Article Figures & Data
Figures
- Figure 1.
CD8α:MyD88 expression enhances T-cell responses to tumor antigen. A, Pmel T cells were engineered with the empty vector control, CD8αΔIC, or CD8α:MyD88. T-cell proliferation was measured by 3H-thymidine incorporation after stimulation with gp10025-33-pulsed splenocytes for 48 hours. B, Flow cytometry of pmel T cells cocultured with splenocytes pulsed with 0.12 μg/mL gp10025-33 peptide at 72 hours. C, Proliferation of OT-I T cells cocultured with OVA (SIINFEKL)-pulsed splenocytes measured by 3H-thymidine incorporation at 48 hours. D and E, DMF5 empty vector control, DMF5 CD8α, or DMF5 CD8α:MyD88 human T cells were labeled with proliferation dye and cultured with HLA-A2+ MART-1+ Malme-3M or with HLA-A2+ MART-1− A375 melanoma pulsed or not with 10 μg/mL/106 cells MART-127-35 cells at a 1:1 ratio. T-cell proliferation was measured by flow cytometry after 5 days. F, Intracellular staining of phosphorylated proteins in transduced T cells stimulated with peptide-pulsed splenocytes and fixed at given time points. Values and error bars represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (A and C), one-way ANOVA with Tukey multiple comparison test; n = 3 experimental replicates; representative of at least two independent experiments. D and E are representative of three independent experiments.
- Figure 2.
CD8α:MyD88 T cells exhibit enhanced cytokine production and alter the expression of costimulatory and coinhibitory molecules in response to tumor antigen. A and B, IFNγ and TNFα levels measured by ELISA in supernatants from pmel T cells cocultured with gp10025-33-pulsed splenocytes (A) and OT-I T cells with OVA (SIINFEKL)-pulsed splenocytes at 24 hours (B). C, IFNγ production by engineered pmel T cells cocultured with gp10025-33-pulsed splenocytes. D, Intracellular cytokine staining of pmel T cells stimulated with splenocyte pulsed with 5 μg/mL gp10025-33. E, Cytokine production by DMF5 empty vector, DMF5 CD8α, or DMF5 CD8α:MyD88 human T cells in response to stimulation with MART-127-35–pulsed PBMCs as measured by ELISA 4 days later. Data from three independent experiments with three replicates per reaction are shown. Values, mean ± SEM. ***, P ≤ 0.001, one-way ANOVA. F, Flow cytometry screen of pmel T cells cocultured for 48 hours with splenocytes pulsed with 0.12 μg/mL gp100. The fold change of the median fluorescence intensity of CD8αΔIC and CD8α:MyD88 T cells over control vector T cells from three independent experiments is displayed in a heatmap. Gray boxes represent data not available (na). Representative histograms are shown of the expression of most upregulated and downregulated molecules. Values and error bars represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (A–C), one-way ANOVA with Tukey multiple comparison test at each concentration; n = 3 experimental replicates; representative of at least two independent experiments.
- Figure 3.
CD8α:MyD88 T cells display improved responses to melanoma in vitro and in vivo. A, Proliferation of engineered pmel T cells stimulated with irradiated B16-F1 tumor cells for 72 hours. B and C, ELISA and Luminex analysis of cytokines and chemokines in the supernatant of T cells cocultured with B16-F1 tumor cells for 48 hours. D–I, Mice bearing established B16-F1 tumors were exposed to a sublethal dose of irradiation (550 rads), followed by transfer of 3 × 106 T cells by intravenous injection one day later. Tissues were harvested and analyzed by flow cytometry one week after T-cell transfer. D, The frequency of GFP+ cells in the tumor and spleen. E, The percentage of CD8+GFP+ cells expressing exhaustion markers Tim-3 and Lag-3. F, IFNγ levels in tumor tissue measured by ELISA of tumor homogenate. G, Protein levels of CXCL9 in the tumor and serum. H, Frequency of CD8+ T cells in the tumor and spleen. I, Frequency of CD45+ cells, CD19+ cells, and NK1.1+ cells in the tumor and spleen. Values and error bars represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (A–C), one-way ANOVA with Tukey multiple comparison test at each concentration; n = 3 experimental replicates or n = 5 for D–I.
- Figure 4.
CD8α:MyD88 T cells alter the tumor microenvironment. A–G, Mice bearing an established B16-F1 tumor (∼50 mm2) were exposed to a sublethal dose of irradiation (550 rads), followed by transfer of the CD8α:MyD88 or vector control pmel T cells (3 × 106) by intravenous injection one day later. Tissues were harvested and analyzed by flow cytometry one week after T-cell transfer. Transduction efficiency of CD8α:MyD88 T cells was 50%. Each data point represented one mouse. A, Median fluorescence intensity (MFI) of MHC I on CD45− cells and MHC II and CD86 on CD11c+ DCs. B and C, Frequency of CD11c+ DCs coexpressing activation markers CD86 and MHC class II in the tumor and spleen. D, Frequency of macrophages in the tumor and spleen as defined by CD11c−CD11b+F4/80+ cells. E, Frequency of M1 (CD38+CD206−) and M2 (CD38−CD206+) macrophages in the tumor and spleen. F, Frequency of myeloid-derived suppressor cells (MDSC) in the tumor and spleen as defined by coexpression of Gr1 and CD11b. G, Luminex analysis of CCL2, IL4, and IL5 in tumor homogenate. ns, nonsignificant.
- Figure 5.
CD8α:MyD88 T cells induce tumor regression of established melanoma tumors. A and B, Mice bearing an established B16-F1 tumors were exposed to a sublethal dose of irradiation (550 rads), followed by transfer of 6 × 106 pmel T cells engineered to express CD8α:MyD88 or vector control-GFP by intravenous injection one day and 8 days later. Average tumor volume as well as tumor volumes for individual mice are shown. Values and error bars represent mean ± SEM. *, P ≤ 0.05 starting on day 28 and onward. Tumor growth was analyzed by a mixed model repeated measures with AR(1) covariance structure, followed by Sidak-adjusted comparisons at each time.
- Figure 6.
Proposed cellular processes impacted by CD8α:MyD88–expressing T cells in the tumor microenvironment. A, We observed an increased frequency of CD8α:MyD88 T cells in the tumor, indicating enhanced infiltration, expansion, and/or survival of CD8α:MyD88–transduced T cells. B, CD8α:MyD88 T cells exhibit enhanced proliferation in response to tumor antigen and tumor cells in culture, an effect that we speculate might be occurring in vivo. C, CD8α:MyD88 T cells produce increased levels of IFNγ. D, Increased levels of the IFNγ can lead to induction of the T-cell chemokine CXCL9, which were detected in the tumor and could contribute to the enhanced CD8+ T-cell numbers observed in the tumor. E and F, IFNγ has the ability to influence the antigen presentation machinery and is reflected by an enhanced expression of MHC I on nonhematopoietic (CD45−) cells, presumably tumor cells (E), and increased MHC II and CD86 expression on DCs (F). G, There were decreased levels of CCL2 in the tumor, which is a monocyte chemoattractant protein. Monocytes are the precursors of macrophages, and accordingly, an overall decreased number of macrophages was detected in the tumor of CD8α:MyD88 T cell–treated mice. H, The macrophages that were present was skewed from a protumorigenic phenotype toward a phenotype favoring antitumor immunity.
Additional Files
Supplementary Data
- Figure S1 - These data show the vector constructs verify expression of the intended protein.
- Figure S2 - These data demonstrate that CD8a:MyD88 expression increases IFN gamma production and induces the expression of various co-stimulatory and co-inhibitory molecules on T cells.
- Figure S3 - These data show changes in the levels of cytokines and chemokines induced by CD8a:MyD88 expression in T cells.
- Figure S4 - These data show changes in the levels of cytokines and chemokines in tumors induced by CD8a:MyD88-expressing T cells.
- Figure S6 - These data demonstrate that neither checkpoint blockade using anti-PD-L1 or costimulation using anti-4-1BB antibodies enhance pmel CD8a:MyD88 T cells antitumor activity
- Figure S5 - These data demonstrate that a single injection of CD8a:MyD88 T cells delay tumor progression and can prolong mouse survival.
Volume 77, Issue 24
