Endothelial Cells Activate Alloreactive CD4⁺ T Cells to Initiate Acute Graft-Versus-Host Disease

Allogeneic donor T cells can exert an anti-leukemic response (GvL effect) after hematopoietic cell transplantation (allo-HCT). However, allogeneic donor T cells can also cause life-threatening acute graft-versus-host disease (aGvHD). Therefore, donor T cell priming is considered as a crucial event determining allo-HCT outcome. In aGvHD, donor CD4⁺ T cells are activated by antigen presenting cells (APCs), proliferate, differentiate and migrate to cause tissue damage. However, non-hematopoietic APCs have been shown to drive aGvHD pathology, making hematopoietic APCs dispensable in the initiation of aGvHD (Toubai et al., Blood 2012, Koyama et al., Nat Med 2012). During the aGvHD initiation phase allogenic T cells exclusively migrate to secondary lymphoid organs (SLOs) before they enter target tissues in the aGvHD effector phase (Beilhack et al., Blood 2005, Beilhack et al., Blood 2008). These observations led us to investigate the role of MHC class II mediated antigen presentation by non-hematopoietic lymph node (LN) stromal (SCs) and endothelial cells (ECs) in aGvHD. Recently we learned that alloreactive CD4⁺ T cells are activated in SLOs by non-hematopoietic APCs independently from Ccl19 expressing SCs, while these SCs provide protective niches to donor Tregs mitigating GvHD. Consequently, here we explored MHCII mediated antigen presentation by SLO blood endothelial cells (BECs) and lymph endothelial cells (LECs).

BECs and LECs expressed endogenous MHCII, costimulatory receptors CD80/CD86 and acquired MHCII from dendritic cells via trogocytosis. Upon myeloablative irradiation, BECs and LECs upregulated CD80 and CD86 whereas MHCII expression decreased upon loss of radiosensitive host DCs. scRNA-seq revealed, BECs and LECs subsets expressing genes involved in MHCII antigen presentation (e.g. Cd74, Ctss, Ctsl, Tap2, H2-Aa, H2-Ab1, H2-Eb1) leading to exogenous antigen presentation (DQ-OVA) by EC subsets. To elucidate MHCII antigen presentation in ECs we crossed Cdh5-Cre to H2-Ab1^fl mice to conditionally knockout MHCII on ECs (MHCII^ΔCdh5). Allo-HCT in MHCII^ΔCdh5 recipients utilizing an MHC major mismatched model (FVB→B6) did not alter allogeneic CD4⁺ T cell activation during aGvHD initiation. However, MHCII^ΔCdh5 showed marginally reduced aGvHD and better survival. Notably, MHCII^ΔCdh5 recipients retained functional hematopoietic APCs presenting alloantigens via MHCII. Therefore, we generated bone marrow (BM) chimeras by syngeneic (syn) HCT from MHCII-deficient (MHCII^Δ) donors into B6.WT mice. After allo-HCT of these BM chimeras, donor CD4⁺ T cells got activated in SLOs during the aGvHD initiation phase. However, to our surprise also syngeneic BM chimeric animals spontaneously developed GvHD-like symptoms making them unsuitable for studying MHCII mediated non-hematopoietic antigen presentation after allo-HCT. This striking phenotype could be explained by a defective CD44^hiCD62L^low CD4⁺ T cell memory development in MHCII^Δ BM chimeras (Huang et al., J Immunol. 2014).

Utilizing our scRNA seq dataset we identified Tie2 and Vav1 to be expressed on cells of hematopoietic lineage, however Tie2 expression was observed on ECs of the LNs. Therefore, we generated MHCII^ΔVav1 mice by crossing Vav1-Cre to H2-Ab1^fl. CD45^- LNs cells from these animals activated CD4⁺ T cell in mixed lymphocyte reaction as well as in vivo. MHCII^ΔVav1 mice developed less aGvHD with delayed kinetics.

Subsequently, we surgically transplanted LNs from syngeneic MHCII^ΔVav1 mice into complete MHCII^Δ mice. After MHCII^ΔVav1 LN engraftment, these MHCII^Δ recipients underwent allo-HCT with allogenic CD4⁺ T cells. Alloreactive donor CD4⁺ T cells expressed more IFNγ and TNFα compared to controls. These experiments proved that non-hematopoietic cells in LNs can activate alloreactive CD4⁺ T cells to initiate aGvHD. Ultimately, we crossed MHCII^ΔCdh5 to MHCII^ΔVav1, resulting in mice deficient for MHCII in the entire hematopoietic and endothelial compartments. After allo-HCT MHCII^ΔVav1ΔCdh5 were significantly better protected from aGvHD than littermate controls, revealing a previously unidentified role of ECs in initiating CD4⁺ T cell mediated aGvHD.

Based on these findings we propose ECs as important non-hematopoietic APCs to activate alloreactive CD4⁺ T cells to trigger aGvHD suited for therapeutic interventions after allo-HCT.

Einsele:BMS/Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Other: travel grants.