The Memory B Cell Subset Responsible for the Secretory IgA Response and Protective Humoral Immunity to Rotavirus Expresses the Intestinal Homing Receptor, α₄β₇

Mice, viruses, and virus inoculation

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).

Detection of viral Ag and Abs

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 α₄β₇ (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 × 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-α₄β₇-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 α₄β₇⁻ memory and naive populations were sorted twice, and the α₄β₇^high 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 α₄β₇⁻ memory, α₄β₇^high 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 α₄β₇^high (but not double-sorted α₄β₇⁻) 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 α₄β₇⁻ IgD⁻ B220⁺ cells and 20,000 double-sorted CD4⁺ cells. We also transferred 10,000 single-sorted α₄β₇⁻ 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.

In the experiments in which donor mice were boosted 7 mo before transfer and CD8⁺ cells were depleted, IELs and splenocytes were isolated from recipient mice 22 to 25 days after transfer; stained with mAbs to CD8 (0.5 μg/10⁶ cells), CD4 (0.5 μg/10⁶ cells), and TCR (0.5 μg/10⁶ cells); and visualized by FACS. There were no TCR⁺CD8⁺ cells in any mice with transferred sorted cells. (Since RAG-2 mice have a limited population of CD8⁺ cells (∼3%) (40), but no TCR⁺ cells, we gated on double-positive (TCR⁺ CD8⁺) cells to visualize transferred CD8⁺ cells.)

RV-reactive B cells populate mucosal tissues after oral, but not after systemic, inoculation

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.

Moreover, orally inoculated mice had RV-specific IgA in the stool (data not shown) and RV-specific IgA and IgG in serum (Table II⇓). 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.

Distribution of α₄β₇ on splenic and PP B cells, and analysis of RV-reactive ASCs among α₄β₇-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, α₄β₇, on B cells. To test this hypothesis, we first examined the expression of α₄β₇ 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 α₄β₇ (α₄β₇⁺ IgD⁺) and two subsets of memory/activated phenotype cells, one of which expresses high levels of α₄β₇ (α₄β₇^hi IgD⁻) and the other of which expresses negligible levels of α₄β₇ (α₄β₇⁻ IgD⁻).

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FIGURE 1.

B220⁺ cells in PP and spleen of RV-immunized mice. FACS dot plot shows gated B220⁺ cells in PP (a) and spleen (b) from mice orally inoculated with murine RV (see Materials and Methods). Lymphocytes were stained with Abs to B220, IgD, and α₄β₇. B220⁺ small lymphocytes were gated and analyzed for IgD (x-axis) and α₄β₇ (y-axis) expression. The FACS dot plot shows three populations (delineated on the plot) of B220⁺ small lymphocytes: two memory phenotype populations (α₄β₇^high IgD⁻ and α₄β₇⁻ IgD⁻) and one naive phenotype population (α₄β₇⁺ IgD⁺). Typical gates used to sort α₄β₇^high IgD⁻ B220⁺ (A), α₄β₇⁻ IgD⁻ B220⁺ (B), and α₄β₇⁺ IgD⁺ B220⁺ (C) small lymphocytes for transfer experiments are shown on the plot. The sorted α₄β₇^high IgD⁻ B220⁺, α₄β₇⁻ IgD⁻ B220⁺, and α₄β₇⁺ IgD⁺ B220⁺ cells with these gates represented 1.3 ± 0.5, 8.1 ± 4.3, and 26 ± 7% of the total small B220⁺ cells, respectively (mean ± SE; n = 3).

We sorted memory/activated phenotype (IgD⁻) splenocytes from orally inoculated mice according to expression of α₄β₇ (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 α₄β₇^high memory population, but no detectable RV-reactive cells were in the α₄β₇⁻ memory population. Thus, mature RV-specific ASCs are significantly enriched in the α₄β₇-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).

Memory (IgD⁻) B cells expressing α₄β₇ clear RV infection

We next assessed the ability of α₄β₇-defined B cell subpopulations to clear RV. We sorted B220⁺ spleen cells from orally inoculated mice into α₄β₇^high IgD⁻ (2% of B220⁺ cells), α₄β₇⁻ IgD⁻ (8% of B220⁺ cells), and α₄β₇⁺ 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. 1⇑b) 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 α₄β₇^high IgD⁻ and CD4⁺ T cells clear the virus 12 days after transfer, whereas mice receiving α₄β₇⁻ IgD⁻ or naive phenotype α₄β₇⁺ 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.

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FIGURE 2.

Viral Ag shedding in RAG-2 mice chronically infected with murine RV: effect of transfer of sorted B cell subsets. Ten thousand sorted B cells and 20,000 sorted CD4⁺ T cells from C57BL/6 mice orally inoculated with murine RV were resuspended in sterile saline and injected i.p. into chronically infected recipient RAG-2 mice. C57BL/6 donor mice were orally boosted with murine RV 1 to 2 mo before transfer of sorted cells. Viral shedding was monitored by ELISA of stool samples (see Materials and Methods). The plot shows mean absorbance values (A₄₀₅); error bars represent the SE of absorbance values from two separate experiments with totals of five (α₄β₇^high IgD⁻ B220⁺), six (α₄β₇⁻ IgD⁻ B220⁺), and four (α₄β₇⁺ IgD⁺ B220⁺ and CD4 only) mice receiving each sorted, transferred population, respectively (noted in the upper right corner of the plot).

Anti-RV Abs (IgA) became detectable in the stool of mice receiving α₄β₇^high 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 α₄β₇⁻ IgD⁻ and CD4⁺ cells (n = 6), α₄β₇⁺ 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 α₄β₇^high 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 α₄β₇⁻ IgD⁻ B cells; these α₄β₇⁻ 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 α₄β₇^high IgD⁻ recipient mice. Mice receiving naive (α₄β₇⁺ 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 (α₄β₇⁺ 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 α₄β₇^high IgD⁻ cells.

Memory (IgD⁻) B cells expressing α₄β₇^high 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 α₄β₇^high IgD⁻ cells cleared RV infection (Fig. 3⇓a), 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 α₄β₇^high IgD⁻ sorted B220⁺ cells produced RV-specific IgA in the stool (Fig. 3⇓b).

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FIGURE 3.

Viral Ag shedding and RV-specific intestinal IgA in RAG-2 mice chronically infected with murine RV after transfer of sorted B220⁺ cells from donor mice that had been orally boosted 7 mo before transfer. a, Ten thousand sorted B cells and 20,000 sorted CD4⁺ cells from C57BL/6 mice orally inoculated with murine RV were resuspended in sterile saline and injected i.p. into chronically infected recipient RAG-2 mice. C57BL/6 donor mice were orally boosted with EC virus 7 mo before transfer. Viral shedding was monitored by ELISA of stool samples (see Materials and Methods). The plot shows mean absorbance values (A₄₀₅), and error bars represent the SE of absorbance values from three mice receiving each sorted, transferred population (noted in the upper right corner). b, Stool samples from RAG-2 mice that received sorted cells were analyzed for RV-specific Ig (IgA) by ELISA (see Materials and Methods). The plot shows mean absorbance values (A₄₀₅) on day 0 (the day of transfer) and days 6 to 20 after transfer; error bars represent the SE of the mean absorbance values from three mice receiving each sorted population (noted in the upper right corner.)

Mice receiving α₄β₇^high 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 (α₄β₇⁺ 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. 3⇑b), indicating an initial response to RV. No other recipient mice, including α₄β₇⁻ B cell recipients, had detectable RV-specific Ab in the stool (Fig. 3⇑b) or RV-specific ASCs in BM, spleen, or LP (data not shown; n = 3 for each group of recipient mice).

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FIGURE 4.

RV-specific ASCs in spleen, BM, and LP of RAG-2 mice receiving sorted B220⁺ cells. RAG-2 mice were sacrificed 22 to 25 days after transfer of sorted B220⁺ cells from donor mice that had been orally boosted 7 mo before transfer. RV-specific IgG and IgA ASCs in spleen, BM, and LP were analyzed by ELISPOT (see Materials and Methods). Bar graphs depict RV-specific ASCs in RAG-2 mice that received α₄β₇^high IgD⁻ B220⁺ or α₄β₇⁺ IgD⁺ B220⁺ sorted cells. Values represent the mean number of ASCs per 10⁶ total cells, and error bars represent the SE (n = 3 for each value shown). There were no detectable RV-specific ASCs in spleen, BM, or LP of mice receiving any sorted population of cells other than those shown in the figure.

Levels of total IgA ASCs (per resident cell) were higher in LP than in BM or spleen of α₄β₇^high recipient mice (Table V⇓). Interestingly, total IgG ASCs in spleen and BM of mice transferred with α₄β₇⁻ IgD⁻ or α₄β₇⁺ IgD⁺ cells were comparable to or greater than those of mice receiving α₄β₇^high IgD⁻ cells. We detected serum Abs to RV only in α₄β₇^high recipients and not in recipients of memory α₄β₇⁻ or naive α₄β₇⁺ 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 α₄β₇⁻ 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.