CD69 Role on T Cell Priming Figure 8. CD69 does not affect the primary formation of Vaccinia virus-specific CD8+ T cell populations. A. CD69+/+ or CD692/2 mice were infected with VACV-WR and 7 days later spleen cells were reestimulated with uninfected (background control) or VACV-WR-infected RMA cells. B. CD69+/+ or CD692/2 mice were infected with VACV-OVA and 7 days later spleen cells were reestimulated with or without (background control) SIINFEKL peptide. Pool of two experiments. C. H-2 class I knockout HLA-A*0201-transgenic mice were i.v. treated with 100 mg of anti-CD69 2.2 or left untreated, and were subsequently infected with VACV-WR. After 7 days, spleen cells were reestimulated with uninfected (background control) or VACV-WR-infected HLA-A*0201 transfectant RMA cells. In all cases, cells were stained for intracellular IFNc, and the percentage of IFNc+ CD8+ T cells within total cells was assessed. The background control values were substracted from each reestimulated sample value. doi:10.1371/journal.pone.0048593.g008 II cells in the presence of CpG and various OVA doses did not affect the OT-II proliferative response (Fig. 4B). In the co-culture of anti-CD69 2.2-treated pDC with OT-II cells (Fig. 4C) the proliferation was also unaltered. The treatment of cDC (contain- ing the cross-presenting CD8+ DC subset) with anti-CD69 2.2 in the presence of CpG did not alter their cross-priming of OT-I cells (Fig. 4D). Consistently, treatment of CpG pre-activated Flt3l- derived BMDC with anti-CD69 2.2 did not affect the surface expression of CD86 and CD40 (Fig. 4A). Thus, CD69 expression or CD69 targeting on DC does not affect their costimulation or Ag presentation capacity to Ag-specific T cells. CD69 deficiency on T cells does not affect Ag-specific T cell proliferation in Ag-draining LN in vivo CD69 has been well documented to be upregulated on T cell in response to the encounter with specific Ag. We thus wanted to assess whether CD69 could function as a costimulatory molecule on T cells. To analyze the effect of T cell CD69 deficiency on Ag priming in vivo, we transferred CD69+/+ or CD692/2 OT-I cells into recipients receiving OVA subcutaneously into the footpad. In this setting, CD69 deficiency on T cells did not affect T cell proliferation in the popliteal LN of recipients injected with various OVA doses plus LPS (Fig. 5A) or 10 mg of OVA with or without LPS (Fig. 5B). A similar observation was made using the DO10.11 transfer model, in which CD69 deficiency did not affect proliferation of transferred OVA-specific transgenic DO10.11 CD4+ T cells in popliteal LN of recipients receiving OVA subcutaneously in the footpad (Fig. 5C). These results suggest that CD69 deficiency on T cells does not affect the extent of their Ag- specific priming in vivo. CD69 MAb-targeting or gene deficiency on Ag-specific CD8+ T cells do not alter the activation threshold at varying peptide agonistic levels in vitro We further investigated the effect of CD69 targeting and deficiency on the extent of proliferation in in vitro cultures of transgenic CD8+ T cells with different agonist peptides. The anti-CD69 2.2 MAb has been shown to bind to TCR- activated T cells [28]. However, the addition of this antibody to whole LN and spleen OT-I RAG12/2 cultures with growing SIINFEKL peptide doses did not affect the dose from which T cell proliferation and CD25 upregulation started (Fig. 6A). This is consistent with our recently published in vivo results, in which anti- CD69 2.2 treatment does not alter proliferation of OT-I transferred T cells in Ag-draining LN of OVA-bearing mice [34]. Reasoning that SIINFEKL might be a too strong agonist and PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e48593