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.

The non-canonical (NC) NF-κB pathway controls peripheral B-cell survival and follicular organization, pointing to a regulatory role during active adaptive immune responses. Here, we show that NC NF-κB activity is selectively attenuated in human germinal center B cells (GCBs), the population required to generate high-affinity antibodies, while remaining elevated in effector B cells. Accordingly, enforced NC NF-κB activation in murine B cells resulted in near-complete loss of GCB and high-affinity antibody production following immunization. These effects were primarily driven by NC NF-κB-induced reshaping of the immune microenvironment: activated B cells secreted IL-10, triggering premature expansion of regulatory T cells, limiting T cell help, and suppressing germinal center responses. Blocking IL-10 signaling was sufficient to normalize the niche and restore GCB. Concurrently, NC NF-κB activation promoted rapid B-cell differentiation into low-affinity antibody-secreting cells. Together, these findings identify NC NF-κB as a tunable rheostat linking B-cell signaling, immune-niche control, and humoral immunity.

INTRODUCTION: Polatuzumab vedotin (Pola) is an anti-CD79b antibody-drug conjugate. Clinical evidence supports the combination efficacy of Pola with mosunetuzumab or glofitamab, both anti-CD20/CD3 bispecific antibodies (anti-CD20/CD3 BsAbs). However, the influence of Pola on the tumor microenvironment and its impact on the efficacy of anti-CD20/CD3 BsAb remains unclear. RESULTS: In a syngeneic mouse model, Pola significantly increased tumor-infiltrating T cells and innate immune cells. Moreover, Pola also upregulated the levels of cytokine and chemokine involved in T-cell infiltration. The Pola + anti-CD20/CD3 BsAb combination showed superior anti-tumor efficacy versus each monotherapy. CONCLUSION: These findings suggest Pola enhances immune cell infiltration into tumors and could enhance the efficacy of anti-CD20/CD3 BsAb. TRIAL REGISTRATION: The authors have confirmed clinical trial registration is not needed for this submission.