Induction of CD8 T Cells against a Novel Epitope in TB10.4: Correlation with Mycobacterial Virulence and the Presence of a Functional Region of Difference-1

Abstract

Although infection with Mycobacterium tuberculosis (M.tb) induces a robust CD8 T cell response, the role of CD8 T cells in the defense against M.tb, and the mechanisms behind the induction of CD8 T cells, is still not clear. TB10.4 is a recently described Ag that is expressed by both bacillus Calmette-Guérin (BCG) and M.tb. In the present study, we describe a novel CD8 T cell epitope in TB10.4, TB10.43-11. We show that TB10.43-11-specific CD8 T cells are induced at the onset of infection and are present throughout the infection in high numbers. TB10.43-11 CD8 T cells were recruited to the site of infection and expressed CD44, TNF-α, and IFN-γ. In addition, TB10.43-11 CD8 T cells showed an up-regulation of FasL and LAMP-1/2 (CD107A/B), which correlated with a strong in vivo cytolytic activity. The induction of TB10.43-11-specific CD8 T cells was less pronounced following infection with BCG compared to infection with M.tb. By using a rBCG expressing the genetic region of difference-1 (RD1), we show that the presence of a functional RD1 region increases the induction of TB10.43-11-specific CD8 T cells as well as the bacterial virulence. Finally, as an M.tb variant lacking the genetic region RD1 also induced a significant amount of TB10.43-11-specific CD8 T cells, and exhibited increased virulence compared with BCG, our data suggest that virulence in itself is also involved in generating a robust CD8 T cell response against mycobacterial epitopes, such as TB10.43-11.

Difference in TB10.4 T‐cell epitope recognition following immunization with recombinant TB10.4, BCG or infection with Mycobacterium tuberculosis

“…administration of BCG at high doses ($1 Â 10 6 CFU/mouse), which does give a significant CD8 response specific for TB10.4) (Fig. 2) [15,24,25]. The recombinant BCG::RD1-strain expressing the ESAT-6 secretion system showed similar TB10.4 epitope recognition patterns as virulent M.tb, both recognizing the MHC-I restricted epitopes in P1 and P2 and the MHC-II restricted epitope in P8 (data not shown), corresponding to earlier described epitopes [24,26,27].…”

“…2) [15,24,25]. The recombinant BCG::RD1-strain expressing the ESAT-6 secretion system showed similar TB10.4 epitope recognition patterns as virulent M.tb, both recognizing the MHC-I restricted epitopes in P1 and P2 and the MHC-II restricted epitope in P8 (data not shown), corresponding to earlier described epitopes [24,26,27]. As it has been suggested that the RD1 region enables M.tb to escape the phagosome [28], it could be speculated that altered intracellular trafficking of BCG might lead to a different epitope pattern and/ or to new protective epitopes.…”

“…Experiments were conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate Permit 2004-561-868 (of January 7, 2004, and in compliance with European Community Directive 86/609. Antigens rTB10.4 was produced in E. coli as described earlier [24]. Native TB10.4 and the native complex of TB10.4/TB9.8 (Rv0288/ Rv0287) was homologously overexpressed in M. smegmatis using the pMyNT vector (Arie Geerlof, EMBL-HH) and purified using a NiNTA column.…”

“…Intracellular cytokine staining of T cells was done as described previously [24]. Briefly, cells were stimulated for 1 h with 2 mg/ mL Ag and subsequently incubated for 5 h with 10 mg/mL brefeldin A (Sigma-Aldrich) at 371C and then stained for surface markers, permeabilized and subsequently stained for intracellular cytokine expression.…”

Peripheral Blood CD8 T Cell Response in Different Phases of MTB Infection

“…In the last years, flow cytometry evaluation of cytokine secretion profiles and cell surface phenotypes of peripheral blood T cells have been employed in the attempt to differentiate active and latent TB infection [ 5,11,12 ]. A large body of evidence suggests the important contribution of CD8 T cells in the MTB-specific immune response [6][7][8]13 ]. The latter display specific effector functions performed upon recognition of mycobacterial antigens -production of IFN-γ, lysis of infected host cells, and direct killing of MT bacteria [ 14 ].…”