Potent immunosuppressive mechanisms within the tumor microenvironment contribute to the resistance of aggressive human cancers to immune checkpoint blockade (ICB) therapy. One of the main mechanisms for myeloid-derived suppressor cells (MDSCs) to induce T cell tolerance is through secretion of reactive nitrogen species (RNS), which nitrates tyrosine residues in proteins involved in T cell function. However, so far very few nitrated proteins have been identified. Here, using a transgenic mouse model of prostate cancer and a syngeneic cell line model of lung cancer, we applied a nitroproteomic approach based on chemical derivation of 3-nitrotyrosine and identified that lymphocyte-specific protein tyrosine kinase (LCK), an initiating tyrosine kinase in the T cell receptor signaling cascade, is nitrated at Tyr394 by MDSCs. LCK nitration inhibits T cell activation, leading to reduced interleukin 2 (IL2) production and proliferation. In human T cells with defective endogenous LCK, wild type, but not nitrated LCK, rescues IL2 production. In the mouse model of castration-resistant prostate cancer (CRPC) by prostate-specific deletion of ,, and , CRPC is resistant to an ICB therapy composed of antiprogrammed cell death 1 (PD1) and anticytotoxic-T lymphocyte-associated protein 4 (CTLA4) antibodies. However, we showed that ICB elicits strong anti-CRPC efficacy when combined with an RNS neutralizing agent. Together, these data identify a previously unknown mechanism of T cell inactivation by MDSC-induced protein nitration and illuminate a clinical path hypothesis for combining ICB with RNS-reducing agents in the treatment of CRPC.

Development of a peptide-based affinity matrix and detection reagent is important for biomedical research and the biopharmaceutical industry. In the present work, we designed and synthesized an immunoglobin G (IgG)-binding peptide ligand, Fc-III-4C. Fc-III-4C is composed of 15 residues, and the 4 cysteine residues form 2 disulfide bonds to generate a double cyclic structure. The binding affinity of the Fc-III-4C peptide toward human IgG was determined to be 2.45 nM (Kd), which is higher than that of IgG with Protein A/G (Pro-A/G). Importantly, the Fc-III-4C peptide displayed high affinity to various IgGs from different species. Fc-III-4C immobilized agarose beads exhibited high stability and reusability when compared with that of the Pro-A/G-immobilized beads. The conjugate of Fc-III-4C with FITC was demonstrated to be suitable for immunofluorescence detection of proteins expressed in cells. These results demonstrate that the Fc-III-4C peptide is a useful affinity ligand for antibody purification and as a protein detection reagent.