CD69 Does Not Affect the Extent of T Cell Priming Elisenda Alari-Pahissa1,2, Laura Notario1, Elena Lorente1, Javier Vega-Ramos3, Ana Justel4, Daniel Lo´ pez1, Jose´ A. Villadangos3, Pilar Lauzurica1* 1 Instituto de Salud Carlos III, Majadahonda, Madrid, Spain, 2 Departament de Fisiologia, Universitat de Barcelona, Barcelona, Spain, 3 Department of Microbiology and Immunology, and Department of Biochemistry and Molecular Biology (Bio21 Institute), University of Melbourne, Melbourne, Australia, 4 Facultad de Matema´ticas, Universidad Auto´ noma de Madrid, Madrid, Spain Abstract CD69 is rapidly upregulated on T cells upon activation. In this work we show that this is also the case for CD69 expression on dendritic cells (DC). Thus, the expression kinetics of CD69 on both cell types is reminiscent of the one of costimulatory molecules. Using mouse models of transgenic T cells, we aimed at evaluating the effect of monoclonal antibody (MAb)- based targeting and gene deficiency of CD69 expressed by either DC or T cells on the extent of antigen (Ag)-specific T cell priming, which could be the result of a putative role in costimulation as well as on DC maturation and Ag-processing and presentation. CD69 targeting or deficiency of DC did not affect their expression of costimulatory molecules nor their capacity to induce Ag-specific T cell proliferation in in vitro assays. Also, CD69 targeting or deficiency of transgenic T cells did not affect the minimal proliferative dose for different peptide agonists in vitro. In in vivo models of transgenic T cell transfer and local Ag injection, CD69 deficiency of transferred T cells did not affect the extent of the proliferative response in Ag-draining lymph nodes (LN). In agreement with these results, CD69 MAb targeting or gene deficiency of Vaccinia-virus (VACV) infected mice did not affect the endogenous formation of virus-specific CD8+ T cell populations at the peak of the primary immune response. Altogether our results argue against a possible role in costimulation or an effect on Ag processing and presentation for CD69. Citation: Alari-Pahissa E, Notario L, Lorente E, Vega-Ramos J, Justel A, et al. (2012) CD69 Does Not Affect the Extent of T Cell Priming. PLoS ONE 7(10): e48593. doi:10.1371/journal.pone.0048593 Editor: Kim J. Hasenkrug, National Institute of Allergy and Infectious Diseases, United States of America Received August 8, 2012; Accepted September 28, 2012; Published October 30, 2012 Copyright: 2012 Alari-Pahissa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: E. Alari-Pahissa was supported by predoctoral fellowships from the Spanish Ministry of Education and Science. This work was supported by grants (SAF2007-64310 and SAF2010-15649) from the Ministerio de Educacio´ n, Ciencia y Tecnolog´ıa and La Fundacio´ La Marato´ de TV3 2004. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lauzurica@isciii.es Introduction CD69 is a type II C-type lectin of unknown ligand specificity encoded in the NK-complex. It is known as a very early activation marker, since it is promptly upregulated on all leukocytes upon activation [1–2]. Importantly, it is upregulated on T cells by IFNa/b [3], and upon Ag encounter [4–5], during the first kinetics phase of brief contacts between T cells and antigen presenting cells, either in the presence or absence of adjuvant [6]. CD69 expression has been reported in infections [7–10], autoimmune diseases [11–16], and tumor infiltrates [17–18]. Some C-type lectins are upregulated on T cells upon activation and have costimulatory or coinhibitory effects, influencing the extent of TCR-mediated T cell activation [19–20]. Apart from that, most C-type lectin receptors are expressed by DC [21], and some of them have been shown to induce signaling or to influence Toll-like receptors (TLR)-induced signaling, modulating the maturation status of the DC [22]. This can affect their Ag processing and presentation activity as well as surface expression of co-stimulatory molecules and cytokine production, all of which can influence the capacity of the DC for priming Ag-specific T cells. Traditionally, a costimulatory role was attributed to CD69, since anti-CD69 monoclonal antibody (MAb) treatment of pre- activated human leukocytes led to further activation. In the case of T cells, the addition of anti-CD69 MAbs enhanced anti-CD3 and PMA-induced proliferation [23–25] through increased interleukin (IL)-2 and IL-2 receptor expression [26] [4]. However a later study using CD692/2 mice argued against such a role, since Ag-specific T cell proliferation was unaffected in vivo [27]. Even more in contrast, our group proposed a negative regulatory role for CD69, since tumor-bearing CD692/2 mice showed increased anti-tumor immunity to NK sensible tumors [28]. That was consistent with later in vivo results showing that CD692/2 mice had increased incidence and severity of different T cell-dependent autoimmune and inflammatory diseases such as Collagen II Induced Arthritis [29], allergic asthma, skin contact hypersensitivity [30] and autoimmune myocarditis [31]. CD692/2 mice also showed increased susceptibility to Listeria monocytogenes (Lm) infection, associated with enhanced type I and II interferon (IFN) responses [10]. Interestingly, in the tumor, arthritis and contact hypersen- sitivity models, the in vivo treatment with the anti-CD69 2.2 MAb also led to increased anti-tumor [32], autoimmune [33] and inflammatory responses [30]. However, this antibody has agonist activity, since it induces a variety of downstream functional outcomes in purified cell types, like IFNc secretion in NK cells [32], IL-2 secretion in plasmacytoid DC [34], CD25 upregulation in IL-2-treated T cells [34] and TGFb secretion when crosslinked on anti-CD3-activated T cells [28]. In vivo, anti-CD69 2.2 MAb treatment, but not CD69 gene deficiency, induced bystander proliferation of memory-phenotype T cells [34]. PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e48593