The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here autoimmunity ͉ cancer ͉ therapy ͉ hypoxia ͉ inflammation T he coexistence of tumors and antitumor immune cells is currently explained by the inhibition of immune cells in a poorly understood ''hostile'' tumor microenvironment (1-3). This unidentified immunosuppressive mechanism limits promising cancer therapies using antitumor T cells (4-14). We hypothesized that cancerous tissues are protected from antitumor T cells because of immunosuppressive signaling via T cell A2A adenosine receptor (A2AR) (15-17) activated by extracellular adenosine produced from hypoxic tumor (Fig. 1a). Indeed, hypoxic cancerous tissues may be protected by the same hypoxia3adenosine3A2AR pathway that was recently shown to be critical and nonredundant in preventing excessive damage of normal tissues by overactive immune cells in vivo (18). It is well established that some areas of solid tumors often have transient or chronic hypoxia (19,20), which is conducive to extracellular adenosine accumulation (21). Hypoxia has been implicated in mechanisms of tumor protection against ionizing radiation and some chemotherapeutic agents (19) and is associated with poor prognosis (20).T cells, including antitumor T cells, do predominantly express cAMP-elevating Gs protein-coupled high-affinity A2AR and͞or low-affinity A2B adenosine receptors (A2BR) (16,17,(22)(23)(24); the number of A2AR per T cell may determine the intensity of maximal T cell response to adenosine (25, 26). Whereas we focused on A2AR, others have discounted A2 receptors and suggested the A3 adenosine receptors as responsible for inhibition of antitumor killer T cells (27,28). Here we report that genetic deletion of A2AR accomplishes the complete rejection of immunogenic tumors by antitumor CD8 ϩ T cells in the majority (Ϸ60%) of mice, whereas the antagonists of A2 receptors facilitate CD8 ϩ T cell-mediated retardation of tumor growth. Results The Gradient of T Cell-Inhibiting Extracellular Adenosine in Tumors.It was important to confirm the presence of elevated extracellular adenosine levels in cancerous tissues using a reliable method (29). The HPLC analysis and the use of equilibrium dialysis probes demonstrated higher levels of extracellular adenosine (Fig. 1b), increased adenosine metabolism, and the concomitant increase in cAMP (29) in a solid tumor microenvironment (Fig. 7, which is published as supporting information on the PNAS web site). We also confirmed that antitumor CD8 ϩ T cells used in this study do express the cAMP-elevating functional A2AR and A2BR (Fig. 1c). To directly test whether A2AR inhibit antitumor T cells in vivo, we studied the effects of A2AR gene deletion or competitive antagonists on tumor growth in mice using different CD8 ϩ T celldependent cancer immunosurveillance and ad...

Highlights d Type I interferon drives differentiation of inf-cDC2s that closely resemble MCs d Inf-cDC2s prime CD4 + and CD8 + T cells, whereas MCs lack APC function d Inf-cDC2s internalize antibody-complexed antigen via Fc receptors d IRF8 controls maturation gene module in inf-cDC2s

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The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable ∼40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in “memory” mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.

The functions and fate of antigen-experienced T cells isolated from lymph node or nonlymphoid tissues were analyzed in a system involving adoptive transfer of in vitro–activated T cells into mice. Activated T cells present in the lymph nodes could be stimulated by antigen to divide, produce effector cytokines, and migrate to peripheral tissues. By contrast, activated T cells that had migrated into nonlymphoid tissues (lung and airway) produced substantial effector cytokines upon antigen challenge, but were completely unable to divide or migrate back to the lymph nodes. Therefore, activated T cells can undergo clonal expansion in the lymph node, but are recruited and retained as nondividing cells in nonlymphoid tissues. These distinct regulatory events in lymph node and nonlymphoid tissues reveal simple key mechanisms for both inducing and limiting T cell immunity.

The lifespan and survival of dendritic cells (DC) in vivo are potentially critical to the expansion of T cell immune responses. We have previously reported that DC loaded with specific antigen are rapidly eliminated by cytotoxic T lymphocytes (CTL) in vivo, but the site, mechanism, and consequences of DC elimination were not defined. In this article we show that DC elimination in vivo occurs in a perforin-dependent manner and does not require IFN-␥ or the presence of CD4 ؉ CD25 ؉ regulatory T cells. Most importantly, failure to eliminate DC had profound consequences on the CTL immune response. Perforin-deficient mice showed a progressive increase in the numbers of antigen-specific CD8 ؉ T cells after repeated immunizations with DC. In contrast, in control mice the number of antigen-specific CD8 ؉ T cells did not notably increase with repeated immunizations. Lastly, we also show that CTLmediated elimination of DC occurs in peripheral tissues but not in the lymph node. Our data suggest that CTL act as ''gatekeepers'' that control access of antigen-loaded DC into the lymph node, thereby preventing continued expansion of antigen-specific T cells.cytotoxic T cells ͉ immunoregulation ͉ killing D endritic cells (DC) are powerful antigen-presenting cells that are critical for the initiation of CD4 ϩ and CD8 ϩ T cell responses. DC reside in peripheral tissues where they take up antigens from the external environment, then migrate to the lymph nodes where they interact with antigen-specific T cells and induce their activation to proliferation and effector function (1, 2). The lifespan of DC once in the lymph node is thought to be relatively brief, but experimental estimates have not yielded a consistent figure (3-5).The mechanism and regulation of DC survival and death (6, 7) are likely to be important in maintaining the homeostatic balance of the immune system. A few reports have linked extended survival of DC to enhanced or dysregulated T cell immune responses and lymphoproliferative disease (8-10). In contrast, reduced survival of DC has been associated with impaired immune responses (7). It is presently unclear whether T cells also influence the lifespan of DC in an antigen-specific fashion. In lymph nodes of mice adoptively transferred with CD4 ϩ T cell receptor (TCR) transgenic T cells, DC presenting specific antigen disappear more rapidly than DC not presenting antigen (11), suggesting that T cells may have a role in regulating DC numbers and survival. Experiments using infection with Listeria or malaria also suggest that the number of antigen-presenting cells becomes limiting during the early phases of CD8 ϩ immune responses and prevents the continued expansion of antigen-specific T cells (12, 13). Together, these experiments suggest the attractive hypothesis that T cells may be able to regulate their own responses in a feedback fashion, by affecting the survival of antigen-presenting DC.We have previously reported that DC loaded with antigen and injected s.c. into immune mice are rapidly eliminated by CD8 ϩ T cel...

cross-presentation ͉ dendritic cells ͉ epidermis ͉ mouse or murine ͉ epicutaneous

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