Isoflow sheath fluid

5 × 10⁶ leukocytes were washed in PBS and then incubated in 500 µL PBS with 10% goat serum (S-1000, Vector Laboratories) for 30 min to block non-specific binding. The cells were resuspended in 100 µL PBS with 10% goat serum for 1 h at room temperature with primary or without (control) rabbit antibodies against SLC5A5/NIS (SAB2102220, Sigma) or SLC26A4/PENDRIN (MBS9215961, MyBioSource). The cells were washed and then stained for 30 min with secondary F(ab′)2 goat anti-rabbit (Invitrogen, A21246) at a 1:100 dilution and CD45 Krome orange (Beckman Coulter, A96416) at a 1:20 dilution at room temperature. Other experiments included CD14 FITC staining (BD Pharmingen, 555397) to identify monocytes. Cells were washed, resuspended in IsoFlow sheath fluid (Beckman Coulter), and then loaded onto BD FACSCanto II where 25,000 events were collected in the lymphocyte gate. The resulting data and the median fluorescence intensity (MFI) were analyzed utilizing FlowJo software.

To determine the percentage of different T cell subsets, at least 1 × 10⁶ PBMCs were suspended in isoflow sheath fluid (Beckman Coulter) and stained in a DuraClone IM T cell tube (Beckman Coulter, Miami, FL) according to the manufacturer's protocol. The T cell tube contained anti-CD45, CD3, CD8, CD4, CD45RA, and CCR7. Samples were measured by use of the Navios flow-cytometer (Beckman Coulter).
To determine the Tfh, at least 1 × 10⁶ PBMC were washed by Fascflow (BD Biosciences, New Jersey, US) and stained with CD3 BV510 (Biolegend, California, US), CD4 BV421 (Biolegend, California, US), CXCR5 Alexa Fluor 647 (BD Biosciences, New Jersey, US), and PD1 APC-Cy7 (Biolegend, California, US) for 30 min at room temperature in the dark.

A library of DPEase of C. cellulolyticum mutants was generated following the error-prone PCR protocol (35 ) using the OneTaq DNA Polymerase (New England Biolabs), the forward primer 5′-GCCGTCTCGGATGAAACACGGTATCTACTAC-3′, the reverse primer 5′-GCCGTCTCCCGCTTTAAGAGTGTTTGTGGCATTC-3′ and as template a gBlock encoding the C. cellulolyticum DPEase. A control library was performed with the Q5^® High-Fidelity DNA Polymerase (New England Biolabs). Each library was inserted in the Mutant Drop Zone (MDZ) downstream of the psicose biosensor (BBa_K2448057) by Golden Gate, using the BsmBI restriction enzyme (Thermo Fisher Scientific). Ten microliters of the Golden Gate reaction were used to transform chemically competent E. coli DH5α cells. After overnight culturing in LB media supplemented with 35 µg/ml chloramphenicol, transformed cells were centrifuged, washed with IsoFlow Sheath Fluid (Beckman Coulter) and resuspended in this same isotonic fluid at a concentration of 10⁶ cells/ml. Flow cytometric measurements were performed at Genoscope on a MoFlo Astrios cell sorter (Beckam Coulter), using a single laser operating at 488 nm for excitation and a channel of 576/21 nm for detection of the mEmerald fluorescence. The selection was triggered by fluorescence (threshold 0.05%). The data were analyzed using the Summit V6.2 Software (Beckam Coulter).