Analyzing human CD4⁺ T cells activated in response to macrophages infected with Mycobacterium tuberculosis

M1- and M2-like macrophages infected with Mycobacterium tuberculosis (Mtb) have been found to differ in their capacity to elicit memory CD4⁺ T cell activation. Here, we present a protocol to quantify and isolate the subset of human memory CD4⁺ T cells activated in response to autologous monocyte-derived macrophages (MDMs) infected with virulent Mtb. We describe steps for CD14⁺ monocyte isolation, generating MDMs, culturing Mtb and infection of macrophages, and identifying activated CD4⁺ T cells by flow cytometry. For complete details on the use and execution of this protocol, please refer to Gail et al.¹.