## 3 Results
### Aged-Associated B Cells (ABCs)  
At baseline, the NR group showed significantly higher circulating ABC counts compared to R group (\(p\) = 0.04), although there was no statistical significance compared to HDs (\(p\) = 0.06) (Figure 3a, Supplemental Table S8). Moreover, at timepoint 4, responders had a significant decrease compared to HDs (\(p\) = 0.02). Contrary, comparing NR's ABCs at timepoints 1-3 to the ABC counts of HDs, they had constantly significantly increased levels (\(p_{1}\) = 0.01, \(p_{2}\) = 0.02, \(p_{3}\) = 0.03). Notably, in either group, there were no significant deviations in ABC counts during the treatment period (one-way ANOVA test, responders: \(p\) = 0.48; nonresponders: \(p\) = 0.45). When we analyzed the mean fluorescence intensity (MFI) of Tbet, we found a significant difference between Rs and NRs at baseline (\(p\) = 0.0002), but there was no difference at any other timepoint (Figure 3b, Supplemental Table S9). However, regarding the nonresponders' group, the repeated-measures one-way ANOVA revealed a statistically significant impact of immunotherapy on ABC counts (F = 7.451, \(p\) = 0.001), showing a significant increase 3 months after a numerical decrease in the preceding timepoints. Differences were significant between timepoints 1 and 3 (95% C.I. = [\(-\)1.410, \(-\)0.3147]) and 2 and 3 (95% C.I. = [\(-\)1.370, \(-\)0.2577]). Based on these results, the ABC subpopulation may play a major role in the PD-1 inhibitor therapy response. Patients with steadily increased counts of ABCs have inadequate outcomes from ICI therapy. Moreover, the fact that in the NR group a significant eleva  
Figure 3: ABCs and Tbet levels show significant differences during treatment. The graphs show the alterations in the percentage of ABC cells and change in MFI of Tbet among all participants enrolled in the study at each timepoint (HD, healthy donors; R-tp0-4, responder groups at every timepoint of the study subsequentially from 0 to 4; NR-tp0-4, nonresponder groups at every timepoint of the study from 0 to 4; * if \(p\) < 0.05, ** if \(p\) < 0.001). (**a**) Nonresponders (NR) had a significantly elevated ABC count compared to responders (R) at the baseline. At the same time, similar alterations of NRs’ ABC counts were noticed compared to healthy donors (HDs) at all three following timepoints examined. At timepoint (tp) 4, responders (Rs) had significantly decreased counts compared to healthy donors. (**b**) Only at baseline did nonresponders have significantly higher levels of MFI Tbet compared to responders. At timepoint (tp) 3, nonresponders revealed a significant elevation of MFI Tbet. tion in their ABCs counts at timepoint 3 was noted suggests that ABCs could potentially serve as new negative prediction markers, but further investigation is needed to come to a definite conclusion. Immune-Related Adverse Events (irAEs)  
We then examined the possible alterations of the B cell subpopulations analyzed between patients who developed adverse events and those who did not. Firstly, as far as total CD19+ B cells are concerned, there was no statistically significant difference among the groups of HDs, patients who developed irAEs (irAE group), and those who did not develop immune-related adverse events (non-irAE group). However, at timepoint 4, total CD19+ B cells were remarkably decreased in both groups of patients compared to the HD group (\(p_{\text{irAEs}}\) = 0.01, \(p_{\text{non-irAEs}}\) = 0.009), as they also were for the non-irAE group at timepoint 3 (\(p\) = 0.04) (Figure 4a, Supplemental Table S10). Figure 4: \(Cont.\)  
The subpopulation of mature naive B cells seemed to follow a similar motif in both groups (patients who developed irAEs and those who did not) when they were compared to healthy donors (Figure 4b, Supplemental Table S11). Especially, both groups confirmed a significant elevation compared to HDs only at timepoints 2 (\(p_{\text{irAEs}}=0.0103\), \(p_{\text{non-irAEs}}=0.0292\)) and 3 (\(p_{\text{irAEs}}=0.005\), \(p_{\text{non-irAEs}}=0.0\)).