Comparative analysis of T lymphocytes recovered from the lungs of mice genetically susceptible, resistant, and hyperresistant to Mycobacterium tuberculosis-triggered disease.

Genetic control of susceptibility to tuberculosis (TB) is being intensively studied, and immune responses to mycobacteria are considerably well characterized. However, it remains largely unknown which parameters of response distinguish resistant and susceptible TB phenotypes. Mice of I/St and A/Sn inbred strains and (A/Sn x I/St)F(1) hybrids were previously categorized as, respectively, susceptible, resistant, and hyperresistant to Mycobacterium tuberculosis-triggered disease. In the present work we compared parameters of lung T cell activation and response following M. tuberculosis challenge. In all mice, the disease progression was accompanied by a marked accumulation in the lungs of activated CD4(+) (CD44(high)/CD45RB(low)) and CD8(+) (CD44(high)/CD45RB(+)) T cells capable of secreting IFN-gamma and of activating macrophages for NO production and mycobacterial growth inhibition. However, significantly more CD8(+) T cells were accumulated in the lungs of resistant A/Sn and F(1) compared with I/St mice. About 80% A/Sn and F(1) CD8(+) cells expressed CD44(high)/CD45RB(+) phenotype, while about 40% I/St CD8(+) cells did not express CD45RB marker at week 5 of infection. In contrast, in susceptible I/St mice lung CD4(+) cells proliferated much more strongly in response to mycobacterial sonicate, and a higher proportion of these cells expressed CD95 and underwent apoptosis compared with A/Sn cells. Unseparated lung cells and T cells of I/St origin produced more IL-5 and IL-10, respectively, whereas their A/Sn and F1 counterparts produced more IFN-gamma following infection. F(1) cells overall expressed an intermediate phenotype between the two parental strains. Such a more balanced type of immune reactivity could be linked to a better TB defense.

Characterization of T cell clones derived from lymph nodes and lungs of Pseudomonas aeruginosa-susceptible and resistant mice following immunization with heat-killed bacteria.

Pseudomonas aeruginosa-resistant BALB/c and susceptible C57Bl/6 (B6) mice were immunized with heat-killed Pseudomonas either in the foot pad or via the trachea, and panels of Pseudomonas-specific T cell clones were developed from lymph nodes and lungs. All clones from either strain, whether of lymph node or lung origin, were CD3+CD4+CD8-TCRalphabeta+. The efficacy of cloning from lymph node cells was comparable between BALB/c and B6 mice. All lymph node BALB/c clones proliferated in response to Pseudomonas antigen in a dose-dependent manner, and this response was MHC class II-restricted. Vigorous proliferation by a considerable proportion of B6 T cell clones occurred in the absence of specific antigen. Lymph node clones from either strain could be categorized as either Th1 or Th0 on the basis of interferon-gamma (IFN-gamma)/IL-4 production. In either mouse strain the efficacy of cloning from lung tissue was substantially lower than from lymph nodes, but the efficacy of cloning from BALB/c compared with B6 lungs was higher. Four lung T cell clones from BALB/c and two from B6 mice were expanded for further analyses, and an interstrain difference was observed in cytokine production. Both B6 lung T cell clones were Th1-like and produced IFN-gamma but not IL-4 and IL-10, whereas four BALB/c lung T cell clones were Th2-like and produced IL-4 and IL-10 but not IFN-gamma. These observations suggest that differences in the CD4+ Th response in the lung may contribute to differences among inbred mouse strains in the level of resistance to bronchopulmonary Pseudomonas infection.

The 19-kD antigen and protective immunity in a murine model of tuberculosis.

The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic mycobacteria-M. vaccae and M. smegmatis-resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response to the 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.

Deletion of the 19kDa antigen does not alter the protective efficacy of BCG.

Expression of the Mycobacterium tuberculosis 19kDa lipoprotein in saprophytic mycobacteria has been found to reduce their ability to prime a protective response to subsequent virulent challenge in the mouse model. The present study was designed to test whether 19kDa expression has an analogous detrimental effect on the efficacy of BCG vaccination. In contrast to the results in saprophytes, neither overexpression of the 19kDa antigen, nor deletion of the endogenous 19kDa gene altered the ability of BCG to protect against M. tuberculosis challenge in a mouse model.

An ex vivo study of T lymphocytes recovered from the lungs of I/St mice infected with and susceptible to Mycobacterium tuberculosis.

I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10(3) or 10(5) CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44(-) CD45RB+ cells disappeared within 2 weeks postinfection, while the number of CD44(+) CD45RB-/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3(+) CD4(+) CD8(-) T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-gamma] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-gamma+ IL-4(-)) clone and one Th0-like (IFN-gamma+ IL-4(+) IL-10(+)) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-gamma responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-gamma production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

Induction of a type 1 immune response to a recombinant antigen from Mycobacterium tuberculosis expressed in Mycobacterium vaccae.

A 19-kDa lipoprotein from Mycobacterium tuberculosis was expressed as a recombinant antigen in the nonpathogenic mycobacterial host strain M. vaccae. Immunization of mice with the recombinant M. vaccae resulted in induction of a strong type 1 immune response to the 19-kDa antigen, characterized by immunoglobulin G2a (IgG2a) antibodies and gamma interferon (IFN-gamma) production by splenocytes. Immunization with the same antigen in incomplete Freund's adjuvant induced a strong IgG1 response with only low levels of IFN-gamma. Subsequent intravenous and aerosol challenges of immunized mice with virulent M. tuberculosis demonstrated no evidence of protection associated with the response to the 19-kDa antigen; in fact, the presence of the recombinant 19-kDa antigen abrogated the limited protection conferred by M. vaccae (vector control). The recombinant M. vaccae system is a convenient approach to induction of type 1 responses to M. tuberculosis antigens. However, the unexpected reduction in protective efficacy of M. vaccae expressing the 19-kDa antigen highlights the complexity of testing recombinant subunit vaccines and the need for a better understanding of the immune mechanisms required for effective vaccination against tuberculosis.

Influence of the mouse Bcg, Tbc-1 and xid genes on resistance and immune responses to tuberculosis infection and efficacy of bacille Calmette-Guérin (BCG) vaccination.

We have studied the role of three mouse distinct non-H-2 genes (Bcg, Tbc-1, xid) in several phenomena of antituberculosis immunity and resistance. On the basis of median survival time (MST) of mice following infection with virulent Mycobacterium tuberculosis H37Rv, Bcg gene did not control resistance to the lethal dose of H37Rv infection in non-vaccinated and Myco. bovis (BCG)-vaccinated mice. However, Bcgr allele, in comparison with Bcgs allele, determined more effective suppression of an early multiplication in spleens of H37Rv mycobacteria after a low dose (5x10(4) colony-forming units (CFU)) injection. CBA/N mice, which are not protected efficiently against tuberculous challenge by BCG vaccination, were characterized by a decreased in vitro proliferation of immune lymph node cells, both spontaneous and stimulated with mycobacterial antigens. The decreased proliferation was due to immunosuppression caused by interactions between responding T cells and CBA/N antigen-presenting cells (APC). We have confirmed that the defective response to BCG-vaccination in CBA/N mice is linked with the X-chromosome and thus is presumably determined by the xid gene itself. I/St mice (Tbc-1s), supersusceptible to H37Rv infection, were not able to restrict the growth of H37Rv mycobacteria in spleens, even following infection with a low dose (5x10(4)), but restricted the growth of Myco.bovis BCG more effectively than Bcgs mice.