Soluble LAG3: A Potential Marker for the Development and Activity of Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (aGVHD), usually developing within 100 days after hematopoietic stem cell transplantation (HSCT), is mainly attributed to donor T cell pro-inflammatory responses against mismatched host histocompatibility antigens. The persistence of increased effector T cell responses following HSCT is partially related to an altered function of T regulatory cells (Tregs) and their failure to prevent GVHD. Certain immune checkpoint molecules, such as cytotoxic T-lymphocyte antigen 4, programmed cell death protein 1 (PD1) and lymphocyte activation gene-3 (LAG3), highly expressed on Tregs, are involved in maintaining immune tolerance and preventing immune-mediated inflammation. LAG3 is a newly identified inhibitory receptor, found on most immune cells, including CD4+ and CD8+ T cells, Tregs, and dendritic cells. Previous studies have demonstrated a role of LAG3 expression on T cells in GVHD suppression. Its soluble isoform sLAG3, released from the cell membrane, has been recently reported to also contribute to enhanced immune-mediated responses in solid tumors and autoimmune diseases, such as anti-PD1-resistant melanoma, rheumatoid arthritis (RA), and ANCA-associated vasculitis. Given this evidence, the current study aimed to explore a potential role of sLAG3 in GVHD development.

Serum samples of 25 patients (≥18 years old) who underwent allogeneic HSCT at Rambam from 01.2021 to 01.2023 were drawn on days -7, +100, +180 of transplant. Twelve serum samples obtained from the Biobank were used as healthy control (HC). All samples were assessed for the sLAG3 levels using a commercial ELISA kit. The median level measured in HC (56pg/mL) was considered a cutoff to define the “low and high level sLAG3 groups”.

Twenty-three patients were included in the analysis [median age at transplant - 59.5 years (range 19-75); 52% males]. Seventeen patients had acute myeloid leukemia, 3 - myelodysplastic syndrome, 2 - acute lymphoblastic leukemia and 1 had primary myelofibrosis. Fourteen patients had a matched unrelated donor, 6- matched related donors, and 3 - haploidentical donors. Nineteen patients received calcineurin inhibitor (CNI) and methotrexate-based GVHD prophylaxis (13 with ATG); 4 patients received post-transplant cytoxan-based prophylaxis, with CNI and mycophenolate-mofetil. Overall, 17/23 (74%) patients developed GVHD: 9 - aGVHD and 8 - chronic GVHD (cGVHD). A low pre-transplant sLAG3 level (<56pg/mL) was found in 8/23 patients, while it was high (>56pg/mL) in 15/23. Notably, 9/15 (60%) patients in the “high sLAG3 group”, but none in the “low sLAG3 group” were diagnosed with aGVHD, suggesting a high sLAG3 level as a possible indicator for patient-pre-transplant susceptibility to aGVHD development, irrespective of transplant factors, such as conditioning regimen, donor type, etc. Four patients in each group developed cGVHD. Furthermore, there was a significant increase in sLAG3 levels between the baseline and GVHD diagnosis in patients who developed aGVHD (mean: from 156.1±172.8, to 323.9±201.3), but not in those who either had cGVHD (mean: from 119.71±90.5 to 150.1±147.1) or did not have GVHD (mean: from 49.7±36 to 52.1±22.2). Overall, 11/23 patients were in complete remission (CR) at the time of HSCT; 9/11 were in the high-sLAG3 group and 6 of them developed aGVHD. In the low-sLAG3 group, most patients (6/8) had some degree of active disease (persistent disease or minimal-residual disease). These findings may reflect high T-effector cell and/or low Treg activity, possibly contributing to both anti-leukemic effect and GVHD development.

In conclusion: The current study demonstrates, for the first time, that the pre-transplant sLAG3 level is significantly increased and continues to further increase in patients who develop aGVHD following HSCT compared to those who don’t. These findings suggest a potential role of sLAG3 as a predicting factor for aGVHD development as well as a marker of treatment response that could guide therapeutic decisions in this patient setting.