Patrick Smith

A2A adenosine receptor protects tumors from antitumor T cells

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 we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in ≈60% of A2AR-deficient mice with no rejection observed in control WT mice. The use of antagonists, including caffeine, or targeting the A2 receptors by siRNA pretreatment of T cells improved the inhibition of tumor growth, destruction of metastases, and prevention of neovascularization by antitumor T cells. The data suggest that effects of A2AR are T cell autonomous. The inhibition of antitumor T cells via their A2AR in the adenosine-rich tumor microenvironment may explain the paradoxical coexistence of tumors and antitumor immune cells in some cancer patients (the “Hellstrom paradox”). We propose to target the hypoxia→adenosine→A2AR pathway as a cancer immunotherapy strategy to prevent the inhibition of antitumor T cells in the tumor microenvironment. The same strategy may prevent the premature termination of immune response and improve the vaccine-induced development of antitumor and antiviral T cells. The observations of autoimmunity during melanoma rejection in A2AR-deficient mice suggest that A2AR in T cells is also important in preventing autoimmunity. Thus, although using the hypoxia→adenosine→A2AR pathway inhibitors may improve antitumor immunity, the recruitment of this pathway by selective drugs is expected to attenuate the autoimmune tissue damage.

Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury.

Acute respiratory distress syndrome (ARDS) usually requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the recent discovery of a critical tissue-protecting mechanism predicts that administration of oxygen to ARDS patients with uncontrolled pulmonary inflammation also may have dangerous side effects. Oxygenation may weaken the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism and thereby further exacerbate lung injury. Here we report experiments with wild-type and adenosine A2AR-deficient mice that confirm the predicted effects of oxygen. These results also suggest the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2AR-mediated lung tissue-protecting pathway. We show that this potential complication of clinically widely used oxygenation procedures could be completely prevented by intratracheal injection of a selective A2AR agonist to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous adenosine. The identification of a major iatrogenic complication of oxygen therapy in conditions of acute lung inflammation attracts attention to the need for clinical and epidemiological studies of ARDS patients who require oxygen therapy. It is proposed that oxygen therapy in patients with ARDS and other causes of lung inflammation should be combined with anti-inflammatory measures, e.g., with inhalative application of A2AR agonists. The reported observations may also answer the long-standing question as to why the lungs are the most susceptible to inflammatory injury and why lung failure usually precedes multiple organ failure.

Mouse model recapitulating human Fcγ receptor structural and functional diversity

The in vivo biological activities of IgG antibodies result from their bifunctional nature, in which antigen recognition by the Fab is coupled to the effector and immunomodulatory diversity found in the Fc domain. This diversity, resulting from both amino acid and glycan heterogeneity, is translated into cellular responses through Fcγ receptors (FcγRs), a structurally and functionally diverse family of cell surface receptors found throughout the immune system. Although many of the overall features of this system are maintained throughout mammalian evolution, species diversity has precluded direct analysis of human antibodies in animal species, and, thus, detailed investigations into the unique features of the human IgG antibodies and their FcγRs have been limited. We now report the development of a mouse model in which all murine FcγRs have been deleted and human FcγRs, encoded as transgenes, have been inserted into the mouse genome resulting in recapitulation of the unique profile of human FcγR expression. These human FcγRs are shown to function to mediate the immunomodulatory, inflammatory, and cytotoxic activities of human IgG antibodies and Fc engineered variants and provide a platform for the detailed mechanistic analysis of therapeutic and pathogenic IgG antibodies.

Targeted Deletion of HIF-1α Gene in T Cells Prevents their Inhibition in Hypoxic Inflamed Tissues and Improves Septic Mice Survival

Background Sepsis patients may die either from an overwhelming systemic immune response and/or from an immunoparalysis-associated lack of anti-bacterial immune defence. We hypothesized that bacterial superantigen-activated T cells may be prevented from contribution into anti-bacterial response due to the inhibition of their effector functions by the hypoxia inducible transcription factor (HIF-1α) in inflamed and hypoxic areas. Methodology/Principal Findings Using the Cre-lox-P-system we generated mice with a T–cell targeted deletion of the HIF-1α gene and analysed them in an in vivo model of bacterial sepsis. We show that deletion of the HIF-1α gene leads to higher levels of pro-inflammatory cytokines, stronger anti-bacterial effects and much better survival of mice. These effects can be at least partially explained by significantly increased NF-κB activation in TCR activated HIF-1 α deficient T cells. Conclusions/Significance T cells can be recruited to powerfully contribute to anti-bacterial response if they are relieved from inhibition by HIF-1α in inflamed and hypoxic areas. Our experiments uncovered the before unappreciated reserve of anti-bacterial capacity of T cells and suggest novel therapeutic anti-pathogen strategies based on targeted deletion or inhibition of HIF-1 α in T cells.

FcγRIV deletion reveals its central role for IgG2a and IgG2b activity in vivo

Cellular Fcγ receptors are essential for IgG-dependent effector functions in vivo. There is convincing evidence that selective activating Fcγ receptors are responsible for the activity of individual IgG subclasses. Thus, IgG1 activity is absent in FcγRIII-deficient mice, and several studies suggest that the activity of the most potent IgG subclasses, IgG2a and IgG2b, might be dependent on either individual or a combination of activating FcγRs. To study the role of individual activating FcγRs for IgG subclass activity, we generated an FcγRIV-deficient mouse and showed that a variety of IgG2a- and IgG2b-dependent effector functions are impaired in the absence of this activating Fc receptor in models of autoimmunity and antibody-dependent cellular cytotoxicity.

Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling

Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA(-/-)) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA(-/-) mice. T cell apoptosis was abundant in thymi of ADA(-/-) mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA(-/-) mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA(-/-) T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA(-/-) mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.

Structure of the Absent in Melanoma 2 (AIM2) Pyrin Domain Provides Insights into the Mechanisms of AIM2 Autoinhibition and Inflammasome Assembly

Background: AIM2 binds dsDNA and associates with ASC through their PYDs to form an inflammasome. Results: The AIM2 PYD structure illustrates distinct charge distribution and a unique hydrophobic patch. Conclusion: The AIM2 PYD may bind the ASC PYD and the AIM2 HIN domain through overlapping surface. Significance: These findings provide insights into the mechanisms of AIM2 autoinhibition and inflammasome assembly. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (dsDNA) sensor essential for innate immune responses against DNA viruses and bacteria such as Francisella and Listeria. Upon dsDNA engagement, the AIM2 amino-terminal pyrin domain (PYD) is responsible for downstream signaling to the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through homotypic PYD-PYD interactions and the assembly of an inflammasome. Toward a better understanding of the AIM2 signaling mechanism, we determined the crystal structure of the human AIM2 PYD. The structure reveals a death domain fold with a short α3 helix that is buttressed by a highly conserved lysine residue at the α2 helix, which may stabilize the α3 helix for potential interaction with partner domains. The surface of the AIM2 PYD exhibits distinct charge distribution with highly acidic α1-α2 helices and highly basic α5-α6 helices. A prominent solvent-exposed hydrophobic patch formed by residues Phe-27 and Phe-28 at the α2 helix resembles a similar surface involved in the death effector domain homotypic interactions. Docking studies suggest that the AIM2 PYD may bind the AIM2 hematopoietic interferon-inducible nuclear (HIN) domain or ASC PYD using overlapping surface near the α2 helix. This may ensure that AIM2 interacts with the downstream adapter ASC only upon release of the autoinhibition by the dsDNA ligand. Our work thus unveils novel structural features of the AIM2 PYD and provides insights into the potential mechanisms of the PYD-HIN and PYD-PYD interactions important for AIM2 autoinhibition and inflammasome assembly.

Analysis of A2a receptor-deficient mice reveals no significant compensatory increases in the expression of A2b, A1, and A3 adenosine receptors in lymphoid organs

Although recent genetic and pharmacologic in vivo studies of acute inflammation models in mice demonstrated that the cyclic AMP-elevating A2a receptor plays a non-redundant role in protection from excessive acute inflammatory tissue damage and in the down-regulation of proinflammatory cytokine production, it remained to be established whether genetic deficiency of the A2a receptor is accompanied by a compensatory up-regulation of the cAMP-elevating A2b receptor and/or other adenosine receptors. Here, we show that most of the cAMP response to adenosine is abolished in lymphoid tissues of A2a receptor-deficient mice, although some response remains in splenocytes. No significant changes were observed in A2b, A1, and A3 mRNA levels in the thymus or lymph nodes of A2a receptor-deficient mice, but small increases in mRNA expression of these receptors were detected in the spleen. These data suggest that regulation of the expression of A2b, A1, and A3 receptors is not affected significantly by the absence of A2a receptors and may provide further explanation of earlier in vivo observations of increased tissue damage and of longer persistence of proinflammatory cytokines in animals with inactivated A2a receptors.

Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli.

The Toll/IL-1 receptor (TIR) domains are crucial signaling modules during innate immune responses involving the Toll-like receptors (TLRs) and IL-1 receptor (IL-1R). Myeloid differential factor 88 (MyD88) is a central TIR domain-containing adapter molecule responsible for nearly all TLR-mediated signaling and is targeted by a TIR domain-containing protein C (TcpC) from virulent uropathogenic Escherichia coli, a common human pathogen. The mechanism of such molecular antagonism has remained elusive. We present the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. Our structural analyses shed light on the genetic mutations at these loops as well as the Poc site. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE, and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR signaling by the uropathogenic E. coli virulence factor TcpC and furnish a framework for the design of novel therapeutic agents that modulate immune activation.

Crystal structures of the Toll/Interleukin-1 receptor (TIR) domains from the Brucella protein TcpB and host adaptor TIRAP reveal mechanisms of molecular mimicry.

Background: The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Results: The crystal structures of the TIR domains from TcpB and TIRAP reveal similar folds and distinct features. Conclusion: TcpB may mimic the function of TIRAP through their similar TIR domain structures. Significance: These findings suggest mechanisms of bacterial mimicry of host signaling adaptor proteins. The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys89 and Cys134. A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.