Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

<Emphasis Type="Italic">Vegf-A</Emphasis> mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation

Aims/hypothesis

The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment.

Methods

Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice.

Results

At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean?±?SEM: 0.118?±?0.01 [n?=?3] in Vegf-A mRNA transfected group (VEGF) vs 0.056?±?0.01 [n?=?3] in no RNA [p?<?0.05] vs 0.063?±?0.02 [n?=?4] in Gfp mRNA transfected group (GFP) [p?<?0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085?±?0.02 [n?=?4] in VEGF vs 0.030?±?0.004 [n?=?4] in no RNA [p?<?0.05] vs 0.034?±?0.01 [n?=?5] in GFP [p?<?0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048?±?0.013 [n?=?3] in VEGF vs 0.015?±?0.0051 [n?=?4] in no RNA [p?<?0.01] vs 0.013?±?0.0046 [n?=?4] in GFP [p?<?0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049?±?0.0042 [n?=?8] in VEGF vs 0.029?±?0.0052 [n?=?5] in no RNA [p?<?0.05] vs 0.027?±?0.0056 [n?=?4] in GFP [p?<?0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292?±?0.0032 μl [n?=?7] in VEGF vs 0.0178?±?0.0021 μl [n?=?5] in no RNA [p?<?0.01] vs 0.0129?±?0.0012 μl [n?=?4] in GFP [p?<?0.001]).

Conclusions/interpretation

Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.

     

Development and first data of a customized short tracheal cannula based on digital data

Purpose

At the moment, there is an inadequate margin fit of commercially available stoma buttons. The aim of the present study was to develop a customized short tracheal cannula based on digital data. Furthermore, the applied material has to be evaluated considering germ colonization and appropriate cleaning procedures.

Methods

Computed tomographies of 53 patients who underwent laryngectomy were surveyed. Based on the digital data, a customized short tracheal cannula was created and manufactured from silicone. The new cannula was incorporated in ten patients and worn for 4 weeks. A clinical examination of an otolaryngologist and subjective assessment of the patients were carried out. Furthermore, microbiological test considering germ colonization was performed.

Results

The customized short tracheal cannula could be incorporated in all patients. The clinical results showed no irritation or mucosal lesions. The subjective individual evaluation by the patients was promising. The proposals for improvement could be considered. The microbiological examination revealed a higher contamination of the silicone compared to the silver cannulas. Both chemical and mechanical decontamination showed sufficient results.

Conclusion

A workflow for development and manufacturing of a customized short tracheal cannula from digital data could be established. The cannula is compatible to standard equipment and routine cleaning procedures. Clinical studies are required to evaluate the potential benefit for patients.