**4. ETiCS substudies**

#### **4.1 Immunobiology of heart-directed autoreactivity**

This substudy attempts to unravel the step-by-step changes occurring in a patients' immune system whilst autoantibody-mediated immune cardiomyopathy develops after inflammatory or ischemic myocardial injury, including the time course and cell types (T and B cell subpopulations) involved in the generation of cardiac aabs. The reactivity and prevalence of receptor (auto)antigen-specific T and B cell populations will be assessed by

event. Follow-up visits at 3, 6, and 12 months comprise a detailed patient history including medication, physical examination, a standardized health questionnaire, echocardiography, ECG, Holter-ECG, and blood sampling (**Fig. 2**). The sequentially obtained blood samples will serve to determine the time course of formation and the titre-course (clearance/persistence) of distinct cardiac aabs at 0, 3, 6, and 12 months after inflammatory or ischemic cardiac injury (AMitis, FAMI). Three-hundred healthy subjects with normal blood pressure, ECG, and exercise-stress test will serve as a control collective (male to female ratio 1:1, n=50 per five-years age range, no history of myocardial infarction, diabetes, or peripheral vascular disease). The primary endpoint of the planned cross-sectional analyses is the association of a specific cardiac aab status at diagnosis (aab-positive/aabnegative) with (**a**) the change in cardiac function as derived from sequential echocardiograms and cMRI (baseline *versus* 12 months), and (b) the severity and clinical course of the index disease. Longitudinal primary endpoints are titre-changes of a given cardiac aab over time, conversion rates (persistence/clearance), and the "time to first cardiovascular event" (see table 2 for definition of composite endpoints). Pre-specified secondary endpoints for each of the different cardiac aabs at diagnosis are time to

cardiovascular death and time to all-cause death (table 2).

Titre course of conformational cardiac aabs

Resuscitation (successful or not successful)

Death due to progressive heart failure Death due to myocardial infarction Table 2. Pre-defined endpoints of the ETiCS study.

**4.1 Immunobiology of heart-directed autoreactivity** 

This substudy attempts to unravel the step-by-step changes occurring in a patients' immune system whilst autoantibody-mediated immune cardiomyopathy develops after inflammatory or ischemic myocardial injury, including the time course and cell types (T and B cell subpopulations) involved in the generation of cardiac aabs. The reactivity and prevalence of receptor (auto)antigen-specific T and B cell populations will be assessed by

Change in left ventricular enddiastolic diameter Change in left ventricular ejection fraction Occurrence of ventricular arrhythmias Change of clinical NYHA classification

Severity of inflammation as assessed by EMB/cMRI Time to cardiovascular death/all-cause death

Primary endpoint: prospective ETiCS study

Composite endpoints

**4. ETiCS substudies** 

Cardiovascular Death Myocardial infarction

ICD discharge, if appropriate Cardiac transplantation Sudden cardiac death

Secondary endpoints: prospective ETiCS study

antigenic recall assays as well as FACS and Elispot analyses. In addition, the respectively activated immunologic paths will be derived from the Th1/Th2/Th17 cytokine profiles determined in the sera from cardiac autoantibody-positive patients with acute myocarditis (including an analysis of the corresponding EMBs (Noutsias et al, 2011)) or acute transmural myocardial infarction. Since the cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a potent (indirect) suppressor of the immune system, its mutation or hampered expression might promote hyperreactivity to autoantigens (Golden et al., 2005). Thus, in all ETiCS patients CTLA-4 expression and CTLA-4 alleles will be determined by FACS and by PCR, respectively. In addition, in all patients the HLA-DR/DQ and MHC class II haplotypes (Limas et al., 2004; Caforio & Iliceto, 2008) will be determined and correlated with the formation and titre-course of distinct cardiac aabs in order to unravel the individual genetic susceptibility for heart-directed autoimmune reactions.
