The Membrane Attack Complex (MAC or C5b-9) is an important effector of the immune system to kill invading microbes. MAC formation is initiated when complement enzymes on the bacterial surface convert complement component C5 into C5b. Although the MAC is a membrane-inserted complex, soluble forms of MAC (sMAC, or terminal complement complex (TCC)) are often detected in sera of patients suffering from infections. Consequently, sMAC has been proposed as a biomarker, but it remains unclear when and how it is formed during infections. Here, we studied mechanisms of MAC formation on different Gram-negative and Gram-positive bacteria and found that sMAC is primarily formed in human serum by bacteria resistant to MAC-dependent killing. Surprisingly, C5 was converted into C5b more potently by MAC-resistant compared to MAC-sensitive Escherichia coli strains. In addition, we found that MAC precursors are released from the surface of MAC-resistant bacteria during MAC assembly. Although release of MAC precursors from bacteria induced lysis of bystander human erythrocytes, serum regulators vitronectin (Vn) and clusterin (Clu) can prevent this. Combining size exclusion chromatography with mass spectrometry profiling, we show that sMAC released from bacteria in serum is a heterogeneous mixture of complexes composed of C5b-8, up to 3 copies of C9 and multiple copies of Vn and Clu. Altogether, our data provide molecular insight into how sMAC is generated during bacterial infections. This fundamental knowledge could form the basis for exploring the use of sMAC as biomarker.

Transforming growth factor β (TGFβ) is an important differentiation factor for cytotoxic T lymphocytes (CTLs) and alters the expression levels of several of homing-receptors during infection. SMAD4 is part of the canonical signaling network used by members of the transforming growth factor family. For this study, genetically-modified mice were used to determine how SMAD4 and TGFβ receptor II (TGFβRII) participate in transcriptional-programing of pathogen-specific CTLs. We show that these molecules are essential components of opposing signaling mechanisms, and cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissues. TGFb uses a canonical SMAD-dependent signaling pathway to down-regulate Eomesodermin (EOMES), KLRG1 and CD62L, while CD103 is induced. Conversely, in vivo and in vitro data show that EOMES, KLRG1, CX₃CR1 and CD62L are positively-regulated via SMAD4, while CD103 and Hobit are downregulated. Intravascular staining shows that signaling via SMAD4 promotes formation of terminally-differentiated CTLs that localize in the vasculature. Our data shows that inflammatory molecules play a key role in lineage-determination of pathogen-specific CTLs, and use SMAD-dependent signaling to alter the expression levels of multiple homing-receptors and transcription factors with known functions during memory formation.