Liposome-mediated combinatorial cytokine gene therapy induces localized synergistic immunosuppression and promotes long-term survival of cardiac allografts

Localized gene transfer has the potential to introduce immunosuppressive molecules only into the transplanted allograft, which would limit systemic side effects, and prolong allograft survival. However, an applicable gene transfer strategy is not available, and the feasible therapeutic gene(s) has not yet been determined. We developed an ex vivo liposome-mediated gene therapy strategy that is able to intracoronary deliver the combination of IL-4 and IL-10 cDNA expression vectors to the allograft simultaneously. We examined the efficiency, efficacy, and cardiac adverse effects of this combinatorial gene therapy protocol using a rabbit functional cervical heterotopic heart transplant model. Although the efficiency was moderate, the expression of both transgenes was long lasting and localized only in the target organ. The mean survival of cardiac allograft was prolonged from 7 to >100 days. Synergism of overexpressed IL-4 and IL-10 in the inhibition of T lymphocyte infiltration and cytoxicity, and modulation of Th1/Th2 cytokine production promote long-term survival of cardiac allografts.     

The alternatively spliced domain TnFnIII A1A2 of the extracellular matrix protein tenascin-C suppresses activation-induced T lymphocyte proliferation and cytokine production.

Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A-D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.     

Redox modulation inhibits CD8 T cell effector function

The evolutionary preservation of reactive oxygen species in innate immunity underscores the important roles these constituents play in immune cell activity and as signaling intermediates. In an effort to exploit these pathways to achieve control of aberrant immune activation we demonstrate that modulation of redox status suppresses cell proliferation and production of IL-2, IFN-gamma, TNF-alpha, and IL-17 in two robust CD8 T-cell-dependent in vitro mouse models: (1) response to alloantigen in an mixed leukocyte reaction and (2) CD8 T cell receptor transgenic OT-1 response to cognate peptide (SIINFEKL). To correlate these findings with cytotoxic lymphocyte (CTL) function we performed cytotoxicity assays and found that redox modulation diminishes the ability of alloantigen-specific and antigen-specific OT-1 CTLs to kill their corresponding antigen-expressing target cells. To further examine the mechanisms of redox-mediated repression of CTL target cell lysis, we analyzed the expression of the effector molecules IFN-gamma, perforin, and granzyme B and the degranulation marker CD107a (LAMP-1). In both models, redox modulation reduced the expression of these effector components by at least fivefold. These results demonstrate that redox modulation quells the CD8 T cell response to alloantigen and the T cell receptor transgenic CD8 T cell response to its cognate antigen by inhibiting proliferation, proinflammatory cytokine synthesis, and CTL effector mechanisms.     

7'-(3',4'-dihydroxyphenyl)-N-[(4-methoxyphenyl)ethyl]propenamide (Z23), an effective compound from the Chinese herb medicine Fissistigma oldhamii (Hemsl.) Merr, suppresses T cell-mediated immunity in vitro and in vivo

Fissistigma oldhamii (Hemsl.) Merr [F. oldhamii], a traditional Chinese herb medicine, is widely used for treating rheumatoid arthritis (RA) in China. Following bioactivity-guided isolation, a representative immunosuppressive compound with low cytotoxicity, 7'-(3',4'-dihydroxyphenyl)-N-[(4-methoxyphenyl)ethyl]propenamide (Z23), was been identified in this herb medicine. We investigated the immunosuppressive effects of Z23 on T cells in vitro and in vivo. The results showed that Z23 in a dose-dependent manner significantly inhibited the proliferation of splenocytes induced by concanavalin A (ConA) and by the mixed lymphocyte culture reaction (MLR), with half inhibitive concentration (IC(50)) values of 6.22 microM and 0.78 microM, respectively. Z23 also dose-dependently inhibited the proliferation and type 1 cytokine (IFN-gamma and IL-2) production of primary T cells stimulated by anti-CD3/CD28 mAbs, but did not affect IL-12 production by mouse peritoneal macrophages (pMphi) stimulated with LPS plus IFN-gamma in vitro. Administration of Z23 (6.25 mg/kg, 12.5 mg/kg, 25 mg/kg, i.p.) dose-dependently suppressed 2,4-dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) reactions. Furthermore, administration of Z23 (25 mg/kg, i.p.) significantly reduced the incidence and severity of type II bovine collagen (CII)-induced arthritis (CIA), which was associated with the inhibition of CII-specific T cell proliferation and type 1 cytokine (IFN-gamma and IL-2) production. In this study, we report that a representative immunosuppressive compound from F. oldhamii, Z23, effectively inhibits murine immune responses in vitro and in vivo, and that the immunosuppressive effects of Z23 might be attributed to suppression of T cell activation and function and Th1 type cytokine production.     

2'Deoxycoformycin and deoxyadenosine affect IL 2 production and IL 2 receptor expression of human T cells

Congenital deficiency of the enzyme adenosine deaminase (ADA) leads to severe combined immunodeficiency. 2'Deoxycoformycin (dCF), a tightly binding inhibitor of ADA, can induce the metabolic state of ADA deficiency. In vivo, the drug causes specific impairment of lymphocyte function and shows strong immunosuppressive properties. However, to decide whether inhibition of the enzyme ADA offers an attractive approach for immunosuppressive therapy, more information is needed about the immunologic mechanisms affected. In human T cells, we investigated the effect of dCF and deoxyadenosine (AdR) on cell activation, interleukin 2 (IL 2) production, and IL 2 receptor induction after allogeneic and lectin-induced stimulation. After allogeneic stimulation, dCF and AdR affected several events in T cellular immune response. Early events in T cell activation showed to be most sensitive to the drugs. Primary MLC was completely inhibited by concentrations as low as 1 microM dCF and 1 microM AdR. The addition of human recombinant IL 2 (rIL 2) could not abrogate the inhibitory effect of the drugs. Apart from activation of T cells, the drugs interfered with proliferation of activated T cells. Two events in activated T cells were affected: IL 2 production and IL 2 receptor expression. In secondary MLC, IL 2 production was markedly reduced in the presence of 9 microM dCF and 60 microM AdR. These concentrations appeared also to affect IL 2 receptor expression in 12-day primary MLC cells stimulated with rIL 2. Lectin stimulation was also affected by the drugs. In phytohemagglutinin (PHA)-stimulated cultures, 9 microM dCF and 60 microM AdR resulted in inhibition of proliferation and IL 2 receptor expression, whereas IL 2 production was normal. It is concluded that dCF and AdR interfere with several events in T cellular immune response such as cell activation, IL 2 production, and IL 2 receptor expression. According to these results, inhibition of the enzyme ADA seems an attractive approach to immunosuppressive therapy.     

Janus kinase inhibition suppresses PKC-induced cytokine release without affecting HIV-1 latency reversal ex vivo

Background

Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation.

Design and methods

We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants.

Results

We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo.

Conclusion

The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.

     

Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors

STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3(flox/flox) mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNA were also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.     

Heterogeneous cytokine production by acutely stimulated bronchoalveolar T lymphocytes in reovirus 1/Lang-infected mice

While the in vitro properties of CD4(+) and CD8(+) cytokine-producing lymphocytes have been well studied, the in vivo cytokine production patterns and relative roles of CD4(+) and CD8(+) T lymphocytes during a primary in vivo immune response remain unclear. In this study, mice were inoculated intranasally with reovirus 1/L, and respiratory T lymphocyte populations were analyzed using multicolor flow cytometric analysis for the production of cytokine within and between classical type 1/type 2 patterns. Cytokine production observed in vivo following infection did not correlate with classical T cell cytokine expression patterns; instead, multiple types of lymphocyte populations that produced one of several possible cytokine combinations were present. Cytokine production by CD4(+) lymphocytes appears in the early and middle stages of the immune response, while CD8(+) lymphocytes produce more cytokine in the later stages. Early cytokine responses occurred predominantly in the whole lung and lung-associated lymph node populations. The complex patterns of cytokine expression seen in this study likely influence local cell-mediated immunity as well as the complex interaction of T cell subsets and the interaction of T cells with B cells which are necessary for the generation of cell-mediated and humoral immune responses required for effective broad-spectrum immunity.     

Engineering T lymphocyte antigen specificity.

Targeting of immune cells by bispecific antibodies has proven to be a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely, the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction, are the major topics of this review.     

Potentiation of delayed-type hypersensitivity response to syngeneic tumors in mice prevaccinated with cells modified by hydrostatic pressure and crosslinking

Summary Targeting of immune cells by bispecific antibodies has proven a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction are the major topics of this review.     

Two Inhibitors of DNA‐Synthesis Lead to Inhibition of Cytokine Production via a Different Mechanism

Methotrexate (MTX) and mycophenolic acid (MPA) are used in the clinic for their immunosuppressive properties. MTX is widely used for the treatment of rheumatoid arthritis (RA). MPA is used to prevent graft rejection and is now experimentally used in systemic lupus erythematosis and RA. It is known that both drugs interfere with DNA synthesis. However, the precise mechanism of action is still debated. We have analysed the effect of the drugs on cytokine production in whole blood during short cultures. The production of T‐cell cytokines was inhibited by both drugs. MTX inhibits cytokine production because MTX induces apoptosis in activated T‐cells. MPA inhibits cytokine production by preventing T‐cells to progress to the S‐phase of the cell cycle. Cytokine production by monocytes was slightly decreased by the drugs. The reason for this inhibition is not clear. These results indicate that T‐cells are the main target cells of the immunosuppressive drugs MPA and MTX.     

Mycophenolic acid and methotrexate inhibit lymphocyte cytokine production via different mechanisms

Mycophenolic acid (MPA) and methotrexate (MTX) are immunosuppressive drugs used for the treatment of various immunological disorders. MPA is an inhibitor of inosine monophosphate dehydrogenase and MTX is a folate antagonist that inhibits tetrahydrofolate reductase. Production of T cell cytokines in whole blood cultures, as well as in PBMC cultures, is inhibited by a low concentration of both drugs. Inhibition of cytokine production after monocyte stimulation was less evident. The mechanism by which inhibition is achieved is different for both drugs. Inhibition of T cell cytokine production by MPA was more profound and started earlier compared to the inhibition by MTX. MTX induced apoptosis in T cells that became activated, whereas MPA prevented activation of T cells by arresting the cell cycle in the G0/G1 phase. Addition of guanosine and adenosine can overcome this cell cycle arrest, even after several days. Furthermore MPA inhibited the expression of activation markers HLA-DR and CD71 on T cells. The observation that MTX cannot prevent T cell activation but induces apoptosis in activated T cells, and that MPA reversibly prevents activation of T cells could explain the immunosuppressive effects of both these drugs.     

Ixodes scapularis: effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice.

Schoeler, G. B., Manweiler, S. A., and Wikel, S. K. 1999. Ixodes scapularis: Effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice. Experimental Parasitology 92, 239-248. Ixodes scapularis is the principal vector in the United States of Borrelia burgdorferi, the causative agent of Lyme borreliosis, the human granulocytic ehrichiosis agent, and Babesia microti. Infestation with I. scapularis nymphs has previously been shown to modulate host T lymphocyte cytokine production. Tick-induced host immunomodulation is increasingly recognized as a contributing factor in successful transmission and/or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on the production of the macrophage cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha and the T lymphocyte cytokines IL-2, IL-4, IL-10, and interferon-gamma in both BALB/c and C3H/HeN mice. The pattern of T lymphocyte cytokine production was evaluated to determine if repeated tick infestation polarizes the immune response toward a Th-1 or Th-2 cytokine profile. Female BALB/c and C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-day tick-free period between each exposure. After each infestation, tick biology parameters were measured and macrophage and T lymphocyte cytokine production was assessed. Elaboration of T lymphocyte and macrophage cytokines was quantitated by antigen capture enzyme-linked immunosorbent assay. Acquired resistance to I. scapularis feeding was not developed by either mouse strain. Significant differences in cytokine production were observed between infested and noninfested mice, as well as between the two mouse strains, following tick infestation. Infestation of both strains with pathogen-free I. scapularis results in a polarization of the host immune response toward a Th-2, anti-inflammatory pattern, with a corresponding suppression of Th-1 responses.     

p66Shc is involved in promoting HIF-1alpha accumulation and cell death in hypoxic T cells

Hypoxia results in adaptationally appropriate alterations of gene expression through the activation of hypoxia-inducible factor (HIF)-1 to overcome any shortage of oxygen. Peripheral blood mononuclear cells may be exposed to low oxygen tensions for different times as they migrate between blood and various tissues. We and others have previously shown that T-cell adaptation to hypoxia is characterized by a modulation of cytokine expression and an inhibition of T-cell activation. We have recently demonstrated that the adaptor protein p66Shc negatively regulates T-cell activation and survival. We here show that hypoxia enhances HIF-1alpha accumulation and vascular endothelial growth factor production in T cells. Hypoxic T cells expressed high levels of p21(WAF1/CIP1), of the pro-apoptotic molecules BNIP3, a classic HIF target gene, and BAX, as well as low levels of the anti-apoptotic molecule BCLxl, associated with an induction of cell death. We found out that hypoxic T cells expressed p66Shc. Furthermore, using T-cell transfectants expressing p66Shc, as well as T cells derived from mice p66Shc-/-, we defined a role of p66Shc in T-cell responses to hypoxia. Of interest, hypoxic p66Shc-positive transfectants expressed higher level of HIF-1alpha than negative controls. Thus, p66Shc may play an important role in downstream hypoxic signaling, involving HIF-1alpha protein accumulation and cell death in T lymphocytes.     

The Novel Calcineurin Inhibitor CN585 Has Potent Immunosuppressive Properties in Stimulated Human T Cells

The Ca²⁺/calmodulin-dependent protein phosphatase calcineurin is a key mediator in antigen-specific T cell activation. Thus, inhibitors of calcineurin, such as cyclosporin A or FK506, can block T cell activation and are used as immunosuppressive drugs to prevent graft-versus-host reactions and autoimmune diseases. In this study we describe the identification of 2,6- diaryl-substituted pyrimidine derivatives as a new class of calcineurin inhibitors, obtained by screening of a substance library. By rational design of the parent compound we have attained the derivative 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2-phenyl-pyrimidine (CN585) that noncompetitively and reversibly inhibits calcineurin activity with a K_i value of 3.8 μm. This derivative specifically inhibits calcineurin without affecting other Ser/Thr protein phosphatases or peptidyl prolyl cis/trans isomerases. CN585 shows potent immunosuppressive effects by inhibiting NFAT nuclear translocation and transactivation, cytokine production, and T cell proliferation. Moreover, the calcineurin inhibitor exhibits no cytotoxicity in the effective concentration range. Therefore, calcineurin inhibition by CN585 may represent a novel promising strategy for immune intervention.     

Design of bifunctional siRNAs: combining immunostimulation and gene-silencing in one single siRNA molecule

Active suppression of T lymphocyte activation can limit the efficacy of immune surveillance and immunotherapy. Here we have explored the possibility of designing bifunctional small interfering RNAs (siRNAs) capable of inducing innate immunity through Toll-like receptors and simultaneously inhibiting the expression of immunosuppressive factors. Using interleukin (IL) 10 as a model, we found that liposomal delivery of IL10 siRNAs could efficiently activate the expression of cytokines (e.g. TNF-alpha, IL6, and IL12) and interferons (e.g. IFN-alpha) in peripheral blood mononuclear cells (PBMCs) and immature monocyte-derived dendritic cells (iMoDCs). Moreover, the designed siRNAs inhibited IL10 gene expression. Transfection of iMoDCs with either chemically or in vitro transcribed IL10 siRNAs induced their differentiation into mature MoDCs (mMoDCs) characterized by the expression of costimulatory molecules CD80/CD86 and the chemokine receptor CCR7. Lipid delivery of either chemically synthesized or T7-transcribed immunostimulatory siRNAs induced cytokine production. However, in contrast to chemically synthesized siRNAs, electroporation of in vitro transcribed siRNAs also induced cytokine production in iMoDCs. Interestingly, IL10 siRNA-transfected iMoDCs were capable for enhancing the response of allogeneic T cells, providing support for the rational design of bifunctional siRNAs as immune modulating therapy.