Tumor Specific Regulatory T Cells in the Bone Marrow of Breast Cancer Patients Selectively Upregulate the Emigration Receptor S1P1
T regulatory cells, or Treg cells, are a subtype of CD4+ cells. They are responsible for inducing self-tolerance by a host of immune suppressive actions mediated by various immune cells, especially the autoreactive T effector cells. However, they can also harm the body via anti-tumor T-cell responses in neoplastic processes. Treg cells infiltrate tumors to promote tumor growth which causes the prognosis to worsen.
For this reason, depleting Treg cell populations via antibody-dependent cell-mediated cytotoxicity (ADCC)-proficient antibodies directed against CTLA-4, which is a Treg-associated molecule, or alternatively, low-dose cyclophosphamide treatment, is associated with better anti-tumor responses by T cells and overall patient survival.
On the other hand, Treg infiltration of tumors associated with chronic inflammation is associated with reduction of inflammatory mediators and a better prognosis.
In either case, the available data shows that Treg cells infiltrating tumors have prognostic significance. In breast cancer, many patients show effector T-cell responses specific for tumor antigens, but also tumor-specific Treg. This buildup of Treg cells in such tumors is known to be linked with impaired overall and relapse-free survival and may thus independently prognosticate a higher risk of relapse in some patients.
Treg cells enter peripheral tissues from lymphoid organs where they stay until they are mobilized by signals ranging from specific antigens that activate them, to chemokine gradients and other associated molecular changes. A high percentage of Treg cells are in the bone marrow, where they are accumulated, recirculated and from where they leave to infiltrate tumors in response to signals that are still poorly understood.
The current experiment aims to demonstrate how S1P1, a molecule that regulates the exit of immune cells from the lymphoid tissues, can control Treg exit from the bone marrow into the peripheral cells of breast cancer tissue. A study by Sawicka et al showed that an S1P1 agonist could be administered to produce Treg cell accumulation within the peripheral blood and the spleen but not inside lymph nodes, however naïve CD4+ cells did accumulate in the latter.
This differential accumulation points to a possible effect of S1P1 as an emigration receptor. The presence of this molecule on the cell surface is thus vital in deciding whether the cell leaves the bone marrow and is closely dependent on the levels of this receptor in the microenvironment which thus regulates the patterns of cell movement.
When Treg are activated, they migrate from the lymph nodes towards S1P, but Treg retain S1P1 on their surface for a longer time compared to Tcon. The aim of this research is to clarify whether S1P1 could play a role in Treg emigration from the bone marrow in breast cancer patients.
The material used included peripheral blood and bone marrow samples taken from breast cancer patients. The areas studied included the expression, induction, and function of S1P1 and its ligand, S1P, in relation to Treg in this disease, both in bulk and in antigen-specific Treg cells. The results seem to indicate that S1P1 expression is enhanced on Treg with tumor antigen specificity, which causes them to leave the bone marrow rather than other Treg cells.
Multicolor Immunofluorescence Staining and Data Acquisition using TissueFAXS
The T-cell subpopulations within the tumor tissue were first detected using a mixture of primary antibodies such as anti-CD3 (Dako, A0452, Host-rabbit), anti-CD8 (Clone YTC182.20, Abcam, Ab60076, Host-rat), and anti-FOXP3 (Clone 236 A/E7, Abcam, Ab20034, Host-mouse) applied to breast tumor cryosections fixed with acetone.
Primary specific secondary antibodies (anti-rabbit Alexafluor 647, A21245; anti-rat Alexa Fluor 488, A11006; anti-mouse Alexa Fluor 555, A31570) were sourced from Life Technologies The nuclei were then stained using DAPI (Invitrogen, D1306).
An Olympus IX51 microscope equipped with a F-View II camera (both Olympus) was used to scan the total tissue slides, and these images were then analyzed using the TissueQuest Cell Analysis Software package (version 4.0.1.0137, TissueGnostics GmbH). DAPI staining was used purely to identify cells based on their nuclei, as a master marker, in this automated analysis.
The H&E slides used as reference were marked with regions of interest, ROI, to differentiate the tumor and non-tumor regions which were closely associated. All analysis parameters were identical, with T cells being detected based on the size of the nuclei, the mean intensity of staining, and the background threshold. The cells were visualized using scatter grams, and the positive-negative gated cell cutoff was validated by manual backward gating using the original image.
It is established that Treg often infiltrates solid tumors, but what drives such emigration is not yet clear. Peters et al has suggested that the patterns of Treg movement might be significantly disrupted in patients with solid tumors. Whereas Zhao et al have described a possible role of the bone marrow in such recirculation of Treg cells in patients.
The current experiment intended to demonstrate the first ever proof of significant changes in the number of Treg cells that are found in the bone marrow, the peripheral blood, and the tumor respectively. The number found within the peripheral blood was markedly increased, while it was reduced in the bone marrow, but simultaneously increased within tumor tissue. These findings suggest that the intratumoral and peripheral blood Treg infiltration came from the bone marrow.
It’s important to note that Treg cell numbers vary markedly between individual patients with breast cancer, perhaps because of variations in the production rate. Also, the period in which recirculation of activated Treg cells mobilize into the peripheral blood is so short that these preclude any conclusion that the elevated number of cells in the blood came from the bone marrow.
However, it is clear that in breast cancer patients, the number of Treg cells in the bone marrow is inversely proportional to that in the tumor tissue.
Rathinasamy A, Domschke C, Ge Y, et al. Tumor specific regulatory T cells in the bone marrow of breast cancer patients selectively upregulate the emigration receptor S1P1. Cancer Immunol Immunother. 2017;66(5):593-603. doi:10.1007/s00262-017-1964-4