Abstract
While cancer immunotherapies have primarily focused on activation of cytotoxic CD8 cells, CD4 T cell activity is also associated with survival and immunotherapeutic response in numerous cancers. We applied integrated single-cell RNA sequencing and multiplexed protein epitope profiling to breast cancer samples to resolve the complexity of immune cell states within the tumor microenvironment. This approach enhanced phenotypic resolution, identifying three distinct states within the CD4 T follicular helper-like (Tfh) cell cluster. A CXCR4(high) progenitor state gave rise to two differentiated states: an IGFL2(high) subset resembling conventional Tfh cells and localised to B cell-rich lymphoid aggregates, and a CD103+ subset, exhibiting features of tissue residency, exhaustion, and cytotoxicity, which co-localised with tumor foci. CD103+ Tfh-like cells were found to interact with CXCL10+ macrophages through production of CCL chemokines and CSF1. A higher CD103+ Tfh to IGFL2(high) Tfh ratio, together with the selective clonal expansion of the CD103+ subset, was strongly associated with improved tumour immunity and superior responses to anti-PD-1 checkpoint blockade, surpassing the predictive value of exhausted CD8 T cells. These findings integrate Tfh and CD4 with cytotoxic potential in breast cancer, offering new insight into anti-tumor immunity and response to checkpoint blockade.