Objective
Type 1 diabetes (T1D) is an autoimmune disease in which the body’s own immune system mistakenly destroys insulin-producing cells in the pancreas, called β cells. Once these cells are gone, people with T1D must rely on daily insulin for life. Scientists know that a type of immune cell called CD8⁺ T cells plays a major role in this process by attacking and killing β cells. However, we still don’t fully understand how these T cells become harmful in the first place.
Some treatments can delay the onset of T1D, such as Teplizumab, but they don’t stop the disease entirely or restore healthy immune function. To develop better therapies, we need to understand the early steps in the immune process specifically, how CD8⁺ T cells are “trained” or programmed to become destructive before they even reach the pancreas.
This project focuses on a molecule called NKG2D, found on the surface of CD8⁺ T cells. NKG2D is known to help immune cells respond to infections and cancer, but its role in autoimmune diseases like T1D is less clear. Our early findings suggest that NKG2D may play a critical role in “teaching” CD8⁺ T cells how to become aggressive and capable of killing β cells.
The goal of this research is to understand how NKG2D affects the development, function, and energy use of CD8⁺ T cells in both mice and humans. By uncovering how this molecule shapes harmful immune responses, we hope to find new ways to stop T cells before they attack. This could lead to more effective and targeted therapies for T1D interventions that prevent the disease or slow its progress without shutting down the entire immune system.
Background Rationale
Type 1 diabetes (T1D) is an autoimmune disease that develops when the immune system, which normally protects us from infections, turns against the body’s own insulin-producing cells in the pancreas, called beta (β) cells. This attack leads to a lifelong need for insulin therapy and can result in serious health complications. Despite advances in our understanding of the disease, we still don’t know exactly how or why this harmful immune response begins, which limits our ability to stop it before lasting damage occurs.
One of the key players in this process is a type of immune cell called a CD8⁺ T cell, sometimes referred to as a “killer T cell.” These cells are designed to destroy infected or abnormal cells, like those infected with viruses or transformed into cancer. However, in T1D, some of these CD8⁺ T cells become autoreactive, meaning they mistake healthy β cells for dangerous targets and destroy them.
Current treatments, like insulin injections, only manage blood sugar levels they do not stop the immune attack. Recently, a drug called Teplizumab was approved by the FDA to delay the onset of T1D in people at high risk. While this is a major breakthrough, the drug only postpones the disease and does not correct the root cause. To truly prevent or cure T1D, we need to better understand the earliest steps that lead CD8⁺ T cells to become harmful.
This study focuses on a molecule called NKG2D, which sits on the surface of CD8⁺ T cells. NKG2D helps immune cells respond more strongly to threats, and it has been studied extensively in cancer and infection. However, we don’t yet know how it affects CD8⁺ T cells in autoimmune diseases like T1D. Our preliminary findings suggest that NKG2D could be playing a key role in shaping the harmful behavior of CD8⁺ T cells early in their development well before they reach the pancreas.
We believe that NKG2D acts like an amplifier, helping to program naïve (inactive) CD8⁺ T cells into aggressive cells capable of killing β cells. If this is true, blocking or modifying NKG2D signals early on could prevent these T cells from becoming pathogenic in the first place. This opens a window of opportunity to intervene before any damage is done, potentially delaying or even preventing T1D altogether.
To test this, we will compare CD8⁺ T cells with and without NKG2D signaling both in mice and in human cells. We will look at how they develop, what genes they express, how they use energy, and how well they can kill β cells. This will give us a detailed picture of how NKG2D influences their behavior.
Understanding this process could lead to more precise therapies that target only the harmful immune cells, leaving the rest of the immune system intact. In the long run, this could move us closer to stopping T1D at its source by preventing the immune system from launching its attack in the first place.
Description of Project
Type 1 diabetes (T1D) is a serious lifelong condition that occurs when the body’s immune system mistakenly attacks and destroys the insulin-producing cells in the pancreas, called beta cells. Without insulin, the body can’t regulate blood sugar, and people with T1D must rely on daily insulin injections for survival. While we’ve known for a long time that this is an autoimmune disease, we still don’t fully understand why the immune system turns against the body in the first place. Researchers believe that a combination of inherited genes and environmental triggers like viruses or gut microbes can lead to this breakdown in the immune system's ability to tell “self” from “non-self.” In T1D, a type of immune cell called a CD8⁺ T cell becomes reactive to beta cells. These T cells normally help the body fight infections, but in T1D they become harmful, infiltrating the pancreas and killing the very cells needed to regulate blood sugar. Even though treatments like insulin therapy manage blood sugar, they don’t address the underlying immune dysfunction. Recently, the FDA approved a new immunotherapy drug called Teplizumab, which delays the onset of T1D in people at high risk. This is a big step forward, but the drug only postpones the disease it doesn’t cure it. That means we need better therapies that can stop the autoimmune attack before it causes irreversible damage.
My research focuses on understanding how these harmful CD8⁺ T cells become activated in the first place, particularly looking at a molecule on their surface called NKG2D. This molecule helps T cells communicate and respond to signals, especially when they’re about to attack a target. While NKG2D is well-studied in infections and cancer, we don’t know much about how it works during the early stages of autoimmune diseases like T1D.
I believe that NKG2D plays a key role in helping CD8⁺ T cells develop into aggressive "killer" cells that destroy beta cells. If this is true, then blocking NKG2D during this early stage might stop or slow down the immune system’s attack, offering a new way to treat or even prevent T1D.
To test this idea, I’ve designed two research aims:
Aim 1: Understanding how NKG2D shapes T cell responses
In this aim, I will study how NKG2D affects the genetic, molecular, and metabolic programming of CD8⁺ T cells. Using advanced tools like single-cell sequencing and metabolic profiling, I will compare T cells from normal mice and mice that lack NKG2D. This will help us understand how the presence or absence of NKG2D changes T cell behavior. I’ll also repeat these studies in human T cells both from healthy individuals and people with T1D by removing the NKG2D gene using CRISPR gene editing.
Aim 2: Testing how these T cells function and contribute to disease
Next, I’ll test how well these T cells can kill beta cells. I’ll compare T cells with and without NKG2D to see whether they are equally capable of destroying mouse and human beta cells in laboratory experiments. I’ll also use a mouse model of T1D to determine whether blocking NKG2D during the early stages of T cell development changes the course of the disease. This will tell us whether NKG2D is only important during T cell development, or if it also affects their behavior after they’ve matured.
This research will help us pinpoint when and how the immune system goes wrong in T1D. By identifying how NKG2D contributes to the harmful behavior of T cells, we may uncover new ways to intervene before the damage is done.
Anticipated Outcome
Through the experiments outlined in this proposal, I anticipate uncovering key molecular and functional differences in CD8⁺ T cell differentiation and cytotoxicity that are dependent on NKG2D signaling. These findings will provide critical insight into how NKG2D contributes to the generation of autoreactive, β cell–destructive cytotoxic T lymphocytes (CTLs) in the context of type 1 diabetes (T1D).
From Aim 1, I expect that single-cell transcriptomic and epigenetic profiling of autoreactive CD8⁺ T cells derived from NKG2D knockout (KO) versus wild-type (WT) NOD mice will reveal distinct differentiation trajectories. Specifically, NKG2D-deficient cells may fail to fully activate canonical effector pathways and instead adopt a less inflammatory or memory-like transcriptional profile. This could be characterized by reduced expression of genes encoding key effector molecules such as Gzmb, Ifng, and Prf1, along with decreased accessibility at their regulatory regions. In parallel, I expect that metabolic profiling will demonstrate lower glycolytic activity and/or impaired mitochondrial function in KO CTLs, consistent with a less activated metabolic phenotype. This would support the hypothesis that NKG2D signaling drives metabolic commitment necessary for full effector differentiation.
In human CD8⁺ T cells, I anticipate that CRISPR-Cas9–mediated deletion of KLRK1 (the gene encoding NKG2D) will recapitulate the murine phenotype, resulting in impaired acquisition of effector features, including reduced cytokine production and altered expression of activation markers. These effects may be more pronounced in T cells from individuals with T1D, potentially indicating disease-specific sensitivity to NKG2D signaling. Such findings would strengthen the translational relevance of this pathway and highlight the importance of patient-specific immunophenotyping.
For Aim 2, I anticipate that CTLs generated in the absence of NKG2D signaling—either through genetic knockout, antibody blockade, or gene editing will demonstrate reduced cytolytic activity against both murine (NIT-1) and human (EndoC-βH5) β cell targets in vitro. I expect that these differences will be evident across a range of effector-to-target (E:T) ratios and will be accompanied by reduced expression of degranulation and cytotoxicity markers (e.g., CD107a, IFNγ). Importantly, I predict that blocking NKG2D signaling only during the effector phase (rather than during differentiation) will have minimal impact on CTL function, suggesting a critical window during early T cell activation where NKG2D signaling programs long-term cytotoxic potential.
Using the NOD-RAG adoptive transfer model, I anticipate that mice receiving CTLs generated without NKG2D signaling will exhibit delayed or reduced diabetes incidence, confirming that NKG2D plays a pivotal role during CTL development, rather than simply during target engagement. This in vivo validation will be essential to support the relevance of our in vitro findings and to establish the timing and necessity of NKG2D signaling in T1D pathogenesis.
Overall, these results will elucidate the molecular and functional roles of NKG2D in shaping autoreactive CD8⁺ T cell fate. They will also provide the mechanistic foundation for future studies aimed at selectively targeting this pathway to prevent or delay T1D progression without broadly suppressing immune function.
Relevance to T1D
Type 1 diabetes (T1D) develops when the body’s own immune system destroys the insulin-producing β cells in the pancreas, leaving people dependent on lifelong insulin therapy. Central to this attack are CD8⁺ T cells, which normally defend us against infections but, in T1D, mistakenly identify β cells as harmful and eliminate them. Although treatments like insulin replacement manage blood sugar, they have some limitation to stop the underlying autoimmune attack.
Our research focuses on a molecule called NKG2D found on CD8⁺ T cells and other many immune cells in our immune system. Past studies in infection and cancer have shown that NKG2D amplifies T cell responses, making them more aggressive. We believe that, in T1D, this same signaling pathway primes naïve CD8⁺ T cells to become potent killers of β cells. By understanding and interrupting this process early, we hope to prevent or slow the autoimmune destruction that causes T1D.
To explore this, we will first compare how CD8⁺ T cells develop with or without NKG2D signaling. In mice prone to diabetes (called as NOD mice), we will profile gene activity, chromatin accessibility, and metabolic function in T cells that carry or lack NKG2D. These experiments will reveal whether NKG2D is required for T cells to adopt a fully armed state capable of killing β cells. We will then extend these studies to human T cells, using gene editing to remove NKG2D and observing how this affects their behavior in both healthy individuals and people with T1D.
Next, we will directly test whether blocking NKG2D reduces the ability of these T cells to kill β cells in laboratory assays, and whether it delays or prevents diabetes in a mouse model. If successful, this approach could identify NKG2D signaling as a precise on switch for the harmful autoimmune response.
By pinpointing how NKG2D drives the earliest steps of β cell destruction, our work aims to uncover new targets for T1D therapies interventions that block the disease at its source rather than merely managing its symptoms.