Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes

Abstract

Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3^-CD21^-) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3⁺CD5^dimCD21^- lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3⁺CD5^dimCD21^- (cytotoxic large granular lymphocytes) and CD3^-CD5^-CD21^- NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3⁺CD5^dimCD21^- lymphocytes can be differentiated into non-B, non-T NK (CD3^-CD5^-CD21^-TCRαβ^-TCRγδ^-GranzymeB⁺) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3⁺CD5^dimCD21^- cells showed that CD3^-CD5^-CD21^- cells are derived from CD3⁺CD5^dimCD21^- cells through phenotypic modulation. CD3⁺CD5^dimCD21^- cells share more NK cell functional characteristics compared with CD3^-CD5^-CD21^- cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3⁺CD5^dimCD21^- and CD3^-CD5^-CD21^- cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.

Keywords: canine; cytotoxic large granular lymphocytes; natural killer cells; non-B non-T lymphocytes; phenotypic modulation.

Figure 1

Analysis of lymphocyte subpopulations during ex vivo expansion in the presence of 100-Gy-irradiated K562 cells, interleukin (IL)-2, IL-15, and IL-21. (A) Representative flow cytometry data (n = 8), and (B) lymphocyte subpopulation dynamics showing changes in the frequency of CD3⁻CD5⁻CD21⁻, CD3⁺CD5^dimCD21⁻, and CD3⁺CD5^brightCD21⁻ cell populations during proliferation in culture of peripheral blood mononuclear cells (PBMCs) for 21 days. The results are shown as the mean ± SD measured from eight different donors.

Figure 2

Intracellular expression of Ki-67 and Granzyme B in CD3⁻CD5⁻CD21⁻, CD3⁺CD5^dimCD21⁻, and CD3⁺CD5^brightCD21⁻ cell compartments, and analysis of cell death frequency during PBMC culture for 21 days. (A) Flow cytometric analysis of Ki-67 expression (n = 9). (B) Changes in the frequency of each cell population within Ki-67-expressing cells during culture for 21 days (n = 9). (C) Changes in the frequency of Granzyme B intracellular expression in CD3⁻CD5⁻CD21⁻, CD3⁺CD5^dimCD21⁻, and CD3⁺CD5^brightCD21⁻ cell populations during PBMC culture for 21 days. Expression level of Granzyme B represents relative mean fluorescence intensity (rMFI) (n = 9). (D) Representative flow cytometry data of the frequency of Granzyme B intracellular expression in each cell population during culture for 21 days (n = 9). (E) The frequency of cell death during culture for 21 days. Whole cells were stained with annexin V/propidium iodide (PI) on days 7, 10, 14, and 21 of culture. The population of cells that were negative for both annexin V and PI was defined as living cells. Annexin V⁺/PI⁻ cells were classified as early apoptotic cells, and double-positive cells were classified as late apoptotic cells. Annexin V⁻/PI⁺ cells were classified as dead cells (n = 5). (F) Representative flow cytometry data of annexin V/PI staining of cells during culture to evaluate the frequency of cell death frequency among the total cell population (n = 5).

Figure 3

Phenotypic changes of purified CD3⁺CD5^dimCD21⁻ cells during culture for 21 days. (A) CD3⁺CD5^dimCD21⁻ cells were purified from freshly isolated PBMCs using a cell sorter. The purity of CD3⁺CD5^dimCD21⁻ cells after sorting was more than 98% (n = 5). (B) Phenotypic analysis of the purified CD3⁺CD5^dimCD21⁻ cells during culture for 21 days. CD3⁻CD5⁻CD21⁻ cells were proliferated from purified CD3⁺CD5^dimCD21⁻ cells, and the majority of expanded cells in culture were CD3⁻CD5⁻CD21⁻ after 21 days. The phenotype of most of these expanded CD3⁻CD5⁻CD21⁻ cells was CD4⁻ CD8^+/− after 21 days of stimulation. The results shown are representative results from one of five different donors. (C) The rate of proliferation was calculated by counting the total number of viable cells in culture. Values represent the mean ± SD (n = 5).

Figure 4

Expression of NK-associated receptor and function of purified CD3⁺CD5^dimCD21⁻ and CD3⁻CD5⁻CD21⁻ lymphocytes. (A) mRNA expression of NK cell-related molecules including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, and Granzyme B in canine PBMCs and purified CD3⁺CD5^dimCD21⁻ (CD5^dim), CD3⁺CD5^brightCD21⁻ (CD5^bright), and CD3⁻CD5⁻CD21⁻ (CD5⁻) lymphocytes 2 (2w) or 3 weeks (3w) after expansion (n = 11). Levels were determined by real-time reverse transcription-polymerase chain reaction. The mRNA levels were expressed relative to β-actin. (B) Production of interferon (IFN)-γ in purified CD3⁺CD5^dimCD21⁻ and CD3⁻CD5⁻CD21⁻ lymphocytes (n = 10). IFN-γ production was assessed in the culture supernatants by enzyme-linked immunosorbent assay. Control conditions consisted of the culture supernatant from canine thyroid adenocarcinoma (CTAC) cells, CD3⁺CD5^dimCD21⁻ (CD5^dim) or CD3⁻CD5⁻CD21⁻ cells (CD5⁻) cultured alone for 24-h without cytokines. IFN-γ production levels in each subset of lymphocytes are shown as the mean ± SD. *p < 0.01, **p < 0.005, and ***p < 0.001. (C) The natural cytotoxicity against CTAC cells of purified CD3⁺CD5^dimCD21⁻ (CD5^dim) and CD3⁻CD5⁻CD21⁻ (CD5⁻) cells 2 weeks after culture. The 4-h cytotoxicity was measured at 10:1 effector to target ratio. The results are shown as mean ± SD percentages of cytotoxicity (n = 5, each tested in duplicate).

Figure 5

Expression of the T-box transcription factors, T-box expressed in T cells (T-bet) and Eomesosermin (Eomes) in each lymphocyte subset in relation to the expression of CD3, CD5, and CD21 before and after culture of PBMCs. (A) Expression of T-bet and Eomes in CD3⁺CD5^dimCD21⁻ (red), CD3⁺CD5^brightCD21⁻ (blue), and CD3⁻CD5⁻CD21⁻ (green) lymphocytes before culture. The results shown represent those obtained from one of nine different donors. (B) The percentage of cells expressing T-bet, Eomes, and both factors in CD3⁺CD5^dimCD21⁻ (CD5^dim), CD3⁺CD5^brightCD21⁻ (CD5^bright), and CD3⁻CD5⁻CD21⁻ (CD5⁻) lymphocytes before culture (n = 9). (C) Expression of T-bet and Eomes in CD3⁺CD5^dimCD21⁻ (CD5^dim, red), CD3⁺CD5^brightCD21⁻ (CD5^bright, blue), and CD3⁻CD5⁻CD21⁻ (CD5⁻, green) lymphocytes 2 weeks (2w) after culture, and in CD3⁻CD5⁻CD21⁻ cells 3 weeks (3w) after culture. The results shown represent those obtained from one of nine different donors. (D) The percentage of cells expressing T-bet, Eomes, and both factors in CD3⁺CD5^dimCD21⁻ (CD5^dim), CD3⁺CD5^brightCD21⁻ (CD5^bright), and CD3⁻CD5⁻CD21⁻ (CD5⁻) lymphocytes 2 (2w) or 3 weeks (3w) after culture (n = 9) (*p < 0.01, **p < 0.005, and ***p < 0.001).

Figure 6

Comparison of the proliferative capacity in CD3⁻CD5⁻CD21⁻ non-B, non-T (CD5⁻), CD3⁺CD5^dimCD21⁻ (CD5^dim), and CD3⁺CD5^brightCD21⁻ (CD5^bright) lymphocytes. PBMCs were stained with Violet Cell Trace Dye before culture. To evaluate the proliferation of the different lymphocyte subsets, cells were stimulated with IL-15; IL-2; and IL-15; canine NK cell-sensitive canine thyroid adenocarcinoma (CTAC) cells; CTAC cells and IL-15; CTAC cells, IL-2, and IL-15; or concanavalin A for 7 days. Cells cultured in medium alone served as a negative control. Results are representative of data from five different donors. The percentage of proliferating cells within the respective subsets is indicated.