Figure 1.
CD8+ T cell proliferation following stimulation.
(A) CFSE-labeled STZ-diabetic, STZ-non-diabetic and control naïve CD8+ T cells from C57BL/6 mice were stimulated by anti-CD3/CD28 antibodies in vitro at the indicated time (24, 48, 72, 96 and 120 hours) and cell divisions were analyzed by flow cytometry. (B) In vivo CD8+ T cell priming model. (C) CFSE-labeled STZ-diabetic or STZ-non-diabetic 2C CD8+ T cells were co-administered with QL9-pulsed B10.A B blast cells into the spleen of healthy CD45.1 mice. Forty eight hours after priming, the splenocytes of CD45.1 mice were stained with PE anti-mouse CD45.2 antibody and cell divisions were analyzed by flow cytometry. (D) Five-day antigen-stimulated 2C CD8+ T cells from STZ-diabetic (red line) and STZ-non-diabetic (black line) mice were cultured in IL-2-containing medium for 24 hours, followed by MTT assay. Mit C, mitomycin C. APC, antigen-presenting cells. The data represent three independent experiments.
Figure 2.
Effector functions of STZ-diabetic CD8+ T cells.
(A, B) Naïve CD8+ T cells from STZ-diabetic (red line), STZ-non-diabetic (blue line) and control (gray-filled) C57BL/6 mice were stimulated by anti-CD3/CD28 antibodies in vitro. At 24 and 72 hours after stimulation, the production of perforin and TNFα was checked by intracellular staining and analyzed by flow cytometry. (C) At 120 hours of antigenic stimulation, 2C CD8+ T cells from STZ-diabetic (red line) and STZ-non-diabetic (gray-filled) mice were harvested and surface CD103 expression was checked and analyzed by flow cytometry. Staining of isotype control for the three experimental groups was shown: STZ-non-diabetic (blue dot), STZ-diabetic (red dash) and control (black line) mice. (D) CD103-positive population was gated in 5 day-stimulated 2C CD8+ T cells from STZ-non-diabetic, STZ-diabetic and control mice. (E) CD103-positive percentage in 5 day-stimulated 2C CD8+ T cells was analyzed from the three experimental groups. Data are representative of three independent experiments with three to five mice per time point.
Figure 3.
Impaired anti-tumor immunity of STZ-diabetic CTLs.
(A) Murine model of B16.gp33 melanoma with adoptive transfer of P14 CD8+ T effector cells. (B) After adoptive transfer of STZ-diabetic (n = 22) or STZ-non-diabetic (n = 21) P14 CD8+ T effector cells into B16.gp33 melanoma-bearing C57BL/6 mice, the survival time of the mice was recorded. Tumor-bearing mice with PBS but not T cells injection were considered as the controls (n = 20). (C) Tumor size of the mice that survived on day 30 after tumor inoculation. The increased fold of tumor size was calculated as tumor size at day 30 divided by that at day 7.
Figure 4.
Infiltration and cytotoxicity of STZ-diabetic CTLs in tumor.
(A, B) Assessment of the tumor-specific CTL infiltration in the tumor. Sixteen hours after adoptive transfer of STZ-diabetic or STZ-non-diabetic P14 CD8+ T effector cells into tumor-bearing CD45.1 mice, the tumors were processed for cryosections and stained by APC anti-mouse CD45.2 antibody and Hoechst 33342. (C) The production of TNFα, granzyme B and perforin by STZ-diabetic or STZ-non-diabetic P14 CTLs was monitored by immunostaining the tumors with specific antibodies and Hoechst 33342, and were visualized by a Zeiss LSM 510 confocal microscope. Scale bar: 50 µm.