Pulmonary Vaccination Research Articles

Generation of effector memory T cell-based mucosal and systemic immunity with pulmonary nanoparticle vaccination.

Many pathogens infiltrate the body and initiate infection via mucosal surfaces. Hence, eliciting cellular immune responses at mucosal portals of entry is of great interest for vaccine development against mucosal pathogens. We describe a pulmonary vaccination strategy combining Toll-like receptor (TLR) agonists with antigen-carrying lipid nanocapsules [interbilayer-crosslinked multilamellar vesicles (ICMVs)], which elicit high-frequency, long-lived, antigen-specific effector memory T cell responses at multiple mucosal sites. Pulmonary immunization using protein- or peptide-loaded ICMVs combined with two TLR agonists, polyinosinic-polycytidylic acid (polyI:C) and monophosphoryl lipid A, was safe and well tolerated in mice, and led to increased antigen transport to draining lymph nodes compared to equivalent subcutaneous vaccination. This response was mediated by the vast number of antigen-presenting cells (APCs) in the lungs. Nanocapsules primed 13-fold more T cells than did equivalent soluble vaccines, elicited increased expression of mucosal homing integrin α₄β₇⁺, and generated long-lived T cells in both the lungs and distal (for example, vaginal) mucosa strongly biased toward an effector memory (T(EM)) phenotype. These T(EM) responses were highly protective in both therapeutic tumor and prophylactic viral vaccine settings. Together, these data suggest that targeting cross-presentation-promoting particulate vaccines to the APC-rich pulmonary mucosa can promote robust T cell responses for protection of mucosal surfaces.

TLR2-targeted secreted proteins from Mycobacterium tuberculosis are protective as powdered pulmonary vaccines

Despite considerable research efforts towards effective treatments, tuberculosis (TB) remains a staggering burden on global health. Suitably formulated sub-unit vaccines offer potential as safe and effective generators of protective immunity. The Mycobacterium tuberculosis antigens, cutinase-like proteins (Culp) 1 and 6 and MPT83, were conjugated directly to the novel adjuvant Lipokel (Lipotek Pty Ltd), a TLR2 ligand that delivers antigen to immune cells in a self-adjuvanting context. Protein-Lipokel complexes were formulated as dry powders for pulmonary delivery directly to the lungs of mice by intra-tracheal insufflation, leading to recruitment of neutrophils and antigen presenting cell populations to the lungs at 72h, that persisted at 7 days post immunisation. Significant increases in the frequency of activated dendritic cells were observed in the mediastinal lymph node (MLN) at 1 and 4 weeks after homologous boosting with protein-Lipokel vaccine. This was associated with the increased recruitment of effector CD4+ and CD8+ T-lymphocytes to the MLN and systemic antigen-specific, IFN-γ producing T-lymphocyte and IgG responses. Notably, pulmonary immunisation with either Culp1-6-Lipokel or MPT83-Lipokel powder vaccines generated protective responses in the lungs against aerosol M. tuberculosis challenge. The successful combination of TLR2-targeting and dry powder vaccine formulation, together with important practical benefits, offers potential for pulmonary vaccination against M. tuberculosis.

Engineering of an Inhalable DDA/TDB Liposomal Adjuvant: A Quality-by-Design Approach Towards Optimization of the Spray Drying Process

The purpose of this study was to identify and optimize spray drying parameters of importance for the design of an inhalable powder formulation of a cationic liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6'-dibehenate (TDB). A quality by design (QbD) approach was applied to identify and link critical process parameters (CPPs) of the spray drying process to critical quality attributes (CQAs) using risk assessment and design of experiments (DoE), followed by identification of an optimal operating space (OOS). A central composite face-centered design was carried out followed by multiple linear regression analysis. Four CQAs were identified; the mass median aerodynamic diameter (MMAD), the liposome stability (size) during processing, the moisture content and the yield. Five CPPs (drying airflow, feed flow rate, feedstock concentration, atomizing airflow and outlet temperature) were identified and tested in a systematic way. The MMAD and the yield were successfully modeled. For the liposome size stability, the ratio between the size after and before spray drying was modeled successfully. The model for the residual moisture content was poor, although, the moisture content was below 3% in the entire design space. Finally, the OOS was drafted from the constructed models for the spray drying of trehalose stabilized DDA/TDB liposomes. The QbD approach for the spray drying process should include a careful consideration of the quality target product profile. This approach implementing risk assessment and DoE was successfully applied to optimize the spray drying of an inhalable DDA/TDB liposomal adjuvant designed for pulmonary vaccination.

Mucosal and Systemic Immune Responses to Mycobacterium tuberculosis Antigen 85A following Its Co-Delivery with CpG, MPLA or LTB to the Lungs in Mice

Pulmonary vaccination is a promising route for immunization against tuberculosis because the lung is the natural site of infection with Mycobacterium tuberculosis. Yet, adjuvants with a suitable safety profile need to be found to enhance mucosal immunity to recombinant antigens. The aim of this study was to evaluate the immunogenicity, the safety and the protective efficacy of a subunit vaccine composed of the immunodominant mycolyl-transferase antigen 85A (Ag85A) and one of three powerful mucosal adjuvants: the oligodeoxynucleotide containing unmethylated cytosine-phosphate-guanine motifs (CpG), the monophosphoryl lipid A of Salmonella minnesota (MPLA) or the B subunit of heat-labile enterotoxin of Escherichia coli (LTB). BALB/c mice were vaccinated in the deep lungs. Our results showed that lung administration of these adjuvants could specifically induce different types of T cell immunity. Both CpG and MPLA induced a Th-1 type immune response with significant antigen-specific IFN-γ production by spleen mononuclear cells in vitro and a tendency of increased IFN-γ in the lungs. Moreover, MPLA triggered a Th-17 response reflected by high IL-17A levels in the spleen and lungs. By contrast, LTB promoted a Th-2 biased immune response, with a production of IL-5 but not IFN-γ by spleen mononuclear cells in vitro. CpG did not induce inflammation in the lungs while LTB and MPLA showed a transient inflammation including a neutrophil influx one day after pulmonary administration. Pulmonary vaccination with Ag85A without or with MPLA or LTB tended to decrease bacterial counts in the spleen and lungs following a virulent challenge with M. tuberculosis H37Rv. In conclusion, CpG and MPLA were found to be potential adjuvants for pulmonary vaccination against tuberculosis, providing Th-1 and Th-17 immune responses and a good safety profile.

Pulmonary vaccination against influenza virus induces a protective CD8 T cell response, which is regulated by APCs within the lungs (P6154)

Abstract Recently there has been a renewed interest in the development of novel pulmonary influenza A virus (IAV) vaccines. CD8 T cells play an important role in clearance of IAV, and our prior studies demonstrate a key role for dendritic cells (DCs) in activation of IAV-specific CD8 T cells in the lymph nodes and maintenance in the lungs during IAV infections. Herein, we determined the role of pulmonary vaccine-induced CD8 T cells in protection from IAV challenge and the requirements for generation and maintenance of such CD8 T cell immunity. Our data demonstrates vaccination with IAV virus-like particles (VLPs) containing IAV HA and M1 leads to a significant increase in HA533-specific CD8 T cells. These VLP-specific CD8 T cells mediate protection of mice challenged with lethal doses of either homo- or heterosubtypic IAVs containing the HA533 epitope. Our preliminary results suggest interactions between DCs and effector T cells within the lungs are required following vaccination for maximal pulmonary VLP-specific CD8 T cell responses. Currently, we are investigating the contribution of inflammation and the APC subsets required in generation, boosting, and maintenance of vaccine-specific CD8 T cells during pulmonary vaccination. Collectively, our results highlight an important role for CD8 T cell mediated protection after i.n. vaccination and the seminal role pulmonary APCs play in activating and maintaining local vaccine-specific CD8 T cell responses against IAV in the lungs.

Stabilized lipid nanocapsules eliciting potent mucosal immune response (P4509)

Abstract We investigated the use of a novel lipid nanocapsule, Interbilayer-Crosslinked Multilamellar Vesicles (ICMV), for mucosal vaccine delivery. ICMV co-encapsulated with MPLA and/or polyI:C as adjuvants, was used to vaccinate mice using model antigen and SIV-gag epitopes. When delivered via the lungs, we found that ~60-fold more antigen transported to the draining lymph nodes than equivalent subcutaneous vaccination, mediated by the vast number of antigen presenting cells in the lungs. Potent CD8 T-cell response was elicited by ICMV pulmonary vaccine and established a substantially greater memory population than a soluble vaccine, with 5.2-fold and 4.8-fold higher frequencies of antigen-specific cells in lungs and spleen, at 11 weeks post-priming. T-cells primed by ICMV pulmonary vaccination were also found to be strongly biased toward an effector memory phenotype, and a 5-7-fold higher number of effector memory cells were found in both the local and distal mucosa compared to soluble vaccines. Protection against a viral challenge was observed in animals given ICMV pulmonary vaccine while animals that received subcutaneous ICMV or pulmonary soluble vaccines showed steady weight loss that led to 100% mortality by day 5 post challenge. These particles can be prepared using aqueous solution processing techniques currently used for production of commercial liposomal products. They can also be freeze-dried for long term storage and have shown to retain immunogenicity after 1 month.

Differing Effects of Interleukin-10 on Cutaneous and Pulmonary Francisella tularensis Live Vaccine Strain Infection

We investigated the role of interleukin-10 (IL-10) in cutaneous and pulmonary infection with Francisella tularensis. We found that after intradermal challenge of mice with the live vaccine strain (LVS) of F. tularensis, splenic IL-10 levels increased rapidly and reached a peak 5 days after infection. However, IL-10 expression after infection was detrimental, since IL-10(-/-) mice showed increased bacterial clearance and were resistant to an infectious dose (>10(6) CFU/mouse) that was uniformly lethal for IL-10(+/+) mice. Furthermore, IL-10(+/+) mice treated with neutralizing anti-IL-10R monoclonal antibody were able to survive lethal cutaneous LVS challenge. The presence of IL-10 appeared to restrain the expression of IL-17, since high levels of splenic IL-17 were observed after intradermal LVS infection only in IL-10(-/-) mice. Furthermore, treatment with neutralizing anti-IL-17R antibody ablated the enhanced survival observed in IL-10(-/-) mice. However, neutralization of IL-10 activity in IL-17R(-/-) mice failed to provide protection. Thus, IL-10 suppresses a protective IL-17 response that is necessary for resistance to cutaneous LVS infection. Surprisingly, however, IL-10(-/-) mice were significantly more susceptible to pulmonary infection with LVS. Finally, although IL-10 is a critical and novel regulator of immunity to F. tularensis LVS infection, its effects were masked during infection with the highly virulent SchuS4 strain. Taken together, these findings suggest that differentially regulating expression of the IL-10 pathway in various tissues could ultimately have prophylactic and therapeutic benefits for protection against tularemia.

Targeting the deep lungs, Poloxamer 407 and a CpG oligonucleotide optimize immune responses to Mycobacterium tuberculosis antigen 85A following pulmonary delivery

The current Bacille Calmette–Guérin vaccine provides variable protection against tuberculosis and new vaccination approaches are urgently needed. Pulmonary vaccination could be the best way to induce a protective immunity against Mycobacterium tuberculosis as it targets its natural site of infection. The aim of this study was to investigate the potential of Poloxamer 407 (P407) combined with a CpG oligonucleotide (CpG) to enhance immune responses to M. tuberculosis antigen 85A (Ag85A) following pulmonary delivery in BALB/c mice. An additional goal of this study was to localize the optimal delivery site of Ag85A within the lungs for generating the most intense immunity. We also investigated the capacity of P407 to prolong the residence time of the antigen within the lungs and we studied the safety of the adjuvants following pulmonary delivery. Targeting the antigen to the deep lungs produced more intense specific immune responses than targeting it to the upper airways. P407 and CpG further increased humoral immune responses and splenocyte proliferation in vitro. CpG strongly increased the Th-1 immune response with high IgG2a/IgG1 ratio, high IFN-γ and TNF-α productions by spleen mononuclear cells in vitro. P407 tended to induce a Th-2 response, as indicated by the slight decrease in IgG2a/IgG1 ratio and the slight increase in IL-5 levels. The combination of P407 and CpG produced the highest Th-1 and Th-17 responses by generating IFN-γ, TNF-α, IL-2, and IL-17A cytokines. Targeting the antigen to the deep lungs and the presence of P407 increased the residence time of the antigen within the lungs. This might explain the enhancement of immune responses induced by these factors. CpG did not induce inflammation in the lungs while P407 produced a reversible alteration of the alveolo-capillary barrier. Adding CpG to P407 did not further increase this alteration of the alveolo-capillary barrier. In conclusion, delivery of Ag85A formulated in a combination of P407 and CpG to the deep lungs induced strong immune responses, with a polyfunctional T cells phenotype.

Which chronic obstructive pulmonary disease care recommendations have low implementation and why? A pilot study.

BackgroundClinical care components for people with COPD are recommended in guidelines if high-level evidence exists. However, there are gaps in their implementation, and factors which act as barriers or facilitators to their uptake are not well described. The aim of this pilot study was to explore implementation of key high-evidence COPD guideline recommendations in patients admitted to hospital with a disease exacerbation, to inform the development of a larger observational study.MethodsThis study recruited consecutive COPD patients admitted to a tertiary hospital. Patient demographic, disease and admission characteristics were recorded. Information about implementation of target guideline recommendations (smoking cessation, pulmonary rehabilitation referral, influenza vaccination, medication use and long-term oxygen use if hypoxaemic) was gained from medical records and patient interviews. Interviews with hospital-based doctors examined their perspectives on recommendation implementation.ResultsFifteen patients (aged 76(9) years, FEV1%pred 58(15), mean(SD)) and nine doctors participated. Referral to pulmonary rehabilitation (5/15 patients) was underutilised by comparison with other high-evidence recommendations. Low awareness of pulmonary rehabilitation was a key barrier for patients and doctors. Other barriers for patients were access difficulties, low perceived health benefits, and co-morbidities. Doctors reported they tended to refer patients with severe disease and frequent hospital attendance, a finding supported by the quantitative data.ConclusionsThis study provides justification for a larger observational study to test the hypothesis that pulmonary rehabilitation referral is low in suitable COPD patients, and closer investigation of the reasons for this evidence-practice gap.

Nanocarriers Targeting Dendritic Cells for Pulmonary Vaccine Delivery

Pulmonary vaccine delivery has gained significant attention as an alternate route for vaccination without the use of needles. Immunization through the pulmonary route induces both mucosal and systemic immunity, and the delivery of antigens in a dry powder state can overcome some challenges such as cold-chain and availability of medical personnel compared to traditional liquid-based vaccines. Antigens formulated as nanoparticles (NPs) reach the respiratory airways of the lungs providing greater chance of uptake by relevant immune cells. In addition, effective targeting of antigens to the most 'professional' antigen presenting cells (APCs), the dendritic cells (DCs) yields an enhanced immune response and the use of an adjuvant further augments the generated immune response thus requiring less antigen/dosage to achieve vaccination. This review discusses the pulmonary delivery of vaccines, methods of preparing NPs for antigen delivery and targeting, the importance of targeting DCs and different techniques involved in formulating dry powders suitable for inhalation.

Pulmonary Vaccine Delivery: A Realistic Approach?

Pulmonary vaccine delivery has gained increasing attention during the last decade because this vaccination method combines potential advantages such as the fact that it omits the use of needles and may elicit immunity at the port of entry for many pathogens. In this review the current status of pulmonary vaccination, the potential advantages of pulmonary vaccine delivery, the hurdles to overcome in the future, and the overall perspectives of this vaccination strategy are described.

Eliciting mucosal and systemic CD8+ T-cell responses via pulmonary vaccination with synthetic nanoparticles (113.21)

Abstract Mucosal vaccines offer needle-free administration and can stimulate immune responses systemically and at mucosal sites. “Vectorless” vaccines based on antigens packaged in synthetic nanoparticles (NPs) offer potential advantages in manufacturing, enhanced safety, and lack of anti-vector immune responses. To this end, we tested the ability of lipid NPs encapsulating ovalbumin or SIV-gag epitopes with Toll-like receptor agonists to stimulate mucosal immunity following pulmonary vaccination in mice. CD11c+B220+MHC-II+CD205+ DCs and CD11c+MHC-IIloF4/80+CD11b+ macrophages were the major cell types acquiring antigen in the lungs. Following prime and boost, a dramatic expansion of endogenous antigen-specific CD8+ T-cells to ~15% of CD8+ cells in blood was observed-twice the level seen with soluble antigen vaccines and 3 times the level achieved by s.c. immunization. Antigen-specific T-cell expansion was detected in the local lung tissue (~60% of CD8 cells), as well as in the gut and vaginal tract (~40% CD8 cells). These T-cells produced effector cytokines, and lasting CD127hi KLRG1lo effector memory cells were detected at 11wks after priming in both systemic and mucosal compartments. ELISA analysis of serum, fecal pellets and vaginal washes demonstrated that humoral responses were also stimulated. This data provides evidence that mucosal delivery of synthetic NPs can safely and effectively stimulate CD8+ T cells at sites relevant for protection against mucosal pathogens such as HIV.

Pulmonary Vaccination as a Novel Treatment for Lung Fibrosis

Pulmonary fibrosis is an untreatable, uniformly fatal disease of unclear etiology that is the result of unremitting chronic inflammation. Recent studies have implicated bone marrow derived fibrocytes and M2 macrophages as playing key roles in propagating fibrosis. While the disease process is characterized by the accumulation of lymphocytes in the lung parenchyma and alveolar space, their role remains unclear. In this report we definitively demonstrate the ability of T cells to regulate lung inflammation leading to fibrosis. Specifically we demonstrate the ability of intranasal vaccinia vaccination to inhibit M2 macrophage generation and fibrocyte recruitment and hence the accumulation of collagen and death due to pulmonary failure. Mechanistically, we demonstrate the ability of lung Th1 cells to prevent fibrosis as vaccinia failed to prevent disease in Rag−/− mice or in mice in which the T cells lacked IFN-γ. Furthermore, vaccination 3 months prior to the initiation of fibrosis was able to mitigate the disease. Our findings clearly demonstrate the role of T cells in regulating pulmonary fibrosis as well as suggest that vaccinia-induced immunotherapy in the lung may prove to be a novel treatment approach to this otherwise fatal disease.

The Skeleton Key for Intracellular Pathogens: Nanoparticles and Pulmonary Vaccination

A nanoparticle-linked antigen induces cytotoxic T cell responses and protective immunity against influenza in mice.

Long-Term Antibody and Immune Memory Response Induced by Pulmonary Delivery of the Influenza Iscomatrix Vaccine

Pulmonary delivery of an influenza Iscomatrix adjuvant vaccine induces a strong systemic and mucosal antibody response. Since an influenza vaccine needs to induce immunological memory that lasts at least 1 year for utility in humans, we examined the longevity of the immune response induced by such a pulmonary vaccination, with and without antigen challenge. Sheep were vaccinated in the deep lung with an influenza Iscomatrix vaccine, and serum and lung antibody levels were quantified for up to 1 year. The immune memory response to these vaccinations was determined following antigen challenge via lung delivery of influenza antigen at 6 months and 1 year postvaccination. Pulmonary vaccination of sheep with the influenza Iscomatrix vaccine induced antigen-specific antibodies in both sera and lungs that were detectable until 6 months postimmunization. Importantly, a memory recall response following antigenic challenge was detected at 12 months post-lung vaccination, including the induction of functional antibodies with hemagglutination inhibition activity. Pulmonary delivery of an influenza Iscomatrix vaccine induces a long-lived influenza virus-specific antibody and memory response of suitable length for annual vaccination against influenza.

Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Immunization of heat-killed Klebsiella pneumoniae promotes strong mucosal Th17 responses and protects mice from live infection (59.4)

Abstract Bacterial pneumonia is a leading cause of mortality and one major pathogen associated with this disease is Klebsiella pneumoniae (KP). Thus, a vaccine against KP is in great need especially for immunocompromised patients. Here we show that intranasal immunization of mice with heat-killed KP strongly induces IL-17 production in the lung. Using an Il17f reporter mouse, we were able to sort IL-17F positive cells after immunization by FACS. Further analysis reveals that these cells are conventional CD4+TCR-beta+ Th17 cells and they express Il17a, Il17f, Il22, Rora and Rorc. These Th17 cells are antigen specific as they proliferate and produce IL-17 upon re-stimulation with heat-killed KP but not heat-killed Escherichia coli or Staphylococcus aureus. These responses ultimately lead to the protection of immunized mice from live KP infection. This immunization strategy also confers protection in B cell-deficient (mu-MT) mice, demonstrating the vaccination can be antibody independent. We also show that other mucosal routes of vaccination such as intragastric immunization also reduces bacterial burden during pulmonary infection, suggesting that Th17 cells can be primed or recruited from other mucosal sites distal to the lung and oral vaccine can be effective in pulmonary infection. These data suggest that the Th17 pathway can be a target of pulmonary vaccine development through intranasal or intragastric immunization.

Opportunities and challenges of the pulmonary route for vaccination

Introduction: The respiratory tract is an attractive target for the delivery of vaccine antigens. Potential advantages of drug delivery by means of the pulmonary route include accessibility, non-invasiveness, ease of administration, and the possibility to reach an elaborate mucosal network of antigen-presenting cells.Areas covered: This review discusses current pulmonary vaccination strategies and their advantages and disadvantages.Expert opinion: To improve efficiency of vaccination and develop new strategies, a well-founded knowledge about composition and characterization of antigen-presenting cell populations throughout the respiratory tract is essential. In particular, respiratory tract dendritic cells, as key antigen-presenting cells in the lung, constitute an ideal target for vaccine delivery. Furthermore, particle size is a key factor when designing new inhalable vaccines, as size determines not only deposition in different respiratory tract compartments, but also how an antigen and its carrier will interact with lung tissue components and immune cells. An increased knowledge of different respiratory tract antigen-presenting cell populations and their interactions with other components of the immune system will enable new targeting strategies to improve the efficacy of pulmonary vaccination.

New developments in dry powder pulmonary vaccine delivery

Pulmonary immunization has gained increased recognition as a means of triggering both a mucosal and systemic immune response without the use of needles. The appropriate formulation of antigens in a dry, solid state can result in improved stability, thereby removing cold-chain storage complications associated with conventional liquid-based vaccines. The particulate nature of dry powder vaccines could also induce a better immune response. This review describes our current understanding of pulmonary immunization, including possible barriers facing the development of pulmonary vaccines, and discusses recent advances in spray-drying technologies applicable to the production of dry powder formulations for pulmonary vaccine delivery.

Lung CD4−CD8− Double-Negative T Cells Are Prominent Producers of IL-17A and IFN-γ during Primary Respiratory Murine Infection with Francisella tularensis Live Vaccine Strain

For several intracellular infections, pulmonary vaccination provides measurably better protection against pulmonary challenge. The unique factors that contribute to pulmonary immune responses are not well characterized. In this study, we show that CD4(-)CD8(-) double negative (DN) T cells are a major responding T cell subset in the lungs of mice during pulmonary Francisella tularensis live vaccine strain (LVS) infection. DN T cells were a minor (<2%) subset in spleens and lungs of mice during sublethal intradermal infection with LVS. In contrast, they were a major responding T cell subset in lungs during pulmonary LVS infection, producing large quantities of IFN-gamma and IL-17A. The numbers of IL-17A(+) DN T cells in the lungs exceeded that of CD4(+) and CD8(+) T cells on day 7 postinfection; by day 14 postinfection, all three IL-17A-producing T cell subsets were present in equivalent numbers. CD4(+), CD8(+), and DN T cell production of IL-17A was not observed in the spleens of pulmonary-infected mice or the lungs and spleens of intradermally infected mice. Correspondingly, IL-17A knockout mice were more susceptible to respiratory than intradermal LVS infection, with delayed clearance 1-3 wk postinfection. Finally, in vitro treatment of LVS-infected macrophages and alveolar type II epithelial cells with IFN-gamma and IL-17A affected significantly greater LVS growth control than treatment with either cytokine alone. The data presented in this study demonstrate that DN cells contribute to production of IL-17A and IFN-gamma in the lungs during inhalational Francisella infection and that these cytokines additively activate host cells to control LVS intracellular growth.