Innate signals compensate for the absence of PKC-{theta} during in vivo CD8(+) T cell effector and memory responses

Abstract

PKC- is central to T-helper (Th) 2 cell differentiation and effector function; however, its importance for antiviral effector, and in particular memory CD8(+) T cell responses, remains unclear. We have investigated the role of PKC- during in vivo and in vitro responses against influenza virus, lymphocytic choriomeningitis virus, vaccinia virus, and replication-deficient virus-like particles. In the absence of PKC-, antiviral CD8(+) T cells presented an unresponsive phenotype in vitro, which could be restored with exogenous IL-2 or by Toll-like receptor ligand-activated dendritic cells. In striking contrast, PKC- appeared to be superfluous for in vivo antiviral responses irrespective of whether the virus infected systemically, was localized to the lung, or did not replicate. In addition, CD8(+) CCR7-effector memory responses were normal in PKC--deficient mice, both in lymphoid and peripheral tissues. Our data show that increased activation signals delivered in vivo by highly activated dendritic cells, as present during viral infections, overcome the requirement for PKC- during CD8(+) T cell antiviral responses.

Fig. 1.

PKC-θ is not required for in vivo immune responses against LCMV. (A) PKC-θ-deficient and C57BL/6 control mice were infected in the hind footpad with 200 pfu of LCMV (WE). At the indicated time points, the inflammatory response was monitored by using metric calipers to determine footpad swelling. Mice were infected with LCMV (WE) i.v., and on day 7 postinfection the proportion of LCMV-p33-peptide-specific CD8+ T cells circulating in the blood (B) and the total number of p33-specific cells in the spleen (C) were determined by tetramer staining and flow cytometry. (D) Splenocytes were isolated and cultured in the presence of brefeldin A for 3 h immediately ex vivo, and the frequency of IFN-γ+ CD8+ T cells was determined by flow cytometry. (E) Splenocytes were cultured in the presence of p33-peptide-pulsed ⁵¹Cr-labeled target cells directly ex vivo for 5 h, and specific cytotoxicity was determined by a conventional ⁵¹Cr-release assay. (F) On day 12 postinfection, LCMV-specific IgG2a was measured in serum by ELISA. Data are from representative experiments with three to four mice per group.

Fig. 2.

PKC-θ is required for CD4+ and CD8+ T cell LCMV-antigen recall responses in vitro. Groups of mice were infected in the hind footpad with 200 pfu of LCMV (WE). On day 17 postinfection (A), CD4+ T cells were purified by MACS bead separation and cultured together with irradiated splenocytes and LCMV for 72 h. [³H]Thymidine incorporation during the final 16 h of culture was measured as an indicator of cell proliferation. Spleens were removed, and isolated splenocytes were cultured in the presence of LCMV-p33 peptide for 5 days in the absence (B) or presence (C) of exogenous IL-2. Cytotoxicity was determined by using a conventional ⁵¹Cr-release assay measuring lysis of p33 peptide- and ⁵¹Cr-pulsed EL4 target cells. Data are from representative experiments with three to four mice per group.

Fig. 3.

PKC-θ is not required for secondary immune responses in vivo. C57BL/6 and PKC-θ-deficient mice were immunized s.c. with 150 μg of VLP-p33 or PBS in the hind-leg flank. (A) Seven days after the immunization, blood was taken, and the proportion of p33-specific CD8+ T cells was determined by tetramer staining and flow cytometry. Forty days after immunization, mice were infected i.p. with 2 × 10⁶ pfu of VV-p33, and 7 days later, the proportion of p33-specific CD8+ T cells in the ovaries was determined by tetramer staining and flow cytometry (B); CCR7 expression on p33-specific CD8+ T cells in the ovaries was determined by flow cytometry (C); expression of the indicated surfaces markers and intracellular levels of Bcl-2 were measured on p33-specific CD8+ T cells by flow cytometry (D); and lymphocytes isolated from the ovaries were restimulated with PMA and ionomycin for 4 h, and the frequency of IFN-γ+ CD8+ T cells was determined by intracellular cytokine staining and flow cytometry (E). Data are from representative experiments with three to five mice per group. Numbers indicate the percentage of cells staining positive for the indicated markers.

Fig. 4.

TLR-ligand activation of DC is necessary for the induction of CD8+ T cell immune responses in the absence of PKC-θ. C57BL/6 and PKC-θ-deficient mice were infected with LCMV, and 12 days later, CD8+ T cells were isolated from pooled splenocytes. CD11c+ cells from naïve spleens were isolated and used as antigen-presenting cells. (A) CD8+ T cell proliferation, as determined by dilution of CFSE, was determined after 3 days of stimulation with p33-pulsed resting DC (Left), p33-pulsed resting DC and IL-2 (Center), or p33-pulsed CpG-activated DC (Right). (B and C) Percentage of IL-2-(B) or IFN-γ-producing (C) cells for each CFSE peak was assessed, for the conditions described, by flow cytometry. (D) After 4 days of stimulation, cytotoxic activity was assessed by using a conventional Cr-release assay. Data are from representative experiments with pooled cells from four to six mice per group.