Thymic Independence of Adaptive Immunity to the Intracellular Pathogen Shigella flexneri Serotype 2a

Abstract

Shigella flexneri is a facultative intracellular pathogen. While immunity to several intracellular pathogens is mediated by T lymphocytes, it is unknown whether cellular immune responses are important to adaptive immunity to S. flexneri. We show that vaccination with S. flexneri serotype 2a confers protection to mice that lack T lymphocytes or gamma interferon (IFN-γ), specific depletion of T lymphocytes does not alter the protection, and adoptive transfer of splenocytes from vaccinated mice does not confer protection to naive mice. In contrast, vaccination conferred no protection to mice that lack B lymphocytes and adoptive transfer of immune sera conferred partial protection to naive mice. These data demonstrate that in the mouse bronchopulmonary model, adaptive immunity to S. flexneri 2a is an antibody-mediated, B-lymphocyte-dependent process and can be generated in the absence of T lymphocytes or IFN-γ.

Shigella spp. are gram-negative bacteria that cause diarrhea and dysentery in humans. Disease due to Shigella involves bacterial invasion of colonic enterocytes and uptake by M cells (30, 55), with induction of an intense acute inflammatory response (30, 50). Within minutes of uptake into mammalian cells, shigellae lyse the phagocytic vacuole and are thereby released into the cell cytoplasm, where they divide and use actin-based motility to move and spread directly into adjacent cells (3, 49, 55). In this way, once within an intestinal epithelial cell, shigellae are able to spread from one infected cell into adjacent cells without reentering the extracellular environment. Shigellae are also taken up by phagocytic cells, including macrophages and polymorphonuclear leukocytes (29, 69). Thus, during the course of intestinal disease, shigellae reside within the intracellular compartment of M cells, enterocytes, macrophages, and polymorphonuclear leukocytes.

Epidemiological studies have found that disease due to endemic species of Shigella occurs primarily in children while disease due to epidemic species is equally prevalent in all age groups, which suggests that protection following natural infection can occur (63). Partial Shigella species-specific and serotype-specific protection has been described following naturally acquired Shigella infection in humans or following experimental vaccination with live attenuated strains in humans and animals (9, 12, 24, 31–33). In humans, Shigella infection results in increased concentrations of Shigella-specific immunoglobulin G (IgG) and IgA in both intestinal secretions and serum (6, 18, 54) and in increased numbers of IgG and IgA antibody-producing cells in peripheral blood and in the intestinal mucosa (17, 46). Furthermore, anti-Shigella antibodies are bactericidal in complement fixation or opsonization assays in vitro (51–53). In mice, secretory IgA provided via “backpack” hybridoma tumors or mixed with the bacterial inoculum is able to protect against disease caused by Shigella of the homologous serotype (48), although mice that lack IgA (IgA^−/− mice) have no increase in susceptibility to challenge after vaccination (67). These data suggest that an antibody response to Shigella is an important component of adaptive immunity and that under certain experimental conditions, antibody can protect against disease. Furthermore, since serotype specificity of Shigella is defined by the composition of the oligosaccharide repeating unit of the O antigen of bacterial lipopolysaccharide (LPS) (26), the serotype specificity of the response seen in certain studies indicates that an antibody response to bacterial LPS is an important component of protection. The ability of vaccination to confer protection to IgA^−/− mice suggests that IgA is not essential for protection and that non-IgA isotypes are likely to be important in this process.

As described above, during much of the course of disease, Shigella resides within the cytoplasm of host cells and is thereby protected from opsonizing antibodies and immune cell surface Igs but is susceptible to cytotoxic T-cell activity and professional phagocytic cell-mediated killing. This intracellular localization and the mechanism of Shigella cell-to-cell spread are strikingly similar to those of the gram-positive pathogen Listeria monocytogenes. In mice, adaptive immunity to L. monocytogenes is mediated by T lymphocytes. Primary T lymphocytes harvested from Listeria-vaccinated mice or T-lymphocyte clones that recognize Listeria immunodominant peptide epitopes are able to confer protection to naive mice (4, 13–16, 22, 27, 41). In this model, CD8⁺ T lymphocytes are the predominant protective cell type (14, 34, 35), although CD4⁺ T lymphocytes also provide protective effects (4, 16, 21, 27). CD8⁺ and CD4⁺ T lymphocytes confer protection via different mechanisms, the former via a perforin-dependent, gamma interferon (IFN-γ)-independent mechanism (14, 16, 20) and the latter via an IFN-γ-mediated mechanism (16, 27).

Given the similar intracytoplasmic localization of Shigella and L. monocytogenes during infection, we wished to address the role of the cellular immune response in adaptive immunity to Shigella. Further, we wished to address whether T-lymphocyte help was required for the generation of protective antibodies. Therefore, in the present study, we examined the contribution of T lymphocytes and IFN-γ to adaptive immunity in Shigella infection.

Mice do not normally acquire intestinal disease following oral inoculation with Shigella. For this reason, as well as the utility of murine gene knockout technology, we used the murine bronchopulmonary model of Shigella infection (28, 64, 66). Following a lethal intranasal inoculation, mice die of an acute pneumonitis; shigellae are observed intracellularly in both the bronchiolar and alveolar epithelia (64, 66). Following inoculation with an identical dose of either a noninvasive derivative of wild-type Shigella or wild-type Shigella killed by exposure to UV light, mice do not become ill or die (66), suggesting that endotoxin-mediated toxicity is not a cause of death. Vaccination with sublethal doses of either wild-type or live attenuated strains of S. flexneri confers significant protection to subsequent challenge with wild-type S. flexneri (28, 64, 67). Thus, the murine bronchopulmonary model is a useful model for the study of adaptive immunity.

In this study, we used a vaccination-challenge system for the study of adaptive immunity to S. flexneri serotype 2a that consists of bronchopulmonary vaccination of mice with two sublethal inoculations of an attenuated strain of S. flexneri serotype 2a followed by bronchopulmonary challenge 20 days later with an inoculum of the wild-type strain of the homologous serotype that is lethal for naive mice (28, 64, 67). Following challenge, mice were monitored for survival and/or time to death. With this system, 93% of vaccinated C57BL/6 mice are protected against challenge (67), a level of protection similar to that observed with other attenuated strains of Shigella for vaccination of BALB/c mice by the bronchopulmonary route (28). The high levels of protection that are generated suggests that this approach can be applied to the evaluation of the mediators of adaptive immunity to S. flexneri serotype 2a in this model.

MATERIALS AND METHODS

Bacterial strains.

The wild-type S. flexneri serotype 2a strain 2457T and serotype 5a strain M90T have been described previously (11, 56). S. flexneri serotype 2a strain SSW202 is 2457T cydC::Tn10 (68). Strain SSW202 expresses cytochrome bd at markedly reduced levels (68). Consequently, while it enters mammalian cells normally, it has decreased survival in the cytoplasm of infected cells and has a 2-log-unit higher lethal dose for mice than strain 2457T has (68). Due to these properties, SSW202 was chosen as the vaccination strain for the experiments conducted in this study.

Mouse strains.

C57BL/6, TCR-β^−/− TCR-δ^−/− (C57BL/6) (39), and μ^−/− (C57BL/6) (23) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). TCR-β^−/− TCR-δ^−/− mice lack all T lymphocytes by virtue of disruptions of both the T-cell receptor β and δ subunits. IFN-γ-deficient mice (129Sv × C57BL/6) (7) were kindly provided by B. R. Bloom. BALB/c mice with severe combined immunodeficiency (SCID mice) were kindly provided by H. Goldstein and B. Diamond. All mice were housed in a specific-pathogen-free environment at the Albert Einstein College of Medicine. Care of animals was in accordance with guidelines set by the American Association for Accreditation of Laboratory Animal Care and the Albert Einstein College of Medicine. All animal studies were approved by the Institutional Review Board at Albert Einstein College of Medicine.

Mouse infections and vaccinations.

Murine intranasal inoculation with S. flexneri strains or saline control was performed as described previously (64, 66). The lethal dose of wild-type S. flexneri 2457T or M90T for naive C57BL/6, TCR-β^−/− TCR-δ^−/−, or μ^−/− mice is 10⁷ bacteria (reference 66 and data not shown), which is not significantly different from the previously published lethal dose of wild-type S. flexneri strains for BALB/c mice (28, 64). The vaccination schedule for C57BL/6, TCR-β^−/− TCR-δ^−/−, and μ^−/− mice was as follows. Beginning at 6 to 8 weeks of age, mice were inoculated with the first of two doses of 1 × 10⁷ to 2 × 10⁷ SSW202 or saline; the second dose was administered 21 days after the first. Mice that received saline inoculations as a control (mock-vaccinated mice) were age and sex matched with SSW202-vaccinated mice. Challenge of vaccinated and mock-vaccinated mice was performed, 20 days after the second inoculation, with a dose of wild-type S. flexneri 2457T or M90T that was equivalent to the lethal dose for naive mice, i.e., 10⁷ bacteria. The survival outcome of C57BL/6, TCR-β^−/− TCR-δ^−/−, and μ^−/− mice was observed for up to 21 days postchallenge.

The lethal dose of 2457T for naive IFN-γ^−/− mice is 10² organisms (66). The vaccination and challenge schedules for IFN-γ^−/− mice was as for C57BL/6, TCR-β^−/− TCR-δ^−/−, and μ^−/− mice, except that the challenge dose was 10³ or 10⁵ wild-type S. flexneri 2457T or M90T. The survival of IFN-γ^−/− mice was observed for up to 40 days postchallenge.

Hybridomas and antibodies.

Hybridoma clones GK1.5 (rat anti-mouse CD4), 30H12 (rat anti-mouse CD3), and 2.43 (rat anti-mouse CD8) were obtained from the American Type Culture Collection (Rockville, Md.). Antibodies were generated from the hybridoma clones as ascites in SCID mice. Nonspecific rat IgG was obtained from Sigma Immunochemicals. For fluorescence-activated cell sorter (FACS) analysis, phycoerythrin-conjugated anti-mouse CD45, fluorescein-conjugated anti-mouse CD3, fluorescein-conjugated anti-mouse CD8, and fluorescein- or phycoerythrin-conjugated anti-mouse CD4 (PharMingen, San Diego, Calif.) was used.

Depletion of T lymphocytes.

In vivo depletion of T lymphocytes in vaccinated mice was performed by intraperitoneal injection of GK1.5 (rat anti-mouse CD4), 30H12 (rat anti-mouse CD3), or 2.43 (rat anti-mouse CD8) antibody 3 days prior to challenge (400 to 500 μg), on the day of challenge (200 to 250 μg), and on days 2, 4, and 6 postchallenge (200 to 250 μg/injection) as described previously (34, 35). The efficacy of depletion was assessed by FACS analysis of peripheral blood and spleen 3 days after the first injection of antibody, just prior to the second. Flow cytometry was performed on a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). LYSIS II was used for instrument control, data acquisition, and data analysis.

Transfer of splenocytes.

Spleens were harvested from vaccinated and mock-vaccinated mice 20 days after the last inoculation of the vaccination schedule. Splenocyte suspensions were prepared by passage of spleens through nylon mesh and lysis of erythrocytes as described previously (4). Splenocytes were resuspended in saline at a concentration of 2.3 × 10⁸ to 4.0 × 10⁸ splenocytes/ml. A 200-μl volume of this suspension, representing 4.5 × 10⁷ to 8.0 × 10⁷ splenocytes, was given intravenously to each recipient naive mouse. The viability of splenocytes was verified by trypan blue dye exclusion prior to transfer.

Transfer of serum.

Peripheral blood from vaccinated or mock-vaccinated mice was collected by retro-orbital bleeding or cardiac puncture 20 days after the last inoculation of the vaccination schedule. Serum was separated from clotted blood and filtered through a 0.22-μm-pore-size membrane. Each recipient naive mouse received 200 μl of this serum intravenously 3 h prior to challenge; surviving mice received an additional 200 μl of serum intravenously at 24 h and 150 μl of serum intraperitoneally at 48 h after administration of the challenge dose. Mice were challenged with a dose of wild-type S. flexneri 2457T that was equivalent to the lethal dose for naive mice, i.e., 10⁷ bacteria.

Histological analysis.

At the indicated times after infection, mice were sacrificed by cervical dislocation. Lungs were harvested, fixed in 10% buffered formalin, and embedded in paraffin. Hematoxylin-and-eosin staining was performed on 5-μm-thick sections.

Determination of the antibody concentration and serotype specificity of antibodies.

For analysis of the antibody response, sera were drawn 20 days after the last of two inoculations with 1 × 10⁷ to 2 × 10⁷ SSW202 or saline. The concentration of antibody of each isotype was determined by enzyme-linked immunosorbent assay. Initially, a standard concentration curve for each isotype was established by coating plates with 1.0 μg of goat anti-murine IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA per ml and determining the titers of known concentrations of commercially available antibody for each isotype (Southern Biotechnology Associates, Inc.). The concentration of S. flexneri 2a-specific antibody in each isotype class present in experimental sera was then determined by enzyme-linked immunosorbent assay, as follows. Plates were coated with 1.2 × 10⁷ 2457T (serotype 2a) or M90T (serotype 5a) in saline overnight under UV light. Serial dilutions of experimental sera were then plated, and isotype-specific alkaline phosphatase-conjugated secondary antibody was added. Antibody concentrations were then calculated from optical density measurements by using the previously established standard concentration curves. Standard deviations were calculated on antibody concentrations, which were determined for three to five mice per group.

Statistics.

The difference in survival between groups of mice was evaluated by using a censored log-rank analysis. The difference between mean antibody concentrations in groups of mice was evaluated by the parametric independent Student t test and the nonparametric Wilcoxon rank sum test. For all analyses, P < 0.05 was taken as indicating statistical significance.

RESULTS

Protection in T-lymphocyte-depleted vaccinated mice.

The vaccination-challenge system used for the study of adaptive immunity to S. flexneri serotype 2a, which consisted of bronchopulmonary vaccination of mice with an attenuated strain followed by bronchopulmonary challenge 20 days later with an inoculum of the wild-type strain of the homologous serotype that is lethal for naive mice (28, 64), induces protection in 93% of vaccinated C57BL/6 mice (67). To address the contribution of T lymphocytes to the survival of vaccinated mice after challenge, the effect of T-lymphocyte depletion was investigated. At 20 days after vaccination, groups of 4 to 13 mice were depleted of CD4⁺, CD8⁺, both CD4⁺ and CD8⁺, or CD3⁺ T lymphocytes or were treated with nonspecific rat IgG and then were challenged with S. flexneri 2a wild-type strain 2457T. The survival of mice depleted of CD4⁺, CD8⁺, both CD4⁺ and CD8⁺, or CD3⁺ T lymphocytes was not significantly different from the survival of mice that had not been depleted or that had been treated with nonspecific rat IgG (Fig. 1A). FACS analysis demonstrated greater than 94% depletion of the appropriate cell type in both peripheral blood and spleens isolated from mice depleted in an identical manner to mice that were subsequently challenged (Fig. 1B and data not shown).

FIG. 1.

Effect of T-lymphocyte depletion on the survival of vaccinated mice upon challenge. (A) Survival of mice treated with antibody to CD4 (n = 6), antibody to CD8 (n = 11), antibody to both CD4 and CD8 (n = 4), antibody to CD3 (n = 10), nonspecific rat IgG (n = 11), or nothing (n = 13) prior to challenge. The observed difference in survival of CD4-depleted mice and other mice was not statistically significant (P > 0.2 for the comparison of CD4-depleted mice with each of the other data sets). (B) Efficacy of in vivo CD3⁺-T-lymphocyte depletion. FACS analysis of peripheral blood from each of two representative CD3⁺-T-lymphocyte-depleted mice (top), a mouse that was not depleted (bottom left), and a TCR-β^−/− TCR-δ^−/− mouse (bottom right). After in vivo depletion, less than 2% of CD45⁺ (leukocyte common antigen) cells expressed the T-lymphocyte marker CD3. Similar levels of depletion were observed by FACS analysis of both splenocytes and peripheral blood for each depletion experiment (data not shown).

Protection following adoptive transfer of splenocytes.

To further evaluate the contribution of cell-mediated immunity to protection, the ability of splenocytes harvested from vaccinated mice to confer protection on naive mice was examined. The spleens of donor vaccinated mice were approximately twofold larger than those of either donor mock-vaccinated or naive mice (data not shown), suggesting that a systemic immune response to vaccination had occurred. Groups of eight naive C57BL/6 mice were given 4.5 × 10⁷ to 8.0 × 10⁷ splenocytes that had been harvested from either vaccinated or saline mock-vaccinated mice and then were challenged with wild-type strain 2457T. No difference in either survival (0% in each group) or mean time to death (3.4 days postchallenge for each group) was observed between mice receiving splenocytes from vaccinated mice and mice receiving splenocytes from mock-vaccinated mice (data not shown).

Adaptive immunity in the absence of T lymphocytes.

To further examine the contribution of T lymphocytes to protection against S. flexneri 2a infection, the ability of vaccination to confer protection to TCR-β^−/− TCR-δ^−/− mice, which lack all T lymphocytes, was examined. We have previously shown that there is no difference in the lethal dose of S. flexneri for naive TCR-β^−/− TCR-δ^−/− and naive C57BL/6 mice (66). Groups of TCR-β^−/− TCR-δ^−/− mice that had been vaccinated with attenuated S. flexneri or mock vaccinated with saline were challenged with wild-type S. flexneri. The survival of vaccinated (n = 15) and mock-vaccinated (n = 8) TCR-β^−/− TCR-δ^−/− mice was 87 and 0%, respectively (Table 1). The 87% protection observed for TCR-β^−/− TCR-δ^−/− mice was not significantly different from the 93% protection observed previously for vaccinated C57BL/6 mice.

TABLE 1.

Protective efficacy of vaccination with serotype 2a S. flexneri against homologous challenge in C57BL/6, TCR-β^−/− TCR-δ^−/− and μ^−/− mice

Mouse strain	Vaccination serotype or saline	Challenge serotype	No. of mice	% Survival	Protective efficacya
C57BL/6	Saline	2a	16	0
	2a	2a	15	93	93
TCR-β−/− TCR-δ−/−	Saline	2a	8	0
	2a	2a	15	87	87
μ−/−	Saline	2a	8	13
	2a	2a	15	20	9

^aProtective efficacy was calculated as [(percent death of controls − percent death of vaccinated mice)/percent death of controls] × 100, as described previously (64). 

Following challenge, the lungs of mock-vaccinated C57BL/6 and mock-vaccinated TCR-β^−/− TCR-δ^−/− mice showed similar patterns of diffuse pneumonitis (Fig. 2A and B), with interstitial infiltration of polymorphonuclear leukocytes. The lungs of vaccinated C57BL/6 and vaccinated TCR-β^−/− TCR-δ^−/− mice were also similar, showing inflammation only in a peribronchiolar distribution (bronchiolitis) with a marked absence of alveolar involvement (Fig. 2C and D).

FIG. 2.

Pulmonary histology of C57BL/6 and TCR-β^−/− TCR-δ^−/− mice following challenge. Representative fields of hematoxylin-and-eosin-stained sections of lung from mock-vaccinated (A and B) or vaccinated (C and D) C57BL/6 (A and C) or TCR-β^−/− TCR-δ^−/− (B and D) mice 3 days after challenge. In mock-vaccinated mice (A and B), there was diffuse alveolitis with a predominance of polymorphonuclear leukocytes in the alveolar cellular infiltrate. In vaccinated mice (C and D), there was minimal alveolar involvement but acute bronchiolitis was present (black arrows). Peribronchiolar lymphoid aggregates were present in vaccinated C57BL/6 mice (C, white arrow) but absent in vaccinated TCR-β^−/− TCR-δ^−/− mice (D). Magnification, ×50.

Role of IFN-γ in adaptive immunity.

IFN-γ is essential for innate immunity to S. flexneri 2a infection (66). The lethal dose of S. flexneri for naive mice that lack IFN-γ is 5 orders of magnitude lower than the lethal dose for naive congenic immunocompetent mice (10² versus 10⁷ bacteria) (66). To evaluate the contribution of IFN-γ to adaptive immunity, the ability of vaccination to confer protection on mice that lack IFN-γ was examined. Following vaccination, mice were challenged with an inoculum (10³ bacteria) of S. flexneri 2457T that was 10-fold greater than the lethal dose for naive IFN-γ^−/− mice. All the vaccinated IFN-γ^−/− mice survived challenge (n = 18), while none of the mock-vaccinated IFN-γ^−/− mice survived (n = 19) (Fig. 3). The mean time to death of the mock-vaccinated mice was 18.9 days. Furthermore, at 21 days after challenge, no S. flexneri could be recovered from the lungs or spleens of vaccinated IFN-γ^−/− mice (n = 4). Thus, vaccination conferred significant protection in the absence of IFN-γ.

FIG. 3.

Effect of vaccination on survival of IFN-γ^−/− mice upon challenge. Results for vaccinated (squares) and mock-vaccinated (triangles) mice following challenge with either 10⁵ (solid symbols) or 10³ (open symbols) S. flexneri 2457T are shown.

To determine the degree of protection conferred on IFN-γ^−/− mice by vaccination, vaccinated IFN-γ^−/− mice were challenged with 10⁵ wild-type S. flexneri 2457T, which is 1,000-fold greater than the lethal dose for naive IFN-γ^−/− mice. In this experiment, 14% of the vaccinated IFN-γ^−/− mice survived (n = 21), with a censored mean time to death of 15.8 days, while 0% of the mock-vaccinated IFN-γ^−/− mice survived (n = 14), with a mean time to death of 7.1 days (Fig. 3). The increase in mean time to death of vaccinated over mock-vaccinated IFN-γ^−/− mice was highly significant (P = 0.0014).

Requirement of B lymphocytes for specific immunity.

The observed protection conferred by vaccination in the absence of T lymphocytes and the failure of splenocytes from vaccinated mice to transfer protective effects suggested that B-lymphocyte-mediated processes were likely to be essential in protection against S. flexneri 2a infection. The contribution of B lymphocytes was examined by evaluating the ability of vaccination to confer protection on B-cell-deficient (μ^−/−) mice. Survival following challenge of vaccinated (n = 15) and mock-vaccinated (n = 8) μ^−/− mice was 20 and 13%, respectively (Table 1), giving a protective efficacy of vaccination (9.0%) significantly lower than that observed for TCR-β^−/− TCR-δ^−/− mice (87%) or C57BL/6 mice (93%) (Table 1).

After challenge, the lungs of both vaccinated and mock-vaccinated μ^−/− mice demonstrated diffuse alveolar and interstitial inflammation (Fig. 4A and C), with polymorphonuclear leukocyte infiltration in the distal airspaces (Fig. 4B and D). Bronchiolitis, which was observed for vaccinated C57BL/6 and vaccinated TCR-β^−/− TCR-δ^−/− mice, was not observed for either vaccinated or mock-vaccinated μ^−/− mice. Of note, peribronchiolar lymphoid aggregates containing mature lymphocytes were present in vaccinated (Fig. 4C and D) but not in mock-vaccinated (Fig. 4A and B) μ^−/− mice.

FIG. 4.

Pulmonary histology of TCR-μ^−/− (antibody-deficient) mice following challenge. Representative fields of hematoxylin-and-eosin-stained sections of lung from vaccinated (C and D) or mock-vaccinated (A and B) mice 2 days after challenge are shown. Diffuse alveolar and interstitial inflammation (A and C) and infiltration of the distal air spaces with polymorphonuclear leukocytes (B and D) that are similar in vaccinated (C and D) and mock-vaccinated (A and B) mice were found. Expansion of peribronchiolar lymphoid aggregates occurred in vaccinated (C and D, arrows), but not in mock-vaccinated mice. Magnifications, ×10 (A and C) and ×50 (B and D).

Protection following passive transfer of immune serum.

The requirement for B lymphocytes, in conjunction with the lack of requirement for T lymphocytes, suggested that protection against S. flexneri 2a infection was mediated by antibody. To directly evaluate the protective effects of antibody, the ability of serum from vaccinated C57BL/6 mice to confer protection on naive C57BL/6 mice was examined. Groups of eight naive C57BL/6 mice were given a passive transfer of serum from either vaccinated or mock-vaccinated C57BL/6 mice prior to challenge. Upon challenge, all mice in each group died; however, the mean time to death for mice that had been given immune serum was 4.1 days, while that for mice that had been given mock-vaccinated serum was 2.4 days, a difference that was highly significant (P = 0.0017). Thus, in this system, systemic administration of immune serum conferred protective effects to naive mice.

Production of specific antibodies following vaccination.

The protective effect conferred on naive mice by passive transfer of immune serum suggested that antibody may be the major mediator of protection to S. flexneri 2a infection in this model system. To determine the magnitude of the S. flexneri 2a-specific antibody response and whether T lymphocytes or IFN-γ is required for its induction, the concentrations and serotype specificity of serum antibody specific for S. flexneri antigens in vaccinated and mock-vaccinated C57BL/6, TCR-β^−/− TCR-δ^−/−, and IFN-γ^−/− mice were examined (Fig. 5). In C57BL/6 mice, vaccination induced a significant 42-fold increase in serotype-specific IgM and IgG1 production (P = 0.004 and 0.008 respectively), a 4-fold increase in IgG2a production (P = 0.004), a 23-fold increase in IgG2b production (P = 0.009), a 7.6-fold increase in IgG3 production (P = 0.05), and a 10-fold increase in IgA production (P < 0.0001) (Fig. 5). The concentration of antibody specific to antigens of the heterologous serotype increased for each isotype, but to a substantially lesser extent than did the concentrations of antibody specific to antigens of the homologous serotype (Fig. 5), reaching statistical significance only for IgA.

FIG. 5.

Specific serologic response in S. flexneri 2a-vaccinated C57BL/6, IFN-γ^−/−, and TCR-β^−/− TCR-δ^−/− mice. Concentrations of IgM (A), IgG1 (B), IgG2a (C), IgG2b (D), IgG3 (E), and IgA (F) that recognize S. flexneri antigens either of the homologous serotype (serotype 2a) or of a heterologous serotype (serotype 5a) are shown. Sera were obtained from saline mock-vaccinated mice (open bars) or vaccinated mice (dark stippled bars). Data represent the mean concentrations in three to five mice per group. The absence of bars indicates antibody levels that were below the limits of detection. Asterisks indicate that the concentration was significantly different from the concentration of antibody in mock-vaccinated mice. Error bars indicate 1 standard deviation.

In TCR-β^−/− TCR-δ^−/− mice, vaccination induced a significant ninefold increase in serotype-specific IgM (P = 0.02) but no increase in IgM to antigens of the heterologous serotype (Fig. 5A). As expected, given the absence of isotype switching in these animals, there were no detectable amounts of any IgG isotype (Fig. 5B to E). Very low concentrations of anti-S. flexneri IgA antibodies were detected in mock-vaccinated TCR-β^−/− TCR-δ^−/− mice, which did not increase with vaccination (Fig. 5F); the detected binding could be nonspecific binding above background.

For IFN-γ^−/− mice, vaccination induced increases in the concentrations of serotype-specific antibody in all isotypes; these increases were statistically significant for IgG2b (P = 0.0002) and were clinically significant for IgG2a, IgG3, and IgA (since the levels for mock-vaccinated mice were undetectable, statistical analysis could not be performed [Fig. 5]). While increases in antibody concentration to antigens of both the homologous and heterologous serotype were observed for all antibody isotypes, there was a relatively greater increase in the concentrations of antibody to antigens of the homologous serotype (Fig. 5); these increases reached statistical significance for IgG2b (P = 0.002), IgG3 (P = 0.02), and IgA (P = 0.03).

Adaptive immunity of C57BL/6, TCR-β^−/− TCR-δ^−/−, and IFN-γ^−/− mice to S. flexneri 2a is serotype specific.

Since these data indicate that T lymphocytes are not required for adaptive immunity and the antibody response is predominantly serotype specific, it was of interest to examine whether protection was also serotype specific. Groups of 7 to 14 C57BL/6, TCR-β^−/− TCR-δ^−/−, and IFN-γ^−/− mice that had been vaccinated or saline mock vaccinated were challenged with the heterologous serotype 5 wild-type strain M90T. The survival of vaccinated (n = 14) and mock-vaccinated (n = 10) C57BL/6 mice was 7 and 0%, respectively; thus, the protective efficacy of vaccination with respect to heterologous challenge was only 7% (Table 2), compared with the protective efficacy with respect to homologous challenge of 93% (Table 1). The survival of vaccinated (n = 9) and mock-vaccinated (n = 7) TCR-β^−/− TCR-δ^−/− mice was 11 and 0%, respectively; thus, the protective efficacy of vaccination with respect to heterologous challenge was only 11% (Table 2), compared with the protective efficacy with respect to homologous challenge of 87% (Table 1). For IFN-γ^−/− mice, despite the observed increases in concentrations of IgM and IgA to antigens of the heterologous serotype, upon challenge with the heterologous serotype there was no significant difference in either survival or time to death between vaccinated and mock-vaccinated mice (data not shown). Thus, protection was serotype specific in all three strains of mice tested.

TABLE 2.

Protective efficacy of vaccination with serotype 2a S. flexneri against heterologous challenge with serotype 5a S. flexneri in C57BL/6 and TCR-β^−/− TCR-δ^−/− mice

Mouse strain	Vaccination serotype or saline	Challenge serotype	No. of mice	% Survival	Protective efficacya
C57BL/6	Saline	5a	10	0
	2a	5a	14	7	7
TCR-β−/− TCR-δ−/−	Saline	5a	7	0
	2a	5a	9	11	11

^aProtective efficacy was calculated as in Table 1, footnote a. 

DISCUSSION

Vaccination-induced protection against several intracellular pathogens is mediated by T lymphocytes. For murine infection with L. monocytogenes, both CD4⁺ and CD8⁺ T lymphocytes contribute to the adaptive response (14–16, 21, 22, 25, 27). CD8⁺ T lymphocytes are absolutely essential for protection, while CD4⁺ T lymphocytes are not (14, 34, 35). Similarly, for Leishmania major infection, CD4⁺ and CD8⁺ T lymphocytes can both confer protection against subsequent challenge (8, 58, 59); however, depletion of CD8⁺ T lymphocytes alone reverses the protective effects of vaccination (8). Following murine infection with Mycobacterium bovis BCG, either CD4⁺ or CD8⁺ T lymphocytes are able to confer protection to subsequent challenge with M. tuberculosis (42, 44, 45); additional work suggests that the BCG-induced protective effects are mediated predominantly by CD4⁺ T lymphocytes (43).

The data presented in this study indicate that, unlike experimental immunity to each of the intracellular pathogens mentioned above, protection against S. flexneri 2a in the model system used here is independent of T lymphocytes. We have shown here that in vivo depletion of T lymphocytes from vaccinated mice did not reduce protection, adoptive transfer of splenocytes from vaccinated mice to naive mice did not transfer protection, and mice that lack T lymphocytes were protected by vaccination to the same extent as congenic immunocompetent mice. The observed protective effects of vaccination in TCR-β^−/− TCR-δ^−/− mice were not due to activation of nonspecific immune mechanisms, since vaccination induced only baseline levels of protection against challenge with a heterologous serotype of S. flexneri. These data suggest that even if T-cell responses to Shigella antigens develop, they do not play an essential role in protection, and that the lack of protection in μ^−/− mice is not due to the absence of antigen-presenting cells. It is not surprising that B cells derived from the spleen did not provide protection in the adoptive-transfer experiments, since essentially all antibody is produced by mature plasma cells, and while some plasma cells are found in the red pulp of the spleen, the majority of mature plasma cells are located at other sites, including the bone marrow, the medullary cords of lymph nodes, and other tissues.

An obvious difference between the approach used in this study and that used in studies describing the mediators of immunity to L. monocytogenes, M. tuberculosis, and Leishmania sp. is the route of vaccination and infection. In this study, mice were vaccinated and infected via the mucosal surfaces of the lungs, whereas in studies of the immune response to these other pathogens, mice were vaccinated and infected predominantly by intravenous, intraperitoneal, or subcutaneous routes (8, 14, 16, 34, 36, 37, 42, 43, 45, 58, 59). Infiltration of peribronchiolar lymphoid aggregates with mature B and T lymphocytes occurs following intranasal vaccination or infection with Shigella (67). Shigella antigens may be initially presented within these peribronchiolar lymphoid aggregates, which may lead to a different type of immune response from that found if the organisms are taken up by cells of the reticuloendothelial system following intravenous inoculation or by cells in other nonmucosal sites following intraperitoneal or subcutaneous inoculation.

IFN-γ is essential for innate immunity to Shigella infection (66). The data presented in this study indicate that IFN-γ is not required for acquired protection to Shigella infection, since mice that lack IFN-γ developed significant protection upon vaccination. However, vaccination does not obviate the need for this cytokine, since the lethal dose for vaccinated IFN-γ-deficient mice was significantly lower than the lethal dose for nonvaccinated immunocompetent mice (10⁵ and 10⁷ wild-type S. flexneri 2a, respectively). It is possible that nonspecific immune defenses contribute in a significant way to the protective response following challenge and that IFN-γ is important to this nonspecific response. Alternatively, IFN-γ may be important to the adaptive response by virtue of its effects on antibody isotype preference. Of note, in the IFN-γ^−/− mouse, we observed significant increases in the concentrations of serotype-specific IgG1 and IgG2b antibodies, a response thought to be mediated by IL-4 (10, 60). In addition, in IFN-γ^−/− mice, the increases we observed in the concentrations of serotype-specific IgG2a and IgG3 antibodies were greater than the vaccination-induced increases observed for C57BL/6 mice (Fig. 5), which could reflect alternative pathways for the induction of these antibody isotypes (10, 62, 65).

A consistent histological finding in groups of mice that had developed adaptive immunity was the presence of plasma cells within peribronchiolar lymphoid aggregates. While plasma cells are readily obscured by mature lymphocytes also present within these aggregates, they were clearly present, as demonstrated in the lungs of both vaccinated TCR-β^−/− TCR-δ^−/− mice and CD3⁺-T-lymphocyte-depleted, vaccinated C57BL/6 mice (data not shown). Although the vaccination-induced peribronchiolar aggregates also contained T lymphocytes, these were not associated with adaptive immunity, as discussed above. A second consistent histological finding in groups of mice that had developed protection was the lack of alveolar and diffuse interstitial inflammation following challenge. Where present, inflammation in protected animals was mild and was located in a peribronchiolar distribution; moreover, mice that developed this pattern of inflammation were consistently protected against challenge. These findings indicate that the presence of increased numbers of plasma cells within these aggregates is associated with a marked diminution in both diffuse interstitial disease and death. Finally, since adaptive immunity is serotype specific, these data suggest that infiltrating plasma cells may produce protective antibody that is serotype specific. The importance of a localized (i.e., peribronchiolar) immune response from such aggregates of plasma cells could be tested by performing the analyses with animals that are either immunized or challenged by a route other than the intranasal one.

The inability of mice with a selective B-lymphocyte deficiency to develop protection upon vaccination, in conjunction with the ability of immune serum to confer partial protection to naive mice, suggests that antibodies secreted by B lymphocytes mediate adaptive immunity to S. flexneri 2a infection in the model used in this study. The failure of passively transferred immune serum to confer complete protection to naive mice probably reflects the inability of antibody that is transferred by this means to attain concentrations at the bronchial mucosa comparable to those engendered by direct intranasal vaccination. As discussed above, the peribronchiolar lymphoid infiltration present in all protected mouse strains contains plasma cells, which may secrete protective antibody. An additional factor that may contribute to the incompleteness of the protection conferred by passive serum transfer is that, due to technical limitations, recipient mice receive less serum than is present in a whole immune animal. Future analyses will characterize the isotype specificity of protection and of the peribronchiolar plasma cells.

We have recently shown that vaccinated IgA^−/− mice are protected to the same extent as congenic IgA^+/+ mice against challenge with wild-type S. flexneri 2a (67), which suggests that specific IgA is not essential to the adaptive response. In the present study, serum IgA was also not absolutely required for protection, since vaccination conferred protection to TCR-β^−/− TCR-δ^−/− mice, which, because CD40 ligand and T-cell-secreted cytokines are important for isotype switching, produce a predominantly IgM response. Of note, TCR-β^−/− TCR-δ^−/− mice were protected in the presence of a significant increase in the concentration of only serotype-specific IgM and the greatest increase of serotype-specific antibody in C57BL/6 mice also occurred for IgM, which suggests that serotype-specific IgM can be sufficient for protection. It will be of interest to test directly which isotype mediates protection in vaccinated immunocompetent mice; additional studies will be performed to address this question. Since the intracellular compartment of cells is inaccessible to antibody, protection mediated by antibody would necessarily involve only antibody-bacterium interactions that occur extracellularly. These interactions might include blocking of initial bacterial entry into host cells and/or scavenging following Shigella-induced lysis of infected cells. Further investigations are necessary to address these possibilities.

The ability to develop full protection in the complete absence of T lymphocytes demonstrates that T-lymphocyte help is not required for the activation of B lymphocytes in this system. Interestingly, the LPS component of gram-negative bacteria, including Escherichia coli and Salmonella, is a thymus-independent antigen (1, 2, 19, 38, 40, 47, 57, 61). Since the Shigella serotype is determined by the O-antigen component of the LPS, which is a carbohydrate and therefore cannot be recognized by T lymphocytes, a response directed against this antigen may well be both immunodominant and thymus independent. In all strains of mice tested in the present study, adaptive immunity was almost entirely serotype specific, while the Shigella-specific antibody response was largely but not entirely serotype specific. The absence of significant cross-serotype protection in the presence of a modest increase in the concentration of antibody that recognizes the heterologous serotype suggests that either (i) antibody to antigens other than LPS is relatively nonprotective, which is consistent with LPS being the predominant protective antigen, or (ii) the magnitude of the increase in antibody concentration is important and was insufficient among non-serotype-specific antibody. The recent analysis of protection following vaccination with an LPS conjugate vaccine in humans is consistent with LPS being a protective antigen (5).