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

AbstractTuberculosis (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

Phenotypic characterization of bovine memory cells responding to mycobacteria in IFNγ enzyme linked immunospot assays

AbstractBovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells. Using expression of CD45RO and CD62L we purified by cytometric cell sorting four distinct CD4+ populations: CD45RO+CD62Lhi, CD45RO+CD62Llo, CD45RO−CD62Lhi and CD45RO−CD62Llo (although due to low and inconsistent cell recovery, this population was not considered further in this study), in BCG vaccinated and Mycobacterium bovis infected cattle. These populations were then tested in the cELISPOT assay. The main populations contributing to production of IFNγ in the cELISPOT were of the CD45RO+CD62Lhi and CD45RO+CD62Llo phenotypes. These cell populations have been described in other species as central and effector memory cells, respectively. Following in vitro culture and flow cytometry we observed plasticity within the bovine CD4+ T-cell phenotype. Populations switched phenotype, increasing or decreasing expression of CD45RO and CD62L within 24h of in vitro stimulation. After 14 days all IFNγ producing CD4+ T cells expressed CD45RO regardless of the original phenotype of the sorted population. No differences were detected in behavior of cells derived from BCG-vaccinated animals compared to cells derived from naturally infected animals. In conclusion, although multiple populations of CD4+ T memory cells from both BCG vaccinated and M. bovis infected animals contributed to cELISPOT responses, the dominant contributing population consists of central-memory-like T cells (CD45RO+CD62Lhi)

The Duration of Antigen-Stimulation Significantly Alters the Diversity of Multifunctional CD4 T Cells Measured by Intracellular Cytokine Staining

The assessment of antigen-specific T cell responses by intracellular cytokine staining (ICS) has become a routine technique in studies of vaccination and immunity. Here, we highlight how the duration of in vitro antigen pre-stimulation, combined with the cytokine accumulation period, are critical parameters of these methods. The effect of varying these parameters upon the diversity and frequency of multifunctional CD4 T cell subsets has been investigated using a murine model of TB vaccination and in cattle naturally infected with Mycobacterium bovis. We demonstrate a substantial influence of the duration of the antigen pre-stimulation period on the repertoire of the antigen-specific CD4 T cell responses. Increasing pre-stimulation from 2 to 6 hours amplified the diversity of the seven potential multifunctional CD4 T cell subsets that secreted any combination of IFN-γ, IL-2 and TNF-α. However, increasing pre-stimulation from 6 to 16 hours markedly altered the multifunctional CD4 T cell repertoire to a dominant IFN-γ+ only response. This was observed in both murine and cattle models

Systemic BCG immunization induces persistent lung mucosal multifunctional CD4 T_EM cells which expand following virulent mycobacterial challenge

To more closely understand the mechanisms of how BCG vaccination confers immunity would help to rationally design improved tuberculosis vaccines that are urgently required. Given the established central role of CD4 T cells in BCG induced immunity, we sought to characterise the generation of memory CD4 T cell responses to BCG vaccination and M. bovis infection in a murine challenge model. We demonstrate that a single systemic BCG vaccination induces distinct systemic and mucosal populations of T effector memory (T(EM)) cells in vaccinated mice. These CD4(+)CD44(hi)CD62L(lo)CD27(−) T cells concomitantly produce IFN-γ and TNF-α, or IFN-γ, IL-2 and TNF-α and have a higher cytokine median fluorescence intensity MFI or ‘quality of response’ than single cytokine producing cells. These cells are maintained for long periods (>16 months) in BCG protected mice, maintaining a vaccine–specific functionality. Following virulent mycobacterial challenge, these cells underwent significant expansion in the lungs and are, therefore, strongly associated with protection against M. bovis challenge. Our data demonstrate that a persistent mucosal population of T(EM) cells can be induced by parenteral immunization, a feature only previously associated with mucosal immunization routes; and that these multifunctional T(EM) cells are strongly associated with protection. We propose that these cells mediate protective immunity, and that vaccines designed to increase the number of relevant antigen-specific T(EM) in the lung may represent a new generation of TB vaccines

The effect of different culture schedules on the viability of CD4 T cells.

<p>Mouse splenocytes 6 weeks following a BCG immunization (A) and PBMC from cattle naturally infected with <i>M. bovis</i> (B) were isolated, stimulated with protein cocktail or PPD-B, respectively, stained by intracellular cytokine staining (ICS) and interrogated by flow cytometry. Graphs represent the percentage of live CD4⁺ lymphocytes detected in the analysis, as determined by the inclusion of a LIVE/DEAD fixable Violet dead cell stain. Data are representative of one of three independent experiments (n = 6). Bars represent the mean ± SEM; **p<0.01; ANOVA.</p

The effect of different culture schedules on antigen-specific bovine CD4 T cell responses.

<p>PBMC from cattle naturally infected with <i>M. bovis</i> were isolated, stimulated with PPD-B (A) or ESAT-6/CFP-10 peptide pools (B), stained by intracellular cytokine staining (ICS) and interrogated by flow cytometry. Plots were gated on live CD4⁺ lymphocytes and analyzed for all combinations of simultaneous IFN-γ, IL-2 and TNF-α productivity. The stacked bar graphs represent the percentage of antigen-specific CD4⁺ cells in the seven individual cell subsets, data is representative of one of three independent experiments (n = 6). Bars represent the mean ± SEM.</p

Identification of CD4⁺ T cells producing IFN-γ, IL-2 & TNF-α.

<p>Mouse splenocytes 6 weeks following a BCG immunization (A) and PBMC from cattle naturally infected <i>M. bovis</i> (B) were isolated, stimulated with antigen, stained by intracellular cytokine staining (ICS) and interrogated by flow cytometry. Plots were gated on live CD4⁺ lymphocytes and analyzed for all combinations of simultaneous IFN-γ, IL-2 and TNF-α productivity. Numbers indicate percentage of CD4⁺ cells in the seven individual cell subsets, data are representative of one of three independent experiments.</p