Longitudinal multiproteomic analysis reveals persistent underlying inflammation in acute graft-versus-host disease patients responding to corticosteroid treatment Free

Background: Acute graft-versus-host disease (aGvHD) is a severe immune complication that can occur following allogeneic hematopoietic stem cell transplantation (HSCT). aGvHD arises when T cells from the donor graft recognize the recipient's tissues as foreign and initiate an immune response against the recipient. This attack typically occurs within the first 100 days post-transplant, leading to tissue damage in various organs, including the skin, gastrointestinal tract and liver.

Methods: To further elucidate the pathobiology of aGvHD and response to corticosteroid (CS) treatment, we conducted a comprehensive multiproteomic analysis of HSCT recipients who developed aGvHD post-transplantation (n=10; samples collected pre-HSCT, at time of aGvHD onset and 30 days after the onset of aGvHD) and healthy controls (n=10). All aGvHD patients received CS as first-line treatment, and 8 of 10 patients achieved clinical remission (median of 16 days from aGvHD onset to remission [range: 7-56 days]). We performed plasma proteomic analyses using Olink and conducted immunophenotyping by flow cytometry using peripheral blood mononuclear cells (PBMCs).

Results: Immunophenotyping of PBMCs from HSCT recipients at onset of aGvHD showed a significant increase in the percentage of circulating monocytes and monocyte-derived dendritic cells (mDCs) compared to pre-HSCT, and those populations were normalized with CS treatment by 30 days post aGvHD onset. Deeper phenotypic analysis of monocytic cells at aGVHD onset revealed that circulating monocytes, and to a lesser extent mDCs, exhibited an activated phenotype with increased expression of CCR7 (a chemokine receptor essential for cell migration to lymph nodes), CD38 (involved in tissue recruitment) and components of the receptor for IL15 (IL2Rγ and IL15Rα). This phenotypic change at aGvHD onset was accompanied by increased plasma levels of macrophage colony-stimulating factor (M-CSF), as well as APRIL and BAFF, which are two cytokines primarily expressed by monocytes and DCs. Intriguingly, CS treatment only partially reverted the overall activation of monocytic cells, with surface IL15Rα remaining elevated on both monocytes and mDCs at day 30 post aGvHD onset. Similarly, APRIL plasma levels remained significantly elevated in CS-treated patients.

Reductions in the percentage of circulating T cells at aGvHD onset compared to pre-HSCT were also observed, consistent with delayed reconstitution of adaptive immunity compared to innate immunity post-HSCT. Despite these relative reductions, extensive phenotypic analysis showed that CD8⁺ T cells, and to a lesser extent CD4⁺ T cells, were highly proliferative and presented with an effector phenotype (e.g. increased surface expression of CD38, CD95 [Fas], IL2Rβ and IL15Rα). Granzyme B expression was also highly expressed in CD8⁺ T cells at aGvHD onset. These phenotypic changes were associated with increased plasma levels of the effector molecules Granzyme B, IFNγ and IFNγ-induced chemokines (CXCL9 and CXCL10), as well as multiple γ chain (γc) cytokines and soluble receptors (IL2, IL2Rα, IL7 and IL15Rα). As expected, CS treatment partially reduced T cell proliferation and activation. However, Granzyme B expression in CD8⁺ T cells was remarkably unaffected by CS treatment. Similarly, other cell surface (e.g. IL15Rα) and plasma (e.g. CXCL10) markers remained elevated at day 30 post aGvHD onset.Conclusion: We demonstrated that aGvHD onset is characterized by relative increases in circulating monocytic cells displaying an activated phenotype. Despite relative reductions in circulating T cells, both CD4⁺ and CD8⁺ T cells also exhibited a highly activated and proliferative phenotype. As expected, the overall activation state associated with aGvHD was reduced by CS treatment, and this was associated with clinical response, as most of the patients (8/10) were steroid-sensitive and achieved remission. However, several activation markers associated with both T cells and monocytic cells remained elevated in CS-treated aGvHD patients at day 30 post aGvHD diagnosis (e.g. plasma APRIL and CXLC10; IL15Rα on both T and monocytic cells, and Granzyme B in CD8⁺ T cells). Our data suggest that aGvHD onset involves innate cell activation followed by an adaptive response that is partially resistant to CS, with underlying inflammation persisting in treated patients.