BackgroundMost current cell‐based regenerative therapies are based on the indirect induction of the affected tissues repair. Xenogeneic cell‐based treatment with expanded human placenta stromal cells, predominantly from fetal origin (PLX‐RAD cells), were shown to mitigate significantly acute radiation syndrome (ARS) following high dose irradiation in mice, with expedited regain of weight loss and haematopoietic function. The current mechanistic study explores the indirect effect of the secretome of PLX‐RAD cells in the rescue of the irradiated mice.MethodsThe mitigation of the ARS was investigated following two intramuscularly (IM) injected 2 × 106 PLX‐RAD cells, 1 and 5 days following 7.7 Gy irradiation. The mice survival rate and their blood or bone marrow (BM) cell counts were followed up and correlated with multiplex immunoassay of a panel of related human proteins of PLX‐RAD derived secretome, as well as endogenous secretion of related mouse proteins. PLX‐RAD secretome was also tested in vitro for its effect on the induction of the migration of BM progenitors.ResultsA 7.7 Gy whole body mice irradiation resulted in ~25% survival by 21 days. Treatment with two IM injections of 2 × 106 PLX‐RAD cells on days 1 and 5 after irradiation mitigated highly significantly the subsequent lethal ARS, with survival rate increase to nearly 100% and fast regain of the initial weight loss (P < 0,0001). This was associated with a significant faster haematopoiesis recovery from day 9 onwards (P < 0.01). Nine out of the 65 human proteins tested were highly significantly elevated in the mouse circulation, peaking on days 6–9 after irradiation, relative to negligible levels in non‐irradiated PLX‐RAD injected mice (P < 0.01). The highly elevated proteins included human G‐CSF, GRO, MCP‐1, IL‐6 and lL‐8, reaching >500 pg/mL, while MCP‐3, ENA, Eotaxin and fractalkine levels ranged between ~60–160pg/mL. The detected radiation‐induced PLX‐RAD secretome correlated well with the timing of the fast haematopoiesis regeneration. The radiation‐induced PLX‐RAD secretome seemed to reinforce the delayed high levels secretion of related mouse endogenous cytokines, including GCSF, KC, MCP‐1 and IL‐6. Additional supportive in vitro studies also confirmed the ability of cultured PLX‐RAD secretome to induce accelerated migration of BM progenitors.ConclusionsA well‐regulated and orchestrated secretion of major pro‐regenerative BM supporting secretome in high dose irradiated mice, treated with xenogeneic IM injected PLX‐RAD cells, can explain the observed mitigation of ARS. This seemed to coincide with faster haematopoiesis regeneration, regain of severe weight loss and the increased survival rate. The ARS‐related stress signals activating the IM injected PLX‐RAD cells for the remote secretion of the relevant human proteins deserve further investigation.

We previously showed that the T cell activation inhibitor, mitochondrial (Tcaim) is highly expressed in grafts of tolerance-developing transplant recipients and that the encoded protein is localized within mitochondria. In this study, we show that CD11c+ dendritic cells (DCs), as main producers of TCAIM, downregulate Tcaim expression after LPS stimulation or in vivo alloantigen challenge. LPS-stimulated TCAIM-overexpressing bone marrow–derived DC (BMDCs) have a reduced capacity to induce proliferation of and cytokine expression by cocultured allogeneic T cells; this is not due to diminished upregulation of MHC or costimulatory molecules. Transcriptional profiling also revealed normal LPS-mediated upregulation of the majority of genes involved in TLR signaling. However, TCAIM BMDCs did not induce Il2 mRNA expression upon LPS stimulation in comparison with Control-BMDCs. In addition, TCAIM overexpression abolished LPS-mediated Ca2+ influx and mitochondrial reactive oxygen species formation. Addition of IL-2 to BMDC–T cell cocultures restored the priming capacity of TCAIM BMDCs for cocultured allogeneic CD8+ T cells. Furthermore, BMDCs of IL-2–deficient mice showed similarly abolished LPS-induced T cell priming as TCAIM-overexpressing wild type BMDCs. Thus, TCAIM interferes with TLR4 signaling in BMDCs and subsequently impairs their T cell priming capacity, which supports its role for tolerance induction.

Human cytomegalovirus infections have a major negative effect on morbidity and mortality of immunosuppressed allograft recipients and indirectly on graft function and survival. The adoptive antiviral T-cell therapy is a novel therapeutic tool to restore immune competence after solid organ transplantation. Till now, the antiviral T-cell products mainly focused on cytotoxic CD8(+) T cells, whereas CD4(+) T cells played a minor role. Here, we demonstrate the importance of CD4(+) T cells within T-cell lines specific for human cytomegalovirus besides its essential support for the quality of CD8(+) T-cell memory. Virus-specific CD4(+) T cells elicit profound functionality after rechallenge (multicytokine secretors, CD137, CD154, and CD107a expression and killing of infected target cells). The CD4(+) T cells show predominantly a Th1 phenotype with cytolytic properties that is mainly perforin-dependent. The data demonstrate the significance of CD4(+) T cells within T-cell products to achieve a successful adoption with enhanced efficacy.