transferred mice that had been fed OVA or
OVA/CT or immunized with OVA/CFA s.c. 19 days previously. Mice fed OVA
alone did not produce any detectable OVA-specific IgG1 (Fig. 1
A) or IgG2a (Fig. 1B). In contrast, those fed OVA/CT or immunized with OVA/CFA
produced anti-OVA Abs of both the IgG1 and IgG2a subclasses (Fig. 1). OVA/CFA-immunized animals produced higher levels of both isotypes
than the OVA/CT-fed animals. No anti-OVA Abs of either subclass
were observed in unimmunized controls (data not shown).
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To further confirm that feeding OVA vs OVA/CT resulted in
tolerance or priming, respectively, the response to antigenic challenge
was assessed by serum Ab production. Adoptively transferred mice were
fed OVA or OVA/CT, or immunized s.c. with OVA/CFA. These animals were
challenged 12 days later with OVA/IFA and serum was collected 7 and 14
days after challenge. As CT is reported to preferentially induce Th2
responses (29), serum from these animals was analyzed for
OVA-specific IgG1 to assess the humoral immune response to challenge.
Seven days after challenge the OVA/CT- and OVA/CFA-primed groups
produced significantly more OVA-specific IgG1 compared with the OVA-fed
(tolerized) and PBS-fed (primary response) groups (Fig. 2A). Fourteen days after
challenge the primed responses remained significantly higher than those
of the tolerized group. At this time point, the primary T cell response
had begun to support Ab production with PBS-fed animals displaying
OVA-specific IgG1 levels similar to the OVA/CT group. The amount of Ab
produced by the OVA-fed group had also increased slightly but still
remained significantly less than that of the PBS-fed primary immune
response group at the higher titers analyzed (Fig. 2B).
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Having confirmed our recent studies that our feeding regimens
induced priming or tolerance in adoptively transferred mice
(30), we initially examined the effects of oral priming
and tolerance on Ag-specific T cells by assessing their clonal
expansion. The response of previously primed or tolerized T cells to
challenge was analyzed by flow cytometry. Adoptively transferred mice
were primed or tolerized as described above. Nine days later, all
animals received tg B cells before being challenged with cOVA-HEL/IFA
s.c. Control groups were injected with PBS, as opposed to OVA/IFA, and
results from these groups at each time point were averaged and are
represented as day 0. The primary T cell response (represented by the
PBS-fed group) peaked on day 3 with a 7-fold increase in tg T cell
numbers and declined thereafter (Fig. 3),
as described previously (24, 25). All other groups, which
had previously been exposed to Ag before challenge, failed to achieve
the same level of T cell clonal expansion as naive cells at any time
point examined. Following challenge the OVA-tolerized and OVA/CT-primed
tg T cells displayed similar levels of clonal expansion 3 days after
challenge. However, the OVA/CT-primed T cell clonal expansion continued
to rise and peaked on day 5, whereas the OVA response declined after
day 3 (Fig. 3). Animals initially primed with OVA/CFA showed a peak
clonal expansion 3 days after challenge, which remained at a similar
level until day 5 (Fig. 3). There were more tg T cells present after
challenge in OVA/CFA-primed animals than in those primed with OVA/CT or
tolerized with OVA alone (Fig. 3). However, the starting frequency of
tg T cells in OVA/CFA-primed animals was higher than in other
experimental groups. Thus, following challenge, all groups that had
previously been exposed to OVA exhibited only a 2- to 3-fold clonal
expansion relative to their respective starting levels. Since TCR tg T
cells previously exposed to Ag in immunogenic or tolerogenic forms do
not clonally expand as well in response to OVA-HEL/IFA as the tg T
cells in PBS-fed animals, these results imply that T cells in
Ag-experienced animals do not have the same capacity to expand in
response to challenge as naive cells, as has previously been suggested
(31, 32, 33, 34), and that an apparent defect in clonal expansion
is not indicative of tolerance.
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As primed or tolerized T cells displayed similar clonal expansion
in response to challenge, we next directly examined their capacity to
provide help for humoral immune responses. Previously, naive tg T cells
(PBS-fed group) helped B cells with the same kinetics as previously
described (24, 25), with the B cell response first
apparent at 3 days but peaking 5 days after challenge (Fig. 4) and declining thereafter. Primed
animals (exposed to OVA/CFA or OVA/CT) also exhibited B cell clonal
expansion following challenge (Fig. 4). tg T cells previously primed by
OVA/CFA injection were able to help B cells clonally expand more
quickly than T cells undergoing a primary immune response, with B cell
proliferation peaking 3 days after challenge. However, the
OVA/CT-primed group helped B cell clonal expansion with similar
kinetics as T cells undergoing a primary immune response (Fig. 4), with
the B cell response peaking 5 days after challenge and declining
thereafter. Tolerized tg T cells appeared to support some clonal
expansion of B cells on day 3. However, the initial B cell clonal
expansion supported by tolerized T cells was impaired and at subsequent
time points tolerized T cells do not help B cells to expand. Therefore,
in sharp contrast to previously primed T cells or T cells undergoing a
primary immune response, tolerized T cells display a marked defect in
this ability.
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The functional ability of primed and tolerized T cells was also
assessed by comparing whether the two types of T cells could support Ab
production by tg B cells. T cells undergoing a primary immune response
took 5 days to help B cells to produce maximal anti-HEL
IgMa titers (Fig. 5B) as previously described
(24, 25). However, tg T cells previously primed with
OVA/CFA or OVA/CT were able to support maximal Ab production by tg B
cells 3 days after challenge (Fig. 5A). By day 5 the OVA/CT
primed Ab responses had greatly diminished, whereas the OVA/CFA-primed
responses remained high. In contrast to all other experimental groups,
tolerized T cells were unable to support Ab production by tg B cells at
any time point examined.
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To confirm the flow cytometric analysis (Fig. 4
) which indicated
that tolerized T cells were defective in providing support for B cell
clonal expansion in response to challenge, IgMa+
tg B cells were visualized directly by immunohistochemistry.
Before challenge, all groups (data not shown) exhibited tg B cell
accumulation similar to that of OVA-fed animals (Fig. 6A). However, following
challenge, tg B cell accumulation appeared to increase only in the PBS,
OVA/CFA, and OVA/CT groups (Fig. 6, B, C, and
E). In confirmation of FACS data (Fig. 4), there
was little accumulation of tg B cells in the OVA-fed group in response
to challenge (Fig. 6D).
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Because primed or tolerized tg T cells were capable of achieving
similar levels of clonal expansion in response to challenge but
displayed marked differences in their ability to support B cell
responses, we examined whether these cells traffic to different
locations during primary and secondary immune responses. Inguinal lymph
nodes were taken from the mice described above and examined for the
presence of tg T cells. In animals fed PBS (data not shown) or OVA
(Fig. 7), tg T cells were primarily
localized within the paracortical areas of PLN (Fig. 7A) or
MLN (Fig. 7B) before challenge with few, if any, cells
apparent in B cell follicles. However, accumulation of tg T cells was
apparent in the paracortex of OVA-fed animals. In OVA/CT-fed (Fig. 7A, B) and OVA/CFA (data not shown)-primed animals, the situation observed
was different. After both of these treatments, tg T cells accumulated
in the paracortex and were visible in B cell follicles in the PLN (Fig. 7A) and MLN (Fig. 7B) during the primary
response. Following challenge in the periphery, tg T cells from
OVA/CFA-primed, OVA/CT-primed, and OVA-tolerized mice expanded in
paracortical regions (Fig. 8), confirming
FACS data. Interestingly, both primed and tolerized tg T cells were
visible within B cell follicles after challenge (Fig. 8, B–D). Therefore, during the primary response T cells primed
with Ag plus adjuvant are able to migrate to B cell follicles, and this
property is not lost during the secondary response. However, tolerized
T cells are unable to enter into B cell follicles during their primary
exposure to Ag but are able to migrate to these locations following
subsequent challenge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms of oral tolerance of T and B cells are unknown. However, it is widely believed that the B cell unresponsiveness induced occurs because of a lack of T cell help and that the B cell remains potentially active. In support of this hypothesis, only T cell-dependent Ags induce oral tolerance and after feeding hapten-protein conjugates the tolerance generated shows complete carrier specificity (1, 4, 7, 35). Furthermore, tolerance in vivo can be bypassed by stimulating OVA-reactive B cells with Ag plus LPS or an unrelated carrier (7, 36). In this study, we have provided direct in vivo evidence that orally tolerized T cells are incapable of providing cognate help for B cells, suggesting that the B cell unresponsiveness observed after feeding may indeed result from a lack of T cell help.
Since we have shown that our feeding regimens induce systemic priming and tolerance (30), we decided to examine whether primed and tolerized tg T cells were functionally different in their ability to support B cell responses. Initially, we assessed the clonal expansion of primed, tolerized, or naive tg T cells in response to challenge. T cells undergoing a primary immune response displayed a 7-fold clonal expansion, whereas tg T cells primed via the oral route with OVA/CT or systemically with OVA/CFA s.c. had expanded only 2- to 3-fold. Somewhat surprisingly, OVA-tolerized T cells also showed a 2-fold increase in tg cell numbers, similar to primed cells. These results are in accordance with previous studies of secondary responses of systemically primed or i.v. tolerized T cells. Merica et al. (32) showed that OVA/CFA-primed T cells clonally expand poorly upon re-exposure to Ag in adjuvant, while other workers demonstrated that i.v. tolerized T cells exhibited a previously activated phenotype and were capable of as much clonal expansion in response to challenge as T cells primed with Ag in adjuvant (33). It has long been hypothesized that T cells tolerized by feeding Ag do not elicit productive immune responses because they proliferate poorly in response to subsequent antigenic challenge (6, 7, 37, 38). Importantly, we have shown directly that in vivo orally tolerized T cells are capable of as much clonal expansion as orally primed T cells. However, although the latter T cells also display reduced clonal expansion in comparison to naive T cells activated in a primary immune response they are able to support immune responses equally well. Thus, our data suggest that orally tolerized T cells are qualitatively distinct from primed cells but the critical difference is not in their ability to clonally expand in response to subsequent challenge.
Because orally primed and tolerized T cells displayed similar abilities to clonally expand in response to challenge, we next examined their functional capacities directly in vivo. Studies have demonstrated that although primed and tolerized T cells do not differ in their clonal expansion, the ability of the former to make effector cytokines is greatly increased in comparison to the latter (33). Since one of the functions of such T cell-derived cytokines is to support B cell responses (39), we examined this ability directly in vivo. We used an adoptive transfer system whereby the response of BcR tg B cells to a coupled Ag is exquisitely dependent upon TCR tg T cells (24, 25). However, we modified this system so that initial priming of tolerizing treatments had no effect upon the B cells because they were physically absent and do not recognize the uncoupled T cell epitope. T cells undergoing a primary response or T cells previously primed with OVA/CFA or OVA/CT were capable of supporting fulminant B cell clonal expansion and Ab production, whereas orally tolerized T cells were unable to support either of these functions adequately. The B cell response was also directly visualized in vivo. Again, it was noted that tg B cell numbers increase in PLN after challenge of both naive and primed groups. However, very little increase in tg B cells in tolerized groups following challenge was observed.
It has long been hypothesized that after tolerance induction the B cell unresponsiveness observed is due to functional defects in the capacity of tolerized T cells to provide B cell help (40). However, this assumption has been difficult to prove. Using several assays based upon the double adoptive transfer system described above, we have been able to follow cognate interactions among naive, primed, and tolerized T cells and fully competent B cells directly in vivo. These studies have shown that previously naive and primed T cells are capable of providing help for B cell clonal expansion and Ab production but tolerized T cells are unable to provide such help. These results support the hypothesis that the B cell unresponsiveness observed after feeding results from a defect in tolerized tg T cells to provide cognate help for B cells. However, whether fed Ag also directly inactivates B cells will require further study. This may be an important consideration if oral tolerance is to be used therapeutically to treat B cell-mediated autoimmune diseases such as myasthenia gravis.
An alternative explanation for the defective capacity of orally tolerized T cells to provide B cell help may lie in their relative ability to migrate to B cell follicles. Previous studies have shown that during primary immune responses T cells proliferate in T cell areas then move toward B cell follicles to help B cells (24, 31, 41, 42). T and B cells specific for the same Ag then meet at the border between the T and B cell areas (24, 43, 44). Ag-specific T cells subsequently provide cognate help for B cells which clonally expand and produce Ab (24). Interestingly, studies of i.v. administration of Ag have indicated that TCR tg T cells tolerized in this way are unable to undergo follicular migration (31). However, in contrast to the findings reported here, the previous studies did not examine follicular migration of tolerized T cells upon challenge. Following oral or systemic priming, we observed significant numbers of TCR tg T cells in follicular regions of local and PLN but such localization was not apparent following the induction of oral tolerance, probably explaining the lack of a primary Ab response in this group. However, following systemic challenge with Ag in adjuvant, T cells were apparent in B cell follicles of all experimental groups. These data not only suggest that tolerized T cells display an initial defect in migration which is overcome by subsequent challenge but also that the effects of orally administered Ag are apparent systemically in both situations.
The migration of T cells is controlled by chemokines. Naive T cells express the chemokine receptor CCR7, the ligands of which, secondary lymphoid tissue chemokine and EBI1 ligand chemokine, are produced by stromal cells in the T cell area (45). The CXCR5 chemokine receptor is expressed by Ag-specific T cells after exposure to Ag plus adjuvant but not Ag alone (46). The ligand for CXCR5, B lymphocyte chemoattractant, is produced by follicular stromal cells (45) and may be responsible for recruiting Ag-stimulated T cells to B cell areas. Interestingly, signals through CD28 and OX40 are required for CXCR5 induction on T cells and subsequent migration to follicles. Furthermore, the ligands for CD28 and OX40 (B7 and OX40 ligand) are induced on dendritic cells by inflammation (47). These findings may explain why tolerized T cells do not migrate into B cell follicles during primary immune responses (as no inflammation is present), whereas following challenge, tolerized T cells acquire the ability to enter B cell follicles, as a consequence of acquiring CXCR5 expression after interaction with adjuvant activated dendritic cells. However, despite being able to migrate into B cell follicles, tolerized T cells remain unable to support fulminant B cell clonal expansion and Ab production. The defect in tolerized T cells remains unclear but may lie in their inability to produce effector lymphokines. Indeed, we have previously shown that the production of Th2 cytokines proposed to be important in B cell help is dramatically reduced in orally tolerized animals (48), and we are now investigating this at the single cell level.
Our studies have begun to directly dissect the defects which may underlie the inability of orally tolerized T cells to provide B cell help in vivo. We have found that orally tolerized T cells display a reduced capacity to provide B cell help and to migrate to B cell follicles following initial exposure to Ag. However, although following challenge in the periphery tolerized T cells remain unable to support B cell responses, this is not a result of defective T cell clonal expansion or follicular migration, which are similar to primed cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Garside, Department of Immunology & Bacteriology, University of Glasgow, Western Infirmary, Glasgow, G11 6NT, Glasgow, U.K. E-mail address: pg3b{at}clinmed.gla.ac.uk
3 Abbreviations used in this paper: tg, transgenic; CT, cholera toxin; HEL, hen egg lysozyme; PLN, peripheral lymph node; MLN, mesenteric lymph node; cOVA, chicken OVA.
Received for publication November 9, 2001. Accepted for publication February 21, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or 100 mg OVA plus 20 µg CT (OVA/CT,
) or s.c. injection with 100 µg OVA/CFA (
). Nineteen days
later, serum was collected and analyzed for OVA-specific IgG1
(A) and IgG2a (B) by ELISA. No
OVA-specific IgG1 or IgG2a was detected in PBS-fed animals. Results
represent two animals per group and are presented as the mean OD plus
range. Similar results were obtained in one further experiment.
), 100 mg OVA (
) s.c. Results represent
two animals per group and are represented by the mean plus range.
Similar results were obtained in two additional experiments.
