Non-specific Cytokine Production Research Articles

Dysfunction of CD8\u2009+\u2009PD-1\u2009+\u2009T cells in type 2 diabetes caused by the impairment of metabolism-immune axis

The metabolic changes and dysfunction in CD8 + T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8 + splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8 + splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8 + PD-1 + T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8 + PD-1 + T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity.

Combining anti-IL-7Rα antibodies with autoantigen-specific immunotherapy enhances non-specific cytokine production but fails to prevent Type 1 Diabetes.

Autoantigen-specific methods to prevent and treat Type 1 Diabetes (T1D) carry high hopes to permanently cure this disease, but have largely failed in clinical trials. One suggested approach to increase the efficacy of islet antigen-specific vaccination is to combine it with a modulator of the T cell response, with the goal of reducing effector differentiation and promoting regulatory T cells (Tregs). Here we asked if addition of antibodies that block the IL-7/IL-7Rα pathway altered the T cell response to islet antigen vaccination and prevented T1D in non-obese diabetic (NOD) mice. Anti-IL-7Rα monoclonal antibodies (mAbs) reduced the numbers of islet antigen-specific T cells generated after vaccination with islet peptides and alum. However, addition of anti-IL-7Rα antibodies to peptide/alum vaccination unexpectedly increased non-specific IFN-γ, IL-2 and IL-10 cytokine production and did not result in improved prevention of T1D onset. In a second approach, we used a conjugate vaccine to deliver islet autoantigens, using Keyhole Limpet Hemocyanin (KLH) as a carrier. Islet antigen-KLH vaccination led to a significant expansion of antigen-specific Tregs and delayed diabetes onset in NOD mice. These outcomes were not further improved by addition of anti-IL-7Rα antibodies. To the contrary, blocking IL-7Rα during vaccination led to non-specific cytokine production and reduced the efficacy of a KLH-conjugated vaccine to prevent T1D. Our study thus revealed that adding anti-IL-7Rα antibodies during autoantigen immunization did not improve the efficacy of such vaccinations to prevent T1D, despite altering some aspects of the T cell response in a potentially advantageous way. Further refinement of this approach will be required to separate the beneficial from the adverse effects of anti-IL-7Rα antibodies to treat autoimmune disease.

The differential short- and long-term effects of HIV-1 latency-reversing agents on T cell function.

Despite the extraordinary success of HIV-1 antiretroviral therapy in prolonging life, infected individuals face lifelong therapy because of a reservoir of latently-infected cells that harbor replication competent virus. Recently, compounds have been identified that can reverse HIV-1 latency in vivo. These latency- reversing agents (LRAs) could make latently-infected cells vulnerable to clearance by immune cells, including cytolytic CD8+ T cells. We investigated the effects of two leading LRA classes on CD8+ T cell phenotype and function: the histone deacetylase inhibitors (HDACis) and protein kinase C modulators (PKCms). We observed that relative to HDACis, the PKCms induced much stronger T cell activation coupled with non-specific cytokine production and T cell proliferation. When examining antigen-specific CD8+ T cell function, all the LRAs except the HDACi Vorinostat reduced, but did not abolish, one or more measurements of CD8+ T cell function. Importantly, the extent and timing of these effects differed between LRAs. Panobinostat had detrimental effects within 10 hours of drug treatment, whereas the effects of the other LRAs were observed between 48 hours and 5 days. These observations suggest that scheduling of LRA and CD8+ T cell immunotherapy regimens may be critical for optimal clearance of the HIV-1 reservoir.

CD4+CD25+ T regulatory cells do not transfer oral tolerance to peanut allergens in a mouse model of peanut allergy

Recent studies have implicated CD4(+) CD25(+) regulatory T cells (nTregs) in the maintenance of tolerance to oral antigens and in the regulation of the food allergic IgE response. The objective was to assess if nTregs can transfer allergen-specific oral tolerance to naïve, non-TCR transgenic mice and regulate peanut extract (PE)-specific hypersensitivity responses. Additionally, the role of the regulatory cytokines IL-10 and TGF-β in the modulation of peanut-allergic sensitization was studied. CD25-enriched T cells from PE-tolerant mice were adoptively transferred to recipient mice, which were subsequently sensitized to PE. Depletion of CD25(+) cells and neutralization of IL-10 and TGF-β were compared in a CH3/HeOuJ mouse model of peanut-allergic sensitization. Transfer of CD25(+) Tregs-enriched cell populations did not affect the PE-specific cytokine production or PE-specific antibody levels compared with control mice but interestingly resulted in a decrease of mast cell responsiveness. On the contrary, transfer of CD25(+) Tregs-depleted cells caused an increase in non-specific cytokine production, in the absence of changes in PE-specific responses. TGF-β neutralization resulted even in a larger increase in spontaneous release of all cytokines measured (IL-4, IL-5, IL-10, IL-13, and IFN-γ), but surprisingly also to a higher PE-specific Th2-associated (IL-4, IL-5, IL-13) cytokine production compared with depletion of CD25 cells or neutralization of IL-10. Similarly, depletion of CD25 cells and TGF-β neutralization but not of IL-10 neutralization lead to an increase in PE-specific antibody levels and elevated mast cell degranulation following a PE challenge. We conclude that CD4(+) CD25(+) Tregs from non-transgenic-tolerant mice cannot transfer specific oral tolerance of exogenous antigens to naïve mice and are more involved in general immune suppressive mechanisms. However, we found evidence that TGF-β secreting Tregs (Th3) may play an important role.

Comparison of the Type of Liposome Involving Cytokine Production Induced by Non-CpG Lipoplex in Macrophages

To improve the transfection efficiency of plasmid DNA (pDNA) into cells, various types of cationic liposome have been used to prepare pDNA/cationic liposome complexes (lipoplexes). It is well-known that lipoplexes induce a large amount of proinflammatory cytokines because unmethylated CpG dinucleotides (CpG motifs) abundantly present in pDNA are recognized by Toll-like receptor-9 (TLR9) expressed in immune cells such as macrophages and dendritic cells. This nonspecific cytokine production is problematic in nonviral gene therapy. Moreover, recent studies have demonstrated that lipoplexes induce not only proinflammatory cytokines but also another type of cytokine, type I interferons (IFNs), irrespective of the frequency of CpG motifs in DNA and the expression of TLR9. To gain more insight into the CpG motif- and TLR9-independent induction of type I IFNs and proinflammatory cytokines by lipoplex, macrophage activation was evaluated in vitro using various cationic liposomes complexed with pDNA containing no CpG motifs. The production of IFN-beta, TNF-alpha and IL-6 by lipoplex was confirmed to be induced independently of the interaction between CpG DNA and TLR9 in macrophages from TLR9-knockout mice. Then, the release of the cytokines, the mRNA expression of Z-DNA binding protein-1 (Zbp1), a cytosolic double-stranded DNA sensor, and the cellular uptake of pDNA were examined in a macrophage-like cell line, RAW264.7. The level of cytokine production and the increase in the Zbp1 mRNA varied depending on the type of cationic liposome used. A good correlation was observed between the cytokine level and the Zbp1 mRNA. A confocal microscopic study using fluorescently labeled pDNA complexes showed that the complexes that released a lot of cytokines showed an enhanced distribution of pDNA-derived fluorescence into the cytosol. These results suggest that different intracellular trafficking derived from the type of liposomes determines the recognition of pDNA by ZBP1 after uptake of lipoplexes by the macrophages, followed by the release of type I IFNs and inflammatory cytokines. The present study demonstrates that cationic liposomes should be selected based on these findings for optimization of DNA-based therapies using lipoplexes.

Mycophenolate mofetil reduces production of interferon-dependent major histocompatibility complex induction during allograft rejection, probably by limiting clonal expansion

The immunosuppressive drug mycophenolate mofetil (MMF) acts by releasing mycophenolic acid (MPA), which inhibits the enzyme inosine monophosphate dehydrogenase (IMPDH) and thus inhibits de novo purine synthesis. Unlike cyclosporine (CsA), MMF has no direct effect on cytokine gene expression in vitro. We examined the effect of MMF, in comparison to CsA, on invivo production of interferon-γ (IFN-γ) in mice. Two stimuli for IFN-γ induction were used: (1) allogeneic P815 mastocytoma ascites tumour cells and (2) bacterial lipopolysaccharide (LPS). The allogeneic response is dependent on clonal expansion of T cells, while the LPS response is polyclonal and T cell independent. Since major hsitocompatibility complex (MHC) induction in mouse kidney in IFN-γ dependent, we assessed the in vivo induction of IFN-γ indirectly by measuring MHC induction in mouse kidneys in three systems: radiolabelled antibody binding assay, immunoperoxidase staining in tissue section, and Northern blotting for steady-state MHC mRNA levels. IFN-γ steady-state mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). In the allogeneic response, MMF (40–160 mg/kg/day) reduced the production of IFN-γ in a dose-dependent fashion. MHC class I and II induction was reduced by 35% to 74% and 30% to 74%, respectively. However, MMF had less effect on the induction of MHC by a nonimmune stimulus, bacterial LPS, whereas CsA reduced the induction of IFN-γ in both responses. We conclude that MMF reduces the IFN-dependent induction of MHC in vivo during specific immune responses, probably by limiting clonal expansion, while preserving nonspecific cytokine production in response to LPS.