Objective
Islet autoantibodies can arise years before diabetes onset and serve as important predictive biomarkers in T1D. These autoantibodies are thought to arise from germinal centers, anatomical sites within lymphoid tissues where CD4+ T and B lymphocytes signal or “talk” to one another. This causes the B cells to become antibody secreting cells, while the CD4+ T cells also acquire new inflammatory potential to drive islet attack. One theory is that B lymphocyte communication with CD4+ T cells induces CD4+ T cells to subsequently communicate with CD8+ T cells, instructing CD8+ T cells to attack and destroy the insulin-producing beta cells within the pancreas. Germinal center formation is dependent upon the protein Bcl6, and loss of Bcl6 reduces antibody production. My preliminary data from our new mouse model of T1D suggests that blocking Bcl6 expression in CD4+ T cells protects mice from T1D development and germinal center T and B lymphocyte formation. My preliminary data suggest that Bcl6 is necessary for T1D development. Small-molecule inhibitors of Bcl6 have been developed by other research teams, however, whether the lack of T1D development in our mice is due to loss of Bcl6 in T cells or inability of germinal center B lymphocyte formation and subsequent “talking” to CD4+ T cells is not known.
Mouse models enable characterization of immune cell changes in the pancreas over time during the early stages of autoimmune attack. Using mouse models, I will correlate immune cells changes in tissue with immune cells changes within peripheral blood, to help guide human studies using peripheral blood monitoring as a practical biospecimen. This type of pre-human work is integrated with translational work using human samples to assess changes in gene expression in T and B lymphocytes in the blood of at-risk T1D patients at different stages of pre-T1D progression, with the goal to identify dysregulated pathways in T and B lymphocytes that can be targeted to prevent T1D progression.
Our objectives are therefore:
1) Identify which T cell subsets (CD4 or CD8) require Bcl6 to drive T1D development in mice
2) Determine B lymphocyte dependence on Bcl6 for their ability to communicate with T cells and support murine T1D
3) Identify transcriptional differences within B lymphocytes in tandem with CD4+ T cell subsets in at-risk T1D individuals who progress towards dysglycemia.
Achieving these objectives will support our long-term goals to 1) Determine the specific germinal center B and T lymphocyte contributions to T1D development, and 2) Identify potential future drug targets and biomarkers that predict T1D development.
Background Rationale
Type 1 Diabetes (T1D) results from the autoimmune destruction of insulin-producing beta cells in the pancreas, however the mechanisms behind this attack are not fully understand. Two types of immune cells, T and B lymphocytes, are known to be vital in promoting the autoimmune attack that drives T1D. During the early stages of the autoimmune attack, certain types of T and B lymphocytes communicate with one another to promote autoantibody production, a known biomarker that can predict T1D. This T-B lymphocyte communication is also implicated in promoting the destruction of the insulin-producing beta cells in the pancreas. Therefore, understanding and blocking T and B lymphocyte communication in T1D is vital for both diagnostic and therapeutic intervention.
Sites where T and B lymphocytes communicate with each other are known as germinal centers. These are anatomical sites within lymphoid tissue that promote the maturation of special subsets of T and B lymphocytes, enabling these cells to better recognize invading pathogens, or, in the case of autoimmunity, self-proteins. Germinal centers are sites where B lymphocytes mature to promote antibody production, and where T cells mature to provide support to other immune cells beyond B lymphocytes. My preliminary data show that blocking function of the protein Bcl6 in CD4+ T cells prevents T1D development in diabetes-prone mice that model many key features of human T1D. Inhibitors of Bcl6 have been developed to treat other diseases, and blocking Bcl6 does not completely block protective immune responses, further supporting Bcl6 as a promising T1D target. Mouse models allow us to study how immune cells behave at the site of attack, which is quite difficult in humans given that pancreas tissue is typically only accessible post-mortem. Preclinical studies in mice also allow us to vet new ideas to ensure only the most promising candidates move on to human clinical trials. This proposal will utilize the advantages of mouse models to further our understanding of how Bcl6 functions in key, specific, immune cell subsets. Furthermore, we will compare tissue changes that occur in mouse pancreas over time with those that are measurable in mouse peripheral blood, as this is a practical biospecimen that could be surveyed in the clinic. This will support translation of our findings in mice to humans, which is further supported by our experiments to address similar immune changes found in the blood of at-risk T1D individuals as they progress towards diabetes.
From our current data, we don’t know whether disrupted Bcl6 altered CD4+ T cell, CD8+ T cell, or B lymphocyte function prevents disease. We also did not explore the impact that loss of Bcl6 had on islet-specific lymphocytes. We will therefore use our unique mouse models of T1D that allow us to examine insulin-reactive B and T lymphocytes directly, and experimental designs that allow us to specifically isolate which immune cell compartment(s) mentioned above depend on Bcl6 for their pathogenic function in T1D.
Finally, we will investigate how peripheral blood T and B lymphocyte “pathologic wiring” changes, in tandem, with T1D progression in humans. This will identify new dysregulated pathways we may be able to therapeutically correct in the future to prevent T1D.
Description of Project
The immune system is vital to protect our bodies from infection against germs. B and T lymphocytes are key immune system players in mounting such protective responses. Unfortunately, B and T lymphocytes can, under the wrong circumstance, turn against self, to destroy cells like pancreatic beta cells and ultimately cause Type 1 Diabetes (T1D). This immune cell attack on beta cells can occur years before people are diagnosed with T1D. This long pre-symptomatic period provides clinicians an opportunity to favorably change the course of the autoimmune response before too many beta cells are lost. The FDA-approved drug teplizumab targets T lymphocytes to delay diabetes onset by two years, but undesirably suppresses protective immune responses, which are needed, especially in children to fight germs. While teplizumab represents a major advance, there is currently no durable way to prevent or cure T1D. Furthermore, children need to mount protective immune responses to vaccination and infection, which a drug like teplizumab disrupts. Therefore, my goal is to identify more selective T1D drug targets that do not limit protective immune responses but prevent immune-mediated attack on beta cells in T1D individuals.
T follicular helper (Tfh) cells are a specific type of T cell that is associated with T1D progression. Tfh cell formation requires communication with B lymphocytes in sites called germinal centers. Their communication within germinal centers drives B and Tfh cells to become more specific to their target (i.e., the beta cell in T1D) and to create more copies of themselves, thereby increasing the army of B and Tfh cells carrying out beta cell attack. The protein, Bcl6, is critical for the development of germinal center B and Tfh cells. We hypothesized that Bcl6 might thus represent a good target for preventing T1D. To test this hypothesis, we genetically deleted Bcl6 in CD4+ cells in mice that develop T1D and found they were completely protected from diabetes. In these mice, we do not know if the protection from diabetes development is due to the loss of Bcl6 in T cells or a lack of germinal center B lymphocyte formation. In this proposal, we will define which specific T cell subsets require Bcl6 disruption for T1D prevention, and additionally determine if disrupting Bcl6 function in B lymphocytes will similarly prevent T1D. Inhibitors of Bcl6 have been developed for use in other diseases. Our proposed preclinical studies will provide a rational basis for testing Bcl6 inhibitors in future T1D clinical trials.
Islet autoantibodies predict T1D development, however, autoantibody expression does not always correlate T1D development. This highlights a need to discover additional immune markers linked with T1D onset. Changes in the frequency of specific types of Tfh and B lymphocytes are associated with disease severity in other autoimmune diseases. We will track gene expression changes in Tfh and B lymphocytes as at-risk T1D individuals progress through pre-T1D to determine how they become re-wired to promote T1D. Our goal is to generate new knowledge about this “pathogenicity evolution” of these T and B lymphocytes that can be leveraged to prevent T1D progression in the future.
Anticipated Outcome
To our knowledge we are the first to demonstrate the importance of the germinal center protein, Bcl6, in Type 1 Diabetes (T1D) development. Inhibitors of Bcl6 have been tested for other diseases, but additional pre-clinical research is needed to identify the mechanisms behind Bcl6-dependent lymphocyte functions that drive T1D. These studies will take advantage of the unique resources and knowledge base here at Vanderbilt including: 1) new and existing preclinical mouse models that allow dissection of Bcl6 functions in T cells and within autoreactive, anti-insulin B lymphocytes, 2) computational programs and pipelines already established in my lab to study autoreactive lymphocytes, and 3) Hundreds of Type 1 Diabetes TrialNet bio-banked samples collected by my lab from different stages of pre-symptomatic and new-onset T1D. We therefore expect my proposed work will produce the following:
Aim 1 will define the role of Bcl6 in CD4+ and CD8+ T cells in driving T1D development. These studies will determine whether Bcl6 function in germinal center associated CD4+ T cells or beta-cell destructive CD8+ T cells is necessary for T1D development. Completion of this aim will determine the specific T cell subsets that depend on Bcl6 to drive diabetes in diabetes-prone mice.
Aim 2 experiments will take advantage of several unique genetic mouse models developed by our lab to address the importance of Bcl6 in B lymphocyte functions and T1D development. For example, insulin autoantigen-specific B lymphocytes are difficult to study in people or mice at risk for T1D. This is because they are typically present at low numbers. To overcome this challenge, we will use our mouse model that has an easily detectable population of insulin autoantigen-specific B lymphocytes, and which I recently used to elucidate important mechanisms of germinal center B lymphocyte tolerance in T1D (PMID: 37261716). We will introduce Bcl6-deficiency in these anti-insulin B lymphocytes and assess their ability to communicate with CD4+ T cells, a process that is known to promote T1D onset. In addition, we will utilize B lymphocyte depletion of Bcl6 to determine if the lack of germinal center B lymphocytes is necessary for T1D development. We will also monitor downstream changes in islet infiltration by B lymphocytes and other immune cells and ultimately diabetes development. The studies outlined here will
provide justification for the use of inhibitors that limit Bcl6 function in B lymphocytes, but not T cells, as a new putative therapy for T1D.
Aim 3 experiments will characterize changes in gene expression profiles of autoreactive-prone B lymphocyte populations, in tandem with CD4+ T cells in the same participant. These studies will generate profiles of T and B lymphocytes that track with progressing from stage 1 of pre-T1D (autoantibody positive, normal blood glucose control) to stage 2 of pre-T1D (autoantibody positive and dysregulated blood glucose control). Completion of this aim will provide important foundational knowledge that can guide both diagnosis of T1D, and potentially enable earlier intervention to prevent T1D.
In addition to supporting advances in T1D immunology, completion of this proposed research will enhance my training in computational biology and in translational research via critical experience working with human T1D samples. These skills, as well as advances in career development (i.e., grant writing, networking, etc.) will set me up to become an independent investigator who studies T1D autoimmunity.
Relevance to T1D
Type 1 Diabetes (T1D) is an autoimmune disease that arises when autoreactive immune cells enter the pancreas and kill insulin-producing beta cells. It is primarily diagnosed in children, and there is currently no permanent cure. Therapies that deplete or alter immune cells temporarily, such as teplizumab, delay diabetes development but their broadly immunosuppressive characteristics limit their chronic use, which may be necessary for durable T1D protection. In addition, while anti-islet cell autoantibodies can predict T1D development in at-risk T1D patients, other markers that correlate with early autoimmune attack are needed to improve diagnosis and facilitate earlier intervention.
The autoimmune attack in T1D is primarily orchestrated by T and B lymphocytes, which are types of immune cells. Within the T and B lymphocyte pool there are specialized subsets that are vital for generating protective immune responses. Some of these subsets depend on a protein called Bcl6 for their transition into effective “germ fighters”. My preliminary studies using genetically deficient mice identify Bcl6 as a promising target for disrupting the pathologic immune processes that cause beta cell death and eventual diabetes. The outlined work will determine which immune cells depend on Bcl6 function to drive T1D development, paving the way for the repurposing of Bcl6 inhibitors that have been developed to treat other diseases. Translation of new, more selective drugs like Bcl6 inhibitors to the clinic might prolong the transient protection conferred by teplizumab or might serve as a less immunosuppressive alternative. In addition, the proposed work will also characterize how activated T and B lymphocyte “beta cell fighters” change transcriptionally in at-risk T1D individuals as they progress towards diabetes. This will potentially identify additional targets beyond Bcl6 that may serve to limit T1D autoimmune attack on beta cells in the future.
I was recently awarded a one-year American Association of Immunologists Intersect Fellowship in Computational Immunology that was granted to only ten postdoctoral fellows in the nation. I will continue to build on the computational experience I gained from this fellowship and apply my experience working with mouse models of diabetes to define the mechanisms by which Bcl6 disrupts the function of key lymphocyte populations in T1D. My mentor, Dr. Bonami, is a Type 1 Diabetes TrialNet Investigator at Vanderbilt and leads both mechanistic studies using mouse models and translational studies using blood samples donated by people at risk for T1D. The tools within our lab and outstanding resources at Vanderbilt position me to perform translational research that advance our understanding of an important immune axis in T1D, as well as support my development towards an independent research career in T1D.