Combination blockade of OX40L and CD30L inhibits allergen-driven memory TH2 cell reactivity and lung inflammation

Abstract

Background: The selective reduction of memory T_H2 cell responses could be key to affording tolerance and protection from the recurrence of damaging allergic pathology.

Objective: We asked whether TNF family costimulatory molecules cooperated to promote accumulation and reactivity of effector memory CD4 T cells to inhaled complex allergen, and whether their neutralization could promote airway tolerance to subsequent reexposure to allergen.

Methods: Mice were sensitized intraperitoneally or intranasally with house dust mite and challenged with intranasal allergen after memory had developed. We assessed whether single or combined blockade of OX40L/CD252 and CD30L/CD153 inhibited memory T cells from driving acute asthmatic lung inflammation and protected mice following exposure to allergen at a later time.

Results: OX40- or CD30-deficient animals showed strong or partial protection against allergic airway inflammation; however, neutralizing either molecule alone during the secondary response to allergen had little effect on the frequency of effector memory CD4 T cells formed and acute lung inflammation. In contrast, a significant reduction in eosinophilic inflammation was observed when OX40L and CD30L were simultaneously neutralized, with dual blockade inhibiting effector memory T_H2 cell expansion in the lungs, whereas formation of peripherally induced regulatory T cells remained intact. Moreover, dual blockade during the secondary response resulted in a tolerogenic state such that mice did not develop a normal tertiary memory T_H2 cell and lung inflammatory response when challenged weeks later with allergen.

Conclusion: Memory T-cell responses to complex allergens are controlled by several TNF costimulatory interactions, and their combination targeting might represent a strategy to reduce the severity of inflammatory reactions following reexposure to allergen.

Keywords: Allergen; CD30L; OX40L; T(H)2 cell; TNF family; asthma; memory.

Fig. 1.. OX40-deficiency prevents memory Th2-driven airway inflammation.

WT or OX40−/−mice were sensitized with HDM in alum i.p., rested for three weeks, and then challenged intranasally with HDM for 4 days. A, Kinetics of mRNA for OX40L and OX40 in the lungs after allergen challenge. B, Surface expression of OX40 on CD44^hi (black line) and CD44^lo (shaded) CD4⁺ T cells in the lungs on day 5 after challenge (left panel) and MFI fold-increase (right panel). A-B, 3–6 mice/group, from 2 experiments. C-D, Numbers of BAL CD45⁺ leukocytes (C) and eosinophils (D). E-F, H&E stained lung tissue sections (E), and histological scoring of lung inflammation (F). G-J, BAL numbers of CD4⁺ T cells (G), and levels of IL-4 (H), IL-5 (I), and IL-13 (J). C, D, G, individual mice, with 10–11 mice/group from 4 experiments; F, 4–5 mice/group, from 2 experiments; and H-J, 3–8 mice/group, from 3 experiments.

Fig. 2.. Inhibition of OX40L is not sufficient to block memory Th2-driven airway inflammation.

WT mice were sensitized and challenged with HDM as in Fig. 1. 2h prior to the first and third intranasal challenge, anti-OX40L or isotype control were injected i.p. A-B, BAL numbers of CD45⁺ leukocytes (A) and eosinophils (B). C-F, BAL numbers of CD4⁺ T cells (C), and levels of IL-4 (D), IL-5 (E), and IL-13 (F). A, B, C, individual mice, 8–9 mice/group, from 3 experiments; D-F, 8 mice/group, from 3 experiments.

Fig. 3.. CD30-deficiency impairs memory Th2 cytokine production but not airway inflammation.

WT or CD30−/− mice were sensitized and challenged with HDM as in Fig. 1. A, Kinetics of mRNA for CD30L and CD30 in the lungs after challenge. B, Surface expression of CD30 on lung CD44^hi (black line) and CD44^lo (shaded) CD4⁺ T cells in WT mice on day 5 after challenge (top panel) and MFI fold-increase (bottom panel). C, Co-expression of CD30 and OX40 on lung CD44^hi (red) and CD44^lo (blue) CD4⁺ T cells. A-C, 3–6 mice/group, from 2 experiments. D-E, Numbers of BAL CD45⁺ leukocytes (D) and eosinophils (E). F-G, H&E stained lung tissue sections (F), and histological scoring of lung inflammation (G). H-K, BAL numbers of CD4⁺ T cells (H), and levels of IL-4 (I), IL-5 (J), and IL-13 (K). D, E, H, individual mice, 8–9 mice/group, from 3 experiments; G, 4–5 mice/group, from 2 experiments; I-K, 7–8 mice/group, from 3 experiments.

Fig. 4.. Memory Th2-driven airway inflammation is ameliorated by simultaneous blockade of OX40L and CD30L.

WT mice were sensitized and challenged with HDM as in Fig. 1. 2h prior to the first and third challenge, anti-OX40L, anti-CD30L, their combination, or isotype controls, were injected i.p. A-B, Numbers of BAL CD45⁺ leukocytes (A) and eosinophils (B). C-D, H&E stained lung tissue sections (C), and histological scoring of lung inflammation (D). A-B, individual mice, 10–12 mice/per group, from 4 experiments; D, 6–8 mice/group, from 3 experiments. E, AHR to methacholine. 7–8 mice/group, from 3 experiments.

Fig. 5.. Dual blockade of OX40L and CD30L inhibits accumulation of lung allergen-reactive effector memory CD4⁺ T cells.

WT mice were sensitized, challenged, and treated as in Fig. 4. A-D, BAL numbers of CD4⁺ T cells (A), and levels of IL-4 (B), IL-5 (C), and IL-13 (D). E, CD11a and CD49d expression in CD44^hi (black) and CD44^lo (grey) CD4⁺ T cells (left), and numbers of CD11a⁺CD49d⁺ CD44^hi CD4⁺ T cells (right) in the lungs. F-G, Proliferation (BrdU stain) of lung CD4+ T cells after in vitro stimulation with HDM. Representative flow plots (F) and mean % BrdU⁺CD4⁺ T cells (G). A, individual mice, 10–12 mice/group, from 4 experiments; B-D, 6–11 mice/group, from 3 experiments; E-G, 4–6 mice/group, from 2 experiments.

Fig. 6.. Co-blockade of OX40L and CD30L inhibits recall effector memory Th2 cell accumulation following airway sensitization.

WT mice were sensitized intranasally with HDM given on days 0, 1 and 2. Mice were challenged i.n. with HDM on days 14–17 and treated with antibodies to OX40L and CD30L during this time. A-B, Numbers of BAL CD45⁺ leukocytes (A) and eosinophils (B). C, H&E stained lung tissue sections from two 2 individual mice. D-G, BAL numbers of CD4⁺ T cells (D), and levels of IL-4 (E), IL-5 (F), and IL-13 (G). H, Numbers of CD11a⁺CD49d⁺ CD44^hi CD4⁺ T cells in the lungs. 5 mice/group.

Fig. 7.. Co-inhibition of OX40L and CD30L reduces expansion of antigen-specific effector memory CD4⁺ T cells and improves the Th2/Foxp3⁺ balance.

A-D, In vitro derived memory-like OT-II Th2 T cells were labelled with CTV and adoptively transferred into congenic recipients. Mice were given i.n. OVA and HDM for 4 days and treated with anti-OX40L, anti-CD30L, or their combination. A, Proliferation of donor OT-II cells determined by dilution of CTV. Rat IgG (thick grey line), anti-OX40L (dotted black line), anti-CD30L (dotted grey line) and combination (thick black line). B, Number of OT-II T cells in the lungs. C-D, Numbers of IL-5⁺ (C) and IL-13⁺ (D) OT-II cells in the lungs calculated from data in B combined with intracellular cytokine staining of CD4⁺ T cells after stimulation in vitro with OVA. B-D, 3–6 mice/group, from 2 experiments. E-F, Naïve Foxp3⁻ OT-II Foxp3-GFP CD4 T cells were transferred with memory-like OT-II Th2 cells and recipients treated with allergen and antibodies as in A-D. Frequency of Foxp3/GFP⁺ OT-II cells in the lungs (E). Th2/Foxp3⁺ T cell ratio calculated for each group (F). 3–6 mice/group, from 2 experiments.

Fig. 8.. Co-inhibition of OX40L and CD30L during secondary allergen challenge results in an inability to mount a normal tertiary recall airway inflammatory response.

WT mice were sensitized, challenged, and treated with antibodies, as in Fig. 4. Mice were then rested for 3 weeks, and subsequently challenged repetitively during a tertiary response with HDM. A-B, Numbers of BAL CD45⁺ leukocytes (A) and eosinophils (B). C-D, H&E stained lung tissue sections (C), and histological scoring of lung inflammation (D). E-H, BAL numbers of CD4⁺ T cells (E), and levels of IL-4 (F), IL-5 (G), and IL-13 (H). A, B, E, individual mice, 6–10 mice/group, from 3 experiments; D, 4–7 mice/group, from 2 experiments; F-H, 5–10 mice/group, from 3 experiments.