A Single Bout of Exercise Enhances the Efficacy of Rituximab Against Autologous Chronic Lymphocytic Leukaemia B Cells Ex Vivo By Transiently Increasing Natural Killer Cell Frequency in Blood, and Simultaneously Mobilises CD5+CD19+CD20+ B Cells into Blood

Chronic Lymphocytic Leukaemia (CLL) is characterised by the proliferation and accumulation of clonal B cells (B-CLL cells) and is often treated with anti-CD20 monoclonal antibody immunotherapies, including rituximab. One of the mechanism-of-action of rituximab is antibody-dependent cellular cytotoxicity (ADCC) which occurs when natural killer (NK) cells detect rituximab bound to CD20⁺ target cells. Rituximab often fails to induce stringent disease eradication, due in part to the diffuse distribution of clonal cells across multiple lymphoid and non-lymphoid tissues where NK cell frequency can be low. It is well established that an individual bout of aerobic exercise induces a transient relocation of lymphocytes - including NK cells and B cells – into peripheral blood. We hypothesised that this exercise-induced lymphocytosis could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab into blood.

In this pilot study n = 20 treatment naïve patients with CLL (mean ± SD: age = 62 ± 10 years; height = 174.0 ± 7.5 cm; body mass = 83.3 ± 16.8 kg; body fat = 31.7 ± 9.8 %; blood leukocytes = 30.70 ± 22.21 ×10⁹/L; anaerobic threshold = 14.1 ± 2.9 mL.kg^-1.min^-1) participated. Participants cycled at a moderate intensity (15% above their anaerobic threshold) for ~30-minutes, with blood samples collected pre-, post-, and 1-hour post-exercise.

Given the importance of CD16⁺ NK cells in evoking ADCC, we enumerated NK cell subsets in blood samples collected pre, post-, and 1-hour post-exercise by flow cytometry. As expected, exercise induced a preferential increase of CD56⁺CD16⁺ (+255%, p < 0.001) and mature, cytotoxic CD56⁺CD57⁺CD16⁺ NK cells (+322%, p < 0.001) pre- to post-exercise. Next, using immunomagnetic negative separation, NK cells and primary B-CLL cells were isolated from blood pre- and post-exercise and incubated together with heat inactivated foetal calf serum, with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab mediated cell lysis increased by +129% following exercise (p < 0.001), with no change in antibody independent NK cell lysis of B-CLL cells – independent of rituximab – following exercise (p = 0.25). Blocking CD16 on NK cells – vital for ADCC – blunted the effects of exercise on B-CLL cell lysis (p = 0.84). In a subset of patients (n = 9) we also explored the effects of autologous time-point matched plasma (instead of heat inactivated foetal calf serum) on rituximab mediated ADCC, which evoked a +92% increase in lysis pre- to post-exercise (p = 0.038). Collectively, our results suggest that augmented efficacy of rituximab mediated ADCC was driven by an increase in CD16⁺ NK cells.

We posited that the enhancement to rituximab mediated ADCC would have greater, clinically relevant implications if there was also a concomitant exercise-induced mobilisation of B-CLL cells expressing CD20 into blood from different body tissues. Flow cytometry revealed a +63% increase in CD5⁺CD19⁺CD20⁺ B-CLL cells (p = 0.002) in blood after exercise. Further analyses revealed that CD5⁺CD19⁺CD20⁺ B-CLL cells with a phenotype consistent with recent egress from lymphoid tissue (CD5^brightCXCR4^dim; 70%, p = 0.004) and B-CLL cells with a propensity to migrate to peripheral tissues (CD5^dimCXCR4^bright; 67%, p = 0.002) were mobilised, with no change to overall CD20 surface antigen density (p = 1.0) – determined by median fluorescence intensity. Furthermore, exercise evoked a +69% (p = 0.022) increase in CD5⁺CD19⁺CD20⁺ B-CLL cells expressing CD49d, which is considered one of the strongest predictors of CLL prognosis. Taken together, these data demonstrate that exercise increased the frequency of CD20⁺ B-CLL cells with lymphoid origins and prognostic relevance into the blood, therefore rendering them susceptible to rituximab mediated ADCC.

Our results show that individual bouts of moderate intensity aerobic exercise temporarily increased the number of cytotoxic CD16⁺ NK cells, and CD20⁺ B-CLL cells in blood. Additionally, our ex vivo investigations demonstrated enhanced rituximab mediated ADCC following exercise. Thus, exercise could be explored as a means of improving clinical responses in patients receiving rituximab, and/or other anti-CD20 monoclonal antibodies such as, Obinutuzumab.