Correction of T-Cell Repertoire and Autoimmune Diabetes in NOD Mice by Non-myeloablative T-Cell Depleted Allogeneic HSCT.

The induction of partial tolerance toward pancreatic autoantigens in the treatment of type 1 diabetes mellitus (T1DM) can be attained by autologous hematopoietic stem cell transplantation (HSCT). However, most patients treated by autologous HSCT eventually relapse. Furthermore, allogeneic HSCT which could potentially provide a durable non-autoimmune T-cell receptor (TCR) repertoire is associated with a substantial risk for transplant-related mortality. We have previously demonstrated an effective approach for attaining engraftment without graft versus host disease (GVHD) of allogeneic T-cell depleted HSCT, following non-myeloablative conditioning, using donor-derived anti-3rd party central memory CD8 veto T cells (Tcm). In the present study, we investigated the ability of this relatively safe transplant modality to eliminate autoimmune T-cell clones in the NOD mouse model which spontaneously develop T1DM. Our results demonstrate that using this approach, marked durable chimerism is attained, without any transplant-related mortality, and with a very high rate of diabetes prevention. TCR sequencing of transplanted mice showed profound changes in the T-cell repertoire and decrease in the prevalence of specific autoimmune T-cell clones directed against pancreatic antigens. This approach could be considered as strategy to treat people destined to develop T1DM but with residual beta cell function, or as a platform for prevention of beta cell destruction after transplantation of allogenic beta cells.

Implantation of an Impedance Sensor for Early Detection of Gastrointestinal Anastomotic Leaks.

INTRODUCTION: Accurate early diagnosis of a gastrointestinal anastomotic leak remains a challenge. When an anastomotic leak develops, the electrical properties of the tissue undergoing inflammatory processes change, resulting from the extravasation of inflammatory fluid and cellular infiltration. The method described here intends to provide a novel early anastomotic leak warning system based upon measurable changes in tissue impedance nearby an acute inflammatory process. METHODS: A biodegradable Mg-alloy was compared with a nonabsorbable stainless steel (STS) electrode connected to a wireless recording system for impedance measurement. In vitro measurements were made in physiological solutions and small animal (eight mice) and large animal (eight pigs) models with an anastomotic leak simulated by an open colotomy. Measurements were made at 10 mm intervals from the open colon at baseline and up to 120 min comparing these with a sutured colonic wound and normal tissue. RESULTS: In-vitro biodegradable magnesium electrode impedance evaluation showed good sensitivity to different media due to its environmental corrosion properties. The impedance of an acidic environment (1.06 ± 0.02 kΩ for citric acid) was twice that of phosphate-buffered saline (PBS) (0.64 ± 0.008 kΩ) with a distinction between Normal Saline (0.42 ± 0.013 kΩ) and PBS (0.64 ± 0.008 kΩ). This was in contrast to the performance characteristics of the control STS electrodes, where impedance in an acidic environment was lower than saline or PBS (citric acid:0.76 ± 0.01 kΩ versus PBS: 1.32 ± 0.014 kΩ). In a mouse model simulating an anastomotic leak, there was a significant increase in impedance after 120 min when compared with controls (99.7% increase versus 9.6% increase, respectively; P < 0.02). This effect was confirmed in a pig model when relative impedance measurements of the leak and control groups were compared (1.86 ± 0.46 versus 1.07 ± 0.02, respectively; P < 0.027). CONCLUSIONS: Electrophysiological measurement shows diagnostic sensitivity for a gastrointestinal leak with potential clinical utility in the postoperative detection of early intra-abdominal sepsis. Further investigation of biodegradable tissue sensors capable of monitoring an early anastomotic leak is required.

Lung Regeneration by Transplantation of Allogeneic Lung Progenitors Using a Safer Conditioning Regimen and Clinical-grade Reagents.

Over the last decades, several studies demonstrated the possibility of lung regeneration through transplantation of various lung progenitor populations. Recently, we showed in mice that fetal or adult lung progenitors could potentially provide donor cells for transplantation, provided that the lung stem cell niche in the recipient is vacated of endogenous lung progenitors by adequate conditioning. Accordingly, marked lung regeneration could be attained following i.v. infusion of a single cell suspension of lung cells into recipient mice conditioned with naphthalene (NA) and 6Gy total body irradiation (TBI). As clinical translation of this approach requires the use of allogenic donors, we more recently developed a novel transplantation modality based on co-infusion of hematopoietic and lung progenitors from the same donor. Thus, by virtue of hematopoietic chimerism, which leads to immune tolerance toward donor antigens, the lung progenitors can be successfully engrafted without any need for post-transplant immune suppression. In the present study, we demonstrate that it is possible to replace NA in the conditioning regimen with Cyclophosphamide (CY), approved for the treatment of many diseases and that a lower dose of 2 GY TBI can successfully enable engraftment of donor-derived hematopoietic and lung progenitors when CY is administered in 2 doses after the stem cell infusion. Taken together, our results suggest a feasible and relatively safe protocol that could potentially be translated to clinical transplantation of lung progenitors across major MHC barriers in patients with terminal lung diseases.

Novel immunoregulatory role of perforin-positive dendritic cells.

The recently described generation of a highly defined population of dendritic cells which express perforin and granzyme A (termed "perf-DCs") and their ability to selectively delete cognate CD8+ T cell has raised the possibility that these cells play a role in the maintenance of peripheral tolerance. Using bone marrow transplantation, we generated mice selectively lacking perforin expressing dendritic cells. These mice progressively gain weight and exhibit features resembling metabolic syndrome as well as an enhanced susceptibility to autoimmunity induction. Interestingly, these pathological phenotypes were reversed upon treatment with CD4/CD8 neutralizing antibodies. Thus, it appears that this rare subpopulation of dendritic cells (perf-DCs) displays a major regulatory role in adipose tissue inflammatory processes and in autoimmunity.

Perforin-Positive Dendritic Cells Exhibit an Immuno-regulatory Role in Metabolic Syndrome and Autoimmunity.

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.