Research Highlights

Regulatory T-Cell Function in Autoimmunity

Evaluation of: Zanin-Zhorov A, Ding Y, Kumari S et al.: Protein kinase C-θ mediates negative feedback on regulatory T cell function. Science 328, 372–376 (2010).

Regulatory T-cell (Treg) function is decreased in autoimmune disorders, including rheumatoid arthritis (RA). This occurs despite the fact that Treg numbers are identical to those observed in healthy controls, and this appears to be secondary to negative regulation of Tregs by the surrounding inflammatory milieu [1]. TNF-α seems to be a key inflammatory cytokine that initiates negative signals for Treg activity, but inhibitory signals transmitted via the T-cell receptor (TCR) can also reduce Treg function [2]. TCR signaling in Tregs leads to the formation of the immunological synapse (IS) and causes negative feedback to inhibit Treg-mediated suppression. In addition, as it has been suggested, the differences in TCR signaling in Tregs may emerge at the level of the IS, which is the interface between T cells and antigen-presenting cells (APCs) where TCR signalosomes are assembled [3].

In a recent issue of Science, Zanin-Zhorov et al. have presented evidence that protein kinase C (PKC)-θ mediates negative feedback on Treg function [4]. In a series of elegant and well designed in vitro and in vivo experiments to study signaling in the human Treg IS, the authors first developed a model system on supported planar bilayers containing labeled ICAM-1, antigen surrogate anti-CD3 antibodies and CD4⁺CD25⁺ effector T cells (Teffs) or CD4⁺CD25⁺ Tregs isolated from peripheral blood. Teffs and Tregs both formed IS, defined by a symmetric pattern consisting of a central cluster of anti-CD3 surrounded by a ring of ICAM-1. Ex vivo-expanded, human umbilical cord blood Tregs displayed similar characteristics to adult peripheral blood Tregs. Recruitment of TCR proximal signaling molecules to the IS by staining with phospho-Src kinase activation loop and Zap70 kinase interdomain A tyrosine 319 antibodies and imaging with total internal reflection fluorescence microscopy revealed no significant differences. Next, they explored the PKC-θ pathway, which is downstream of Src family kinases and mediates IS breakage [4]. PKC-θ recruitment to the IS leads to recruitment of Carma-1, which enables the assembly of a Carma-1–Bcl10–Malt1 complex necessary for transcription factor NF-κB activation and subsequent Teff activation. Quantification of PKC-θ and its downstream target to the IS was shown to be reduced in Tregs. In addition, TCR triggering in Tregs was found to downregulate PKC-θ recruitment to the IS compared with basal recruitment by LFA-1 engagement alone.

The authors then proceeded to investigate whether inhibition of PKC-θ with compound 20 (C20; a potent and selective PKC-θ inhibitor) [5] may affect the suppressive function of human CD4⁺CD25^-Tregs. Teff function was measured as cytokine secretion and cell proliferation. C20 treatment of only the Tregs upregulated their suppressive ability, even in the presence of CD28-mediated costimulation, but did not induce suppressive activity in treated Teffs. In addition, pretreatment of CD4⁺CD25^high T cells with C20 significantly enhanced their suppression of IFN-γ secretion.

In a series of experiments, it was also shown that C20 inhibited NF-κB activation, and antibodies against TGF-β receptor II completely blocked the suppressive function of Tregs induced by inhibition of PKC-θ cocultures either with or without APCs, suggesting the possible involvement of TGF-β presented by Tregs as a suppressive mechanism. Finally, PKC-θ gene expression was silenced by using RNA interference. It was concluded that PKC-θ activity induced by TCR signaling mediates a negative feedback loop that reduces the activity of CD4⁺CD25^high Tregs to suppress cytokine secretion and proliferation of Teffs in vitro.

The authors then turned their attention to RA. Using Tregs purified from the peripheral blood of 25 RA patients with different degrees of disease activity, they found that despite anergic state and comparable Treg numbers with healthy donors, RA Tregs demonstrated reduced suppression of IFN-γ from autologous CD4⁺CD25^- Teffs. The loss of function was due to defective intrinsic function of Tregs from RA patients and not due to increased resistance of Teffs. Treatment with C20 significantly increased the suppressive function of Tregs purified from all 25 RA patients to levels comparable with those of healthy donor-derived Tregs. Furthermore, the defective Treg function in RA patients was inversely correlated with the disease activity score, and the shift in IFN-γ secretion was similar across the disease score spectrum. Thus, inhibition of PKC-θ boosts the suppressive function of Tregs isolated from RA patients independent of the severity of disease.

Treatment of Tregs with TNF-α inhibits their activity and downregulates expression of the transcription factor Foxp3 [6]. C20 prevented TNF-α-induced downregulation of Foxp3 in Tregs.

Finally, they determined the ability of C20 to increase Treg function in vivo by using a colitis model in TCR-α^-/-B^-/- mice induced by transfer of the CD4⁺CD25^- CD45RB^high Teffs [7]. Treatment of murine Tregs with C20 upregulated their suppressive function in vitro. Furthermore, C20-treated CD4⁺CD25⁺ Tregs provided significant protection from colitis, as demonstrated by normal weight gain and normal histology of the distal colon. Thus, PKC-θ inhibition significantly increased the suppressive effect of Treg in vivo.

The presented evidence provides strong support of the notion that targeting the PKC-θ-mediated negative feedback loop enhances the activity of Tregs, and that inhibition of PKC-θ in Tregs may be of value in Treg adoptive immunotherapy to treat autoimmune disorders such as RA.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Dysregulated Humoral Immunity in HIV Infection

Evaluation of: MacLennan CA, Gilchrist JJ, Gordon MA et al.: Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults. Science 328, 508–512 (2010).

Nontyphoidal salmonellae (NTS), especially Salmonella enteric serovars typhimurium and enteritidis, commonly induce self-limited gastroenteritis in healthy individuals, but are a major cause of systemic bacteremia in Africa, affecting young children as well as HIV-infected adults [1,2]. Cell-mediated immunity was long thought to play a major role in controlling infection, but recent observations have shown an important protective role for antibody-induced complement-mediated killing of NTS in African children [3]. In addition, lack of bactericidal activity resulting from dysregulated antibody production against S. typhimurium lipopolysaccharide (LPS) has been demonstrated in HIV-infected African adults, and it has also been demonstrated that antibodies against S. typhimurium outer membrane protein induce killing of NTS in HIV-uninfected African adults.

In a recent issue of Science, MacLennan et al. presented a series of studies aimed at determining whether HIV infection affects humoral immunity to NTS [4]. First, they were able to demonstrate that all sera from HIV-uninfected adults induced killing of two invasive S. typhimurium isolates. By contrast, there was considerable variability in the ability of sera from HIV-infected adults to kill both isolates. CD4 counts of HIV-infected individuals with impaired serum killing of one of the isolates were lower than those from normal killing. S. enteritidis IgG titer correlated with impaired killing of S. enteritidis, but failure to deposit complement is not responsible for a lack of Salmonella-killing by HIV-infected serum. Further experiments demonstrated that an inhibitor factor present in HIV-infected serum, subsequently shown to be LPS IgG, blocks killing. In another series of experiments, they showed that the addition of small amounts of LPS to HIV-infected sera with partially impaired Salmonella-killing ability restored normal killing.

The presence of elevated LPS IgG in a high proportion of HIV-infected individuals suggests that high titers are not the consequence of random expansion of antigen-specific B-cell clones. This indicates that immune dysregulation, not immune deficiency, accounts for impaired humoral immunity to NTS. In view of these findings, they hypothesized that LPS antibodies prevent the killing of Salmonella by two possible mechanisms. One would act by diverting complement deposition away from the bacterial membrane, thereby preventing insertion of membrane attack complexes into the membrane, and a second potential mechanism would impede the access of antibody and/or complement to the outer membrane by cross-linking O-antigen, the distal portion of the LPS molecule. The latter proved to be correct when it was demonstrated that the sera of HIV-infected individuals contain inhibitory antibodies that target O-antigen. Findings also indicate that individual sera contain antibodies that can kill Salmonella, by targeting outer membrane proteins and block killing of Salmonella leading to the suggestion that killing of Salmonella by inhibitory HIV-infected sera could be restored by adding IgG from HIV-uninfected donors to inhibitory HIV-infected sera. Indeed, further experiments conclusively demonstrated that killing of Salmonella in antibody-deficient sera could be induced or prevented by adding combinations of IgG from HIV-uninfected and inhibitory HIV-infected sera.

This study provides strong evidence for dysregulated humoral immunity to NTS in HIV-infected African individuals, and may have significant clinical implications such as the development of an outer membrane protein-based vaccine that could induce protective antibodies [5].

Psoriasis pathophysiology

Evaluation of: Guilloteau K, Paris I, Pedretti N et al.: Skin inflammation induced by the synergistic action of IL-17A, IL-22, oncostatin  M, IL-1α and TNF-α recapitulates some features of psoriasis. J. Immunol. 184, 5263–5270 (2010).

Psoriasis is a prevalent inflammatory skin disorder with significant systemic involvement including metabolic syndrome, arthritis and cardiovascular morbidity and mortality. Its pathogenesis is complex and multifactorial and involves the interplay of environmental, genetic and immunological factors. Whether the epidermis, dermis or a combination of both sites is primarily affected is a matter of considerable debate. More recently, increasing attention has been focused on the keratinocyte and provides solid evidence that keratinocytes are direct targets for a specific set of cytokines, leading to the regulation of their biological properties, such as secretion of cytokines, chemokines and antimicrobial peptides, and their differentiation and migration capacities [1–3]. Psoriasis is now considered the paradigm of an inflammatory disease involving the proinflammatory Th17 subset [4]. Moreover, in vitro studies have characterized certain cytokines as being able to induce specific expression patterns related to innate immune response, such as IL-1a, TNF-α, IL-17A and oncostatin M (OSM). In addition, some of these cytokines can induce skin inflammation reminiscent of psoriasis in animal models [5].

In a recent study published in the Journal of Immunology, Guilloteau et al. aimed to identify an optimal and relevant cytokine combination able to synergize in order to generate in vitro an inflammatory keratinocyte model recapitulating some features of lesional psoriatic skin [6]. In vitro and in vivo analyses were performed using both normal and psoriatic skin samples, keratinocyte cell cultures, murine models of skin inflammation, macroarrays, real-time PCR, and western blotting analyses, chemotaxis and antibacterial activity assays and histological and immunochemical studies for Gr-1 were performed.

Following screening of 36 different cytokines that have been previously shown to have an effect in the skin or for their involvement during regulation of the immune/inflammatory response, several cytokines including IL-22, IL-24, IL-6, OSM, IL-1a, IL-1b, TNF-α and IL-17A were identified as able to modify the expression of at least five genes. Among these, IL-1a, IL-17A, IL-22, OSM and TNF-α showed strong synergistic activity on the production of B-defensin (BD2) and CXCL8. IL-17A and TNF-α were shown to be more critical to the activity of the combination. In vivo intradermal injection of the five cytokines in mice increased CXCL1, CXCL2, CXCL3, S100A9 and BD3 expression and associated with neutrophilic infiltration. These findings were subsequently confirmed by using real-time PCR analysis. Production of the chemokines CXCL, CXCL5 and CXCL8 by keratinocytes stimulated in the presence of this cytokine combination was associated with increased neutrophil chemotactic activity. Similarly, high production of BD2, BD3 and S100A7 was associated with an increased antimicrobial activity. Finally, the authors evaluated the pathophysiological relevance of the inflammatory phenotype observed in vitro when stimulating keratinocytes with the five cytokine combination, and quantified the gene expression of several proinflammatory cytokines and their potential targets. The transcriptional profile observed correlated with the one of lesional psoriatic skin. Their data demonstrate the important synergistic activities of several cytokines on keratinocytes and may have important practical implications. These findings provide a theoretical framework for the potential use of combination anticytokine therapy in psoriasis and similar inflammatory disorders.