doi: 10.1126/science.1159806.
Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin
Affiliations
- PMID: 18635804
- PMCID: PMC2807624
- DOI: 10.1126/science.1159806
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Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin
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Abstract
When intracellular pathogens invade mammalian hosts, naïve CD8+ T cells differentiate into cytotoxic killers, which lyse infected target cells and secrete cytokines that activate intracellular microbicides. We show that CD8+ T cells deficient in the transcription factors T-bet and eomesodermin (Eomes) fail to differentiate into functional killers required for defense against lymphocytic choriomeningitis virus. Instead, virus-specific CD8+ T cells lacking both T-bet and Eomes differentiate into an interleukin-17-secreting lineage, reminiscent of the helper T cell fate that has been implicated in autoimmunity and extracellular microbial defense. Upon viral infection, mice with T cells lacking both T-bet and Eomes develop a CD8+ T cell-dependent, progressive inflammatory and wasting syndrome characterized by multi-organ infiltration of neutrophils. T-bet and Eomes, thus, ensure that CD8+ T cells adopt an appropriate course of intracellular rather than extracellular destruction.
Figures
Mice with T cells lacking both T-bet and Eomes fail to control LCMV infection. (A) Quantification of LCMV virus in spleen and kidney from mice of the indicated genotype 23d after infection. Detection threshold of limiting dilution assay is approximately 2×102 PFU/gram; * indicates undetectable. Values represent mean ±SEM of at least 3 mice per genotype. (B, C) Defective IFN-γ expression and cytolysis of target cells by CD8+ T cells lacking T-bet and Eomes. Splenocytes from mice of the indicated genotypes were isolated 8d after infection with LCMV. (B) Flow cytometry of IFN-γ production by CD8+ T cells in response to stimulation with LCMV-derived peptide GP33–41. % of live cells expressing IFN-γ is indicated. (C) Cytolytic activity of virus-specific CD8+ T cells. Equalized numbers of GP33- or NP396-specific CD8+ T cells were added to target cells pulsed with LCMV-derived peptides GP33–41 or NP396–404. % specific lysis was determined after 24h (12). Virus-specific CD8+ T cells from Double KO mice also exhibited impaired accumulation and abnormal expression of numerous surface receptors, reflecting both persistent antigen exposure and abnormal differentiation (12). Results are representative of at least 3 independent experiments.
Virus-induced wasting disease in mice with T cells lacking both T-bet and Eomes. (A) Progressive weight loss of Double KO mice after LCMV infection. Percent of weight before infection is depicted graphically over time. Values represent mean ±SEM of at least 3 mice per genotype. (B) Virus-induced neutrophilia in Double KO mice. Values represent % neutrophils in spleen 23d after infection with LCMV. (C) Virus-induced pathology in multiple organs (tissue, magnification) of Double KO mice. H&E staining of Brain (meninges), 60x; Lung (airway with associated vessel), 10x; Heart (base), 5x; Liver (portal vessel), 5x; Thymus (C = cortex, M = medulla), 5x. Spleen (F = follicle, R = red pulp), 5x; Kidney (hilar vessel near calyx), 5x; Pancreas, 5x. Arrowheads indicate neutrophils; arrows indicate immune cell infiltration. All organs from d28 post-infection, except for kidney and pancreas (d23). (D) High power magnification (100x) demonstrating neutrophilic infiltration of Liver, Spleen, and Pancreas, and mixed monocytic/neutrophilic infiltration of Lung. Results are representative of 2 independent experiments with >6 mice per group.
Type 17 effector differentiation of CD8+ T cells lacking both T-bet and Eomes. (A) IFN-γ and IL-17 production by LCMV-specific CD8+ T cells from Double KO mice. Splenocytes from mice of the indicated genotypes were isolated 8d after LCMV infection and given no stimulation (top row) or stimulated with LCMV-derived peptides GP33–41 (middle row) or NP396–404 (bottom row); cytokine production was assessed by intracellular staining; plots show CD8+ events; numbers indicate % of CD8+ T cells expressing the indicated cytokine. Expression of IL-17 by Double KO CD8+ T cells in the absence of re-stimulation (upper right plot) could be due to persistent virus in vivo (Fig. 1A) and/or constitutive expression of this cytokine (12). In addition to the abnormal CD8+ T cell response, CD4+ T cells from both T-bet KO and Double KO expressed IL-17 in response to stimulation with LCMV-derived peptide GP61–80 (12). Additional controls were performed to implicate the CD8+ T cell-intrinsic loss of T-box factors as the cause of anomalous differentiation: Analysis of IFN-γ- and perforin-deficient mice suggested that IL-17 production by CD8+ T cells from Double KO mice was not solely attributable to impaired cytotoxic effector differentiation (12). In separate experiments, we determined that IL-17 expression by CD8+ T cells was not contingent on T-bet deficiency in CD4+ T cells or cells of the innate immune system (12). (B, C) Lack of IL-4, IL-10, or Foxp3 expression by CD8+ T cells from Double KO mice. Splenocytes from indicated genotypes were isolated 8d after LCMV infection. Cells were either stimulated with anti-CD3 (B) or examined directly (C). Cytokine production (B) or Foxp3 expression (C) were assessed by intracellular staining. Numbers indicate % of CD8+ T cells expressing the indicated cytokine or lineage marker. Despite their defective cytotoxocity and IFN-γ expression, virus-specific CD8+ T cells from Double KO mice were capable of producing TNF-α and IL-2 (12). (D) Type 17 transcriptional profile of LCMV-specific CD8+ T cells from Double KO mice. GP33-specific CD8+ T cells were isolated using peptide-MHC tetramers from indicated genotypes 8d after LCMV infection. mRNA expression was assessed by quantitative real-time PCR. * indicates undetectable signal. Lowest detectable sample was arbitrarily set at a value of 1, except for IL-22, where only Double KO had detectable signal. Values represent mean ±SEM of triplicate determinations normalized to HPRT. Results are representative of at least 2 independent experiments.
CD8+ T cell depletion prevents virus-induced wasting disease in Double KO mice. (A) Double KO mice were treated with PBS (“Control Tx”), anti-CD4 (“CD4 deplete”, mAb GK1.5, 0.5 mg i.p., left), or anti-CD8 (“CD8 deplete”, mAb 2.43, 0.5 mg i.p., right) 1d before, 1d after, and every 3d following LCMV infection. Weight at indicated day post-infection is displayed as a percentage of weight before infection. (B) Neutrophils were quantified in the blood of control or anti-CD8 treated mice by automated complete blood count with differential (right). Number of neutrophils per microliter of blood is depicted graphically. (C) H&E staining of organs from control-treated or CD8+ T cell-depleted Double KO mice at d18 post-infection. Lung (airway with associated vessel), 10x; Heart (base), 5x; Liver (portal vessel), 5x; Spleen (F = follicle, R = red pulp), 5x; Kidney (hilar vessel near calyx), 5x; Pancreas, 5x. Arrows indicate immune cell infiltration. Values for (A) and (B) represent mean ±SEM of 3 individual mice per treatment group. Results are representative of 2 independent experiments.
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