Interleukin-12 and Interleukin-18 Associate with Chronic Graft-Versus-Host Disease but with Inverse Clinical Outcomes

Background: Chronic graft-versus-host disease (cGVHD) is an immune-mediated complication after allogeneic stem cell transplantation (alloSCT). It associates with graft-versus-leukemia effects (GVL) and lower relapse rates, but in its severe forms also with high morbidity and mortality. The interferon-gamma (IFNg) pathway is involved in its pathophysiology.

The interferon-response marker CXCL9 was shown to associate with any grade cGVHD, activation of IDO and increased activity of the kynurenine (Kyn) pathway. High interleukin (IL)-18 deviated the Kyn pathway to anthranilic and kynurenic acid, and this associated with severe fibrosing GVHD (1).

Hypothesis: We hypothesized that CXCL9 and IL18 are part of a cytokine network that modulates anti-leukemic immunity and tissue fibrosis, whereby specific players may mediate either graft-versus-leukemia effects or GVHD.

Methods: Included in this analysis were 350 consecutive patients who were allografted from matched related (n=103), matched unrelated (n=190), mismatched unrelated (n=43) or haploidentical donors (n=13) for hematologic malignancies (standard indications) between 10/2002 and 10/2018 at our institution. Only patients without relapse until d180 and without refractory acute GVHD were included who had sera available on day+180 (controls) or at onset of cGVHD. Using R&D ELISAs we measured cytokines that belonged to the IFNg pathway (IFNg, CXCL9, IL12p40, IL12EBI3, IL17, IL18, IL23), or to innate inflammatory pathways (TNFa, IFNa, IL1b, activin, follistatin). Cytokine networks were analyzed by Pearson correlation coefficient and visualized as correlation matrix. Mild, severe fibrosing, and severe gastro-intestinal (GI) cGVHD were diagnosed according to simplified NIH criteria (2). Overall survival (OS) was calculated by Cox regression analyses. Multiparameter flow cytometry of peripheral blood mononuclear cells . Patients for flow cytometry (n=100) were selected according to the serum levels of IL12 and IL18.

Results: Median time of blood sampling at onset of cGVHD was day+168 (range 41-723). Control patients (n=169) were collected on day+180 (171-183). Mild cGVHD was recorded in 133 patients, severe fibrosing cGVHD in 39 patients, and isolated GI-cGVHD in 8 patients. Cytokine clustering revealed a large cluster of intercorrelating innate inflammatory mediators, including TNFa, IL1b, IFNg, IFNa, IL17, IL23, Follistatin and Activin. IL12p40 clustered only with CXCL9, whereas IL18 and IL12EBI3 stood apart. As expected, IL23 correlated with IL17 (r=0.375, p<0.001).

Patients with any grade cGVHD had significantly higher serum levels of CXCL9, IL12p40, IFNg, TNFa, and IL12B3 as compared to patients who did not develop cGVHD within the observation period. Severe cGVHD correlated only with CXCL9, IL18 and IL12p40 but not with any cytokine of the innate inflammatory cluster. At cGVHD onset, only IL18 was associated with severe cGVHD, whereas CXCL9 and IL12p40 were correlated with any grade cGVHD.

In multivariable analyses including IL12p40, IL18, age, mismatch, recipient and donor gender, recipient CMV status, disease type, disease stage, ATG, MTX and conditioning intensity, IL18 associated with poor OS after blood sampling (HR 1.31 per log2 increase, p=0.008) due to increased NRM, whereas IL12p40 associated with improved OS (HR for cut>0 ng/ml: 0.50, p=0.002) because of both reduced NRM and reduced TTR. This is visualized in Figure 1.

Flow cytometry revealed that IL12p40 correlated with increased counts of all CD3+ lymphocyte subsets, whereas IL18 did not associate with cellular reconstitution. A positive association with lower relapse rates within the 18 months following blood tests was observed exclusively for Vd1-neg gd-T-cells (AUC 0.67 (0.50-0,85, p=0.047).

Conclusion: Six months after alloSCT, the IFNg pathway (CXCL9, IL12, IL18) associated with severe cGVHD. IL12 and IL18 are the first molecular markers to potentially separate the GVL effects from mortality/morbidity in cGVHD.

1) Orsatti et al, Cell Rep Med 2022

2) Lehners et al, Transplantation 2021

Figure 1: OS after d180/onset cGVHD IL18 and IL12p40 measured at onset of cGVHD or on day+180

IL18 shown as quartiles, IL12p40 above or below lower limit of sensitivity (ELISAs)

Luft:JAZZ Pharmaceuticals: Honoraria. Dreger:AbbVie, AstraZeneca, Gilead, Novartis, Riemser, and Roche: Speakers Bureau; AbbVie, AstraZeneca, Bluebird Bio, Gilead, Janssen, Novartis, Riemser, and Roche: Consultancy.