## 3 Results
### Aged-Associated B Cells (ABCs)
No significant difference was observed during the comparison between the irAE group and non-irAE group at any timepoint. Additionally, when a repeated-measures one-way ANOVA test was performed, none of these groups were affected by immunotherapy during our study (irAEs: F = 1.625, \(p=0.2036\); non-irAEs: F = 1.1, \(p=0.3657\)). Moreover, the subpopulation of memory B cells was notably diminished in both irAE and non-irAE groups compared to HDs at almost all timepoints (\(p_{\text{irAEs(1)}}=0.04\), \(p_{\text{irAEs(2)}}=0.01\), \(p_{\text{irAEs(3)}}=0.03\), \(p_{\text{irAEs(4)}}=0.02\)) (\(p_{\text{non-irAEs(0)}}=0.03\), \(p_{\text{non-irAEs(1)}}=0.006\), \(p_{\text{non-irAEs(2)}}=0.0004\), \(p_{\text{non-irAEs(3)}}=0.002\), \(p_{\text{non-irAEs(4)}}=0.01\)) (Figure 4c, Supplemental Table S12). However, it seemed that there was no difference between irAE and non-irAE groups in memory B cell counts, as we did not observe any impact of immunotherapy in these groups for the particular subpopulation when we regarded a repeated-measures one-way ANOVA test (memory irAEs: F = 0.41, \(p=0.79\); memory non-irAEs: F = 1.49, \(p=0.21\)). Regarding Bregs pattern, it seemed that in both groups there was a numerical decline in their count at timepoints 2 and 3, whereas significant changes were noted at time point 2 in the non-irAE group compared to HDs (\(p=0.025\)) (Figure 4d, Supplemental Table S13). Additionally, no noticeable impact of immunotherapy was found during the first 6 months of our observation in the Bregs count in each group. Moreover, in ABCs subpopulation counts, we observed an increase only in timepoint 1 (\(p=0.03\)) and 2 (\(p=0.04\)) of the non-irAE group when compared to HDs (Figure 4f,g, Supplemental Table S14). However, at timepoint 4 in irAE and non-irAE groups, there was a numerical decrease; regarding a repeated-measures one-way ANOVA test, no significant alterations were noticed (irAEs: F0.55, \(p=0.69\); non-irAEs: F = 0.77, \(p=0.55\)). Finally, it is important to mention that no  
Figure 4: Alterations in different B cell populations in patients who developed immune-related adverse events (HD, healthy donors; irAEs (0)–(4), the patient group that developed irAEs at every timepoint of the study subsequential from 0 to 4; N-irAEs (0)–(4), the patient group that did not develop irAEs at every timepoint of the study from 0 to 4; * if \(p<0.05\), ** if \(p<0.001\)). (**) CD19+ alterations were similar between patients who developed irAEs and those who did not. (**) An increase in mature naïve B cell counts at timepoints 2 and 3 in both groups was observed. (**) Similar alterations in memory B cell subpopulation between both groups compared to HDs were noticed at almost all timepoints. (**) Significant changes were noted only at timepoint 2 in the non-irAE group compared to healthy donors, despite the numerical decline of Bregs counts in both groups at timepoints 2 and 3. (**) No significant variations of ASC count among irAEs, non-irAEs, and healthy donors during the period of immunotherapy. (**f,g**) ABC subpopulation of the non-irAE group was significantly increased compared to healthy donors only at timepoints 1 and 2.  
significant differences were observed when comparing the count of ASC B cells of irAE, non-irAE, and HD groups at every timepoint separately, as long as they had variation during the period of treatment (Figure 4e). There is a strong involvement of almost all subpopulations of circulating B cells in the possibility of a patient developing, or not, irAEs due to ICI administration. Nevertheless, even if the differences were significant in both groups compared to HDs, important alterations between the irAE and non-irAE groups were not found. As PD-1 inhibitors trigger the patient's immune system against tumors, our facts empower the suggestion that circulating B cells are strongly involved in this procedure. However, from these data, we could not extract a specific marker that would determine whether or not a patient is going to develop irAEs.