B and T lymphocyte attenuator tempers early infection immunity

Abstract

Coinhibitory pathways are thought to act in later stages of an adaptive immune response, but whether coinhibition contributes to early innate immunity is unclear. We show that engagement of the newly discovered coinhibitory receptor B and T lymphocyte attenuator (BTLA) by herpesvirus entry mediator (HVEM) is critical for negatively regulating early host immunity against intracellular bacteria. Both HVEM(-/-) and BTLA(-/-), but not LIGHT(-/-), mice are more resistant to listeriosis compared with wild-type mice, and blockade of the BTLA pathway promotes, while engagement inhibits, early bacterial clearance. Differences in bacterial clearance were seen as early as 1 day postinfection, implicating the initial innate response. Therefore, innate cell function in BTLA(-/-) mice was studied. We show that innate cells from BTLA(-/-) mice secrete significantly more proinflammatory cytokines upon stimulation with heat-killed Listeria. These results provide the first evidence that a coinhibitory pathway plays a critical role in regulating early host innate immunity against infection.

FIGURE 1

HVEM-BTLA interactions play an essential role in regulating early LM clearance. Age and sex matched WT (Open bar) and knockout mice (Closed bar) including HVEM^-/- (A), LIGHT^-/- (B) and BTLA^-/- (C) mice were infected with 5×10⁵ CFU rLM-OVA by i.p. injection. (D) WT mice were infected with 5×10⁵ CFU rLM-OVA and treated with 100 mg 6A6 (closed bar) or hamster IgG (hIgG, open bar) as control. Four days later, the bacterial burden in the spleen and liver was tested. Bar graphs depict mean±SEM of bacterial loads from six to nine mice per group.

FIGURE 2

Immobilized HVEM-Ig inhibits T cell proliferation in a BTLA-dependent but LIGHT-independent manner. LN cells isolated from WT (A), LIGHT^-/- (B) or BTLA^-/- mice (C) were stimulated with immobilized anti-CD3 (2 μg/ml) in the absence or presence of various doses of soluble mIgG (s-mIgG, closed triangle), soluble HVEM-Ig (S-HVEM-Ig, open triangle), plate bound mIgG (C-mIgG, closed circle) or plate bound HVEM-Ig (c-HVEM-Ig, open circle) for two days (A), or in the presence of plate bound mIgG (solid circle) or HVEM-Ig (open circle) for two days (B & C), then pulsed with [³H]thymidine for 18 hours. Data are expressed as the mean [³H]thymidine incorporation of triplicate cultures (+SD). The results are representative of three independent experiments.

FIGURE 3

HVEM-Ig treatment enhances host susceptibility to listeria infection in a LIGHT-independent manner. WT (A & B) and LIGHT^-/- (C) mice were infected with 5×10⁵ CFU rLM-OVA and treated with 200 to 300 μg of HVEM-Ig (closed bars) or mIgG (open bars). Four days later, bacterial burdens of spleens and livers (A and C, respectively) were tested. Survival was observed in a separate experiment (B). Bar graphs depict mean±SEM of bacterial load from six to twelve mice per group.

FIGURE 4

Kinetics of LM infection in WT and BTLA^-/- mice, and WT mice treated with anti-BTLA and HVEM-Ig. (A) WT (open circles) and BTLA^-/- mice (closed circles), (B) WT mice treated with 100 μg of 6A6 (closed circles) or hIgG (open circles), and (C) WT mice treated with 200 μg of HVEM-Ig (closed circles) or mIgG (open circles), were infected with 5×10⁵ CFU rLM-OVA. On days 1, 3, and 5, the bacterial burden of the spleen and liver was tested. Bar graphs depict mean±SEM of bacterial load from four to five mice per group.

FIGURE 5

Increased innate response to LM in the absence of BTLA. (A) WT and BTLA^-/- splenocytes were incubated in triplicate wells with HKLM for 5 minutes (for TNF-α and IL-6 determination) or 15 minutes (for IFN-γ determination), washed, and cultured in vitro for 24 hours before supernatant cytokine levels were determined. (B) WT and BTLA^-/- mice were injected i.v. with 1×10⁹ HKLM and serum cytokine levels were determined at indicated times; five to ten mice per group. (C) WT and BTLA^-/- mice were injected i.v. with 1.5×10⁹ HKLM and survival was monitored; five mice per group. Bar graphs depict mean±SEM; ND, not detected.