Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications.

Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics.

Update on Gender Equity in Immunology, 2001 to 2016.

In 2001, The American Association of Immunologists Committee on the Status of Women conducted a survey examining the percentage of women faculty members within immunology departments or women in immunology graduate programs across 27 institutions in the United States, comparing it to the percentage of women receiving a Ph.D. Here, we examine the representation of women across these same 27 immunology departments and programs to examine changes in gender equity over the last 15 years.

Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is the most common cause of bacterial, seafood-related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro- and anti-inflammatory cytokines by V. parahaemolyticus-infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL-1α, IL-6, TNF-α and IL-10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T-cell co-stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.

Intranasal prophylaxis with CpG oligodeoxynucleotide can protect against Yersinia pestis infection.

Immunomodulatory agents potentially represent a new class of broad-spectrum antimicrobials. Here, we demonstrate that prophylaxis with immunomodulatory cytosine-phosphate-guanidine (CpG) oligodeoxynucleotide (ODN), a toll-like receptor 9 (TLR9) agonist, confers protection against Yersinia pestis, the etiologic agent of plague. The data establish that intranasal administration of CpG ODN 1 day prior to lethal pulmonary exposure to Y. pestis strain KIM D27 significantly improves survival of C57BL/6 mice and reduces bacterial growth in hepatic tissue, despite paradoxically increasing bacterial growth in the lung. All of these CpG ODN-mediated impacts, including the increased pulmonary burden, are TLR9 dependent, as they are not observed in TLR9-deficient mice. The capacity of prophylactic intranasal CpG ODN to enhance survival does not require adaptive immunity, as it is evident in mice lacking B and/or T cells; however, the presence of T cells improves long-term survival. The prophylactic regimen also improves survival and reduces hepatic bacterial burden in mice challenged intraperitoneally with KIM D27, indicating that intranasal delivery of CpG ODN has systemic impacts. Indeed, intranasal prophylaxis with CpG ODN provides significant protection against subcutaneous challenge with Y. pestis strain CO92 even though it fails to protect mice from intranasal challenge with that fully virulent strain.

Fibrin facilitates both innate and T cell-mediated defense against Yersinia pestis.

The Gram-negative bacterium Yersinia pestis causes plague, a rapidly progressing and often fatal disease. The formation of fibrin at sites of Y. pestis infection supports innate host defense against plague, perhaps by providing a nondiffusible spatial cue that promotes the accumulation of inflammatory cells expressing fibrin-binding integrins. This report demonstrates that fibrin is an essential component of T cell-mediated defense against plague but can be dispensable for Ab-mediated defense. Genetic or pharmacologic depletion of fibrin abrogated innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild-type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin to withstand Y. pestis encounters and effectively clear bacteria.

Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus in seawater and oysters.

This study shows that naturally occurring Vibrio predatory bacteria (VPB) exert a major role in controlling pathogenic vibrios in seawater and shellfish. The growth and persistence of Vibrio parahaemolyticus and Vibrio vulnificus were assessed in natural seawater and in the Eastern oyster, Crassostrea virginica. The pathogens examined were V. vulnificus strain VV1003, V. parahaemolyticus O1:KUT (KUT stands for K untypeable), and V. parahaemolyticus O3:K6 and corresponding O3:K6 mutants deficient in the toxRS virulence regulatory gene or the rpoS alternative stress response sigma factor gene. Vibrios were selected for streptomycin resistance, which facilitated their enumeration. In natural seawater, oysters bioconcentrated each Vibrio strain for 24 h at 22°C; however, counts rapidly declined to near negligible levels by 72 h. In natural seawater with or without oysters, vibrios decreased more than 3 log units to near negligible levels within 72 h. Neither toxRS nor rpoS had a significant effect on Vibrio levels. In autoclaved seawater, V. parahaemolyticus O3:K6 counts increased 1,000-fold over 72 h. Failure of the vibrios to persist in natural seawater and oysters led to screening of the water samples for VPB on lawns of V. parahaemolyticus O3:K6 host cells. Many VPB, including Bdellovibrio and like organisms (BALOs; Bdellovibrio bacteriovorus and Bacteriovorax stolpii) and Micavibrio aeruginosavorus-like predators, were detected by plaque assay and electron microscopic analysis of plaque-purified isolates from Atlantic, Gulf Coast, and Hawaiian seawater. When V. parahaemolyticus O3:K6 was added to natural seawater containing trace amounts of VPB, Vibrio counts diminished 3 log units to nondetectable levels, while VPB increased 3 log units within 48 h. We propose a new paradigm that VPB are important modulators of pathogenic vibrios in seawater and oysters.

The Vibrio parahaemolyticus ToxRS regulator is required for stress tolerance and colonization in a novel orogastric streptomycin-induced adult murine model.

Vibrio parahaemolyticus, a marine bacterium, is the causative agent of gastroenteritis associated with the consumption of seafood. It contains a homologue of the toxRS operon that in V. cholerae is the key regulator of virulence gene expression. We examined a nonpolar mutation in toxRS to determine the role of these genes in V. parahaemolyticus RIMD2210633, an O3:K6 isolate, and showed that compared to the wild type, ΔtoxRS was significantly more sensitive to acid, bile salts, and sodium dodecyl sulfate stresses. We demonstrated that ToxRS is a positive regulator of ompU expression, and that the complementation of ΔtoxRS with ompU restores stress tolerance. Furthermore, we showed that ToxRS also regulates type III secretion system genes in chromosome I via the regulation of the leuO homologue VP0350. We examined the effect of ΔtoxRS in vivo using a new orogastric adult murine model of colonization. We demonstrated that streptomycin-treated adult C57BL/6 mice experienced prolonged intestinal colonization along the entire intestinal tract by the streptomycin-resistant V. parahaemolyticus. In contrast, no colonization occurred in non-streptomycin-treated mice. A competition assay between the ΔtoxRS and wild-type V. parahaemolyticus strains marked with the ß-galactosidase gene lacZ demonstrated that the ΔtoxRS strain was defective in colonization compared to the wild-type strain. This defect was rescued by ectopically expressing ompU. Thus, the defect in stress tolerance and colonization in ΔtoxRS is solely due to OmpU. To our knowledge, the orogastric adult murine model reported here is the first showing sustained intestinal colonization by V. parahaemolyticus.

B cell-deficient mice display markedly enhanced resistance to the intracellular bacterium Brucella abortus.

BACKGROUND: Brucella species are facultative intracellular bacteria that cause lifelong infections in humans and livestock. METHODS: Here we evaluated the contribution of B cells in control of murine brucellosis in the more susceptible BALB/c and the more resistant C57BL/6 mice by infecting B cell-deficient mice. RESULTS: Strikingly, in the absence of B cells in both C57BL/6 and BALB/c mice, 99% and 99.5% of the infection found in wild type mice was cleared, respectively. This augmented clearance was not reversed in either strain by passive transfer of immune serum. In C57BL/6 mice, the clearance of infection coincided with an increase in interferon γ (IFN-γ)-producing CD4 and CD8 T cells and a reduction in interleukin 10 (IL-10)-producing cells. In BALB/c mice, this clearance was IFN-γ-dependent, as B cell/IFN-γ dual knockout mice were unable to clear the infection, and was inversely related to the levels of transforming growth factor ß (TGF-ß). Furthermore, B cells were found to produce TGF-ß and IL-10 during early stages of infection in BALB/c wild-type and C57BL/6 wild-type mice, respectively. CONCLUSIONS: Thus, we demonstrate that the establishment of the high plateau phase of infection is dependent on non-antibody-mediated B cell effector mechanisms, including B regulatory functions, during murine brucellosis.

Modulation of responses of Vibrio parahaemolyticus O3:K6 to pH and temperature stresses by growth at different salt concentrations.

Vibrio parahaemolyticus inhabits marine, brackish, and estuarine waters worldwide, where fluctuations in salinity pose a constant challenge to the osmotic stress response of the organism. Vibrio parahaemolyticus is a moderate halophile, having an absolute requirement for salt for survival, and is capable of growth at 1 to 9% NaCl. It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia. We determined whether growth in differing NaCl concentrations alters the susceptibility of V. parahaemolyticus O3:K6 to other environmental stresses. Vibrio parahaemolyticus was grown at a 1% or 3% NaCl concentration, and the growth and survival of the organism were examined under acid or temperature stress conditions. Growth of V. parahaemolyticus in 3% NaCl versus that in 1% NaCl increased survival under both inorganic (HCl) and organic (acetic acid) acid conditions. In addition, at 42 degrees C and -20 degrees C, 1% NaCl had a detrimental effect on growth. The expression of lysine decarboxylase (encoded by cadA), the organism's main acid stress response system, was induced by both NaCl and acid conditions. To begin to address the mechanism of regulation of the stress response, we constructed a knockout mutation in rpoS, which encodes the alternative stress sigma factor, and in toxRS, a two-component regulator common to many Vibrio species. Both mutant strains had significantly reduced survival under acid stress conditions. The effect of V. parahaemolyticus growth in 1% or 3% NaCl was examined using a cytotoxicity assay, and we found that V. parahaemolyticus grown in 1% NaCl was significantly more toxic than that grown in 3% NaCl.

D27-pLpxL, an avirulent strain of Yersinia pestis, primes T cells that protect against pneumonic plague.

Vaccinating with live, conditionally attenuated, pigmentation (Pgm)-deficient Yersinia pestis primes T cells that protect mice against pneumonic plague. However, Pgm-deficient strains are not considered safe for human use because they retain substantial virulence in animal models. Y. pestis strains engineered to express Escherichia coli LpxL are avirulent owing to constitutive production of lipopolysaccharide with increased Toll-like receptor 4-activating ability. We generated an LpxL-expressing Pgm-deficient strain (D27-pLpxL) and demonstrate here that this avirulent strain retains the capacity to prime protective T cells. Compared with unvaccinated controls, mice immunized intranasally with live D27-pLpxL exhibit a decreased bacterial burden and increased survival when challenged intranasally with virulent Y. pestis. T cells provide a substantial degree of this protection, as vaccine efficacy is maintained in B-cell-deficient muMT mice unless those animals are depleted of CD4 and CD8 T cells at the time of challenge. Upon challenge with Y. pestis, pulmonary T-cell numbers decline in naive mice, whereas immunized mice show increased numbers of CD44(high) CD43(high) effector T cells and T cells primed to produce tumor necrosis factor alpha and gamma interferon; neutralizing these cytokines at the time of challenge abrogates protection. Immunization does not prevent dissemination of Y. pestis from the lung but limits bacterial growth and pathology in visceral tissue, apparently by facilitating formation of granuloma-like structures. This study describes a new model for studying T-cell-mediated protection against pneumonic plague and demonstrates the capacity for live, highly attenuated, Y. pestis vaccine strains to prime protective memory T-cell responses safely.

Genomic islands are dynamic, ancient integrative elements in bacterial evolution.

Acquisition of genomic islands plays a central part in bacterial evolution as a mechanism of diversification and adaptation. Genomic islands are non-self-mobilizing integrative and excisive elements that encode diverse functional characteristics but all contain a recombination module comprised of an integrase, associated attachment sites and, in some cases, a recombination directionality factor. Here, we discuss how a group of related genomic islands are evolutionarily ancient elements unrelated to plasmids, phages, integrons and integrative conjugative elements. In addition, we explore the diversity of genomic islands and their insertion sites among Gram-negative bacteria and discuss why they integrate at a limited number of tRNA genes.

Antibodies and cytokines independently protect against pneumonic plague.

Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFalpha) and gamma-interferon (IFNgamma) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFalpha and IFNgamma confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFalpha and IFNgamma upon exposure to Y. pestis.

Molecular analysis of the emergence of pandemic Vibrio parahaemolyticus.

BACKGROUND: Vibrio parahaemolyticus is abundant in the aquatic environment particularly in warmer waters and is the leading cause of seafood borne gastroenteritis worldwide. Prior to 1995, numerous V. parahaemolyticus serogroups were associated with disease, however, in that year an O3:K6 serogroup emerged in Southeast Asia causing large outbreaks and rapid hospitalizations. This new highly virulent strain is now globally disseminated. RESULTS: We performed a four-way BLAST analysis on the genome sequence of V. parahaemolyticus RIMD2210633, an O3:K6 isolate from Japan recovered in 1996, versus the genomes of four published Vibrio species and constructed genome BLAST atlases. We identified 24 regions, gaps in the genome atlas, of greater than 10 kb that were unique to RIMD2210633. These 24 regions included an integron, f237 phage, 2 type III secretion systems (T3SS), a type VI secretion system (T6SS) and 7 Vibrio parahaemolyticus genomic islands (VPaI-1 to VPaI-7). Comparative genomic analysis of our fifth genome, V. parahaemolyticus AQ3810, an O3:K6 isolate recovered in 1983, identified four regions unique to each V. parahaemolyticus strain. Interestingly, AQ3810 did not encode 8 of the 24 regions unique to RMID, including a T6SS, which suggests an additional virulence mechanism in RIMD2210633. The distribution of only the VPaI regions was highly variable among a collection of 42 isolates and phylogenetic analysis of these isolates show that these regions are confined to a pathogenic clade. CONCLUSION: Our data show that there is considerable genomic flux in this species and that the new highly virulent clone arose from an O3:K6 isolate that acquired at least seven novel regions, which included both a T3SS and a T6SS.

Gamma interferon suppresses erythropoiesis via interleukin-15.

Impaired erythropoiesis causes anemia during genetic disorders, chronic disease, and infection. In studies of the underlying mechanisms researchers have increasingly focused on gamma interferon (IFN-gamma). Here, we identified a previously unrecognized role for interleukin-15 (IL-15) in red blood cell homeostasis and demonstrated that IFN-gamma and signal transducer and activator of transcription protein 1-dependent pathways up-regulate expression of IL-15 in vivo. These findings identified new therapeutic targets for anemia.

Brucella abortus bacA mutant induces greater pro-inflammatory cytokines than the wild-type parent strain.

The inner-membrane protein BacA affects Brucella LPS structure. A bacA deletion mutant of Brucella abortus, known as KL7 (bacA(mut)-KL7), is attenuated in BALB/c mice and protects against challenge. Thus, bacA mutation was a candidate for incorporation into live attenuated vaccines. We assessed bacA(mut)-KL7 in 2 additional mouse strains: the more resistant C57BL/6 that produces interferon-gamma throughout the infection and the highly susceptible interferon-gamma-deficient C57BL/6 in which brucellae exhibit continual exponential growth. While it was hypothesized that bacA(mut)-KL7 would exhibit even greater attenuation relative to its parent strain B. abortus 2308 in C57BL/6 mice than it did in BALB/c mice, this was not the case. Moreover, it was more pathogenic in C57BL/6 interferon-gamma-deficient mice than 2308 causing abscesses and wasting even though the splenic loads of bacA(mut)-KL7 were significantly lower. These 2 observations were correlated, respectively, with an ability of IFNgamma-activated macrophages to equivalently control strains 2308 and bacA(mut)-KL7 and the ability of bacA(mut)-KL7 organism and its LPS to induce greater amounts of pro-inflammatory cytokines than 2308. We conclude that attenuation properties of bacA mutation are dependent upon the nature of the host but more importantly that bacterial gene deletion can result in increased host pathology without an increase in bacterial load, crucial considerations for vaccine design.

Gamma interferon, tumor necrosis factor alpha, and nitric oxide synthase 2, key elements of cellular immunity, perform critical protective functions during humoral defense against lethal pulmonary Yersinia pestis infection.

Pulmonary infection by Yersinia pestis causes pneumonic plague, a rapidly progressing and often fatal disease. To aid the development of safe and effective pneumonic plague vaccines, we are deciphering mechanisms used by the immune system to protect against lethal pulmonary Y. pestis infection. In murine pneumonic plague models, passive transfer of convalescent-phase sera confers protection, as does active vaccination with live Y. pestis. Here, we demonstrate that protection by either protocol relies upon both gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) cytokines classically associated with type 1 cellular immunity. In both protocols, abrogating IFN-gamma or TNF-alpha activity significantly decreases survival and increases the bacterial burden in pulmonary, splenic, and hepatic tissues. Neutralization of either cytokine also counteracts challenge-induced, vaccination-dependent upregulation of nitric oxide synthase 2 (NOS2). Moreover, genetic depletion of NOS2 suppresses protection conferred by serotherapy. We conclude that IFN-gamma, TNF-alpha, and NOS2, key elements of cellular immunity, perform critical protective functions during humoral defense against lethal pulmonary Y. pestis challenge. These observations strongly suggest that plague vaccines should strive to maximally prime both cellular and humoral immunity.

Tumor necrosis factor alpha and gamma interferon, but not hemorrhage or pathogen burden, dictate levels of protective fibrin deposition during infection.

While coagulation often causes pathology during infectious disease, we recently demonstrated that fibrin, a product of the coagulation pathway, performs a critical protective function during acute toxoplasmosis (L. L. Johnson, K. N. Berggren, F. M. Szaba, W. Chen, and S. T. Smiley, J. Exp. Med. 197:801-806, 2003). Here, we investigate the mechanisms regulating the formation of this protective fibrin. Through comparisons of Toxoplasma-infected wild-type and cytokine-deficient mice we dissociate, for the first time, the relative fibrin-regulating capacities of pathogen products, host cytokines, and infection-stimulated hemorrhage. Remarkably, neither the pathogen burden nor hemorrhage is a primary regulator of fibrin levels. Rather, two type 1 cytokines exert dominant and counterregulatory roles: tumor necrosis factor alpha (TNF-alpha), acting via the type 1 TNF-alpha receptor, promotes fibrin deposition, while gamma interferon (IFN-gamma), acting via STAT1 and IFN-gamma receptors expressed on radioresistant cells, suppresses fibrin deposition. These findings have important clinical implications, as they establish that cytokines known to regulate pathological coagulation also dictate levels of protective fibrin deposition. We present a novel model depicting mechanisms by which the immune system can destroy infected tissue while independently restraining hemorrhage and promoting tissue repair through the deliberate deposition of protective fibrin.

Cell-mediated protection against pulmonary Yersinia pestis infection.

Pulmonary infection with the bacterium Yersinia pestis causes pneumonic plague, an often-fatal disease for which no vaccine is presently available. Antibody-mediated humoral immunity can protect mice against pulmonary Y. pestis infection, an experimental model of pneumonic plague. Little is known about the protective efficacy of cellular immunity. We investigated the cellular immune response to Y. pestis in B-cell-deficient microMT mice, which lack the capacity to generate antibody responses. To effectively prime pulmonary cellular immunity, we intranasally vaccinated microMT mice with live replicating Y. pestis. Vaccination dramatically increased survival of microMT mice challenged intranasally with a lethal Y. pestis dose and significantly reduced bacterial growth in pulmonary, splenic, and hepatic tissues. Vaccination also increased numbers of pulmonary T cells, and administration of T-cell-depleting monoclonal antibodies at the time of challenge abrogated vaccine-induced survival. Moreover, the transfer of Y. pestis-primed T cells to naive microMT mice protected against lethal intranasal challenge. These findings establish that vaccine-primed cellular immunity can protect against pulmonary Y. pestis infection and suggest that vaccines promoting both humoral and cellular immunity will most effectively combat pneumonic plague.

Infection-stimulated fibrin deposition controls hemorrhage and limits hepatic bacterial growth during listeriosis.

Bacterial infections are major causes of human mortality. The activation of coagulation pathways leading to the deposition of insoluble fibrin frequently accompanies bacterial infection, and much attention has focused upon the pathological attributes of infection-stimulated fibrin deposition. Nevertheless, here we present conclusive evidence that infection-stimulated fibrin deposition can perform critical protective functions during bacterial infection. Specifically, we demonstrate that coagulation-impaired fibrin(ogen)-deficient mice, in comparison with genetically matched control mice, display increased mortality upon peritoneal infection with the gram-positive facultative intracellular bacterium Listeria monocytogenes. To distinguish effects of fibrinogen from those of fibrin, we treat wild-type mice with warfarin, an anticoagulant that suppresses fibrin formation without impacting fibrinogen levels. Warfarin treatment exacerbates listeriosis, suggesting that fibrin is the key mediator of protection. With regard to the underlying protective mechanisms, we demonstrate that fibrin(ogen) suppresses anemia, reduces hemorrhagic pathology, and limits bacterial growth during listeriosis. Despite confirming a prior report that fibrin(ogen) promotes the peritoneal clearance of the extracellular bacterium Staphylococcal aureus, we demonstrate that fibrin(ogen) plays little role in controlling peritoneal numbers of L. monocytogenes bacteria or the dissemination of L. monocytogenes bacteria from the peritoneal cavity. Rather, fibrin(ogen) primarily limits the growth of these intracellular bacteria within hepatic tissue. While the pathological potential of excessive infection-stimulated fibrin deposition is well appreciated, our findings reveal that fibrin can function protectively, via multiple mechanisms, during bacterial infection.

Yersinia pestis V protein epitopes recognized by CD4 T cells.

Pneumonic plague, an often-fatal disease for which no vaccine is presently available, results from pulmonary infection by the bacterium Yersinia pestis. The Y. pestis V protein is a promising vaccine candidate, as V protein immunizations confer to mice significant protection against aerosolized Y. pestis. CD4 T cells play central roles during vaccine-primed immune responses, but their functional contributions to Y. pestis vaccines have yet to be evaluated and optimized. Toward that end, we report here the identification of three distinct epitopes within the Y. pestis V protein that activate CD4 T cells in C57BL/6 mice. To our knowledge, these are the first identified CD4 T-cell epitopes in any Y. pestis protein. The epitopes are restricted by the I-A(b) class II major histocompatibility complex molecule and are fully conserved between Y. pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. Immunizing mice with a V protein-containing vaccine or with short peptides containing the identified epitopes primes antigen-specific production of interleukin 2 and gamma interferon by CD4 T cells upon their restimulation in vitro. Consistent with prior studies documenting protective roles for CD4 T cells during Y. enterocolitica infection, vaccinating mice with a 16-amino-acid peptide encoding one of the epitopes suffices to protect against an otherwise lethal Y. enterocolitica challenge. The identification of these epitopes will permit quantitative assessments of V-specific CD4 T cells, thereby enabling researchers to evaluate and optimize the contribution of these cells to vaccine-primed protection against pneumonic plague.