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SummaryT cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses. Graphical abstract Highlights•Isolation of leukocytes from murine BM, liver, heart and kidneys•Non-enzymatic dissociation of kidney and heart tissue•Protocol for immunomagnetic enrichment of T cells•Flow cytometry analysis of T cell purity and viability Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. T cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses.

Background: Natural killer (NK) cells are key effector lymphoid cells involved in both innate and adaptive immunity and are capable of clearing abnormally aggregated α-synuclein (αSyn). In preclinical Parkinson’s disease (PD) models, NK cell depletion worsens motor deficits and increases insoluble αSyn accumulation, suggesting a neuroprotective role. However, the therapeutic potential of NK cell transfer in modulating αSyn pathology and neurodegeneration remains unexplored. Methods: To assess the efficacy of NK cell therapy, we administered biweekly systemic injections of untouched NK cells isolated from B6C3H donor mice into 2-month-old presymptomatic homozygous M83 transgenic mice injected with human αSyn preformed fibrils. Neurological function was assessed via clasping behavior and clinical scoring. αSyn pathology and dopaminergic neurodegeneration were evaluated via immunohistochemistry. CyTOF-based immune profiling and multiplex ELISA were performed to characterize central and peripheral immune responses. Results: Adoptive NK cell transfers improved motor function and reduced αSyn pathology in a region- and dose-dependent manner, with significant reductions in phosphorylated-αSyn inclusions and tyrosine hydroxylase-positive neuronal loss in the substantia nigra. NK cell transfer modulated the CNS immune landscape by reducing CD11b+CD45high and MHCII+ activated microglial, CD4⁺ T cells, and neutrophil infiltration, while promoting CD19+ B and CD8+ T cells. Similar immunomodulatory effects were observed in the periphery, including restoration of follicular B cells and reduced neutrophil frequencies. Mechanistically, αSyn exposure downregulated activating NK ligands and upregulated inhibitory receptor ligand mQa1b, along with p21 induction in microglia, suggesting a senescence-associated, immune-evasive phenotype that may contribute to reduced therapeutic efficacy at later disease stages. Conclusions: Our study provides direct evidence of NK cells exerting neuroprotective and immunomodulatory effects in a preclinical model of synucleinopathy. These findings support NK cell transfer as a novel therapeutic strategy for PD and related neurodegenerative disorders.

Zhang et al. demonstrate that the expression of a mutated CARMIL2 protein in CD28-deficient mice induces most of the developmental and functional consequences known to result from CD28 costimulation and in turn triggers potent tumor-specific T cell responses resistant to PD-1 and CTLA-4 blockade. Naive T cell activation requires both TCR and CD28 signals. The CARMIL2 cytosolic protein enables CD28-dependent activation of the NF-κB transcription factor via its ability to link CD28 to the CARD11 adaptor protein. Here, we developed mice expressing a mutation named Carmil2QE and mimicking a mutation found in human T cell malignancies. Naive T cells from Carmil2QE mice contained preformed CARMIL2QE-CARD11 complexes in numbers comparable to those assembling in wild-type T cells after CD28 engagement. Such ready-made CARMIL2QE-CARD11 complexes also formed in CD28-deficient mice where they unexpectedly induced most of the functions that normally result from CD28 engagement in a manner that remains antigen-dependent. In turn, tumor-specific T cells expressing Carmil2QE do not require CD28 engagement and thereby escape to both PD-1 and CTLA-4 inhibition. In conclusion, we uncovered the overarching role played by CARMIL2-CARD11 signals among those triggered by CD28 and exploited them to induce potent solid tumor–specific T cell responses in the absence of CD28 ligands and immune checkpoint inhibitors.