Cytoprotective effects of high dose of α-galactosylceramide against activation-induced CD4+ T and CD8+ T cell death as an adjuvant.

OBJECTIVE: To investigate the cytoprotective effects of high dose of α-galactosylceramide (α-GC) on the activation-induced CD4+ T and CD8+ T cell death. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced using adoptive transfer of MOGCD4+ cells treated using α-GC into recipient C57BL/6 mice while the MOGCD4+ cells treated using 0.5% polysorbate were set as vehicle group, based on which to investigate the effects of α-GC on activation induced CD4+ T cell death. Additionally, an EG7 tumor-bearing mice model is established using adoptive transfer of CD8+ T cells, based on which to investigate the effect of α-GC on the apoptosis of CD8+ T cells. RESULTS: A higher induction rate was noticed after adoptive transfer of MOGCD4+ cells treated using α-GC together with the severity of EAE compared with the conventional methods. Longer survival duration was noted in the green fluorescent protein (GFP) labeled MOGT in the α-GC group compared with the vehicle group (P < 0.05). Severe inflammatory cell infiltration and myelinoclasis was noted in the white matter of nervous system in the α-GC group. In the EG7 tumor model, more adoptive CD8+ T cells were survived in α-GC group compared with that of vehicle group. The growth of tumor mass was significantly inhibited in α-GC group. CONCLUSIONS: high dose of α-GC could be used as an adjuvant for inhibiting activation-induced CD4+ T and CD8+ T cell death. Our study could provide helpful information for the development of adoptive cell therapy with reduced programmed cell death.

Association of thrombomodulin Ala455Val dimorphism and inflammatory cytokines with carotid atherosclerosis in the Chinese Han population.

BACKGROUND AND METHODS: It has been reported that C/T dimorphism at position 1418 of the thrombomodulin gene causes a cytosine (C) transition to thymidine (T), resulting in an alanine (A) to valine (V) substitution at amino acid position 455 (TM455). TM455 had been found not only in African American and American whites, but also in whites in The Netherlands and Sweden. Among these populations, the C/C genotype is predominant, although the distribution of this dimorphism is different. Thrombomodulin is an important anticoagulant protein that is downregulated in endothelial cells overlying atherosclerotic plaques and is also an anti-inflammatory molecule. TM455 is located in the last epidermal growth factor-like repeat of thrombomodulin, which is functionally important for protein C activation and thrombin binding. The distribution of thrombomodulin polymorphism and association between TM455, inflammatory cytokines, and carotid atherosclerosis in the Chinese Han population is unclear. METHODS: This thrombomodulin dimorphism was analyzed by allele-specific amplification in 144 patients with carotid atherosclerosis and in 384 healthy controls. TM455 was found in the Chinese Han population, but the genotype frequency and distribution of each genotype in this population differed substantially from that in other ethnic subgroups. The C/T and T/T genotypes were predominant in the Chinese Han population, and the frequency of the T allele in this population (63.8%) was much higher than that in whites in The Netherlands (18%), Sweden (26.1%), and the US (18.4%), and in blacks in the US (7.6%). The frequencies of these single nucleotide polymorphisms complied well with the Hardy-Weinberg equilibrium in healthy individuals. The C allele was significantly more common among patients with carotid atherosclerosis than in controls (P < 0.05). The frequency of the C allele was 45.5% in patients and 36.2% in controls. The thrombomodulin Ala455 genotypes C/C and C/T were significantly more common than the T/T genotype in patients with carotid atherosclerosis in the Chinese Han population. In addition, higher baseline levels of tumor necrosis factor alpha (55.45 ± 11.58 pg/mL versus 52.70 ± 10.74 pg/mL; P < 0.05), interleukin-6 (31.53 ± 10.51 pg/mL versus 27.73 ± 8.37 pg/mL; P < 0.01), and C-reactive protein (6.65 ± 2.01 mg/L versus 4.06 ± 1.03 mg/L; P < 0.01) were observed in patients with carotid atherosclerosis than in controls. Interestingly, compared with baseline inflammatory cytokine levels in those with the Val/Val genotype, higher baseline tumor necrosis factor alpha, interleukin-6, and C-reactive protein levels were observed for the Ala/Ala genotype in both patients with carotid atherosclerosis and healthy controls. CONCLUSION: Our results support a significant association between thrombomodulin Ala455Val dimorphism, inflammatory cytokines, and carotid atherosclerosis in the Chinese Han population.

High doses of alpha-galactosylceramide potentiate experimental autoimmune encephalomyelitis by directly enhancing Th17 response.

Alpha-galactosylceramide (alpha-GC) is widely known to activate invariant natural killer T (iNKT) cells to suppress myelin antigen-specific Th1 responses, protecting susceptible mice against experimental autoimmune encephalomyelitis (EAE). Here, we demonstrate an unexpected finding that high doses of alpha-GC exacerbated, rather than ameliorated, EAE. Similar results were observed when MOG(35-55)-specific T cells treated with high-dose alpha-GC were transferred into naïve syngeneic recipient mice. Further study showed that high doses of alpha-GC directly enhance the Th17 and Th1 response by activation of CD4(+)CD44(+) memory T cells through phosphorylation of STAT3 and activation of NF-kappaB. Unlike the activation of iNKT cells by low doses of alpha-GC, high doses of alpha-GC directly interacted with CD1d expressed on T cells and activated Th17 and Th1 cells. Furthermore, antigen-presenting cells (APCs) predominantly express CD1d1, whereas the majority of CD4(+) T cells express CD1d2. Knockdown of CD1d1 or CD1d2 gene expression by RNAi interfered with the activation of iNKT or Th17/Th1 cells, respectively. Therefore, alpha-GC treatment could improve or worsen EAE by engaging either APCs or Th17/Th1 cells depending on the dose used.

SOCS1 silencing can break high-dose dendritic cell immunotherapy-induced immune tolerance.

Dendritic cells (DCs) play a pivotal role in T cell-mediated immunity and have been shown to induce strong anti-tumor immune responses. As of yet, only a limited number of objective tumor regressions have been observed in clinical studies using a DC vaccine. Suppressor of cytokine signaling-1 (SOCS1) is a key negative regulator of the JAK/STAT signal pathway and plays an essential role in suppressing systemic autoimmunity that is mediated by DCs. The aim of this study was to investigate whether SOCS1-silenced DCs can break the vaccine-induced immune tolerance stimulated by high-dose DC, thereby enhancing anti-tumor activity. In the mouse melanoma model, we found that a 2x106 TRP2-pulsed DC vaccine was able to induce immune tolerance, while a 2x106 SOCS1-silenced DC/TRP2 vaccine prevented immune tolerance. Further experiments revealed that activation-induced T cell death (AICD) through the Fas/Fas-L pathway may play a crucial role in immune tolerance induced by 2x106 TRP2-pulsed DC. SOCS1-silencing in DCs could prevent immune tolerance by inhibiting Fas and Fas-L expression, induced by an increase in IL-12p70 and IL-6 production. In addition, in 2x106 SOCS1-silenced DC/TRP2 immunized mice, higher levels of IL-12p70 and IFN-γ and lower IL-17 production may inhibit tumor angiogenesis and therefore assist in breaking immune tolerance. In conclusion, high-doses of DCs can inhibit the vaccine-induced AICD of T cells and cytokine regulation in tumor angiogenesis. These results indicate that SOCS1-silenced DC vaccines may greatly enhance anti-tumor activity by breaking self-tolerance.