Canine B Cell Lymphoma- and Leukemia-Derived Extracellular Vesicles Moderate Differentiation and Cytokine Production of T and B Cells In Vitro

Abstract

Extracellular vesicles (EVs) are formed in physiological and pathological conditions by almost all mammalian cells. They are known as submicron "molecules" that transport and horizontally transfer their cargo from maternal cells to donor cells. Moreover, cancer cells produce tumor-derived EVs (TEVs), which are present in blood of patients with solid tumors and those with hematological malignancies. Their role in evading immune system surveillance and induction of immunosuppression in hematological cancer is limited. According to the authors' best knowledge, there is no information about the impact of TEVs from canine lymphoma (CLBL-1) and leukemia (CLB70) on lymphocytes isolated from peripheral blood mononuclear cells (PBMCs). In conclusion, we demonstrate in in vitro experiments that CLBL-1 EVs and CLB70 EVs are effectively taken up by T and B lymphocytes. TEVs decrease the percentage of B lymphocytes and increase that of T lymphocytes, and change T cells' phenotype into the effector memory (EM) or terminally differentiated effector memory (TEMRA) subtype after in vitro co-culturing. Moreover, CLBL70 EVs have pro-tumorogenic properties by inhibiting the production of CD8⁺IL-17⁺ cells.

Keywords: CLB70; dog; extracellular vesicles; leukemia; lymphocytes; lymphoma; oncosomes; proliferation.

Figure 1

Time-dependent internalization of TEVs by lymphocytes. Flow cytometric analysis of the fluorescence transferred by CLBL-1 EVs and CLB70 EVs into lymphocytes. The staining of directly labeled cells and the negative control cells (gray lines) after 30, 50, and 120 min of incubation are shown. (A) the kinetics of two concentrations of CLBL-1 EVs taken up by: CD3⁺ T lymphocytes, CD21⁺ B lymphocytes, CD4⁺ T lymphocytes, and CD8⁺ T lymphocytes. (B) The kinetics of two concentrations of CLB70 EVs taken up by: CD3⁺ T lymphocytes, CD21⁺ B lymphocytes, CD4⁺ T lymphocytes, and CD8⁺ T lymphocytes.

Figure 2

Influence of TEVs on the proliferation of activated lymphocytes. The figures show the proliferation intensity of CD3⁺ and CD3⁻CD21⁺ lymphocytes after 5 days of culture of PBMC in a 37 °C, 5% CO₂ environment with ConA and IL-2, in the presence of CLBL-1 EVs (A) and CLB70 EVs (B) in two concentrations or without TEVs (n = 7). The results are presented as the mean ± SEM.

Figure 3

Influence of TEVs on the percentage of subpopulations of activated lymphocytes. The percentage of CD3⁺, CD4⁺, CD8⁺, CD4⁺CD8⁺, and CD3⁻CD21⁺ lymphocytes after 5 days of culture of PBMC in a 37 °C, 5% CO₂ environment with ConA and IL-2 in the presence of CLBL-1 EVs (A) and CLB70 EVs (B) in two concentrations or without TEVs (n = 7). The results are presented as the mean ± SEM. The significance levels are: ** p < 0.01 and *** p < 0.001.

Figure 4

Influence of TEVs on the percentage of activated memory CD4⁺ lymphocytes. Changes in frequencies of canine CD4⁺ lymphocytes after 5 days culture of PBMCs in a 37 °C, 5% CO₂ environment with ConA and IL-2 in the presence of CLBL-1 EVs (A) and CLB70 EVs (B) in two concentrations or without TEVs (n = 7). The results are presented as the mean ± SEM. The significance level is: * p < 0.05.

Figure 5

Influence of TEVs on the percentage of activated memory CD8⁺ lymphocytes. Changes in frequencies of canine CD4⁺ and CD8⁺ lymphocytes after 5 days culture of PBMCs in a 37 °C, 5% CO₂ environment with ConA and IL-2 in the presence of CLBL-1 EVs (A) and CLB70 EVs (B) in two concentrations or without TEVs (n = 7). The results are presented as the mean ± SEM. The significance level is: * p < 0.05.

Figure 6

Influence of TEVs on the percentage of IL-17⁺ and IFN-γ⁺ CD4⁺ and CD8⁺ activated lymphocytes. Changes in frequencies of canine CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ and CD4⁺IL-17⁺ lymphocytes after 5 days of culture of PBMCs in a 37 °C, 5% CO₂ environment with ConA and IL-2 in the presence of CLBL-1 EVs (A) and CLB70 EVs (B) in two concentrations or without TEVs (n = 7). The results are presented as the mean ± SEM. The significance level is: * p < 0.05.