Maria Letizia Giardino Torchia

Outer Membrane Vesicles of a Human Commensal Mediate Immune Regulation and Disease Protection

Commensal bacteria impact host health and immunity through various mechanisms, including the production of immunomodulatory molecules. Bacteroides fragilis produces a capsular polysaccharide (PSA), which induces regulatory T cells and mucosal tolerance. However, unlike pathogens, which employ secretion systems, the mechanisms by which commensal bacteria deliver molecules to the host remain unknown. We reveal that Bacteroides fragilis releases PSA in outer membrane vesicles (OMVs) that induce immunomodulatory effects and prevent experimental colitis. Dendritic cells (DCs) sense OMV-associated PSA through TLR2, resulting in enhanced regulatory T cells and anti-inflammatory cytokine production. OMV-induced signaling in DCs requires growth arrest and DNA-damage-inducible protein (Gadd45α). DCs treated with PSA-containing OMVs prevent experimental colitis, whereas Gadd45α(-/-) DCs are unable to promote regulatory T cell responses or suppress proinflammatory cytokine production and host pathology. These findings demonstrate that OMV-mediated delivery of a commensal molecule prevents disease, uncovering a mechanism of interkingdom communication between the microbiota and mammals.

The CD8+ memory T-cell state of readiness is actively maintained and reversible

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.

Optineurin Insufficiency Impairs IRF3 but Not NF-κB Activation in Immune Cells

Optineurin is a widely expressed polyubiquitin-binding protein that has been implicated in regulating cell signaling via its NF-κB essential modulator–homologous C-terminal ubiquitin (Ub)-binding region. Its functions are controversial, with in vitro studies finding that optineurin suppressed TNF-mediated NF-κB activation and virus-induced activation of IFN regulatory factor 3 (IRF3), whereas bone marrow–derived macrophages (BMDMs) from mice carrying an optineurin Ub-binding point mutation had normal TLR-mediated NF-κB activation and diminished IRF3 activation. We have generated a mouse model in which the entire Ub-binding C-terminal region is deleted (Optn470T). Akin to C-terminal optineurin mutations found in patients with certain neurodegenerative diseases, Optn470T was expressed at substantially lower levels than the native protein, allowing assessment not only of the lack of Ub binding, but also of protein insufficiency. Embryonic lethality with incomplete penetrance was observed for 129 × C57BL/6 Optn470T/470T mice, but after further backcrossing to C57BL/6, offspring viability was restored. Moreover, the mice that survived were indistinguishable from wild type littermates and had normal immune cell distributions. Activation of NF-κB in Optn470T BMDM and BM-derived dendritic cells with TNF or via TLR4, T cells via the TCR, and B cells with LPS or anti-CD40 was normal. In contrast, optineurin and/or its Ub-binding function was necessary for optimal TANK binding kinase 1 and IRF3 activation, and both Optn470T BMDMs and bone marrow–derived dendritic cells had diminished IFN-β production upon LPS stimulation. Importantly, Optn470T mice produced less IFN-β upon LPS challenge. Therefore, endogenous optineurin is dispensable for NF-κB activation but necessary for optimal IRF3 activation in immune cells.

Lack of the T-cell–specific alternative p38 activation pathway reduces autoimmunity and inflammation

Stimulation via the T-cell receptor (TCR) activates p38α and p38β by phosphorylation of p38 Tyr-323 (p38(Y323)). Here we characterize knockin mice in which p38α and/or β Tyr-323 has been replaced with Phe. We find that p38α accounts for two-thirds and p38β the remainder of TCR-induced p38 activation. T cells from double knockin mice (p38αβ(Y323F)) had defects in TCR-mediated proliferation and Th1 and Th17 skewing, the former corresponding with an inability to sustain T-bet expression. Introduction of p38α(Y323F) into Gadd45α-deficient mice, in which the alternative p38 pathway is constitutively active, reversed T-cell hyperproliferation and autoimmunity. Furthermore, p38αβ(Y323F) mice had delayed onset and reduced severity of the inflammatory autoimmune diseases collagen-induced arthritis and experimental autoimmune encephalomyelitis. Thus, T cell-specific alternative activation of p38 is an important pathway in T-cell proliferation, Th skewing, and inflammatory autoimmunity, and may be an attractive tissue-specific target for intervention in these processes.

CD70 Is Downregulated by Interaction with CD27

Engagement of the receptor CD27 by CD70 affects the magnitude and quality of T cell responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic cells (DCs), T cells, and B cells only upon activation. In this article, we show that a second level of regulation also is present. First, although undetectable on the cell surface by flow cytometry, immature DCs have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DCs and T cells were higher in mice deficient in CD27, or on DCs for which the interaction between CD70 and CD27 was precluded by blocking Abs. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor.

Balance between NF-κB p100 and p52 Regulates T Cell Costimulation Dependence

c-IAP1 and c-IAP2 are ubiquitin protein ligases (E3s) that repress noncanonical NF-κB activation. We have created mice that bear a mutation in c-IAP2 that inactivates its E3 activity and interferes, in a dominant-negative fashion, with c-IAP1 E3 activity (c-IAP2H570A). The immune response of these animals was explored by infecting them with the Th1-inducing parasite Toxoplasma gondii. Surprisingly, c-IAP2H570A mice succumbed because of T cell production of high levels of proinflammatory cytokines. Unlike naive wild-type (WT) cells, which require signals generated by the TCR and costimulatory receptors to become fully activated, naive c-IAP2H570A T cells proliferated and produced high levels of IL-2 and IFN-γ to stimulation via TCR alone. c-IAP2H570A T cells had constitutive noncanonical NF-κB activation, and IκB kinase inhibition reduced their proliferation to anti-TCR alone to WT levels but had no effect when costimulation via CD28 was provided. Notably, T cells from nfkb2−/− mice, which cannot generate the p52 component of noncanonical NF-κB, were also costimulation independent, consistent with the negative role of this unprocessed protein in canonical NF-κB activation. Whereas T cells from nfkb2+/− mice behaved like WT, coexpression of a single copy of c-IAP2H570A resulted in cleavage of p100, upregulation of p52, and T cell costimulation independence. Thus, p100 represses and p52 promotes costimulation, and the ratio regulates T cell dependence on costimulatory signals.

The TBK1-binding domain of optineurin promotes type I interferon responses.

Pathogen‐associated molecular pattern (PAMP) recognition leads to TANK‐binding kinase (TBK1) polyubiquitination and activation by transautophosphorylation, resulting in IFN‐β production. Here, we describe a mouse model of optineurin insufficiency (OptnΔ157) in which the TBK1‐interacting N‐terminus of optineurin was deleted. PAMP‐stimulated cells from OptnΔ157 mice had reduced TBK1 activity, no phosphorylation of optineurin Ser187, and mounted low IFN‐β responses. In contrast to pull‐down assays where the presence of N‐terminus was sufficient for TBK1 binding, both the N‐terminal and the ubiquitin‐binding regions of optineurin were needed for PAMP‐induced binding. This report establishes optineurin as a positive regulator TBK1 via a bipartite interaction between these molecules.

c-IAP1 and c-IAP2 Redundancy Differs between T and B Cells

Cellular Inhibitors of Apoptosis 1 and 2 (c-IAP1 and c-IAP2) are ubiquitin protein ligases (E3s) that constitutively ubiquitinate and induce proteasomal-mediated degradation of NF-κB Inducing Kinase (NIK) and repress non-canonical NF-κB activation. Mice expressing an E3-inactive c-IAP2 mutant (c-IAP2H570A) have constitutive activation of non-canonical NF-κB, resulting in B cell hyperplasia and T cell costimulation-independence. If, and if so to what extent, c-IAP1 and c-IAP2 are redundant in NF-κB regulation in these mice is not known. Here we have generated mice expressing a mutant c-IAP1 that lacks E3 activity (c-IAP1H582A). These mice were phenotypically normal and did not have constitutive NF-κB activation in B cells or MEFs. siRNA-mediated knockdown of c-IAP2 showed that accumulated c-IAP2, resulting from lack of c-IAP1-dependent degradation, compensated for absent c-IAP1 E3 activity. Surprisingly, c-IAP1H582A T cells had a lower p100/p52 ratio than wild type T cells, and in the absence of costimulation proliferated to a degree intermediate between wild type and c-IAP2H570A T cells. Therefore, although c-IAP1 and c-IAP2 both can repress constitutive NF-κB activation, the relative importance of each varies according to cell type.

c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8+ memory T-cell survival

Cellular inhibitor of apoptosis proteins (c‐IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4‐1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c‐IAPs to upregulate NF‐κB and ERK, and has been implicated in memory T‐cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3‐inactive c‐IAP2 (c‐IAP2H570A) have impaired signaling downstream of 4‐1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c‐IAPs were acutely downregulated by c‐IAP antagonists, the primary response of c‐IAP2H570A mice was normal. However, the number of antigen‐specific CD8+ but not CD4+ T cells declined more rapidly and to a greater extent in c‐IAP2H570A mice than in WT controls. Studies with T‐cell adoptive transfer demonstrated that the enhanced decay of memory cells was T‐cell intrinsic. Thus, c‐IAP E3 activity is required for 4‐1BB coreceptor signaling and maintenance of CD8+ T‐cell memory.

Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70T293 and destabilization of the signalosome

Significance Activation of T cells via their antigen receptor (TCR) and the formation of a multimolecular signaling complex in or near the plasma membrane is a key initial event in the generation of an immune response. Here we find that p38, a serine/threonine kinase activated by TCR signaling, in turn phosphorylates its upstream tyrosine kinase ZAP-70. This results in a decrease in the size and longevity of the TCR signaling complex, limiting T cell effector responses. Therefore, cross-talk between an upstream and downstream kinase in the signaling complex itself generates a negative feedback loop that may limit excessive T cell responses. ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70–activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.