About

Work in the Bettini labs is focused on T cell biology. We ask questions related to T cell development and function in the context of autoimmunity, cancer, and intestinal homeostasis. We leverage basic immunology knowledge to develop new therapeutic approaches for treatment of autoimmunity and cancer.

Research topics

• Biology
• Immunology
• Genetics
• Medicine
• Cell biology
• Endocrinology
• Computational biology
• Bioinformatics

Selected publications

• Bond lifetime under force as a potential mechanism underlying the pathogenicity of low affinity CD8 T cells in experimental cerebral malaria 3182

  The Journal of Immunology · 2025-11-01

  articleOpen access

  Abstract Description Despite the presence of anti-malarial treatments, cerebral malaria persists as one of the most lethal malaria complications. CD8 T cells mediate the development of experimental cerebral malaria (ECM), which is characterized by breakdown of the blood-brain barrier (BBB). During a Plasmodium infection, CD8 T cells recognize different Plasmodium epitopes and expand, leading to polyclonal populations with T cell receptors (TCRs) of different reactivity and affinity. Generally, T cells harboring high affinity TCRs are believed to dominate any polyclonal T cell response and to be the most potent cells during an immune response. We have made the paradigm-shifting discovery that pathogenicPlasmodium-reactive CD8 T cells with low affinity TCRs dominate the CD8 T cell response during ECM. In adoptive transfer experiments, high affinity CD8 T cells are unable to drive BBB dysfunction to the same extent as low affinity cells. We have found that high and low affinity Plasmodium-reactive CD8 T cells display different phenotypes. High affinity CD8 T cells might be dysfunctional or undergoing apoptosis, consistent with their inability to mediate BBB breakdown. TCR affinity can impact functional outcome by inducing a different bond lifetime under force. We have identified TCRs specific for the Plasmodium GAP50 epitope and transfected these TCRs into 58TCR-ve cells. High affinity GAP50-specific TCRs induced longer bond lifetimes under force compared to low affinity GAP50-specific TCRs. Funding Sources NIH 5R01AI167422-04 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

  PublisherOA PDFDOI

• Flt3L-Derived Antigen-Presenting Cell Transfer in Neonatal NOD Mice Reduces the Incidence of Type 1 Diabetes

  Diabetes · 2025-11-06

  articleOpen access

  Type 1 diabetes (T1D) is an autoimmune disease characterized by progressive stages culminating in T-cell-mediated destruction of the β-cells at the islets of Langerhans. The immune mechanisms that initiate T1D are not fully resolved but likely involve an interaction between proinflammatory antigen-presenting cells (APCs) and autoreactive T cells that initiate immune infiltration and activation. Previous studies have tested the use of tolerogenic APCs in adult female NOD mice to delay or prevent T1D with only slight to intermediate success. Moreover, immune infiltration begins as early as age 4 weeks; therefore, targeting autoreactive T cells with tolerogenic APCs in adult mice may not impact later stages of diabetes. Thus, we hypothesize that the transfer of tolerogenic APCs at the neonatal stage prior to priming and immune infiltration will result in effective protection from autoimmunity. Our studies demonstrate that immature APCs travel to the pancreatic draining lymph nodes, alter the cytokine milieu in young mice, divert autoreactive CD4+ T cells to anergy, and drastically decrease proliferation and function of cytotoxic lymphocytes in adult prediabetic mice, leading to a significant reduction in the incidence of T1D. ARTICLE HIGHLIGHTS: Neonatal transfer of immature dendritic cell-enriched Flt3L splenocytes significantly reduces the incidence of type 1 diabetes in female NOD mice. Early time points are associated with accumulation of anergic T cells. In adult mice, there is a reduction in CD4 T helper 1 cells and reduced proliferation and perforin of CD8 T cells. Our work demonstrates how targeting the neonatal window of tolerance alters autoimmunity outcome.

  PublisherOA PDFDOI

• Abstract A024: CD3ζ ITAM diversity determines chimeric antigen receptor force and function

  Cancer Immunology Research · 2025-02-23

  article

  Abstract The Chimeric antigen receptor (CAR) T cell therapy is a powerful treatment for hematologic malignancies; however, clinical responses vary depending on CAR T cell function and persistence. The optimization of CAR design has gained considerable interest with a new focus on calibrating CAR intracellular signals via the immunoreceptor tyrosine-based activation motifs (ITAMs) to increase the fitness of CAR T cells. Evidence suggests that unique ITAM motifs at positions 1, 2, or 3 (ITAMs-A, B, and C respectively) of the CD3ζ signaling domain may have non-redundant roles in signal strength, T cell functionality, persistence, and memory formation. However, the individual contributions of each CD3ζ ITAM in transmitting force and activating mechanosensitive signals are yet unexplored. We have found that human CARs expressing specific ITAM sequences (ITAM-A, ITAM-B, or ITAM-C only) generate differing amounts of force and bond lifetimes after interacting with their target antigen, CD19. Therefore, a CAR may act as a mechanosensor similar to a TCR, discriminating the quality of the interaction with antigen as cellular-derived forces are applied to the bond. The higher transmission of force directly correlates with better anti-tumor efficacy in vitro and in vivo. Hence, understanding the mechanotransduction in CARs has important implications in “tuning” the activation of a CAR T cell without changing specificity. Citation Format: Hilda Echelibe, Shubhabrata Majumdar, Elizabeth Kolawole, Baoyu Liu, Maria Bettini, Brian Evavold, Matt Bettini. CD3ζ ITAM diversity determines chimeric antigen receptor force and function [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A024.

  PublisherDOI

• Flt3L-derived Antigen Presenting Cell transfer in neonatal NOD mice delays the incidence of Type 1

  2025-11-06

  articleOpen access

  <p dir="ltr">Type 1 Diabetes (T1D) is an autoimmune disease characterized by progressive stages culminating in T cell mediated destruction of the b cells at the islets of Langerhans. The immune mechanisms that initiate T1D are not fully resolved but likely involve interaction between pro-inflammatory antigen presenting cells and autoreactive T cells that initiate immune infiltration and activation. Previous studies have tested the use of tolerogenic antigen presenting cells (APCs) in adult non-obese-diabetic (NOD) female mice to delay or prevent T1D with only slight to intermediate success. Moreover, immune infiltration begins as early as 4 weeks old, therefore targeting autoreactive T cells with tolerogenic APCs in adult mice may not impact late stages of diabetes. Thus, we hypothesize that the transfer of tolerogenic APCs at the neonatal stage prior to priming and immune infiltration will result in effective protection from autoimmunity. Our studies demonstrate that immature APCs travel to the pancreatic draining lymph nodes, alter the cytokine milieu in young mice, divert autoreactive CD4<sup>+</sup> T cells to anergy and drastically decrease proliferation and function of CTLs in adult pre-diabetic mice leading to a significant reduction in the incidence of T1D.</p>

  PublisherDOI

• The impact of CD3ζ ITAM multiplicity and sequence on CAR T-cell survival and function

  Frontiers in Immunology · 2025-01-16 · 4 citations

  articleOpen access

  Introduction Chimeric antigen receptor (CAR) expressing T-cells have shown great promise for the future of cancer immunotherapy with the recent clinical successes achieved in treating different hematologic cancers. Despite these early successes, several challenges remain in the field that require to be solved for the therapy to be more efficacious. One such challenge is the lack of long-term persistence of CD28 based CAR T-cells in patients. Although, CD28 based CAR T-cells elicit a robust acute anti-tumor response, they are more prone to early exhaustion, terminal differentiation and cell death due to their strong signaling patterns. Hence attenuation of signaling strength in CD28 based CARs is an accepted strategy to improve long-term CAR T-cell function and persistence in patients. Previous studies with the conventional T-cell receptor (TCR) have suggested that manipulation of CD3 immunoreceptor tyrosine-based activation motif (ITAM) sequences can alter TCR signaling strength. Based on these studies, we have designed 2 nd generation murine anti-CD19 CD28 based CARs with restricted CD3ζ ITAM sequence diversity while maintaining a multiplicity of three. They are called ζAAA, ζBBB and ζCCC based on which CD3ζ ITAM they express. The goal of the study is to understand the non-redundant signaling properties of the individual CD3ζ ITAMs and their effect on CAR T-cell function. We hypothesized that the individual CD3ζ ITAMs will exhibit unique signaling properties in the ITAM restricted CARs which may allow for optimization of CAR signaling and improve CAR T-cell persistence and function. Method We subjected the ITAM restricted CAR T cells to various conditions of in vitro stimulation using CD19+ tumor cells or CD19-coated magnetic beads. Immunoblotting and flow cytometry based Ca2+ signaling assays were used to quantify signaling differences. Functional differences were studied using in vitro cytotoxicity, degranulation and cytokine expression assays. CAR T cell exhaustion and differentiation were studied using an in vitro exhaustion assay. Results We observed that ζAAA CARs had stronger signaling strength compared to ζBBB and ζCCC CARs. The signaling differences were reflected in their functional activation profiles with T-cells expressing ζAAA CARs having a strong activation profile and ζCCC CARs having a weak activation profile. ζCCC CAR T cells were less prone to differentiation and exhaustion. Discussion Since, weaker signaling ζCCC CARs favored less cell death, exhaustion and differentiation, they might be better candidates for improving long term survival and persistence of CAR T cells in patients.

  PublisherOA PDFDOI

• Flt3L-derived Antigen Presenting Cell transfer in neonatal NOD mice delays the incidence of Type 1

  2025-11-06

  articleOpen access

  <p dir="ltr">Type 1 Diabetes (T1D) is an autoimmune disease characterized by progressive stages culminating in T cell mediated destruction of the b cells at the islets of Langerhans. The immune mechanisms that initiate T1D are not fully resolved but likely involve interaction between pro-inflammatory antigen presenting cells and autoreactive T cells that initiate immune infiltration and activation. Previous studies have tested the use of tolerogenic antigen presenting cells (APCs) in adult non-obese-diabetic (NOD) female mice to delay or prevent T1D with only slight to intermediate success. Moreover, immune infiltration begins as early as 4 weeks old, therefore targeting autoreactive T cells with tolerogenic APCs in adult mice may not impact late stages of diabetes. Thus, we hypothesize that the transfer of tolerogenic APCs at the neonatal stage prior to priming and immune infiltration will result in effective protection from autoimmunity. Our studies demonstrate that immature APCs travel to the pancreatic draining lymph nodes, alter the cytokine milieu in young mice, divert autoreactive CD4<sup>+</sup> T cells to anergy and drastically decrease proliferation and function of CTLs in adult pre-diabetic mice leading to a significant reduction in the incidence of T1D.</p>

  PublisherDOI

• Autoimmune CD4 T cell exhaustion is counteracted by peripheral circulating T cell population with stem-like potential

  The Journal of Immunology · 2024-05-01

  articleSenior author

  Abstract Stem-like antigen specific T cells serve as a reservoir for autoimmune CD8 T cell responses. However, it is unclear whether CD4 T cells depend on a similar population to counteract depletion through terminal differentiation and exhaustion. Single cell RNAseq and flow cytometric analysis of NOD islet-infiltrating T cells revealed the heterogeneity of CD4 cells, with subpopulations displaying features of exhaustion. Blockade of T cell entry with FTY720 reduced T cell numbers in the tissue, and protected NOD mice from diabetes. Moreover, islet CD4 T cells progressively lose TCF1 expression overtime. Transfer experiments and T cell tracking in photoconvertible NOD.KikGR mice showed peripheral circulating T cells with stem-like TCF1+ phenotype as an important source for autoimmune T cells. We observed that islet infiltrating CD62L+ naïve-like T cells include low affinity antigen specific cells, show activation of TCR regulated genes, and exhibit epigenetic signature distinct from lymph node bona fide naïve T cells. Similarly, BDC2.5 islet antigen specific T cells circulating in peripheral non-draining lymphoid organs exhibit phenotypic and epigenetic signature of pre-primed, but largely undifferentiated T cells. Collectively, our data indicates that autoimmune CD4 T cell persistence is sustained through continuous recruitment of new T cells from circulating non-differentiated stem-like compartment.

  PublisherDOI

• Autoimmune CD4+ T cells fine-tune TCF1 expression to maintain function and survive persistent antigen exposure during diabetes

  Immunity · 2024-10-12 · 26 citations

  articleOpen accessSenior author
  PublisherOA PDFDOI

• Immunogen-Specific Strengths and Limitations of the Activation-Induced Marker Assay for Assessing Murine Antigen-Specific CD4+ T Cell Responses

  The Journal of Immunology · 2023-02-17 · 10 citations

  articleOpen access

  The activation-induced marker (AIM) assay is a cytokine-independent technique to identify Ag-specific T cells based on the upregulated expression of activation markers after Ag restimulation. The method offers an alternative to intracellular cytokine staining in immunological studies, in which limited cytokine production makes the cell subsets of interest difficult to detect. Studies of lymphocytes in human and nonhuman primates have used the AIM assay to detect Ag-specific CD4+ and CD8+ T cells. However, there is a lack of validation of the strengths and limitations of the assay in murine (Mus musculus) models of infection and vaccination. In this study, we analyzed immune responses of TCR-transgenic CD4+ T cells, including lymphocytic choriomeningitis virus-specific SMARTA, OVA-specific OT-II, and diabetogenic BDC2.5-transgenic T cells, and measured the ability of the AIM assay to effectively identify these cells to upregulate AIM markers OX40 and CD25 following culture with cognate Ag. Our findings indicate that the AIM assay is effective for identifying the relative frequency of protein immunization-induced effector and memory CD4+ T cells, whereas the AIM assay had reduced ability to identify specific cells induced by viral infection, particularly during chronic lymphocytic choriomeningitis virus infection. Evaluation of polyclonal CD4+ T cell responses to acute viral infection demonstrated that the AIM assay can detect a proportion of both high- and low-affinity cells. Together, our findings indicate that the AIM assay can be an effective tool for relative quantification of murine Ag-specific CD4+ T cells to protein vaccination, while demonstrating its limitations during conditions of acute and chronic infection.

  PublisherOA PDFDOI

• The Amphiregulin/EGFR axis has limited contribution in controlling autoimmune diabetes

  Research Square · 2023-08-03 · 3 citations

  preprintOpen accessSenior authorCorresponding

  Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. Whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. We postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin and both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum (ER) stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited.

  PublisherOA PDFDOI

Recent grants

• Self-Reactive T Cell Development in Type 1 Diabetes

  NIH · $158k · 2015–2017

• TCR PARAMETERS OF TREG FUNCTION IN AUTOIMMUNITY

  NIH · $1.9M · 2017–2023

• Parameters T-Cell Receptor avidity during thymic selection in autoimmune diabetes

  NIH · $269k · 2014–2017

• Self-Reactive T Cell Development in Type 1 Diabetes

  NIH · $1.9M · 2019–2024

• Biophysical parameters of self-reactive TCR engagement in T1D

  NIH · $2.7M · 2023–2028

Frequent coauthors

• Matthew L. Bettini

  University of Utah

  89 shared

• Maran L. Sprouse

  Baylor College of Medicine

  45 shared

• Yuelin Kong

  Huazhong University of Science and Technology

  43 shared

• Jing Yi

  Air Force Medical University

  37 shared

• Thomas Lee

  Northumbria Healthcare NHS Foundation Trust

  23 shared

• Malgorzata Borowiak

  Adam Mickiewicz University in Poznań

  22 shared

• Matthew L. Bettini

  University of Utah

  18 shared

• Dario A.A. Vignali

  University of Pittsburgh

  16 shared

Labs

• Bettini labPI

Education

• Ph.D., Microbiology and Immunology

  Emory University School of Medicine

  2006