## 4 Discussion  
In this prospective study, we tried to understand the impact of ICIs in circulating B cells, with the potential of creating in the future an easy and accessible marker for ICI response. Starting from ICI initiation up to six months, including five subsequent timepoints of blood sampling and clinical evaluation, we focused on B cells as the main mediator of autoimmunity and their crucial subpopulations, whose role is understudied. We examined the circulating CD19+, mature naive, memory, regulatory, antibody-secreting, and age-associated B cells and how they correlate with alterations in ICI response and the development of irAEs. Our aim was to explore the potential effect of the ICI treatment on different circulating B cell populations. Our analysis revealed nuanced variations between responders, non-responders, and healthy donors; nevertheless, it depicted distinct alterations in various B cell subpopulations during the course of immunotherapy. While CD19+ B cell counts remained relatively stable within all groups and timepoints, suggesting a low impact of ICIs on them, significant differences were observed in specific B cell subsets. At the treatment onset, responders revealed a notable rise in mature naive B cells and a decline in memory cells compared to HDs, showcasing a potential correlation with positive treatment responses. This assumption was reinforced by the fact that responders' mature naive B cells exhibited, during the period of the study, elevated counts compared to HDs, with the timepoints 2 and 3 to be the ones with a statistically significant result; in addition, there was a significant reduction in memory B cell subpopulation at time point 1 compared to nonresponders. Notwithstanding, nonresponders exhibited a numerical decrease in the memory B cell subset, with no statistical significance to be obtained, in combination with the fact that in the mature naive B cell subset, nonresponders showed a significant increase only at time point 2 when compared to HDs, weakening the potential functional discrepancy in the immunomodulatory effects of ICIs between the two groups. The detected elevation in ABCs in nonresponders at baseline compared to responders, although not significant compared to HDs, enhanced by similar results of Tbet MFI analysis, suggests a possible treatment resistance. In the procedure of investigating a possible pattern of irAEs incidence, the similar behavior of both irAE and non-irAE groups compared to HDs did not reveal a possible prognostic factor. However, CD19+ and memory B cells showed a decrease, while mature naive B cells obtained an increase in both groups compared to HDs at multiple timepoints, indicating no significant difference between irAE and non-irAE groups. Regarding the non-irAE group, the significant elevation of ABCs at timepoints 1 and 2 and the decline of B regs cells at timepoint 2 compared to HDs showcased a possible correlation with irAEs induction, though further investigation is needed. Our findings align with the existing literature, supporting that there is no significant association of B cell count and their subtypes between responders and nonresponders at the baseline of the treatment [40; 57; 58; 59], though some data revealed a lower CD19+ count in patients with partial response or stable disease compared to nonresponders [60]. Moreover, according to a published similar study, elevated levels of nonswitched memory cells at baseline were associated with a higher proportion of response as well as an improvement in OS and PFS [61]. When a study for the behavior of B cells in melanoma and RCC was demonstrated, it revealed that responders had a higher count of B cell receptors (BCRs) and higher expression of memory and activated B cells [40; 62].