This study provides another context by which the TNFR family is important for the function of low affinity T cells by supporting the recall response of low affinity primed memory CD8+ T cells to a high affinity graft. immunomodulation of pathogenic T cell responses during transplantation. activation (7), and during protective responses, tumor immunity, AC-264613 and autoimmunity (9C13). However, little is known about the ability of TNF and TNFR2 signals to provide costimulatory signals during effector or memory CD8+ T cell responses in the context of transplantation. Here we report an important role for TNFR2 on low affinity primed secondary effector CD8+ T cells. These results demonstrate the importance of TNF signaling in low affinity, cross-reactive CD8+ T cell responses during heterologous immunity and spotlight the role of TCR affinity in dictating costimulation requirements of T cell responses. Materials and Methods Mice C57BL/6 Ly5.2-Cr (CD45.1, H-2b), OT-I (14) transgenic mice, (Taconic Farms), and mOVA (N4 OVA) mice (C57BL/6 background, H-2b, a gift from M. Jenkins, University or college of Minnesota, Minneapolis, MN), were utilized in accordance with Emory University or college Institutional Animal Care and Use Committee guidelines. Generation of OT-I Memory and Secondary Effectors Thy1.1+ OT-I cells (1.0 104) were transferred i.v. and mice were infected with 104 CFU strains expressing OVA APL epitope (LM-OVA APLs) (15) 24 h later. Mouse monoclonal to CD14.4AW4 reacts with CD14, a 53-55 kDa molecule. CD14 is a human high affinity cell-surface receptor for complexes of lipopolysaccharide (LPS-endotoxin) and serum LPS-binding protein (LPB). CD14 antigen has a strong presence on the surface of monocytes/macrophages, is weakly expressed on granulocytes, but not expressed by myeloid progenitor cells. CD14 functions as a receptor for endotoxin; when the monocytes become activated they release cytokines such as TNF, and up-regulate cell surface molecules including adhesion molecules.This clone is cross reactive with non-human primate Secondary effectors were generated in 4 week post-infection mice by immunizing with 50 g N4 OVA peptide (GenScript) emulsified in IFA (Gibco) in both hind foot pads. Five days later, draining popliteal LNs were collected and pooled for analysis. Serum was analyzed using Ready-Set-Go ELISA kit according to manufacturers instructions (eBiosciences). Memory OT-I T Cell Enrichment To assess resting OT-I memory cells, at week 4 post contamination (day 28C35), spleen and lymph nodes (popliteal, inguinal, mesenteric, brachial, axial, and cervical) were pooled and enriched for Thy1.1 cells using magnetic beads (16). Briefly, single cell suspensions were incubated with anti-Thy1.1 PE and anti-PE microbeads (Miltenyi), following by enrichment over LS columns. AC-264613 The unbound column flow-through and wash portion was routinely absent of OT-I cells. Memory OT-I cells were assessed as CD45.2+CD19?CD11c?CD4?CD8+CD44hiThy1.1+. In some experiments, 200 L of 2 mg/mL BrdU was given intraperitoneally on day 4 post graft and splenic OT-I cells were enriched for analysis 18 h later. Absolute cell figures were decided using AccuCheck beads (Invitrogen). Generation of Tm Cells Spleen and mesenteric lymph node cells from OT-I mice were processed to single cell AC-264613 suspension and 3106 splenocytes were plated in 24 well plates in total RPMI supplemented with 0.1 M OVA APL peptide (N4 OVA or V4 OVA), 0.1 g/mL anti-CD28 (37.51, Biolegend), and 10 ng/mL IL-2 (Biolegend) for 3 days. Dead cells were removed using Lymphocyte Separation Medium (CellGro) and cells were cultured in media made up of 10 ng/mL IL-15 (Biolegend) overnight, followed by circulation cytometry. On day 4, lifeless cells were removed again and 5106 N4 OVA or V4 OVA Tm cells were transferred i.v. Skin Transplantation Full-thickness tail and ear skins were transplanted as previously explained (17). Mice were treated with 500 g of CTLA-4 Ig on days 0, 2, 4, 6, or with 500 g anti-TNFR2 (T75, BioXCell) on days 2, 4, 6, 8 post transplant. Circulation Cytometry and Intracellular Cytokine Staining Splenic and lymph node cells were stained with Abs from BD Biosciences or Biolegend. TNFR2 expression was analyzed using biotin main (TR75C89 or IgG)/streptavidin secondary (Biolegend). For N4 OVA-specific tetramer staining, monomers were obtained from the NIH Tetramer Core Facility and 180 g of monomer (90% biotinylation) was tetramerized with streptavidin APC using standard techniques. Tetramer staining was performed on splenic CD8+Thy1.1? cells for 20C30 min at room temperature. Cytokine production was assessed following activation with 1 M SIINFEKL peptide and 10 g/ml GolgiPlug for 5 h at 37 C. Data were analyzed using FlowJo software (Tree Star) and GraphPad Prism software (GraphPad Software Inc.). Results Low affinity AC-264613 CD8+ T cell priming efficiently generates memory cells The affinity of TCR interactions during priming impacts CD8+ T cell programming during effector and memory phases (1, 18, 19). However, the functional effects of low TCR affinity priming on subsequent CD8+ T cell memory responses are poorly understood. We utilized the OVA-based TCR transgenic system in which congenically labeled OT-I T cells are primed during contamination with an acutely cleared strain engineered to express the high affinity OT-I epitope SIINFEKL (N4 OVA) or its altered peptide ligand (APL) variant SIIVFEKL (V4 OVA, Physique 1A), which has a 680-fold lower function avidity for OT-I T cells (15). We found that both high and low affinity.