Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1+ KLRG1- CD4+ T cells.

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4+ T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4+ T cells compared to systemic vaccination. Tissue-resident CD4+ T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4+ T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1+ KLRG1- cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4+ T cells.

Induction and maintenance of a phenotypically heterogeneous lung tissue-resident CD4+ T cell population following BCG immunisation.

Tuberculosis (TB) is the biggest cause of human mortality from an infectious disease. The only vaccine currently available, bacille Calmette-Guérin (BCG), demonstrates some protection against disseminated disease in childhood but very variable efficacy against pulmonary disease in adults. A greater understanding of protective host immune responses is required in order to aid the development of improved vaccines. Tissue-resident memory T cells (TRM) are a recently-identified subset of T cells which may represent an important component of protective immunity to TB. Here, we demonstrate that intradermal BCG vaccination induces a population of antigen-specific CD4+ T cells within the lung parenchyma which persist for >12 months post-vaccination. Comprehensive flow cytometric analysis reveals this population is phenotypically and functionally heterogeneous, and shares characteristics with lung vascular and splenic CD4+ T cells. This underlines the importance of utilising the intravascular staining technique for definitive identification of tissue-resident T cells, and also suggests that these anatomically distinct cellular subsets are not necessarily permanently resident within a particular tissue compartment but can migrate between compartments. This lung parenchymal population merits further investigation as a critical component of a protective immune response against Mycobacterium tuberculosis (M. tb).

Persistent BCG bacilli perpetuate CD4 T effector memory and optimal protection against tuberculosis.

Tuberculosis (TB) remains one of the most important infectious diseases of man and animals, and the only available vaccine (BCG) requires urgent replacement or improvement. To facilitate this, the protective mechanisms induced by BCG require further understanding. As a live attenuated vaccine, persistence of BCG bacilli in the host may be a crucial mechanism. We have investigated the long term persistence of BCG following vaccination and the influence on the induced immune response and protection, using an established murine model. We sought to establish whether previously identified BCG-specific CD4 TEM cells represent genuine long-lived memory cells of a relatively high frequency, or are a consequence of continual priming by chronically persistent BCG vaccine bacilli. By clearing persistent bacilli, we have compared immune responses (spleen and lung CD4: cytokine producing T effector/TEM; TCR-specific) and BCG-induced protection, in the presence and absence of these persisting vaccine bacilli. Viable BCG bacilli persisted for at least 16 months post-vaccination, associated with specific CD4 T effector/TEM and tetramer-specific responses. Clearing these bacilli abrogated all BCG-specific CD4 T cells whilst only reducing protection by 1log10. BCG may induce two additive mechanisms of immunity: (i) dependant on the presence of viable bacilli and TEM; and (ii) independent of these factors. These data have crucial implications on the rational generation of replacement TB vaccines, and the interpretation of BCG induced immunity in animal models.

An improved assay of 3-hydroxy-3-methylglutaryl-CoA reductase activity in Reuber H35 hepatoma cells without microsomes isolation.

The optimal conditions for measuring 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity in Reuber H35 hepatoma cells are described in this paper. Cells in the exponential phase of growth were lysed by incubation with Brij 97 detergent for 30 min. We used imidazole buffer supplemented with EDTA and leupeptine, two inhibitors of proteases. Disrupted cells were then centrifuged at 12,000 g. Although microsomes are usually reported as enzyme preparations for measuring HMG-CoA reductase, our data showed that hepatoma cells may be used without previous isolation of microsomes. The 12,000 g supernatant showed similar levels of total and specific activities to those found in the microsomal fraction obtained after 105,000 g centrifugation. The soluble fraction showed less than 10% of reductase activity. Reductase activity from Reuber H35 hepatoma cells increased proportionally to the reaction time from 30 to 90 min and to the amount of protein added in a range of 50-500 micrograms. Our modified method was very sensitive and reproducible, because very low specific activity (about 15-100 pmol min-1 per mg protein) could be quantified in different assay conditions obtaining similar values.

Metabolism of palmitic and docosahexaenoic acids in Reuber H35 hepatoma cells.

In this work, we have modified the fatty acid composition of Reuber H35 hepatoma cells by supplementation of the culture medium with a saturated (palmitic) or a polyunsaturated (docosahexaenoic) acid. These fatty acids were incorporated into total lipids and phospholipids of hepatoma cells. Palmitic acid readily increased the percentage of its monounsaturated derivative (16:1 n-7). When both fatty acids were supplemented at the same concentration, the percentage of docosahexaenoic acid in the total lipids and phospholipids of Reuber H35 cells increased more than that of palmitic acid. Although the levels of 16:0 increased, the addition of docosahexaenoic acid to the culture medium decreased the percentages of monoenoic acids. From our results, it can be concluded that palmitic and docosahexaenoic acids modify the fatty acid composition of Reuber H35 hepatoma cells. The profound changes induced by docosahexaenoic acid, especially those in the phospholipid fraction, may be of great interest given the main role of these components in the regulation of chemical and physical properties of biological membranes and/or membrane systems.