Ann E. Herman

CD4+CD25+ T Regulatory Cells Dependent on ICOS Promote Regulation of Effector Cells in the Prediabetic Lesion

CD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69− cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69− cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory–specific profile. Thus, CD4+CD25+69− Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.

Where CD4+CD25+ T reg cells impinge on autoimmune diabetes

Foxp3 is required for the generation and activity of CD4+CD25+ regulatory T (T reg) cells, which are important controllers of autoimmunity, including type-1 diabetes. To determine where T reg cells affect the diabetogenic cascade, we crossed the Foxp3 scurfy mutation, which eliminates T reg cells, with the BDC2.5 T cell receptor (TCR) transgenic mouse line. In this model, the absence of T reg cells did not augment the initial activation or phenotypic characteristics of effector T cells in the draining lymph nodes, nor accelerate the onset of T cell infiltration of the pancreatic islets. However, this insulitis was immediately destructive, causing a dramatic progression to overt diabetes. Microarray analysis revealed that T reg cells in the insulitic lesion adopted a gene expression program different from that in lymph nodes, whereas T reg cells in draining or irrelevant lymph nodes appeared very similar. Thus, T reg cells primarily impinge on autoimmune diabetes by reining in destructive T cells inside the islets, more than during the initial activation in the draining lymph nodes.

Gene expression microarrays: glimpses of the immunological genome.

Successful microarray experimentation can generate enormous amounts of data, potentially very rich but also very unwieldy. Bold outlooks and new methods for data analysis and presentation should yield additional insight into the complexities of the immune system.

Cathepsin L is essential for onset of autoimmune diabetes in NOD mice

Lysosomal proteases generate peptides presented by class II MHC molecules to CD4+ T cells. To determine whether specific lysosomal proteases might influence the outcome of a CD4+ T cell-dependent autoimmune response, we generated mice that lack cathepsin L (Cat L) on the autoimmune diabetes-prone NOD inbred background. The absence of Cat L affords strong protection from disease at the stage of pancreatic infiltration. The numbers of I-A(g7)-restricted CD4+ T cells are diminished in Cat L-deficient mice, although a potentially diabetogenic T cell repertoire persists. Within the CD4+ T cell compartments of Cat L-deficient mice, there is an increased proportion of regulatory T cells compared with that in Cat L-sufficient littermates. We suggest that it is this displaced balance of regulatory versus aggressive CD4+ T cells that protects Cat L-deficient mice from autoimmune disease. Our results identify Cat L as an enzyme whose activity is essential for the development of type I diabetes in the NOD mouse.

A Conserved Salt Bridge in the G Loop of Multiple Protein Kinases Is Important for Catalysis and for In Vivo Lyn Function

The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

Development of an Automated, High-Throughput Bactericidal Assay That Measures Cellular Respiration as a Survival Readout for Neisseria meningitidis

ABSTRACT Complement-mediated bactericidal activity has long been regarded as the serological correlate of protective immunity against Neisseria meningitidis. This was affirmed in 2005 at a WHO-sponsored meningococcal serology standardization workshop. The assay currently employed by most laboratories involves determining surviving bacterial colony counts on agar as a readout which is labor-intensive, time-consuming, and not amendable to rapid data analysis for clinical trials. Consequently, there is an acute need to develop a sensitive, high-throughput bactericidal assay to enable a rapid and robust assessment of the effectiveness of vaccine candidates. To this end, we have developed an automated, kinetic assay based on the fluorescent respiration product of resazurin which reduces assay volume, shortens assay time, and facilitates automation of data analysis. We demonstrate proof of concept for applicability of this high-throughput system with multiple meningococcal strains and utilizing different lots of human complement. The assay is robust and highly reproducible. Titers obtained by the fluorescence readout method are strongly correlated with the data obtained using the conventional, agar plate-based assay. These results demonstrate that the detection of bacteria that have survived the bactericidal reaction by measuring metabolic activity using a fluorescent dye as an alternative readout is a promising approach for the development of a high-throughput bactericidal assay.

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of Autoimmune Diabetes

Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy.

Integrating traditional and postgenomic approaches to investigate lymphocyte development and function.

The sequencing of the human and mouse genomes to over 95% coverage revealed that more than three quarters of the ~30,000 genes have not yet been assigned biological functions. Thus, a strong need exists to functionally annotate over 20,000 genes. This challenge provides the opportunity to discover novel genes essential for disease pathology. If their encoded proteins are druggable, such genes represent promising targets for the development of new and improved therapies for severe human disorders including autoimmunity (rheumatoid arthritis, systemic lupus erythematosus, diabetes, etc.), allergies (asthma, dermatitis, etc.), and other inflammatory diseases. Traditional approaches to investigate gene function use results from biochemical, cell biological, molecular biological, and genetic studies, often accumulated over many years of intense research, to formulate a hypothesis that is then tested via targeted gene disruption, or more recently siRNA-mediated targeted knockdown for increased invivo relevance. This approach has been impressively powerful and productive. For example, over 100 genes involved in development and function of the adaptive immune system have been identified. On the other hand, the traditional approach is clearly slow and highly labor intensive, as many thousands of graduate students, postdocs, and technicians can attest.

Inhibition of c-Kit Is Not Required for Reversal of Hyperglycemia by Imatinib in NOD Mice

(1) Aim/Hypothesis Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice. (2) Methods The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-KitT670I mice and NOD.c-Kitwt littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-KitT670I allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored. (3 )Results In vitro expansion of HSCs from NOD.c-Kitwt mice was sensitive to imatinib, while expansion of HSCs from NOD.c-KitT670I mice was insensitive to imatinib. However, in vivo treatment with imatinib lowered blood glucose levels in both strains of mice. (4) Conclusions/Interpretation The HSC experiment confirmed that, in NOD.c-KitT670I mice, c-Kit is resistant to imatinib. As both NOD.c-KitT670I and NOD.c-Kitwt mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.