Emerging evidence indicates an inverse relationship between the percentage of regulatory T cells (Tregs) and poor prognosis in cancer patients [1,2]. It is acknowledged that Tregs and tumor-initiating cancer stem cells (CSCs) collaborate to endorse tumor immune tolerance and encourage tumor progression [2,3]. CSCs are instrumental in tumor initiation, metastasis, and recurrence as well as in mediating drug resistance [[4], [5], [6]]. However, the liaison between Tregs and CSCs remains mostly unexplored. Being the tumor-initiating cells [2,7], CSCs face the challenge of overcoming the host immune system's resistance in order to establish, proliferate, and sustain the tumor [2,7]. The tumor microenvironment (TME) contains a diverse array of immune cells, which are manipulated by tumor cells to create an immunosuppressive environment for their own growth and development [3,8,9]. Nevertheless, the mechanisms behind the survival of tumor-initiating CSCs throughout the early ‘elimination phase’, remains elusive. Hence, comprehending the process by which CSCs evade the immune system is crucial for devising potent treatments that can eliminate these highly resistant cells responsible for tumor initiation and immune evasion.

CSCs possess altered surface proteins (intrinsic) and also release secretory soluble factors (extrinsic) that aid in immune evasion during initial stages [10] of cancer development. The reciprocal communication between CSCs and tumor-infiltrating immune cells is a paradigm for these cell extrinsic and intrinsic interactions. Regarding this, the production of pro-tumor Tregs produced by CSCs may have a crucial role in enabling tumors to escape immune-mediated destruction [2,11]. Within the TME, various tumor secreted-substances, such as TGFβ, IL10, and adenosine, govern generation of pro-tumor Treg cells [3]. Among these, exosomes which are 30-100 nm membrane vesicles derived from the endosomal pathway, containing lipids, proteins, and RNA, are also significant mediators of cell-to-cell communication in this environment [12,13]. CSC-derived exosomes (CDEs) have been implicated in facilitating tumor growth by increasing angiogenesis and migration [14]. However, the role of CDEs in the case of T cell-immunosuppression have been studied limitedly.

Here, we investigated the role of CSCs in reprogramming CD4+ T cells to immunosuppressive Treg cells through exosomes. Our search revealed that CSCs and FOXP3 demonstrate a positive correlation, and these CSCs overexpress FOXP3 within them. Moreover, depleting exosomes from conditioned medium (CM) of CSCs lowered their Treg generating potential from CD4+ T cells, thereby, demonstrating the role of CDEs in Treg formation. Consequently, isolated and characterized CDEs were found to be capable of generating immunosuppressive Tregs at an early time-point of 24 h. Detection of FOXP3 protein in CDEs hinted towards the possibility of FOXP3 being gifted by CDEs towards Treg generation. Indeed, we observed that blocking of translation machinery in T cells failed to abolish FOXP3 generation in CDE-exposed T cells at 12 h. Moreover, exosomes isolated from FOXP3 attenuated-CSCs led to lower FOXP3 levels in T cells than control CDEs at this time-point. These FOXP3 attenuated-CDEs also generated lower tumor volume and Treg percentage, while showing higher effector T cell percentage in our in-vivo murine model, when compared to control CDEs. These findings indicate the hitherto un-explored role of CSC-FOXP3 in generating immunosuppressive Treg cells that might help in tumor immune-escape at the initial stages of tumor formation.