Methotrexate Exhibits a Dual Role in Regulation of CD4 ⁺ T Follicular Helper (Tfh) Cell Differentiation and Activity

Methotrexate (MTX) is one of the most prevalent drugs used in the treatment of autoimmune disorders as it has an established role in preventing T and B cell proliferation which reduces the severity of autoimmunity. However, MTX in combination with several other drugs is commonly used as chemotherapeutic regimens for the treatment of breast cancer (BC). Given the critical role of immune cells—particularly T and B cells—in recognising and eliminating tumor cells, we sought to investigate the impact of MTX on immune cell regulation in the context of breast cancer. CD4 + T follicular helper (Tfh) cells are recognised for their anti‐tumor potential during BC progression, as they support B cell differentiation and proliferation within germinal centres. In this study, we specifically aimed to observe the regulatory role of MTX during in vitro human Tfh cell differentiation under normal circumstances, and within in vivo 4T1 metastatic tumour‐bearing mice. Our findings reveal that MTX exerts a dual and context‐dependent role in modulating Tfh cell differentiation and function across the two models. Furthermore, we explored the influence of the AMPK activator AICAR in combination with MTX in both the human and mouse models. MTX when used in combination with AICAR, rescued human Tfh cell differentiation in vitro from MTX‐mediated suppression. In the 4T1 mouse model, the synergistic administration of MTX and AICAR significantly enhanced the proliferation of Tfh cells, along with increases in germinal centre B cells, memory B cells, and plasma cells in both circulation and tumor‐draining lymph nodes.