194P - The impact of BTK inhibitors on ?d T cell fitness: Lesson for immunotherapy

Background

γδ T cells are a small subset (0.5-5%) of peripheral blood T cells playing a key role in both defense against a wide range of pathogens and anticancer immunity. In recent years, Bruton’s tyrosine kinase (BTK) inhibitors have revolutionized the treatment of B-cell malignancies. Although primarily expressed in B-cell lineage cells, BTK expression has also been identified in other cell types. However, the role of BTK expression and the impact of BTK inhibitors on non-B cells, including γδ T cells, remain unclear.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using density gradient centrifugation. Selective γδ T cell expansion was achieved by adding zoledronic acid and IL-2. On day 14 of culturing, ibrutinib, acalabrutinib, or zanubrutinib were introduced. After 48 hours of co-incubation, cytokine production and γδ T cell activation were assessed. To evaluate the impact of BTK inhibitors on γδ T cell activation, HMBPP or the CLL cell line Duller was used as stimulation agents. Cytokine production was evaluated by stimulating the cells with M. tuberculosis lysate, PHA, HMBPP, or anti-CD3 [OKT3], followed by cell permeabilization, staining with specific antibodies, and analysis using flow cytometry.

Results

Ibrutinib significantly reduced the production of IFN-γ and TNF-α by γδ T cells stimulated with HMBPP, but not with Duller cells. This suggests that TCR-independent stimulation pathways, such as through the NK family of receptors, may restore cytokine production to normal levels. No significant differences were found in the production of granzyme B, IL-6, IL-9, IL-10, or IL-17a. All inhibitors reduced the early activation of γδ T cells, as indicated by decreased CD69 expression, while having no effect on late-stage activation. Interestingly, this inhibitory effect was least pronounced with ibrutinib.

Conclusions

BTK inhibitors seem to modulate γδ T cell function, with their impact being pathway-specific and potentially partially reversible depending on the type of stimulation. Moreover, newer inhibitors appear to have a lesser impact on γδ T cells, likely due to their higher specificity for BTK. From an immunotherapeutic perspective, concomitant treatment with BTK inhibitors and in vitro expanded γδ T cells could be a viable option.

Legal entity responsible for the study

Medical University of Lublin.

Funding

DKMS Foundation.

Disclosure

All authors have declared no conflicts of interest.