Stéphane Temmerman

Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Differential T and B Cell Responses against Mycobacterium tuberculosis Heparin-Binding Hemagglutinin Adhesin in Infected Healthy Individuals and Patients with Tuberculosis

Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.

Optimal kinetics for quantification of antigen-induced cytokines in human peripheral blood mononuclear cells by real-time PCR and by ELISA.

Real-time polymerase chain reaction (PCR) has recently been described as a new tool to measure and accurately quantify mRNA levels. In this study, we have applied this technique to evaluate cytokine mRNA synthesis induced by antigenic stimulation with purified protein derivative (PPD) or heparin-binding haemagglutinin (HBHA) in human peripheral blood mononuclear cells (PBMC) from Mycobacterium tuberculosis-infected individuals. Whereas PPD and HBHA optimally induced IL-2 mRNA after respectively 8 and 16 to 24 h of in vitro stimulation, longer in vitro stimulation times were necessary for optimal induction of interferon-gamma (IFN-gamma) mRNA, respectively 16 to 24 h for PPD and 24 to 96 h for HBHA. IL-13 mRNA was optimally induced by in vitro stimulation after 16-48 h for PPD and after 48 to 96 h for HBHA. Comparison of antigen-induced Th1 and Th2 cytokines appears, therefore, valuable only if both cytokine types are analysed at their optimal time point of production, which, for a given cytokine, may differ for each antigen tested. Results obtained by real-time PCR for IFN-gamma and IL-13 mRNA correlated well with those obtained by measuring the cytokine concentrations in cell culture supernatants, provided they were high enough to be detected. We conclude that real-time PCR can be successfully applied to the quantification of antigen-induced cytokine mRNA and to the evaluation of the Th1/Th2 balance, only if the kinetics of cytokine mRNA appearance are taken into account and evaluated for each cytokine measured and each antigen analysed.

Effector functions of heparin-binding hemagglutinin-specific CD8+ T lymphocytes in latent human tuberculosis

BACKGROUND Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN)- gamma responses during latent, but not active, TB. Because of the recently recognized importance of CD8(+) T lymphocytes in anti-TB immunity, we characterized the CD8(+) T lymphocyte responses to HBHA in subjects with latent TB. RESULTS HBHA-specific CD8(+) T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN- gamma . They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8(+) T lymphocytes were distinct from the IFN- gamma -producing CD8(+) T lymphocytes. CONCLUSION During latent TB, the HBHA-specific CD8(+) T lymphocyte population expresses all 3 effector functions associated with CD8(+) T lymphocyte-mediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8(+) T lymphocyte responses may be useful in the monitoring of protection.

The cationic lipid, diC14 amidine, extends the adjuvant properties of aluminum salts through a TLR-4- and caspase-1-independent mechanism.

Adjuvant efficiency is critical for inducing a protective and long-lasting immune response against weak immunogenic antigens. Discovered more than 70 years ago, aluminum salts remain the most widely used adjuvant in human vaccine. Prone to induce a strong humoral response, alum fails to drive a cell-mediated immunity, which is essential to fight against intracellular pathogens. Adjuvant systems that contain more than one component may represent an excellent alternative for completing the lack of T cell immunity associated with the injection of alum-based vaccine. In this work, we demonstrated that the adjuvant effects of alum strongly benefited from combining with a cationic lipid, the diC14 amidine. Indeed, we measured a significant improvement of alum-driven IL-1β release when human macrophages were co-cultured with a mixed suspension of alum and the diC14 amidine. Morphological analysis suggested that diC14 amidine improved the alum uptake by phagocytes. Furthermore, the addition of diC14 amidine to alum efficiently enhanced antigen processing and cross-presentation by antigen presenting cells. The biological relevance of these in vitro data was assessed by measuring the in vivo development of a cytotoxic activity and the enhanced synthesis of antigen-specific immunoglobulins after immunization with alum combined to diC14 amidine. Mechanistically, we demonstrated that diC14 amidine supported the alum adjuvanticity independently of the TLR-4 and caspase-1 agonist activities of the cationic lipid. Based on our findings, we conclude that diC14 amidine works synergistically with alum to achieve higher immune protection after vaccination.