IL-7 and IL-10 Serum Levels Are Potential Immune Biomarkers for Acute Graft-Versus-Host Disease Following Unrelated Hematopoietic Stem Cell Transplantation

Acute GVHD (aGVHD) remains a major cause of morbidity in patients who undergo allogeneic hematopoietic stem cell transplantation (HSCT). Although numerous studies have investigated potential immunologic parameters that could predict for aGVHD, there are no validated immune biomarkers presently used in clinical practice.

We are currently performing a prospective analysis of immune reconstitution after allogeneic HSCT in different patient cohorts. The goal of the present study is to detect abnormalities of immune reconstitution associated with an inability to maintain T cell tolerance after allogeneic HSCT and to identify specific immunologic biomarkers predictive of aGVHD.

We have thus far accrued 36 unrelated HSCT recipients with a median age of 48 years old (16 females; 20 males). The degree of HLA-matching at the allele level between patients and donors was: 10/10 (n=20), 9/10 (n=15), 8/10 (n=1). Graft source consisted of unmanipulated bone marrow (n=18) or peripheral blood stem cells (n=18). All patients received a reduced-intensity conditioning regimen with fludarabine, melphalan and thymoglobulin (dose dependent on the degree of HLA matching), in association with cyclosporine and mycophenolate mofetil for GVHD prophylaxis. A total of 21 patients (58%) developed grades II-IV aGVHD in the first six months post-transplant.

Whole blood and serum samples were collected from HSCT recipients 1, 2, 3 and 6 months post-HSCT. To assess the impact of different immune markers on the occurrence of aGVHD, patients were categorized into 2 groups based on the development or not of grade II-IV aGVHD in the first six months post-HSCT. Our comprehensive 7-colour flow cytometry panel encompasses phenotypic markers for relevant T, B, NK, NKT and dendritic cell subsets, further including intracellular markers for the assessment of proliferation and susceptibility to apoptosis within regulatory (Treg), conventional (Tcon) CD4 T cells and CD8 T cell subsets. Serum concentrations of IL-2, IL-7, IL-10, IL-15, IFN-γ and TNF-α were evaluated with a multiplex cytokine assay.

Absolute counts of lymphocytes, total T cells, CD4 Tcon, CD4 Treg and CD8 T cells display similar reconstitution patterns in unrelated HSCT recipients with and without aGVHD. However, we have observed a clear trend for higher levels of γδ T cells, both in terms of frequency and absolute counts, in patients who develop aGVHD, particularly at 1 month post-HSCT (Mann Whitney test, P = 0.0699). Furthermore, we found a significantly higher frequency of proliferating cells, as assessed by Ki67 expression, within the CD4 T cell population in patients who do not develop aGVHD, both at 2 and 3 months (Mann Whitney test, P = 0.0400 and P = 0.0177, respectively) post-HSCT. When dissected into proliferating CD4 Tcon and Treg, the frequency of both Ki67⁺ Tcon and Ki67⁺ Treg within the CD4 T cell population is higher in patients who do not develop aGVHD in all time-points analysed, reaching statistical significance at months 2 and 3 for Tcon (Mann Whitney test: P = 0.0280 and P = 0.0051, respectively) and at month 6 for Treg (Mann Whitney test, P = 0.0043).

We are also quantifying the serum levels of relevant pro-inflammatory, suppressive and homeostatic cytokines in the two patient groups. We have observed that IL-7 serum levels (pg/ml) in the first 3 months post-transplant are significantly lower in patients who develop aGVHD compared to those who do not (Mann Whitney test: Month 1 P = 0.0051; Month 2 P = 0.0177; Month 3 P = 0.0303), whereas IL-10 levels are significantly higher in aGVHD patients (Mann Whitney test: Month 1 P = 0.0442; Month 2 P = 0.0051; Month 3 P = 0.0480). None of the other cytokines analyzed has revealed significant differences between the two groups of patients, although there seems to be a trend for higher IL-15 serum levels in patients who develop aGVHD, particularly in the first two months post-HSCT.

These results indicate that in our patient population sensitive methods for the detection of IL-7 and IL-10 serum levels can identify patients at high risk for grades II-IV aGVHD early post-transplant. The accrual of more patients will clarify whether the levels of γδ T cells post-transplant, as well as the frequency of proliferating CD4 Tcon and Treg populations, can also be used as an early indicator for the development of aGVHD.