Enza Piccolella

The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis

Psoriasis is a common T-cell-mediated skin disease with 2-3% prevalence worldwide. Psoriasis is considered to be an autoimmune disease, but the precise nature of the autoantigens triggering T-cell activation remains poorly understood. Here we find that two-thirds of patients with moderate-to-severe plaque psoriasis harbour CD4(+) and/or CD8(+) T cells specific for LL37, an antimicrobial peptide (AMP) overexpressed in psoriatic skin and reported to trigger activation of innate immune cells. LL37-specific T cells produce IFN-γ, and CD4(+) T cells also produce Th17 cytokines. LL37-specific T cells can infiltrate lesional skin and may be tracked in patients blood by tetramers staining. Presence of circulating LL37-specific T cells correlates significantly with disease activity, suggesting a contribution to disease pathogenesis. Thus, we uncover a role of LL37 as a T-cell autoantigen in psoriasis and provide evidence for a role of AMPs in both innate and adaptive immune cell activation.

Multispecific T cell response and negative HCV RNA tests during acute HCV infection are early prognostic factors of spontaneous clearance

Background/Aims: Hepatitis C virus (HCV) infection results in a high frequency of chronic disease. The aim of this study was to identify early prognostic markers of disease resolution by performing a comprehensive analysis of viral and host factors during the natural course of acute HCV infection. Methods: The clinical course of acute hepatitis C was determined in 34 consecutive patients. Epidemiological and virological parameters, as well as cell mediated immunity (CMI) and distribution of human leukocyte antigens (HLA) alleles were analysed. Results: Ten out of 34 patients experienced self-limiting infection, with most resolving patients showing fast kinetics of viral clearance: at least one negative HCV RNA test during this phase predicted a favourable outcome. Among other clinical epidemiological parameters measured, the self-limiting course was significantly associated with higher median peak bilirubin levels at the onset of disease, and with the female sex, but only the latter parameter was independently associated after multivariate analysis. No significant differences between self-limiting or chronic course were observed for the distribution of DRB1 and DQB1 alleles. HCV specific T cell response was more frequently detected during acute HCV infection, than in patients with chronic HCV disease. A significantly broader T cell response was found in patients with self-limiting infection than in those with chronic evolving acute hepatitis C. Conclusion: The results suggest that host related factors, in particular sex and CMI, play a crucial role in the spontaneous clearance of this virus. Most importantly, a negative HCV RNA test and broad CMI within the first month after onset of the symptoms represent very efficacious predictors of viral clearance and could thus be used as criteria in selecting candidates for early antiviral treatment.

Vav cooperates with CD28 to induce NF-κB activation via a pathway involving Rac-1 and mitogen-activated kinase kinase 1

CD28‐delivered costimulatory signals are required to induce NF‐κB activation in response to TCR stimulation. We have recently demonstrated that the mitogen‐activated kinase kinase 1 (MEKK1), a kinase known to regulate the c‐jun N‐terminal kinase (JNK) pathway, is also involved in the CD28‐ and TCR‐induced inhibitor of κB factor (IκB) kinases (IKK) and NF‐κB activation. Searching for molecules that couple TCR and CD28 to MEKK1, we found that the guanine nucleotide exchange factor Vav synergized with CD28 stimulation in Jurkat cells to induce NF‐κB transcriptional activity through the activation of IKKα and IKKβ. Dominant negative mutants of Vav inhibited TCR‐ and CD28‐NF‐κB‐dependent transcription by interfering with the activation of the IKK complex. Blocking Rac signaling downstream of Vav by dominant negative RacN17 exerts similar effects on IKK and NF‐κB activation after TCR/CD28 stimulation. Finally, Vav‐induced NF‐κB activation in CD28 costimulated cells was inhibited by dominant negative MEKK(KM). These results identify Vav, Rac‐1 and MEKK1 as components of a common pathway regulating both NF‐κB and AP‐1 that contributes to full activation of the CD28 response element (CD28RE).

Early impairment of hepatitis C virus specific T cell proliferation during acute infection leads to failure of viral clearance

Background and aims: Cellular mediated immunity (CMI) is thought to play a key role in resolution of primary hepatitis C virus (HCV) infection. However, CD4+ and CD8+ T cell responses are also generated during acute infection in individuals who become chronic, suggesting that they developed a defective CMI. The aim of this study was to verify if and when such immune dysfunction is established by measuring the breadth, magnitude, function, and duration of CMI in a large cohort of subjects during the natural course of acute HCV infection. Methods: CMI was comprehensively studied by prospective sampling of 31 HCV acutely infected subjects enrolled at the onset of infection and followed for a median period of one year. Results: Our results indicated that while at the onset of acute HCV infection a measurable CMI with effector function was detected in the majority of subjects, after approximately six months less than 10% of chronically infected individuals displayed significant CMI compared with 70% of subjects who cleared the virus. We showed that progressive disappearance of HCV specific T cells from the peripheral blood of chronic patients was due to an impaired ability to proliferate that could be rescued in vitro by concomitant exposure to interleukin 2 and the antigen. Conclusion: Our data provide evidence of strong and multispecific T cell responses with a sustained ability to proliferate in response to antigen stimulation as reliable pharmacodynamic measures of a protective CMI during acute infection, and suggest that early impairment of proliferation may contribute to loss of T cell response and chronic HCV persistence.

Human Anergic CD4+ T Cells Can Act as Suppressor Cells by Affecting Autologous Dendritic Cell Conditioning and Survival

T cell suppression exerted by regulatory T cells represents a well-established phenomenon, but the mechanisms involved are still a matter of debate. Recent data suggest that anergic T cells can suppress responder T cell activation by inhibiting Ag presentation by dendritic cells (DC). In this study, we focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and analyzed the fate of DC and responder T cells. To address this issue, we have cocultured human alloreactive or Ag-specific CD4+ T cell clones, rendered anergic by incubation with immobilized anti-CD3 Ab, with autologous DC and responder T cells. We show that anergic T cells affect either Ag-presenting functions or survival of DC, depending whether immature or mature DC are used as APC. Indeed, MHC and costimulatory molecule expression on immature DC activated by responder T cells is inhibited, while apoptotic programs are induced in mature DC and in turn in responder T cells. Ligation of CD95 by CD95L expressed on anergic T cells in the absence of CD40-CD40L (CD154) interaction are critical parameters in eliciting apoptosis in both DC and responder T cells. In conclusion, these findings indicate that the defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cells could be the primary event in determining T cell suppression and support the role of CD40 signaling in regulating both conditioning and survival of DC.

Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection.

Cellular immune responses are induced during hepatitis C virus (HCV) infection and acute‐phase CD8+ T cells are supposed to play an important role in controlling viral replication. In chimpanzees, failure of CD8+ T cells to control HCV replication has been associated with acquisition of mutations in MHC class I‐restricted epitopes. In humans, although selection of escape mutations in an immunodominant CTL epitope has been recently described, the overall impact of immune escape during acute HCV infection is unclear. Here, by performing an in depth analysis of the relationship between early cellular immune responses and viral evolution in a chronically evolving HCV acutely infected individual, we demonstrate: (i) the presence of a potent and focused CD8+ T cell response against a novel epitope in the NS3 protein, (ii) the elimination of the quasi‐species harboring the original amino acid sequence within this epitope, and (iii) the selection for a virus population bearing amino acid changes at a single residue within the cytotoxic T cell epitope that strongly diminished T cell recognition. These results support the view that acute‐phase CD8+ T cell responses exert a biologically relevant pressure on HCV replication and that viruses escaping this host response could have a significant survival advantage.

Vav-1 and the IKKα subunit of IκB kinase functionally associate to induce NF-κB activation in response to CD28 engagement

We have recently observed that CD28 engagement initiates a signaling pathway leading to the activation of IκB kinase (IKK) complex and, consequently, to NF-κB activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKKα in both Jurkat and primary CD4+ T cells. Vav-1/IKKα association is mediated by their helix-loop-helix domains, does not involve IKKβ, and is functionally relevant in that Vav-1-associated IKKα kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-κB activation is augmented by both IKKα and Vav-1, but not IKKβ. Confocal microscopy showed that endogenous Vav-1 and IKKα, but not IKKβ, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-κB pathway by targeting IKKα in the T cell membrane and favoring its activation in response to CD28 stimulation.

Mitogen-activated kinase kinase kinase 1 regulates T cell receptor- and CD28-mediated signaling events which lead to NF-κB activation

Optimal activation of Rel / NF‐κB transcription factors in T lymphocytes requires a CD28‐delivered co‐stimulatory signal in addition to TCR engagement. Although, Rel / NF‐κB transcription factors are critical regulators of many T cell functions, the mechanisms and molecules, which link the surface receptors to their activation, are poorly characterized. Using Jurkat T cells stimulated with superantigen presented on B7‐positive APC, we showed that CD28‐ and TCR‐stimulated NF‐κB‐dependent transcription is associated to the activation of IκB kinase β (IKKβ) and, to a lesser extent, of IκB kinase α (IKKα). A dominant negative mutant of the MAP3 kinase MEKK1, a kinase known to regulate the JNK pathway and to activate NF‐κB‐dependent transcription in many cell types, strongly inhibits CD28‐ and TCR‐induced IKK activity, whereas the dominant negative mutants of the NF‐κB‐inducing kinase (NIK) did not exert any significant effects. In addition, TCR / CD28 stimulation results in the recruitment and autophosphorylation of endogenous MEKK1, whereas endogenous NIK was not detectably activated. Our data identify MEKK1 as a critical step in coupling signals initiated by TCR and CD28 to the downstream pathways which lead to both AP‐1 and NF‐κB activation in T lymphocytes.

Engagement of CD4 Before TCR Triggering Regulates Both Bax- and Fas (CD95)-Mediated Apoptosis

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (ΔΨm) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.

Characterization of an IS-like element from Mycobacterium tuberculosis

A DNA sequence, present in members of the Mycobacterium tuberculosis complex, has been identified and characterized. The distribution of this DNA sequence among mycobacterial species was analysed by DNA hybridization and PCR experiments. As the sequence was detected only in bacteria belonging to the M. tuberculosis complex, it may be useful for the rapid discrimination of mycobacteria. Interestingly, the sequence has some characteristics of an insertion element (IS) and codes for a hypothetical protein with significant homologies to proteins encoded by several IS elements of other organisms, namely IS427 and IS869 from Agrobacterium tumefaciens, IS402 from Pseudomonas cepacia, Tn4811 from Streptomyces lividans and ISRm4 from Rhizobium meliloti. Together, these elements form a previously unrecognized family of transposable elements. This finding suggests the possibility of horizontal gene transfer between pathogenic mycobacteria and other organisms including Gram-negative plant-pathogenic bacteria.