Systemic recovery and therapeutic effects of transplanted allogenic and xenogenic mesenchymal stromal cells in a rat blunt chest trauma model.

BACKGROUND: Effective therapy of Acute Lung Injury (ALI) is still a major scientific and clinical problem. To define novel therapeutic strategies for sequelae of blunt chest trauma (TxT) like ALI/Acute Respiratory Distress Syndrome, we have investigated the immunomodulatory and regenerative effects of a single dose of ex vivo expanded human or rat mesenchymal stromal cells (hMSCs/rMSCs) with or without priming, immediately after the induction of TxT in Wistar rats. METHODS: We analyzed the histological score of lung injury, the cell count of the broncho alveolar lavage fluid (BAL), the change in local and systemic cytokine level and the recovery of the administered cells 24 h and 5 days post trauma. RESULTS: The treatment with hMSCs reduced the injury score 24 h after trauma by at least 50% compared with TxT rats without MSCs. In general, TxT rats treated with hMSCs exhibited a lower level of pro-inflammatory cytokines (interleukin [IL]-1B, IL-6) and chemokines (C-X-C motif chemokine ligand 1 [CXCL1], C-C motif chemokine ligand 2 [CCL2]), but a higher tumor necrosis factor alpha induced protein 6 (TNFAIP6) level in the BAL compared with TxT rats after 24 h. Five days after trauma, cytokine levels and the distribution of inflammatory cells were similar to sham rats. In contrast, the treatment with rMSCs did not reveal such therapeutic effects on the injury score and cytokine levels, except for TNFAIP6 level. CONCLUSION: TxT represents a suitable model to study effects of MSCs as an acute treatment strategy after trauma. However, the source of MSCs has to be carefully considered in the design of future studies.

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.

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.

KIR2DS4 and Its Variant KIR1D in KIR-AA Genotype Donors Showed Differential Survival Impact in Patients with Lymphoid Disease after HLA-Matched Unrelated Hematopoietic Stem Cell Transplantation.

Previous studies have illustrated associations between the presence of activating killer cell immunoglobulin-like receptor (KIR) genes and lower susceptibility to hematologic malignancies in humans. In addition, favorable hematopoietic stem cell transplantation (HSCT) outcomes have been reported in patients who received transplants from donors with KIR genotypes dominant for activating KIR receptors. However, the association of activating KIR genes on an allelic level with disease and their impact on HSCT outcome has been little investigated to date. To this end, we genotyped a large transplantation cohort for KIR 2 Ig domains and short cytoplasmic tail 4 (KIR2DS4) polymorphisms and investigated their association with disease. We next investigated the impact of KIR-AA genotype donor KIR2DS4 polymorphisms (AA/KIR2DS4 versus AA/ KIR 1 Ig domain [KIR1D]) on clinical outcomes of HSCT in myeloid versus lymphoid patient subgroups. Among 2810 transplantation donor-recipient pairs, 68.8% (n = 1934) were 10/10 HLA-matched and 31.2% (n = 876) were 9/10 HLA-matched. The distribution of KIR1D was equal in patients and donors (P = .205). Multivariate analysis in 10/10 HLA-matched patients with lymphoid disease showed improved HSCT outcomes when they received grafts from AA/KIR1D donors (overall survival: hazard ratio [HR], .62, P = .002; disease free survival: HR, .70, P = .011; graft-versus-host disease-free and relapse-free survival: HR, .67, P = .002; nonrelapse mortality: HR, .55, P < .001). This effect was not seen in either 9/10 HLA-matched patients with lymphoid disease or patients with myeloid disease. Our study indicates that the presence of KIR1D alleles is not associated with disease in patients, and, interestingly, using grafts from AA/KIR1D donors translated into beneficial survival outcomes in 10/10 HLA-matched patients with lymphoid disease.

Higher risk for chronic graft-versus-host disease (GvHD) in HLA-G mismatched transplants following allogeneic hematopoietic stem cell transplantation: A retrospective study.

INTRODUCTION: Graft-versus-host disease (GvHD) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is highly influenced by the degree of HLA matching between recipient and donor. The HLA-class Ib molecule HLA-G has been shown to promote tolerogenicity through its interaction with inhibitory receptors found on several immunocompetent cells. We hypothesized that in an allo-HSCT setting, HLA-G mismatches may negatively impact the HLA-G-mediated tolerogenicity either due to inefficient interaction with the inhibitory receptors of the transplanted immune cells or due to direct allorecognition of mismatched HLA-G on host cells by the immune cells of the donor. METHODS: In order to explore this hypothesis, we investigated the impact of HLA-G mismatching in 2.083 10/10 matched high resolution HLA-typed allo-HSCT transplants. RESULTS: We found that the risk of chronic GvHD was significantly higher in HLA-G-mismatched transplant cases as compared with the HLA-G-matched control group (HR: 1.46, 95%CI = 1.11-1.91, p = 0.006). Sub-analysis of the mismatch vector revealed that this effect was only detectable in the GvH (HR: 1.89, 95%CI 1.39-2.57, p < 0.001) but not the HvG direction (HR: 1.01, 95%CI = 0.63-1.63, p = 0.967). In addition, the negative impact of HLA-G mismatching on chronic GvHD was only significant in younger patients (<30y HR: 3.02, 95%CI = 1.25-7.28, p = 0.014; >29y HR: 1.28, 95%CI = 0.94-1.72, p = 0.113). DISCUSSION: Our results indicate that HLA-G mismatches may contribute to the onset of chronic GvHD, especially in younger patients and should therefore be avoided when possible.

Inflammatory response of mesenchymal stromal cells after in vivo exposure with selected trauma-related factors and polytrauma serum.

Polytrauma (PT) is a life-threatening disease and a major global burden of injury. Mesenchymal stromal cells (MSC) might be a therapeutic option for PT patients due to their anti-inflammatory and regenerative potential. We hypothesised that the inflammatory response of MSC is similar after exposure to selected trauma-relevant factors to sera from PT patients (PTS). Therefore, we investigated the effects of a mixture of defined factors, supposed to play a role on MSC in the early phase of PT. Additionally, in a translational approach we investigated the effect of serum from PT patients on MSC in vitro. MSC were incubated with a PT cocktail in physiological (PTCL) and supra-physiological (PTCH) concentrations or PTS. The effect on gene expression and protein secretion of MSC was analysed by RNA sequencing, ELISA and Multiplex assays of cell culture supernatant. Stimulation of MSC with PTCH, PTCL or IL1B led to significant up- or downregulation of 470, 183 and 469 genes compared to unstimulated MSC at 6 h. The intersection of differentially expressed genes in these groups was very high (92% overlap with regard to the PTCL group; treated for 6 h). Cytokine secretion profile of MSC revealed that IL1B mimics the effect of a more complex PT cocktail as well. However, there was only a minor proportion of overlapping differentially expressed genes between the MSC group stimulated with early times of PTS and the MSC group stimulated with PTCH, PTCL and IL1B. In conclusion, the effect of sera from PT patients on MSC activation cannot be simulated by the chosen trauma-relevant factors. Furthermore, we conclude that while IL1B might be useful to prime MSC prior to therapeutic application, it might not be as useful for the in vitro study of functional properties of MSC in the context of PT.

Experimental blunt chest trauma-induced myocardial inflammation and alteration of gap-junction protein connexin 43.

OBJECTIVE: Severe blunt chest trauma in humans is associated with high mortality rates. Whereas lung tissue damage and lung inflammation after blunt chest trauma have extensively been investigated, the traumatic and posttraumatic effects on the heart remain poorly understood. Therefore, the purpose of this study was to define cardiac injury patterns in an experimental blunt chest trauma model in rats. METHODS: Experimental blunt chest trauma was induced by a blast wave in rats, with subsequent analysis of its effects on the heart. The animals were subjected either to a sham or trauma procedure. Systemic markers for cardiac injury were determined after 24 h and 5 days. Postmortem analysis of heart tissue addressed structural injury and inflammation 24 h and 5 days after trauma. RESULTS: Plasma levels of extracellular histones were elevated 24 h and 5 days after blunt chest trauma compared to sham-treated animals. In the heart, up-regulation of interleukin-1ß 24 h after trauma and increased myeloperoxidase activity 24 h and 5 days after trauma were accompanied by reduced complement C5a receptor-1 expression 24 h after trauma. Histological analysis revealed extravasation of erythrocytes and immunohistochemical analysis alteration of the pattern of the gap-junction protein connexin 43. Furthermore, a slight reduction of α-actinin and desmin expression in cardiac tissue was found after trauma together with a minor increase in sarcoplasmatic/endoplasmatic reticlulum calcium-ATPase (SERCA) expression. CONCLUSIONS: The clinically highly relevant rat model of blast wave-induced blunt chest trauma is associated with cardiac inflammation and structural alterations in cardiac tissue.

Effect of high-dose irradiation on human bone-marrow-derived mesenchymal stromal cells.

Cell therapy using multipotent mesenchymal stromal cells (MSCs) is of high interest in various indications. As the pleiotropic effects mediated by MSCs rely mostly on their unique secretory profile, long-term persistence of ex-vivo-expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long-term persistence of nucleated cells and could therefore also be applied to MSCs. We have exposed human bone-marrow-derived MSCs to 30 or 60 Gy of γ-irradiation and assessed cell proliferation, clonogenicity, differentiation, cytokine levels in media supernatants, surface receptor profile, as well as expression of proto-oncogenes/cell cycle markers, self-renewal/stemness markers, and DNA damage/irradiation markers. Irradiated MSCs show a significant decrease in proliferation and colony-forming unit-fibroblasts. However, a subpopulation of surviving cells is able to differentiate, but is unable to form colonies after irradiation. Irradiated MSCs showed stable expression of CD73 and CD90 and absence of CD3, CD34, and CD45 during a 16-week follow-up period. We found increased vascular endothelial growth factor (VEGF) levels and a decrease of platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB in culture media of nonirradiated cells. Irradiated MSCs showed an inverse pattern, that is, no increase of VEGF, and less consumption of PDGF-AA and PDGF-AB/BB. Interestingly, interleukin-6 (IL-6) levels increased during culture regardless of irradiation. Cells with lower sensitivity toward γ-irradiation showed positive ß-galactosidase activity 10 days after irradiation. Gene expression of both irradiated and nonirradiated MSCs 13-16 weeks after irradiation with 60 Gy predominantly followed the same pattern; cell cycle regulators CDKN1A (p21) and CDKN2A (p16) were upregulated, indicating cell cycle arrest, whereas classical proto-oncogenes, respectively, and self-renewal/stemness markers MYC, TP53 (p53), and KLF4 were downregulated. In addition, DNA damage/irradiation markers ATM, ATR, BRCA1, CHEK1, CHEK2, MDC1, and TP53BP1 also mostly showed the same pattern of gene expression as high-dose γ-irradiation. In conclusion, we demonstrated the existence of an MSC subpopulation with remarkable resistance to high-dose γ-irradiation. Cells surviving irradiation retained their trilineage differentiation capacity and surface marker profile but changed their cytokine secretion profile and became prematurely senescent.