Id-neoantigen vaccine induces therapeutic CD8+ T cells against multiple myeloma: H chain-loss escapees cause FLC MM.

BACKGROUND: Multiple myeloma (MM) cancers originate from plasma cells that have passed through the germinal center reaction where somatic hypermutation of Ig V regions takes place. Myeloma protein V regions often express many mutations and are thus a rich source of neoantigens (traditionally called idiotopes (Id)). Therefore, these are highly tumor-specific and excellent targets for immunotherapy. METHODS: We have developed a DNA Id vaccine which as translated protein targets conventional dendritic cells (cDC) for CCL3-mediated delivery of myeloma protein V regions in a single-chain fragment variable (scFv) format. Vaccine efficacy was studied in the mouse MM model, mineral oil-induced plasmacytoma 315.BM. RESULTS: The Id vaccine protected mice against a challenge with MM cells. Moreover, the vaccine had a therapeutic effect. However, in some of the vaccinated mice, MM cells not producing H chains escaped rejection, resulting in free light chain (FLC) MM. Depletion of CD8+ T cells abrogated vaccine efficacy, and protection was observed to be dependent on cDC1s, using Batf3-/- mice. Modifications of scFv in the vaccine demonstrated that CD8+ T cells were specific for two mutated VH sequences. CONCLUSIONS: VH neoantigen-specific CD8+ T cells elicited by CCL3-containing Id vaccines had a therapeutic effect against MM in a mouse model. MM cells could escape rejection by losing expression of the H chain, thus giving rise to FLC MM.

A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza.

Immunogenicity of DNA vaccines can be increased by constructing the DNA in such a way that it encodes secreted homodimeric fusion proteins that target antigen-presenting cells (APCs). In this study, we have developed novel APC-targeting vaccine molecules with an increased flexibility due to introduction of a heterodimerization motif. The heterodimeric proteins permit four different fusions within a single molecule, thus allowing expression of two different APC-targeting moieties and two different antigens. Two types of heterodimeric fusion proteins were developed that employed either the ACID/BASE or the Barnase/Barstar motifs, respectively. The ACID/BASE heterodimeric vaccines conferred protection against challenges with either influenza virus or tumor cells in separate preclinical models. The ACID/BASE motif was flexible since a large number of different targeting moieties and antigens could be introduced with maintenance of specificity, antigenicity, and secretion. APC-targeting ACID/BASE vaccines expressing two different antigens induced antibody and T cell responses against either of the two antigens. Heterodimeric ACID/BASE DNA vaccines were of approximately the same potency as previously reported homodimeric DNA vaccines. The flexibility and potency of the ACID/BASE format suggest that it could be a useful platform for DNA vaccines that encode APC-targeting fusion proteins.

B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2.

Autoantibodies to transglutaminase 2 (TG2) are hallmarks of celiac disease. To address B cell tolerance and autoantibody formation to TG2, we generated immunoglobulin knock-in (Ig KI) mice that express a prototypical celiac patient-derived anti-TG2 B cell receptor equally reactive to human and mouse TG2. We studied B cell development in the presence/absence of autoantigen by crossing the Ig KI mice to Tgm2-/- mice. Autoreactive B cells in Tgm2+/+ mice were indistinguishable from their naive counterparts in Tgm2-/- mice with no signs of clonal deletion, receptor editing, or B cell anergy. The autoreactive B cells appeared ignorant to their antigen, and they produced autoantibodies when provided T cell help. The findings lend credence to a model of celiac disease where gluten-reactive T cells provide help to autoreactive TG2-specific B cells by involvement of gluten-TG2 complexes, and they outline a general mechanism of autoimmunity with autoantibodies being produced by ignorant B cells on provision of T cell help.

Comparison of dendritic cells obtained from autoimmunty-prone and resistant rats.

Dendritic cells (DC) are responsible for the initiation and shaping of the adaptive immune response and are in the focus of autoimmunity research. We were interested in comparison of DC obtained from autoimmunity-prone Dark Agouti (DA) rats and autoimmunity-resistant Albino Oxford (AO) rats. DC were generated from bone marrow precursors and matured (mDC) by lipopolysaccharide. Tolerogenic DC (tolDC) obtained by vitamin D3 treatment were studied in parallel. Profile of cytokine production was different in AO and DA mDC and tolDC. Expression of MHC class II molecules and CD86 were higher in DA DC, while vitamin D3 reduced their expression in dendritic cells of both strains. Allogeneic proliferation of CD4+ T cells was reduced by AO tolDC, but not with DA tolDC in comparison to respective mDC. Finally, expression of various genes identified as differentially expressed in human mDC and tolDC was also analyzed in AO and DA DC. Again, AO and DA DC differed in the expression of the analyzed genes. To conclude, AO and DA DC differ in production of cytokines, expression of antigen presentation-related molecules and in regulation of CD4+ T proliferation. The difference is valuable for understanding the divergence of the strains in their susceptibility to autoimmunity.

In vitro effects of binuclear (η (6)-p-cymene)ruthenium(II) complex containing bridging bis(nicotinate)-polyethylene glycol ester ligand on differentiation pathways of murine Th lymphocytes activated by T cell mitogen.

T cell differentiation into distinct T helper (Th) subpopulations is crucial in governing acquired immune responses as well as some inflammatory and autoimmune disorders. This study investigated potential of the novel neutral binuclear ruthenium(II) complexes 1-8 with general formula [{RuCl2(η(6)-p-cym)}2µ-(N(∩)N)] (N(∩)N = bis(nicotinate)- and bis(iso-nicotinate)-polyethylene glycol esters; (3-py)COO(CH2CH2O) n CO(3-py) and (4-py)COO(CH2CH2O) n CO(4-py); n = 1-4), as well as [RuCl2(η(6)-p-cym)(nic)] (R1, nic = nicotinate) and [RuCl2(η(6)-p-cym)(inic)] (R2, inic = isonicotinate) as an immunomodulatory agents capable to direct Th cell differentiation. From all investigated complexes, [{RuCl2(η(6)-p-cym)}2µ-{(3-py)COO(CH2CH2O)4CO(3-py)}] (4) was selected for further study because it did not affect splenocyte viability (in concentration up to 50 µM), but significantly reduced secretion of representative Th1 cytokine, IFN-γ induced by T cell mitogen. Besides IFN-γ, 4 inhibited dose dependently expression and production of representative Th17 cytokine, IL-17, in these cells. Otherwise, the production of anti-inflammatory cytokines IL-4 and IL-10 was upregulated. Also, 4 significantly increased CD4(+)CD25(+)FoxP3(+) Treg cell frequency in the activated splenocytes. Moreover, ConA-induced expression of Th1 transcription factors, T-bet and STAT1, as well as of Th17-related protein STAT3 was attenuated upon exposure to 4, while the expression of Th2-related transcription factor GATA3 remained stable. In conclusion, ruthenium(II) complex 4 modulates immune system cell functions in vitro by inhibiting T cell differentiation towards pathogenic Th1/Th17 phenotype and inducing a regulatory phenotype characterized by IL-10 and IL-4 production, which may provide novel therapeutic opportunities for immune-inflammatory and/or autoimmune disorders.

Tumor necrosis factor stimulates expression of CXCL12 in astrocytes.

It has been increasingly appreciated that tumor necrosis factor (TNF) performs various protective and anti-inflammatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Recently, CXCL12 has been identified as a key inhibitor of leukocyte entry into the central nervous system (CNS) and as a regulator of inflammation resulting from the invasion. Here, a positive correlation between expression of TNF and CXCL12 in the CNS samples of EAE rats is presented. Also, it is shown that TNF potentiates CXCL12 expression in astrocytes. These results contribute to a view that TNF produced within the CNS plays a protective role in neuroinflammation.

A comparative analysis of multiple sclerosis-relevant anti-inflammatory properties of ethyl pyruvate and dimethyl fumarate.

Dimethyl fumarate (DMF), a new drug for multiple sclerosis (MS) treatment, acts against neuroinflammation via mechanisms that are triggered by adduct formation with thiol redox switches. Ethyl pyruvate (EP), an off-the-shelf agent, appears to be a redox analog of DMF, but its immunomodulatory properties have not been put into the context of MS therapy. In this article, we examined and compared the effects of EP and DMF on MS-relevant activity/functions of T cells, macrophages, microglia, and astrocytes. EP efficiently suppressed the release of MS signature cytokines, IFN-γ and IL-17, from human PBMCs. Furthermore, the production of these cytokines was notably decreased in encephalitogenic T cells after in vivo application of EP to rats. Production of two other proinflammatory cytokines, IL-6 and TNF, and NO was suppressed by EP in macrophages and microglia. Reactive oxygen species production in macrophages, microglia activation, and the development of Ag-presenting phenotype in microglia and macrophages were constrained by EP. The release of IL-6 was reduced in astrocytes. Finally, EP inhibited the activation of transcription factor NF-κB in microglia and astrocytes. Most of these effects were also found for DMF, implying that EP and DMF share common targets and mechanisms of action. Importantly, EP had in vivo impact on experimental autoimmune encephalomyelitis, an animal model of MS. Treatment with EP resulted in delay and shortening of the first relapse, and lower clinical scores, whereas the second attack was annihilated. Further studies on the possibility to use EP as an MS therapeutic are warranted.

Nitric oxide inhibits CXCL12 expression in neuroinflammation.

Chemokine CXCL12 (C-X-C motif chemokine ligand 12) restricts immune cell invasion of the central nervous system (CNS) and limits neuroinflammation in experimental autoimmune encephalomyelitis (EAE), an animal model of inflammatory and demyelinating disease of the CNS, multiple sclerosis (MS). Nitric oxide (NO), by contrast, predominantly contributes to CNS tissue destruction in MS and EAE. Thus, the influence of NO on CXCL12 in the inflamed CNS was investigated. Excess expression of inducible NO synthase was inversely correlated to CXCL12 gene expression in spinal cord homogenates of rats immunized to develop EAE. NO inhibited gene expression of CXCL12 in astrocytes and endothelial cells in vitro. The inhibition was paralleled with reduction of p38 mitogen-activated protein kinase (MAPK) phosphorylation and it was mimicked with inhibitors of p38 MAPK activation in astrocytes. In vivo suppression of nitric generation recovered CXCL12 expression in the CNS and attenuated EAE in Dark Agouti rats. On the contrary, in vivo NO donation decreased CXCL12 expression in the CNS of EAE-resistant Albino Oxford (AO) rats. However, the effect was not paralleled with induction of EAE in AO rats. It is suggested that NO acting through suppression of p38 MAPK inhibits CXCL12 expression in neuroinflammation. These results imply that downregulation of NO release and protection of CXCL12 expression within the CNS might present the potential approaches in MS therapy.

CXCL12-γ expression is inhibited in neuroinflammation.

CXCL12 plays a protective role in CNS autoimmunity. Expression of CXCL12-γ, which has distinct structural and functional properties than the other isoforms of CXCL12, was determined in spinal cords of rats immunized to develop experimental autoimmune encephalomyelitis (EAE). CNS expression of CXCL12-γ was markedly lower in EAE-prone Dark Agouti rats than in EAE-resistant Albino Oxford rats, both in spinal cord homogenates and micro-blood vessels isolated from spinal cords. Inhibition of nitric oxide (NO) synthesis in DA rats upregulated, while donation of NO in AO rats downregulated CNS expression of CXCL12-γ. NO inhibited CXCL12-γ expression in astrocytes in vitro. A splice variant of CXCL12-γ which migrates into nucleolus was not detected in spinal cord or astrocytes. Thus, CXCL12-γ is expressed in the CNS after EAE induction, but its expression is markedly suppressed in spinal cord affected with full blown inflammation. NO is an important regulator of CXCL12-γ expression in neuroinflammation.

High interleukin-10 expression within the central nervous system may be important for initiation of recovery of Dark Agouti rats from experimental autoimmune encephalomyelitis.

Dark Agouti (DA) rats are highly susceptible to induction of experimental autoimmune encephalomyelitis (EAE), still they completely recover from the disease. Here, we were interested to determine contribution of major anti-inflammatory cytokines transforming growth factor (TGF)-ß and interleukin (IL)-10 to the recovery of DA rats from EAE. To that extent we determined CNS expression of these cytokines in DA rats at different phases of EAE and compared data to those obtained in EAE-resistant Albino Oxford (AO) rats. Higher expression of TGF-ß was persistently observed in the CNS of AO rats, even if rats were not immunized. This implied that high TGF-ß within the CNS is important for resistance of AO rats to EAE induction. On the contrary, IL-10 expression was consistently higher in DA than in AO rats and it culminated at the peak of EAE. Methylprednisolone suppressed EAE and expression of IL-10 in spinal cord homogenates, while IL-10 was increased in CNS-infiltrating immune cells. This implied that IL-10 might have a significant role in recovery of DA rats from the disease. Thus, we next explored effects of IL-10 on astrocytes, glial cells that largely contribute to control of CNS inflammation. IL-10 stimulated astrocytic expression of an important regulator of neuroinflammation, CXCL12. Thus, IL-10 might contribute to recovery of DA rats from EAE through induction of CXCL12 expression in astrocytes.

Saquinavir-NO inhibits S6 kinase activity, impairs secretion of the encephalytogenic cytokines interleukin-17 and interferon-gamma and ameliorates experimental autoimmune encephalomyelitis.

NO-hybridization of the HIV protease inhibitor Saquinavir generates a new chemical entity named Saq-NO, that retains the anti-viral activity and exerts lower toxicity. We show that Saq-NO inhibited the generation of various cytokines in ConA-stimulated unfractionated murine spleen cells and rat lymph nodes stimulated with ConA as well as in purified CD4(+) T cells in vitro and reduced the circulating levels of cytokines in mice challenged with anti-CD3 antibody. Furthermore, Saq-NO reduced IL-17 and IFN-γ production in myelin basic protein (MBP)-specific cells isolated from rats immunized with MBP. These findings translated well into the in vivo setting as Saq-NO ameliorated the course of the disease in two preclinical models of multiple sclerosis. Our results demonstrate that Saq-NO exerts immunomodulatory effects that warrant studies on its application in autoimmune diseases.

Betulinic acid regulates generation of neuroinflammatory mediators responsible for tissue destruction in multiple sclerosis in vitro.

AIM: To investigate the influences of betulinic acid (BA), a triterpenoid isolated from birch bark, on neuroinflammatory mediators involved in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis in vitro. METHODS: Encephalitogenic T cells were prepared from draining lymph nodes and spinal cords of Dark Agouti rats 8 to 10 d after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Macrophages were isolated from the peritoneal cavity of adult untreated rats. Astrocytes were isolated from neonatal rat brains. The cells were cultured and then treated with different agents. IFN-γ, IL-17, iNOS and CXCL12 mRNA levels in the cells were analyzed with RT-PCR. iNOS and CXCL12 protein levels were detected using immunoblot. NO and ROS generation was measured using Griess reaction and flow cytometry, respectively. RESULTS: In encephalitogenic T cells stimulated with MBP (10 µg/mL), addition of BA inhibited IL-17 and IFN-γ production in a dose-dependent manner. The estimated IC(50) values for IL-17 and IFN γ were 11.2 and 63.8 µmol/L, respectively. When the macrophages were stimulated with LPS (10 ng/mL), addition of BA (50 µmol/L) significantly increased ROS generation, and suppressed NO generation. The astrocytes were stimulated with ConASn containing numerous inflammatory mediators, which mimicked the inflammatory milieu within CNS; addition of BA (50 µmol/L) significantly increased ROS generation, and blocked ConASn-induced increases in iNOS and CXCL12 mRNA levels, but did not affect iNOS and CXCL12 protein levels. Importantly, in both the macrophages and astrocytes, addition of BA (50 µmol/L) inhibited lipid peroxidation. CONCLUSION: Besides inhibiting encephalitogenic T cell cytokines and reducing NO generation, BA induces tissue-damaging ROS generation within CNS.

Novel octahedral Pt(IV) complex with di-n-propyl-(S,S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoato ligand exerts potent immunomodulatory effects.

We have recently reported that a novel octahedral Pt(IV) complex with di-n-propyl-(S,S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoato ligand has a potent cytotoxic effect on glioma, melanoma and fibrosarcoma cell lines. In this work, we investigated the influence of the Pt(IV) compound on immune cells. We determined its effect on the viability of spleen cells and lymph node cells and on their capability to produce interferon (IFN)-γ and interleukin (IL)-17. Also, we researched the compound's impact on peritoneal macrophages and generation of NO in these cells. Our results show that the complex has limited influence on cell viability of immune cells, but profound inhibitory effect on the production of examined immune mediators. These results are valuable as they show that the novel Pt(IV) complex applied in concentrations which are effective against tumor cells do not affect immune cell viability. Moreover, they also imply that the complex has immunomodulatory properties.