Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients.

SARS-CoV-2 infection triggers extensive host immune reactions, leading to severe diseases in certain individuals. However, the molecular basis underlying the excessive yet non-productive immune responses in severe COVID-19 remains incompletely understood. In this study, we conducted a comprehensive analysis of the peripheral blood mononuclear cell (PBMC) proteome and phosphoproteome in sepsis patients positive or negative for SARS-CoV-2 infection, as well as healthy subjects, using quantitative mass spectrometry. Our findings demonstrate dynamic changes in the COVID-19 PBMC proteome and phosphoproteome during disease progression, with distinctive protein or phosphoprotein signatures capable of distinguishing longitudinal disease states. Furthermore, SARS-CoV-2 infection induces a global reprogramming of the kinome and phosphoproteome, resulting in defective adaptive immune response mediated by the B and T lymphocytes, compromised innate immune responses involving the SIGLEC and SLAM family of immunoreceptors, and excessive cytokine-JAK-STAT signaling. In addition to uncovering host proteome and phosphoproteome aberrations caused by SARS-CoV-2, our work recapitulates several reported therapeutic targets for COVID-19 and identified numerous new candidates, including the kinases PKG1, CK2, ROCK1/2, GRK2, SYK, JAK2/3, TYK2, DNA-PK, PKCδ, and the cytokine IL-12.

Peptide Blockers of PD-1-PD-L1 Interaction Reinvigorate PD-1-Suppressed T Cells and Curb Tumor Growth in Mice.

The programmed cell death protein 1 (PD-1) plays a critical role in cancer immune evasion. Blocking the PD-1-PD-L1 interaction by monoclonal antibodies has shown remarkable clinical efficacy in treating certain types of cancer. However, antibodies are costly to produce, and antibody-based therapies can cause immune-related adverse events. To address the limitations associated with current PD-1/PD-L1 blockade immunotherapy, we aimed to develop peptide-based inhibitors of the PD-1/PD-L1 interaction as an alternative means to PD-1/PD-L1 blockade antibodies for anti-cancer immunotherapy. Through the functional screening of peptide arrays encompassing the ectodomains of PD-1 and PD-L1, followed by the optimization of the hit peptides for solubility and stability, we have identified a 16-mer peptide, named mL7N, with a remarkable efficacy in blocking the PD-1/PD-L1 interaction both in vitro and in vivo. The mL7N peptide effectively rejuvenated PD-1-suppressed T cells in multiple cellular systems designed to recapitulate the PD-1/PD-L1 interaction in the context of T-cell receptor signaling. Furthermore, PA-mL7N, a chimera of the mL7N peptide coupled to albumin-binding palmitic acid (PA), significantly promoted breast cancer cell killing by peripheral blood mononuclear cells ex vivo and significantly curbed tumor growth in a syngeneic mouse model of breast cancer. Our work raises the prospect that mL7N may serve as a prototype for the development of a new line of peptide-based immunomodulators targeting the PD-1/PD-L1 immune checkpoint with potential applications in cancer treatment.

Agreement of LC-MS assays for IGF-1 traceable to NIST and WHO standards permits harmonization of reference intervals between laboratories.

OBJECTIVES: In this study, we aimed to determine the feasibility of transferring IGF-1 reference intervals between two liquid chromatography-mass spectrometry assays with distinct assay formats and calibration traceability. DESIGN AND METHODS: To adopt a reference interval (RI) for our new assay we have conducted RI transference and verification studies according to the CLSI EP28-A3c and EP9c guidelines. Specifically, the analytical agreement between the assays was evaluated using the linear model and the appropriateness of the linear model for RI transference was assessed using Deming regression, correlation coefficients, Q-Q plot, difference plot and studentized residues for the LC-MS/MS against DiaSorin LiaisonXL IGF-1 immunoassay and the liquid chromatography-high resolution mass spectrometry (LC-MS/HRMS) IGF-1 assay. Both Diasorin immunoassay and LC-MS/HRMS assays are traceable to WHO, 02/254. RESULTS: Our study showed a strong correlation (R2 > 0.93) and agreement (slope = 1.006, negligible intercept) between LC-MS/MS and LC-MS/HRMS regardless of their traceability and all statistical criteria were met per CLSI guidelines. Conversely, while the LC-MS/MS and Diasorin immunoassay results showed a strong correlation (R2 > 0.97, slope = 1.055), they failed to meet all statistical criteria for RI transference due to the bias (-44.91) and non-normal distribution of the residues. The RI verification study showed that 90% of the local LC-MS results fell within the RIs transferred from the reference LC-MS method, thus meeting CLSI EP28-A3c guidelines and permitting the transference of the reference LC-MS RIs. CONCLUSIONS: Taken together, this study provides data to suggest excellent agreement between assays traceable to distinct reference standards for IGF-1.