LIGHT contributes to early but not late control of Mycobacterium tuberculosis infection.

The TNF superfamily member, LIGHT, contributes to optimal T-cell activation in vitro through co-stimulation of dendritic cell cytokine production; however, its role in T-cell-mediated control of intracellular bacterial infections is unknown. Protective immunity against Listeria monocytogenes and Mycobacterium tuberculosis infection requires both antigen-specific CD4(+) and CD8(+) T cells. Using LIGHT-deficient mice we determined that LIGHT was necessary for optimal re-stimulation of anti-listerial CD8(+) T cells in vitro. By contrast, LIGHT(-/-) mice infected with L. monocytogenes generated equivalent T-cell responses and controlled the infection as effectively as normal C57BL/6 mice. Following M. tuberculosis infection, LIGHT(-/-) mice showed a significant increase in bacterial replication in the lungs at 4 weeks, but by 6 weeks had controlled the infection. Analysis of T-cell responses in vivo revealed that LIGHT was dispensable for the activation of primary T-cell responses and the production of IL-12 and IFN-gamma. In addition, LIGHT was not required for the induction of memory T-cell responses to anti-mycobacterial DNA or BCG vaccines and for subsequent protection against tuberculosis challenge. Therefore, LIGHT contributes to the optimal co-stimulation of anti-listerial CD8(+) T-cell responses in vitro and to the early control of M. tuberculosis infection; however, other mechanisms compensate for LIGHT deficiency in the control of these pathogens in vivo.

Differential requirements for soluble and transmembrane tumor necrosis factor in the immunological control of primary and secondary Listeria monocytogenes infection.

The relative contributions of transmembrane tumor necrosis factor (memTNF) and soluble tumor necrosis factor (solTNF) in innate and adaptive immunity are poorly defined. We examined the capacities of wild-type (WT) mice, TNF-/- mice, and memTNF mice, which express only transmembrane TNF, to control primary and secondary Listeria monocytogenes infections. Soluble TNF was not required for induction or maintenance of protective immunity against a low-dose (200-CFU) Listeria infection. In contrast to TNF-/- mice, both WT and memTNF mice cleared the bacilli within 10 days and were fully protected against rechallenge with a lethal infective dose. Furthermore, T cells transferred from immune mice, but not from naïve, WT, and memTNF mice, protected TNF-/- recipients against an otherwise lethal infection. By contrast, infection with a higher dose of Listeria (2,000 CFU) clearly demonstrated that solTNF is required to coordinate an optimal protective inflammatory response. memTNF mice were more susceptible to a high-dose infection, and they exhibited delayed bacterial clearance, increased inflammation, and necrosis in the liver that resulted in 55% mortality. The dysregulated inflammation was accompanied by prolonged elevated expression of mRNAs for several chemokines as well as the macrophage effector molecules inducible nitric oxide synthase and LRG-47 in the livers of memTNF mice but not in the livers of WT mice. These data demonstrated that memTNF is sufficient for establishing protective immunity against a primary low-dose Listeria infection but that solTNF is required for optimal control of cellular inflammation and resistance to a primary high-dose infection. By contrast, memTNF alone is sufficient for resolution of a secondary, high-dose infection and for the transfer of protective immunity with memory T cells.

Increasing the foreignness of an antigen, by coupling a second and foreign antigen to it, increases the T helper type 2 component of the immune response to the first antigen.

It has been proposed that the degree of an antigen's foreignness is important in determining the Th1/Th2 phenotype of the immune response it generates. We test this hypothesis here and partially dissect the underlying mechanism. Immunization of C57BL/6 and hen egg lysozyme (HEL)-transgenic mice, tolerant to HEL at the T-cell level, with low doses of sheep red blood cells (SRBC), generated a predominant T helper type 1 (Th1) response in both mouse strains. However, substantial numbers of SRBC-specific Th2 cells were generated when normal, but not HEL-transgenic, mice were immunized with a low dose of the conjugate HEL-SRBC. The generation of these anti-SRBC Th2 cells in normal mice required that HEL be coupled to SRBC, since HEL was ineffective in deviating the response to SRBC when present but coupled to another, non-cross-reacting, xenogeneic RBC. This Th2 deviation of the anti-SRBC response by HEL thus requires the operational recognition of HEL epitopes linked to SRBC. Thus increasing the foreignness of an antigen increases its ability to generate Th2 cells. Our findings, in the context of previous observations in related systems, support the proposal that more CD4(+) T-cell/CD4(+) T-cell interactions, mediated by the operational recognition of linked epitopes, are required to generate Th2 cells than Th1 cells.

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection.

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.

The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette-Guèrin) in protecting against systemic Mycobacterim avium infection.

Sub-unit vaccines utilizing purified mycobacterial proteins or DNA vaccines induce partial protection against mycobacterial infections. For example, immunization with DNA vaccines expressing the gene for the immunodominant 35000 MW protein, common to Mycobacterium avium and Mycobacterium leprae but absent from the Mycobacterium tuberculosis complex, conferred significant protection against infection with either virulent M. avium or M. leprae in mice. However, the level of protection was equivalent to that obtained with the viable, attenuated vaccine, Mycobacterium bovis, bacille Calmette-Guèrin (BCG). The cytokine, interleukin (IL)-12, is essential for priming naïve CD4+ T lymphocytes to differentiate into interferon-gamma (IFN-gamma)-secreting T cells. We have used a novel self-splicing vector expressing both chains of murine IL-12 to determine if plasmid IL-12 would increase the efficacy of a vaccine expressing the M. avium 35000 MW protein (DNA-Av35). Co-immunization with p2AIL-12 and DNA-Av35 led to a significant increase in the number of antigen-specific IFN-gamma secreting cells and total amount of IFN-gamma released, but a concomitant fall in the antibody response to the 35000 MW protein. This pattern of response was associated with enhanced clearance of M. avium from the liver and spleen of coimmunized mice, and was significantly more effective than BCG or DNA-Av35. alone. Following M. avium challenge there was significant increase in the expansion of the 35000 MW antigen-reactive T cells in the coimmunized mice. Therefore, plasmid-delivered IL-12 acts as an effective adjuvant to increase the protective efficacy of a single DNA vaccine against M. avium infection above that achieved by BCG, and this strategy may improve the efficacy of subunit vaccines against M. leprae and M. tuberculosis.

Characterization of immune responses during infection with Mycobacterium avium strains 100, 101 and the recently sequenced 104.

Mycobacterium avium strain 104 was chosen as the M. avium isolate to sequence, as it is virulent to humans, stable and readily transfectable. As this strain has not been widely studied we sought to investigate the pattern of 104 infection in mice. Bacterial growth and the immune response generated were compared with infection with the low virulence M. avium strain 100, and the high virulence common laboratory strain, 101. Mycobacterium avium strains 104 and 101 grew progressively within mice, while strain 100 was gradually cleared. Strains 104 and 101 induced strong T cell activation and spleen cell cultures produced similar levels of IFN-gamma. In mice infected with strain 100 no significant T cell activation or IFN-gamma production was measured. Further, mice infected with strain 104 or 101 also displayed comparable inflammatory responses and similar granuloma formation, while only minimal inflammation was seen in mice infected with strain 100. Strains 101 and 104 also grew in a similar fashion in bone-marrow-derived macrophages and induced significant levels of TNF and nitric oxide. Thus infection with M. avium strain 104 induced an immunological response comparable to M. avium strain 101 and, with the availability of its sequence, should be a useful tool for designing new vaccines or drugs therapies to treat the increasing incidence of M. avium infection in humans.

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection.

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.