Provided is a method for the dual culture of immune cells used for immunotherapy, and more specifically, to a dual culture method in which immune cells, e.g., immune cells (PBMCs), natural killer cells, and gamma delta T cells, which are highly effective in treating malignant tumors and the like, are selectively and efficiently amplified and activated by culturing lymphocytes derived from human peripheral blood, wherein the culture efficiency and activity can be increased when immune cells are cultured in combination after applying different stimuli for a predetermined period of time.

The present disclosure is directed, in some aspects to methods and compositions for the assessment of regulatory T cell stability over time. In other aspects, the present disclosure provides methods and compositions for expanding regulatory T cells in culture.

As disclosed herein, SIRP is integral to immuno-evasion by many different cancer types as well as cancer resistance to therapies, and reducing SIRP levels on can bolster antigen acquisition, processing, and presentation, decrease TME immunosuppression and thereby promote tumor-specific T cell activation to eliminate tumors and generate an adaptive immune response consisting of memory T cells, circulating antibodies, and plasma cells, all of which may be specific for neo-antigens in the original cancer. Therefore, disclosed are activated SIRP.sup.low macrophages that are useful for treating cancers.

The disclosure provides methods of making CAR-expressing immune effector cells (e.g., T cells, or NK cells), and compositions and reaction mixtures comprising the same. The disclosure further provides methods of using said CAR-expressing immune effector cells.