Klebsiella pneumoniae is a Gram-negative, opportunistic pathogen that commonly causes nosocomial pneumonia, urinary tract infection, and septicemia. Our recent work utilizing a murine model of respiratory tract infection with classical K. pneumoniae demonstrated leukocyte aggregates in the lungs of mice at 28 days postinfection.

Infections with classical strains of the Gram-negative bacterium Klebsiella pneumoniae pose a significant clinical challenge due to rising antibiotic resistance. We previously established a lung inoculation plus challenge model using live, classical K. pneumoniae in order to study host protection. Here, we employ this model to dissect adaptive immune responses to this critical pathogen. First, we performed convalescent serum transfers from inoculated mice to naïve recipients and found that classical K. pneumoniae infection outcomes, unlike hypervirulent K. pneumoniae infection outcomes, were not improved. This suggests that circulating antibody responses alone are not sufficient to mediate protection against this classical strain. Hence, we evaluated the role of T cells in protection against classical K. pneumoniae reinfection and demonstrated that mice lacking T cells are unable to establish a protective response. However, mice individually deficient in either of the major T cell subsets, γδ or αβ (classical T cells), effectively mount a protective response, indicating either subset alone is sufficient to mediate protection. Sequestration of T cells in secondary lymphoid organs during the challenge infection did not ablate protection, indicating the circulating T cell pool is not required for the protective phenotype. Finally, we demonstrate that depletion of T cells during initial infection eliminates protection against challenge. Collectively, these experiments demonstrate the imperative contribution of T cells to protective immunity against classical K. pneumoniae and will guide further inquiries into host effector responses required to control this infection.

Klebsiella pneumoniae is a worrisome nosocomial pathogen given the widespread emergence of antibiotic resistant strains. Little is known about the host immune response to K. pneumoniae; however, it has recently been shown that aggregates of lymphoid cells can be found in murine lungs 4 weeks after surviving K. pneumoniae pulmonary infection. Others have characterized pulmonary collections of lymphoid cells in response to other pathogens and coined them induced bronchus-associated lymphoid tissue (iBALT). iBALT is believed to assist in protection against re-infection. In our nonlethal model of K. pneumoniae, mice that survive the primary infection and exhibit these structures are indeed protected from K. pneumoniae challenge. To further characterize the development and organization of these structures, pulmonary immune cell populations were analyzed post-infection by flow cytometry, histology, and immunofluorescence. As expected, pulmonary neutrophil populations increase over the first 10 days post-infection. T cells appear to rapidly expand between 7 and 10 days post-infection and both CD4+ and γδ T cell subsets make up the majority of this population. Immunofluorescent imaging of lymphoid aggregates demonstrate that CD4+ and γδ T cells are found within these structures at early time-points. Experiments using TCRβδ−/− mice lacking T cells reveals the absence of lymphoid aggregates 28 days post-infection, suggesting that T cells are essential in the initial formation and development of these structures.

Due to emerging antibiotic resistance, the nosocomial pathogen Klebsiella pneumoniae is an increasing public health threat. The extent to which survivors are protected against re-infection, or potential correlates of such protection, are poorly understood. We have explored the immunological mechanisms required for protection against K. pneumoniae re-infection using a nonlethal murine model of pulmonary K. pneumoniae challenge. Mice that survive K. pneumoniae TOP52 infection are protected from subsequent re-infection by an adaptive immune response. Genetically modified mice lacking mature lymphocytes or mature T cells were equally susceptible to primary and secondary infection, suggesting that T cells are required for protection. Intriguingly, neither CD4+ nor CD8+ T cells were absolutely required for either the establishment or execution of a protective memory response as depletion of CD4+ and/or CD8+ T cells during either primary or secondary infection did not impair protection against re-infection. However, we observe an expansion of γδ T cells following primary exposure to K. pneumoniae and a profound amplification of this population following secondary infection. Experiments using genetically modified mice deficient in γδ T cells or intact mice treated with γδ T cell depleting antibodies suggest that this T cell subpopulation is important for protection against re-infection. Thus, we hypothesize that K. pneumoniae exposure drives the development of a resident memory-like population of γδ T cells that are maintained in the lung and contribute to the efficient control of secondary K. pneumoniae infection.

Klebsiella pneumoniae (Kp) is a versatile pathogen capable of causing numerous infections that are becoming increasingly difficult to treat due to increasing antibiotic resistance. Kp strains are categorized into two distinct pathotypes: classical and hypervirulent, which exhibit critical differences in pathogenesis. Despite causing the majority of clinical cases, adaptive immunity resulting from classical Kp infections is poorly understood. Our laboratory has recently developed a model of adaptive immunity to live infection with classical Kp in which wild-type mice surviving primary infection are protected from morbidity and mortality upon rechallenge. Passive transfer of convalescent serum from surviving mice to naïve recipients does not confer protection against cKp infection, suggesting antibody responses alone are inadequate to protect against cKp. CD69+CD4+ T cells and γδ T cells expand dramatically in the lung following primary cKp infection and mice deficient in T cells are not protected from reinfection, indicating a requirement for T cells in the protective response. Further analysis of mice deficient in either classical αβ T cells or γδ T cells indicates that either T cell subset is capable of mediating protective immunity against Kp reinfection. Production of IL-17A by both CD4 and γδ T cells is enhanced during secondary infection. Together these data indicate that a natural course of infection with classical Kp elicits populations of classical T cells and γδ T cells that mediate protection against reinfection, likely through the rapid production of IL-17A. These results will ultimately inform rational design of vaccines and immunotherapies to prevent and treat Kp infection. Supported by AAI Careers in Immunology Fellowship