Column purification

After isolation, cells were resuspended in phosphate-buffered saline (PBS) containing 2 mM EDTA and spun for the removal of contaminating platelets. Prior to sorting, peripheral CD4⁺ T cells were enriched with the use of magnetic beads and column purification (Miltenyi Biotec). Enriched peripheral CD4⁺ T cells were then stained with previously determined volumes of the following fluorescently conjugated MAbs: CD3-APC-Cy7 or CD3-AF700 (clone SP34-2), CCR7-PE-Cy7 (clone 3D12), CD8-APC-Cy7 (clone SK1), CD45RA-APC (clone 5H9), CD95-PE-Cy5 (clone DX2), and CD62L-PE (clone SK11) from BD Bioscience; CD28-ECD (clone CD28.2) from Beckman Coulter, and CD4-BrilliantViolet650 (clone OKT4) and CD8-BV421 (clone RPA-T8) from BioLegend. Circulating populations for sorting were defined as follows: naive, CD45RA⁺ CCR7⁺ CD95⁻ SCM, CD45RA⁺ CCR7⁺ CD95⁺ CD28⁺ CD62L⁺; CM, CD45RA⁻ CD95⁺ CCR7⁺ CD62L⁺; and EM, CD95⁺ CCR7⁻. Sorting was performed on a FACSAria LSR II (BD Biosciences) equipped with FACSDiva software.

Human monocytes were purified from human PBMC using CD14-labeled magnetic beads, followed by column purification (Miltenyi Biotec) and then cultured in RPMI complete medium with 10% FCS in the absence or presence of either a Borrelia burgdorferi sonicate (10 μg/ml) or LPS (1 μg/ml; Sigma-Aldrich) for 18 h. To some cultures were added TNF-α (10 ng/ml) (BioLegend), anti-TNF-α (10 μg/ml) (BioLegend), IL-1β (10 pg/ml) (Invitrogen), or anti–IL-1β (5 μg/ml) (R&D Systems). Cells were then stained with the sTCR-γδ tetramer. T cells from PBMC were either used fresh or were activated with anti-CD3/anti-CD28 (each 10 μg/ml; BioLegend) + IL-2 (50 U/ml; Cetus) and propagated for 3 d. Cells were then stained with the sTCR-γδ tetramer. Human PBMC were obtained using an approved protocol from The University of Vermont Human Studies Committee. Verified cell lines were obtained from American Type Culture Collection. CHO cells deficient for glycosaminoglycans (GAGs) were derived as previously described (34 (link)).