Fig 2: LILRB2 antagonism enhances inflammatory cytokines in Mtb-infected MDSCs. CD14+/CD33+ bead-purified M-MDSCs derived from GM-CSF/IL-6 cultured PBMCs from donors were infected for 4 h with Mtb H37Rv (MOI = 1), washed, and activated using either a monoclonal antibody to LILRB2 (mab-B2; 1 µg/ml/10*6 cells) or its IgG isotype (1 µg/ml/10*6 cells) and SHP 1/2 inhibitors (1 µg/ml of each per 10*6 cells). Cells were lysed in trizol for qPCR and supernatants were tested for cytokines. (A) qPCR mRNA quantitation after mab-B2 or IgG treatment of Mtb-infected MDSCs at 18 h post infection (*p < 0.02 **, unpaired t-test). (B) Supernantants were collected at 18 h post infection and cytokines were assayed using sandwich ELISA (*p < 0.01 **, unpaired t-test).

Fig 3: Prior treatment of MDSCs with monoclonal antibodies to leukocyte immunoglobulin-like receptors B2 and B3 (LILRB2/3) enhances their bactericidal function against Mtb. (A, B) CD33+ flow sorted MDSCs from donor PBMCs (A) or CD14+/CD33+ bead-purified M-MDSCs derived from GM-CSF/IL-6 cultured PBMCs from donors (B) were activated or not with monoclonal antagonist antibodies to LILRB2 (mab-B2) or LILRB3 (mab-B3) followed by infection with Mtb H37Rv (MOI = 1) and incubation over time. Lysates were plated at indicated time points for Mtb CFUs on 7H11 agar (*, **p < 0.007 1-way ANOVA and Tukey’s posttest; 1 of 2 similar experiments shown).

Fig 4: Mab-B2 antagonism of LILRB2 and pharmacological inhibition of SHP1/2 synergistically boost killing of Mtb in MDSCs. CD14+/CD33+ bead-purified M-MDSCs derived from GM-CSF/IL-6 cultured PBMCs from donors were infected for 4 h with Mtb H37Rv (MOI = 1), washed, and activated using either a monoclonal antibody to LILRB2 (mab-B2) or its IgG isotype with or without added SHP-1 and SHP-2 inhibitors (1 µg/ml each per 10*6 cells). (A) MDSC lysates collected 18 h after infection were plated for Mtb CFUs on 7H11 agar (**p < 0.002 by 1- way ANOVA with Tukey’s posttest; 1 of 2 similar experiments shown).(B) Lysates collected from replicate cultures of MDSCs were analyzed using Wes simple Western blot for phosphorylated ERK1/2 and p-38 kinases, and densitometry is shown to the right (*p < 0.02, unpaired t-test).

Fig 5: LILRB2 antagonism effects on the bactericidal function of the MDSCs are independent of autophagy. (A) CD14+/CD33+ bead-purified M-MDSCs derived from GM-CSF/IL-6 cultured PBMCs from donors were subjected to autophagy blockade using siRNA vs. beclin1 (ATG6) or its scrambled control, followed by infection with Mtb H37Rv (MOI = 1), washed, and incubated after activation with mab-B2 or isotype. MDSC lysates were plated on day 3 for Mtb CFUs on 7H11 agar (triplicate wells per point; p < 0.008, 1-way ANOVA and Tukey’s posttest; 1 of 2 similar experiments shown). (B) Lysates of cells from panel A were collected at 18 h and analyzed using Western blot for the autophagy marker, microtublue-associated light chain-3 (LC3). Lipidation is indicated by LC3-II. (C) Densitometry of cells from panel B indicates the expression of LC3 normalized to GAPDH. (*p < 0.01 t-test). (D) M-MDSCs were infected with gfpMtb (4 h MOI = 1) followed by fixation in 3.7% paraformadehyde and staining using mab to LC3 followed by Alexafluor590 anti-IgG conjugate. Images were acquired using an N90 Nikon scope with Metaview deconvolution software. (E) Data show quantitation of gfpMtb phagosomes colocalizing with mab-LC3 staining (*p < 0.05, **p < 0.005, t-test; 1 of 2 similar experiments shown).