Chantale Lacelle

Ex Vivo Expansion of CD4+CD25+FoxP3+ T Regulatory Cells Based on Synergy between IL-2 and 4-1BB Signaling

Naturally occurring CD4+CD25+FoxP3+ T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-β, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4+CD25− T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.

CD4+CD25+ T Regulatory Cells Dominate Multiple Immune Evasion Mechanisms in Early but Not Late Phases of Tumor Development in a B Cell Lymphoma Model

Tumors use a complex set of direct and indirect mechanisms to evade the immune system. Naturally arising CD4+CD25+FoxP3+ T regulatory (Treg) cells have been implicated recently in tumor immune escape mechanism, but the relative contribution of these cells to overall tumor progression compared with other immune evasion mechanisms remains to be elucidated. Using the A20 B cell lymphoma as a transplantable tumor model, we demonstrate that this tumor employs multiple direct (expression of immunoinhibitory molecule PD-L1, IDO, and IL-10, and lack of expression of CD80 costimulatory molecule) and indirect (down-regulation of APC function and induction of Treg cells) immune evasion mechanisms. Importantly, Treg cells served as the dominant immune escape mechanism early in tumor progression because the physical elimination of these cells before tumor challenge resulted in tumor-free survival in 70% of mice, whereas their depletion in animals with established tumors had no therapeutic effect. Therefore, our data suggest that Treg cells may serve as an important therapeutic target for patients with early stages of cancer and that more vigorous combinatorial approaches simultaneously targeting multiple immune evasion as well as immunosurveillance mechanisms for the generation of a productive immune response against tumor may be required for effective immunotherapy in patients with advanced disease.

Senescent fibroblasts resist apoptosis by downregulating caspase-3

In replicative senescence, cells undergo permanent exit from cell cycle traverse; this is traditionally thought to occur at the end of a cultures in vitro life span, after serial passaging. In general, the checkpoint for replicative senescence is found at the G(1)/S border, controlled by the modulation of a battery of proteins, typified by gaining inhibitors of cell cycle traverse, such as cyclin-dependent kinases or RB hyperphosphorylation, and losing pro-proliferation gene expressions such as c-fos, c-myc, and a cadre of proliferation-dependent kinases. Here, we present evidence that replicatively senescent fibroblasts are resistant to apoptotic death, associated with a lack of key enzyme activities, caspase-3 being the chief executioner. This observation, coupled with our earlier report that senescent fibroblasts maintain persistently high levels of pro-survival factor Bcl-2, suggests that the molecular signaling program present in fibroblasts at the end of their in vitro life span may not only cater to the state of permanent exit from cell cycle traverse, but also dictate an inability to commit cellular suicide. Future experiments will reveal whether replicatively senescent fibroblasts that can neither proliferate nor die contribute to organismic aging, and whether their accumulation over time in tissue becomes detrimental to the normal aging process.

Alterations in TNF- and IL-related gene expression in space-flown WI38 human fibroblasts

Spaceflight, just like aging, causes profound changes in musculoskeletal parameters, which result in decreased bone density and muscular weakness. As these conditions decrease our ability to conduct long‐term manned space missions, and increase bone frailty in the elderly, the identification of genes responsible for the apparition of these physiological changes will be of great benefit. Thus, we developed and implemented a new microarray approach to investigate the changes in normal WI38 human fibroblast gene expression that arise as a consequence of space flight. Using our microarray, we identified changes in the level of expression of 10 genes, belonging to either the tumor necrosis factor‐ (TNF) or interleukin‐ (IL) related gene families in fibroblasts when WI38 cells exposed to microgravity during the STS‐93 Space Shuttle mission were compared with ground controls. The genes included two ligands from the TNF superfamily, TWEAK and TNFSF15; two TNF receptor‐associated proteins, NSMAF and PTPN13; three TNF‐inducible genes, ABC50, PTX3, and SCYA13; TNF‐α converting enzyme, IL‐1 receptor antagonist, and IL‐15 receptor alpha chain. Most of these are involved in either the regulation of bone density, and as such the development of spaceflight osteopenia, or in the development of proinflammatory status.

Donor-specific HLA alloantibodies: Impact on cardiac allograft vasculopathy, rejection, and survival after pediatric heart transplantation

BACKGROUND There is increasing evidence that donor-specific anti-HLA antibodies (DSA) are associated with poor outcomes after cardiac transplantation in adults, but data are limited in children. The objective of this study was to examine the development and consequences of de novo DSA in pediatric recipients of heart transplants. METHODS We analyzed 105 pediatric patients who received heart transplants at our center from January 2002 to December 2012. All patients had negative T-cell and B-cell post-transplant crossmatches. Patients underwent HLA antibody screening at 1, 2, 3, 6, and 12 months post-transplant and annually thereafter unless there was suspicion for rejection. HLA class I and II antibodies were identified using Luminex assay. Coronary angiography was performed at 1 year and annually thereafter. Acute cellular rejection, antibody-mediated rejection, and treated clinical rejections were included together as rejection events. RESULTS Of 105 patients, 45 (43%) developed de novo DSA. DSA-positive patients had significantly higher rates of coronary artery vasculopathy (CAV) compared with DSA-negative patients (36% vs 13%). CAV-free survival at 1 year and 5 years post-transplant for DSA-negative patients was 90% and 25%, respectively, compared with 70% and 0%, respectively, for DSA-positive patients (p < 0.01). DSA-positive patients had 2.5 times more rejection events per year than DSA-negative patients. The 5-year graft survival rate was 72.4% for DSA-negative patients and 21% for DSA-positive patients (p < 0.001). CONCLUSIONS De novo DSA has a strong negative impact on CAV, rejection, and graft survival in pediatric recipients of heart transplants.

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice.

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL–engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL–engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4+CD25+Foxp3+ regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.

Induction of Tolerance to Cardiac Allografts Using Donor Splenocytes Engineered to Display on Their Surface an Exogenous Fas Ligand Protein

The critical role played by Fas ligand (FasL) in immune homeostasis renders this molecule an attractive target for immunomodulation to achieve tolerance to auto- and transplantation Ags. Immunomodulation with genetically modified cells expressing FasL was shown to induce tolerance to alloantigens. However, genetic modification of primary cells in a rapid, efficient, and clinically applicable manner proved challenging. Therefore, we tested the efficacy of donor splenocytes rapidly and efficiently engineered to display on their surface a chimeric form of FasL protein (SA-FasL) for tolerance induction to cardiac allografts. The i.p. injection of ACI rats with Wistar-Furth rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. Tolerance was associated with apoptosis of donor reactive T effector cells and induction/expansion of CD4+CD25+FoxP3+ T regulatory (Treg) cells. Treg cells played a critical role in the observed tolerance as adoptive transfer of sorted Treg cells from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary Wistar-Furth grafts. Immunomodulation with allogeneic cells rapidly and efficiently engineered to display on their surface SA-FasL protein provides an effective and clinically applicable means of cell-based therapy with potential application to regenerative medicine, transplantation, and autoimmunity.

Identification of high caspase-3 mRNA expression as a unique signature profile for extremely old individuals.

Apoptosis, or programmed cell death, is important for maintaining tissue homeostasis, as it permits the elimination of damaged, functionless or unwanted cells. As we age, our immune system undergoes constant remodeling, during which age-associated changes in immune parameters, including decreased naïve and increased memory T cells, have been reported. However, excessive immune cell loss, rendering the elderly more vulnerable to infections, and inappropriate deletion of damaged or functionless lymphocytes, can contribute to the development of age-associated diseases. As such, we studied the mRNA expression of cell death (specifically caspase) genes in nonagenarians and centenarians, successful models of ageing who have survived or avoided age-associated diseases, as well as in their younger counterparts and found that population composed of extremely old individuals shows a unique pattern of caspase mRNA expression, characterized by high levels of caspase-1 and -3, and low levels of caspase-8, mRNA while those composed of old individuals are characterize by high level of caspase-8 mRNA expression. Furthermore, we show that the described changes in caspases mRNA do not appear to results from age-related changes in PBMC composition, such as decreases in CD24. Therefore, we suggest that unique patterns of caspase mRNA results from the regulation of message abundance on a per cell basis, via a putative regulation of caspase genes at the transcription or RNA processing level, rather than changes in immune profiles.

Establishing lymphoblastoid cell lines from frozen blood of extremely old individuals.

In population studies involving peripheral blood samples from nonagenarian and centenarian donors, the amount of biological material available for research is restricted, as only a few millilitres of blood can be obtained from extremely old donors without significantly compromising their health. Here we describe a protocol to immortalize small amounts of total frozen blood from extremely old donors (90+) using the Epstein-Barr virus, despite the low level of circulating B cells present in nonagenarian and centenarian blood samples. This methodology provides a unique way to maximize resources of biological material available for research.

Complement (C1q) Binding de Novo Donor-Specific Antibodies and Cardiac-Allograft Vasculopathy in Pediatric Heart Transplant Recipients

Background We hypothesized C1q binding de novo donor-specific antibody (DSA) after heart transplant (HT) is a higher risk for development of coronary artery vasculopathy (CAV) in children. Methods A retrospective analysis of 127 pediatric HT recipients transplanted between January 2005 and December 2014 was used to determine complement (C1q)-binding de novo DSA on the outcomes of HT and the ability of the C1q assay to predict CAV development. Results Of 127 patients, 59 (46.4%) developed de novo DSA, 37 of those had C1q+ DSA. There was no difference in baseline characteristics except patients who developed C1q+ DSA more often received a donor heart from a female compared with C1q− DSA group (P = 0.034). The DSA median fluorescent intensity (MFI) value of 7000 or greater had 80% sensitivity and 80% specificity (C statistics 0.89, P <0.05) for predicting positive C1q binding. Multivariate analyses identified C1q binding DSA as an independent risk for CAV with a hazard ratio (HR) of 3.25 (95% confidence interval [CI], 1.33-7.93; P = 0.0095). In multivariable Cox proportional hazard models, the covariates associated with graft loss included: C1q+ DSA (HR, 3.2; 95% CI, 1.34-7.86; P < 0.009), pre-HT renal insufficiency (HR, 11.3; 95% CI, 3.71-34.29; P < 0.0001), and pre-HT ventilator support (HR, 3.3; 95% CI, 1.39-7.81; P = 0.007). Conclusions The DSA strength in MFI correlates with positive C1q-binding activity and hence functional capabilities of DSA. Close monitoring of DSA strength in MFI and function (C1q assay) may be useful for identifying pediatric HT recipient at risk for development of CAV.