About

Janis K. Burkhardt, Ph.D., is a Professor of Pathology and Laboratory Medicine at the Perelman School of Medicine, University of Pennsylvania. He is a member of multiple research institutes and centers, including the CHOP Research Institute, the University of Pennsylvania Cancer Center, the Pennsylvania Muscle Institute, the Nano/Bio Interface Center, the ITMAT Program in Translational Biomechanics, the Institute for Immunology, the Penn Orphan Disease Center, and the CHOP normal and malignant hematopoiesis Research Affinity Group. Dr. Burkhardt's research focuses on the role of the cytoskeleton in T cell and dendritic cell function, particularly how cytoskeletal dynamics influence immune responses, antigen presentation, cell migration, and immune signaling. His long-term goal is to understand how receptor-ligand interactions trigger cytoskeletal remodeling and how this affects immune responses, with specific interest in proteins such as WASP, HS1, and Crk family adapter proteins. He has contributed to understanding the regulation of cytoskeletal dynamics at the immune synapse and its implications for immunodeficiency, autoimmunity, vaccine development, and cancer immunotherapy.

Research topics

• Genetics
• Immunology
• Cell biology
• Biology

Selected publications

• Moesin controls cell–cell fusion and osteoclast function

  The Journal of Cell Biology · 2025-08-20 · 6 citations

  articleOpen access

  Cell-cell fusion is an evolutionarily conserved process that is essential for many functions, including the formation of bone-resorbing multinucleated osteoclasts. Osteoclast multinucleation involves dynamic interactions between the actin cytoskeleton and the plasma membrane that are still poorly characterized. We found that moesin, a cytoskeletal linker protein member of the Ezrin, radixin, and moesin (ERM) protein family, plays a critical role in both osteoclast fusion and function. Moesin inhibition favors osteoclast multinucleation as well as HIV-1- and inflammation-induced cell fusion. Accordingly, moesin depletion decreases membrane-to-cortex attachment and enhances the formation of tunneling nanotubes, F-actin-based intercellular bridges triggering cell-cell fusion. In addition, moesin regulates the formation of the sealing zone, a key structure determining osteoclast bone resorption area, and thus controls bone degradation via a β3-integrin/RhoA/SLK pathway. Finally, moesin-deficient mice have reduced bone density and increased osteoclast abundance and activity. These findings provide a better understanding of cell-cell fusion and osteoclast biology, opening new opportunities to specifically target osteoclasts in bone disease therapy.

  PublisherDOI

• S1P induces bleb-based T cell motility via S1PR1-dependent activation of RhoA and WNK1

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-04

  preprintOpen accessSenior author

  In vivo, the chemokine CCL19 and its receptor CCR7 control T cell retention in lymph nodes, while the lipid chemoattractant spingosine-1-phosphate (S1P) drives T cell egress from lymphoid organs. CCL19 is known to activate actin polymerization at the leading edge of migrating cells, generating a mode of motility driven by lamellipodial protrusions. In contrast, we showed recently that S1P induces a transient lamellipodial response, followed by pressure-driven bleb-based motility. Here, we elucidate the mechanisms controlling S1P responses in naïve T cells. We show that S1P signals through S1PR1, with coupling through Gai. In contrast to CCR7, which signals through Gai to induce sustained Rac1 activation, S1PR1 engagement yields only weak and transient Rac1 activation; the dominant response is sustained activation of RhoA. This pathway, together with a pathway involving phospholipase C and myosin light chain kinase, results in phosphorylation of myosin regulatory light chain (MLC) and enhanced myosin contractility. Inhibition of mTORC2 blocks MLC phosphorylation, consistent with evidence that tension sensing by mTORC2 can couple Rac1 and RhoA signaling during leukocyte migration. Surprisingly, although RhoA pathway inhibitors blocked S1P-induced MLC phosphorylation and blebbing, they failed to block S1P-dependent chemotaxis. This led to the identification of a second arm of the S1P response: WNK1-dependent phosphorylation of SPAK1 and OSXR1, proteins that regulate ion channels and water influx. Partial WNK1 inhibition, together with inhibition of myosin contractility, was sufficient to block S1P-induced blebbing and chemotaxis, indicating that S1P-driven T cell migration involves coordinate activation of myosin contractility and water influx.

  PublisherOA PDFDOI

• Modeling the HLH immune synapse uncovers critical roles for IS termination, cytokine intensity, and target cell death

  Blood · 2025-05-13 · 6 citations

  articleOpen access

  ABSTRACT: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory syndrome arising in many contexts. Its underlying mechanisms are often unclear, but defective granule-mediated cytotoxicity (familial HLH) and excess interleukin-18 (IL-18; macrophage activation syndrome) provide clues. Mounting evidence suggests the causes of HLH converge on cytotoxic T lymphocyte (CTL) hyperactivation and interferon gamma (IFN-γ) overproduction. We refined an in vitro system to simultaneously quantify multiple parameters of the murine CTL immune synapse (IS). Even in haploinsufficiency, perforin deficiency prolonged IS duration and increased IFN-γ/tumor necrosis factor production. Similarly, both target cell immortalization and inhibition of apoptotic caspases impaired IS termination and increased cytokine production. Strong CTL activation, through T-cell receptor or IL-18 signaling, also increased IFN-γ secretion but accelerated target cell death. Impaired IS termination synergized with strong CTL activation in driving IFN-γ production. Visually, both typical and Prf1-/- CTL-IS terminated with apoptotic contraction. Serendipitously, we observed many IL-18-exposed CTL-IS terminated by target cell ballooning. Both IL-18-activated CTL and IFN-γ pretreatment caused up to half of target cells to die by receptor-interacting protein kinase 1 (RIPK1)-dependent necroptosis. In vivo, RIPK1 inhibition ameliorated virus-triggered HLH in Il18tg more than Prf1-/- mice. By quantifying CTL-IS duration, cytokine production, and mode of cell death, we modeled multiple HLH contributors and their interactions and identified 3 HLH mechanistic categories: impaired IS termination, intense CTL cytokine production, and inflammatory target cell death. Integrating the inputs and outcomes of a hyperinflammatory CTL-IS may provide a useful framework for understanding, predicting, or treating HLH in its many forms.

  PublisherOA PDFDOI

• Splenic fibroblasts control marginal zone B cell movement and function via two distinct Notch2-dependent regulatory programs

  Immunity · 2024-12-27 · 6 citations

  articleOpen access
  PublisherOA PDFDOI

• Moesin controls cell-cell fusion and osteoclast function

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-05-15 · 2 citations

  preprintOpen access

  ABSTRACT Cell-cell fusion is an evolutionarily conserved process that is essential for many functions, including fertilisation and the formation of placenta, muscle and osteoclasts, multinucleated cells that are unique in their ability to resorb bone. The mechanisms of osteoclast multinucleation involve dynamic interactions between the actin cytoskeleton and the plasma membrane that are still poorly characterized. Here, we found that moesin, a cytoskeletal linker protein member of the Ezrin/Radixin/Moesin (ERM) protein family, is activated during osteoclast maturation and plays an instrumental role in both osteoclast fusion and function. In mouse and human osteoclast precursors, moesin inhibition favors their ability to fuse into multinucleated osteoclasts. Accordingly, we demonstrated that moesin depletion decreases membrane-to-cortex attachment and enhances the formation of tunneling nanotubes (TNTs), F-actin-based intercellular bridges that we reveal here to trigger cell-cell fusion. Moesin also controls HIV-1- and inflammation-induced cell fusion. In addition, moesin regulates the formation of the sealing zone, the adhesive structure determining osteoclast bone resorption area, and thus controls bone degradation, via a β3-integrin/RhoA/SLK pathway. Supporting our results, moesin - deficient mice present a reduced density of trabecular bones and increased osteoclast abundance and activity. These findings provide a better understanding of the regulation of cell-cell fusion and osteoclast biology, opening new opportunities to specifically target osteoclast activity in bone disease therapy.

  PublisherOA PDFDOI

• Long range mutual activation establishes Rho and Rac polarity during cell migration

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-10-02 · 7 citations

  preprintOpen access

  In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are appropriately positioned at the opposite poles of migrating cells is unknown. Here we leverage optogenetics, manipulation of cell mechanics, and mathematical modeling to reveal a surprising mechanochemical long-range mutual activation of the front and back polarity programs that complements their well-known local mutual inhibition. Rac-based protrusion stimulates Rho activation at the opposite side of the cell via membrane tension-based activation of mTORC2. Conversely, Rho-based contraction induces cortical-flow-based regulation of phosphoinositide signaling to trigger Rac activation at the opposite side of the cell. We develop a minimal unifying mechanochemical model of the cell to explain how this long-range facilitation complements local inhibition to enable robust Rho and Rac partitioning. We show that this long-range mutual activation of Rac and Rho is conserved in epithelial cells and is also essential for efficient polarity and migration of primary human T cells, indicating the generality of this circuit. Our findings demonstrate that the actin cortex and plasma membrane function as an integrated mechanochemical system for long-range partitioning of Rac and Rho during cell migration and likely other cellular contexts.

  PublisherDOI

• Stroma-Driven Notch2 Signaling Controls Naïve B Cell Fate By Regulating Microenvironmental Positioning within the Spleen

  Blood · 2023-11-02

  articleOpen access

  Marginal zone (MZ) B cells are naïve innate-like B cells that function as a first line of defense in the spleen within the blood-rich marginal zone. Previous research revealed a critical role for Notch2 receptors in B cells and Delta-like1 (Dll1) ligands in Ccl19-Cre + fibroblastic stromal cells to support the development and maintenance of MZ B cells in mice. Yet, little is known about how MZ B cells integrate extracellular signals and Notch-regulated transcriptional programs to support their positioning and function. To understand the basis of Dll1/Notch2's specificity, we used monoclonal antibodies to acutely block Notch2 receptors vs. Dll1 or Dll4 Notch ligands in vivo. Dll1 and Notch2 blockade, but not Dll4 inhibition, rapidly decreased MZ B cell numbers and Notch2-regulated transcription in B cells. Analysis of fluorescent reporter alleles revealed co-expression of Ccl19-Cre;ROSA26 YFP and Dll1-mCherry or Dll4-mCherry within white pulp follicles, showing that both Delta-like ligands are available in fibroblastic stromal cells, although with differential abundance and spatial distribution. To determine if the reliance of MZ B cells on Dll1 was due to ligand availability in the appropriate stromal niche, we inactivated endogenous Dll1 and Dll4 via Ccl19-Cre-mediated recombination, and restored Dll1 or Dll4 expression selectively via separate Cre-inducible expression alleles in the Hprt locus. Dll4 could not sustain Notch2-dependent MZ B cells even when expressed in the correct stromal niche, suggesting that Dll1/Notch2 interactions have unique biochemical and functional properties. As MZ B cells are known to shuttle between the follicle and the MZ across the marginal sinus, we assessed where Dll1/Notch2-mediated signals are delivered to MZ B cells. High abundance of intracellular Notch2 and expression of the Notch target gene, Hes1 (as revealed via a Hes1-GFP reporter) was apparent in CD1d high MZ B cells across the marginal sinus, including prominent signaling within B cell follicles (and not only in the MZ) (Figure 1A). We next evaluated the transcriptional programs controlled by Notch in MZ B cells. Notch2/Dll1-regulated genes included both Myc-dependent and Myc-independent cohorts, with the latter highly enriched for integrin and chemotactic receptor genes including S1pr1, shown previously to guide B cells towards the MZ across the marginal sinus. Myc itself was dispensable for MZ B cell positioning and retention in the spleen. Without S1pr1, B cells experienced Notch signaling within B cell follicles even without entering the MZ. Unlike wild-type B cells, mislocalized S1pr1-deficient MZ-like B cells were retained in the spleen upon Notch deprivation (Figure 1B). These findings identify splenic B cell follicles and not the MZ as a central hub for stroma-driven Dll1/Notch2 signaling, with Notch empowering subsequent B cell migration and positioning to the MZ through a Myc-independent transcriptional program. As many Notch-regulated transcriptional targets are conserved in human Notch-driven B cell lymphomas, we speculate that principles of stroma-driven Notch signaling and its downstream effects in B cells have been conserved during evolution from mouse to human B cells - with the Notch signature tagging the B cell subsets that rely on conserved Notch programs and the B cell malignancies that hijack them.

  PublisherOA PDFDOI

• A Murine Model of X-Linked Moesin-Associated Immunodeficiency (X-MAID) Reveals Defects in T Cell Homeostasis and Migration

  Frontiers in Immunology · 2022-01-06 · 4 citations

  articleOpen accessSenior authorCorresponding

  X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesin R171W ). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesin R171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesin R171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesin R171W . Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesin R171W , and will provide new insights into how the actin cortex regulates lymphocyte function.

  PublisherDOI

• Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells

  Journal of Clinical Investigation · 2022-05-17 · 12 citations

  articleOpen access

  In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2-/- mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone-like B cells when transferred into Rag2-/- lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

  PublisherOA PDFDOI

• Viral interference with class II function

  Molecular Immunology · 2022-10-01

  article
  PublisherDOI

Recent grants

• NIH Grant P01CA093615

  NIH · $9.6M · 2013

• NIH Grant R01AI065644

  NIH · $2.0M · 2013

• NIH Grant R21AI088376

  NIH · $458k · 2013

• NIH Grant R01AI050098

  NIH · $1.9M · 2007

• NIH Grant R01GM104867

  NIH · $1.3M · 2018

Frequent coauthors

• Yair Argon

  Children's Hospital of Philadelphia

  60 shared

• Nathan H. Roy

  59 shared

• Susan Hester

  Research Triangle Park Foundation

  36 shared

• Tanner F. Robertson

  University of Wisconsin–Madison

  33 shared

• Yanping Huang

  Tongren Hospital

  33 shared

• Esteban Carrizosa

  29 shared

• Shuixing Li

  29 shared

• Meredith H. Shaffer

  University of Pennsylvania

  26 shared

Labs

• Burkhardt LabPI