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Migration of Antigen-Presenting B Cells from Peripheral to Mucosal Lymphoid Tissues May Induce Intestinal Antigen-Specific IgA Following Parenteral Immunization¹

Susan E. Coffin^2,*,, Stephanie L. Clark^*, Nico A. Bos, Jeffery O. Brubaker^* and Paul A. Offit^*,,§

^* Division of Immunologic and Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; University of Pennsylvania School of Medicine, Philadelphia, PA 19104; University of Groningen, Groningen, The Netherlands; and ^§ Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104

	Abstract

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Parenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymphocytes. We have now used adoptive transfer studies to identify the cell types responsible for the generation of virus-specific Ab production by gut-associated lymphoid tissue (GALT) after i.m. immunization. Three days after i.m. immunization with rotavirus, cells obtained from the draining peripheral lymph nodes of donor mice were transferred into naive recipient mice. We found that intestinal lymphocytes produced rotavirus-specific Igs (IgM, IgA, and IgG) 2 wk after transfer of either unfractionated cells, or unfractionated cells rendered incapable of cellular division by mitomycin C treatment. Additional studies demonstrated that rotavirus-specific IgA, but not IgG, was produced by intestinal lymphocytes after transfer of purified B cells. Ig allotype analysis revealed that rotavirus-specific IgA was produced by intestinal B cells of recipient origin, suggesting that migration of Ag-presenting B cells from peripheral lymphoid tissues to GALT may contribute to the generation of mucosal IgA responses after parenteral immunization. Strategies that promote Ag uptake and presentation by B cells may enhance mucosal IgA production following parenteral immunization.

	Introduction

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For decades parenteral immunization has been used to stimulate protection from a variety of infectious organisms whose site of replication is limited solely to mucosal surfaces (e.g., influenza virus and Bordetella pertussis). Ag-specific Abs (1, 2, 3, 4) and CTL (5, 6, 7) that protect against mucosal challenge have been detected at intestinal and respiratory mucosal surfaces after parenteral inoculation.

Little is known about the mechanisms by which mucosal Ab-secreting cells (ASC)³ (3) are induced by nonmucosal immunization. Studies by several groups have shown that Ag-specific IgA-secreting cells that bear the mucosal homing receptor ₄ß₇ can be detected in the circulation shortly after parenteral inoculation (8, 9). These findings demonstrate that nonmucosal immunizations can induce effector B cells capable of homing to mucosal tissues. However, how and where ₄ß₇-bearing ASC are induced by nonmucosal immunization remain obscure. Understanding the mechanisms by which nonmucosal immunizations induce IgA-secreting cells that bear mucosal homing receptors may allow for rational design of vaccines administered by the parenteral route.

We recently developed an animal model to examine the mechanisms by which nonmucosal immunization induces protection from mucosal infection (10). Our initial studies demonstrated that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced complete protection from challenge. Protection against challenge correlated with the production of virus-specific IgA by gut-associated lymphoid tissues (GALT). In addition, we found that protective mucosal immunity after i.m. inoculation with rotavirus was not due to 1) dissemination of free virus from the site of inoculation to the intestine, 2) dissemination of virus-specific IgA-secreting cells from the draining peripheral lymph node to the intestine, or 3) transudation of serum-derived Ab onto mucosal surfaces (10). Using lymphoid cultures of intestinal and nonintestinal tissues, we also evaluated the site and kinetics of virus-specific Ab production following i.m. inoculation of mice with rotavirus. These studies revealed that virus-specific IgA was initially produced by lymphocytes resident in the inductive tissues of GALT (i.e., Peyer’s patches (PP) and mesenteric lymph nodes (MLN)) and later by lamina propria (LP) lymphocytes. Virus-specific IgA was not produced by systemic lymphoid tissues during the first 14 days after primary i.m. immunization. In this report, we extend these findings by determining the cell types resident in the draining peripheral lymph node immediately after i.m. inoculation with rotavirus that lead to the production of virus-specific Igs by intestinal lymphocytes.

	Materials and Methods

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Mice

Conventionally reared, 5- to 8-wk-old BALB/c (H-2^d, Igh^a), C.B-17 (H-2^d, Igh^b), or C57BL/6 (H-2^b, Igh^b) female mice (Taconic Breeding Laboratories, Germantown, NY), or C.B-17-SCID (H-2^d) female mice (Wistar Institute, Philadelphia, PA) were housed in individual isolation units. Before inoculation, sera from these mice did not contain rotavirus-specific Abs as determined by ELISA.

Virus

Murine rotavirus strain EDIM was originally obtained from Richard Ward (Children’s Hospital of Cincinnati, Cincinnati, OH) and passaged in infant mice as previously described (10).

Immunization of donor mice

Adult BALB/c mice were inoculated i.m. bilaterally in the quadriceps femoris muscle with 200 µl per hind leg of EDIM. Mice were inoculated with 2.4 x 10⁵ 50% shedding dose (equivalent to 18 ng of viral Ag).

Experimental design

Three days after i.m. inoculation of donor mice, draining inguinal lymph nodes (ILN) were harvested and disrupted. Unfractionated or FACS-purified populations of cells were transferred either i.v. (via tail vein infusion) or i.p. in a volume of 200–300 µl to seronegative, adult recipient mice. Two or 6 wk after adoptive transfer, recipient mice were sacrificed and intestinal and nonintestinal lymphoid cultures were established. Supernatants from lymphoid cultures were subsequently tested for the presence of rotavirus-specific Abs by ELISA.

Isolation of cells

ILN were harvested and then disrupted with 21-gauge needles and 200-µm wire mesh (Small Parts, Miami Lakes, FL) in IMDM (Life Technologies, Grand Island, NY) with 10% FBS (Life Technologies). Cells were passed through a 125-µm cell sieve (Thomas Scientific, Swedesboro NJ), washed three times, and resuspended in IMDM with 10% FBS.

Cell preparation and sorting

Treatment of cells with mitomycin C to block cellular division. Unfractionated ILN cells were diluted to a concentration of 5 x 10⁷ cells/ml in PBS and incubated with 50 µg/ml mitomycin C (Sigma, St. Louis, MO) for 20 min at 37°C. Cells were washed three times in IMDM with 10% FBS.

Purification of B cells. Unfractionated ILN cells were diluted to a concentration of 2 x 10⁷ cells/ml and incubated for 30 min at room temperature (RT) with 4 µl/ml of mouse anti-mouse Thy1.2 (Biosource International, Camarillo, CA). Cells were washed with IMDM with 10% FBS, 0.3% (w/v) BSA (Sigma), and 20 mM HEPES buffer solution (Life Technologies) (lysis buffer). Cells were then resuspended in 900 µl lysis buffer and 100 µl Low-Tox rabbit complement (Accurate Chemical and Scientific, Westbury, NY) per 2 x 10⁷ cells and incubated for 1 h at 37°C. Cells were placed on ice for 5 min, washed with cold lysis buffer, and resuspended in IMDM with 5% FBS. The remaining cells were incubated for 30 min at RT with PE-conjugated rat anti-mouse CD19 (PharMingen, San Diego, CA) at a concentration of 10 µg/10⁷ cells. The cells were washed and resuspended in IMDM with 5% FBS. CD19⁺ cells were purified by FACS using an EPICS Elite Flow Cytometer (Coulter, Hialeah, FL). This method yielded 99.9% CD19⁺ cells.

Depletion of macrophages

Macrophages were stained by incubating unfractionated ILN cells with PE-conjugated rat anti-mouse CD11b (PharMingen) for 30 min at RT at a concentration of 2 µg/10⁷ cells. Cells were washed and resuspended in IMDM with 5% FBS. Non-CD11b⁺ cells were purified by FACS. This method yielded 99.8% non-CD11b⁺ cells.

Depletion of dendritic cells

Dendritic cells were stained by incubating unfractionated ILN cells with biotin-conjugated mAb 33D1 (a kind gift of Dr. Ralph Steinman, Rockefeller University, New York, NY) (11) for 30 min at RT at a concentration of 2 µg/10⁷ cells. The cells were washed and incubated with PE-conjugated streptavidin (PharMingen) for 15 min at RT at a concentration of 0.5 µg/10⁷ cells. Cells were washed and resuspended in IMDM with 5% FBS. Non-33D1⁺ cells were purified by FACS. This method yielded 99.9% non-33D1⁺ cells.

Intestinal or nonintestinal lymphoid cultures

To assess the production of virus-specific Abs by intestinal or nonintestinal tissues of recipient mice, lymphoid cultures of ILN, PP, MLN, or small intestinal LP fragments were established 2 or 6 wk after adoptive transfer as previously described (12) with the following modifications. In brief, under sterile conditions ILN, PP, MLN, and small intestines were isolated. MLN, ILN, and PP were washed twice in IMDM containing 50 µg/ml of gentamicin (JRH Bioscience, Lenexa, KS). Segments of small intestine 5 cm in size were opened and washed twice in calcium- and magnesium-free HBSS (Life Technologies) containing 50 µg/ml of gentamicin and 25 mM HEPES (Mediatech, Washington, DC), once in HBSS with 0.05% EDTA to remove villous epithelial cells and intraepithelial lymphocytes, and twice in HBSS. Under a dissecting microscope (x30 magnification) fat, mesentery, and connective tissue were removed from small intestinal segments. Eight 1 x 1 mm LP fragments from small intestinal segments of each animal were dissected. One LP fragment, MLN, PP, or ILN was placed in a well of a 48-well plate (Costar Scientific, Braintree, MA) containing 0.5 ml of GALT media (Kennet’s HY media (Life Technologies), 100 µg/ml of streptomycin (JRH Bioscience), 50 µg/ml of gentamicin and 0.25 µg/ml of amphotericin B (Fungizone, JRH Bioscience)). Samples were incubated at 37°C in an atmosphere of 95% O₂ and 5% CO₂ for 5 days. Supernatant fluids were collected and tested for the presence of rotavirus-specific Igs (IgM, IgA, and IgG) by ELISA.

Detection of rotavirus-specific and total Igs by ELISA

Sera and supernatant fluids from intestinal and nonintestinal lymphoid cultures were tested for the presence of rotavirus-specific and total IgM, IgA, and IgG (13). Quantities of total IgM, IgA, and IgG were determined to assure the viability of each lymphoid culture. To determine quantities of rotavirus-specific Abs, individual wells of 96-well, flat-bottom plates (Costar) were coated with either 100 µl PBS (Life Technologies) or 200 ng purified rotavirus diluted in 100 µl of PBS. To determine quantities of total Abs, individual wells of 96-well, flat-bottom plates were coated with either 100 µl PBS or 100 µl goat anti-mouse IgM, IgA, or IgG (Cappel, West Chester, PA) diluted in 1:1,000 in PBS. Plates were incubated for 18 h at 4°C in a humid chamber. Wells were washed 5 times with PBS plus 0.05% Tween-20 (Sigma), blocked with 300 µl of 1% BSA plus 0.025% Tween-20 (BSA-T), and incubated for 1 h at RT. Wells were washed again as above and 50 µl of sera or supernatant fluids diluted in BSA-T were added. Plates were gently rocked for 2 h at RT and washed as above. Fifty microliters of HRP-conjugated goat anti-mouse IgM, IgA, or IgG (Southern Biotechnology Associates, Birmingham, AL) diluted 1:2,000 in BSA-T were added to each well. Plates were incubated for 1 h at RT and then washed as above. Fifty microliters of 0.04% tetramethylbenzedine peroxidase solution (Kirkegaard & Perry, Gaithersburg, MD) were added to each well and incubated for 5 min at RT. Fifty microliters of 85% o-phosphoric acid (Sigma) were added and the OD of each well was determined at 450 nm on a microplate ELISA reader (Dynex Technologies, Chantilly, VA). Samples were considered positive if the OD value for a coated well was both 0.1 OD units and 2-fold greater than OD values for the corresponding uncoated well. Supernatants of lymphoid cultures obtained from BALB/c or C.B-17 mice inoculated with rotavirus were used as controls. Quantities of virus-specific and total Igs were determined using an isotype-specific standard curve that was constructed for each assay based on serial dilutions of purified mouse IgM, IgA, or IgG (Sigma).

Detection of allotype-specific, rotavirus-specific IgA by ELISA

Quantities of rotavirus-specific IgA^a or IgA^b were determined by ELISA using a modification of the technique described above. Following coating, blocking, and addition of sample as described above, 25 ng of biotinylated-mAb specific for mouse IgA^a (HY16) or IgA^b (HISM2) (14) diluted in 50 µl of BSA-T were added to each well and incubated for 1 h at RT. Wells were washed as above and 50 µl of streptavidin-HRP conjugate (Phar-Mingen) diluted 1:2000 in BSA-T was added. Plates were developed, and quantities of Abs were determined as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adoptive transfer of unfractionated cells from draining lymph nodes of i.m.-immunized mice induces rotavirus-specific Ab production by GALT of recipient mice

To determine whether cells resident in draining peripheral lymph nodes after i.m. immunization with rotavirus were capable of inducing mucosal humoral immune responses, 2 x 10⁷ unfractionated cells harvested from draining ILN were transferred into naive syngeneic mice. Repeated analysis by flow cytometry demonstrated that unfractionated cell populations contained 58–73% CD3⁺, 12–21% CD19⁺, 7–11% CD11b⁺, and <1% 33D1⁺ cells (data not shown). Two weeks after adoptive transfer, cultures of intestinal and nonintestinal lymphoid tissues from recipient mice were established. Supernatant fluids from these cultures were tested for the presence of virus-specific Igs. Rotavirus-specific IgM, IgA, and IgG were present in GALT cultures 2 wk after unfractionated cells were transferred i.p. into naive recipient mice (Table I). Previously performed kinetic studies demonstrated that Igs detected in supernatant fluids of lymphoid cultures were generated in vitro and did not arise from passive transudation of serum-derived Abs (12, 15). Likewise, virus-specific IgA was not detected in sera from recipient animals (data not shown). Similar quantities of virus specific IgM, IgA, and IgG were detected in GALT cultures following i.v. adoptive transfer of 2 x 10⁷ unfractionated cells (data not shown). Virus-specific IgM and IgG were also produced by draining ILN. Virus-specific Abs were not produced by intestinal or nonintestinal lymphoid tissues from recipient mice inoculated i.p. with 2 x 10⁷ cells harvested from unimmunized donor mice (data not shown).

Production of rotavirus-specific IgM and IgA by intestinal lymphocytes is short-lived following adoptive transfer; conversely, production of virus-specific IgG persists for at least 6 wk

To determine the duration of virus-specific Ab production by GALT after adoptive transfer, we established lymphoid cultures 6 wk after transfer of 2 x 10⁷ unfractionated cells into naive recipient mice. Six weeks after cell transfer, virus-specific IgG was still produced by intestinal lymphocytes, although at reduced quantities than observed at 2 wk (Table II). However, neither virus-specific IgM nor IgA were produced by LP, PP, or MLN 6 wk after transfer.

To evaluate the role of APCs in the generation of intestinal humoral immunity following i.m. immunization, unfractionated ILN cells from recently i.m.-immunized BALB/c mice were treated with the radiomimetic agent, mitomycin C. Virus-specific IgA was produced by intestinal lymphocytes following adoptive transfer into naive BALB/c recipient mice (Table III). Similarly, no virus-specific IgA was produced by intestinal lymphoid tissues of SCID mice following adoptive transfer of 2 x 10⁷ unfractionated cells harvested from the ILN of recently i.m.-immunized BALB/c mice (data not shown).

To evaluate the contribution of adoptively transferred B cells to the generation of intestinal humoral immunity, 2 x 10⁶ purified B cells derived from the ILN from i.m.-immunized donor mice were transferred into naive recipient mice. Two weeks after transfer, only virus-specific IgA was produced by PP, MLN and LP (Table IV). Rotavirus-specific IgM and IgG were not produced by intestinal lymphocytes.

To determine whether adoptively transferred B cells were expanding and differentiating into virus-specific IgA-secreting cells or functioning as APCs, the following experiment was performed: 2 x 10⁷ unfractionated cells from i.m.-immunized BALB/c mice (H-2^d, IgA^a) were transferred into naive C.B-17 mice (H-2^d, IgA^b). Two weeks after cell transfer, virus-specific IgA^b (recipient phenotype), but not IgA^a (donor phenotype), was produced by intestinal lymphoid tissues of recipient mice (Table V, columns 1 and 2). Similarly, virus-specific IgA^a (recipient phenotype), but not IgA^b (donor phenotype), was produced by intestinal tissues after transfer of 2 x 10⁷ ILN cells from recently immunized C.B-17 mice into naive BALB/c mice (Table V, columns 1 and 2). In addition, adoptive transfer of 4 x 10⁶ purified B cells from i.m.-immunized BALB/c mice into naive C.B-17 mice resulted in production of virus-specific IgA^b (recipient phenotype), not IgA^a (donor phenotype) (Table VI). To exclude the possibility that B cell-associated rotavirus was transferred into recipient mice, purified B cells derived from recently immunized C57BL/6 mice were transferred i.p. into naive BALB/c mice. Virus-specific Abs were not produced by intestinal or nonintestinal tissues of recipient mice (data not shown). Taken together, these results indicate that intestinal rotavirus-specific IgA production is induced by adoptive transfer of B cells. Adoptively transferred B cells obtained from the draining lymph nodes 3 days after i.m. immunization act as APC and do not differentiate to rotavirus-specific ASC.

To determine the contribution of macrophages or dendritic cells to the generation of intestinal humoral immune responses after i.m. immunization, ILN cells from i.m.-immunized mice were depleted of either macrophages (CD11b-bearing cells) or dendritic cells (33D1-bearing cells) by FACS. A total of 1.7 x 10⁷ non-CD11b- or 1.4 x 10⁷ non-33D1-bearing cells were transferred into naive recipient mice, and 2 wk later lymphoid cultures were established. Virus-specific IgG was not detected after transfer of cells depleted of either macrophages (Table VII) or dendritic cells (Table VIII). However, small quantities of virus-specific IgA were produced by GALT of recipient mice despite depletion of either macrophages or dendritic cells from adoptively transferred cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The quantities of virus-specific Igs produced by GALT following transfer of purified cell populations were less than those produced following transfer of unfractionated cells. Likewise, smaller quantities of virus-specific Abs were produced following adoptive transfer of mitomycin C-treated, as compared to untreated, cells. These findings suggest that multiple cell types may be responsible for the generation of mucosal virus-specific IgA following i.m. immunization. Although we have shown that adoptively transferred B cells harvested from donor lymph nodes three days after i.m. immunization can function as APCs and induce intestinal IgA production, the time of cell harvest may have biased against identification of activated B cells that were also capable of inducing mucosal IgA responses. We are currently exploring the contributions of donor-derived CD4⁺ T cells and activated B cells in this system.

Using IgA-allotype-specific reagents we found that intestinal rotavirus-specific IgA responses were induced by Ag-presenting B cells present in adoptively transferred cells. Following adoptive transfer of congenic purified B cells, virus-specific IgA produced by intestinal tissues was of the recipient, not donor, phenotype. In addition, adoptive transfer of MHC-incompatible B cells failed to induce production of virus-specific Abs, indicating that rotavirus does not bind nonspecifically to murine B cells. In this system, B cells, resident in draining peripheral lymph nodes 3 days after primary i.m. immunization, appeared to be capable of virus uptake, processing, and presentation to naive CD4⁺ T cells. To be effective APCs, B cells must meet three requirements: uptake of Ag, presentation of peptide in the context of MHC molecules, and expression of costimulatory molecules. Although the efficiency of Ag presentation by Ag-primed B cells has been described (16, 17, 18, 19, 20), naive B cells may also function as APCs (21). Naive B cells may take up Ag either specifically, by endocytosis of Ag bound to membrane Ig (mIg), or nonspecifically, by pinocytosis. Although in a naive animal the B cell precursor frequency for a given Ag may be presumed to be low, Milich et al (21) found that less than 3 x 10³ activated, or 2 x 10⁴ resting, splenic B cells were capable of activating hepatitis B core Ag-specific T cell hybridomas in the presence of hepatitis B core Ag. Low-affinity binding of multivalent Ag to mIg may explain the unexpectedly high frequency of Ag-presenting naive B cells observed (21). Similarly, recent work by DalPorto et al. (22) demonstrated that B cells with low-affinity mIg may bind Ag and participate in humoral immune responses. The mechanism by which rotavirus is taken up by naive B lymphocytes is unknown. Rotavirus is not known to infect lymphocytes. The redundant expression of two viral surface proteins on intact rotavirus capsids may facilitate virus uptake by mIg on nonimmune B cells. Alternatively, Ag uptake by pinocytosis may occur. However, nonspecific uptake of Ag has been shown to be less efficient than receptor-mediated binding and internalization, and requires much higher concentrations of Ag and longer times for Ag uptake (21). Using limiting dilutions of B cells and virus, we are currently examining the mechanism by which naive B cells resident in peripheral lymph nodes take up rotavirus. In addition to providing cognate interaction between the TCR and surface MHC-peptide complexes, B cells must also express costimulatory molecules to activate naive CD4⁺ T cells and initiate T cell-dependent humoral immune responses (23). Expression of CD80 and CD86 by naive B cells has been shown to be induced by Ag-specific binding to mIg in vivo (17) and in vitro (21, 24, 25). Thus, i.m. immunization with rotavirus may induce expression of costimulatory molecules on naive B cells after binding and internalization of rotavirus through mIg. By adoptive transfer of B cells that do or do not express costimulatory molecules we hope to define the role of B7 expression in the induction of mucosal virus-specific IgA production in this system.

Our findings suggest that intestinal virus-specific ASC generated by i.m. immunization may be induced by Ag-bearing APCs that migrate from the draining peripheral lymph node to GALT. Once in intestinal inductive tissues, such as PP or MLN, these rotavirus-bearing APCs may participate in the activation of naive B and T cells with subsequent generation of virus-specific effector B cells that express the mucosal homing receptor ₄ß₇. Kantele et al. (9) and Quiding-Jabrink et al. (8) demonstrated that Ag-specific ₄ß₇-bearing IgA-secreting cells circulate 7–10 days after parenteral immunization. By isolating ₄ß₇-bearing B cells from intestinal and nonintestinal tissues we hope to identify the site of origin of virus-specific IgA-secreting cells following i.m. immunization.

Although B cells acting as APCs induced the production of virus-specific IgA in recipient mice, either macrophages or dendritic cells or both appeared to be responsible for the induction of intestinal virus-specific IgG production. At present we are unable to distinguish the relative contributions of highly purified populations of macrophages and dendritic cells; adoptive transfer of cell populations depleted of either CD11b⁺ or 33D1⁺ cells resulted in virtually identical profiles of intestinal rotavirus-specific Ab production. Because CD11b is expressed by a subpopulation of dendritic cells resident in peripheral lymph nodes (26), rotavirus-bearing dendritic cells may have been inadvertently excluded from our macrophage-depleted cell preparations.

The differential induction of virus-specific IgA or IgG by different types of APCs suggests that the profile of cytokines produced by individual naive CD4⁺ T cells may be influenced by APC type. Using in vitro models of APC-CD4⁺ interactions, several investigators found that Ag presentation by B cells induced production of Th2 effector cytokines such as IL-4 (27, 28, 29, 30, 31) but not Th1 cytokines such as IL-2 (32) or IFN- (30). Conversely, macrophages and dendritic cells may preferentially induce Th1 responses. T cell clones have been shown to be more responsive to stimulation from splenic macrophages and dendritic cells than B cells (27). However, numerous studies suggest that patterns of cytokine production by CD4⁺ T cells may not be predicted simply by the class of stimulating APC. First, factors such as the nature of the Ag (16, 33, 34), dose of the Ag (35), route of Ag administration (35), and number of CD4⁺ T cell divisions stimulated by antigenic exposure (36) have all been shown to influence the T cell cytokine repertoire. Second, the anatomic source of dendritic cells (37, 38) may influence the outcome of APC-T cell interactions. Schrader et al. (39) demonstrated that both mucosally and systemically derived dendritic cells induced IgA production in vitro, whereas Spaulding et al. (40) found IgA production was preferentially supported by PP as compared with splenic dendritic cells. Finally, differences in cytokine profiles may be influenced by the anatomic site where APC-CD4⁺ interactions occur (39).

We found that production of intestinal rotavirus-specific IgA, as compared with IgG, was short-lived following adoptive transfer of unfractionated cells. Although controversial, persistence of Ag may be necessary to maintain Ag-specific, ASC populations (41, 42). Thus, the transient presence of intestinal virus-specific IgA-secreting B cells may reflect a relative inability of B cells displaying rotavirus peptide-MHC complexes to persist in intestinal lymphoid tissues as compared with either macrophages or dendritic cells. Although Ag-bearing dendritic cells have been shown to persist for over 100 days in germinal centers (43), the life span of Ag-presenting B cells is not known.

Pathogen-specific mucosal IgA plays a critical role in mucosal defense. Nonmucosal inoculation has previously been shown to induce mucosal IgA responses (11, 15, 44, 45). We have extended our understanding of how nonmucosal immunization induces mucosal immunity by demonstrating that intestinal IgA production can be induced by nonimmune, Ag-presenting B cells found in the draining peripheral node after i.m. immunization. However, the transient nature of Ag-specific, intestinal IgA induced by parenteral immunizations may explain a major shortcoming of these vaccines, namely the need for booster dosing to maintain mucosal protection. Strategies that increase the number or functional life span of Ag-presenting B cells may augment the magnitude and duration of mucosal IgA responses and thus enhance the efficacy of parenterally administered vaccines.

	Acknowledgments

 
We thank Jeffery Faust and Jason McCormick for technical assistance, and Jeffrey Bergelson, Kurt Brown, John Cebra, H. Fred Clark, Jenny Kriss, Natasha Kushnir, Kristine Macartney, and Charlotte Moser for helpful discussions.

	Footnotes

 
¹ This work was supported in part by Grants 1 K08 AI01367 (S.E.C.) and 1 R01 AI26251 (P.A.O.) from the National Institutes of Health.

² Address correspondence or reprint requests to Dr. Susan E. Coffin, Division of Immunologic and Infectious Diseases, Children’s Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104. E-mail address:

³ Abbreviations used in this paper: ASC, Ab-secreting cells; GALT, gut-associated lymphoid tissues; ILN, inguinal lymph nodes; LP, lamina propria; mIg, membrane Ig; MLN, mesenteric lymph nodes; PP, Peyer’s patches; RT, room temperature.

Received for publication May 5, 1999. Accepted for publication June 30, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Buscho, R. F., J. C. Perkins, H. L. S. Knopf, A. Z. Kapikian, R. M. Chanock. 1972. Further characterization of the local respiratory tract antibody response induced by intranasal instillation of the inactivated rhinovirus 13 vaccine. J. Immunol. 108:169.[Abstract/Free Full Text]
2. Friedman, M. G., M. Phillip, R. Dagan. 1989. Virus-specific IgA in serum, saliva, and tears of children with measles. Clin. Exp. Immunol. 75:58.[Medline]
3. Kaul, T. N., R. C. Welliver, D. T. Wong, R. A. Udwadia, K. Riddlesberger, P. L. Ogra. 1981. Secretory antibody response to respiratory syncytial virus infection. Am. J. Dis. Child. 135:1013.[Abstract/Free Full Text]
4. Velazquez, F. R., D. O. Matson, J. J. Calva, L. Guerrero, A. L. Morrow, S. Carter-Campbell, R. I. Glass, M. K. Estes, L. K. Pickering, G. M. Ruiz-Palacios. 1996. Rotavirus infections in infants as protection against subsequent infections. N. Engl. J. Med. 335:1022.[Abstract/Free Full Text]
5. Cebra, J. J., C. F. Cuff, D. H. Rubin. 1991. Relationship between /ß T cell receptor/CD8⁺ precursors for cytotoxic T lymphocytes in the murine Peyer’s patches and the intraepithelial compartment probed by oral infection with reovirus. Immunol. Res. 10:321.[Medline]
6. Issekutz, T. B.. 1984. Kinetics of cytotoxic lymphocytes in efferent lymph from single lymph nodes following immunization with vaccinia virus. Clin. Exp. Immunol. 56:515.[Medline]
7. Offit, P. A., S. L. Cunningham, K. I. Dudzik. 1991. Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection. J. Virol. 65:1318.[Abstract/Free Full Text]
8. Quiding-Jabrink, M., I. Nordstrom, G. Granstrom, A. Kilander, M. Jertborn, E. C. Butcher, A. I. Lazarovits, J. Holmgren, C. Czerkinsky. 1997. Differential expression of tissue-specific adhesion molecules on human circulating antibody-forming cells after systemic, enteric, and nasal immunization. J. Clin. Invest. 99:1281.[Medline]
9. Kantele, A., J. M. Kantel, E. Savilahti, M. Westerholm, H. Arvilommi, A. Lazarovits, E. C. Butcher, P. H. Makela. 1997. Homing potential of circulating lymphocytes in humans depend on the site of activation. J. Immunol. 158:574.[Abstract]
10. Coffin, S. E., C. A. Moser, S. Cohen, H. F. Clark, P. A. Offit. 1997. Immunologic correlates of protection against rotavirus challenge after intramuscular immunization of mice. J. Virol. 71:7851.[Abstract]
11. Nussenzweig, M. C., R. M. Steinman, M. D. Witmer, B. Gutchinov. 1982. A monoclonal antibody specific for mouse dendritic cells. Proc. Natl. Acad. Sci. USA 79:161.[Abstract/Free Full Text]
12. Logan, A. C., K. N. Chow, A. George, P. D. Weinstein, J. J. Cebra. 1991. Use of Peyer’s patch and lymph node cultures to compare local immune response to Morganella morganii. Infect. Immun. 59:1024.[Abstract/Free Full Text]
13. Khoury, C. A., K. A. Brown, J. Kim, P. A. Offit. 1994. Rotavirus-specific intestinal immune response in mice assessed by enzyme-linked immunospot assay and intestinal fragment culture. Clin. Diagn. Lab. Immunol. 1:722.[Abstract/Free Full Text]
14. Kroese, F. G. M., J. J. Cebra, M. J. F. van der Cammen, A. B. Kantor, N. A. Bos. 1995. Contribution of B1 cells to intestinal IgA production in the mouse. Methods Companion Methods Enzymol. 8:37.
15. Coffin, S. E., M. Klinek, P. A. Offit. 1995. Induction of virus-specific antibody production by lamina propria lymphocytes following intramuscular inoculation with rotavirus. J. Infect. Dis. 172:874.[Medline]
16. Constant, S., D. Sant’Angelo, T. Pasqualini, T. Taylor, D. Levin, R. Flavell, K. Bottomly. 1995. Peptide and protein antigens require distinct antigen-presenting cell subsets for the priming of CD4⁺ T cells. J. Immunol. 154:4915.[Abstract]
17. Constant, S., N. Schweitzer, J. West, P. Ranney, K. Bottomly. 1995. B lymphocytes can be competent antigen-presenting cells for priming T cells to protein antigens in vivo. J. Immunol. 155:3734.[Abstract]
18. Jr Janeway, C. A., J. Ron, M. E. Katz. 1987. The B cell is the initiating antigen-presenting cell in the peripheral lymph nodes. J. Immunol. 138:1051.[Abstract/Free Full Text]
19. Lanzavecchia, A.. 1990. Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu. Rev. Immunol. 8:773.[Medline]
20. Lanzavecchia, A.. 1985. Antigen-specific interactions between T and B cells. Nature 314:537.[Medline]
21. Milich, D. R., M. Chen, F. Schodel, D. L. Peterson, J. E. Jones, J. L. Hughes. 1997. Role of B cells in antigen presentation of the hepatitis B core. Proc. Natl. Acad. Sci. USA 94:14648.[Abstract/Free Full Text]
22. DalPorto, J. M., A. M. Haberman, M. J. Shlomchik, G. Kelsoe. 1998. Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers. J. Immunol. 161:5373.[Abstract/Free Full Text]
23. Jr Janeway, C. A., K. Bottomly. 1994. Signals and signs for lymphocytes responses. Cell 76:275.[Medline]
24. Lenschow, D. J., A. I. Sperling, M. P. Cooke, G. Freeman, L. Rhee, D. C. Decker, G. Gray, L. M. Nadler, C. C. Goodnow, J. A. Bluestone. 1994. Differential up-regulation of the B7-1 and B7-2 costimulatory molecules after Ig receptor engagement by antigen. J. Immunol. 153:1990.[Abstract]
25. Ho, W. Y., M. P. Cooke, C. C. Goodnow, M. M. Davis. 1994. Resting and anergic B cells are defective in CD28-dependent costimulation of naïve CD4⁺ T cells. J. Exp. Med. 179:1539.[Abstract/Free Full Text]
26. Salomon, B., J. L. Cohen, C. Masurier, D. Klatzmann. 1998. Three populations of murine lymph node dendritic cells with different origins and dynamics. J. Immunol. 160:708.[Abstract/Free Full Text]
27. Gajewski, T. F., M. Pinnas, T. Wong, F. W. Fitch. 1991. Murine Th1 and Th2 clones proliferate optimally in response to distinct antigen-presenting cell populations. J. Immunol. 146:1750.[Abstract]
28. Macaulay, A. E., R. H. DeKruyff, D. T. Umetsu. 1998. Antigen-primed T cells from B cell-deficient JHD mice fail to provide B cell help. J. Immunol. 160:1694.[Abstract/Free Full Text]
29. Macaulay, A. E., R. H. DeKruyff, C. C. Goodnow, D. T. Umetsu. 1997. Antigen-specific B cells preferentially induce CD4⁺ T cells to produce IL-4. J. Immunol. 158:4171.[Abstract]
30. Maecker, H. J., M. S. Do, S. Levy. 1998. CD81 on B cells promotes interleukin 4 secretion and antibody production during T helper type 2 immune responses. Proc. Natl. Acad. Sci. USA 95:2458.[Abstract/Free Full Text]
31. Adorini, L., J. C. Guery, F. Ria, F. Galbiati. 1997. B cells present antigen to CD4⁺ T cells, but fail to produce IL-12: selective APC for Th2 cell development?. Ann. NY Acad. Sci. 815:401.[Medline]
32. Stockinger, B., T. Zal, A. Zal, D. Gray. 1996. B cells solicit their own help from T cells. J. Exp. Med. 183:891.[Abstract/Free Full Text]
33. Guery, J. C., F. Ria, F. Galbiati, S. Smiroldo, L. Adorini. 1996. The mode of protein antigen administration determines preferential presentation of peptide-class II complexes by lymph node dendritic or B cells. Int. Immunol. 9:9.[Abstract/Free Full Text]
34. Pasare, C., V. Morafo, M. Entringer, P. Bansal, A. George, V. Bal, S. Rath, J. M. Durdik. 1998. Presence of activated antigen-binding B cells during immunization enhances relative levels of IFN- in T cell responses. J. Immunol. 160:778.[Abstract/Free Full Text]
35. DeKruyff, R. H., Y. Fang, D. T. Umetsu. 1992. IL-4 synthesis by in vivo primed keyhole limpet hemocyanin-specific CD4⁺ T cells: influence of antigen concentration and antigen-presenting cell type. J. Immunol. 149:3468.[Abstract]
36. Gett, A. V., P. D. Hodgkin. 1998. Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation. Proc. Natl. Acad. Sci. USA 95:9488.[Abstract/Free Full Text]
37. Everson, M. P., W. J. Koopman, K. W. Beagley. 1993. Divergent T cell cytokine profiles induced by dendritic cells from different tissues. ed. Dendritic Cells in Fundamental and Clinical Immunology 47. Plenum Press, New York.
38. Spaulding, D. M., J. A. Griffin. 1986. Different pathways of differentiation of pre-B cell lines are induced by dendritic cells and T cells from different lymphoid tissues. Cell 44:507.[Medline]
39. Schrader, C. E., A. George, R. L. Kerlin, J. J. Cebra. 1990. Dendritic cells support production of IgA and other non-IgM isotypes in clonal microculture. Int. Immunol. 2:564.
40. Spaulding, D. M., W. J. Koopman Williamson, J. R. McGhee. 1984. Preferential induction of polyclonal IgA secretion by murine Peyer’s patch dendritic cell-T cell mixtures. J. Exp. Med. 160:941.[Abstract/Free Full Text]
41. Gray, D., H. Skarvall. 1988. B-cell memory is short-lived in the absence of antigen. Nature 336:70.[Medline]
42. Bachmann, M. F., B. Odermatt, H. Hengartner, R. M. Zinkernagel. 1996. Induction of long-lived germinal centers associated with persisting antigen after viral infection. J. Exp. Med. 183:2259.[Abstract/Free Full Text]
43. Tew, J. G., T. F. Mandel. 1979. Prolonged antigen half-life in the lymphoid follicles of specifically immunized mice. Immunology 37:69.[Medline]
44. Clements, M. L., R. F. Betts, B. R. Murphy. 1985. Comparison of immune response and efficacies of inactivated and live virus vaccines. J. Cell. Biochem. 9C:284.
45. Cripps, A. W., M. L. Dunkley, R. L. Clancy. 1994. Mucosal and systemic immunizations with killed Pseudomonas aeruginosa protects against acute respiratory infection in rats. Infect. Immun. 62:1427.[Abstract/Free Full Text]

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