Changes in the T Helper Cell Compartment during Treatment with the BTK Inhibitor Acalabrutinib

Interactions with T helper cells are part of the supportive lymph node microenvironment promoting chronic lymphocytic leukemia (CLL) cell growth and survival. Treatment of CLL patients with ibrutinib, the first-in-class BTK inhibitor, induces profound changes in the T cell compartment, with normalization of elevated T cell numbers and plasma cytokine levels, and diversification of the T cell receptor (TCR) repertoire (Yin Q et al., J Immunol 2017). Acalabrutinib, a second generation BTK inhibitor, is more selective for BTK, with less off-target effects towards other kinases, including IL-2-inducible T cell kinase (ITK), an important kinase for T cell signaling. To characterize acalabrutinib effects on T cell numbers and function in patients with CLL, we analyzed serial samples from 12 acalabrutinib-treated CLL patients at baseline, after 1 week, and after 1, 3, 6, and 12 months of continuous acalabrutinib treatment (with monthly Obinutuzumab added during months 3-9) in an ongoing Phase 2 trial (NCT04505254). Six age-matched healthy donors (HD) were analyzed for comparison. T cell subsets were characterized by flow cytometry. Functional T cell response were assessed after stimulation with phorbol myristate acetate and ionomycin, and proliferation by Ki-67 expression.

Prior to treatment, untreated CLL patients had elevated numbers of CD4⁺ (1864±32/ml) and CD8⁺ (2533±452/ml, mean T cells which normalized after 12 months of therapy (CD4⁺ 514±65/ml, p<0.01; and CD8⁺ 747±271/ml, p<0.05). Two key subsets, T follicular helper cells (Tfh) and T regulatory cells (Tregs), also decreased significantly after 12 months of treatment (Tfh from 2.9±0.4% to 0.2±0.1%, p<0.05; and Tregs from 2.4±0.4% to 0.9±0.2%, p<0.05). Next, we analyzed expression of Tfh and Treg surface markers and found significant down-modulation of CD69 (on Tfh from 20±3.7% to 3.3±0.6%, p<0.001; on Tregs from 10±1.8% to 1.7±0.5%, p<0.01), ICOS (on Tfh from 33±2.6% to 13±1.6%, p<0.01), PD-1 (on Tregs from 7.8±1.3% to 1.2±0.3%, p<0.01), and CTLA-4 (on Tfh from 16±3.2% to 3.8±1.0%, p<0.05; on Tregs from 14±1.5% to 3.1±0.7%, p<0.001) after 12 months of treatment. Additionally, this therapy resulted in reduced cytokine production by Tfh and Tregs when compared to baseline samples, as quantified by flow cytometry after cytoplasmatic staining. For example, induced IL-21 expression decreased from 23±1.3% in baseline samples to 12±0.5% after 12 months, p<0.05 (for comparison, 5.7%±1.3% in HD), IL-10 decreased from 7.3±1.1% to 0.9±0.2% at 12 months, p<0.05 (0.9%±0.1% in HD), and TGFb (from 6.2±0.9% to 0.8±0.2% at 12 months, p<0.01 (1.6%±0.5% in HD). Moreover, circulating Tfh cells from pre-treatment samples expressed higher levels of the co-stimulatory molecule CD40L upon restimulation at baseline compared to after 12 months of treatment and the levels in HD (from 23.0±1.6% to 11.0±1.1% at 12 months, p<0.05; versus 3.8%±1.0% in HD, p<0.001). Looking at Ki-67 expression, we noted, as expected, a significant decrease in CLL cell proliferation from 3.9±0.8% down to 0.8±0.6% after 1 month of therapy (p<0.001), similar data were seen at later time points. Interestingly, Tregs also had reduced Ki-67 expression, down from 16±0.6% to 7.3±0.9% after 6 months (p<0.01) and to 7.9%±1.1% after 12 months of therapy (p<0.05). Collectively, the reduction in T cells and T helper cell subset numbers, along with changes in surface activation markers, and reduced cytokine and Ki-67 expression suggest that an activated pre-treatment T helper cell compartment normalizes during continuous acalabrutinib therapy. These findings are reminiscent of the T cell data from patients receiving ibrutinib therapy, suggesting that off-target effects of the BTK inhibitor may not dictate T helper cell changes, and these may rather be due to activation by the CLL clone, which diminishes as the underlying disease responds to treatment.

Ferrajoli:Beigene: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Wierda:Gilead Sciences: Research Funding; Pharmacyclics LLC: Research Funding; Miragen: Research Funding; Genzyme: Consultancy; Genentech: Research Funding; Sunesis: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; Loxo Oncology, Inc./Lilly: Research Funding; Kite, a Gilead Company: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Cyclacel: Research Funding; Bristol Meyers Squibb (Juno and Celgene): Research Funding; AstraZeneca/Acerta Pharma. Inc.: Research Funding; Sanofi: Consultancy; Karyopharm: Research Funding; Juno: Research Funding; GSK/Novartis: Research Funding; AbbVie: Research Funding. Burger:Gilead: Consultancy; TG Therapeutics: Consultancy; BeiGene: Consultancy, Research Funding; Pharmacyclics LLC: Consultancy, Research Funding; Janssen: Consultancy, Speakers Bureau; Novartis: Consultancy.