Mobilising Tumour and Immune Cells Via Exercise in Chronic Lymphocytic Leukaemia

Whilst repeated cycles of anti-cluster of differentiation (CD)20 immunotherapy are effective at killing large numbers of chronic lymphocytic leukaemia (CLL) cells, in the peripheral blood circulation, minimal residual disease (MRD) persists diffusely in the bone marrow and secondary lymphoid organs such as the lymph nodes and spleen. This is thought to be for a number of reasons. Firstly, malignant cells sequestered in lymphoid compartments receive protection from their stromal microenvironment. For example, T cells, dendritic cells and macrophages release pro-proliferative and apoptosis-inhibiting signalling pathways, that prolong CLL survival. Consequently, CLL cells in lymphoid tissues are much less susceptible to anti-CD20 immunotherapy, than CLL cells trafficking in the blood. This may be because the circulating CLL cells are not embedded into a protective stromal microenvironment. Secondly, to exert antibody-dependent cellular cytotoxicity (ADCC; i.e., the primary mechanism of action elicited by anti-CD20 immunotherapy against CLL cells) NK cells need to express CD16, a molecule expressed by CD56dim NK cells. However, these potent NK cells are unable to traffic from blood, where NK cells are more abundant, to secondary lymphoid tissues, because NK cells lack lymphoid homing markers. Thus, CLL cells are able to 'hide' from NK cells in lymphoid tissue, ensuring their ongoing survival. In light of the pro-survival features of the CLL microenvironment, new approaches to detect and treat MRD are sought.

It has been repeatedly shown, in one of the most reproduced findings in human physiology, that immune cell frequency in the peripheral blood increases profoundly, by approximately 50-100%, during a bout of aerobic exercise of moderate to vigorous intensity. This response reflects a redistribution of immune cells from marginal pools in the blood vessels, as well as from lymphoid organs, such as the spleen, bone marrow and lymph nodes into the blood, driven by increased haemodynamic forces and increased levels of catecholamines during exercise. In response to increased levels of exercise-induced catecholamines, beta-2 adrenergic receptors on the cell surface of lymphocytes, induce detachment from the vascular endothelium, thus promoting the mobilisation of lymphocytes into the peripheral blood circulation, during a bout of exercise, returning to pre exercise values in the 1 hour following exercise. The migration of immune cells into blood is comprised of all major immune cell types, including neutrophils, monocytes and lymphocytes. Due to the adrenergic mechanisms responsible there is a preferential mobilisation of the subtypes with the highest expression of beta-2 adrenergic receptors on the cell surface. NK cells are the most exercise responsive T lymphocyte population, followed by CD8+ T cells, B cells and CD4+ T cells. B cells express beta-2 adrenergic receptors and are therefore susceptible to mobilisation via exercise-induced catecholamines. In healthy persons, B cell frequency is increased in blood by approximately 50-100% during various different types of exercise including, cycling, running, rowing and resistance exercise. Furthermore, a rodent model of acute psychological stress - which induces similar adrenergic responses as exercise - demonstrated a similar B cell mobilisation pattern. An additional rodent model observed that acute stressors appear to redirect B cells from the bone marrow, suggesting that the B cells mobilised in response to exercise may have been mobilised from lymphoid tissue. Given these changes to immune cell kinetics, exercise may be an effective means of mobilising CLL cells from diffuse lymphoid tissues into the peripheral blood circulation, which may, in turn, facilitate the detection of MRD in CLL.

This exercise-induced mobilisation of CLL cells from lymphoid tissues may also increase the efficacy of anti-CD20 immunotherapy. Indeed, this process may facilitate the removal of CLL cells from protective, pro-survival microenvironments into the blood circulation. CLL cells survive in lymphoid tissue receiving external signals from the microenvironment promoting growth and survival. Furthermore, CD16+ NK cells, the primary effector cell in ADCC, do not express lymphoid homing markers on their cell surface, and cannot traffic to the lymphoid tissue to elicit potent ADCC responses. Therefore, in order to improve the depth and efficacy of treatment with anti-CD20 monoclonal antibodies (mAb), strategies need to be explored that remove CLL cells from their protective stromal microenvironment within lymphoid tissues, and mobilise into the blood circulation where CLL cells will encounter NK cells capable of eliciting ADCC. Exercise has the potential to redistribute B cells, and exercise has also been shown to increase the frequency of CD16+ NK cells, by over 500% into the blood circulation. Therefore, exercise may mobilise both CLL cells from their protective microenvironment and large numbers of effector NK cells capable of ADCC into the blood circulation, simultaneously. Thus, this process has the potential to improve the depth and efficacy of anti-CD20 mAb treatment.

It is of importance to examine the effects of exercise on CLL and CD16+ NK cells at different stages of the CLL survivorship. For instance, people with watch and wait CLL, who have not yet received anti-CLL therapy, enables investigations into the impact of exercise on both CLL tumour cell and immune cell kinetics, without the presence of confounding variables like anti-CLL treatments, or disease associated morbidities. Indeed, people with watch and wait CLL have no disease symptoms and are thus likely to have fewer contra-indications to exercise than CLL patients later in the CLL survivorship continuum.

Investigating the impact of exercise in people receiving anti-CLL treatments is also warranted, as those likely to benefit from the adjuvant effects of exercise on immunotherapy are, by reason, patients undergoing cycles of immunotherapy treatment. Thus, it is essential to explore how acute exercise is tolerated in people undergoing cycles of treatment, and assessment of the safety and acceptability of exercise is warranted. Moreover, chemoimmunotherapy induces immune-suppression which may nullify the immune changes induced by exercise. Accordingly, investigation of the effects of exercise on immune cell kinetics, as well as residual CLL tumour cells, is warranted in people receiving anti-CLL chemoimmunotherapy.

Lastly, examining the impact of exercise in people in CLL remission, following completion of anti-CLL treatment, is required as any exercise induced mobilisation of CLL cells may also facilitate the detection of MRD following anti-CLL treatment. Several studies have demonstrated that patients who achieved a clinical complete remission (CR) but with detectable MRD, can experience a disease relapse due to expansion of the residual CLL cells. Patients with CLL who achieve undetectable MRD (<1 CLL cell per 10,000 leukocytes in blood or bone marrow), following anti-CLL treatment have better clinical outcomes than those with detectable MRD. A meta-analysis of eleven international studies with a total of 2457 patients observed that undetectable MRD status after treatment with chemotherapy or chemoimmunotherapy in newly diagnosed CLL is associated with overall and progression free survival. Therefore, MRD is a robust post-treatment prognostic biomarker of tumour burden, required for predicting disease recurrence. However, due to the multicompartmental nature of CLL, the bone marrow and secondary lymphoid tissue may serve as reservoirs for residual disease. Therefore, exercise may be utilised to facilitate the detection of MRD by mobilising CLL cells from diffuse sites into the blood circulation, thus potentially accelerating the identification of disease progression in patients following anti-CLL treatment. It is also important to assess immune competency in people in remission following completion of anti-CLL therapy, as any immune suppression at this time may hinder any effects of exercise.

The hypotheses of this pilot study are as follows:

• An acute bout of exercise will increase the frequency of CLL tumour cells in the peripheral blood of people with CLL at different stages of CLL survivorship.
• An acute bout of exercise will increase the frequency of immune cells (e.g., CD16+ NK cells) in the peripheral blood of people with CLL at different stages of CLL survivorship.
• Exercise induced mobilisation of immune cells (e.g., NK cells) will improve the efficacy of anti-CD20 immunotherapy against CLL cells ex vivo.