Cutting Edge: DNAX Accessory Molecule 1–Deficient CD8⁺ T Cells Display Immunological Synapse Defects That Impair Antitumor Immunity

DNAM-1 is required for optimal expansion of naive CD8⁺ T cells in response to Ag presentation

Because DNAM-1 has been implicated in impaired T cell responses to infection in vivo (11), we first determined whether naive T cells deficient for DNAM-1 responded normally to Ag presentation. We used naive CD8⁺ T cells from OT-I DNAM-1 wild-type (DNAM-1^+/+) or deficient (DNAM-1^−/−) mice to assess proliferation and cytokine production in an Ag-specific response to SIINFEKL-pulsed syngeneic DCs in vitro. Although naive DNAM-1^−/− T cells displayed a wild-type phenotype (CD62L^hiCD44^medCD25⁻CD69⁻; Supplemental Fig. 1A), they showed reduced proliferation, as measured by dilution of the division tracking dye CFSE, compared with DNAM-1^+/+ T cells when stimulated with peptide-pulsed DCs (Fig. 1A). We also detected lower amounts of two key CD8⁺ T cell effector cytokines, IFN-γ and TNF-α, in the supernatant of DNAM-1^−/− T cell–DC cocultures (Fig. 1B). This was a consequence of reduced T cell numbers, because DNAM-1^−/− and DNAM-1^+/+ T cells produced equivalent levels of intracellular IFN-γ and TNF-α upon stimulation with peptide-pulsed DCs (Supplemental Fig. 1C). This is consistent with previous data demonstrating that during acute lymphocytic choriomeningitis virus priming, CD8⁺ T cells from DNAM-1^−/− mice make equivalent levels of IFN-γ compared with DNAM^+/+ T cells (11). The reduced proliferation was not a result of TCR signaling defects, because anti-CD3/CD28 Ab stimulation resulted in proliferation and cytokine levels equivalent between DNAM-1^+/+ and DNAM-1^−/− T cells (Supplemental Fig. 1D, 1E). These results indicate that expression of DNAM-1 on CD8⁺ T cells contributes to optimal T cell activation and proliferation in response to Ag presentation by DCs.

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FIGURE 1.

DNAM-1–deficient T cells show reduced proliferation, cytokine production, and cytotoxic activity, but normal polarity. (A) CFSE dilution assay of naive OT-1 DNAM-1^+/+ or DNAM-1^−/− T cells in response to DCs pulsed with 1 μM SIINFEKL peptide. FACS plots represent T cells at 72 h after incubation with DCs. (B) Naive T cells were cocultured with DCs pulsed with 1 μM SIINFEKL, and the concentration of IFN-γ and TNF-α in the supernatant at 72 h was measured by cytometric bead array. Data are presented as mean and SD of triplicate samples. (C) Activated T cells were cultured in an eight-well chamber slide overnight, then fixed and permeabilized. The cells were stained for actin, tubulin (red), LFA-1, or CD43 (green) and analyzed by confocal microscopy. The protein of interest is costained with tubulin and DAPI (blue), except for tubulin, which is costained with CD8 (green). Images shown are representative of localization of each protein over at least three independent experiments. Scale bar, 10 μM. (D) Activated T cells were tested in a chromium release killing assay with peptide-pulsed DCs as target cells. Data are presented as mean and SD of triplicate samples. All data in (A)–(C) are representative of at least three independent experiments. *p < 0.05.

DNAM-1–deficient CD8⁺ effector T cells demonstrate normal phenotype and morphology in vitro but reduced cytotoxic activity

Despite a reduction in proliferation and differentiation kinetics, a proportion of DNAM-1^−/− T cells were activated and proliferated in vitro. To determine whether this change in activation kinetics affected the phenotype or polarity of responding T cells, we first used flow cytometry to assess the cell-surface profile of the activated T cells. Activated DNAM-1^−/− T cells upregulated CD44, CD69, and CD25, and downregulated CD62L to equivalent levels as DNAM-1^+/+ T cells (Supplemental Fig. 1B). The activated DNAM-1^−/− T cells also displayed no detectable defects in polarization of actin and LFA-1 to the leading edge, CD43 to the uropod, and localization of the MTOC to the base of the uropod, as assessed by confocal microscopy (Fig. 1C). However, activated DNAM-1^−/− T cells did show impaired cytotoxic activity against peptide-pulsed DCs (Fig. 1D). Both DNAM-1^−/− and DNAM^+/+ T cells expressed equivalent levels of the cytotoxic molecule, Granzyme B, and showed no difference in degranulation after TCR stimulation (Supplemental Fig. 2A, 2B). Together, these data suggest that the DNAM-1 is critical for the cell–cell contact required for efficient killing of target cells by CD8⁺ T cells.

DNAM-1 is required for optimal conjugation and killing of tumor cells by CD8⁺ effector T cells

To explore potential reasons for the defect in killing shown earlier, we next determined whether activated DNAM-1^−/− T cells display normal cytotoxic activity against tumor cells that express the ligands for DNAM-1 (CD155 and CD112) (5). Mice deficient for DNAM-1 have impaired antitumor responses using models dependent on both NK and CD8⁺ T cell activity (5, 20). Furthermore, the association of DNAM-1 with LFA-1 and proteins that mediate cytoskeletal changes in T cells such as Dlg and 4.1G (6, 21) suggests a role for this accessory molecule in cell–cell interactions. We therefore assessed the ability of the DNAM-1^−/− T cells to form conjugates with tumor cells in vitro using a flow cytometry–based conjugation assay. We found that activated DNAM-1^−/− effector T cells formed significantly less conjugates than DNAM-1^+/+ T cells (Fig. 2A, quantitated on right), confirming that the absence of DNAM-1 impairs the stable interaction between T cells and tumor cells. Furthermore, consistent with our previous data showing that DNAM-1^−/− mice do not clear MC38-OVA tumors in vivo (22), the DNAM-1^−/− T cells showed reduced cytotoxic activity against MC38-OVA tumor cells in vitro, as determined by a chromium release assay (Fig. 2B). These results demonstrate that DNAM-1 expression on CD8⁺ T cells is critical for efficient conjugation and killing of Ag-presenting tumor cells.

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FIGURE 2.

Activated DNAM-1–deficient T cells show reduced conjugation, cytotoxic activity, and synapse formation with tumor cells. (A) Activated OT-I DNAM-1^+/+ or DNAM-1^−/− T cells were labeled with Cell Trace and cocultured with CFSE-labeled SIINFEKL-pulsed EL4 tumor cells and analyzed by flow cytometry after 60 min. The graph is of percentage of total T cells conjugated to tumor cells at 60 min and is data pooled from three independent experiments. Data are presented as mean and SD. (B) Activated T cells were tested in a chromium release killing assay with MC38-OVA or empty vector (EV) tumor cells as target cells. Data are representative of at least four independent experiments with mean and SD of triplicate samples from one representative experiment shown. (C) Activated T cells were cocultured with adhered MC38-OVA tumor cells in an eight-well chamber slide. After 1–2 h, the conjugates were fixed, permeabilized, and stained for tubulin (MTOC), actin, LFA-1, and GM1 (lipid rafts). The protein of interest is shown in green, and the merge includes DAPI/nucleus (blue) and tubulin (red), except for tubulin/MTOC, which is costained with actin (red) (D) Quantification of polarization of proteins at the synapse (LFA-1, n = 68; GM1, n = 48; actin, n = 61). (C and D) Images and quantification are pooled from three independent experiments. Scale bar, 10 μM. *p < 0.05.

DNAM-1 deficiency results in impaired immunological synapse formation between CD8⁺ effector T cells and tumor cells

To determine whether reduced cytotoxic activity of the DNAM-1^−/− T cells against MC38-OVA tumor cells was a result of impaired cytotoxic synapse formation between the T cells and tumor cells, we used immunofluorescent microscopy to dissect the polarization of synapse components during T cell–tumor cell interactions. Despite fewer conjugates being formed, in the DNAM-1^−/− T cells that attached to a tumor cell, we found the MTOC was recruited to the synapse, along with enrichment of actin at the interface of the two cells. However, we observed significantly impaired polarization of LFA-1 and lipid rafts (as determined by Ganglioside, GM-1, localization) to the interface between the OT-1 DNAM-1^−/− T cells and tumor cells (Fig. 2C and quantified in Fig. 2D).

Formation of a “suboptimal” cytotoxic synapse, and subsequent inability to secrete effector molecules in a directed manner, may therefore account for the reduced cytotoxic activity of DNAM-1^−/− T cells against tumor cells in vivo. Strong interactions between T cells and target cells is dependent on cell-surface molecules such as TCR and LFA-1, but is also controlled by cytoskeletal dynamics and the integrity of lipid rafts (23). The reduced polarization of both LFA-1 and lipid rafts containing costimulatory molecules such as DNAM-1 (9, 24) would therefore impact on conjugation efficiency and in the activation and cytotoxic activity of the T cells. Indeed, DNAM-1 is important for the LFA-1 costimulatory signal necessary for naive CD4⁺ T cell differentiation and proliferation (8). LFA-1–deficient mice show normal cytotoxic T cell responses to virus but fail to reject immunogenic tumors (25), suggesting that LFA-1 may also play a significant role in the interaction of CD8⁺ T cells with tumor cells.

Together, our results suggest that DNAM-1 deficiency affects T cell function in a 2-fold manner: 1) reduced activation of responding effector T cells, and 2) reduced ability of activated T cells to form an IS with target cells and deliver the cytotoxic payload necessary for elimination of target cells. Furthermore, our results demonstrate that these defects are manifested through suboptimal formation of a cytotoxic synapse involving reduced polarization of LFA-1 and lipid rafts. These results highlight the importance of DNAM-1 in the control of optimal synapse formation required for effective antitumor T cell responses.