Evaluating thymic stromal lymphopoietin (TSLP) as a biomarker in acute graft versus host disease

Abstract

Abstract Background Steroid refractory acute graft versus host disease of the lower gastrointestinal tract (SR-LGI-aGVHD) carries a high morbidity and mortality. Several prognostic biomarkers have been studied, prominent among them being ST2 and Reg3a. However, there are no targetable biomarkers to date. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine known to exist as a long (18kDa) and short (7kDa) isoform. The short isoform is constitutively expressed and mediates homeostatic functions while the long isoform is upregulated during inflammation. Hence, differential expression of the isoforms correlates with normal vs pathogenic responses. TSLP is produced by epithelial cells in the skin, gastrointestinal tract and lungs, overlapping with organs affected by GVHD, in response to environmental and proinflammatory stimuli and plays a key role in mucosal homeostasis and inflammation. The heterodimeric TSLP receptor contains the IL-7Ra subunit and is expressed primarily by myeloid derived dendritic cells (MDDCs). Binding of TSLP to its receptor leads to downstream activation of the JAK-STAT pathway in MDDCs which then drive antigen exposed CD4+ T cells towards Th-2 differentiation with production of IL4, IL5, IL13 and TNFa. TSLP is primarily implicated in type-2 inflammatory conditions but also in Th-17 type immune responses which are known to play a role in GVHD. TSLP levels reported in the literature thus far are measured by ELISA which does not differentiate the isoforms. We measured TSLP isoforms with nano immunoassay (NIA) in plasma and serum of allogeneic stem cell transplant (HCT) patients with and without aGVHD to test the association of TSLP isoform levels with presence or severity of aGVHD. Methods TSLP levels were measured in blood samples obtained from 17 HCT patients without GVHD and 24 HCT patients with aGVHD from Stanford University, and 60 patients with SR-LGI-aGVHD at start of second line therapy obtained from the Mount Sinai Acute GVHD International Consortium (MAGIC) biorepository. To test the association of TSLP with NRM, the MAGIC cohort was divided into 2 groups, one with and the other without 6-month non-relapse mortality (6m-NRM). The two groups were balanced for demographics, underlying disease, donor and graft type, conditioning regimen, GVHD prophylaxis and second line treatment. TSLP was measured using nano immunoassay (NIA) on a PeggySue instrument at the Stanford Translational Applications Service Center (TASC) lab and reported as relative units of chemiluminescence. For each sample, the highest chemiluminescence peak is reported. Two-sample t-tests with Welch's correction were used to compare TSLP levels between the above groups and to test the association between TSLP levels and 6m-NRM. Results Using NIA we discovered novel, previously undescribed, high molecular weight isoforms of TSLP ranging from 29 to 40kDa. Levels normalized for total protein showed correlation with raw measurements. The TSLP isoform with highest peak is reported as relative units of chemiluminescence. In HCT patients, the development of aGVHD led to significantly increased TSLP levels (p<0.001) that further increased with aGVHD severity as follows: Without aGVHD - median 35432 (95% CI 28773, 35837)With aGVHD - median 61869 (95% CI 46017, 78305)With SR-LGI-aGVHD - median 130158 (95% CI 117859, 164324) Among SR-LGI-aGVHD:Survivors had a median TSLP of 141828 (95%CI 123667, 191153)Patients with 6m-NRM had a median TSLP of 128243 (95%CI 98388, 185982) Conclusions and future directionsUsing NIA we have discovered novel, previously undescribed, HMW isoforms of TSLP. In HCT patients, TSLP levels were significantly higher in aGVHD and increased further in SR-LGI-aGVHD, indicating that TSLP may have value as a diagnostic biomarker in aGVHD. The role of TSLP in the pathogenesis of GVHD and the efficacy of targeting TSLP in the treatment of GVHD are not known and warrant investigation. Tezepelumab, a monoclonal antibody that binds to TSLP, indirectly downregulates JAK-STAT-mTOR signaling downstream of the TSLP receptor, has shown a well-tolerated safety profile in clinical trials and is approved for use in severe asthma. Therefore, TSLP has the potential to be a targetable biomarker in aGVHD. Molecular characterization of the newly discovered isoforms, including phosphorylation status, is being studied by our group. PS and IEL contributed equally to this study.