Development of a Transplantable GFP+ B-Cell Lymphoma Tumor Cell Line From MHC-Defined Miniature Swine: Potential for a Large Animal Tumor Model.

The lack of a reliable and reproducible large animal tumor model for the study of hemolymphatic malignancies limits the ability to explore the underlying pathophysiology and testing of novel therapies. The goal of this study was to develop an aggressive, trackable swine tumor cell line in mice for adoptive transfer into MHC matched swine. Two tumor cell lines, post-transplant lymphoproliferative disease (PTLD) 13271 and chronic myelogenous leukemia (CML) 14736, were previously established from the Massachusetts General Hospital (MGH) miniature swine herd. PTLD 13271 is a swine B-cell lymphoma line originating from an animal that developed PTLD following hematopoietic cell transplantation (HCT), while CML 14736 was generated from a swine that spontaneously developed CML. In order to select for aggressive tumor variants, both lines were passage into NOD/SCID IL-2 receptor γ-/- (NSG) mice. Tumor induced mortality in mice injected with CML14736 was 68% while 100% of mice injected with PTLD 13271 succumbed to PTLD by day 70. Based on aggressiveness, PTLD 13271 was selected for further development and re-passage into NSG mice resulting in increased tumor burden and metastasis. Transduction of the PTLD 13271 cell line with a green fluorescent protein (GFP)-expressing lentivirus facilitated tumor tracking when re-passaged in mice. Utilizing a tolerance induction strategy, GFP+ tumors were injected into an MHC matched miniature swine and successfully followed via flow cytometry for 48 h in circulation, although tumor engraftment was not observed. In summary, we report the development of an aggressive GFP+B-cell lymphoma cell line which has the potential for facilitating development of a large animal tumor model.

Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells.

Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25(+) B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins' ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25(+) cells for studying immune regulation.

A truncated diphtheria toxin based recombinant porcine CTLA-4 fusion toxin.

Targeted cell therapies are possible through the generation of recombinant fusion proteins that combine a toxin, such as diphtheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the fusion protein to the cell of interest, the diphtheria toxin is internalized which results in protein synthesis inhibition and subsequent cell death. We have recently expressed and purified the recombinant soluble porcine CTLA-4 both with and without N-glycosylation in yeast Pichia pastoris for in vivo use in our preclinical swine model. The glycosylated and non-N-glycosylated versions of this recombinant protein each bind to a porcine CD80 expressing B-cell lymphoma line (LCL13271) with equal affinity (K(D)=13 nM). In this study we have linked each of the glycosylated and non-N-glycosylated soluble porcine CTLA-4 proteins to the truncated diphtheria toxin DT390 through genetic engineering yielding three versions of the porcine CTLA-4 fusion toxins: 1) monovalent glycosylated soluble porcine CTLA-4 fusion toxin; 2) monovalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin and 3) bivalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin. Protein synthesis inhibition analysis demonstrated that while all three fusion toxins are capable of inhibiting protein synthesis in vitro, the non-N-glycosylated porcine CTLA-4 isoforms function most efficiently. Binding analysis using flow cytometry of the porcine CTLA-4 fusion toxins to LCL13271 cells also demonstrated that the non-N-glycosylated porcine CTLA-4 isoforms bind to these cells with higher affinity compared to the glycosylated fusion toxin. The monovalent non-N-glycosylated porcine CTLA-4 fusion toxin was tested in vivo. NSG (NOD/SCID IL-2 receptor γ(-)/(-)) mice were injected with porcine CD80(+) LCL13271 tumor cells. All animals succumbed to tumors and those treated with the monovalent non-N-glycosylated porcine CTLA-4 fusion toxin survived longer based on a symptomatic scoring system compared to the untreated controls. This recombinant protein may therefore provide a novel approach for in vivo depletion of porcine antigen presenting cells (APCs) for studies investigating the induction of transplantation tolerance, autoimmune disease and cancer treatment.

Lack of antidonor alloantibody does not indicate lack of immune sensitization: studies of graft loss in a haploidentical hematopoietic cell transplantation swine model.

Loss of chimerism is an undesirable outcome of allogeneic hematopoietic cell transplantation (HCT) after reduced-intensity conditioning. Understanding the nature of cellular and humoral immune responses to HCT after graft loss could lead to improved retransplantation strategies. We investigated the immunologic responses after graft loss in miniature swine recipients of haploidentical HCT that received reduced-intensity conditioning. After the loss of peripheral blood chimerism, antidonor cellular responses were present without detectable antidonor antibody. Reexposure to donor hematopoietic cells after graft loss induced a sensitized antidonor cellular response. No induced antidonor antibody response could be detected despite evidence of cellular sensitization to donor cells. In contrast, unconditioned animals exposed repeatedly to similar doses of haploidentical donor cells developed antidonor antibody responses. These results could have important implications for the design of treatment strategies to overcome antidonor responses in HCT and improve the outcome of retransplantation after graft loss.

Cannabinoid exposure in adolescent female rats induces transgenerational effects on morphine conditioned place preference in male offspring.

In the United States, marijuana is one of the drugs most abused by adolescents, with females representing a growing number of users. In previous studies, treatment of adolescent female rats with morphine significantly altered brain reward systems in future offspring. As both cannabinoid and opioid systems develop during adolescence, it was hypothesized that early exposure to cannabinoids would induce similar transgenerational effects. In the current study, female rats were treated with the cannabinoid receptor (CB1/CB2) agonist WIN 55,212-2 or its vehicle for three consecutive days during adolescent development (30 days of age), and were subsequently mated in adulthood (60 days of age). The adolescent and adult male offspring of these WIN 55,212-2 (WIN-F1)- or vehicle (VEH-F1)-treated females were tested for their response to morphine using the conditioned place preference (CPP) paradigm. Both adolescent and adult WIN-F1offspring exhibited greater sensitivity to morphine CPP than their VEH-F1 counterparts. Collectively, the findings provide additional evidence of transgenerational effects of adolescent drug use.

Adolescent opiate exposure in the female rat induces subtle alterations in maternal care and transgenerational effects on play behavior.

The non-medical use of prescription opiates, such as Vicodin(®) and MSContin(®), has increased dramatically over the past decade. Of particular concern is the rising popularity of these drugs in adolescent female populations. Use during this critical developmental period could have significant long-term consequences for both the female user as well as potential effects on her future offspring. To address this issue, we have begun modeling adolescent opiate exposure in female rats and have observed significant transgenerational effects despite the fact that all drugs are withdrawn several weeks prior to pregnancy. The purpose of the current set of studies was to determine whether adolescent morphine exposure modifies postpartum care. In addition, we also examined juvenile play behavior in both male and female offspring. The choice of the social play paradigm was based on previous findings demonstrating effects of both postpartum care and opioid activity on play behavior. The findings revealed subtle modifications in the maternal behavior of adolescent morphine-exposed females, primarily related to the amount of time females' spend nursing and in non-nursing contact with their young. In addition, male offspring of adolescent morphine-exposed mothers (MOR-F1) demonstrate decreased rough and tumble play behaviors, with no significant differences in general social behaviors (i.e., social grooming and social exploration). Moreover, there was a tendency toward increased rough and tumble play in MOR-F1 females, demonstrating the sex-specific nature of these effects. Given the importance of the postpartum environment on neurodevelopment, it is possible that modifications in maternal-offspring interactions, related to a history of adolescent opiate exposure, plays a role in the observed transgenerational effects. Overall, these studies indicate that the long-term consequences of adolescent opiate exposure can impact both the female and her future offspring.