A role for tumor necrosis factor-alpha in remodeling the splenic marginal zone during Leishmania donovani infection.

The development of secondary lymphoid organs is a highly regulated process, mediated by tumor necrosis factor (TNF) family cytokines. In contrast, the mechanisms controlling changes in lymphoid architecture that occur during infectious disease are poorly understood. Here we demonstrate that during infection with Leishmania donovani, the marginal zone of mice undergoes extensive remodeling, similar in extent to developmental abnormalities in mice lacking some TNF family cytokines. This process is selective, comprising a dramatic and rapid loss of marginal zone macrophages (MZMs). As a functional consequence, lymphocyte traffic into the white pulp is impaired during chronic leishmaniasis. Significantly, MZMs were preserved in L. donovani-infected B6.TNF-alpha(-/-) mice or mice that received anti-TNF-alpha antibodies, whereas studies in CD8(+) T-cell-deficient mice and in mice lacking functional CD95L, excluded a direct role for either cytotoxic T lymphocyte activity or CD95-mediated apoptosis in this process. Loss of MZMs was independent of parasite burden, yet could be partially prevented by chemotherapy, which in turn reduced endogenous TNF-alpha levels. This is the first report of an infectious agent causing selective and long-lasting changes to the marginal zone via TNF-alpha-mediated mechanisms, and illustrates that those cytokines involved in establishing lymphoid architecture during development, may also play a role in infection-induced lymphoid tissue remodeling

Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents by multiplex bead array and intracellular cytokine staining

BACKGROUND: The vaccine efficacy reported following Mycobacterium bovis Bacillus Calmette Guerin (BCG) administration to UK adolescents is 77% and defining the cellular immune response in this group can inform us as to the nature of effective immunity against tuberculosis. The aim of this study was to identify which cytokines and lymphocyte populations characterise the peripheral blood cellular immune response following BCG vaccination. RESULTS: Diluted blood from before and after vaccination was stimulated with Mycobacterium tuberculosis purified protein derivative for 6 days, after which soluble biomarkers in supernatants were assayed by multiplex bead array. Ten out of twenty biomarkers measured were significantly increased (p < 0.0025) 1 month after BCG vaccination when compared to paired samples (n = 12) taken prior to vaccination (IFNgamma, TNFalpha, IL-1alpha, IL-2, IL-6, IL-10, IL-17, GM-CSF, MIP1alpha, IP-10). All of these remained detectable by multiplex bead array in samples taken 12 months after BCG vaccination of a partially overlapping adolescent group (n = 12). Intracellular cytokine staining after 24 hour Mycobacterium tuberculosis purified protein derivative stimulation of PBMC samples from the 12 month group revealed that IFNgamma expression was detectable in CD4 and CD8 T-cells and natural killer cells. Polyfunctional flow cytometry analysis demonstrated that cells expressing IFNgamma alone formed the majority in each subpopulation of cells. Only in CD4 T-cells and NK cells were there a notable proportion of responding cells of a different phenotype and these were single positive, TNFalpha producers. No significant expression of the cytokines IL-2, IL-17 or IL-10 was seen in any population of cells. CONCLUSIONS: The broad array of biomarker responses detected by multiplex bead array suggests that BCG vaccination is capable, in this setting, of inducing a complex immune phenotype. Although polyfunctional T-cells have been proposed to play a role in protective immunity, they were not present in vaccinated adolescents who, based on earlier epidemiological studies, should have developed protection against pulmonary tuberculosis. This may be due to the later sampling time point available for testing or on the kinetics of the assays used

Persistence of the immune response induced by BCG vaccination.

BACKGROUND: Although BCG vaccination is recommended in most countries of the world, little is known of the persistence of BCG-induced immune responses. As novel TB vaccines may be given to boost the immunity induced by neonatal BCG vaccination, evidence concerning the persistence of the BCG vaccine-induced response would help inform decisions about when such boosting would be most effective. METHODS: A randomised control study of UK adolescents was carried out to investigate persistence of BCG immune responses. Adolescents were tested for interferon-gamma (IFN-gamma) response to Mycobacterium tuberculosis purified protein derivative (M.tb PPD) in a whole blood assay before, 3 months, 12 months (n = 148) and 3 years (n = 19) after receiving teenage BCG vaccination or 14 years after receiving infant BCG vaccination (n = 16). RESULTS: A gradual reduction in magnitude of response was evident from 3 months to 1 year and from 1 year to 3 years following teenage vaccination, but responses 3 years after vaccination were still on average 6 times higher than before vaccination among vaccinees. Some individuals (11/86; 13%) failed to make a detectable antigen-specific response three months after vaccination, or lost the response after 1 (11/86; 13%) or 3 (3/19; 16%) years. IFN-gamma response to Ag85 was measured in a subgroup of adolescents and appeared to be better maintained with no decline from 3 to 12 months. A smaller group of adolescents were tested 14 years after receiving infant BCG vaccination and 13/16 (81%) made a detectable IFN-gamma response to M.tb PPD 14 years after infant vaccination as compared to 6/16 (38%) matched unvaccinated controls (p = 0.012); teenagers vaccinated in infancy were 19 times more likely to make an IFN-gamma response of > 500 pg/ml than unvaccinated teenagers. CONCLUSION: BCG vaccination in infancy and adolescence induces immunological memory to mycobacterial antigens that is still present and measurable for at least 14 years in the majority of vaccinees, although the magnitude of the peripheral blood response wanes from 3 months to 12 months and from 12 months to 3 years post vaccination. The data presented here suggest that because of such waning in the response there may be scope for boosting anti-tuberculous immunity in BCG vaccinated children anytime from 3 months post-vaccination. This supports the prime boost strategies being employed for some new TB vaccines currently under development

BCG vaccination: a role for vitamin D?

BCG vaccination is administered in infancy in most countries with the aim of providing protection against tuberculosis. There is increasing interest in the role of vitamin D in immunity to tuberculosis. This study objective was to determine if there was an association between circulating 25(OH)D concentrations and BCG vaccination status and cytokine responses following BCG vaccination in infants

Activation-induced cell death in human T cells is a suicidal process regulated by cell density but superantigen induces T cell fratricide.

Repeated ligation of the TCR results in apoptosis (activation-induced cell death; AICD). Superantigens such as Staphylococcal enterotoxin B (SEB) are particularly efficient at inducing AICD in T cells. We investigated whether apoptosis in human T cell subsets was due to fratricide (killing of neighboring cells) or suicide (cell autonomous death). AICD of Th1, Th2, Tc1, and Tc2 effector cells was dramatically enhanced at low cell densities and could be observed in single cell microcultures. AICD was unaffected by adhesion molecules or neighboring cells undergoing AICD, confirming the predominance of a suicidal mechanism. However, SEB was able to induce fratricidal apoptosis of type 1, but not type 2 cells. Fratricide was also observed when unstimulated T cells were exposed to activated Tc1 effector cells. Thus, AICD is tightly regulated to allow clonal T cell expansion and memory cell generation, but superantigens may subvert this process by allowing T cell fratricide

Immunogenicity of Danish-SSI 1331 BCG vaccine in the UK: comparison with Glaxo-Evans 1077 BCG vaccine.

The immunogenicity and reactogenicity, in British schoolchildren, of the newly introduced Danish-SSI 1331 BCG vaccine was compared with that of the previously used Glaxo-Evans 1077 BCG vaccine. Interferon-gamma (IFN-gamma) response to M. tuberculosis purified protein derivative (M.tb PPD) in a 6-day whole blood assay and delayed type hypersensitivity (DTH) to tuberculin PPD were determined before and 1 year after receiving BCG or no vaccination. Scar size was measured 1 year after vaccination. There was no evidence of a difference in immunogenicity (IFN-gamma and DTH conversion rates) but evidence of lower reactogenicity (scar size) with Danish-SSI 1331 compared to Glaxo-Evans 1077 vaccines

The PPD-specific T-cell clonal response in UK and Malawian subjects following BCG vaccination: a new repertoire evolves over 12 months.

Mycobacterium bovis bacille Calmette Guerin vaccination protects against pulmonary tuberculosis in the United Kingdom but not in Malawi. We investigated whether a difference in the clonal T-cell response to BCG vaccination might account for this. The results of clonal analysis were compared to those obtained by skin testing and in a whole blood interferon gamma assay. Pre-vaccination antigen specific T-cell clones were detected, but the majority of clones present 12 months after vaccination were not present earlier. The magnitude of the clonal response did not correlate well with results of the other assays. These data indicate that single assays may not be reliable and that a stable memory T-cell repertoire is slow to develop

Identification of immunological biomarkers which may differentiate latent tuberculosis from exposure to environmental nontuberculous mycobacteria in children.

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic