Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo.

Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.

Outcomes of Haploidentical Transplants with PT-CY versus 10/10 MUD Transplants with ATG in Germany.

Allogeneic stem cell transplantation (alloHSCT) is the best curative treatment modality for many malignant haematological disorders. In the absence of a matched related donor (MRD), matched unrelated donors (MUD) and haploidentical donors (Haplo-Tx) are the most important sources of stem cells. However, multicenter real-life data which compare 10/10 MUD transplantations with Haplo-Tx is still limited. In this registry based retrospective study, we compared the outcomes of alloHSCTs from 10/10 MUD with anti-thymocyte globulin (ATG) based regimens (n=7050) versus Haplo-Tx using post-transplant cyclophosphamide (PT-CY Haplo) (n=487) in adult patients with haematological malignancies between 2010 and 2020. Cox proportional hazard models and competing risks regression models were formed to compare the outcomes of the groups. OS, DFS and GRFS were superior for 10/10 MUD (OS: HR 1.27, CI 1.10- 1.47, p=0.001, DFS: HR 1.17, CI 1.02-1.34, p=0.022, GRFS: HR 1.34, CI 1.19-1.50, p<0.001). Risk for aGVHD grade II-IV, aGVHD grade III-IV and cGVHD was higher in the PT-CY Haplo group compared to the 10/10 MUD group (aGVHD grade II-IV: HR 1.46, CI 1.25- 1.71, p<0.001; aGVHD grade III-IV: HR 1.74, CI 1.37- 2.20, p<0.001 and cGVHD: HR 1.30, CI 1.11-1.51, p=0.001). A lower incidence of relapse was observed in the PT-CY Haplo group (relapse: HR 0.83, CI 0.69-0.99, p=0.038). Unrelated 10/10 matched transplantation with ATG treatment leads to lower GvHD rates and improved survival rates when compared to PT-CY Haplo transplantation in Germany.

The impact of MICB mismatches in unrelated haematopoietic stem cell transplantation.

MICA polymorphisms have been associated with increased incidence of acute GvHD and adverse outcome in allogeneic haematopoietic stem cell transplantation (HSCT). MICB is another expressed member of MHC class I-related chain genes and its impact on HSCT outcome is yet to be fully defined. We typed a large cohort of patients and donors for MICB polymorphisms and investigated the impact of MICB matching on outcome after unrelated HSCT. 69.2% of the patients were 10/10 human leukocyte antigen (HLA) matched and 30.8% were 9/10 HLA matched. MICB typing was performed using a short amplicon-based NGS typing assay on the Illumina MiSeq platform. Differences in proteins were considered as mismatches. MICA polymorphisms were identified as possible confounder and were therefore included as parameter in the multivariate analyses. Due to the strong linkage disequilibrium with the classical HLA-genes, sub-stratification for HLA matching status was necessary, and no effect of MICB mismatches was seen in the 10/10 HLA matched group when compared to the MICB matched cases. However, in the 9/10 HLA matched group, MICB mismatched cases showed significantly worse disease free survival (DFS), GvHD and relapse free survival (GRFS) compared to the MICB matched cases (DFS: HR 1.24, p = 0.011; GRFS: HR 1.26, p = 0.002). MICA mismatches had no impact on any outcome parameter. According to our findings, effects previously attributed to MICA differences may have been confounded by MICB polymorphisms. We show that MICB differences contribute a small but relevant effect in 9/10 HLA-matched transplantations, which in turn highlights the possible usefulness of MICB typing in donor selection among similarly suitable 9/10 matched donors, especially when HLA-B mismatches have to be accepted.

Clostridial C3 Toxins Target Monocytes/Macrophages and Modulate Their Functions.

The C3 enzymes from Clostridium (C.) botulinum (C3bot) and Clostridium limosum (C3lim) are single chain protein toxins of about 25 kDa that mono-ADP-ribosylate Rho-A, -B, and -C in the cytosol of mammalian cells. We discovered that both C3 proteins are selectively internalized into the cytosol of monocytes and macrophages by an endocytotic mechanism, comparable to bacterial AB-type toxins, while they are not efficiently taken up into the cytosol of other cell types including epithelial cells and fibroblasts. C3-treatment results in disturbed macrophage functions, such as migration and phagocytosis, suggesting a novel function of clostridial C3 toxins as virulence factors, which selectively interfere with these immune cells. Moreover, enzymatic inactive C3 protein serves as a transport system to selectively deliver pharmacologically active molecules into the cytosol of monocytes/macrophages without damaging these cells. This review addresses also the generation of C3-based molecular tools for experimental macrophage pharmacology and cell biology as well as the exploitation of C3 for development of novel therapeutic strategies against monocyte/macrophage-associated diseases.