Objective
T1D is an autoimmune disease in which the body’s own immune system mistakenly attacks beta-cells. Despite decades of research, we still do not fully understand how immune cells find, target, and destroy beta-cells—especially in early stages of disease. This knowledge gap has limited our ability to design effective therapies that can block or reverse the autoimmune process. To surmount these obstacles, our project brings together four powerful strategies: human T-cell engineering that mimics T1D immune injury, assessment of islets (beta-cells) in situ in human pancreas slices, state-of-the-art live-cell imaging technologies that spatio-temporally tracks immune-cell/beta-cell interactions, and an avant-garde spatial (single-cell) imaging mass spectrometry strategy. We will use these combined strategies on these Specific Aims:
Aim 1. Visualize human T-cell behavior vs human beta-cell function and injury. We will perform multiplex, large field-of-view, high-throughput 4D visualization of immune cell behavior with minimal phototoxicity. T-cells (mCherry) added to human pancreas slices with beta-cells loaded with fluorescent Ad-GCaMP6s or apoptosis marker Annexin V to allow simultaneous visualization of T-cell activity vs beta-cell functional and survival states. We will quantify T-cell motility patterns to characterize islet infiltration, expecting to observe a transition in T-cell behavior from transient surveillance-like motility in early infiltration stages to more stable antigen-engaged arrest as auto-immunity progresses to severe disease. The islets will likely show heterogeneous infiltration patterns with increased spatial confinement of CD4 or CD8 T-cells around beta-cell clusters undergoing dysfunction. We expect progressive disruption of Ca2+ signaling from normal rhythmic Ca2+ oscillations to disruption towards a sustained basal Ca2+ (of dying beta-cells) along with progressive increase in Ann-V fluorescence.
Aim 2. Assess whether human beta-cell connectivity and spatial heterogeneity of secretion influence T-cell targeting and effector function. We hypothesize that beta-cell-to-beta-cell connectivity and local heterogeneity in secretion can govern spatiotemporal dynamics of T-cell infiltration, positioning and effector activity during autoimmune progression. Secretory failure may precede or promote immune recognition, yet spatiotemporal coupling between Ca2+ dynamics, insulin secretion and T-cell contact has not been directly visualized. From these studies, we expect to observe: 1) Single beta-cell functional decline (loss of Ca2+ oscillations, asynchronous secretion) correlated with beta-cell attrition, providing insight into whether beta-cell dysfunction precedes or coincides with immune attack. 2) beta-cell-to-beta-cell connectivity and secretory behavior may influence T-cell engagement. We expect highly-connected beta-cell networks to exhibit uniform whole-islet Ca2+ pulsatile dynamics and secretion, limiting spatial cues that facilitate T-cell localization and activation. Disruption of beta-cell-to-beta-cell connectivity may generate spatially-confined hyper- or hypo-active beta-cell clusters - acting as “immunological hotspots” drawing T-cells and enhancing effector function. Increased heterogeneity in insulin secretion may correlate with elevated local T-cell activation, even in absence of overt inflammation. Intriguingly, non-connected, functionally dormant (low Ca2+/secretory activity) beta-ells may be resistant to T-cell recruitment and toxic effects.
Aim 3. Apply the PIRL strategy to determine the precise proteomic / transcriptomic changes during the process of human beta-ell injury - in Aims 1 and Aim 2, which includes: a) beta-cells in contact with T-cells showing progressive reduction of normal Ca2+ response to ‘dying’ Ca2+ pattern caused by immune injury; 2) beta-cell Ca2+ response with no T cell contact (i.e. early T1D), 3) beta-cells with no Ca2+ response and no T-cell contact, which are probably dormant survivors resistant to immune injury. These beta-cell samples will be sent for proteomics analysis via PIRL-MS along with sample fractions for transcriptomics. We will correlate the proteomic and transcriptomic data precisely co-registered to various beta-cell function/ dysfunction and survival imaging, which will culminate in a precise ‘functional’ vis-à-vis transcriptomic and proteomic landscapes of the T1D islet.
Background Rationale
T1D is an autoimmune disease characterized by targeted destruction of beta-cells brought about by progressive infiltration of pancreatic islets by immune cells, starting with activation and recruitment of autoreactive CD4+ T cells that recognize insulin epitopes, which activate cytotoxic CD8+ T-cells and macrophages that kill beta-cells. While these disease processes have been well characterized, the final effector phase that unfolds from initiation of beta-cell injury to beta-cell death within the intact pancreas remains poorly understood, particularly with human beta-cells that are not inexorably lost in all T1D subjects, even after many years of established T1D (>3 years post diagnosis). Whether surviving beta-cells are functionally intact and why they are resistant to immune injury are unclear. Autoimmune attack on beta-cells is spatially heterogeneous with some islets heavily infiltrated while others remain unscathed; and within an islet, not all beta-cells are equally susceptible to the immune-mediated killing. It is currently impossible to dissect all these spatial and cellular heterogeneity in beta-cell secretory and survival responses to immune injury because of the inaccessibility of human T1D pancreases, particularly autoantibody-positive (AAb+) and early onset T1D, even with the availability of pancreases (as thin slices) from the BT1D-supported nPOD. We propose the following multi-pronged innovative strategy to surmount these limitations including: human T-cell engineering capable of mimicking human T1D, assessment of islets (beta-cells) in situ in human pancreas slices, state-of-the-art live-cell imaging technologies capable of tracking immune cell/beta-cell interactions (secretory functions, cell injury), and an avant-garde spatial imaging mass spectrometry strategy called picosecond infrared laser (PIRL) that can perform single cell (PIRL) and subcellular (femtosecond, FIRL) ablation for cytosol extraction with no degradation for PIRL-imaging mass spectrometry (proteomics) and transcriptomics. These PIRL/FIRL-omics, when co-registered to the precise visually observed progressive stages of beta-cell secretory dysfunction or survival, will be able determine the precise ‘functional’ vis-à-vis transcriptomic and proteomic landscapes of the human T1D islet.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease in which the body’s own immune system mistakenly attacks insulin-producing beta-cells in the pancreas. This destruction leads to the loss of insulin, a hormone critical for controlling blood sugar and requires patients to rely on lifelong insulin therapy. Despite decades of research, we still do not fully understand how immune cells find, target, and destroy beta-cells—especially in the early stages of disease, wherein we could implement earlier interventions to prevent disease progression. This knowledge gap has indeed limited our ability to design effective therapies that can block or reverse the autoimmune process at the different stages of disease.
To surmount these obstacles, our project brings together four powerful strategies: human T-cell engineering mimicking T1D, assessment of the islet (beta-cells) in situ in human pancreas slices, state-of-the-art live-cell imaging technologies, and an avant-garde spatial (single cell) imaging mass spectrometry (MS) strategy. First, we perfected T-cell receptor (TCR) engineering approaches for human T-cells isolated from human spleens whereby endogenous TCR is deleted, then well-designed TCRs that recognize selected islet antigens and at different affinities and tagged with mCherry to allow visualization of T-cell behavior, all these knocked-in by lentivirus. Second, we perfected a thin human pancreas slice preparation that can last 10 days in culture to allow extensive manipulation such as multi-adenovirus directed cell-specific expression of calcium and insulin granule exocytosis fluorophores into beta-cells, Third, spatio-temporal imaging with the most sophisticated microscopes to visualize the various mCherry-tagged T-cell populations dynamic interactions with beta-cells (expressed green calcium fluorophore GCaMP6s) or injury fluorescent markers (Annexin-V), to precisely track the effects of specific T-cells (CD4 helper, CD8 killer, immune-tolerant regulatory T cells (Treg)) on beta-cell secretory functions and survival. Fourth and most importantly, a novel strategy for imaging mass spectrometry (IMS) proteomic and transcriptomic analysis with the Picosecond Infrared Laser (PIRL), which is the main thrust of this proposal. This powerful technology uses ultrashort picosecond (now upgraded to femtosecond – thus FIRL) laser pulses with the energy fully confined by the exceptionally high absorption of water in the infrared region to drive ablation by which all molecules are excised fully intact within the laser plume, even protein complexes at 90% intact, that is with very little degradation in contrast to very substantial degradation with the use of current IMS strategies.
The most important single application of this PIRL/FIRL technology is mapping out in space and time the extremely complex immune cell-islet cell interactions and fates within the intact human islet micro-environment in a human pancreas slice that genuinely mimics human T1D (at least at the start of disease) during the process of beta-cell injury. We can then correlate/co-register the PIRL/FIRL-obtained molecular signatures to the precise (visually observed) and progressive stages of beta-cell secretory dysfunction or survival inflicted by the specific T-cell populations. Such mechanistic insights can then inform novel and tailored treatment strategies.
Anticipated Outcome
This project is expected to produce important new insights into how and why the immune system attacks beta-cells in people with T1D. By using powerful imaging tools to visualize these processes in real time and within real human pancreatic tissues, we will move beyond static snapshots to uncover the step-by-step progression of disease. The ultimate goal is to identify new ways to stop or even reverse the disease caused by the different T-cells early—before too many beta-cells are destroyed and determine the underlying mechanisms (by PIRL-IMS) by which T-cell inflict injury of beta-cell function and survival.
One major outcome of this work is the technical advancement in the first-of-its-kind imaging platform that allows us to observe immune cell behavior at the tissue level with subcellular detail. This technology will enable us to track how T-cells—the immune cells responsible for attacking beta-cells—move, interact, and make decisions inside living pancreatic tissue. We will be able to see how immune cells’ behavior changes over the course of the disease and how it responds to specific features of the beta-cells in terms of inducing progressive loss of secretory function, and increasing cellular injury towards death, and potentially test drug molecules to mitigate any of these deleterious processes. New concepts in beta-cell dysfunction in T1D will be elucidated. For example, we expect to learn whether certain patterns of insulin secretion or connectivity between beta-cells make them more vulnerable to immune attack. We will have a better understanding of how beta-cells’ own activity and communication influence whether they are targeted or spared. By mapping the dynamics of insulin secretion and calcium signaling across the islet, and how these relate to immune cell behavior, we can uncover what makes certain beta-cells more "visible" or "appealing" to the immune system, and conversely, ‘tolerant’ to immune injury. These insights could inform strategies to make beta-cells less recognizable—or more resilient—to immune attack. We hope that most of these possible outcomes will be revealed in the work in Aims 1 and 2.
We have preliminary data that was not shown in the proposal because of spatial limitations that an engineered Treg accumulated in the human islet in pancreas slice of an Aab+ donor (from nPOD), and in those regions there was less injury indicated by the reduced Annexin-V fluorescence in contrast to surrounding beta-cells which showed more injury pattern. Much further work will be needed to determine the precise protective actions in those Tregs and cellular events in beta-cells they come in contact with. Further questions could be pursued: can these Tregs block injury caused by CD4 and CD8 T-cells. These experiments with Tregs will help refine and optimize next-generation cell-based therapies before they are tested in patients, thus also serving as a pre-clinical tool to test such and other novel therapies.
A second technology advancement is also the first-of-its kind, the PIRL/FIRL strategy of ablating and extracting single-cell cytosols of beta-cells in live islets in pancreas slices that correspond to the T-cell-induced beta-cell dysfunction or threat to survival. This enables an unprecedented determination of the precise ‘functional’ vis-à-vis transcriptomic and proteomic landscapes of the humanT1D islet that can explain all or most of the above deleterious effects of different T-cells (and protection by Tregs) during the progression of beta-cell dysfunction and injury. This achieves an unprecedented precise ‘molecular-functional’ fingerprint of the progressive stages of specific immune-cell/beta-cell injury. This would be the outcome from the work in Aim 3.
Relevance to T1D
Despite the considerable ongoing research and advances in diabetes care, there is still no cure for T1D, and we still do not fully understand why or how the immune system starts to attack the pancreas in the first place. The major impediment to progress is insufficient ‘technology’. This project aims to directly address this severe technological deficit to address what has been very important but unanswerable questions in T1D. Here, we combined four technologies, human T-cell engineering mimicking T1D, assessment of the islet (beta-cells) in situ in human pancreas slices, state-of-the-art live-cell imaging technologies, and an avant-garde spatial imaging mass spectrometry strategy (PIRL/FIRL-omics). The first three have some degree of advancement also used by others but separately, whereas the fourth is entirely novel and is the major thrust in the current application. However, we would be the first to combine all four strategies, enabling us to literally watch in real time how specific T-cell populations of human T1D interact with human beta-cells in pancreas slices during the development of T1D. With the PIRL/FIRL spatial proteomics/transcriptomics strategy applied concurrently with live-cell imaging, we will not only see what is happening but also when, where, and why it happens, and eventually block specific deleterious effects. This work will enable us to resolve these key questions in T1D: (1) Identify the earliest signs of beta-cell dysfunction in T1D inflicted by T-cells before any clinical phenotype occurs. (2) What makes certain beta-cells more likely to be attacked, and whether we can protect them. (3) What makes certain beta-cells resistant to immune attack, which could be those dormant beta-cells often talked about but with little mechanistic insight so far. Much of these insights will be revealed in this one-year innovative grant proposal.
Using this powerful strategy, the NEXT STEPS to pursue include the following, which will require additional funding for several more years if BT1D finds our strategy deserving of support and serves the BT1D mandate. (1) The above protocols and insights will demonstrate dynamic beta-cell dysfunction caused by specific T-cell injury towards which novel drugs could be applied to mitigate and determine the drug action on beta-cells or attacking T-cells, revealed by PIRL/FIRL-omics, thus providing a preclinical model to test new therapeutic strategies. (2) Our preliminary data on an engineered Treg showed protective actions on beta-cell injury in an Aab+ human donor, suggest that Tregs could be considered to be cell-based therapy for T1D. Much more work will be required to design Tregs and determine their precise mechanistic actions in mitigating beta-cell injury. (3) Functionally and mechanistically map how other T-cell populations that recognize different islet antigens (i.e. PPI, GAD IA2, ZnT8, etc.) attack islets at different times in T1D, which might explain the less aggressive responses at the start of disease to various degrees of severity and rapidity of disease progression. (4) Some T-cells were reported to have affinity for alpha-cells (which we have developed tools to target expression of calcium and exocytosis fluorophores), but is it not known whether this contributes to alpha-cell dysfunction that perturbs glucagon secretion in T1D (glucose blindness and hypoglycemia) shown to already occur at early stages of the disease when there is insufficient beta-cell loss to explain paracrine dysfunction (that is currently believed). Addressing these questions is not only fundamental to understanding T1D pathogenesis but also essential for developing targeted therapies that protect beta-cells such as with the mentioned engineered Tregs that can restore immune tolerance or prevent disease progression in at-risk individuals. Mechanistic insights elucidated from pursuing these questions will inform strategies to modulate immune infiltration, enhance beta-cell resilience and ultimately preserve insulin-producing capacity in T1D.