Natural killer cells promote early CD8 T cell responses against cytomegalovirus

Abstract

Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-alpha/beta production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-alpha administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity.

Figure 1. Decreased pDC Cytokine Production in the Presence of Ly49H/m157-Mediated NK Cell Activation

(A) Serum from Klra8 and BALB/c mice was collected on days 0, 1.5, 2, and 3 post–MCMV infection. Cytokine levels of IFN-α and IL-12p70 were determined by ELISA. Results are expressed as mean ± SD of three mice per group. (B) Splenic leukocytes were isolated from Klra8 and BALB/c mice and analyzed for the frequency of IFN-α/β⁺ pDCs and IL-12⁺ pDCs directly ex vivo. Numbers in dot plots represent the percent of IFN-α/β⁺ and percent of IL-12⁺ cells within the total pDC population. One representative animal from groups of three mice for day 1.5 post–MCMV infection is shown. Graphs represent the total numbers of IFN-α/β⁺ pDCs and IL-12⁺ pDCs present in the spleens of Klra8 and BALB/c mice on days 0, 1.5, 2, and 3 post–MCMV infection. Results are expressed as mean ± SD of three mice per group and in ten thousands of cells. One experiment representative of three for (A, B) is shown. *p ≤ 0.05; **p ≤ 0.01; § = not detected.

Figure 2. Antiviral NK Cell Responses Prevent Ablation of the cDC during MCMV Infection

Splenic leukocytes were isolated from Klra8 and BALB/c mice and analyzed for the frequency of pDCs (120G8⁺ CD11c^int), CD11b cDCs (120G8⁻ CD11c^hi CD8α⁻), and CD8α cDCs (120G8⁻ CD11c^hi CD8α⁺) within the DX5⁻ and TCRβ⁻ population. Numbers in dot plots represent percent pDCs, percent CD11b cDCs, and percent CD8α cDCs within the total splenocyte population for one representative animal from groups of three mice for days 0 and 2 post–MCMV infection. Graphs represent the total numbers (in millions) of pDCs, CD11b cDCs, and CD8α cDCs present in the spleens of Klra8 and BALB/c mice on days 0, 1.5, 2, and 3 post–MCMV infection. Results are expressed as mean ± SD of three mice per group. One experiment representative of three is shown. *p ≤ 0.05; **p ≤ 0.01.

Figure 3. Kinetics of Antigen-Specific CD8 T Cells Expansion in Klra8 and BALB/c Mice in Response to MCMV Infection

Splenic leukocytes were isolated from Klra8 and BALB/c mice on days 0, 4, 5, and 7 post–MCMV infection and analyzed for (A) total numbers of CD8 T cells (TCRβ⁺ CD8α⁺), (B) CD43 expression on the total CD8 T cell population, and (C) binding of IE-1-loaded MHC class I tetramers on the total CD8 T cell population. CD43 expression on CD8 T cells and IE-1-loaded MHC class I tetramer binding to CD8 T cells is shown for one representative animal from groups of three mice for days 4, 5, and 7 post–MCMV infection. Numbers in dot plots represent percent CD43^hi or percent IE-1 tetramer⁺ CD8 T cells within the total CD8 T cell population. Graphs represent the total numbers (in millions) of CD8 T cells, CD43^hi CD8 T cells, or IE-1 tetramer⁺ CD8 T cells. Results are expressed as mean ± SD of three mice per group. One experiment representative of four for (A–C) is shown. **p ≤ 0.01.

Figure 4. Accelerated Acquisition of CD8 T Cell Effector Functions in Klra8 Mice in Response to MCMV Infection

Splenic leukocytes were isolated from Klra8 and BALB/c mice on days 0, 4, 5, and 7 post–MCMV infection. (A) The CD8 T cell population was analyzed directly ex vivo for intracellular expression of Ki-67. Intracellular expression of Ki-67 is shown for one representative animal from groups of three mice for days 4, 5, and 7 post–MCMV infection. Numbers in dot plots represent percent Ki-67⁺ CD8 T cells within the total CD8 T cell population. Graphs represent the total numbers (in millions) of Ki-67⁺ CD8 T cells. (B) The CD8 T cell populations were analyzed for their ability to produce IFN-γ after antigen-specific re-stimulation with IE-1 peptide-pulsed P815.B7 cells. Intracellular expression of IFN-γ is shown versus surface CD43 expression for the total CD8 T cell population for one representative animal from groups of three mice. Numbers in dot plots represent percent IFN-γ⁺ CD8 T cells within the total CD8 T cell population. Graphs represent the total numbers (in millions) of IFN-γ⁺ CD8 T cells. (C) The CD8 T cell populations were analyzed for their ability to kill IE-1 and m164 peptide-pulsed target cells in vivo. Target cells were transferred into mice at the time points indicated and the spleens harvested 4 h later. Histograms are gated on PKH26⁺ target cells. Numbers represent the percentage of target cells killed for one representative animal from groups of three mice. Graphs represent the percentage of target cells killed. Results in graphs are expressed as mean ± SD of three mice per group for (A–C). One experiment representative of three for (A, B), and two for (C) are shown. **p ≤0.01.

Figure 5. Kinetics of Antigen-Specific CD8 T Cell Expansion in Response to MCMV Infection in Mice Lacking NK Cells or Ly49H-Mediated NK Cell Activation

Splenic leukocytes were isolated at the time points indicated from (A) Klra8 mice and Klra8 mice depleted of NK cells infected with MCMV, (B) Klra8 and BALB/c mice infected with in vitro–generated BAC-derived wild-type (WT), revertant (REV), or Δm157 viruses, and (C) B10.D2 and B10.D2-DAP12° mice infected with MCMV. Total CD8 T cell numbers, CD43 expression on CD8 T cells, and IE-1-loaded MHC class I tetramer binding to CD8 T cells were analyzed for (A–C). Graphs represent total numbers (in millions) of cells for each parameter as indicated. Results are expressed as mean ± SD of two (A) or three (B, C) mice per group. One experiment representative of two for (A–C) is shown. **p ≤ 0.01.

Figure 6. IFN-α-Driven Loss of cDCs and Delay in CD8 T Cell Activation

(A) Splenic leukocytes were isolated on days 0 and 3 post–MCMV infection from Klra8, Klra8 treated with IFN-α at 30 and 48 h post-infection, and BALB/c mice. The total numbers (in millions) of pDCs, CD11b cDCs, and CD8α cDCs were determined and are presented as in Figure 2. (B) Splenic leukocytes were isolated on days 0 and 4 post–MCMV infection from Klra8, Klra8 treated with IFN-α at 30 and 48 h post-infection, and BALB/c mice. Total numbers (in millions) of CD8 T cells, CD43^hi CD8 T cells, and IE-1 tetramer⁺ CD8 T cells were determined and are presented as in Figure 3. One experiment representative of two for (A, B) is shown. *p ≤ 0.05; **p ≤ 0.01.

Figure 7. Control of Early Viral Load Results in Decreased pDC Cytokine Production, the Maintenance of cDCs, and the Accelerated Expansion of Antigen-Specific CD8 T Cells

(A) Spleens from Klra8, BALB/c, and BALB/c mice treated with doxycycline 20 h post-infection were harvested on days 0, 1.5, 2, and 3 post–DN-SCP-MCMV infection and homogenized. Viral titers were determined and are presented as in Figure S1B. (B) Serum from Klra8, BALB/c, and BALB/c mice treated with doxycycline 20 h post-infection was collected on days 0, 1.5, 2, and 3 post–DN-SCP-MCMV infection. Cytokine levels of IFN-α and IL-12p70 were determined and are presented as in Figure 1. (C) Splenic leukocytes were isolated from on days 0, 1.5, 2, and 3 post–DN-SCP-MCMV infection from Klra8, BALB/c, and BALB/c mice treated with doxycycline 20 h post-infection. The total numbers (in millions) of pDCs, CD11b cDCs, and CD8α cDCs were determined and are presented as in Figure 2. (D) Splenic leukocytes were isolated on days 0, 4, and 5 post–DN-SCP-MCMV infection from Klra8, BALB/c, and BALB/c mice treated with doxycycline 20 h post-infection. Total numbers (in millions) of CD8 T cells, CD43^hi CD8 T cells, and IE-1 tetramer⁺ CD8 T cells were determined and are presented as in Figure 3. One experiment representative of two for (A–C), and four for (D) are shown. *p ≤ 0.05; **p ≤ 0.01; § = not detected.