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.

Mycobacterium tuberculosis impairs human memory CD4+ T cell recognition of M2 but not M1-like macrophages.

Direct recognition of Mycobacterium tuberculosis (Mtb)-infected cells is required for protection by CD4+ T cells. While impaired T cell recognition of Mtb-infected macrophages was demonstrated in mice, data are lacking for humans. Using T cells and monocyte-derived macrophages (MDMs) from individuals with latent Mtb infection (LTBI), we quantified the frequency of memory CD4+ T cell activation in response to autologous MDMs infected with virulent Mtb. We observed robust T cell activation in response to Mtb infection of M1-like macrophages differentiated using GM-CSF, while M2-like macrophages differentiated using M-CSF were poorly recognized. However, non-infected GM-CSF and M-CSF MDMs loaded with exogenous antigens elicited similar CD4+ T cell activation. IL-10 was preferentially secreted by infected M-CSF MDMs, and neutralization improved T cell activation. These results suggest that preferential infection of macrophages with an M2-like phenotype limits T cell-mediated protection against Mtb. Vaccine development should focus on T cell recognition of Mtb-infected macrophages.