The human macrophage cell line U937 as an in vitro model for selective evaluation of mycobacterial antigen-specific cytotoxic T-cell function

Abstract

Despite strong evidence for CD8+ T-cell function in murine mycobacterial infections, their corresponding role in human tuberculosis has proven more difficult to demonstrate. We have evaluated the human macrophage (Mphi) cell line U937 as an in vitro model for human leucocyte antigen (HLA) class I-restricted presentation of mycobacterial antigens, as HLA class I is constitutively expressed at high levels by U937 cells in the absence of detectable HLA class II or CD1 molecules. U937 cells were evaluated for their ability to phagocytose Mycobacterium tuberculosis and for their ability to present mycobacterial antigens to human HLA class I-matched cytotoxic T lymphocytes (CTLs). Differentiated U937 cells were capable of efficient phagocytosis of M. tuberculosis but did not generate a subsequent respiratory burst response, and were permissive for intracellular growth of both bacillus Calmette-Guérin (BCG) and the virulent M. tuberculosis H37Rv strain. CTL activity was restricted to live mycobacterial organisms and was shown to be mediated by M. tuberculosis-specific, HLA class I-matched, purified CD8+ CTL lines and CD8+ T-cell clones. Furthermore, M. tuberculosis-infected U937 targets were more rapidly and strongly lysed by CD8+ CTLs than were infected autologous Mphi. Finally, M. tuberculosis-infected U937 cells simultaneously provided a sensitive indicator for detection of mycobacterial-specific, HLA-unrestricted gammadelta+ CTL activity.

Figure 1

The effect of differentiation with phorbol 12-myristate 13-acetate (PMA), interferon-γ (IFN-γ) or 1,25-dihydroxyvitamin D₃ (VD₃) on the ability of U937 cells to bind (a) and phagocytose (b) Mycobacterium tuberculosis, generate a respiratory burst response following infection (c) and control intracellular mycobacterial growth (d). (a) The ability of untreated, PMA- (5 ng/ml), IFN-γ- (200 IU/ml), or VD₃- (10⁻⁷ m) differentiated U937 cells to bind M. tuberculosis (10 colony-forming units [CFU]/cell) in the absence (top panel) or presence (bottom panel) of fresh human serum was investigated using Zeihl–Neilson (ZN) staining and light microscopy. M.tb, Mycobacterium tuberculosis. (b) Transmission electron micrograph (TEM) photomicrograph of M. tuberculosis contained in membrane-bound phagosomes within PMA-differentiated U937 cells (× 5000 magnification). (c) Untreated, PMA-, IFN-γ- or VD₃-differentiated U937 cells were assessed for their ability to mount a respiratory burst by Nitro-blue tetrazolium (NBT) reduction following stimulation with PMA (20 µg/ml; top panel) or infection with M. tuberculosis H37Rv (50 CFU/cell; bottom panel). *Represents significantly (P < 0·05) increased ability to reduce NBT following differentiation. (d) The permissiveness of PMA-differentiated (○), IFN-γ-differentiated (▴) or VD₃-differentiated (□) U937 for intracellular growth of M. tuberculosis H37Rv (0·1 CFU/cell; top panel) or bacillus Calmette–Guérin (BCG) (0·1 CFU/cell; bottom panel) was assessed over a period of 5 days. Each bar/data point represents the mean percentage (± SD) of at least three independent experiments.

Figure 2

Natural killer (NK) (a) and lymphokine-activated killer (LAK) (b) cell-mediated cytotoxicity against untreated U937 cells (▿), phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells (▵), K562 (•) and Daudi (▪). Cytotoxicity was determined after 4 hr. Each data point represents the mean percentage kill of duplicate experiments.

Figure 3

(A) Mycobacterium tuberculosis-specific cytolysis generated by the (a) human leucocyte antigen (HLA)-A3-matched (n = 4; TS, PB, ES and EC), (b) HLA-B51-matched (n = 2; RG and BR), and (c) HLA-mismatched (n = 6; MH, NP, SG, SJ, MF and WM) donor cytotoxic T lymphocytes (CTL) against U937 (top panel) and autologous macrophage (Mφ) targets (bottom panel). (B) Mycobacterial antigen-specific cytolysis generated by HLA-B51-matched donor CTLs (n = 1; RG) against U937 targets infected with (a) M. tuberculosis H37Rv (5 colony-forming units [CFU]/cell) (b) bacillus Calmette–Guérin (BCG) (5 CFU/cell), or (c) pulsed with the soluble mycobacterial extract purified-protein derivative (PPD) (3 µg/ml). Target cells were infected with M. tuberculosis (5 CFU/cell) (▪) or pulsed with an irrelevant antigen, streptokinase-streptodornase (SK-SD) (○). Cytotoxicity against U937 and autologous Mφ targets was measured after 4 hr. Each data point represents the mean percentage cytolysis (± SEM) of at least three independent experiments.

Figure 4

(A) Characterization of cytolytic T-cell subsets generating mycobacterial-specific cytolysis against U937 target cells. Mycobacterium tuberculosis-primed human leucocyte antigen (HLA)-B51-matched cytotoxic T lymphocytes (CTL) (RG and BR; top panel) and the HLA-mismatched CTL (MF and WM; bottom panel) populations were either (a) not separated, or fractionated into (b) CD8⁺ (c) CD4⁺ or (d) γδ⁺ T-cell subsets, and assessed for their respective cytolytic abilities against U937 target cells. U937 target cells were either infected with M. tuberculosis (5 colony-forming units [CFU]/cell) (▪) or pulsed with an irrelevant antigen, streptokinase-streptodornase (SK-SD) (○). Each data point represents the mean percentage cytolysis (± SEM) of at least three independent experiments. (B) Comparison of CD8⁺ CTL cytolytic activity generated by HLA-B51-matched donors (n = 2; RG and BR) against U937 versus macrophage (Mφ) targets. Mycobacterial-specific cytolysis was calculated according to the following equation: $\begin{array}{c}\text{Mycobacterial-specific cytolysis}=\left[\right(\%\text{cytolysis against} \mathrm{M.\; tuberculosis}\text{-infected targets})\\ \phantom{\text{sdifusdfiu}\text{sdifusdfiu}\text{sdifusdfi}}-(\%\text{cytolysis against SK-SD-pulsed targets})]\end{array}$ Cytotoxicity was measured after 4 hr for U937 targets, and after 4 and 18 hr for Mφ targets (as indicated). Each box-and-whisker plot shows the distribution, median (solid line), mean (dotted line), 10th and 90th percentile of six independent experiments. M.tb, Mycobacterium tuberculosis.

Figure 5

The effect of priming conditions on mycobacterial-specific CD8⁺ cytotoxic T lymphocyte (CTL) cytolytic function. (a) CD25 (interleukin-2 receptor [IL-2R]) expression and (b) mycobacterial-specific cytolytic activity against monocyte-derived macrophages (Mφ) was assessed following different CTL priming procedures. Mycobacterial-specific CTLs were generated from peripheral blood mononuclear cells (PBMC) stimulated with: (i) bacillus Calmette–Guérin (BCG) (1 colony-forming unit [CFU]/ml) (ii), Mycobacterium tuberculosis (M.tb) H37Rv (1 CFU/ml) or (iii) M. tuberculosis H37Rv (1 CFU/ml) in the presence of additional recombinant human IL-2 (M.tb/IL-2) (10 IU/ml) and CD8⁺ cells isolated after 6 days. Alternatively, CTLs were generated by (iv) stimulating PBMC with M. tuberculosis for 24 hr, isolating the CD8⁺ CTLs and then culturing these in the presence of rhIL-2 (50 IU/ml) for the remaining 5 days [M.tb (24 hr)/IL-2]; or (v) primed with M. tuberculosis-infected, osmotically lysed Mφ and then isolating the CD8⁺ T cells after 6 days. Mycobacterial-specific cytolysis was calculated according to the following equation: $\begin{array}{c}\text{Mycobacterial-specific cytolysis}=\left[\right(\%\text{cytolysis against} \mathrm{M.\; tuberculosis}-\text{infected targets})\\ \phantom{\text{sdifusdfiu}\text{sdifusdfiu}\text{sdifusdf}}-(\%\text{cytolysis against SK-SD-pulsed targets})]\end{array}$ Cytotoxicity was measured after 18 hr. Each box-and-whisker plot shows the distribution, median (solid line), mean (dotted line), 10th and 90th percentile of six independent experiments.

Figure 6

Cytotoxic and proliferative responses of mycobacterial antigen-specific cytotoxic T-lymphocyte (CTL) clones generated from a human leucocyte antigen (HLA)-B51-matched donor (RG). (A) Both CD8⁺ (a) and γδ⁺ (b) CTL clones mediate mycobacterial-specific cytolytic activity against U937 target cells. U937 target cells were either infected with Mycobacterium tuberculosis (5 colony-forming units [CFU]/cell) (filled bars) or pulsed with an irrelevant antigen, streptokinase-streptodornase (SK-SD) (open bars). Cytotoxicity was measured after 4 hr. Each data point represents the mean percentage cytolysis (± SD) of at least four independent experiments. (B) Proliferative responses of CD8⁺ T-cell clones to (a) soluble purified-protein derivative (PPD) or (b) M. tuberculosis. CD8⁺ T-cell clones were unstimulated, or stimulated with PPD (3 µg/ml) or M. tuberculosis (5 CFU/cell) in triplicate wells in the presence of autologous irradiated peripheral blood mononuclear cells (PBMCs). After 40 hr, cultures were pulsed with [³H]thymidine (1 µCi/well) for 8 hr. Results are expressed as stimulation indices (SI) (in counts per minute [c.p.m.]) and were calculated as follows: $\begin{array}{c}\text{SI}=\left[\right(\text{mean c.p.m. of antigen-stimulated wells})\\ \phantom{\text{s}}÷\left(\text{mean c.p.m. of unstimulated wells}\right)]\end{array}$ Each bar represents the mean SI (± SD) of triplicate wells.