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Graded Defects in Cytotoxicity Determine Severity of Hemophagocytic Lymphohistiocytosis in Humans and Mice

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Graded Defects in Cytotoxicity Determine Severity of Hemophagocytic Lymphohistiocytosis in Humans and Mice

Birthe Jessen et al. Front Immunol.

Abstract

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease of hyperinflammation resulting from immune dysregulation due to inherited defects in the cytolytic machinery of natural killer and T cells. In humans, mutations in seven genes encoding proteins involved in cytolytic effector functions have so far been identified that predispose to HLH. However, although most affected patients develop HLH eventually, disease onset and severity are highly variable. Due to the genetic heterogeneity and variable time and nature of disease triggers, the immunological basis of these variations in HLH progression is incompletely understood. Several murine models of primary HLH have been established allowing to study HLH pathogenesis under more defined conditions. Here we directly compare the clinical HLH phenotype in six HLH-prone mouse strains with defects in the granule-dependent cytotoxic pathway. A severity gradient of HLH manifestations could be identified that is defined by the genetically determined residual lytic activity of cytotoxic T lymphocytes (CTL) and their ability to control lymphocytic choriomeningitis virus, which was used as a trigger for disease induction. Importantly, analysis of cohorts of HLH patients with severe bi-allelic mutations in the corresponding genes yielded a similar severity gradient in human HLH as reflected by the age at disease onset. Our findings define HLH as a threshold disease determined by subtle differences in the residual lytic activity of CTL.

Keywords: CTL; antigen persistence; cytotoxicity; hemophagocytic lymphohistiocytosis; inflammation; virus persistence.

Figures

Figure 1
Figure 1
Degree of weight loss after LCMV infection of different HLH-prone mouse models depends on the affected gene. Mice were infected with 200 pfu LCMV i.v. Percent weight loss of initial body weight is depicted over the course of 12 days. Body weight of mutant mice (beige, pearl, souris, STX-11KO, ashen/filled circles) in comparison with wild-type (open circle) and PKO (open triangle) mice are shown. The graph depicted on the lower right illustrates a direct comparison of body weight loss of all mouse groups on day 12 after LCMV infection.
Figure 2
Figure 2
Virus control following LCMV infection differs between various HLH-prone mouse models. Mice were infected with 200 pfu LCMV i.v. On day 8 and day 12 after LCMV infection, viral titers in spleen of beige, pearl, souris, STX-11KO, and ashen mice (filled circle) in comparison to wild-type (open circle) and PKO (open triangle) mice were determined.
Figure 3
Figure 3
Cytotoxic T lymphocytes degranulation and cytotoxicity are differently impaired in various HLH-prone mouse models. Mice were infected with 200 pfu LCMV i.v. On day 8 after LCMV infection, spleen cells of mutant mice (beige, pearl, souris, STX-11KO, ashen/filled circles) and wild-type (open circle) and PKO (open triangle) mice were restimulated with gp33 peptide or were left in medium without peptide. Surface expression of CD107a of IFN-γ+CD8+ T cells was determined by flow cytometry as illustrated by (A) representative FACS plots. (B) Percentage of CD107a expressing IFN-γ+CD8+ T cells after restimulation with gp33 peptide. (C) Correlation of percentage of CTL expressing IFN-γ without restimulation ex vivo with the IFN-γ levels in sera of the various HLH-prone mutant mice. (D) CTL cytotoxicity was determined in a 51Cr-release assay by using LCMV-infected MC57 target cells.
Figure 4
Figure 4
Subtle differences in CTL cytotoxicity of various HLH-prone mouse models determine virus control. On day 8 after 200 pfu of LCMV infection, splenic CD8+ T cells of mutant mice (beige, pearl, souris, STX-11KO, ashen/filled circles), wild-type (open circle), and PKO (open triangle) mice were MACS purified and 2 × 106 of MACS purified CD8+ T cells were transferred into wild-type C57BL/6 mice that had been infected with 104 pfu LCMV 10 h before. After 18 h of CD8+ T cell transfer, viral titers in spleen were determined (nil: infected wild-type C57BL/6 without cell transfer).
Figure 5
Figure 5
Delayed HLH onset in patients with SYNTAXIN-11 and LYST deficiency compared with RAB27A- and PRF1-deficient patients. (A) Cumulative incidence and (B) age at onset of HLH in FHL2 (PRF1 deficiency, gray circles; n = 72), GS2 (RAB27A deficiency, gray triangles; n = 61), FHL4 (STX11 deficiency, open squares; n = 30), and CHS (LYST deficiency, black squares; n = 21) patients carrying null bi-allelic mutations, as detailed in Table 3. The incidence of HLH was analyzed with a log-rank test; *p < 0.05 for FHL4 vs. GS2 patients; ***p < 0.001 for GS2 vs. CHS patients; ****p < 0.0001 for FHL2 vs. GS2, FHL2 vs. FHL4 and FHL2 vs. CHS; ND = no statistical differences were observed between FHL4 vs. CHS patients. The onset of HLH was analyzed with a one-way ANOVA. *p < 0.05; ***p < 0.001; ***p < 0.0001. Mutations in PRF1, RAB27A, and STX11 are detailed in Ref. (44), and mutations in CHS1/LYST are detailed in Table 3.
Figure 6
Figure 6
Impact of various genetic defects on threshold of HLH development. (A) Differences in CTL cytotoxicity and their impact on LCMV control determine whether or not HLH develops. (B) Pronounced impairment of CTL cytotoxicity results in loss of virus control and development of HLH.

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