*2.3. DC:CD4+ Re-Stimulation Assay (Epibase®IV, Lonza)*

Monocytes were isolated from frozen PBMC samples by magnetic bead selection using CD14 microbeads (Miltenyi Biotec # 130-050-201 on an AutoMACS Pro system) and differentiated into immature DC (iDC) using 1000 IU/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1000 IU/mL of IL-4 in a serum-free mediun (Cell-Genix # 20805-0500, supplemented with 0.05 mg/mL Gentamicin Lonza # 17-518L) for 5 days at 37 ◦C, 5% CO2. iDC were then harvested, washed and loaded with each test protein/peptide individually for 4 h at 37 ◦C, 5% CO2. A DC maturation cocktail containing TNFα (800 IU/mL) and IL-1β (100 IU/mL) was then added for a further 40–42 h to activate/mature the DC (mDC). The expression of key DC surface markers (CD11c-3.9, CD14-63D3, CD40-5C3, CD80-2D10, CD83-HB15E, CD86-BU63, CD209-9E9A8 and HLA-DR-L243) at both the immature and mature stage were assessed by flow cytometry (Bio-Rad ZE5 Cell Analyzer) to ensure the DC were activated prior to T cell interaction. After a thorough washing procedure, 100,000 mDCs were then co-cultured with 1 million autologous CD4+ T cells (isolated by magnetic bead selection, Miltenyi Biotec # 130-045-101 on an AutoMACS Pro system) in a deep-well plate (final volume of 1.2 mL, Greiner # 780271). The DC:CD4+ T cells ratio is 1:10 and the co-culture is incubated for 6 days at 37 ◦C, 5% CO2 in a humidified atmosphere. On day 6, autologous monocytes were isolated from PBMC using magnetic bead selection (Miltenyi Biotec # 130-050-201 on an AutoMACS Pro system) and loaded with the selected protein or peptide that were initially used to load the DC. After incubation at 37 ◦C, 5% CO2 in a humidified atmosphere for 4 h, the monocytes were washed and then added to anti-IFN-γ/anti-IL-5 pre-coated FluoroSpot plates (Mabtech # FSP-0108-10) along with the corresponding DC:CD4 co-culture in quadruplicate (25,000 monocytes: 250,000 CD4+ T cells in a final volume of 200 μL). The FluoroSpot plates were incubated for 40–42 h at 37 ◦C, 5% CO2 in a humidified atmosphere. After incubation, the FluoroSpot plates were developed according to the manufacturer's procedure (IRIS FluoroSpot reader, Mabtech) and the number of spot-forming cells (SFC) per well were assessed for each test condition in an automated and unbiased manner.

#### *2.4. Data Analysis*

Data management and statistical analysis were performed in the R programming language (https://www.R-project.org/, accessed on 28 October 2022, versions 3.6.1 up to 4.1.2), including essential packages for handling generalized linear models (nlme, emmeans) and carrying out variance component analyses (VCA, version 1.4.3).

The calculation of Stimulation Indices (SI) was performed as follows. Spot forming cells (SFC) from the FluoroSpot assay were transformed to a log2 scale, and a generalized linear model (GLM) was applied to estimate the SI (i.e., the ratio between a treatment condition and the donor-matched blank on a linear signal scale) and associated confidence intervals. Quadruplicate SFC measurements were implicitly aggregated by the GLM to yield one SI value for each combination of a specific test compound, donor, and screen. The screens were analyzed sequentially and independently from each other, with the linear model considering a specific cytokine readout of an entire screen as input. The processing workflow was tailored to address a few peculiarities of the given data. Specifically, we used an exponential type of heteroscedasticity adjustment in the GLM to achieve scaleinvariance of residuals and injected some Gaussian noise at the low end of the SFC scale to support model convergence with the frequent presence of ties of discrete values around zero. (The standard deviation of this normally distributed, zero-centered noise was chosen to correspond to the replicate variability inferred by the GLM in the limit of zero SFC counts at the low end of the SFC scale and drops down exponentially by a factor of exp (−2) = 0.14 for every unit increase of the log2 SFC). Furthermore, we observed a consistent trend in the data to the effect that higher blank values of a donor corresponded to systematically lower SI values for that donor. The relation between 'pre-stimulation' of the blank and observed stimulation indices could be well captured by linear regressions performed for each treatment within a screen. We corrected the raw SI values then for every donor-

treatment pair with the respective linear model, basically extrapolating to the value which would have been observed with a common blank value of 0.

Standard quality control plots were generated for every data set, including the visualization of DC differentiation markers, the reproducibility of reference compound data across studies, and (if possible) the variability of repeated compound testing with the same donor. We also looked at the individual stimulation profile of each donor within a study, as the overall inducibility of T-cell response could vary from person to person; simultaneously, this enabled us to rule out the presence of generally inert sample material. A donor response was recorded as "positive" if a SI fold-change of 2 or above (compared to its blank control) was measured at a statistical significance of *p* < 0.05 (using non-adjusted *p*-values from the GLM). The fraction of positive donor responses (within a cohort of typically 30 healthy donors per screen) provided the response rate for the treatment in a specific screen.

### **3. Results**

#### *3.1. DC:CD4+ T Cell Restimulation Assay Workflow*

The general workflow of the assay is illustrated on Figure 1a. Test items were investigated in independent screens of the DC:CD4+ T cell restimulation assay over a time span of several years. Therefore, various controls were employed to ensure a consistent and comprehensive analysis of the data.

**Figure 1.** The DC:CD4+ T cell restimulation assay. (**a**) Experimental setup of the DC:CD4+ restimulation assay. The assay starts with the isolation of monocytes from healthy donor PBMCs, followed by the loading of the protein of interest and maturation of the monocyte derived Dendritic Cells (moDC). Autologous CD4+ T cells are isolated and co-cultured with the loaded moDCs. After an incubation of 6 days, freshly isolated monocytes are challenged with the same protein and added to the co-culture for an additional 42 h before analyzing the production of IFN-γ by FluoroSpot. (**b**) DC were characterized by the expression of the following cell surface markers: CD11c, CD14, CD80, CD83, CD86, CD209, and HLA-DR before and after DC activation by addition of TNF-α and IL-1β to ensure good cell fitness. Created with BioRender.com, accessed on 27 November 2022.

For each screen, 30 healthy donors were selected based on their HLA-DRB1 alleles to reflect the world population [5] (Supplementary Figure S1). In addition, a characterization of the dendritic cells (DCs) was included in every screen to assess the phenotype of these cells before and after maturation by flow cytometry. Activation of the DCs was determined by upregulation of key maturation markers on the cell surface that are known to be

correlated with T-cell priming capacity: CD40, CD80, CD83, CD86, and HLA-DR [10]. Moreover, CD209, a pathogen-recognition receptor expressed on the surface of immature DCs, is internalized together with other markers, thus resulting in efficient presentation [11]. Accordingly, the downregulation of CD209 is the consequence of a shift from an immature to a mature DC phenotype. A representative distribution of cell surface marker expression at both the immature (iDC) and mature stage (mDC) is shown in Figure 1b. The addition of the DC maturation cocktail, composed of TNF-α and IL-1β, led to a slightly higher expression of CD40, CD80, CD83, and HLA-DR, but also a substantial increase in CD86 expression, resulting in a more than ten-fold increase in the average MFI for this surface marker. In addition, we also observed a moderate decrease in CD209 expression. Altogether, this analysis confirmed that DCs from all donors of the cohort have the potential to be activated prior to their interaction with autologous CD4+ T cells. Moreover, the assay is qualified for a given immunomodulatory protein by treating the DCs together with KLH to assess what impact the protein has on the KLH-induced T cell response. This enables us to highlight proteins that may influence the DC-induced activation of T cells.
