Magnetic bead negative selection

Graft-draining axillary and brachial lymph nodes were processed into single-cell suspensions. Cells were surface stained with their appropriate markers followed by the fixable blue cell viability kit for UV excitation (LIVE/DEAD, Invitrogen) before fixation. Intracellular staining was performed with the use of the Foxp3 Fixation/Permeabilization Buffer Kit (eBioscience, Invitrogen). All antibodies were obtained from Biolegend and BD Biosciences. Samples were run on either LSR Fortessa or FACSymphony (BD Biosciences) and analyzed using FlowJo Software, version 10 (Flowjo, LLC). For sorting, cells were obtained from graft-draining axillary and brachial lymph nodes following skin transplantation. CD4+ T cells were enriched using magnetic bead negative selection (Miltenyi Biotec) and then sorted into CXCR5^– (CD19^–CD4⁺CD44^hiPD1^loCXCR5^–GITR^–) and CXCR5⁺ Tfh (CD19^–CD4⁺CD44^hiPD1^hiCXCR5⁺GITR^–) cell populations.

Total T cells isolated from C57BL/6 wild-type mice by magnetic bead negative selection (Miltenyi Biotec) were transfected with 600 pmol siRNA using the Amaxa Mouse T cell Nucleofector kit and according to the manufacturer’s instructions (Lonza). Transfected cells were rested for 2 h before stimulation and assessment of proliferation as described previously. Chemically modified siRNAs were generated as previously described (Mantei et al., 2008 (link)) and their sequences are as follows: 5′-3′ nontargeting control, sense 5′-GmGmAGCGCACCAUCUUCTdCdAdAmUmUm-3′, antisense 5′-AdUUGAGAAGAUGGUGCGCUCmCm-3′; Usp9X (1) 5′-AmAmCCAAGUAACUCAUGATdCdAdAmGmCm-3′, antisense 5′-TdUGAUCAUGAGUUACUUGGUmUm-3′; Usp9X (2), sense 5′-CmCmCAAAUGAAGAAGUGACdAdAdAmAmAm-3′, antisense 5′-TdUUGUCACUUCUUCAUUUGGmGm-3′, Usp9X (3), 5′-UmUmUGAAUUUCCUCGAGAGdTdTdAmGmAm-3′, antisense 5′-TdAACUCUCGAGGAAAUUCAAmAm-3′; Usp9X (4), 5′-CmUmUGGCAAAGUUAGAUGAdTdAdUmGmAm-3′, antisense 5′-AdUAUCAUCUAACUUUGCCAAmGm-3′. Cm, Um, Am, Tm, or Gm denote a methoxy nucleotide and Gd, Ad, Td, or Cd denote a deoxynucleotide.

Naïve human cells were obtained from benign inguinal lymph nodes of transplant recipients at the time of transplantation. In accordance with previously published methods,^{25 (link),26 (link)} B cells were enriched using magnetic bead negative selection (Miltenyi Biotec), and T cells were FACS sorted into CXCR5^– (CD4⁺TCR⁺CXCR5^–CD45RA⁺) and CXCR5⁺ Tfh (CD4⁺TCR⁺CXCR5⁺CD45RA^–) cells. The prototypical Tfh cell markers ICOS and PD-1 were not used because of the absence of sufficient ICOS^hi or PD-1^hi effector Tfh cells normally present in pathologic or reactive lymph nodes. Cells were then cocultured for 5 d in media containing either CD3/CD28 Dynabeads (Life Technologies) or the superantigen staphylococcal enterotoxin in 96-well round bottom plates and then collected for flow cytometry and RT-PCR as described earlier. Results were equivalent between both T-cell stimulation techniques.