ICOSL, OX40L, and CD30L Control Persistence of Asthmatic CD4 Trm Cells

Tissue-resident memory T cells (Trm) are central to maintaining autoimmune and inflammatory disease. Modulation of their continued replenishment in tissues has significant implications for clinical treatment. In a murine model of lung inflammation, scRNA-seq revealed the complexity of antigen-responding CD4+ Trm, encompassing many pathogenic subpopulations including Th2, Th17, Th1 and CTL, but suggested several targets for therapeutic intervention, with significant co-expression of the costimulatory molecules OX40, ICOS, CD30, and CD30L throughout these T cells. Inhibiting ICOSL alone, or co-blocking OX40L and CD30L, with neutralizing antibodies, only partially suppressed the response of Trm to recall antigen, whereas inhibiting all three molecules strongly reduced the accumulation of Trm-derived effector memory T cells, and ablated all aspects of lung inflammation. Most importantly, transient therapeutic inhibition of these molecules together prevented the continued accumulation and long-term persistence of induced Trm, leading to a state of tolerance such that subsequent exposure to antigen failed to re-establish a pathogenic inflammatory response. These data show that costimulatory molecules are critical for reactivation and persistence of pathogenic CD4+ Trm, and reveal several therapeutic combinations that are applicable for treatment of lung inflammatory disease and potentially multiple autoimmune diseases. Overall design: Mice were sensitized by injecting a mixture of 20 µg HDM (Greer Labs, Lenoir, NC) and 2 mg alum intraperitoneally (i.p.), followed by a resting period of 10 days. Sensitized mice were then exposed to 10 µgHDM via theintranasal (i.n.) route for four consecutive days (10 to 13) in a secondary response. Challenged mice were then rested for another period of 2 weeks and re-exposed to 10 µg HDM i.n. for four consecutive days (27-30) in a tertiary response. Secondary and tertiary challenged mice were rested and subjected a fourth time to 10 µg HDM on days 44 and 45 as a quaternary challenge. To monitor lung-localized memory T cells, mice were subjected to short-term labeling with APC eF780-labelled anti-CD90.2 (2 µg in 100ul of PBS; clone 30-H12; Invitrogen) injected via the retro-orbital sinus, 10 minutes before tissue collection. Memory T cells present within tissues protected from this antibody staining are referred to as extravascular tissue-resident cells. Lungs were analyzed at varying end-points for single-cell RNA sequencing of lung extravascular memory T cells.