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Mouse infection with intracellular bacteria induces a potent inflammatory response that requires protective mechanisms to avoid infection-induced immune pathology. CD69 is expressed in all leukocytes during activation after infection with a wide range of microbial pathogens. This study explores the way in which CD69 affects cell activation after Listeria monocytogenes (Lm) infection and its effects on host protection. We show that infectivity and bacterial clearance capability are unaltered in CD69(-/-) peritoneal macrophages, bone marrow-derived macrophages and dendritic cells. We found no major altered cell populations in splenocytes of Lm-infected CD69(-/-) mice. However, an increase in the expression of Th1 cytokines was observed after infection, with increased production of type I and II interferon (IFN). In addition, CD69(-/-) splenocytes showed increased apoptosis, consistent with IFN enhancement of lymphocyte apoptosis in response to Lm infection. CD69(-/-) mice showed liver and spleen damage, and greatly increased susceptibility to Lm infection, compared with wild-type controls. Lm-specific T cells were decreased in CD69(-/-) mice even if T-cell cross-presentation and T-cell intrinsic priming response were not compromised. As listeriosis was increased as early as day 1 post-infection but CD69(-/-)RAG2(-/-) mice were more efficient at controlling Listeria, we propose that CD69 controls the cross-talk between innate components and lymphocytes. These results highlight a role for CD69 in preventing infection-induced immunopathology.

CD69 expression is induced following activation of leukocytes at inflammatory sites and plays a negative regulatory role in the development of collagen-induced arthritis (CIA). To evaluate potential strategies of CD69 targeting in chronic inflammatory diseases, two different anti-CD69 mAbs were generated and their effects on CIA were studied. Administration of the IgG1 anti-CD69 mAb 2.2 to DBA/1 mice with CIA led to an exacerbation of the disease, correlated with down-modulation of CD69 from the cell surface, and reproduced the phenotype of the CD69(-/-) mouse in wild-type animals. In contrast, treatment with the IgG2a anti-CD69 mAb 2.3 was effective in ameliorating CIA when administered in the early or intermediate phases of the disease, causing a decreased production of proinflammatory cytokines in inflammatory foci. Monoclonal antibody 2.3 induces partial depletion of CD69+ cells in vivo. Moreover, adoptive transfer of type-II collagen (CII)-sensitized cells treated with mAb 2.3 to deplete CD69+ cells did not result in arthritis. The attenuation of inflammation correlates with reduced lymphocyte proliferative response in response to CII and with a reduction in the frequency of CII-specific T cells producing IFN-gamma. We thus conclude that CD69 targeting by mAbs can either enhance or dampen the immune response.

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.

The leukocyte activation marker CD69 is a novel regulator of the immune response, modulating the production of cytokines including transforming growth factor-beta (TGF-beta). We have generated an antimurine CD69 monoclonal antibody (mAb), CD69.2.2, which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell-dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69(-/-) mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-beta production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor-independent manner, resulting in a substantial increase in both NK-cell cytolytic activity and interferon gamma (IFNgamma) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis.

CD69 is induced after activation of leukocytes at inflammatory sites, but its physiological role during inflammation remains unknown. We explored the role of CD69 in autoimmune reactivity by analyzing a model of collagen-induced arthritis (CIA) in WT and CD69-deficient mice. CD69-/- mice showed higher incidence and severity of CIA, with exacerbated T and B cell immune responses to type II collagen. Levels of TGF-beta1 and TGF-beta2, which act as protective agents in CIA, were reduced in CD69-/- mice inflammatory foci, correlating with the increase in the proinflammatory cytokines IL-1beta and RANTES. Local injection of blocking anti-TGF-beta antibodies increased CIA severity and proinflammatory cytokine mRNA levels in CD69+/+ but not in CD69-/- mice. Moreover, in vitro engagement of CD69 induced total and active TGF-beta1 production in Concanavalin A-activated splenocyte subsets, mouse and human synovial leukocytes, and Jurkat stable transfectants of human CD69 but not in the parental CD69 negative cell line. Our results show that CD69 is a negative modulator of autoimmune reactivity and inflammation through the synthesis of TGF-beta, a cytokine that in turn downregulates the production of various proinflammatory mediators.

We investigated the in vivo role of CD69 by analyzing the susceptibility of CD69-/- mice to tumors. CD69-/- mice challenged with MHC class I- tumors (RMA-S and RM-1) showed greatly reduced tumor growth and prolonged survival compared with wild-type (WT) mice. The enhanced anti-tumor response was NK cell and T lymphocyte-mediated, and was due, at least in part, to an increase in local lymphocytes. Resistance of CD69-/- mice to MHC class I- tumor growth was also associated with increased production of the chemokine MCP-1, diminished TGF-beta production, and decreased lymphocyte apoptosis. Moreover, the in vivo blockade of TGF-beta in WT mice resulted in enhanced anti-tumor response. In addition, CD69 engagement induced NK and T cell production of TGF-beta, directly linking CD69 signaling to TGF-beta regulation. Furthermore, anti-CD69 antibody treatment in WT mice induced a specific down-regulation in CD69 expression that resulted in augmented anti-tumor response. These data unmask a novel role for CD69 as a negative regulator of anti-tumor responses and show the possibility of a novel approach for the therapy of tumors.

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.