Modulation of T-Cell Function and Immune Phenotype in the Microenvironment of Emu-TCL1 CLL Bearing Mice By Ibrutinib

Background: Chronic lymphocytic leukaemia (CLL) is associated with global immunodeficiency, hypogammagobulinemia and T-cell exhaustion. We hypothesise that repairing T cell functions would improve cancer immune-editing, improve outcome and decrease infectious complications which cause significant morbidity in CLL patients.

Chronic B-cell receptor (BCR) activation as well as close interactions with the tumour microenvironment promote survival of malignant CLL B-cells, supporting their ability to induce immune suppression. To date, the most clinically successful approach to BCR-signalling inhibition is by the use of BTK inhibitors (BTKi). It has been suggested that the BTKi Ibrutinib has the ability to modulate T-helper cell polarity from Th2 to Th1 and thus would be a step towards repairing CLL associated T-cell defects (1). We examined the impact of Ibrutinib on T cell function and immune phenotype in vivo in Eµ-TCL1 mice with CLL.

Materials and Methods: C57/Bl6 animals 2.5 months of age were injected with 40x10e6 purified CLL B-cells pooled from Eµ-TCL1 mice with CLL. When peripheral blood CLL load reach >10% animals were randomized (mean day 14) to either vehicle treatment (2% HPBCD) or Ibrutinib treatment (0.15.mg/kg in 2% HPBCD) for 21 days. 17 animals per group were analysed. Splenic cells were isolated, the cellular component characterized by CyTOF and T cell function assessed by multi-parameter flow cytometry and T-cell synapse formation assay. We demonstrated that Ibrutinib administration this way led to high levels of BTK occupancy.

Results: Treatment with both Ibrutinib resulted in increased expression of IL2 (p=0.0004) in CD4+ T cells and decreased expression of IL4 among both CD4+ T cells (p=0.0015) and CD8+ T-cells (p<0.0001). Interferon gamma production was reduced in CD4+ (p=0.0056) and CD8+ T-cells (p=0.0020) with Ibrutinib treatment, which also resulted in an increase in CD107a+/CD107a- ratio among both CD44+ (p=0.0002) and CD44- CD8+ cytotoxic T-cells (p=0.0463). Ibrutinib treatment increases T-cell synapse area (p<0.0001) (Figure 1). We find a trend towards less antigen experienced CD44+ T-cells with Ibrutinib treatment with decreased expression of PD-1 in both CD44+ and CD44- negative T-cells but more pronounced in the antigen experienced T-cells. (Figure 2A). In addition, decreased expression of immune checkpoint receptor KLRG-1 on antigen experienced CD44+ T-cells was observed, most pronounced in the CD4+ subsets (Figure 2 B). Among NK 1.1+ NK-cells we find a strong trend towards decreased expression of immune checkpoint receptor KLRG-1 (Figure 2C). In white pulp myelomonocytic splenocytes (WPMC) we find a shift away from Ly6c low macrophage/patrolling monocyte-like cells towards more Ly6C high inflammatory monocyte-like cells. Moreover, we find a trend towards decreased expression of PD-L1, which is highly expressed among Ly6c low cells and shows little to no expression among Ly6C high cells (Figure 2D).

Conclusion: In vivo Ibrutinib treatment in this mouse model resulted in alteration in T cell function with cytokine secretion changes in keeping with a switch away from Th2 towards Th1 polarity as well as increased in cytotoxic T-cell function. The typical exhaustion phenotype of T-cell subsets is significantly ameliorated by Ibrutinib including a decrease in PD-1 expression. Moreover, we demonstrate a decrease in numbers of KLRG-1 high NK1.1+ NK cells. WPMC cells are shifted away from a potentially more tumour promoting Ly6C low PD-L1+ phenotype towards a more inflammatory Ly6c high PD-L1 low phenotype. These findings may point to a potential synergism of the combination of BTK inhibitors with immune checkpoint blockade for the treatment of CLL.

References

1. Dubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Zhong Y, et al. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood. 2013;122(15):2539-49.

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