Patrick De Baetselier

Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell–suppressive activity

The induction of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) is an important immune-evading mechanism used by tumors. However, the exact nature and function of MDSCs remain elusive, especially because they constitute a heterogeneous population that has not yet been clearly defined. Here, we identified 2 distinct MDSC subfractions with clear morphologic, molecular, and functional differences. These fractions consisted of either mononuclear cells (MO-MDSCs), resembling inflammatory monocytes, or low-density polymorphonuclear cells (PMN-MDSCs), akin to immature neutrophils. Interestingly, both MO-MDSCs and PMN-MDSCs suppressed antigen-specific T-cell responses, albeit using distinct effector molecules and signaling pathways. Blocking IFN-gamma or disrupting STAT1 partially impaired suppression by MO-MDSCs, for which nitric oxide (NO) was one of the mediators. In contrast, while IFN-gamma was strictly required for the suppressor function of PMN-MDSCs, this did not rely on STAT1 signaling or NO production. Finally, MO-MDSCs were shown to be potential precursors of highly antiproliferative NO-producing mature macrophages. However, distinct tumors differentially regulated this inherent MO-MDSC differentiation program, indicating that this phenomenon was tumor driven. Overall, our data refine tumor-induced MDSC functions by uncovering mechanistically distinct MDSC subpopulations, potentially relevant for MDSC-targeted therapies.

Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor κB

Cells of the monocyte–macrophage lineage play a central role in the orchestration and resolution of inflammation. Plasticity is a hallmark of mononuclear phagocytes, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic or M1 and alternative or M2. NF-κB is a key regulator of inflammation and resolution, and its activation is subject to multiple levels of regulation, including inhibitory, which finely tune macrophage functions. Here we identify the p50 subunit of NF-κB as a key regulator of M2-driven inflammatory reactions in vitro and in vivo. p50 NF-κB inhibits NF-κB–driven, M1-polarizing, IFN-β production. Accordingly, p50-deficient mice show exacerbated M1-driven inflammation and defective capacity to mount allergy and helminth-driven M2-polarized inflammatory reactions. Thus, NF-κB p50 is a key component in the orchestration of M2-driven inflammatory reactions.

Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages

Alternatively activated macrophages (aaMφ) display molecular and biological characteristics that differ from those of classically activated macrophages (caMφ). Recently, we described an experimental model of murine trypanosomosis in which the early stage of infection of mice with a Trypanosoma brucei brucei variant is characterized by the development of caMφ, whereas in the late and chronic stages of infection, aaMφ develop. In the present study, we used suppression subtractive hybridization (SSH) to identify genes that are expressed differentially in aaMφ versus caMφ elicited during infection with this T. b. brucei variant. We show that FIZZ1 and Ym1 are induced strongly in in vivo‐ and in vitro‐elicited aaMφ as compared with caMφ. Furthermore, we demonstrate that the in vivo induction of FIZZ1 and Ym1 in macrophages depends on IL‐4 and that in vitro, IFN‐γ antagonizes the effect of IL‐4 on the expression of FIZZ1 and Ym1. Collectively, these results open perspectives for new insights into the functional properties of aaMφ and establish FIZZ1 and Ym1 as markers for aaMφ.

Apolipoprotein L-I is the trypanosome lytic factor of human serum.

Human sleeping sickness in east Africa is caused by the parasite Trypanosoma brucei rhodesiense. The basis of this pathology is the resistance of these parasites to lysis by normal human serum (NHS). Resistance to NHS is conferred by a gene that encodes a truncated form of the variant surface glycoprotein termed serum resistance associated protein (SRA). We show that SRA is a lysosomal protein, and that the amino-terminal α-helix of SRA is responsible for resistance to NHS. This domain interacts strongly with a carboxy-terminal α-helix of the human-specific serum protein apolipoprotein L-I (apoL-I). Depleting NHS of apoL-I, by incubation with SRA or anti-apoL-I, led to the complete loss of trypanolytic activity. Addition of native or recombinant apoL-I either to apoL-I-depleted NHS or to fetal calf serum induced lysis of NHS-sensitive, but not NHS-resistant, trypanosomes. Confocal microscopy demonstrated that apoL-I is taken up through the endocytic pathway into the lysosome. We propose that apoL-I is the trypanosome lytic factor of NHS, and that SRA confers resistance to lysis by interaction with apoL-I in the lysosome.

A VSG Expression Site–Associated Gene Confers Resistance to Human Serum in Trypanosoma rhodesiense

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its resistance to a lytic factor present in human serum. In the ETat 1 strain this character was associated with antigenic variation, since expression of the ETat 1.10 variant surface glycoprotein was required to generate resistant (R) clones. In addition, in this strain transcription of a gene termed SRA was detected in R clones only. We show that the ETat 1.10 expression site is the one selectively transcribed in R variants. This expression site contains SRA as an expression site-associated gene (ESAG) and is characterized by the deletion of several ESAGs. Transfection of SRA into T.b. brucei was sufficient to confer resistance to human serum, identifying this gene as one of those responsible for T.b. rhodesiense adaptation to humans.

Efficient Cancer Therapy with a Nanobody-Based Conjugate

Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies are strictly monomeric, very stable, and highly soluble entities. We identified a nanobody with subnanomolar affinity for the human tumor-associated carcinoembryonic antigen. This nanobody was conjugated to Enterobacter cloacae beta-lactamase, and its site-selective anticancer prodrug activation capacity was evaluated. The conjugate was readily purified in high yields without aggregation or loss of functionality of the constituents. In vitro experiments showed that the nanobody-enzyme conjugate effectively activated the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug 7-(4-carboxybutanamido) cephalosporin mustard at the surface of carcinoembryonic antigen-expressing LS174T cancer cells. In vivo studies demonstrated that the conjugate had an excellent biodistribution profile and induced regressions and cures of established tumor xenografts. The easy generation and manufacturing yield of nanobody-based conjugates together with their potent antitumor activity make nanobodies promising vehicles for new generation cancer therapeutics.

Efficient tumor targeting by single‐domain antibody fragments of camels

The variable domain of functional heavy chain antibodies (VHH) devoid of light chains, found in camels, constitute the smallest intact antigen‐binding domain fragment. Two camel single‐domain fragments, cAb‐Lys2 and cAb‐Lys3, recognizing an overlapping epitope of lysozyme with a dissociation constant of 2 nM and 65 nM, respectively, and a bivalent cAb‐Lys3 were investigated for their ability to target transgenic tumors expressing lysozyme on their membrane. Biodistribution studies revealed that these non‐immunogenic monomeric and bivalent camel single‐domain antigen binders specifically target lysozyme‐expressing tumors and metastatic lesions. The excess of antibody is rapidly eliminated from the blood circulation and no cAb retention was observed in normal organs. The tumor to organ cAb‐ratios at 2 and 8 hr were in the (2.1–10.8):1 and (6.2–23.7):1 range, respectively. The degree and specificity of tumor retention is independent of the affinity of the recombinant camel single‐domain fragments for their antigen and from their univalent monomeric (15 kDa) or bivalent format (33 kDa). This study demonstrates the successful and specific in vivo targeting of tumors by camel single‐domain fragments. It may open perspectives for their future use as tumor‐targeting vehicle, due to their small size, soluble behaviour and because they are non‐immunogenic and interact with epitopes that are less antigenic for conventional antibodies.

Arginase-1 and Ym1 are markers for murine, but not human, alternatively activated myeloid cells

In an interesting paper, Scotton et al. ([1][1]) reported the effects of IL-13 on the transcriptional profile of human monocytes. Comparing their results with studies analyzing the profile induced by IL-4 in murine macrophages ([2][2], [3][3]), the authors notice some differences, giving as examples

Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation

Molecular markers, especially surface markers associated with type II, cytokine‐dependent, alternatively activated macrophages (aaMF), remain scarce. Besides the earlier documented markers, macrophage mannose receptor and arginase 1, we demonstrated recently that murine aaMF are characterized by increased expression of found in inflammatory zone 1 (FIZZ1) and the secretory lectin Ym. We now document that expression of the two members of the mouse macrophage galactose‐type C‐type lectin gene family (mMGL1 and mMGL2) is induced in diverse populations of aaMF, including peritoneal macrophages elicited during infection with the protozoan Trypanosoma brucei brucei or the Helminth Taenia crassiceps and alveolar macrophages elicited in a mouse model of allergic asthma. In addition, we demonstrate that in vitro, interleukin‐4 (IL‐4) and IL‐13 up‐regulate mMGL1 and mMGL2 expression and that in vivo, induction of mMGL1 and mMGL2 is dependent on IL‐4 receptor signaling. Moreover, we show that expression of MGL on human monocytes is also up‐regulated by IL‐4. Hence, macrophage galactose‐type C‐type lectins represent novel surface markers for murine and human aaMF.

Nanobodies as novel agents for cancer therapy.

Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. As such, the cloning and selection of antigen-specific nanobodies obviate the need for construction and screening of large libraries, and for lengthy and unpredictable invitro affinity maturation steps. The unique and well-characterised properties enable nanobodies to excel conventional therapeutic antibodies in terms of recognising uncommon or hidden epitopes, binding into cavities or active sites of protein targets, tailoring of half-life, drug format flexibility, low immunogenic potential and ease of manufacture. Moreover, the favourable biophysical and pharmacological properties of nanobodies, together with the ease of formatting them into multifunctional protein therapeutics, leaves them ideally placed as a new generation of antibody-based therapeutics. This review describes the state of the art on nanobodies and illustrates their potential as cancer therapeutic agents.