Immune interferon gamma (IFN-γ) is essential for mammalian host defense against intracellular pathogens. IFN-γ induces nearly 2000 host genes, yet few have any assigned function. Here, we examined a complete mouse 65-kilodalton (kD) guanylate-binding protein (Gbp) gene family as part of a 43-member IFN-γ-inducible guanosine triphosphatase (GTPase) superfamily in mouse and human genomes. Family-wide loss-of-function analysis found that at least four Gbps--Gbp1, Gbp6, Gbp7, and Gbp10--conferred cell-autonomous immunity to listerial or mycobacterial infection within macrophages and gene-deficient animals. These Gbps solicited host defense proteins, including the phagocyte oxidase, antimicrobial peptides, and autophagy effectors, to kill intracellular bacteria. Thus, specific 65-kD Gbps coordinate a potent oxidative and vesicular trafficking program to protect the host from infection.

Macrophage migration inhibitory factor (MIF) is a pivotal regulator of the immune response. Neutralization or genetic deletion of MIF does not completely abrogate activation responses, however, and deletion of the MIF receptor, CD74, produces a more pronounced phenotype than MIF deficiency. We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a probable candidate to be the protein encoded by the homologous, D-dopachrome tautomerase (D-DT) gene. We show that recombinant D-DT protein binds CD74 with high affinity, leading to activation of ERK1/2 MAP kinase and downstream proinflammatory pathways. Circulating D-DT levels correlate with disease severity in sepsis or malignancy, and the specific immunoneutralization of D-DT protects mice from lethal endotoxemia by reducing the expression of downstream effector cytokines. These data indicate that D-DT is a MIF-like cytokine with an overlapping spectrum of activities that are important for our understanding of MIF-dependent physiology and pathology.inflammation | lipopolysaccharide | septic shock M acrophage migration inhibitory factor (MIF) is the first cytokine activity described and a key regulatory mediator that is released upon activation of different cell types (1-3). MIF increases macrophage antimicrobial responses and it is expressed upstream of cytokines such as tumor necrosis factor (TNF)-α, IFN-γ, and IL-1β (4). MIF activates immune cells by binding to CD74, leading to the recruitment of CD44 into a signaling complex, the stimulation of nonreceptor tyrosine kinases, and initiation of the ERK1/2 MAP kinase pathway (5, 6). The chemokine receptors CXCR2 and CXCR4 also become activated by MIF via noncognate interactions that are reinforced in the presence of CD74 (7). Among mesenchymal cell types, MIF binding to cardiomyocyte CD74 stimulates the AMP-activated kinase (AMPK) cascade to mediate protection from ischemic injury (8, 9).Although MIF receptor knockout mice (CD74) phenocopy features of MIF deficiency (10-12), recent observations have led to the hypothesis that there may be a second ligand for CD74. MIF-deficient B cells, for example, are more sensitive to apoptosis than wild-type B cells, but the magnitude of this defect is twofold more pronounced in CD74-deficient cells (13). Intravital microscopy studies also have shown a more pronounced effect of antagonism of CD74 than MIF in monocyte arrest (7). Finally, anti-MIF antibodies, although highly effective in experimental studies, do not completely inhibit CD74-dependent cellular activation responses (14).We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a likely candidate to be the protein encoded by the DDT gene, D-dopachrome tautomerase (D-DT). DDT and MIF show a conserved intron-exon structure and their coding regions are highly homologous. The genes for MIF and D-DT are in close apposition to each other and to two theta-class glutathione S-transferases, suggesting that these gene clusters arose by...

Combination therapy concurrently targeting PD1 and CTLA4 immune checkpoints leads to remarkable anti-tumor effects. While both PD1 and CTLA4 dampen the T cell activation, the in vivo effects of these drugs in humans remain to be clearly defined. In order to better understand biologic effects of therapy, we analyzed blood/tumor tissue from 45 patients undergoing single or combination immune checkpoint blockade. We show that blockade of CTLA4, PD1 or combination of the two leads to distinct genomic and functional signatures in vivo in purified human T cells and monocytes. Therapy-induced changes are more prominent in T cells than in monocytes and involve largely non-overlapping changes in coding genes including alternatively-spliced transcripts, and non-coding RNAs. Pathway analysis revealed that CTLA4-blockade induces a proliferative signature predominantly in a subset of transitional memory T cells, while PD1-blockade instead leads to changes in genes implicated in cytolysis and natural killer cell function. Combination blockade leads to non-overlapping changes in gene expression including proliferation-associated and chemokine genes. These therapies also have differential effects on plasma levels of CXCL10, sIL2R and IL1α. Importantly, PD1 receptor occupancy following anti-PD1 therapy may be incomplete in the tumor T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that in spite of shared property of checkpoint blockade, antibodies against PD1, CTLA4 alone or in combination have distinct immunologic effects in vivo. Improved understanding of pharmacodynamic effects of these agentsin patients will support rational development of immune-based combinations against cancer.

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