Biology subjects

Callahan, A.

Publications and source records attributed to Callahan, A..

6 recordsLinked to original sources

bioRxiv2025

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

{zeta}-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.

immunology↗

bioRxiv2025

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

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 {zeta}-chain in first ({zeta}-CAR), second (28{zeta}-CAR and BB{zeta}-CAR), and third (28BB{zeta}-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 BB{zeta}-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 {zeta}-chain determines the pY signalling profile of CD19-CAR Jurkat T cells and that Itk may drive antigen-independent CD19-CAR activation.

cancer biology↗

bioRxiv2025

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

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 ProtiFis 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/657744v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@13b77e2org.highwire.dtl.DTLVardef@13bb4cforg.highwire.dtl.DTLVardef@dba171org.highwire.dtl.DTLVardef@1440f7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

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

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.

systems biology↗

bioRxiv2024

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

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.

cancer biology↗

bioRxiv2024

Endosymbionts impact ladybird predation rates of aphids in a temperature-dependent manner

Aphids are worldwide pests causing major economic losses to growers. Current management strategies rely heavily on pesticides, but some effective pesticides are being withdrawn and the efficiency of remaining pesticides is also decreasing as aphids build up resistance. Biological control using predators can provide a sustainable alternative to pesticides under some circumstances, while the deliberate introduction of facultative bacterial endosymbionts that induce host fitness costs and reduce plant virus transmission provides another potential future strategy to combat aphid pests. However, new control options should not be antagonistic, with the concern that the effectiveness of biocontrol might be altered by endosymbiont presence in hosts. We, therefore, tested if predation by two aphidophagous ladybirds, Adalia bipunctata and Harmonia conformis, on the green peach aphid, Myzus persicae, and the oat aphid, Rhopalosiphum padi, was affected by transinfected Rickettsiella viridis and both native and transinfected Regiella insecticola endosymbionts at different temperatures. The predation rate of aphids infected by either endosymbiont was higher at 14 {degrees} C than the rate for uninfected aphids of both species, but the opposite pattern was apparent at 20 {degrees}C and for one host-endosymbiont combination at 26 {degrees}C. Overall, the results showed that higher temperatures increased predation, while differences between intermediate and low temperatures were species-dependent. No transmission of endosymbionts from aphid to ladybird through predation was detected. These findings point to a lack of consistent effects of the investigated endosymbionts on predation rates in these major aphid pests. The temperature dependence of endosymbiont-predation interactions suggests that the impact of seasonal climate should be considered when assessing the potential of endosymbionts in a biological control setting. HIGHLIGHTS- Aphid endosymbiont effects on ladybird predation rates are temperature-dependent - Ladybird predation rate of aphids increases as temperature rises - Prey (aphid) endosymbionts are not transferred to predators (ladybirds)

ecology↗