Additional file 2: Figure S1. of Microbial functional genes enriched in the Xiangjiang River sediments with heavy metal contamination

Detrended correspondence analysis (DCA) of all functional genes in three sites. (PDF 8 kb

Macrophage treated with different <i>M</i>. <i>leprae</i> antigens showed committed functional differentiation.

<p>Live <i>M</i>. <i>leprae</i>-infected macrophages or killed <i>M</i>. <i>leprae</i>-treated macrophages were either restimulated with killed <i>M</i>. <i>leprae</i>, or cultured in plain culture medium for 6 days. Cytokine expression during the restimulation period was measured by Luminex assay. **: p < 0.01. ***: p < 0.001.</p

Macrophages incubated with viable <i>M</i>. <i>leprae</i> suppressed CD8⁺ T cell cytotoxicity.

<p>Viable or killed <i>M</i>. <i>leprae</i> were incubated with T cells for 6 days, after which the CD8⁺ T cells were negatively purified from the coculture and were added to chromium-51-labeled target cells at the indicated effector-to-target ratio. (A) Percentage specific lysis using purified autologous monocyte loaded with sonicated <i>M</i>. <i>leprae</i> antigen as the target cells. (B) Percentage specific lysis using in vitro derived, <i>M</i>. <i>leprae</i>-infected macrophages as the target cells. The CD8⁺ T cell-induced cytotoxicity (isolated CD8⁺ T cell + target cell culture minus naive T cell + target cell culture) was shown in (C) and (D), at 27-to-1 effector-to-target ratio. The effectors are (C) CD8⁺ T cells incubated with killed <i>M</i>. <i>leprae</i>-stimulated macrophages and (D) CD8⁺ T cells incubated with live <i>M</i>. <i>leprae</i>-infected macrophages. N = 6, with two independent repetitions. *: p < 0.05. **: p < 0.01. ***: p < 0.001.</p

Viable <i>M</i>. <i>leprae</i> induced M2-type macrophage differentiation.

<p>Peripheral blood monocytes were obtained from healthy volunteers and were differentiated into macrophages in vitro. Viable or heat-killed <i>M</i>. <i>leprae</i> were added to the macrophage culture for 6 days. (A) Cytokine expression by macrophages during coculture, as measured by Luminex assay. N = 6. (B) Mean fluorescence intensity (MFI) of MHC class II and CD163 expression on macrophages after 6-day incubation in all healthy volunteers. N = 6. *: p < 0.05. **: p < 0.01. ***: p < 0.001.</p

Live <i>M</i>. <i>leprae</i>-infected macrophages preferentially primed Treg cell responses.

<p>Autologous CD45RA⁺ naive T cells were incubated with live <i>M</i>. <i>leprae</i>-infected macrophages or killed <i>M</i>. <i>leprae</i>-treated macrophages for 6 days. The T cells were then negatively selected and incubated separately in anti-CD3/CD28-stimulated media for an additional 72 hours, after which the supernatant was collected for ELISA and cells for flow cytometry. (A) Summary of IFN-gamma and IL-10 concentration from all healthy volunteers in the supernatant. N = 6. (B) Ratio of IFN-gamma-to-IL-10 in each individual. (C) Mean fluorescence intensity (MFI) of FoxP3 expression in T cells from all treatment conditions. N = 6. *: p < 0.05. **: p < 0.01.</p

Design and Synthesis of Isoquinolidinobenzodiazepine Dimers, a Novel Class of Antibody–Drug Conjugate Payload

Antibody–drug conjugates (ADCs) represent an important class of emerging cancer therapeutics. Recent ADC development efforts highlighted the use of pyrrolobenzodiazepine (PBD) dimer payload for the treatment of several cancers. We identified the isoquinolidinobenzodiazepine (IQB) payload (D211), a new class of PBD dimer family of DNA damaging payloads. We have successfully synthesized all three IQB stereoisomers, experimentally showed that the purified (<i>S</i>,<i>S</i>)-D211 isomer is functionally more active than (<i>R</i>,<i>R</i>)-D221 and (<i>S</i>,<i>R</i>)-D231 isomers by >50,000-fold and ∼200-fold, respectively. We also synthesized a linker-payload (D212) that uses (<i>S</i>,<i>S</i>)-D211 payload with a cathepsin cleavable linker, a hydrophilic PEG8 spacer, and a thiol reactive maleimide. In addition, homogeneous ADCs generated using D212 linker-payload exhibited ideal physicochemical properties, and anti-CD33 ADC displayed a robust target-specific potency on AML cell lines. These results demonstrate that D212 linker-payload described here can be utilized for developing novel ADC therapeutics for targeted cancer therapy