D1.223 - TNFR2 regulates early PD-L1/PD-L2 induction and Th1-associated cytokine responses during the early events of Allergen-Specific Immunotherapy (AIT) in a preclinical model

Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment for allergic conditions. However, the immunological mechanisms underlying successful tolerance induction are not fully elucidated. TNFR2 is a key immunomodulatory receptor involved in immune regulation and cell survival. Our previous findings demonstrated early TNFR2 upregulation in preclinical AIT and TNFR2 downstream signaling during the up-dosing phase of successful insect venom AIT in patients’ Th2 cells. Nevertheless, its role in regulating immune checkpoint pathways during early phases of AIT remains mechanistically unclear. This study investigates the functional and sequential contribution of TNFR2 in a previously established preclinical murine model of AIT, focusing on early immune responses.

Wild-type (WT-C57BL/6) and TNFR2 knockout (KO-B6.129S2-Tnfrsf1b^tm1Mwm/J) mice were sensitized to the major cat allergen Fel d 1 and treated with AIT using CpG adjuvant (CpG-AIT). Early immune responses were assessed 24 hours after the first AIT injection. Cytokine production and tolerogenic/immune-regulatory molecule expression on immune cells from the AIT injection site (peritoneal cavity/PC), spleen, and serum were evaluated using flow cytometry and multiplex assays. In parallel, WT and TNFR2KO splenocytes were stimulated with CpG in vitro to assess TNFR2-dependent innate and adaptive immune responses.

In WT mice, TNFR2 expression increased in NK, Th1, and B cells in the spleen, and in NK cells and cDCs at the AIT injection site (PC). In parallel, early-phase AIT increased PD-L1 on B cells, NK cells, and cDCs in the spleen, and on NK cells and pDCs in the PC. PD-L2 was upregulated on splenic NK cells and peritoneal cDCs and pDCs. These changes were absent in TNFR2KO mice. WT mice also showed increased serum IFN-γ and IL-12 following AIT, which was not observed in TNFR2KO mice. Consistently, CpG-stimulated TNFR2KO splenocytes exhibited reduced IFN-γ and IL-12 levels in supernatants, along with decreased T1 cytokine expression in Th1, CD8 T cells, NK, and DCs.

TNFR2 is critical for the early induction of the PD-L1/PD-L2 immune-regulatory and the IL-12/IFN-γ Th1 axis during the early events of AIT. TNFR2 deficiency impairs tolerogenic molecule upregulation and Th1-skewed cytokine responses, highlighting TNFR2 as an upstream regulator of early immunoregulatory events during AIT.