Long-Term Treatment with Ibrutinib in Chronic Lymphocytic Leukemia Patients Normalizes CD3⁺ and CD8⁺ T Cells and Immune Checkpoints Expression but Gradually Leads to CD4⁺ T-Cell Depletion

In this study, we analyzed the changes in the peripheral blood mononuclear cells (PBMC) in symptomatic, relapsed or refractory chronic lymphocytic leukemia (CLL) patients (pts) during long-term treatment with the Bruton's tyrosine kinase inhibitor ibrutinib. Peripheral blood (PB) samples were collected before start of treatment with ibrutinib (420 mg/day p.o.) and at week (wk) 4, 10, 16, 22 and at 8, 12 and 24 months (mo) of treatment. Flow-cytometry analyses of PBMC included CLL cells, Natural Killer (NK) cells, T-cell memory subsets, helper subpopulations (Ths) and regulatory T cells (Tregs).

Eleven pts were included in the study, 8 males and 3 females; median age was 73 years. Nine age- and sex-matched healthy individuals were included as controls. Eight pts were still on ibrutinib treatment after 24 mo; for the remaining 3 pts, the last follow-up time was 12 mo. Eight pts had high-risk and 3 intermediate disease according to the modified Rai staging system; 8/11 had 17p deletion. The median lymphocyte count at study entry was 45 x 10⁹/L (range 3.1-109.5). Median number of previous treatment regimens was 1 (range 1-4). All but 2 pts stayed on full-dose ibrutinib during the whole treatment period. In the remaining 2 pts, the dose was decreased to 280 mg/day after approximately 1 year of treatment due to toxicity.

In 9/11 pts, the number of CLL cells initially increased at wk 4 compared to baseline (p=0.03); then a gradual reduction occurred during treatment and became significant at wk 16 (p=0.005). At baseline, CLL pts had significantly higher absolute numbers of CD3⁺ and CD8⁺ cells compared to healthy controls. CD3⁺ normalized (i.e. reached values not significantly different from healthy individuals) by wk 10. In contrast to previously published data (Long M et al, J Clin Invest 2017), we observed that CD8⁺ T cell numbers started to decrease at wk 10 and were normalized by 12 mo. CD4⁺ T cells, which were at start of treatment non-significantly different from healthy, also gradually decreased in 9/11 pts from wk 16 reaching by 24 mo levels below normal (p=0.02) (Fig.1A). At 24 mo of follow-up, CD4⁺ cell numbers had decreased in 7/8 evaluable pts; the median reduction vs baseline was 59% (range 3-92%). Among the helper subsets, Th1 (CCR6^-/ CXCR3⁺) cells, which at baseline were higher compared to healthy (p=0.0003), normalized by 12 mo, while Th2 (CCR6^-/ CXCR3^-) cells, at baseline non-significantly different from healthy, decreased below normal levels at 12 mo (p=0.006). Th17 (CCR6⁺/ CXCR3^-) cells, lower than in healthy controls at baseline (p=0.04), remained unchanged (Fig. 1B). Tregs (CD4⁺/CD25⁺/ CCR4⁺/CD127^low) and NK cells remained also unchanged.

The surface expression of PD-1 and intracellular CTLA-4, which was significantly higher at baseline in pts compared to healthy controls in both CD4⁺ and CD8⁺ cells, decreased also gradually. In the CD4⁺ subset, PD-1 expression normalized by 24 mo, while in the CD8⁺ subset already at 12 mo (Fig.2A). The expression of CTLA-4 normalized in CD4⁺ cells only by wk 22 (Fig.2B). Proliferating (Ki67⁺) cells, which at baseline were higher in pts compared to controls both in CD4⁺ and CD8⁺ cells, also normalized at wk 10 and 12 mo, respectively (Fig.2C). The distribution of the CD4⁺ and CD8⁺ memory cell subsets, abnormal at baseline, normalized as well by 12 mo.

In conclusion, these data indicate that ibrutinib treatment gradually led to normalization of T-cell counts in CLL patients. Down-regulation of immune checkpoints and cell proliferation markers also occurred, which paralleled the reduction of tumour burden. Moreover, the CD4⁺ T-cell counts gradually reached levels significantly lower than normal. This reduction affected both the Th1 and Th2 helper populations. The possible clinical implications remain to be shown during extended therapy.

View largeDownload slide

View largeDownload slide

Close modal