Mannose-Capped Lipoarabinomannan from Mycobacterium tuberculosis Induces CD4+ T Cell Anergy via GRAIL

Abstract

Mycobacterium tuberculosis cell wall glycolipid, lipoarabinomannan, can inhibit CD4(+) T cell activation by downregulating the phosphorylation of key proximal TCR signaling molecules: Lck, CD3ζ, ZAP70, and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an Ag encounter, yet remain viable and hyporesponsive. We tested whether mannose-capped lipoarabinomannan (LAM)-induced inhibition of CD4(+) T cell activation resulted in CD4(+) T cell anergy. The presence of LAM during primary stimulation of P25 TCR-transgenic murine CD4(+) T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25 TCR-transgenic CD4(+) T cells primed in the presence of LAM also exhibited decreased response upon restimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hyporesponsiveness to restimulation was not due to apoptosis, generation of Foxp3-positive regulatory T cells, or inhibitory cytokines. Acquisition of the anergic phenotype correlated with upregulation of gene related to anergy in lymphocytes (GRAIL) protein in CD4(+) T cells. Inhibition of human CD4(+) T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA-mediated knockdown of GRAIL before LAM treatment abrogated LAM-induced hyporesponsiveness. In addition, exogenous IL-2 reversed defective proliferation by downregulating GRAIL expression. These results demonstrate that LAM upregulates GRAIL to induce anergy in Ag-reactive CD4(+) T cells. Induction of CD4(+) T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition.

FIGURE 1

Presence of LAM on CD4⁺ T cell membranes is required for inhibition of CD4⁺ T cell activation after primary stimulation. (A) CD4⁺ T cells (1×10⁶) were pre-treated with LAM (1 μM) or media (untreated) for 1 h at 37°C, washed and incubated with IL-7 (20 ng/ml). Cells were chased for 0, 24, 48 and 96 h. At the end of each chase period, cell aliquots were removed and washed with media, prior to LAM staining. LAM-treated T cells were stained with anti-LAM mAb or isotype control and analyzed by flow cytometry. (B, C) LAM-treated and untreated CD4⁺ T cells at the end of each chase period were activated with plate-bound anti-CD3 (1 μg/ml) and soluble anti-CD28 (1 μg/ml). IL-2 was measured in 24 h culture supernatants by ELISA (B) and T cell proliferation was measured after 48 h by [³H] thymidine incorporation (C). Percent inhibition reflects the ratio of IL-2 (B) or proliferation (C) between LAM-treated and untreated T cells x 100. Error bars indicate mean +/− SD of triplicate wells of one representative experiment (n=3).

FIGURE 2

LAM induces anergy in P25 CD4⁺ T cells. (A) Experimental design. (B) P25 TCR-Tg CD4⁺ T cells (1×10⁶) pretreated with LAM for 1 h were stained with anti-LAM mAb or with an isotype control mAb before priming (left histogram) or 5 d after priming and before re-stimulation (right histogram), and analyzed by flow cytometry. (C) 1 h after pretreatment, untreated (none), LAM-treated or ionomycin-treated T cells were primed with Ag 85B peptide-pulsed APC for 48 h. IL-2 was measured in 24-h culture supernatants by ELISA. (D) Primed P25 TCR-Tg CD4⁺ T cells (in C) were cultured for 5 d in IL-7. Cells were washed and re-stimulated with Ag85B peptide-pulsed APC. IL-2 was measured in 24 h-culture supernatants by ELISA (upper panel) and cell proliferation was determined after 48 h by [³H] thymidine incorporation (lower panel). Data in C through D are means +/− SEM of three independent experiments conducted in triplicate. ***P<0.001 (paired t test)

FIGURE 3

LAM does not induce FoxP3-positive regulatory T cells and/or increase activation-Induced cell death (apoptosis). (A) 1×10⁶ P25 TCR-Tg CD4⁺ T cells left untreated (none) or treated with LAM (as described in Fig. 2) were primed with APC-Ag85B peptide for 48 h. Cells were washed and cultured in IL-7 for 5 d. Cells were washed, and Foxp3-positive T cells were determined by intracellular staining and analyzed by flow cytometry. (B) 1×10⁶ T reg depleted P25 TCR-Tg CD4⁺ T cells were left untreated (none) or pre-treated with LAM (1 μM) for 1 h. Cells were washed and primed with Ag 85B peptide-pulsed (APC + Pep) or mock-pulsed APC (APC). After 48 h cells were washed and rested in media containing IL-7 for 5 days. CD4⁺ T cells were then washed and re-stimulated for 24 h. IL-2 was measured in 24-h culture supernatants by ELISA. (C) P25 TCR-Tg CD4⁺ T cells suppressed through treatment with LAM or ionomycin (as described in Fig. 2) were re-stimulated with APC-Ag85B peptide. After 24 h, T cells were stained with Annexin V eFluor 450 and apoptosis was measured by flow cytometry analysis of annexin V-positive T cells. At re-stimulation, T cells treated with 1 μM of dexamethasone (Dexa) were used as a positive control for apoptosis. Error bars indicate means +/− SD of triplicate wells of one representative experiment (n=3). ***P<0.001 (paired t test).

FIGURE 4

LAM does not affect receptor expression on LAM-anergized P25 TCR-Tg CD4⁺ T cells. (A) 1×10⁶ CD4⁺ T cells were pre-treated with either LAM or left untreated (none) and co-cultured with Ag85B-pulsed APCs. After 48 h, cells were collected, washed and labeled with anti-PD1, anti-Lag3 or anti-Tim 3, anti-CD28, anti-CD40L mAbs. Intracellular staining was performed for CTLA4. Gating was performed on live CD4⁺ T cells. Alternatively, after 48 h cells were collected, washed and cultured in IL-7 for 5 d. Cells were then stained for surface expression of TCR and CD3. Gating was performed on live CD4⁺ T cells. Representative histograms of at least three independent experiments are shown. (B) The mean fluorescence intensity (MFI) of PD-1 staining in LAM-treated (LAM) and untreated (none) T cells was quantified after 48 h of primary stimulation. Data are means +/− SEM of three independent experiments.

FIGURE 5

LAM induces increased GRAIL protein expression both during priming and upon re-stimulation. (A) Untreated and LAM-pre-treated P25 TCR-Tg CD4⁺ T cells were stimulated with Ag85B peptide-pulsed APC for the indicated times. GRAIL protein expression was measured in cell lysates from re-purified CD4⁺ T cells by Western blot. Blots were analyzed with ImageJ software (NIH) and the ratios of band intensities of GRAIL/beta-actin expressed as relative densities (bar graph). (B) P25 TCR-Tg CD4⁺ T cells were left untreated (none) or pretreated with LAM or ionomycin and primed with Ag85B peptide-pulsed APC. Cells were washed and cultured in IL-7 for 5 d. Cells were re-stimulated with Ag85B peptide-pulsed APC for 24 h. T cell lysates were obtained from re-stimulated CD4⁺ T cells, and Western blot performed for GRAIL protein. Data presented are the means +/− SEM of three independent experiments conducted in triplicate. ***P<0.001 (paired t test).

FIGURE 6. Knockdown of GRAIL expression by siRNA prevents inhibition of CD4⁺ T cell activation by LAM

(A, B) A total of 3×10⁶ pre-activated naïve CD4⁺ T cells plated at 3×10⁵ cells per well in a 24-well plate were transfected with 75 nM or 100 nM of siRNAs targeting GRAIL (GRAIL siRNA) or non-targeting control siRNA (NC) using 6 μl HiPerfect dissolved in serum-free Opti-MEM medium. After 6 h of transfection, an additional 400 μl DMEM medium with 10% FBS was added and cells allowed to incubate at 37°C for 24 h. After 24 h, GRAIL was measured by Western blot with β-actin as loading control. (C, D) GRAIL knocked-down or control naïve CD4⁺ T cells were left untreated (None) or treated with LAM for 1 h at 37°C. Cells were washed and re-stimulated with plate-bound anti-CD3ε and soluble anti-CD28 for 48 h. IL-2 was measured in 24 h culture supernatants by ELISA (C) and T cell proliferation after 48 h by [³H] thymidine incorporation (D). For A-D, representative blots and densitometry data are shown. (E, F) A total of 3×10⁶ Th1 effector CD4⁺ T cells plated at 3×10⁵ cells per well in a 24-well plate were transfected and cultured as in (A, B) above. After 24 h, GRAIL was measured by Western blot. (G, H) GRAIL knocked down or control Th1 effector CD4⁺ T cells were left untreated (None) or treated with LAM for 1 h at 37°C. Cells were washed and re-stimulated as above for 48 h. IL-2 (G) and IFN-γ (H) were measured in 24 h and 72 h culture supernatants respectively by ELISA. IL-2 and proliferation data shown are means +/− SEM of three independent experiments. *P< 0.05, **P< 0.01 (paired t test)

FIGURE 7. Exogenous IL-2 down-regulates GRAIL expression and restores T cell proliferation in LAM-anergized CD4⁺ T cells

(A) P25 TCR-Tg CD4⁺ T cells (1×10⁶) were left untreated (none) or treated with LAM (1 μM) or ionomycin (1 μM) for 1 h. Cells were washed and primed with Ag85B peptide-pulsed APC. After 48 h, cells were washed and rested in media containing IL-7 for 5 days. CD4⁺ T cells were washed and re-stimulated with Ag85B peptide-pulsed APC with or without addition of exogenous rIL-2 (20 ng/ml). Cell proliferation was measured after 48 h by [³H] thymidine incorporation. Data are means +/− SEM of three independent experiments conducted in triplicates. *P< 0.05, **P< 0.01 (paired t test). (B) P25 TCR-Tg CD4⁺ T cells were pretreated with LAM and primed with Ag85B peptide-pulsed APC. Cells were washed and cultured in IL-7 for 5 d. Cells were re-stimulated with Ag85B peptide-pulsed APC with or without addition of exogenous rIL-2 (20 ng/ml) for 48 h. T cell lysates were obtained and GRAIL protein measured by Western blot. A representative blot and densitometry for 3 experiments is shown.

FIGURE 8

LAM associates with lipid rafts and CD3 on human CD4⁺ T cells, and up-regulates GRAIL upon activation with anti-CD3/CD28. (A) Human CD4⁺ T cells were incubated with LAM for 1 h at 37°C. Top panels, after incubation with LAM, cells were incubated with Alexa Fluor 647 conjugated-cholera toxin subunit B (CT-B, 1 μg/ml) for 20 min on ice, washed extensively, labeled with anti-LAM mAb (clone Cs35) followed by Alexa Fluor 488 conjugated anti-mouse IgG and fixed with 1% paraformaldehyde. Lower panels, after incubation with LAM, cells were labeled on ice with anti-LAM mAb (clone Cs35) followed by Alexa Fluor 488 conjugated-anti-mouse IgG. Then, Alexa Fluor 647 congugated-anti-CD3 mAb was used to label the CD3-TCR complex. Cells were visualized and images merged in a Leica DM16000B confocal microscope (100X oil immersion lens). (B) Human CD4⁺ T cells isolated from four different Mtb uninfected adult donors were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of indicated LAM concentrations for 24 h, followed by measurement of IL-2 levels by ELISA. (C) Human CD4⁺ T cells (1×10⁶) were left untreated (none) or pretreated with LAM (1 μM), followed by stimulation as above. Cells were stained for surface CD4 and intracellular GRAIL using APC-CD4 mAb and rabbit anti-GRAIL primary antibody, respectively. FITC-labeled anti-rabbit secondary antibody was used, followed by FACS analysis. (D) The mean fluorescence intensity (MFI) of GRAIL staining in LAM-treated (LAM) and untreated (none) T cells was quantified after 48 h of primary stimulation. Error bars indicate mean +/− SD of triplicate wells of one representative experiment.