Agonistic anti-CD27 antibody ameliorates EAE by suppressing IL-17 production.

CD27/CD70 costimulation enhances T-cell survival, memory formation and Th1-cell differentiation and effector function. In addition to promoting Th1 responses, CD27 signaling has been shown to exert a negative regulatory role on IL-17 production, resulting in increased sensitivity of CD27 KO mice to EAE. By inducing EAE in full CD27 KO mice, and in a novel, T-cell specific CD27 KO mouse strain (CD4-Cre x CD27flox/flox ), we demonstrate herein that CD27 engagement by its natural ligand (CD70) suppresses IL-17 production in a cell autonomous fashion. We further show that CD27 engagement by an agonistic antibody given after EAE induction or at symptom onset similarly suppresses IL-17 production by activated CD4+ T cells infiltrating the inflamed CNS while IFN-γ production was unaffected, leading to an amelioration of inflammatory-related symptoms. These findings propose CD27 costimulation as a potential candidate for therapeutic manipulation to treat autoimmune and autoinflammatory diseases characterized by excessive IL-17 production.

Regulatory T cells constrain the TCR repertoire of antigen-stimulated conventional CD4 T cells.

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non-self-antigen, a TCRß transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T-cell repertoire was used. The response of EF4.1 mice to an I-Ab-associated epitope of the F-MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg-depleted EF4.1 mice were immunized, and the extent of the Vα2-bearing, antigen-specific TCR repertoire was characterized by high-throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre-immune repertoire. Injection of anti-CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non-self-antigens.

An intermediate level of CD161 expression defines a novel activated, inflammatory, and pathogenic subset of CD8+ T cells involved in multiple sclerosis.

Several lines of evidence support a key role for CD8+ T cells in central nervous system tissue damage of patients with multiple sclerosis. However, the precise phenotype of the circulating CD8+ T cells that may be recruited from the peripheral blood to invade the CNS remains largely undefined to date. It has been suggested that IL-17 secreting CD8 (Tc17) T cells may be involved, and in humans these cells are characterized by the expression of CD161. We focused our study on a unique and recently described subset of CD8 T cells characterized by an intermediate expression of CD161 as its role in neuroinflammation has not been investigated to date. The frequency, phenotype, and function of CD8+ T cells with an intermediate CD161 expression level were characterized ex-vivo, in vitro, and in situ using RNAseq, RT-PCR, flow cytometry, TCR sequencing, and immunohistofluorescence of cells derived from healthy volunteers (n = 61), MS subjects (n = 90), as well as inflammatory (n = 15) and non-inflammatory controls (n = 6). We report here that CD8+CD161int T cells present characteristics of effector cells, up-regulate cell-adhesion molecules and have an increased ability to cross the blood-brain barrier and to secrete IL-17, IFNγ, GM-CSF, and IL-22. We further demonstrate that these cells are recruited and enriched in the CNS of MS subjects where they produce IL-17. In the peripheral blood, RNAseq, RT-PCR, high-throughput TCR repertoire analyses, and flow cytometry confirmed an increased effector and transmigration pattern of these cells in MS patients, with the presence of supernumerary clones compared to healthy controls. Our data demonstrate that intermediate levels of CD161 expression identifies activated and effector CD8+ T cells with pathogenic properties that are recruited to MS lesions. This suggests that CD161 may represent a biomarker and a valid target for the treatment of neuroinflammation.

Regulatory T Cell-Dependent and -Independent Mechanisms of Immune Suppression by CD28/B7 and CD40/CD40L Costimulation Blockade.

Blocking of costimulatory CD28/B7 and CD40/CD40L interactions is an experimental approach to immune suppression and tolerance induction. We previously reported that administration of a combination of CTLA-4Ig and MR1 (anti-CD40L mAb) for blockade of these interactions induces tolerance in a fully mismatched allogeneic splenocyte transfer model in mice. We now used this model to study whether regulatory T cells (Tregs) contribute to immune suppression and why both pathways have to be blocked simultaneously. Mice were injected with allogeneic splenocytes, CD4(+) T cells, or CD8(+) T cells and treated with MR1 mAb and different doses of CTLA-4Ig. The graft-versus-host reaction of CD4(+) T cells, but not of CD8(+) T cells, was inhibited by MR1. CTLA-4Ig was needed to cover CD8(+) T cells but had only a weak effect on CD4(+) T cells. Consequently, only the combination provided full protection when splenocytes were transferred. Importantly, MR1 and low-dose CTLA-4Ig treatment resulted in a relative increase in Tregs, and immune suppressive efficacy was abolished in the absence of Tregs. High-dose CTLA-4Ig treatment, in contrast, prevented Treg expansion and activity, and in combination with MR1 completely inhibited CD4(+) and CD8(+) T cell activation in a Treg-independent manner. In conclusion, MR1 and CTLA-4Ig act synergistically as they target different T cell populations. The contribution of Tregs to immune suppression by costimulation blockade depends on the concentration of CTLA-4Ig and thus on the degree of available CD28 costimulation.

Protein aggregation as an antibiotic design strategy.

Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.

CD28/CTLA-4/B7 costimulatory pathway blockade affects regulatory T-cell function in autoimmunity.

Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA-4 on effector T (Teff) cells, its immune-modulatory effects are potentially more complex. Here, we used the mouse model of multiple sclerosis (MS), EAE, to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation, and therefore we injected CTLA-4Ig at day 7 and 9 after immunization, when myelin-reactive T cells have been primed and start migrating toward the CNS. Surprisingly, B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination, and was associated with an enhanced production of the inflammatory cytokines IL-17 and IFN-γ. Importantly, CTLA-4Ig treatment resulted in a transient reduction of Ki67 and CTLA-4 expression and function of peripheral Treg cells. Taken together, B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells, leading to a more severe disease.

Foxp3+ regulatory T cells are activated in spite of B7-CD28 and CD40-CD40L blockade.

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)-cell activity. We previously reported that the blockade of the B7-CD28 and CD40L-CD40 interaction efficiently suppresses allogeneic T-cell activation in vivo. This was characterized by an initial rise in Foxp3(+) cells, followed by depletion of host-reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4(+) cell activation. When CTLA-4Ig and anti-CD40L mAb (MR1) were added to the cultures, T-cell proliferation and IL-2 production were strongly reduced. However, Foxp3(+) cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4(+) cells much more efficiently than did freshly isolated Treg cells. CD4(+) cells activated by allogeneic cells in the presence of MR1 and CTLA-4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4(+) cells when Foxp3(+) Treg cells were removed. We conclude that natural Treg cells are less dependent on B7-CD28 or CD40-CD40L costimulation compared with Foxp3(-) T cells. Reduced costimulation therefore alters the balance between Teff and Treg-cell activation in favor of Treg-cell activity.