Processing of Mycobacterium tuberculosis antigen 85B involves intraphagosomal formation of peptide-major histocompatibility complex II complexes and is inhibited by live bacilli that decrease phagosome maturation

Abstract

Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-gamma (IFN-gamma)-activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241-256)-I-A(b) complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241-256)-I-A(b) complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide-MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.

Figure 1.

Requirements for processing of MTB bacilli for presentation of MTB Ag 85B. Macrophages were pulsed with HK MTB for 20 min, chased for 10 min at 37°C, and fixed. BB7 T hybridoma cells were used to detect Ag 85B(241–256)–I-A^b complexes. Supernatants were assessed for IL-2 content using a CTLL-2 proliferation assay that was monitored with Alamar blue, an indicator dye. (A) Effects of cytochalasin D (10 μg/ml) and chloroquine (100 μM) on Ag processing. (B) Comparison of processing of HK MTB by B6D2 and H2-DM^−/− macrophages. Data points are means of triplicate samples ± SD.

Figure 1.

Requirements for processing of MTB bacilli for presentation of MTB Ag 85B. Macrophages were pulsed with HK MTB for 20 min, chased for 10 min at 37°C, and fixed. BB7 T hybridoma cells were used to detect Ag 85B(241–256)–I-A^b complexes. Supernatants were assessed for IL-2 content using a CTLL-2 proliferation assay that was monitored with Alamar blue, an indicator dye. (A) Effects of cytochalasin D (10 μg/ml) and chloroquine (100 μM) on Ag processing. (B) Comparison of processing of HK MTB by B6D2 and H2-DM^−/− macrophages. Data points are means of triplicate samples ± SD.

Figure 2.

Characterization of MTB phagosomes isolated on 27% Percoll density gradients. Macrophages were incubated with soluble OVA (3 mg/ml) for 1 h, incubated with fluorescein-labeled HK MTB and OVA for 20 min, washed, chased for 30 min at 37°C in the presence of soluble OVA, homogenized, and fractionated on 27% Percoll gradients. (A) Distribution of HK MTB phagosomes detected by fluorimetry. (B) Distribution of plasma membrane radioactivity when macrophage plasma membranes were labeled with ¹²⁵I-Y-3P (anti–I-A^b) at 4°C before fractionation. (C) B-Hexosaminidase activity (a marker of lysosomal enzyme distribution). (D) Distribution of OVA(323–339)–I-A^d complexes assessed by DOBW T hybridoma assay.

Figure 3.

Arrest of phagosomal maturation in MTB phagosomes containing live but not HK MTB. Macrophages were incubated with fluorescein-labeled live or HK MTB for a 20-min pulse with a 10- or 100-min chase incubation. Phagosomes were pelleted, purified on 27% Percoll gradients, and prepared for flow organellometry with staining for LAMP-1 and MHC-II (I-A^b/d). Events were gated for fluorescence and scatter parameters consistent with phagosomes. Staining with isotype-matched negative control Abs was used to define the H1 gate. The H2 gate represents positive events. The H3 gate represents all events. MFV values represent the mean for the H3 gate.

Figure 3.

Arrest of phagosomal maturation in MTB phagosomes containing live but not HK MTB. Macrophages were incubated with fluorescein-labeled live or HK MTB for a 20-min pulse with a 10- or 100-min chase incubation. Phagosomes were pelleted, purified on 27% Percoll gradients, and prepared for flow organellometry with staining for LAMP-1 and MHC-II (I-A^b/d). Events were gated for fluorescence and scatter parameters consistent with phagosomes. Staining with isotype-matched negative control Abs was used to define the H1 gate. The H2 gate represents positive events. The H3 gate represents all events. MFV values represent the mean for the H3 gate.

Figure 4.

MTB phagosomes have negligible contamination with other MHC-II expressing membranes. B6D2 and CBA/J macrophages were pulsed with fluorescein-labeled HK MTB for 20 min, washed, and chased for 30 min at 37°C. Half of the MTB-infected B6D2 macrophages were mixed with an equal number of naive CBA/J macrophages before homogenization. Homogenates were prepared from MTB-exposed B6D2 macrophages, MTB-exposed CBA/J macrophages, and MTB-exposed B6D2 macrophages mixed with naive CBA/J macrophages. Phagosomes were pelleted, purified on separate 27% Percoll gradients, fixed, and stained for I-A^d/b or I-A^k. Flow organellometry was performed as in Fig. 3.

Figure 5.

Ag 85B(241–256)–I-A^b complexes are initially found in phagosomes and later appear on the plasma membrane (PM). (A and B) Macrophages were pulsed with HK MTB (MOI = 40) for 20 min, washed, chased for various periods, and fractionated on 27% Percoll density gradients. (C) Macrophages were incubated with soluble OVA for 1 h and then pulsed with HK MTB and OVA for 20 min, washed, chased for 10 min at 37°C in the presence of OVA, and fractionated on 40% Percoll density gradients. Aliquots (50 μl) of each fraction were frozen, thawed, and analyzed for Ag 85B(241–256)–I-A^b and OVA(323–339)–I-A^d complexes using BB7 and DOBW T hybridoma cells, respectively. Diagrams at the top summarize the positions of different compartments in the Percoll gradients.

Figure 6.

HK MTB is processed more rapidly than live MTB. Macrophages were pulsed with HK or live MTB for 20 min, chased for varying periods at 37°C, and fixed. BB7 T hybridoma cells were used to detect presentation of Ag 85B(241–256)–I-A^b complexes (see Fig. 1). (A) Presentation at varying MOI after 0- or 100-min chase. (B) Presentation after varying chase intervals with MOI = 5. A and B are from a single experiment. Data points are means of triplicate samples ± S.D.

Figure 7.

Ag 85B(241–256)–I-A^b complexes are produced in early phagosomes at higher levels with HK MTB than with live MTB. (A and B) Macrophages were pulsed with HK or live MTB for 20 min at a MOI of either 40 or 13 bacteria per cell and fractionated on separate 27% Percoll density gradients. Fractions were processed as in Fig. 5 and analyzed for expression of Ag 85B(241–256)–I-A^b complexes using BB7 T hybridoma cells. (C) B6D2 and CBA/J macrophages were pulsed with live MTB for 20 min at a MOI of 40, washed and chased for 20 min. The MTB-infected CBA/J macrophages were mixed with an equal number of naive B6D2 macrophages before homogenization. The two samples were fractionated on separate 27% Percoll density gradients, and fractions were analyzed for expression of Ag 85B(241–256)–I-A^b complexes using BB7 T hybridoma cells.