About

Our laboratory studies how signaling networks control T cell function in health and disease, with a major focus on T cell receptor and CAR T cell signaling using state-of-the-art quantitative LC-MS proteomic workflows. We investigate the phosphorylation-driven pathways that regulate immune cell activation, feedback control, and signaling crosstalk, because these molecular events shape how T cells respond to cancer, infection, and other disease states. By defining these pathways at high resolution, we aim to uncover mechanisms of immune dysfunction and identify signaling nodes that may be leveraged therapeutically. We also study how CAR T cell design alters signaling in both engineered immune cells and the tumor cells they encounter. This has direct relevance to human health because CAR-driven signaling can influence therapeutic efficacy, persistence, toxicity, and in some cases unintended target-cell responses. Through the integration of advanced phosphoproteomics, biochemical approach

Research topics

• Biochemistry
• Chemistry
• Biology
• Neuroscience
• Chromatography
• Computational biology
• Cancer research
• Cell biology

Selected publications

• Phosphotyrosine proteomics reveals novel Zap70 and Itk pathway targets downstream of TCR and CAR in Jurkat T cells

  Scientific Reports · 2026-04-06

  articleOpen accessSenior author

  Ζ-associated protein of 70 kDa (Zap70) and interleukin-2-inducible T cell kinase (Itk) propagate the primary and CD28-integrated phosphotyrosine (pY) signalling, respectively, to achieve full T cell activation. Despite their canonical roles in T cell activation, our understanding of how each kinase controls canonical and noncanonical pY signalling is incomplete. Here, using three T cell activation methods (soluble antibodies, APC-pMHC/TCR, and CD19-CAR/Raji), we evaluated the effects of two novel inhibitors, RDN2150 (RDN, Zap70) and Soquelitinib (Soq, Itk), on T cell activation. We validated the published working concentrations of RDN and Soq on phosphorylation of key T cell signalling proteins and on T cell activation markers, finding that RDN provides more complete inhibition of T cell signalling and activation. We used LC-MS/MS to evaluate how RDN and Soq treatment affected the phosphotyrosine (pY) signalling and proteome of T cells, finding that RDN, as opposed to Soq, completely downregulated the TCR signalling pathway. Finally, we identified new, noncanonical pY sites responsive to RDN and Soq, providing new insights into the pathways regulated by Zap70 and Itk. Together, our work provides a basis for further study on RDN and Soq, as well as a molecular roadmap for the effects of these inhibitors.

  PublisherDOI

• CSF1R-CAR T cells induce CSF1R signaling and can promote target cell proliferation

  Science Signaling · 2025-11-11 · 2 citations

  articleOpen accessSenior authorCorresponding

  Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented success in treating relapsed or refractory blood cancers. Previous studies of the mechanisms underlying the interactions and responses of CAR T cells and their targets have largely ignored the responses of tumors to CAR ligation. We compared the signaling of a second-generation, ligand-based CAR built from colony-stimulating factor 1 (CSF1) to target the CSF1 receptor (CSF1R) on target cells with a conventional, single-chain variable fragment-based CAR against the B cell antigen CD19. Using SILAC coculture with phosphotyrosine enrichment and LC-MS/MS analysis, we showed that ligation of CSF1R-expressing THP-1 cells with CSF1R-CAR T cells stimulated CSF1R-like signaling in the THP-1 cells. In contrast, no target cell signaling response was observed after the ligation of CD19-CAR T cells with target Raji cells. Using small-molecule inhibitors of the tyrosine kinase Lck, actin polymerization, and CSF1R, we found that CAR-induced CSF1R signaling in THP-1 cells depended exclusively on the kinase activity of CSF1R with no participation from T cell activation. Consistently, CSF1R-CAR T cells promoted THP-1 cell proliferation at low effector-to-target ratios but prevented THP-1 cell proliferation at high effector-to-target ratios. Our data provide evidence for CAR-induced signaling in target cells, an unintended consequence of CARs that may have implications for the choice of CAR antigen for optimal clinical efficacy.

  PublisherOA PDFDOI

• Tyrosine 67 Phosphorylation Controls Respiration and Limits the Apoptotic Functions of Cytochrome c

  Cells · 2025-06-21 · 1 citations

  articleOpen access

  Cytochrome c (Cytc) is a multifunctional protein, essential for respiration and intrinsic apoptosis. Post-translational modifications of Cytc have been linked to physiological and pathophysiologic conditions, including cancer. Cytc tyrosine 67 (Y67) is a conserved residue that is important to the structure and function of Cytc. We here report the phosphorylation of Y67 of Cytc purified from bovine heart mapped by mass spectrometry. We characterized the functional effects of Y67 Cytc modification using in vitro and cell culture models. Y67 was mutated to the phosphomimetic glutamate (Y67E) and to phenylalanyl (Y67F) as a control. The phosphomimetic Y67E Cytc inhibited cytochrome c oxidase (COX) activity, redirecting energy metabolism toward glycolysis, and decreased the pro-apoptotic capabilities of Cytc. The phosphomimetic Y67E Cytc showed a significantly impaired rate of superoxide scavenging and a reduced rate of oxidation by hydrogen peroxide, suggesting a lower ability to transfer electrons and scavenge reactive oxygen species (ROS). Phosphomimetic Y67E replacement led to an almost complete loss of cardiolipin peroxidase activity, pointing to a central role of Y67 for this catalytic function of Cytc. In intact cells, phosphomimetic replacement leads to a reduction in cell respiration, mitochondrial membrane potential, and ROS levels. We propose that Y67 phosphorylation is cardioprotective and promotes cell survival.

  PublisherDOI

• Evaluating First-Pass, High Protein Capacity Desalting Techniques For Phosphoproteomics Applications

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-03 · 2 citations

  preprintOpen access

  Many commercial desalting products exist for pre-MS peptide cleanup, although few exist that can handle the high protein input (≥ 4 mg) required for phosphotyrosine enrichment. For these desalting products, the technical aptitude required for effective and organized desalting is often a barrier to entry for new users. Here, we evaluate four commercially available desalting techniques with varying degrees of automation, operational organization, and chemistries to determine the most cost-effective, user-friendly, and sensitive technique for protein profiling and phosphotyrosine (pY) enrichment. We find that TECAN Narrow Bore Extraction (NBE) products are the most cost effective per sample and least difficult to use, whereas ProtiFi's S-Trap are the most expensive per sample and Pierce C18 spin columns have the worst operational organization. ProtiFi S-Trap vastly outperforms other desalting methods for peptide sequencing and protein profiling applications, uniquely identifying 25,654 unique peptide sequences and 375 unique proteins. Consistently, ProtiFi S-Trap samples show the deepest pY sequencing after Src SH2 superbinder enrichment, leading to the highest identification of significantly changing, biologically relevant pY sites in a Jurkat T cell signalling model. Our data show that ProtiFi S-Trap columns provide high peptide recovery, thus increasing meaningful pY site identification.

  PublisherOA PDFDOI

• The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR

  Scientific Reports · 2025-07-30 · 2 citations

  articleOpen accessSenior author

  The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, LckY394. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.

  PublisherOA PDFDOI

• Inclusion of the ζ-chain drives phosphotyrosine signalling in CD19-CAR T cells

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-07-07 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Although chimeric antigen receptor (CAR) T cell therapy has revolutionised individualised cancer therapies for relapsed/refractory lymphomas, signalling mechanisms underlying CAR T activation remain incompletely understood, especially among the three generations of CAR T exploiting different signalling domains. Here, using Jurkat T cell as a model, we investigate how costimulation influences tyrosine phosphorylation cascades using LC-MS/MS based phosphotyrosine (pY) proteomics and CD69 expression in the presence of small molecule inhibitors of key TCR signalling regulators. We find that including the ζ-chain in first (ζ-CAR), second (28ζ-CAR and BBζ-CAR), and third (28BBζ-CAR) generation CARs largely determines pY signalling, irrespective of costimulation. Further, we show that the phosphatase activity of PTPN22 and SHP-1 are largely negligible for activation of CARs, but indiscriminate inhibition of phosphatases using Pervanadate (PV) selectively activates BBζ-CARs without antigen encounter. Finally, we find that selective, partial inhibition of Itk using Soquelitinib reduces basal CD69 expression in Jurkat CAR T cells while maintaining their ability to activate in response to antigen. Our data suggest that the ζ-chain determines the pY signalling profile of CD19-CAR Jurkat T cells and that Itk may drive antigen-independent CD19-CAR activation.

  PublisherOA PDFDOI

• The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-05-09

  preprintOpen accessSenior authorCorresponding

  Abstract The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, Lck Y394. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.

  PublisherOA PDFDOI

• Phospotyrosine proteomics reveals novel Zap70 and Itk pathway targets downstream of TCR and CAR in Jurkat T cells

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-11-05

  preprintOpen accessSenior authorCorresponding

  Abstract ζ-associated protein of 70 kDa (Zap70) and interleukin-2-inducible T cell kinase (Itk) propagate the primary and CD28-integrated phosphotyrosine (pY) signalling, respectively, to achieve full T cell activation. Despite their canonical roles in T cell activation, our understanding of how each kinase controls canonical and noncanonical pY signalling is incomplete. Here, using three T cell activation methods (soluble antibodies, APC- pMHC/TCR, and CD19-CAR/Raji), we evaluated the effects of two novel inhibitors, RDN2150 (RDN, Zap70) and Soquelitinib (Soq, Itk), on T cell activation. We validated the published working concentrations of RDN and Soq on phosphorylation of key T cell signalling proteins and on T cell activation markers, finding that RDN provides more complete inhibition of T cell signalling and activation. We used LC-MS/MS to evaluate how RDN and Soq treatment affected the phosphotyrosine (pY) signalling and proteome of T cells, finding that RDN, as opposed to Soq, completely downregulated the TCR signalling pathway. Finally, we identified new, noncanonical pY sites responsive to RDN and Soq, providing new insights into the pathways regulated by Zap70 and Itk. Together, our work provides a basis for further study on RDN and Soq, as well as a molecular roadmap for the effects of these inhibitors.

  PublisherOA PDFDOI

• Phosphoproteomic analysis of successive Jurkat CD19-CAR generations reveals TCRζ-driven signalling

  Cellular Signalling · 2025-11-01 · 1 citations

  articleSenior author
  PublisherDOI

• CSF1R-CAR T cells induce CSF1R signalling and promote cancer cell growth

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-12-20 · 3 citations

  preprintSenior authorCorresponding

  Abstract Chimeric antigen receptor (CAR) T cells have transformed the landscape of cancer therapy and demonstrate unprecedented success in treating relapsed/refractory blood cancers. The mechanism underlaying the interactions and responses of CAR T cells and their targets remain incompletely understood. Previous studies focus on the activation of CAR T cells and attempt to optimise CAR design to increase efficacy, meanwhile ignoring tumours and their responses to CAR ligation. Here, we evaluate the signalling capacity of a second generation CSF1-tageted CSF1R CAR compared with a scFv-targeted CD19-CAR using a SILAC co- culture approach coupled with phosphotyrosine (pY) enrichment and LC-MS/MS. We show that ligation of CSF1R-expressing THP1 cells with CSF1R-CAR T cells induces CSF1R-like signalling in THP1 cells, whereas no target cell signalling response is observed after CD19- CAR/Raji B cell ligation. Using small molecule inhibitors of Lck, actin polymerisation, and CSF1R, we find that CAR-induced CSF1R signalling in THP1 cells depends exclusively on CSF1R kinase activity with no participation from T cell activation. Consistently, CSF1R- CAR T cells promote THP1 growth at low effector-to-target (E:T) ratios but prevent THP1 growth at high E:T ratios. Our data provide evidence for an unintended consequence of CARs; CAR-induced signalling in cancer cells. These data may have broad implications for the choice of CAR antigen for optimal clinical efficacy. One Sentence Summary: CSF1R-CAR activates intracellular signalling cascades in THP1 cells, which promote THP1 cell growth.

  PublisherDOI

Recent grants

• NIH Grant R21AI083908

  NIH · $436k · 2011

• Phosphoproteomic Analysis of Feedback Networks in T cell signaling

  NIH · $4.0M · 2010–2024

• NIH Grant P20RR015578

  NIH · $9.0M · 2012

• The role of positive and negative regulation on ligand discrimination by the TCR signaling pathway

  NIH · $54.2M · 2011–2027

• COBRE Center for Cancer Research Development

  NIH · $12.6M · 2021

Frequent coauthors

• Qinqin Ji

  Janssen (United States)

  63 shared

• Kebing Yu

  57 shared

• Nagib Ahsan

  University of Oklahoma

  44 shared

• Philip A. Gruppuso

  Brown University

  41 shared

• Nagib Ahsan

  University of Oklahoma

  36 shared

• Ynes Helou

  Century Therapeutics (United States)

  34 shared

• Icksoo Lee

  Dankook University

  28 shared

• Maik Hüttemann

  Wayne State University

  26 shared

Labs

• Arthur Salomon's LabPI

  Not provided