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4ß71
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*
Laboratory of Immunology and Vascular Biology, Department of Pathology, and
Digestive Disease Center, Stanford University, Stanford, CA 94305;
Departments of Medicine and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; and
§
Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 integrin on lymphocytes with the
vascular mucosal addressin cell adhesion molecule-1. To determine
whether B cell memory for RV correlates with
4ß7 expression, we transferred sorted
B220+ phenotypically defined memory
(IgD-4ß7high and
IgD- 4ß7-) and
naive (IgD+4ß7+)
splenocytes into recombination-activating gene-2 knockout mice (B and T
cell-deficient) that were chronically infected with RV. Only mice
receiving 4ß7high memory
(IgD-) B cells produced RV-specific IgA in the stool,
cleared the virus, and were immune to reinfection.
4ß7high (but not
4ß7-) memory B cells from
donors boosted as much as 7 mo previously also cleared the virus,
indicating that 4ß7high memory
B cells maintain long term functional immunity to RV. Although only
4ß7high memory cells provided
mucosal immunity, 4ß7- cells
from recently boosted donor animals could generate RV-specific serum
IgG, but, like naive (IgD+) B cells, were unable to induce
viral clearance even 60 days after cell transfer. These data indicate
that protective immunity for an intestinal pathogen, RV, resides in
memory phenotype B cells expressing the intestinal homing receptor,
4ß7. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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90% IgA+ in LP, 60%
IgA+ in PP) (3, 4, 5, 6, 7). In both neonatal and adult mice, large
amounts of RV-specific IgA are found in stool samples following
infection with murine virus, and stool IgA persists for up to 1 yr
following primary infection (8, 9). Anti-RV IgA in stool and serum
correlates with protection from reinfection (8, 10). In contrast, while
high serum IgG levels are also observed (10, 11, 12), their role in
immunity is unclear. Experiments using immunodeficient mice have
demonstrated that local Ab is sufficient to clear viral infection in
the absence of T cells (3, 13) and is the principal determinant of
protection from reinfection (3, 14, 15).
The preferential accumulation of RV-specific ASCs in PP and
intestinal LP suggests that these cells are able to traffic
specifically to these sites. Lymphocytes that have previously
encountered Ag display tissue-selective trafficking patterns (16, 17, 18, 19, 20).
Mesenteric lymph node (MLN) B blasts, for example, bind preferentially
to LP endothelium in vitro (21) and preferentially traffic to mucosal
(gut, cervix, vaginal wall, uterus, mammary gland, and MLN) as opposed
to peripheral organs and tissues (22, 23). Selective localization is
thought to be directed in part by the differential expression of homing
receptors on the surface of circulating cells and endothelial cell
adhesion molecules in the target tissues (17, 19, 21, 24). The integrin
4ß7 is essential in lymphocyte homing to
intestinal LP and PP (25, 26, 27). Moreover, recent studies demonstrate
that memory phenotype T lymphocytes expressing
4ß7 recirculate selectively through the
intestine (28), and that
4ß7high, but not
4ß7-, memory T cells home to
the intestinal lymphoid organs, PP (16). Homing of these
4ß7 defined subsets of memory cells to
other mucosal-associated lymphoid tissue (cervix, vaginal wall, uterus,
and mammary gland) has not been examined.
Circulating B cells can be divided into naive and memory/effector cells
based on the expression of cell surface markers such as IgD in mice
(29, 30) and humans (31, 32). Memory phenotype
(IgD-) B cells in human peripheral blood subdivide
into ß7high and
ß7- populations (L. S. Rott, manuscript
in preparation), of which only ß7high cells
bind selectively to the mucosal vascular addressin MAdCAM-1 (33). In
humans, memory phenotype (IgD-) B cell blasts in LP
express high levels of 4ß7 and are
primarily IgA+ (34). Moreover, recent studies of B cells
responding to typhoid vaccination and bacterial enteric pathogens have
shown that in comparison with systemic vaccination, mucosal
presentation of Ags (by oral or rectal vaccination) is associated with
increased numbers of circulating Ag-specific B cells expressing
4ß7 (35, 36, 37, 38).
To determine whether memory for an intestinal virus correlates with
expression of 4ß7, we have assessed the
response of memory phenotype
(4ß7high IgD- and
4ß7- IgD-) and
naive phenotype
(4ß7+IgD+)
B220+ cells to RV. Using in vitro ELISPOT assays with
sorted cells, we show that only the IgD- cells expressing
high levels of 4ß7 produce RV-reactive
Abs. Moreover, in vivo cell transfer experiments demonstrate that
critical effector functions, including anti-RV IgA production,
clearance, and protection against reinfection correlate only with
transfer of memory B cells expressing high levels of
4ß7. Interestingly, mice receiving
4ß7- memory B cells from
recently immunized donors produced RV-specific IgG in serum, but
continue to shed RV in the stool. Thus, following oral immunization,
both 4ß7high and
4ß7- memory B cells can
transfer RV-specific Ab responses, but only the
4ß7high memory subset is
capable of providing protective humoral immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stocks of wild-type murine EC RV (EDIM, Cambridge) were prepared as intestinal homogenates and titrated in mice as previously described (8). Stocks of tissue culture-adapted rhesus rotavirus (RRV) were prepared and titrated to determine plaque-forming units (pfu) per milliliter as previously described (39).
Six-week-old C57BL/6 mice were obtained from the Charles River Laboratories (Hollister, CA) and were bred in the Palo Alto Veterans Administration breeding facility to be used as donors for cell transfer experiments. Mice were orally gavaged with 105 diarrheal dose 50 (DD50) (14) of EC RV after receiving 100 µl of 1.33% sodium bicarbonate to neutralize stomach acid. Viral shedding was monitored by ELISA to assess the progress of infection (8). Following initial infection, immunized mice were boosted by oral gavage with a second dose of virus (105 DD50). Between 1 and 2 mo after boosting, splenocytes and PP were harvested for FACS analysis. Also, between 1 and 2 mo after oral boosting, splenocytes from donor mice were harvested for sorting experiments. For systemic inoculation, mice were injected i.p. with live RRV at a dose of 106 pfu/mouse. One month later, a second dose of 106 pfu/mouse was administered. For cell transfer experiments examining earlier boost time points, 6-wk-old C57BL/6 mice were obtained from the Palo Alto Veterans Administration breeding facility, orally gavaged, and boosted following standard procedures (as described above). Splenocytes from these inoculated mice were harvested for sorting 7 mo after boosting.
Recipient RAG-2 mice (40) were obtained from GenPharm International (Mountain View, CA), bred in the Palo Alto Veterans Administration breeding facility, and infected with 105 DD50 of the murine EC virus as previously described (14). Stool samples from infected RAG-2 mice were assayed for viral Ag 2 wk postinoculation and again before cell transfer to confirm the establishment of a chronic infection. RAG-2 mice used in this study were chronically infected for 1 to 3 mo before study.
Isolation of lymphocytes from blood, spleen, lymphoid organs, LP, intraepithelial lymphocytes (IELs), and bone marrow (BM)
Lymphocytes were isolated from mouse MLN, peripheral lymph nodes, PP, and spleens as previously described (16). LP lymphocytes were isolated by collagenase or by dispase digestion of intestines from which PP had been removed, as previously described (3, 41). IELs were isolated as previously described (14). BM was obtained from the femurs of mice by removing the ends of the bone with a razor blade and flushing the inside of the bone with RPMI supplemented with 10% FCS (RPMI 10) through a 22-gauge needle. BM was dissociated by repeated resuspension through the needle and was washed with 10 ml of RPMI 10.
ELISPOT assay
The ELISPOT assay for detection of ASCs was performed as previously described (3) with the following modifications. Millipore 96-well filtration plates with Immobilon-P membranes (Millipore, Bedford, MA) were coated overnight at 4°C with 1 µg/ml goat anti-mouse Ig (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for detection of total ASCs. For detection of anti-RV ASCs, plates were coated with a 1/1000 dilution of rabbit anti-RRV hyperimmune serum (produced in the laboratory of H. Greenberg); washed with 10 mM Tris (pH 7.5), 100 mM NaCl, and 5 mM CaCl2; and incubated with a 1/5 dilution of RRV stock virus overnight at 4°C. Just before the addition of lymphocytes, plates were washed with 10 mM Tris (pH 7.5), 100 mM NaCl, and 5 mM CaCl2 and blocked for 30 min at 37°C with RPMI 10. Cells were added to plates at various dilutions in RPMI 10 and cultured overnight at 37°C. Secreted Abs were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-IgA, or anti-Ig(A+G+M) (Kirkegaard & Perry Laboratories) and visualized by reaction with the precipitating substrate, 3-amino-9-ethylcarbazole. Spots formed by precipitated substrate were counted using a Bausch and Lomb stereomicroscope at 1.5-fold magnification. Under the conditions used in these assays, the efficiency of capture of ASCs was essentially 100%, since equal total ELISPOTs were obtained when assaying the RV-specific hybridoma, IE11, on plates coated with RV and on plates coated with anti-Ig mAb (data not shown; n = 15).
In the ELISPOT assays described in Figure 4
, VP2 and VP6 virus-like
particles (made from baculovirus recombinants expressing heterologous
bovine RF rotavirus) (42) were used as Ag for coating plates directly.
Since in mice >90% of the anti-RV Ab response is directed to VP2
and VP6 (5) (M. Franco, unpublished observations), the results using
the two protocols (reported in Tables I, II, and III vs Fig. 4) were
similar. As a control for the specificity of the ELISPOT assay, cells
were also incubated in wells from which Ag had been omitted (and
resulted in no detectable spots; n = 27).
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Viral Ag detection was performed by ELISA as described
previously (8). Briefly, microtiter plates (Dynatech, McLean, VA) were
coated with diluted hyperimmune guinea pig anti-RRV serum and
blocked with 5% nonfat milk. Stool samples were made to a 10%
suspension and added to plates for 2 h at 37°C. Ag was detected
with rabbit anti-RRV serum followed by HRP-conjugated goat
anti-rabbit serum (Kirkegaard & Perry Laboratories). Visualization
was performed by incubation of plates with
2,2'-azino-di-[3-ethyl-benzthiazoline sulfonate] (6) substrate
(Kirkegaard & Perry Laboratories), followed by quenching of the
reaction with 10% SDS. Plates were read using an EIA Autoreader
(Bio-Tek Instruments, Burlington, VT) at 405 nm. Clearance was defined
as the point at which ELISA values fell below 0.08 OD. To determine
background absorbance levels, stool samples from mice that had not been
infected with RV (OD, 
0.08) in addition to wells with no stool sample
(OD, 0.04) were assayed by ELISA. Also, samples were assayed in the
absence of rabbit anti-RRV serum (OD, 0.04) in the absence of
HRP-conjugated goat anti-rabbit serum (OD, 0.05) as well as on
sample wells that were not coated with guinea pig anti-RRV serum
(OD = -0.05 to -0.06).
Analysis of RV-specific Abs was performed as described previously (8). Briefly, RV-specific Abs were captured on ELISA plates that were first coated with hyperimmune guinea pig anti-RRV serum and were subsequently treated overnight with a 1/5 dilution of RRV stock virus at 4°C. After three washes, 5% stool samples were added to the plates at 37°C. Ab was detected with HRP-conjugated anti-mouse IgA or IgG (Kirkegaard & Perry Laboratories) and visualized as described above. To determine background absorbance levels, stool samples from animals that were not infected with RV were assayed (OD = 0.011–0.030; n = 6). For determination of total Ig, plates were coated with goat anti-mouse Ig(A+G+M) (Kirkegaard & Perry Laboratories) and blocked with 5% nonfat dry milk.
Analysis of serum Ab was performed as described previously (8).
Briefly, serum samples were diluted serially in 1% nonfat dry milk and
added to plates at 37°C. After 2- to 4-h incubation, Ab was detected
with HRP-conjugated anti-mouse IgA or IgG (Kirkegaard & Perry
Laboratories) and visualized as described. Serial threefold dilutions
of serum were performed. The titer of the serum for each isotype of Ab
was defined as the maximum dilution of serum that had an
OD405 0.1. Background was defined as the mean OD of three
wells incubated without serum and was typically between 0.047 and
0.067. The serum titers were log3 transformed to calculate
the geometric mean titer.
Abs and reagents
For sorting, biotinylated mAb to B220 (CD45R/B220; RA3-6B2),
PE-conjugated mAb to 4ß7 (DATK32),
FITC-conjugated mAb to IgD (217-170), FITC-conjugated mAb to CD4
(RM4-5), and biotin-conjugated mAb to CD8 (53-6.7) were purchased from
PharMingen (La Jolla, CA). Biotinylated mAb to human CD44 (43)
(Hermes-1, produced in the laboratory of E. C. Butcher) and
PE-conjugated anti-rat-IgG2a (PharMingen) were used as isotype
control Abs. Streptavidin Red 613 (SA-Red 613) was purchased from Life
Technologies (Gaithersburg, MD). For staining of IELs of recipient
mice, PE-conjugated mAb to the TCR (H57-597), FITC-conjugated mAb to
CD4 (RM4-5), and biotinylated mAb to CD8 (53-6.7) were purchased from
PharMingen (La Jolla, CA). Streptavidin-conjugated peridinin
chlorophyll protein was purchased from Becton Dickinson (San Jose, CA).
Staining, cell sorting, and FACS analysis
Splenocytes to be sorted from donor RV-infected C57BL/6 mice were harvested, disaggregated by teasing between glass slides, and pressed through stainless steel screen mesh. Pooled cells were washed with 5 to 10 ml of DMEM supplemented with 10% FBS (DMEM-10) and then treated with 5 ml of lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA) for 5 min to lyse erythrocytes. After lysis, splenocytes were washed twice and resuspended in DMEM-10. The yield was typically 1 to 2 x 108/harvested spleen. To maximize purity, CD4+ splenocytes were isolated with anti-CD4 beads (Dynabeads, Dynal, Lake Success, NY) and subsequently detached from beads following the manufacturer’s protocol.
Splenocytes were stained for sorting as follows. Dynabead-isolated
CD4+ splenocytes were incubated on ice with
anti-CD4-FITC mAb (0.5 µg/106 cells) for 20 min,
washed with DMEM-10, stained with SA-Red 613 (0.5 µl/106
cells) for 20 min on ice, washed, resuspended in DMEM-10, and then
sorted. To isolate B cells, the fraction that did not adhere to
anti-CD4 beads was resuspended in DMEM-10 and stained with
anti-B220-biotin (0.5 µg/106 cells),
anti-4ß7-PE (DATK32; 0.2
µg/106 cells), and anti-IgD-FITC (0.5
µg/106 cells). The cells were then washed, stained with
SA-Red 613 (0.5 µl/106 cells), and sorted. In experiments
in which donor mice were boosted 7 mo before transfer, cells that did
not bind the anti-CD4 beads were further depleted of
CD8+ cells by incubation with anti-CD8 beads (Dynabead)
following the manufacturer’s instructions. To isolate CD8 cells,
splenocytes were stained with anti-CD8-FITC (0.5
µg/106 cells), and CD8+ cells were sorted for
transfer into selected recipients (see below).
Cells were sorted on a modified FACStar (Becton Dickinson, San Jose,
CA) with a single 488-nm argon laser and three fluorescence detectors.
Filters for the three fluorochromes were 530/30 for FITC detection,
585/42 for PE detection, and 630/22 for Red 613 detection. In two
experiments (in which donor mice were boosted 1 to 2 mo before
transfer) the 4ß7- memory and
naive populations were sorted twice, and the
4ß7high memory population was
sorted once. In one experiment (in which donor mice were boosted 7 mo
before transfer and CD8+ cells were depleted with
anti-CD8 beads before sorting; see above), the
4ß7- memory,
4ß7high memory, and naive
populations were sorted once. The level of purity after sorting once
(98.7 ± 1.3) was determined using the FACStar. Because of the
scarcity of cells, the purity could not be determined on the
transferred cells that had been sorted twice; however, in parallel
experiments, we determined by FACStar analysis that the purity after
sorting twice was typically at least 99.7%.
All experiments involved cotransfer of CD4+ cells. Because
of the possibility that low levels of CD8+ cells might
contaminate single-sorted
4ß7high (but not double-sorted
4ß7-) B cells in the
experiment described in Figure 2 and might affect viral clearance, in
three animals we transferred 300 double-sorted CD8+ cells
from orally inoculated donors (representing a 3% contamination level,
the maximum level of impurity we observed in single-sorted cells; see
above) along with 10,000 single-sorted
4ß7- IgD-
B220+ cells and 20,000 double-sorted CD4+
cells. We also transferred 10,000 single-sorted
4ß7- IgD-
B220+ cells and 20,000 double-sorted CD4+ cells
(and no CD8+ cells) in three additional animals (see Table V). Intentional addition of CD8+ contaminants to
transferred B cells had no effect on the clearance of RV (data not
shown). CD8+ and CD4+ cells were sorted twice.
Sorted donor cells were resuspended in sterile saline solution at
105/ml and injected i.p. into chronically infected RAG-2
mice.
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3%) (40), but no
TCR+ cells, we gated on double-positive (TCR+
CD8+) cells to visualize transferred CD8+
cells.) |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To compare the localization of RV-specific ASCs after oral vs
parenteral immunization, we inoculated adult mice either orally with
homologous murine RV or i.p. with the heterologous simian RRV.
Heterologous RRV was selected for i.p. inoculation because it shares
major antigenic epitopes with the mouse RV and has very limited
capacity to infect intestinal epithelium or proliferate in the mouse
host (and thus is expected to yield a systemic immune response by not
invoking mucosal presentation) (2). We then compared ASCs in spleen and
GALT of these mice as well as examined localization of ASCs in BM, a
site that has not previously been examined in studies of RV infection.
The results of ELISPOT analyses of tissues from orally and i.p.
inoculated mice are shown in Table I.
Consistent with previous reports (4, 6), oral inoculation led to the
production of large numbers of RV-specific ASCs in LP, moderate levels
in mucosal tissues (PP and MLN), and low levels in spleen. High levels
of RV-specific ASCs were also present in BM, but no detectable
RV-specific ASCs were found in PLN (superficial inguinal lymph nodes;
data not shown). In contrast, i.p. inoculation failed to generate
RV-specific ASCs in mucosal tissues, as there were no detectable
RV-specific ASCs in LP, MLN, or PP. Instead, RV-specific ASCs were
found only in spleen and BM. In one of four mice, RV-specific ASCs (8.5
RV-specific ASCs/100 total ASCs; see Table I) were also detected
in the superficial inguinal lymph nodes (possibly reflecting Ag
drainage from the abdominal site of i.p. injection). These data suggest
that at these doses of homologous and heterologous virus, an efficient
intestinal response is generated using only the oral inoculation
protocol.
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). In contrast, i.p. inoculated mice
had only RV-specific IgG in the serum (Table II) and no detectable
RV-specific Ab in the stool (data not shown). These results agree with
previous observations of the isotypes of secreted Abs of RV-reactive
cells in stool and serum samples of oral vs parenterally inoculated
mice (4, 44). A small, but significant, number of RV-specific
IgA-secreting cells was present in BM of i.p. inoculated mice (Table II) even though anti-RV IgA was not detected in serum.
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4ß7 on splenic and PP
B cells, and analysis of RV-reactive ASCs among
4ß7-defined memory subsets
Since oral inoculation generates RV-specific ASCs primarily in
intestinal sites, we hypothesized that there could be an association
between effector function and expression of the intestinal homing
receptor, 4ß7, on B cells. To test this
hypothesis, we first examined the expression of
4ß7 on splenic and PP B220+
cells isolated from mice orally inoculated with RV (Fig. 1). To distinguish
memory/activated-phenotype from naive phenotype B220+
cells, we stained lymphocytes with Abs to IgD (29, 30). FACS dot plots
show three main populations of B220+ cells: naive phenotype
cells that express low levels of 4ß7
(4ß7+ IgD+) and
two subsets of memory/activated phenotype cells, one of which expresses
high levels of 4ß7
(4ß7hi IgD-) and
the other of which expresses negligible levels of
4ß7
(4ß7- IgD-).
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4ß7 (for typical sort gates, see Fig. 1
b) and analyzed the number of RV-reactive APCs in the
sorted populations using ELISPOT assays (see Materials and
Methods). As shown in Table III,
RV-reactive cells represented 2.3% of the total Ig-secreting cells in
the sorted 4ß7high memory
population, but no detectable RV-reactive cells were in the
4ß7- memory population. Thus,
mature RV-specific ASCs are significantly enriched in the
4ß7-expressing subset after oral RV
inoculation. Due to the limited time of incubation with Ag (4–12 h),
the ELISPOT assay only detects cells actively secreting Abs and
therefore would not be expected to detect germinal center or resting
memory phenotype cells (18, 45, 46) that could potentially exert
effector function after transfer (see below).
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4ß7 clear RV infection
We next assessed the ability of
4ß7-defined B cell subpopulations to clear
RV. We sorted B220+ spleen cells from orally inoculated
mice into 4ß7high
IgD- (2% of B220+ cells),
4ß7- IgD- (8%
of B220+ cells), and
4ß7+ IgD+ (10% of
B220+ cells) subsets for injection into immunodeficient
RAG-2 (B and T cell-deficient) mice that were chronically infected with
RV (for typical sort gates, see Fig. 1b) We gated on small
B220+ lymphocytes to separate presumptive memory B cells
from the blast cell population (which has a larger forward scatter)
(18, 47) and transferred 10,000 sorted small B220+ cells.
In addition to sorted B cells, sorted CD4+ T cells (20,000)
from orally immunized mice were cotransferred to provide T cell help
for reactivation of memory B cells (45).
Figure 2 shows the effects of the
transferred cell populations on viral shedding in chronically infected
RAG-2 mice. Mice receiving
4ß7high IgD- and
CD4+ T cells clear the virus 12 days after transfer,
whereas mice receiving 4ß7-
IgD- or naive phenotype
4ß7+ IgD+ B cells
in conjunction with CD4+ T cells failed to clear the virus
for up to 60 days after transfer (when the experiment was terminated).
Chronically infected RAG-2 mice receiving CD4+ cells alone
also failed to clear the virus.
Anti-RV Abs (IgA) became detectable in the stool of mice receiving
4ß7high IgD-
cells beginning just before viral clearance (day 10)
(A405 = 0.25 ± 0.04; mean ± SE;
n = 4). By day 13, stool IgA Ab levels
(A405 = 1.1 ± 0.1; n = 4)
were comparable to those observed in immunocompetent, orally infected
C57BL/6 mice (A405 = 1.4 ± 0.2;
n = 5). High Ab levels were still observed at 18 days
(A405 = 1.2 ± 0.03; n = 5)
after transfer of sorted cells and remained consistently high for up to
60 days after transfer (when the experiment was terminated). These mice
were completely protected from reinfection with 105
DD50 of EC at 1 mo after clearance (not shown;
n = 3), presumably because of high levels of
anti-RV Ab, which clears the virus before it can replicate in the
animal. Low to negligible levels of RV-specific Ig were detected in the
stool of all recipients of other transferred populations
(A405 0.04 for mice receiving
4ß7- IgD- and
CD4+ cells (n = 6),
4ß7+ IgD+ and
CD4+ cells (n = 4), and CD4+
cells alone (n = 4)).
We examined serum from each of these recipient mice (Table IV). Mice receiving
4ß7high IgD-
cells had significant levels of both RV-specific IgA and IgG in serum.
In contrast, no RV-specific IgA was produced in recipients of
4ß7- IgD- B
cells; these 4ß7- B cells
did, however, give rise to significant levels of RV-specific serum IgG,
even though, as stated above, these animals did not clear the virus.
Furthermore, titers of total IgG in these mice exceeded those of
4ß7high IgD-
recipient mice. Mice receiving naive
(4ß7+ IgD+) B
cells and mice receiving only CD4+ cells did not have
significant levels of anti-RV Ig in serum. No Ab was present in the
sera of RAG-2 mice receiving only CD4+ cells, confirming
the absence of B cells in this sorted population. Only orally infected
immunocompetent C57BL/6 controls and RAG-2 mice receiving naive
(4ß7+ IgD+) cells
had detectable levels of total IgM. Total serum IgG was detected in all
RAG-2 mice receiving B220+ cells, whereas total IgA was
only detected in orally infected C57BL/6 mice and RAG-2 mice receiving
4ß7high IgD-
cells.
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4ß7high maintain long term
immunity to RV
To examine long term immunity provided by RV-specific B cells, we
transferred sorted B220+ splenocytes from donor C57BL/6
mice that had been orally boosted with RV at an earlier time point (7
mo before transfer, as opposed to 1–2 mo in the experimental transfers
depicted in Fig. 2 and Table IV). As in the previous experiments, we
transferred 10,000 sorted B220+ cells along with 20,000
double-sorted CD4+ lymphocytes into chronically infected
RAG-2 recipients. Again, only mice receiving
4ß7high IgD-
cells cleared RV infection (Fig. 3a), although clearance was
slightly delayed compared with that in previous experiments (day 16
rather than day 12). As in previous experiments, only mice that
received 4ß7high
IgD- sorted B220+ cells produced RV-specific
IgA in the stool (Fig. 3b).
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4ß7high
IgD- sorted B220+ cells had RV-specific ASCs
in spleen, BM, and LP 22 to 25 days after transfer of sorted cells
(Fig. 4). There were higher levels of
RV-specific IgA than IgG-producing ASCs in spleen, BM, and LP (4-,
15-, and 100-fold, respectively). Also, there was a significantly
higher frequency of RV-specific IgG and IgA ASCs in LP (per resident
cell) than in spleen and BM (60- and 230-fold more ASCs,
respectively), reflecting the localization of ASCs to the intestinal
compartment where RV proliferates (13) and where RV Ag is retained for
at least 20 days after inoculation (7). (Viral Ag within intestinal
APCs has been evaluated up to but not beyond 20 days after infection
(7).) Mice receiving naive phenotype
(4ß7+ IgD+
B220+) cells had low levels of RV-specific ASCs in LP, BM,
and spleen (by days 22–25, when ASCs were assessed; Fig. 4). Also,
naive B cell recipients had low levels of RV-specific IgA in the stool
by day 18 (Fig. 3b), indicating an initial response to RV.
No other recipient mice, including
4ß7- B cell recipients, had
detectable RV-specific Ab in the stool (Fig. 3b) or
RV-specific ASCs in BM, spleen, or LP (data not shown;
n = 3 for each group of recipient mice).
Levels of total IgA ASCs (per resident cell) were higher in LP than in
BM or spleen of 4ß7high
recipient mice (Table V). Interestingly,
total IgG ASCs in spleen and BM of mice transferred with
4ß7- IgD- or
4ß7+ IgD+ cells
were comparable to or greater than those of mice receiving
4ß7high IgD-
cells. We detected serum Abs to RV only in
4ß7high recipients and not in
recipients of memory 4ß7- or
naive 4ß7+ B cells (data not
shown.) This contrasts with experiments (presented in Table IV and Fig. 2) with sorted cells from donor mice that had been boosted within 1 to
2 mo of transfer, in which recipients of memory
4ß7- B cells had RV-specific
IgG in serum. As in our previous experiments, there was no detectable
Ab in the serum of RAG-2 recipients of CD4+ cells alone
(data not shown), confirming that no B cells were transferred in this
sorted population.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7, we transferred sorted memory B
splenocytes expressing high vs negligible levels of this integrin into
RAG-2 mice chronically infected with RV. For these analyses, B-lineage
cells lacking surface IgD were sorted as the presumptive memory
population, and surface IgD+ B cells were assumed to be
predominantly naive. We show that recipients of 10,000
4ß7high memory B cells, and
not 4ß7- memory or
4ß7+ naive B cells, clear RV.
Moreover, recipients of
4ß7high memory B cells are
protected from reinfection, presumably due to clearance of RV by Ab at
intestinal surfaces before the virus can infect enterocytes.
Interestingly, we detected RV-specific Ig in the sera of both
4ß7high and
4ß7- recipient mice (when
donor mice were boosted 1–2 mo before transfer). Thus, although both
4ß7high and
4ß7- memory B cells from
orally immunized mice produce RV-specific Ig, only
4ß7high cells provide
protective humoral immunity. This is the first direct demonstration
that the ability to confer functional immunity at intestinal
surfaces segregates among memory B cells according to homing receptor
expression. To examine the B cell response to RV in orally vs i.p. inoculated mice, we compared ASCs in spleen, GALT, and BM in mice inoculated via each route. Nonreplicative simian RRV was used for i.p. immunization because murine RV would spread rapidly to infect intestinal epithelia. We confirmed previous reports showing that mice orally inoculated with RV produce virus-specific ASCs in spleen and GALT (MLN, PP, and LP) (4, 6, 7). We also showed that significant numbers of RV-specific ASCs localize to BM, a site that has not previously been examined in studies of RV infection. We demonstrated that in contrast to oral inoculation, if virus was injected i.p., RV-reactive cells localized to spleen and BM, but not to intestinal lymphoid tissue (PP, MLN, and LP). This agrees with previous reports demonstrating that the majority of the response to RRV in i.m. inoculated mice localizes in peripheral lymph nodes (inguinal lymph nodes and draining ipsilateral lymph nodes), with significantly less response in GALT (12, 44).
The route of RV inoculation also plays a role in determining the
isotype and titer of anti-RV Abs. We confirmed the findings of
previous studies showing that high titers of serum anti-RV IgG and
IgA as well as stool anti-RV IgA are observed following oral
infection of mice with both murine and heterologous viruses (9, 10). No
anti-RV IgG is detected in the stool, although anti-RV IgG ASCs
can be detected in the LP of orally infected mice (4, 5). Our studies
also confirm that parenteral administration of heterologous viruses
results in high titers of primarily anti-RV IgG, and not IgA, in
serum (12). In this case, some anti-RV IgG is found in the lumen
and is thought to be due to transport from circulating serum IgG (12, 44, 48). Importantly, the isotypes of RV-specific Ig in serum and stool
in transfer experiments with memory
4ß7high vs
4ß7- cells mimic the isotypes
observed at these sites in orally vs peritoneally inoculated normal
mice. 4ß7high cells (from
orally inoculated donors) generate IgA and IgG in serum and RV-specific
IgA and IgG ASCs in LP, but only IgA, and not IgG in stool. On the
other hand, 4ß7- cells never
give rise to IgA in serum and produce serum IgG only when donor animals
were recently boosted (1–2 mo before transfer). It is possible that
the 4ß7- B cells responsible
for this IgG production are B cells primed outside the intestine, as
viral Ag has been detected in the spleen after infection with RV (7).
Alternatively, it is possible that these
4ß7- cells responsible for
RV-specific IgG production are not mature memory cells, but rather, are
residual germinal center B cells that lack surface homing receptor
expression (49), including ß7.
We have also assessed the anti-RV ASC response in BM of orally vs
parenterally inoculated mice and in RAG-2 recipients of sorted B cell
subsets. As for stool, LP, and serum, our analyses revealed
similarities in the isotypes of BM ASCs in orally inoculated
immunocompetent C57BL/6 mice and in RAG-2 recipients of
4ß7high B cells and, on the
other hand, between i.p. inoculated normal mice and RAG-2 recipients of
4ß7- B cells (even though the
transferred populations were both from orally inoculated donors). We
observed primarily IgA ASCs in BM of mice orally inoculated with RV as
well as in RAG-2 recipients of
4ß7high B cells. Conversely,
systemically inoculated normal mice and RAG-2 recipients of
4ß7- cells produced primarily
IgG ASCs in BM. Furthermore, these data suggest that in intestinal
infection, ASCs localize efficiently not only to LP but also to BM,
which is believed to be a source of long term Ab production (50). The
predominance of IgA ASCs in BM in orally inoculated normal RV-infected
mice contrasts with the predominance of IgG ASCs in the marrow
compartment in most reported models, perhaps reflecting the unusual
degree of site (intestine) selectivity of RV Ag presentation.
Based on studies of adult SCID mice hosting transplanted hybridomas
secreting RV-specific IgA vs IgG, we have hypothesized that for Abs to
mediate effective blocking and clearance of RV infection, the Abs must
be transcytosed by the polyimmunoglobulin receptor (pIgR) into villous
epithelium, where the virus replicates, and then into the lumen, where
neutralization occurs (13). The pIgR transports J chain-containing
dimeric IgA and pentameric IgM from the intestinal LP across the
epithelial barrier into the lumen (51, 52, 53, 54, 55). IgG cannot enter epithelial
cells through direct transport by pIgR. In our transfer experiments,
RV-specific serum IgG, observed in mice transferred with
4ß7- cells, is ineffective at
clearing the virus, most likely due to the lack of transport of IgG
into or through villous epithelium. On the other hand, it has been
shown that in suckling mice and neonatal calves, IgG can protect
against infection by RV and other viruses (such as influenza virus)
whose site of replication is confined to the mucosal surface (56, 57, 58, 59).
However, experiments with neonatal animals may not be relevant to our
transfer model. Unlike our model, which uses adult mice as both
recipients and donors, oral ingestion of Ab (in mother’s milk) results
in localization of substantial amounts of IgG in the neonatal
intestine. Similarly, when rabbits are injected parenterally with
heterologous RV, it has been shown that IgG and not IgA correlates with
protection (60). In these studies high levels of virus-specific IgG
(with negligible levels of IgA) are observed in the intestine of i.m.
inoculated animals. In contrast, we observe no virus-specific IgG in
the stool of any recipient mice, including mice transferred with
4ß7- cells, which may account
for differences in our observations and those with neonatal mice and
i.m. inoculated rabbits.
4ß7high IgD- B
cells maintain long term memory for RV, as mice transferred with sorted
4ß7high cells from donors
boosted as much as 7 mo before sacrifice produce RV-specific Ab and
clear the virus. In contrast to experiments in which donors received a
recent oral boost (1–2 mo before transfer), we did not detect
RV-specific serum Ig in RAG-2 recipients of
4ß7- cells from donors
boosted 7 mo before transfer. Some researchers have argued that Ag must
be intermittently sampled for maintenance of long term memory (45, 46),
and while 4ß7high memory cells
responded and were capable of clearing the virus, there may have been
too few RV-reactive 4ß7-
cells in the transferred population to produce an anti-RV response
as the delay from the oral boost increased. While long term immunity to
RV was maintained by 4ß7high
memory cells from animals boosted 7 mo before transfer, clearance of RV
in 4ß7high recipients is
slightly delayed compared with that in mice receiving donor cells from
animals boosted only 1 to 2 mo before transfer (clearance on day 16
compared with day 13), perhaps reflecting a decrease in the number of
RV-specific cells in this population as well. RV Ag is retained in the
intestine (PP and MLN) of normal mice for at least 20 days
postinfection (7), and probably much longer. The increased exposure of
recirculating RV-specific
4ß7high cells to localized Ag
in PP and intestine may enhance their survival within RAG-2 recipients
compared with RV-specific memory cells that lack the
4ß7 intestinal homing receptor. In fact,
the majority of the ASCs were RV reactive in
4ß7high recipients. Thus, our
data are consistent with the hypothesis that the selective homing of
lymphocyte subsets to specialized microenvironments can help control
lymphocyte life span, function, and population dynamics (or
homeostasis) by determining access to regulatory factors (in this case,
residual RV Ag) that support differentiation and/or survival (20).
4ß7- memory B cells from
donors boosted 7 mo before transfer do not mount a detectable
anti-RV response. In contrast,
4ß7+ IgD+ (naive)
B cells isolated from these same donors produce anti-RV ASCs in LP,
spleen, and BM 22 to 25 days after transfer. Also, production of
virus-specific intestinal IgA in recipients of
4ß7+ naive B cells begins to
increase on days 18 and 19 after transfer, while anti-RV intestinal
IgA does not change in recipients of
4ß7- IgD-
B220+ splenocytes. These data indicate that
4ß7+ naive B cells, apparently
unlike 4ß7- memory B cells,
have the potential to develop immunity to the virus. It should be
pointed out that we did not detect RV-specific Ab in all mice
transferred with 4ß7+ naive B
cells, indicating some variability in the response. Also, while some of
these animals produce virus-specific Ab and ASCs, none clears the virus
60 days after transfer; this may simply reflect the presence of
inadequate numbers of RV-specific B cells among the naive cells
transferred.
What are the implications of these data for RV vaccine development? It
is clear from these and previous studies (10, 14, 61) that production
of anti-RV (IgA) Ab at intestinal surfaces is the most effective
means of clearing and preventing RV infection in the mouse model. Our
studies show that following oral infection, the majority of RV-specific
B cells populate intestinal sites and BM, and that only transferred
4ß7high memory phenotype B
cells can mediate clearance of RV in our RAG-2 model. Interestingly, B
cells making Ab to RV persist in intestinal sites, particularly the LP,
for periods up to 1 yr (6, 9). While this persistence is not observed
in humans (62), immunization strategies optimized to stimulate a strong
anti-RV response in
4ß7high memory B cells will
likely be most effective at preventing or attenuating RV
infection.
In conclusion, our results demonstrate that B cell memory following
oral RV infection correlates with 4ß7
expression, and that while
4ß7- IgD- B
cells can give rise to RV-specific Ig in vivo, these cells primarily
produce serum IgG and are ineffective at clearing the virus. Only cells
with intestinal homing phenotype produce significant levels of stool
and serum IgA and can function in viral clearance and active prevention
of reinfection. Furthermore, we show that
4ß7high IgD- B
cells maintain long term functional immunity to RV, as much as 7 mo
after reinfection with the virus. These results extend and complement
our studies that have demonstrated that CD8+ cells
expressing 4ß7high also carry
memory to RV and clear RV in chronically infected, immunodeficient mice
(63). We conclude that humoral immunity can be segregated according to
homing receptor expression, and that 4ß7
expression is a critical correlate and determinant of humoral as well
as cellular immune responses in the intestinal tract.
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Acknowledgments |
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Footnotes |
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2 M.B.W. and J.R.R. contributed equally to this manuscript, and E.C.B. and H.B.G. contributed equally as senior authors.
3 Address correspondence and reprint requests to Marna B. Williams, Ph.D., Department of Pathology, L235, Stanford University School of Medicine, Stanford, CA 94305-5324. E-mail address:
4 Abbreviations used in this paper: RV, rotavirus; ASC, antibody-secreting cell; LP, lamina propria; PP, Peyer’s patches; MLN, mesenteric lymph node; MAdCAM-1, mucosal addressin cell adhesion molecule-1; ELISPOT, enzyme-linked immunospot; RRV, rhesus rotavirus; pfu, plaque-forming units; DD50, diarrheal dose 50; RAG-2, recombination-activating gene-2; IEL, intraepithelial lymphocyte; BM, bone marrow; HRP, horseradish peroxidase; PE, phycoerythrin; SA-Red 613, streptavidin Red 613; DMEM-10, Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum; GALT, gut-associated lymphoid tissues; pIgR, polyimmunoglobulin receptor.
Received for publication March 20, 1998. Accepted for publication June 22, 1998.
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