Vinh Nguyen

Type 1 diabetes immunotherapy using polyclonal regulatory T cells

Autologous regulatory T cells can be expanded and are well tolerated in patients with recent-onset type 1 diabetes. Regulating type 1 diabetes In patients with type 1 diabetes (T1D), immune cells attack the insulin-producing β cells of the pancreas. The resulting prolonged increase in blood sugar levels can lead to serious complications including heart disease and kidney failure. Regulatory T cells (Tregs) have been shown to be defective in autoimmune diseases. Now, Bluestone et al. report a phase 1 trial of adoptive Treg immunotherapy to repair or replace Tregs in type 1 diabetics. The ex vivo–expanded polyclonal Tregs were long-lived after transfer and retained a broad Treg phenotype long-term. Moreover, the therapy was safe, supporting efficacy testing in further trials. Type 1 diabetes (T1D) is an autoimmune disease that occurs in genetically susceptible individuals. Regulatory T cells (Tregs) have been shown to be defective in the autoimmune disease setting. Thus, efforts to repair or replace Tregs in T1D may reverse autoimmunity and protect the remaining insulin-producing β cells. On the basis of this premise, a robust technique has been developed to isolate and expand Tregs from patients with T1D. The expanded Tregs retained their T cell receptor diversity and demonstrated enhanced functional activity. We report on a phase 1 trial to assess safety of Treg adoptive immunotherapy in T1D. Fourteen adult subjects with T1D, in four dosing cohorts, received ex vivo–expanded autologous CD4+CD127lo/−CD25+ polyclonal Tregs (0.05 × 108 to 26 × 108 cells). A subset of the adoptively transferred Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer. Immune studies showed transient increases in Tregs in recipients and retained a broad Treg FOXP3+CD4+CD25hiCD127lo phenotype long-term. There were no infusion reactions or cell therapy–related high-grade adverse events. C-peptide levels persisted out to 2+ years after transfer in several individuals. These results support the development of a phase 2 trial to test efficacy of the Treg therapy.

A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation

Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

Quantitative Time-Resolved Phosphoproteomic Analysis of Mast Cell Signaling

Mast cells play a central role in type I hypersensitivity reactions and allergic disorders such as anaphylaxis and asthma. Activation of mast cells, through a cascade of phosphorylation events, leads to the release of mediators of the early phase allergic response. Understanding the molecular architecture underlying mast cell signaling may provide possibilities for therapeutic intervention in asthma and other allergic diseases. Although many details of mast cell signaling have been described previously, a systematic, quantitative analysis of the global tyrosine phosphorylation events that are triggered by activation of the mast cell receptor is lacking. In many cases, the involvement of particular proteins in mast cell signaling has been established generally, but the precise molecular mechanism of the interaction between known signaling proteins often mediated through phosphorylation is still obscure. Using recently advanced methodologies in mass spectrometry, including automation of phosphopeptide enrichments and detection, we have now substantially characterized, with temporal resolution as short as 10 s, the sites and levels of tyrosine phosphorylation across 10 min of FcεRI-induced mast cell activation. These results reveal a far more extensive array of tyrosine phosphorylation events than previously known, including novel phosphorylation sites on canonical mast cell signaling molecules, as well as unexpected pathway components downstream of FcεRI activation. Furthermore, our results, for the first time in mast cells, reveal the sequence of phosphorylation events for 171 modification sites across 121 proteins in the MCP5 mouse mast cell line and 179 modification sites on 117 proteins in mouse bone marrow-derived mast cells.

Attenuation of donor-reactive T cells allows effective control of allograft rejection using regulatory T cell therapy

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long‐term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor‐specific transfusion followed by cyclophosphamide treatment deleted 70–80% donor‐reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 106 Tregs with direct donor reactivity or 25 × 106 polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen‐specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8+ T cells and concomitant increase in endogenous Tregs among graft‐infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor‐reactive T cells will be an important component of Treg‐based therapies in transplantation.

Menin determines K-RAS proliferative outputs in endocrine cells.

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors.

An evolving autoimmune microenvironment regulates the quality of effector T cell restimulation and function

Significance Autoimmunity is self-perpetuating within the islets at the time of type 1 diabetes diagnosis. However, little is known about how pathogenic effector T cell functions are stimulated within the islets. Here, we show that the infiltration state of the islet affects T cell interactions with antigen-presenting cells and corresponding T cell effector cytokine production within the islets. This work identifies a restimulation event that is modified by the local islet environment as a critical step in the expression of T cell effector functions and autoimmune tissue destruction. Defining the processes of autoimmune attack of tissues is important for inhibiting continued tissue destruction. In type 1 diabetes, it is not known how cytotoxic effector T cell responses evolve over time in the pancreatic islets targeted for destruction. We used two-photon microscopy of live, intact, individual islets to investigate how progression of islet infiltration altered the behavior of infiltrating islet-specific CD8+ T cells. During early-islet infiltration, T-cell interactions with CD11c+ antigen-presenting cells (APCs) were stable and real-time imaging of T cell receptor (TCR) clustering provided evidence of TCR recognition in these stable contacts. Early T cell–APC encounters supported production of IFN-γ by T effectors, and T cells at this stage also killed islet APCs. At later stages of infiltration, T-cell motility accelerated, and cytokine production was lost despite the presence of higher numbers of infiltrating APCs that were able to trigger T-cell signaling in vitro. Using timed introduction of effector T cells, we demonstrate that elements of the autoimmune-tissue microenvironment control the dynamics of autoantigen recognition by T cells and their resulting pathogenic effector functions.

Polyclonal Regulatory T cell Therapy for Control of Inflammation in Kidney Transplants

Early subclinical inflammation in kidney transplants is associated with later graft fibrosis and dysfunction. Regulatory T cells (Tregs) can reverse established inflammation in animal models. We conducted a pilot safety and feasibility trial of autologous Treg cell therapy in three kidney transplant recipients with subclinical inflammation noted on 6‐month surveillance biopsies. Tregs were purified from peripheral blood and polyclonally expanded ex vivo using medium containing deuterated glucose to label the cells. All patients received a single infusion of ~320 × 106 (319, 321, and 363.8 × 106) expanded Tregs. Persistence of the infused Tregs was tracked. Graft inflammation was monitored with follow‐up biopsies and urinary biomarkers. Nearly 1 × 109 (0.932, 0.956, 1.565 × 109) Tregs were successfully manufactured for each patient. There were no infusion reactions or serious therapy‐related adverse events. The infused cells demonstrated patterns of persistence and stability similar to those observed in non‐immunosuppressed subjects receiving the same dose of Tregs. Isolation and expansion of Tregs is feasible in kidney transplant patients on immunosuppression. Infusion of these cells was safe and well tolerated. Future trials will test the efficacy of polyclonal and donor alloantigen‐reactive Tregs for the treatment of inflammation in kidney transplants.

Therapeutic Regulatory T Cells Subvert Effector T Cell Function in Inflamed Islets To Halt Autoimmune Diabetes

Therapeutic regulatory T cells (Tregs) can reverse pre-established autoimmune pathology. In this study, using a mouse model of autoimmune diabetes, we aimed to determine the means by which therapeutic Tregs control islet inflammation. Islet Ag-specific Tregs infiltrated inflamed islets soon after infusion into prediabetic mice, which was quickly followed by a selective reduction of mRNA associated with effector T cells in the islets. This change was partially due to decreased CD8+ T cell accumulation in the tissue. CD8+ T cells that remained in the islets after Treg treatment were able to engage dendritic cells in a manner similar to that found in untreated mice, consistent with the retention of an activated phenotype by islet dendritic cells shortly after Treg treatment. Nonetheless, Treg treatment abrogated IFN-γ production by intraislet CD8+ and CD4+ T cells at the protein level with minimal effect on IFN-γ mRNA. Sustained expression of IFN-γ protein by effector T cells was dependent on common γ-chain cytokine activation of the mTOR pathway, which was suppressed in islet CD8+ T cells in vivo after Treg treatment. These multifaceted mechanisms underlie the efficacy of therapeutic Treg subversion of effector T cell functions at the site of inflammation to restore normal tissue homeostasis.

Mitigating Ischemic Injury of Stem Cell-Derived Insulin-Producing Cells after Transplant

Summary The advent of large-scale in vitro differentiation of human stem cell-derived insulin-producing cells (SCIPC) has brought us closer to treating diabetes using stem cell technology. However, decades of experiences from islet transplantation show that ischemia-induced islet cell death after transplant severely limits the efficacy of the therapy. It is unclear to what extent human SCIPC are susceptible to ischemia. In this study, we show that more than half of SCIPC die shortly after transplantation. Nutrient deprivation and hypoxia acted synergistically to kill SCIPC in vitro. Amino acid supplementation rescued SCIPC from nutrient deprivation, likely by providing cellular energy. Generating SCIPC under physiological oxygen tension of 5% conferred hypoxia resistance without affecting their differentiation or function. A two-pronged strategy of physiological oxygen acclimatization during differentiation and amino acid supplementation during transplantation significantly improved SCIPC survival after transplant.

Cutting Edge: Origins, Recruitment, and Regulation of CD11c + Cells in Inflamed Islets of Autoimmune Diabetes Mice

In NOD mice, CD11c+ cells increase greatly with islet inflammation and contribute to autoimmune destruction of pancreatic β cells. In this study, we investigated their origin and mechanism of recruitment. CD11c+ cells in inflamed islets resembled classical dendritic cells based on their transcriptional profile. However, the majority of these cells were not from the Zbtb46-dependent dendritic-cell lineage. Instead, monocyte precursors could give rise to CD11c+ cells in inflamed islets. Chemokines Ccl5 and Ccl8 were persistently elevated in inflamed islets and the influx of CD11c+ cells was partially dependent on their receptor Ccr5. Treatment with islet Ag-specific regulatory T cells led to a marked decrease of Ccl5 and Ccl8, and a reduction of monocyte recruitment. These results implicate a monocytic origin of CD11c+ cells in inflamed islets and suggest that therapeutic regulatory T cells directly or indirectly regulate their influx by altering the chemotactic milieu in the islets.