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Memory Th2 Effector Cells Can Develop in the Absence of B7-1/B7-2, CD28 Interactions, and Effector Th Cells After Priming with an Intestinal Nematode Parasite¹

Melinda J. Ekkens^*, Zhugong Liu^*, Qian Liu^*, Anthony Foster^*, Jeannette Whitmire^*, John Pesce^*, Arlene H. Sharpe, Joseph F. Urban and William C. Gause^2,*

^* Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and Nutrient Requirements and Functions Laboratory, Beltsville Human Nutrition Research Center, U.S. Department of Agriculture, Beltsville, MD 20705

	Abstract

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B7-1/B7-2 interactions are required for many Th2-cell mediated primary immune responses including the response that follows infection with the intestinal nematode parasite, Heligmosomoides polygyrus. However, few studies have examined the role of B7-1/B7-2/CD28 interactions in the development of a Th2 memory immune response. We examined the development of the memory Th2 response to H. polygyrus in BALB/c mice deficient in both B7-1 and B7-2 (B7-1/B7-2^-/-) and in BALB/c mice deficient in CD28 (CD28^-/-). Following primary inoculation with H. polygyrus, adult worms in the gut were cleared with an anti-helminthic drug and mice were subsequently challenge-inoculated with H. polygyrus larvae. The memory Th2 response is readily distinguished by its inhibitory effect on adult worm maturation, resulting in marked reductions in adult worm egg production that are not observed during the primary immune response. Following H. polygyrus challenge inoculation, comparable decreases in egg production and similar increases in mesenteric lymph node cell IL-4 production were observed in B7-1/B7-2^-/- and B7-1/B7-2^+/+ mice. However, elevations in total serum IgG1 and IgE were reduced, while increases in serum Ag-specific IgG1 and IgE and germinal center formation were blocked in H. polygyrus-challenged B7-1/B7-2^-/- mice. In contrast, in H. polygyrus-challenged CD28^-/- mice, marked elevations in Ag-specific IgG1 and IgE and increased germinal center formation were observed. The results of these studies demonstrate that effector Th2 memory cells that produce IL-4 and mediate host defense can develop when B7-1/B7-2 interactions, and associated effector Th2 cell development, are blocked during priming. However, humoral immunity is impaired and differentially affected in B7-1/B7-2^-/- mice and CD28^-/- mice following H. polygyrus challenge.

	Introduction

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The development of CD4⁺ Th effector cells during the Th2 primary immune response to infectious pathogens is associated with B7-dependent maturation of naive CD4⁺ T cells into IL-4-producing cells. Previous studies have shown that blocking B7 interactions during the primary response with either the chimeric fusion protein CTLA4Ig or with blocking anti-B7 Abs inhibits the development of IL-4-producing CD4⁺ T cells and associated increases in serum IgE and IgG1, following infection of mice with a number of different parasites including the gastrointestinal parasite Heligmosomoides polygyrus (1, 2), the protozoan parasite Leishmania major (3), and the nematode parasite Schistosoma mansoni (4, 5).

In contrast, the role of B7 interactions in the development of memory Th2 cells is not well-defined. CD28 ligation can increase T cell survival by up-regulating the antiapoptotic gene, Bcl-x_L (6), suggesting that this costimulatory molecule could play a role in the survival and maintenance of memory CD4⁺ T cells. Several pathways have been proposed for the development of memory T cells, with one model suggesting that memory T cells may develop directly from naive T cells following priming (7, 8) and others indicating that memory T cells develop primarily from differentiated effector T cells (9, 10), suggesting that if B7 blockade inhibited effector T cell development during the primary response, the development of memory T cells would also be blocked. A study of CD8⁺ memory T cell development following influenza virus infection suggested that CD28 is not required for memory T cell development but that CD28 signaling is required for effector T cell function during both the primary and memory responses (11). In contrast, recent studies suggest that CD28 interactions are not required for CD8⁺ CTL responses following challenge immunization with lymphocytic choriomeningitis virus (LCMV)³ (12). During the LCMV immune response, B7 interactions are not required for generating primary CTL responses (12, 13), consistent with other studies suggesting that CD8⁺ T cells are less dependent on B7 signaling than CD4⁺ T cells (14).

Infection of mice with intestinal nematode parasites induces the development of a Th2 immune response that can control infection. The murine nematode parasite, Heligmosomoides polygyrus, is a useful model for studying the development of the Th2 memory immune response during infectious disease. The parasite has a strictly enteral life cycle in which infective third stage larvae invade the intestinal mucosa after ingestion and develop there into mature adults that enter the gut lumen 8 days later. The host response is characterized by elevations of Th2 cytokines, with CD4⁺'/TCR⁺ T cells being the primary source of IL-4 elevations during the primary (15) and the secondary immune responses (16). Pronounced CD4⁺ T cell-dependent elevations in serum IgE and IgG1 and increased germinal center (GC) formation in the mesenteric lymph node (MLN) are observed. During the primary response, adult worms mature and produce eggs, inhabiting the gut lumen for at least several months in BALB/c mice. However, after clearance of worms from the gut with an antihelminthic drug, a secondary challenge inoculation triggers a CD4- and IL-4-dependent memory response that effectively limits adult worm maturation and egg production (17). We have previously shown that B7 blockade inhibits the development of the Th2 primary response to H. polygyrus (1, 2), but that B7 blockade at the time of challenge is ineffective in inhibiting memory cell activation (16, 18). In this investigation, we examined whether memory Th2 cells could develop when B7 interactions are inhibited during priming. Our results show that although effector T cell development is inhibited during the primary immune response to H. polygyrus in BALB/c B7-1/B7-2^-/- mice, memory T cells can still develop that produce IL-4, and that mediate effective reductions in adult worm egg production, but cannot provide effective Ag-specific B cell help or support increased GC formation. Furthermore, parallel studies of the memory response in BALB/c CD28^-/- mice show similar IL-4 elevations and decreases in adult worm egg production; however, the Ag-specific Ab response and increased GC formation were significantly restored.

	Materials and Methods

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Mice

All experiments were performed using BALB/c mice genetically deficient for both B7-1 and B7-2 (B7-1/B7-2^-/-), CD28 (CD28^-/-), or normal BALB/c controls. Breeding pairs of BALB/c B7-1/B7-2^-/- mice were generated in the laboratory of Dr. A. Sharpe and breeding pairs of BALB/c CD28^-/- mice were the generous gift of Dr. S. Reiner (University of Pennsylvania, Philadelphia, PA), while BALB/c controls were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in a specific pathogen-free, virus Ab-free facility administered by the Laboratory of Animal Medicine, Unformed Services University of the Health Sciences (Bethesda, MD), which is an institution accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. The experiments in this study were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education and Welfare (National Institutes of Health) 78-23.

Antibodies

BALB/c B7-1/B7-2^-/- mice infected with H. polygyrus were treated with anti-CD4 mAb (GK1.5; generous gift from F. Finkelman, University of Cincinnati School of Medicine, Cincinnati, OH) at days 0 and 7 postchallenge.

Parasites

Mice (five per treatment group) were inoculated per os with 200 infective third stage H. polygyrus using a rounded gavage tube, and adult worm numbers and egg production were quantitated as described previously (19). An anti-helminthic, pyrantel pamoate (1–2 mg), was administered orally to expulse H. polygyrus adults from the gut after primary infection and before challenge infection (19).

Quantitation of serum Igs

Total serum IgG1, IgG2a, and IgE levels were quantitated by ELISA (20). Ag-specific Ab levels were measured using a modified ELISA. Briefly, individual wells of Immulon IV plates (Thermo Labsystems, Franklin, MA) were coated with diluted (5 µg/ml) H. polygyrus Ag (Ag-specific IgG1, H. polygyrus extract; Ag-specific IgE, H. polygyrus excretory/secretory Ag), and incubated overnight at 4°C. After three to five washes with double-distilled (dd) H₂O, plates were blocked with 1% FBS + 10% sodium azide for 30 min at room temperature. Following three to five washes with ddH₂O, serum samples were added to coated plates in 4-fold serial dilutions, and incubated for 2 h at room temperature. Plates were then washed three to five times with ddH₂O, and incubated for 30 min (room temperature) with borate-buffered saline (BBS) + 1% Tween 20. After three washes with ddH₂O, anti-mouse IgG1-alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL), diluted 1/2000 in BBS-Tween 20, was added to individual wells, and the plates were incubated for 30 min at room temperature. Following three washes with ddH₂O, plates were incubated with BBS-Tween 20 for 30 min at room temperature and washed three times with ddH₂O, before adding the substrate (5% 4-methylumbelliferyl phosphate free acid buffer). Alternatively, following five washes with ddH₂O, biotinylated anti-mouse IgE (BD PharMingen, San Diego, CA), diluted 1/250 in BBS-Tween 20, was added to individual wells, and the plates were incubated for 1 h at room temperature. After five washes with ddH₂O, plates were treated with BBS-Tween 20, and washed five times with ddH₂O, before adding the secondary Ab, (streptavidin-alkaline phosphatase; BD PharMingen) diluted 1/1000 in BBS-Tween 20. Following a 1-h incubation at room temperature, plates were washed five times with ddH₂O, incubated with BBS-Tween 20 for 30 min at room temperature, and washed five times with ddH₂O, before adding the substrate (5% 4-methylumbelliferyl phosphate free acid buffer). Following incubation at room temperature, fluorescence of the samples was read using a µQuant ELISA plate reader (Bio-Tek Instruments, Winooski, VT).

ELISPOT

The frequency of IL-4-producing cells was determined by an ELISPOT assay as previously described (20, 21).

Flow cytometry

Lymph node cells were harvested and 1 x 10⁶ cells were blocked with Fc Block (BD PharMingen) and then incubated with FITC-anti-CD4 (BD PharMingen), and PE-anti-CD69 (BD PharMingen). After washes, cells were fixed with 1% paraformaldehyde (Fisher, Pittsburgh, PA) and analyzed on flow cytometry using an EPICS XL-MCL (Beckman Coulter, Fullerton, CA). Twenty thousand events were collected and analysis of activation marker expression was performed by gating on the CD4⁺ population.

Immunohistological analysis

The procedure used for immunohistological staining and GC quantitation was as described previously (2, 22). Alternatively, 8-µm frozen tissue sections were dual-stained with PE-anti-CD4 (BD PharMingen), and biotinylated peanut lectin (agglutinin) (PNA; Sigma-Aldrich, St. Louis, MO) followed by streptavidin Alexa-488 (Molecular Probes, Eugene, OR) for fluorescence analysis. Tissues were stained and digitally photographed, on the same day, at magnification of x150 magnification with a Zeiss Auxiophot microscope, using Slidebook software (Intelligent Imaging Innovations, Denver, CO).

Statistical analysis

Statistical differences (significance level of p < 0.05) between groups were assessed using ANOVA and Tukey’s t test for pairwise comparisons. The software program SigmaStat (Jandel, San Rafael, CA) was used for all statistical analyses.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increases in IL-4 producing lymphocytes and corresponding decreases in adult worm egg production are comparable in B7-1/B7-2^-/- and B7-1/B7-2^+/+ mice following challenge with H. polygyrus

Previous studies have shown that initial infection with H. polygyrus stimulates a chronic Th2 CD4-dependent immune response (17). If infected mice are administered an anti-helminthic at day 14 after infection, complete worm expulsion occurs, as demonstrated by a decline to zero in fecal egg counts and an absence of worms in killed animals. Following anti-helminthic treatment, if mice are subsequently challenged with H. polygyrus, a memory response develops that impairs adult worm development resulting in reduced fecundity. Although B7 blockade inhibits the primary response, administration of the B7 antagonist, CTLA4Ig, at the time of challenge, does not impair the memory response (1, 2, 16). To examine whether B7 blockade during the primary H. polygyrus response also inhibits memory T cell development and the subsequent H. polygyrus memory response, B7-1/B7-2^-/- mice and B7-1/B7-2^+/+ mice were orally infected with H. polygyrus larvae. At 2 wk postinfection, both groups were treated with the anti-helminthic pyrantel pamoate to expulse all worms, and 30 days later were administered a challenge oral dose of H. polygyrus. Two additional control groups (B7-1/B7-2^-/- and B7-1/B7-2^+/+ mice) received an initial inoculum of H. polygyrus at the time of challenge, allowing direct comparison between the primary and secondary immune responses to H. polygyrus. All mice were sacrificed at day 10 after H. polygyrus inoculation and various immune parameters were assessed. In vivo anti-CD4 Ab administration inhibited elevations in IL-4 production and serum Ig levels and blocked decreased worm fecundity in both H. polygyrus-challenged B7-1/B7-2^-/- and H. polygyrus-challenged B7-1/B7-2^+/+ mice (data not shown), indicating that the secondary immune response remains CD4-dependent in B7-1/B7-2^-/- mice.

Previous studies have shown that the primary source of IL-4 production in the MLN of H. polygyus-inoculated mice during the primary (15) and challenge (16) immune response is CD4⁺'/TCR⁺ cells. This cell population directly mediates host protection during the challenge response (19), resulting in an inhospitable enteric environment in which egg production by adult worms is inhibited and, in some cases, worms are expulsed (16, 19); in contrast, the primary response is associated with chronic infection (17). Adult worm egg production or fecundity was monitored at day 10 after challenge H. polygyrus inoculation because fecundity is a sensitive and reproducible indicator of an ongoing immune response against adult nematodes in the intestine. As shown in Fig. 1A, egg production was blocked in H. polygyrus-challenged B7-1/B7-2^+/+ mice, while egg production was pronounced in B7-1/B7-2^+/+ mice receiving only a primary H. polygyrus inoculation. Unexpectedly, adult worm egg production was also significantly reduced (p < 0.05), though not completely blocked, in H. polygyrus-challenged B7-1/B7-2^-/- mice compared with egg production in B7-1/B7-2^-/- mice given a primary immunization only. These results demonstrate that a memory Th2 response capable of impairing intestinal adult worm egg production can develop in H. polygyrus-inoculated B7-1/B7-2^-/- mice.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Increased IL-4 secretion and decreased adult worm egg production are observed in B7-1/B7-2-/- and B7-1/B7-2+/+ mice after challenge with H. polygyrus. B7-1/B7-2+/+ control mice and B7-1/B7-2-/- mice were orally infected with 200 third-stage H. polygyrus larvae, and then treated with the anti-helminthic, pyrantel pamoate, at 2 wk postinfection. Thirty days later (44 days after initial inoculation), the mice were administered a challenge dose of H. polygyrus; as a control, two additional groups of mice (B7-1/B7-2+/+ and B7-1/B7-2-/- mice) received the initial inoculum of H. polygyrus at the time of challenge, allowing direct comparison between the primary (Hp1') and secondary (Hp2') immune responses to H. polygyrus. A, Ten days after the challenge infection, the total number of eggs recovered from the intestine was determined in all groups (five mice per treatment group), as described in Materials and Methods. B, At the same time-point, MLNs were removed from individual mice (five mice per treatment group), and single-cell suspensions were analyzed for IL-4 protein secretion using the ELISPOT assay. A value of "0" represents nondetectable levels. The results are expressed as the mean and SE for each treatment group, and similar results were obtained in two experiments.

We also examined T cell activation after priming with H. polygyrus in B7-1/B7-2^-/- and B7-1/B7-2^+/+ mice. At day 5 after inoculation, comparable increases in CD4⁺ T cell CD69 expression were detected in B7-1/B7-2^-/- as well as B7-1/B7-2^+/+ mice (data not shown), suggesting that T cell activation did occur during priming in the absence of B7 interactions, but that further differentiation to IL-4-producing T cells required B7 costimulation.

Elevations in total IgE and IgG1 are reduced while elevations in Ag-specific IgG1 and IgE and GC formation are blocked in H. polygyrus-challenged B7-1/B7-2^-/- mice

Elevations in IL-4 and decreased adult worm egg production in H. polygyrus-challenged B7-1/B7-2^-/- mice suggested that memory Th2 cells could develop in the absence of B7 interactions. To examine whether T cell help leading to B cell Ig class switching and Ab secretion still occurred in B7-1/B7-2^-/- mice during the memory response to H. polygyrus, total serum IgE and IgG1 levels were measured by ELISA. Previous studies have shown that serum IgE and IgG1 elevations are CD4⁺ T cell-dependent following H. polygyrus-challenge (19) and that increases in serum IgE, but not IgG1, are IL-4 dependent (23, 24, 25). At day 10 after H. polygyrus challenge, significant increases (p < 0.01) in total serum IgE and IgG1 were detected in B7-1/B7-2^+/+ mice compared with untreated controls. In H. polygyrus-challenged B7-1/B7-2^-/- mice, total serum IgE and IgG1 elevations were significantly reduced (p < 0.01) compared with H. polygyrus-challenged B7-1/B7-2^+/+ mice (Fig. 2, A and B), but elevations in serum IgE levels in H. polygyrus-challenged B7-1/B7-2^-/- mice were significantly increased (p < 0.01) over IgE elevations in B7-1/B7-2^-/- mice receiving only a primary immunization.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Elevations in total IgE and IgG1 are reduced, while elevations in Ag-specific IgG1 and Ag-specific IgE are blocked in H. polygyrus-challenged B7-1/B7-2-/- mice. B7-1/B7-2-/- and B7-1/B7-2+/+ mice were primed and challenged with H. polygyrus as described in Fig. 1. At day 10 postchallenge, individual mice (five mice per treatment group) were bled and examined for increases in total and Ag-specific serum Ig levels. Total serum IgE (A) and IgG1 (B) and H. polygyrus (Hp) Ag-specific IgE (C) and IgG1 (D) levels were determined using the ELISA, as described in Materials and Methods. A value of "0" represents nondetectable levels. Results are expressed as the mean and SE for each treatment group, and similar results were obtained in two experiments.

The GC provides a microenvironment for T helper/B cell interactions resulting in Ig class switching, affinity maturation, and development of memory B cells. MLNs were collected from H. polygyrus-infected B7-1/B7-2^-/-, and B7-1/B7-2^+/+ mice, and increases in GC formation were quantified as described in Materials and Methods. As shown in Table I, increased GC formation was significantly reduced (p < 0.01) during both the primary and secondary H. polygyrus response in B7-1/B7-2^-/- mice as compared with B7-1/B7-2^+/+ mice. These findings indicate that GC formation is profoundly impaired in the absence of B7 interactions, suggesting that although effector T cells can develop to produce IL-4 and mediate decreased adult worm egg production, CD4⁺ T cell-dependent GC formation and specific Ab production is abrogated.

Although few studies have examined the effects of CD28 blockade on the development of the memory Th2 response, CD28 is generally thought to provide a positive signal required for the development of Th2 cells; in contrast, a number of studies have suggested that the other known B7-1/B7-2 ligand, CTLA-4, provides a negative signal that can trigger T cell tolerance in the absence of CD28 signaling during the primary response (26, 27, 28, 29, 30, 31). To examine the effects of CD28 blockade on the development of the memory Th2 response, CD28^-/- mice were immunized using a regimen similar to that used to immunize B7-1/B7-2^-/- mice. CD28^-/- and CD28^+/+ mice were inoculated with H. polygyrus, treated with pyrantel pamoate at day 14 after inoculation, and then challenged with H. polygyrus 50 days after primary immunization. Two additional control groups (CD28^-/- and CD28^+/+ mice) received an initial inoculum of H. polygyrus at the time of challenge, allowing direct comparison between the primary and secondary immune responses to H. polygyrus. Twelve days after challenge, adult worm egg production was determined and mice were analyzed for immune parameters.

H. polygyrus-challenged CD28^-/- mice showed significantly reduced (p < 0.05) adult worm egg production compared with CD28^-/- mice receiving only a primary immunization (Fig. 3A). The frequency of MLN IL-4-secreting lymphocytes was measured using the ELISPOT assay, without restimulation. As shown in Fig. 3B, H. polygyrus-challenged CD28^+/+ and CD28^-/- mice demonstrated significant (p < 0.05) increases in IL-4 production, as compared with CD28^+/+ and CD28^-/- mice receiving a primary dose of H. polygyrus, which showed comparable increases in IL-4 production. In a separate experiment, similar decreases in adult worm egg production and increases in IL-4 secretion were obtained at day 10 after H. polygyrus challenge. These findings indicate that blocking CD28 during priming, without inhibiting CTLA-4, does not trigger Th2 unresponsiveness following H. polygyrus challenge.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Blocking CD28 alone results in the development of a pronounced IL-4 response and decreased adult egg production during the H. polygyrus memory response. CD28+/+ control mice and CD28-/- mice were orally infected with H. polgyrus, treated with pyrantel pamoate, and challenged with H. polygyrus at day 50, as described in Fig. 1. At day 12 postchallenge, all treatment groups were necropsied, and the total number of eggs recovered from the intestine was determined in all groups (five mice per treatment group), as described in Materials and Methods (A). At the same timepoint, MLNs were collected from individual mice (five mice per treatment group) and IL-4 protein secretion was measured using the ELISPOT assay (B). A value of "0" represents nondetectable levels, and results are expressed as the mean and SE for each treatment group; similar results were obtained in three experiments.

Our findings of pronounced increases in IL-4 and decreased adult worm egg production suggested that Th2 effector cells had developed that could mediate host protective responses to H. polygyrus in the absence of CD28 interactions. Therefore, we next examined T-dependent elevations in total serum IgG1 and IgE and H. polygyrus Ag-specific serum IgG1 and IgE. As shown in Fig. 4, A and B, pronounced (p < 0.01) total IgE and IgG1 elevations were detected in H. polygyrus-challenged CD28^-/- mice compared with CD28^-/- mice given a primary immunization only. However, these elevations were significantly reduced (p < 0.01) compared with those observed in H. polygyrus-challenged CD28^+/+ mice. H. polygyrus Ag-specific serum IgE (Fig. 4C) and H. polygyrus Ag-specific serum IgG1 (Fig. 4D) levels were also markedly elevated in H. polygyrus-challenged CD28^-/- mice, although they were still significantly reduced (p < 0.01) compared with H. polygyrus-challenged CD28^+/+ mice. Similar results were also obtained at day 10 after H. polygyrus challenge inoculation of CD28^-/- mice.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Total and Ag-specific serum Ig levels are elevated in H. polygyrus-challenged CD28-/- mice. CD28+/+ control mice and CD28-/- mice were inoculated with H. polygyrus as described in Fig. 3, and at day 12 postchallenge, individual mice (five mice per treatment group) were bled and examined for increases in total and Ag-specific serum Ig levels. Total serum IgE (A) and IgG1 (B) and H. polygyrus (Hp) Ag-specific IgE (C) and IgG1 (D) levels were determined using the ELISA, as described in Materials and Methods. A value of "0" represents nondetectable levels. Results are expressed as the mean and SE for each treatment group, and similar results were obtained in three experiments.

View larger version (163K): [in this window] [in a new window]   FIGURE 5. GC formation is elevated in H. polygyrus-challenged CD28-/- mice. CD28+/+ mice and CD28-/- mice were inoculated with H. polygyrus as described in Fig. 3. At day 12 postchallenge, MLNs were obtained from individual mice (five mice per treatment group), frozen in liquid nitrogen, and sectioned at 8 µm. Tissue sections were dual-stained with PE-anti-CD4 (red stain) and biotinylated peanut lectin (agglutinin) followed by streptavidin Alexa-488 (green stain). Tissues were digitally photographed at a magnification of x150 with a Zeiss Auxiophot microscope, using Slidebook software (Intelligent Imaging Innovations, Denver, CO), and each panel is representative of an average field.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous studies using B7-1/B7-2 antagonists have shown that B7 interactions are required for the development of the primary H. polygyrus immune response, including increases in CD4⁺ T cell IL-4 production, GC formation, and serum IgG1 and IgE elevations (1, 2). Furthermore, blocking B7 interactions at the time of challenge immunization does not affect the memory response with elevations in both CD4⁺ T cell IL-4 production and serum Ig levels remaining comparable to H. polygyrus-challenged mice administered a control fusion protein (16, 18). In the studies reported in this work, we examined the effects of B7 blockade on memory Th2 cell development when B7 interactions are blocked throughout the primary, as well as the secondary, immune response.

Few studies have examined the role of B7-1/B7-2 interactions in the development and maintenance of memory CD4⁺ T cells, particularly during the Th2 response. CTLA4Ig administration following infection with the intestinal nematode parasite, Nippostrongylus brasiliensis, did not block T cell IL-4 production or the associated host protective primary or secondary Th2 response (33). However, a caveat in this model is that, although the primary response to N. brasiliensis is CD4-dependent, the secondary host-protective immune response is CD4-independent (33, 34). Furthermore, the primary response to N. brasiliensis, resulting in CD4-dependent worm expulsion, is B7-independent (33). In contrast, the primary immune response to H. polygyrus is B7-dependent, while the memory response is CD4⁺ T cell-dependent (1, 2, 19). Studies of the H. polygyrus memory immune response can thus address whether memory Th2 cells can develop in the absence of B7-dependent effector T cell development during the primary immune response. A number of models have suggested that memory T cells are survivors of effector cells that revert to a resting state (10, 35). However, more recent studies have suggested heterogeneity in memory T cell populations and in their development (7, 36, 37). Indeed, central memory cells, the memory T cell population that generates secondary responses in the lymph node, may not require an intermediate effector cell stage during the primary response (8, 37). Our studies are consistent with this model of memory T cell development, because they indicate that the CD4⁺ memory T cells that respond in the MLN following H. polygyrus challenge can develop in the absence of B7-1/B7-2 costimulatory molecules and during the associated blockade of CD4⁺ T effector cell function during the primary response.

Significant elevations in total serum IgE and IgG1 were obtained in H. polygyrus-challenged B7-1/B7-2^-/- mice, although the Ab levels were still reduced compared with H. polygyrus-challenged B7-1/B7-2^+/+ mice. In contrast, elevations in serum H. polygyrus Ag-specific IgG1 and H. polygyrus Ag-specific IgE were abrogated in H. polygyrus-challenged B7-1/B7-2^-/- mice. A number of studies have suggested that parasites can trigger polyclonal B cell responses (25, 38, 39, 40) and that much of the consequent total Ab produced is nonspecific (41, 42). However, elevations in total serum IgE and IgG1 are CD4-dependent in both the primary (25, 43) and secondary (19) H. polygyrus immune response, and elevations in total Igs are B7-dependent during the primary response (1). Our findings suggest that when B7 interactions are blocked during the primary response, a population of memory CD4⁺ T cells develops that provides B cell help leading to Ig class switching and plasma cell development during challenge inoculation. However, development of Ag-specific plasma cells is preferentially blocked, perhaps due to a specific impairment in memory CD4⁺ T cell function, or alternatively, a defect in memory B cell development. Previous studies with B7 antagonists have also suggested that elevations in Ag-specific serum Igs are inhibited during the memory response when B7 interactions are inhibited during priming (44).

Both CD28 and its homologue, CTLA-4, bind to B7-1 and B7-2 during in vivo immune responses, although CTLA-4 binds B7-1 and B7-2 with higher avidity than does CD28 (45, 46), and has been shown to transmit a negative signal independently of CD28 (31, 47). CTLA-4 may provide key regulatory signals at the initiation phase as well as the effector phase of the immune response, and previous studies have suggested that CTLA-4 signaling in the absence of CD28 signaling during priming can induce T cell unresponsiveness following restimulation (26). Previous studies of BL/6 CD28^-/- mice have indicated that the CD4⁺ T cell responses and associated elevations in serum Igs are intact following primary H. polygyrus inoculation (48). We have now extended these studies to an analysis of the memory response. We hypothesized that CD28 blockade during priming might cause memory T cell unresponsiveness in H. polygyrus-challenged mice. Instead, a pronounced memory T cell response was still observed, with IL-4 elevations and reduced adult worm egg production that was comparable to H. polygyrus-challenged CD28^+/+ mice. Furthermore, a substantial Ab response was also observed in H. polygyrus-challenged CD28^-/- mice, including pronounced elevations in H. polygyrus Ag-specific IgG1 and Ag-specific IgE and increases in GC formation. These findings are in marked contrast to the abrogation of Ag-specific IgG1 and Ag-specific IgE and GC formation in H. polygyrus-challenged B7-1/B7-2^-/- mice, suggesting that CTLA-4, or some other as yet unidentified B7-1/B7-2 ligand, can sustain the T-dependent memory Ab response. Alternatively, other differences may exist between B7-1/B7-2^-/- and CD28^-/- mice that effect the development of memory cells. These may include differential expression of other costimulatory molecules or perhaps cell populations. For example, recent studies have indicated a role for inducible costimulator protein in memory cell activation following challenge immunization (49, 50, 51); however, its function in memory cell development during priming is not known.

Taken together, our results indicate that memory Th2 effector cells that mediate host defense following H. polygyrus challenge are primed in the absence of B7-1/B7-2 interactions and effector CD4⁺ T cell development. However, memory humoral immunity is suppressed, with H. polygyrus Ag-specific IgG1 and H. polygyrus Ag-specific IgE production and GC formation abrogated. In contrast, blockade of CD28 during priming is associated with significant H. polygyrus Ag-specific IgG1 and H. polygyrus Ag-specific IgE response and pronounced GC formation during the memory H. polygyrus response.

	Acknowledgments

 
We thank Dr. Fred D. Finkelman for his review of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI31678. The opinions or assertions contained within are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or the Department of Defense.

² Address correspondence and reprint requests to Dr. William C. Gause, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address: wgause{at}usuhs.mil

³ Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; PNA, peanut lectin (agglutinin); GC, germinal center; MLN, mesenteric lymph node; dd, double distilled; BBS, borate-buffered saline.

Received for publication January 29, 2002. Accepted for publication April 4, 2002.

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