Abstract The KRASG12D mutation is an ideal target for anti-cancer therapies as its expression is typically clonal, restricted to cancer tissue, and is among the most common oncogenic drivers in solid tumors. TCR-T cell therapies have demonstrated clinical activity in some solid cancers but have been limited by heterogeneous antigen expression and unfavorable tumor microenvironments. By targeting the KRASG12D mutation for which the cancer has established genetic dependency, AFNT-212 is designed to selectively target all cancer cells while avoiding on-target/off-tumor toxicities. AFNT-212 is non-virally engineered to knock-in a 5-transgene cassette expressing a high-avidity TCR specific for the KRASG12D mutation, a CD8α/β coreceptor, and a chimeric cytokine receptor. Transgene insertion at the TRAC locus disrupts expression of the endogenous TCRα, further enhancing the expression/activity of the transgenic KRASG12D TCR. Primary human CD8+ and CD4+ T cells were genetically engineered by a novel CRISPR-Cas nuclease system to integrate AFNT-212 transgenes within the TRAC locus. A cGMP compatible scale-up process for non-viral knock-in was established to support AFNT-212 clinical manufacturing. The activity of AFNT-212 was assessed against a panel of human KRASG12D tumor cell lines in vitro and established mouse xenograft models in vivo. The preclinical safety profile of AFNT-212 was evaluated by X-scan and crossreactivity assessment, alloreactivity studies, and cytokine independent growth studies. The specificity of gene-editing (GE) was assessed by an unbiased oligo-capture method followed by targeted sequencing. AFNT-212 TCR-T cells demonstrated potent in vitro anti-tumor activity against endogenously expressing HLA-A*11:01 KRASG12D tumor cells, including during chronic exposure to viable tumor cells. AFNT-212 TCR-T cells showed robust antitumor activity in established xenograft mouse models in vivo. No cross-reactivity was identified for the KRASG12D TCR against potential self-peptides even at supraphysiological levels, demonstrating high specificity of the TCR. No alloreactivity or cytokine-independent proliferation was observed. GE safety evaluations did not reveal any off-target activity using high sensitivity (~0.1%) NGS-based analyses or any GE-associated chromosomal rearrangements. The manufacturing of AFNT-212 consistently delivered >50-fold expansion of engineered TCR-T cells to meet expected clinical dose levels and exhibit memory/stemness phenotypes and negligible markers of immunologic exhaustion. AFNT-212, a novel TCR T cell therapy targeting KRASG12D mutant tumors, demonstrates robust activity against KRASG12D mutant tumors in vitro and in vivo. The robust manufacturing process developed using non-viral gene editing in the TRAC locus will support future clinical development of AFNT-212. Citation Format: Allison Drain, Nicholas Rouillard, Nathaniel Swanson, Martina Canestraro, Santosh Narayan, Tyler Warner, Nicole Danek, Ken Gareau, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Iqraa Priyata, Sarah Vidyasagar, Taylor Riggins-Walker, Hui-Wen Liu, Klaus Pechhold, Lauren Brown, Joshua Francis, Xingyue He, Patrick Browne, Rebecca Lamothe, Meghan Storlie, Gregory Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Christopher A. Klebanoff, Hubert Lam, Ankit Gupta, Damien Hallet, Gary Shapiro, Kim Nguyen, Loïc Vincent. AFNT-212: A TRAC-knocked-in KRASG12D-specific TCR-T cell product enhanced with CD8αβ and a chimeric cytokine receptor for treatment of solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 9.

2543 Background: KRAS is the most common oncogenic driver mutation in solid tumors, promoting the initiation and progression of many uncurable cancers, including colorectal, pancreatic and lung cancer. While small molecule inhibitors to KRAS G12C mutations have been approved, there are no targeted therapies available for patients with highly prevalent KRAS G12V mutations. TCR-T cell therapies have demonstrated remarkable responses in clinical trials, but their durability has been limited by the immunosuppressive tumor microenvironment (TME). AFNT-211 is an autologous T cell therapy engineered to express an HLA-A*11:01 KRAS G12V -specific TCR, further enhanced with CD8α/β coreceptor and a FAS-41BB switch receptor to drive T cell persistence and durable clinical responses. CD8α/β coreceptor enables a coordinated CD4+/CD8+ T cell response and FAS-41BB converts the FAS ligand (FASL) TME death signal into a costimulatory signal through 41BB activation. Methods: AFNT-211 was assessed for efficacy in vitro against a panel of KRAS G12V -expressing tumor cell lines and in vivo using human xenograft mouse models. In vitro safety studies were performed to assess potential cross-reactivity, alloreactivity, and cytokine-independent growth. The clinical manufacturing process consists of autologous CD4+/CD8+ T cells transduced with lentivirus and expanded using culture conditions that drive robust expansion while preserving stem-like properties. Phenotypic and functional analyses of AFNT-211 were performed using flow cytometry and cell-based assays. Results: Coculture of AFNT-211 with a panel of KRAS G12V -expressing tumor cell lines led to significant effector cytokine secretion, T cell proliferation, and tumor cell killing. The CD8α/β coreceptor enabled CD4+ T cell recognition of KRAS G12V and greater overall cytotoxicity. The FAS-41BB switch receptor dramatically augmented the magnitude and durability of the anti-tumor response against FASL-expressing tumor cells. XScan mutagenesis and potential off-target peptide testing revealed no significant cross-reactivities. No alloreactivity was observed against a panel of lymphoblastoid cell lines presenting the most frequent HLA types in the US population. Potent anti-tumor response was observed in vivo in a mouse xenograft model. Affini-T’s Thrive manufacturing platform used for the production of AFNT-211 consistently delivers >30-40e 9 TCR-engineered T cells with a high frequency of naïve and central memory T cells expressing negligible markers of exhaustion. Conclusions: The AFNT-211 manufacturing process generates a large number of TCR-T cells with desirable stem-like properties optimized for clinical dosing. Preclinical data demonstrated a potent and safe profile for AFNT-211 that supports clinical development in HLA-A*11:01 patients with advanced/metastatic solid tumors harboring a KRAS G12V mutation.

Somatic hypermutation (SHM) is necessary for Ab diversification and involves error-prone DNA repair of activation-induced cytidine deaminase-induced lesions in germinal center (GC) B cells but can also cause genomic instability. GC B cells express low levels of the DNA repair protein apurinic/apyrimidinic (AP) endonuclease (APE)1 and high levels of its homolog APE2. Reduced SHM in APE2-deficient mice suggests that APE2 promotes SHM, but these GC B cells also exhibit reduced proliferation that could impact mutation frequency. In this study, we test the hypothesis that APE2 promotes and APE1 suppresses SHM. We show how APE1/APE2 expression changes in primary murine spleen B cells during activation, impacting both SHM and class-switch recombination (CSR). High levels of both APE1 and APE2 early after activation promote CSR. However, after 2 d, APE1 levels decrease steadily with each cell division, even with repeated stimulation, whereas APE2 levels increase with each stimulation. When GC-level APE1/APE2 expression was engineered by reducing APE1 genetically (apex1+/-) and overexpressing APE2, bona fide activation-induced cytidine deaminase-dependent VDJH4 intron SHM became detectable in primary B cell cultures. The C terminus of APE2 that interacts with proliferating cell nuclear Ag promotes SHM and CSR, although its ATR-Chk1-interacting Zf-GRF domain is not required. However, APE2 does not increase mutations unless APE1 is reduced. Although APE1 promotes CSR, it suppresses SHM, suggesting that downregulation of APE1 in the GC is required for SHM. Genome-wide expression data compare GC and cultured B cells and new models depict how APE1 and APE2 expression and protein interactions change during B cell activation and affect the balance between accurate and error-prone repair during CSR and SHM.

Next Generation Sequencing (NGS) workflows for transcriptionally profiling cells involved in complex tissue functions have been limited in identifying and isolating rare cells, preparing suitable samples, and sequencing assays. TempO-Seq® provides a robust, quantitative, and specific gene expression platform that does not require RNA extraction or reverse transcription. It targets short RNA-sequences, is not biased, or require polyadenylation, and is compatible with fixed cell samples. Assays are available for human, rat, mouse. We implemented an integrated cell suspension workflow that combines surface and intracellular (ic) staining with WT TempO-Seq assay (∼27,000 mouse genes) followed by FACS-purification of selected cells and sequencing. We assayed suspensions of individual mouse Peyer Patch (PP) cells (6-8 PP, ∼3x106 cells/mouse), profiling gene expression of GC B-cells. After sequential surface labeling, fixation and permeabilization, ic-staining, and TempO-Seq, GC B-cells (∼2-3% of total cells) were FACS-purified based on B220+CD95+GL7+ staining, then sequencing-ready libraries prepared of 100-cell aliquots/sample. The assay has an excellent replicate repeatability (R2=0.94 detecting >11,000 genes in cell lines). Sorted, GC B-cells appeared more heterogeneous, with repeatability R2=0.73-0.78 and 7,000-8,000 different genes measured. Data integrity was demonstrated by concordance of selected protein markers (B220, CD95) and RNA expression (B-cell lineage genes, Fas). We confirmed selective expression of genes associated with the GC including BCL6, CXCR4, immunoglobulin class-switched isotypes, and downregulation of the GL7 repressor CMP-Neu5Ac hydroxylase. Using ic-staining for the cell cycle progression biomarker Ki67, we found that only 40-45% of PP GC B-cells stained positive, presumably undergoing affinity maturation through somatic hypermutation. We measured differential profiles and these and other data will be presented.

Abstract Accurate, sensitive and robust multiplexed measurements of gene expression from formalin fixed paraffin embedded (FFPE) tissue or paraformaldehyde fixed samples are ideally required for clinical diagnostic tests and retrospective analysis of archived samples. We evaluated the measurement of gene expression from FFPE tissues using HTG Molecular's qNPA™ assay and made comparisons to qPCR. The results show that qNPA provides a highly sensitive, accurate, quantitative, and robustly reliable automated multiplexed assay of gene expression from FFPE, permitting the rapid development and launch of new diagnostic and research assays. qNPA uses a lysis-only, extraction-free protocol which measures up to 47 genes/sample. In contrast, qPCR requires RNA extraction/reverse transcription. Accuracy was determined by correlating measurements from matched frozen and fixed samples, determining the R2 correlation coefficient. Comparing frozen/FFPE cell pellets qNPA R2 = 0.97. Comparing frozen/FFPE pancreas tissue qNPA R2= 0.97 with a present call rate of 91%, compared to qPCR R2= 0.86 and a present call rate of just 17%, even using 26 times more FFPE sample amount than qNPA. Comparing fresh/paraformaldehyde fixed Islet cells, qNPA R2= 0.98, qPCR R2= 0.02, after staining for sorting by cytometry qNPA R2= 0.96, qPCR R2= 0.12. Thus, qNPA fixed tissue measurements were much more accurate than qPCR. In an additional comparison using matched breast cancer frozen compared with FFPE samples, qNPA measurements averaged R2=0.81, with 6% CV for triplicate measurements of separately processed samples. For >95% of genes the expression levels measured by qNPA were independent of cold ischemic times of 0 to 16 hr. Measured levels are also independent of fixation from 4 to 72 hr. The robust performance of qNPA measurements from FFPE translated into quantitative consistency that has previously not been achievable using qPCR. Data from clinical FFPE samples submitted by a large number of HTG's clients was reviewed and for each set the number of samples tested, the average area tested/sample (cm2 area of a 5 micron thick section), the failure rate (FR), and the avg reproducibility (%CV for samples independently processed in triplicate) was determined for breast FFPE (300 samples, 0.3 cm2/sample, 1% FR, 10.5%CV), lung FFPE (700 samples, 0.3 cm2/sample, 0.5% FR, 9.5%CV), prostate FFPE (15 samples, 0.3 cm2/sample, 0% FR, 11%CV), colon FFPE (15 samples, 0.3 cm2/sample, 0% FR, 9%CV), ovary FFPE (15 samples, 0.3 cm2/sample, 0% FR, 10%CV) and lymphoma FFPE (300 samples, 0.3 cm2/sample, 0.5% FR, 8%CV). Thus, qNPA provides a highly accurate, reproducible, robust multiplexed measurement of gene expression from FFPE tissue that is not affected by wide variations in ischemic or fixation time. Fully automated qNPA on the EDGE platform is expected in Jan 2013. This system, with a turn-around time of less than 24 hours, will improve even further the robustness of extraction-free gene expression. Citation Format: Matthew B. Rounseville, Klaus Pechhold, Debrah Thompson, Mark Schwartz, Bruce Seligmann. Comparison of qNPA vs qPCR on FFPE tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4133. doi:10.1158/1538-7445.AM2013-4133

Pancreatic endocrine cells are co-located into clusters called the islets of Langerhans that are comprised of glucagon producing alpha cells, insulin secreting beta cells, somatostatin generating delta cells, and other cell types. Type 1 diabetes results from an autoimmune process in which autoreactive T cells destroy the insulin producing beta cells, requiring the patient to inject insulin to regulate their blood glucose levels. Thus far, attempts to cure diabetes via islet transplantation have been limited by insufficient donor supply, inconsistent isolated islet quality, continued autoimmunity, alloimmune rejection, and limited beta cell regeneration. Diabetes research has focused on preventing the autoimmune response, promoting stem cell to beta cell differentiation, and defining the factors that influence beta cell proliferation. Islet research, in turn, has been limited to whole islet studies since, isolating the islet cell subtypes has not been possible. Using a method recently developed for mouse islet cells (Pechhold et al. Nat Biotechnol. 2009 Nov; 27(11):1038-42), that uses intracellular hormone staining and flow cytometry, we are able to sort human islets into populations uniquely expressing glucagon, insulin, or somatostatin. Further, we have developed a human gene array to measure candidate gene expression using a quantitative nuclease protection assay (qNPA). This technique uses 50 base oligomers that specifically recognize RNA from each gene of interest, overcoming limitations caused by the harsh conditions required for intracellular staining. We report gene expression analysis for specific hormones and transcription factors expressed in each islet cell population. We are further modifying this technique to study nonhuman primate islets, and investigate the specific proteome and miRNA profiles for individual islet cell populations. The goal of these studies is to characterize the genetic differences between the islet cell populations and understand which factors control beta cell regeneration and The adult human islet is comprised of alpha, beta, and delta cells. The cells are dispersed in a heterogenous manner throughout the islet.

The pancreatic alpha- and beta-cells are critical components in regulating blood glucose homeostasis via secretion of glucagon and insulin, respectively. Both cell types are typically localized in the islets of Langerhans. However, little is known about the roles of paracrine interactions that contribute to their physiological functions. The lack of suitable cell lines to study alpha- and beta-cells interactions have led us to develop an alpha-cell-specific Cre-expressing transgenic line utilizing a glucagon promoter sequence, the Glu-Cre transgenic mouse. Here, we demonstrate that the Glu-Cre could specifically and efficiently excise floxed target genes in adult islet alpha-cells. We further showed that deletion of the tumor suppressor gene, multiple endocrine neoplasia type 1 (Men1), in alpha-cells led to tumorigenesis. However, to our surprise, the lack of Men1 in alpha-cells did not result in glucagonomas but rather beta-cell insulinomas. Because deletion of the Men1 alleles was only present in alpha-cells, our data suggested that cross communication between alpha- and beta-cells contributes to tumorigenesis in the absence of Men1. Together, we believed that the new model systems described here will allow future studies to decipher cellular interactions between islet alpha- and beta-cells in a physiological context.

Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1 , retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.

OBJECTIVE Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by β-cell–specific killing by autoreactive T-cells. Less is known about β-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents. RESEARCH DESIGN AND METHODS We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell–mediated β-cell–destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse. RESULTS The proportion of pancreatic insulin-positive β-cells to glucagon-positive α-cells was about 4:1 in nondiabetic mice. Islets isolated from newly diabetic mice exhibited the expected severe β-cell depletion accompanied by phenotypic β-cell changes (i.e., hypertrophy and degranulation), but they also revealed a substantial loss of α-cells, which was further confirmed by quantitative immunohistochemisty. While maintaining normal randomly timed serum glucagon levels, newly diabetic mice displayed an impaired glucagon secretory response to non–insulin-induced hypoglycemia. CONCLUSIONS Systematically applying multicolor flow cytometry and immunohistochemistry to track declining β-cell numbers in recently diabetic mice revealed an altered endocrine cell composition that is consistent with a prominent and unexpected islet α-cell loss. These alterations were observed in induced and spontaneous autoimmune diabetes models, became apparent at diabetes onset, and differed markedly within islets compared with sub–islet-sized endocrine cell clusters and among pancreatic lobes. We propose that these changes are adaptive in nature, possibly fueled by worsening glycemia and regenerative processes.

Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet-cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in α-cells or an insulin promoter in β-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated α-cells or β-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases.

Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic beta-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of beta-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ss-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation.We adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5'-bromo-2'-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that beta-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the beta-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ss endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of delta-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (alpha-cells).We conclude that murine beta-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic beta-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient beta-cell mass to restore normal glucose homeostasis.

The biology and properties of dendritic cells (DCs) have been intensely studied in the research areas of infectious diseases, tumor immunology, and vaccine development. This unique subset of immune cells has recently also moved to the center of interest for basic and clinical research in autoimmunity, owing not only to the extraordinary importance of DCs in the initiation and sustenance of adaptive immune responses, but also to more recent discoveries about their profound ability to control and downregulate ongoing T-cell responses. We review current progress of using DCs in mice for induction and propagation of autoimmune T-cell responses and their therapeutic potential to dampen or even stop β-cell-specific autoimmunity. Finally, we offer our perspective on how basic research progress in DC technology, mostly from mouse models, may translate into emerging diagnostic and therapeutic applications for human type 1 diabetes.

Rodent immune-mediated diabetes model studies have advanced understanding of beta cell-specific T cell responses, and the testing of therapeutic approaches. We have used an inducible diabetes model based on rat insulin promotor (RIP)-driven expression of CD80 (B7-1) on pancreatic beta cells. Using these mice, we have established that immunizing with a single autoantigen can promote progressive islet inflammation and eventually T cell-mediated diabetes. We now describe a potent immunization protocol using peptide-pulsed mature dendritic cells (DCs) to examine peptide epitopes derived from endogenous (preproinsulin) and transgenically expressed beta cell antigens, namely lymphocytic choriomeningitis virus glycoprotein (LCMV-GP). LCMV-GP epitopes efficiently promote beta cell destruction, and the autoantigenic peptide concentration used to load the DCs correlates directly with diabetes onset. The system allowed us to assess cytotoxic T cell (CTL) fine specificity by immunizing with DCs presenting altered peptide ligands (APLs) of the dominant LCMV-GP epitope, gp33. Finally, using an adoptive transfer system, we tested alternative in vitro T cell activation conditions, including APLs and mitogens, for their impact on T cell effector function and diabetes onset. Our studies revealed a marked discrepancy between (inflammatory) effector functions and diabetes progression, thus emphasizing the importance of structural identity between sensitizing and target epitope and the context of initial T cell activation.

We used cre/loxP-based genetic lineage tracing analysis to test a previously proposed hypothesis that in vitro cultured adult pancreatic β-cells undergo epithelial-mesenchymal transition (EMT) to generate a highly proliferative, differentiation-competent population of mesenchymal islet “progenitor” cells. Our results in the mouse that are likely to be directly relevant to the human system show that adult mouse β-cells do not undergo EMT in vitro and that the mesenchymal cells that arise in cultures of adult pancreas are not derived from β-cells. We argue that these cells most likely originate from expansion of mesenchymal cells integral to the heterogeneous pancreatic islet preparations. As such, these mesenchymal “progenitors” might not represent the best possible source for generation of physiologically competent β-cells for treatment of diabetes.

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.