Identification of a highly suppressive Treg subset associated to immunotherapy response

Background Cancer immunotherapy has shown surprising efficacy in several types of advanced and incurable tumors, particularly, malignant melanoma. There are several immunotherapeutic strategies aimed at enhancing immunological defenses against tumor. Among these, monoclonal antibodies against the so-called “immune checkpoint inhibitors”, that counteract tumor-induced immune-disarming pathways, have shown the best outcomes. Regulatory T lymphocytes or Tregs are a subset of lymphocytes involved in immune-surveillance and maintenance of self-tolerance. Tumor often exploits Tregs to allow tolerance to its own antigens and avoid immune system attack. Tregs are usually increased in melanoma patients. It is noticeable that Tregs is a heterogeneous population with respect to their immunosuppressive capability. Lymphocytes are particularly rich in FKBP51 (FKBP5 gene), an immunophilin better known as the intracellular receptor for FK506 and rapamycin. Melanoma aberrantly expresses this immunophilin, which supports cancer resistance and invasion. Recently, our group has shown that melanoma interaction with immune cells, through PD-L1/PD1, bidirectionally generated the splicing of FKBP5 gene inducing a lower molecular weight form of FKBP51, termed FKBP51s, in both melanoma and lymphocyte. A study performed on PBMC of 64 patients with advanced melanoma (stage III/IV) showed that FKBP51s marks a Treg subset which was correlated, as an independent variable, to anti-CTLA4 (ipilimumab) response. More precisely, a low frequency of Treg FKBP51spos (<1% of total CD3/CD4 lymphocytes) was associated with unresponsiveness to ipilimumab (Chi-square=9.916, p=0.002). Aim of the present study was to assess the role of Treg FKBP51s+ as potential biomarker of response in a different cohort of patients subjected to anti-PD1 treatment. In addition, the suppressive potential of Treg FKBP51s+ in comparison with that of Treg FKBP51s- is investigated. Methods Treg FKBP51s+ were measured in peripheral blood by flow cytometry. To date, we have outcomes of 11 patients. For 6 patients, we have collected from 4 up to 16 blood samples, before each anti-PD1 administration (every 2- 3 weeks), with a total of 80 sample analysis. iTregs were generated by purified CD4+ T lymphocytes, from normal donor, stimulated with CD3+CD28+beads. The suppressive capacity was assessed according to the parameters CD25high, Ki67high and p70S6khigh. Results In 5 responder patients, Treg FKBP51s+ was >1.2 and < 4.8%; in 5 non responder patients, the count was >0.04 and < 0.8%. After a transient increase registered following the first administration, the count decreased to 0.3+0.2% in responder patients. Interestingly, a patient with count = 0.72% developed autoimmune side effects that led to therapy discontinuation. Resolution of side effects was accompanied by an increase in Treg FKBP51s+ value to 9.9%; thenafter, anti-PD1 re-administration produced a successful and objective response. In vitro iTreg generation suggested that FKBP51s was induced in Treg CD25high, Ki67high and p70S6khigh , corresponding to a highly metabolically active profile associated with strong suppressive capability. Conclusion Our data reinforce the hypothesis that melanoma patients that benefit from immune checkpoint targeted therapy are recognizable by an expansion of a Treg subset which plays a central role in de-activation of stimulatory co-signalling pathways, in support of tumor immune evasion. Such a Treg subset is marked by FKBP51s, a splicing protein isoform generated by triggering of surface antigens (PD-L1, PD1) that are abundantly expressed on highly suppressive Tregs.