Targeting poly(I:C) to the TLR3-independent pathway boosts effector CD8 T cell differentiation through IFN-alpha/beta

Abstract

Poly(I:C) is an adjuvant used for antitumor treatment and vaccines because of its prominent effects on CD8 T cells and NK cells. Poly(I:C) binds TLR3 and this interaction is thought to be central for driving cell-mediated immune responses. We investigated the importance of TLR3 in poly(I:C)-mediated endogenous CD8 T cell responses using the pathogenic T cell stimulant Staphylococcus aureus enterotoxin A. While the responsive CD8 T cells expanded comparably in both wild-type and TLR3(-/-) mice, differentiation of effector CD8 T cells was enhanced by poly(I:C) in the TLR3(-/-) mice. A higher percentage of Ag-specific CD8 T cells became IFN-gamma and TNF-alpha producers in the absence of TLR3 signaling. Consistent with this boosted response was the observation that TLR3-deficient cells synthesized less IL-10 compared with TLR3-sufficient cells in response to poly(I:C). Ultimately, however, the fundamental mechanism of CD8 effector T cell differentiation through the TLR3-independent pathway was shown to be completely IFN-alpha/beta-dependent. Administration of IFN-alpha/beta-neutralizing Abs abolished the poly(I:C) effects in TLR3(-/-) mice. These findings reveal specific roles of how dsRNA receptors shape CD8 T cell responses, which should be considered as poly(I:C) is authenticated as a therapeutic adjuvant used in vaccines.

Figure 1

Poly I:C enhances CD8 and CD4 T cell expansion in vivo independently of TLR3 and TRIF. Wild type, TLR3^−/− or TRIF-deficient mice were immunized with 1μg SEA with or without 40 μg poly I:C. Five days post immunization, spleens were harvested and red blood cells lysed. Frequency of antigen-specific T cells (Vβ3⁺) or non-antigen-specific T cells (Vβ6⁺) were analyzed by gating on CD8⁺ (A) or CD4⁺ population (B). Wild type (N=14), TLR3^−/− (N=5−13), TRIF-deficient (N=6). Other time points were analyzed and day 5 appeared as the peak response. Data presented as mean values + standard error of means. * P<0.05, ** P<0.01.

Figure 2

Enhanced CD8 effector differentiation in TLR3^−/− mice correlated with absence of immunosuppressive cytokines production after poly I:C treatment. (A) Wild type, TLR3^−/− or TRIF-deficient mice were immunized with 1μg SEA with 40μg poly I:C. Five days post immunization, spleens were harvested and red blood cells lysed. One million total splenocytes were restimulated with SEA in the presence of brefeldin A for 5 hours in vitro. Intracellular staining for the presence of IFNγ and TNFα was analyzed by gating on CD4⁻Vβ3⁺ T cells. Wild type (N=6), TLR3^−/− (N=13), TRIF-deficient (N=6). (B) Naïve wild type or TLR3^−/− splenocytes were cultured with increasing doses of poly I:C or CpG for 24 hours in vitro. Supernatant was collected and IL-10 or IFNγ production was measured by ELISA. N=4−6. CpG treatment data is representative of 2 experiments. (C) Naïve wild type or TLR3^−/− mice were i.p. injected with 40μg of poly I:C. 1, 2, or 4 hours later serum was collected and TNFα or IL-6 was measured by ELISA (N=3−4). Data presented as mean values +/− standard error of means. ** P<0.01.

Figure 3

CD8 T cell expansion in the absence of TLR3 signaling is mediated by type I IFNs. (A) Naïve wild type or TLR3^−/− mice were injected I.P. with 40 μg of poly I:C. One, 2, or 4 hours later mice were sacrificed and spleens were harvested. After red blood cell lysis, 3×10⁶ total splenocytes were collected for total RNA extraction, reverse transcription and RT-PCR. Data shown is representative of 4 experiments. (B) TLR3^−/− mice were I.P. injected with control or anti-IFNα/β serum before immunization with 1μg SEA and 40μg poly I:C. Neutralization of type I IFNs was validated by staining for Ly6A/E on peripheral blood CD8 T cells at 48 hours post immunization. (C) Type I IFN neutralization and immunization were carried out as in (B). On day 1−5 post immunization, mice were bled and frequency of Vβ3⁺ T cells was analyzed by gating on CD8⁺ cells in the peripheral blood lymphocyte population. N=2−5 for each data point. (D) Type I IFN neutralization and immunization were carried out as in (B). On day 5 post immunization, mice were sacrificed and frequency of Vβ3⁺ T cells was analyzed by gating on CD8⁺ cells in each tissue indicated. N=4−8 and data presented as mean values ± standard error of means. ** P<0.01.

Figure 4

Poly I:C-induced CD8 effector differentiation in TLR3^−/− mice requires type I IFNs. Type I IFN neutralization and immunization were carried out as in figure 3B. Mice were sacrificed on day 5 post immunization. (A) Total liver lymphocytes were fixed, permeabilized and stained with anti-granzyme B antibodies. Granzyme B staining was analyzed after gating on CD8⁺Vβ3⁺ cells. Numbers on the upper right corner indicate mean fluorescence intensity of granzyme B stain. (B) Five hundred thousand liver lymphocytes were restimulated with SEA for 2 hours in vitro and were immediately stained with CD107a- and CD107b-FITC antibodies. FACS plots shown were gated on CD8⁺Vβ3⁺ cells. Numbers on the upper right corner indicate mean fluorescence of CD107 staining for samples restimulated with SEA. (C) One million total splenocytes were restimulated with SEA in the presence of brefeldin A for 5 hours in vitro. Intracellular staining of IFNγ and TNFα was analyzed by gating on CD4⁻Vβ3⁺ T cells. Data are representative of 4−8 mice from 4 experiments.

Figure 5

Poly I:C-induced CD8 T cell activation is NK1.1⁺ population-independent. (A) Wild type or TLR3^−/− mice were i.p. injected with 1μg SEA with or without 40 μg poly I:C. A day later mice were sacrificed and lung lymphocytes extracted. NK cell activation is defined by upregulation of CD69 surface expression on NK1.1⁺D×5⁺ cells. Data shown are representative of 2 experiments. (B) TLR3^−/− mice were i.p. injected with control or anti-IFNα/β serum before immunization with 1μg SEA and 40 μg poly I:C. On day 1−4 post immunization, mice were bled and frequency of CD69⁺ NK cells analyzed by gating on NK1.1⁺D×5⁺ cells in the peripheral blood lymphocyte population. N=2−5 for each data point. (C) Wild type mice were injected i.p. with control Ig or PK136 antibody to deplete NK1.1⁺ population 18 hours before immunization with 1μg SEA with or without 40 μg poly I:C. On day 5 post immunization, the frequency of NK cells in the spleen were examined by staining for D×5 and CD94 markers. (D) NK1.1⁺ depletion and immunization were carried out as in (C). On day 5-post immunization the frequency of splenic Vβ3⁺ T cells was analyzed by gating on CD8⁺ cells. N=5. (E) One million total splenocytes were restimulated with SEA in the presence of brefeldin A for 5 hours in vitro. Intracellular staining of IFNγ and TNFα was analyzed by gating on CD4⁻Vβ3⁺ T cells. N=5. * P<0.05.

Figure 6

TLR3 deficiency results in decreased CD8 memory T cells. (A) Wild type or TLR3^−/−mice were i.p. immunized with 1μg SEA with or without 40 μg poly I:C or 50 μg CpG. Three months post immunization, mice were sacrificed and liver and lung lymphocytes extracted. Frequency of gated memory CD8⁺ T cells expressing CD11a^hiVβ3⁺ was analyzed. Data shown is representative of 5−9 mice (B) Frequency and numbers of memory population (CD11a^hiVβ3⁺) in lungs, gated on CD8⁺ cells. (C) Frequency and numbers of memory population (CD11a^hiCD8⁺Vβ3⁺)