Tissue-resident memory T cells (T(RM) cells) provide superior protection against infection in extralymphoid tissues. Here we found that CD103(+)CD8(+) T(RM) cells developed in the skin from epithelium-infiltrating precursor cells that lacked expression of the effector-cell marker KLRG1. A combination of entry into the epithelium plus local signaling by interleukin 15 (IL-15) and transforming growth factor-β (TGF-β) was required for the formation of these long-lived memory cells. Notably, differentiation into T(RM) cells resulted in the progressive acquisition of a unique transcriptional profile that differed from that of circulating memory cells and other types of T cells that permanently reside in skin epithelium. We provide a comprehensive molecular framework for the local differentiation of a distinct peripheral population of memory cells that forms a first-line immunological defense system in barrier tissues.

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-β production, and decreased lymphocyte apoptosis. Moreover, the in vivo blockade of TGF-β in WT mice resulted in enhanced anti–tumor response. In addition, CD69 engagement induced NK and T cell production of TGF-β, directly linking CD69 signaling to TGF-β 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.

SummaryThe rearrangement and expression of T cell receptor (TCR.) gene segments occurs in a highly ordered fashion during thymic ontogeny of T lymphocytes. To study the regulation of gene rearrangement within the TCR c~/~ locus, we generated transgenic mice that carry a germline human TCR 5 minilocus that includes V~I, V~2, D~3, J~l, J~3, and C~ segments, and either contains or lacks the TCR ~ enhancer. We found that the enhancer-positive construct rearranges stepwise, first V to D, and then V-D to J. Construct V-D rearrangement mimics a unique property of the endogenous TCR 5 locus. V-D-J rearrangement is T cell specific, but is equivalent in c~/3 and 3"/6 T lymphocytes. Thus, either there is no commitment to the c~//~ and 7/~5 T cell lineages before TCR 5 gene rearrangement, or if precommitment occurs, it does not operate directly on TCR ~ gene cis-acting regulatory elements to control TCR 5 gene rearrangement. Enhancer-negative mice display normal V to D rearrangement, but not V-D to J rearrangement. Thus, the V-D to J step is controlled by the enhancer, but the V to D step is controlled by separate elements. The enhancer apparently controls access to J~l but not D~3, suggesting that a boundary between two independently regulated domains of the minilocus lies between these elements. Within the endogenous TCR ot/~5 locus, this boundary may represent the 5' end of a chromatin regulatory domain that is opened by the TCR ~ enhancer during T cell development. The position of this boundary may explain the unique propensity of the TCR di locus to undergo early V to D rearrangement. Our results indicate that the TCR ~ enhancer performs a crucial targeting function to regulate TCR (5 gene rearrangement during T cell development.T he ability of the immune system to recognize a diverse universe of antigens results in large part from the process of V-D-J recombination that assembles the genes encoding antigen receptors on T and B lymphocytes (1-3). Studies of lymphocyte development reveal that the assembly of antigen receptor genes is under stringent developmental control. The rearrangement of Ig genes occurs in a stepwise fashion during B cell maturation, with initial D to J joining followed by V to D-J joining at the H chain locus, and sub-

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-β1 and TGF-β2, which act as protective agents in CIA, were reduced in CD69 -/-mice inflammatory foci, correlating with the increase in the proinflammatory cytokines IL-1β and RANTES. Local injection of blocking anti-TGF-β 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-β1 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-β, a cytokine that in turn downregulates the production of various proinflammatory mediators.