Bovine Immunoinhibitory Receptors Contribute to Suppression of Mycobacterium avium subsp. paratuberculosis-Specific T-Cell Responses

Abstract

Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-γ) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-γ responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-γ responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-γ production from M. avium subsp. paratuberculosis-specific CD4(+) and CD8(+) T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses.

FIG 1

Reactivity of the anti-LAG-3 MAb with LAG-3-expressing cells and bovine lymphocytes. (a) Flow cytometric analysis of bovine LAG-3. Cos-7 cells expressing LAG-3-EGFP (white area) and EGFP (shaded area) were stained with eight anti-LAG-3 MAb clones. Rat IgG1 (for 71-1A1, 71-1B3, 71-1H12, 71-2D8, 104-1G3, and 104-2F6), rat IgG2a (for 104-1F11), and rat IgG2b (for 104-2C9) were used as negative controls. (b) Western blotting of bovine LAG-3 protein from Cos-7 cells. Anti-LAG-3 MAb:71-2D8 recognized a LAG-3-EGFP protein band of ∼93 kDa. Anti-EGFP and antiactin antibodies were used as positive and loading controls, respectively. (c) Flow cytometric analysis of LAG-3 expression in CD4⁺ and CD8⁺ T cells. Freshly isolated bovine PBMCs were stained with anti-LAG-3:71-2D8 (white area), CD4, and CD8 MAbs. Rat IgG1 (gray area) was used as a negative-control stain. PBMCs were cultured with PBS (no stimulation) or PMA/ionomycin for 48 h and analyzed as described above.

FIG 2

IFN-γ responses to J-PPD in blood and fecal shedding of M. avium subsp. paratuberculosis in cattle experimentally infected with M. avium subsp. paratuberculosis. The dates of sample collection are indicated on the horizontal axis. The left vertical axis (gray circle) indicates the M. avium subsp. paratuberculosis (Map) DNA quantity in fecal samples determined using IS900 real-time PCR. The detection limit of this assay was 0.0001 pg/2.5 μl (19). Thus, the sample in which Map DNA was not detected is shown as “<0.0001 pg” on the graph. The right vertical axis (black diamond) shows IFN-γ production in whole blood cultures stimulated with J-PPD.

FIG 3

Expression levels of PD-1 and LAG-3 on T cells in M. avium subsp. paratuberculosis-infected cattle. (a) Gating strategy and representative dot plots for expression analyses of PD-1 and LAG-3 on IgM⁻ CD3⁺ CD4⁺ and IgM⁻ CD3⁺ CD8⁺ T cells from the blood of M. avium subsp. paratuberculosis-infected cattle. Values in the quadrant indicate the percentages of cells. (b and c) Percentages of PD-1-expressing (b) and LAG-3-expressing (c) cells in IgM⁻ CD3⁺ CD4⁺ and IgM⁻ CD3⁺ CD8⁺ T cells of peripheral blood, spleen, ileocecal LN, ileal and jejunal mLNs, and ileal and jejunal PPs isolated from uninfected cattle (n = 3) and M. avium subsp. paratuberculosis-infected cattle (n = 7). Statistical comparisons between groups were performed using Welch's t test. Differences were considered significant when P < 0.05 (*, P < 0.05; **, P < 0.01). See also Table S1 in the supplemental material for individual expression data.

FIG 4

Expression levels of PD-L1 and MHC-II on macrophages in M. avium subsp. paratuberculosis-infected cattle. (a) Gating strategy and representative dot plots for expression analysis of PD-L1 and MHC-II on CD14⁺ CD11b⁺ macrophages from the blood of M. avium subsp. paratuberculosis-infected cattle. Values in the quadrant indicate the percentages of the cells. (b and c) Percentages of PD-L1-expressing (b) and MHC-II-expressing (c) cells among CD14⁺ CD11b⁺ cells of peripheral blood, spleen, ileocecal LNs, and ileal and jejunal mLNs from uninfected cattle (n = 3) and M. avium subsp. paratuberculosis-infected cattle (n = 7). Comparisons of groups were performed using Welch's t test. Differences were considered significant when P < 0.05 (*, P < 0.05; **, P < 0.01). See also Table S2 in the supplemental material for individual expression analyses.

FIG 5

PD-L1 expression and localization of M. avium subsp. paratuberculosis in ileal mucosa of M. avium subsp. paratuberculosis-infected cattle. (A and B) Immunohistochemical staining of PD-L1 (A) and Ziehl-Neelsen staining for acid-fast bacilli (B) in ileum tissues. Immunohistochemical staining was performed using anti-PD-L1 MAb (6C11). Arrowheads and arrows indicate M. avium subsp. paratuberculosis-infected macrophages and epithelioid cells, respectively, both of which express PD-L1.

FIG 6

Reactivation of J-PPD-specific IFN-γ responses by LAG-3 blockade. Whole blood cells were cultured with blocking MAbs (anti-PD-1, anti-PD-L1, and anti-LAG-3 MAbs; 10 μg/ml) or rat IgG control in the presence of J-PPD (5 μg/ml). IFN-γ production in plasma was measured using ELISA (uninfected, n = 3; infected, n = 7). Comparisons of blocking MAbs in animal groups were performed using Tukey's test. Differences were considered significant when P < 0.05, indicated by an asterisk.

FIG 7

Reactivation of J-PPD-specific T cells by LAG-3 blockade. PBMCs from M. avium subsp. paratuberculosis-infected cattle (n = 7) and uninfected cattle (n = 3) were cultured with blocking MAbs (anti-PD-1, anti-PD-L1, and anti-LAG-3 MAbs; 10 μg/ml) or rat IgG control in the presence of J-PPD (10 μg/ml). (A) Representative dot plots for CD4⁺ CD69⁺ IFN-γ⁺ and CD8⁺ CD69⁺ IFN-γ⁺ T cells of an infected animal after treatment with B-PPD (left panels) or J-PPD with or without LAG-3 blockade (right and central panels, respectively). Values in the quadrant indicate the percentages of cells evaluated by flow cytometry. (B and C) Percentages of CD69⁺ IFN-γ⁺ cells among CD4⁺ (B) and CD8⁺ (C) T cells after treatment with J-PPD and blocking MAbs. Comparisons of blocking MAbs in animal groups were performed using Tukey's test. Differences were considered significant when P < 0.05, indicated by an asterisk.