Ets1 Promotes the Differentiation of Post-Selected iNKT Cells through Regulation of the Expression of Vα14Jα18 T Cell Receptor and PLZF.

The transcription factor Ets1 is essential for the development/differentiation of invariant Natural Killer T (iNKT) cells at multiple stages. However, its mechanisms of action and target genes in iNKT cells are still elusive. Here, we show that Ets1 is required for the optimal expression of the Vα14Jα18 T cell receptor (TCR) in post-selected thymic iNKT cells and their immediate differentiation. Ets1 is also critical for maintaining the peripheral homeostasis of iNKT cells, which is a role independent of the expression of the Vα14Jα18 TCR. Genome-wide transcriptomic analyses of post-selected iNKT cells further reveal that Ets1 controls leukocytes activation, proliferation differentiation, and leukocyte-mediated immunity. In addition, Ets1 regulates the expression of ICOS and PLZF in iNKT cells. More importantly, restoring the expression of PLZF and the Vα14Jα18 TCR partially rescues the differentiation of iNKT cells in the absence of Ets1. Taken together, our results establish a detailed molecular picture of how Ets1 regulates the stepwise differentiation of iNKT cells.

Combination Therapy of Pulsed-Wave Ultrasound Hyperthermia and Immunostimulant OK-432 Enhances Systemic Antitumor Immunity for Cancer Treatment.

PURPOSE: In this study, we hypothesized that systemic antitumor immunity might be enhanced by combining pulsed-wave ultrasound hyperthermia (pUSHT) with OK-432 and that the induced antitumor immunity could confer protection against tumorigenesis. These hypotheses were tested in bilateral and rechallenged tumor models. METHODS AND MATERIALS: Bilateral and rechallenged tumor models were applied in the studies. In the bilateral tumor model, BALB/c mice were inoculated in both flanks with CT26-luc tumor cells. The tumors in the right flank were treated with 4 courses of pUSHT with or without OK-432. In the rechallenged tumor model, tumor cells were implanted into the right flank. Once formed, the tumors were treated with pUSHT with OK-432, followed by surgical resection. New tumor cells were then implanted into the contralateral flank. The antitumor response was evaluated via infiltrated immune cells and the severity of necrosis/apoptosis in tumors. RESULTS: In the bilateral tumor model, the tumor growth rate and growth activity of both treated (100% reduction) and untreated tumors (90.5% reduction) were significantly inhibited with the combination treatment compared with the sham control group, and the systemic antitumor effect was prolonged. The survival rate was significantly enhanced (sham control, 8 days; OK plus pUSHT, >20 days). IFNγ+ CD4 (treated tumor, 8.6-fold; untreated tumor, 4-fold), IFNγ+ CD8 (treated tumor, 6.7-fold; untreated tumor, 2.6-fold), and T cell and NK cell (treated tumor, 4-fold; untreated tumor, 2.5-fold) infiltration was increased in the combination group compared with the control group. In the rechallenged tumor model, new tumors failed to form with the combination treatment. CONCLUSION: This experimental study combining pUSHT and OK-432 explored a new therapeutic strategy for controlling colon cancer metastasis. The results show that the combination treatment may produce an effective antitumor immune response.

Pulsed-wave Ultrasound Hyperthermia Enhanced Nanodrug Delivery Combined with Chloroquine Exerts Effective Antitumor Response and Postpones Recurrence.

Autophagy is found to serve as a surviving mechanism for cancer cells. Inhibiting autophagy has been considered as an adjuvant anti-cancer strategy. In this study, we investigated the anti-tumor effect of combining pulsed-wave ultrasound hyperthermia (pUH) enhanced PEGylated liposomal doxorubicin (PLD) delivery with an autophagy inhibitor chloroquine (CQ). BALB/c mice bearing subcutaneous 4T1 tumor received intravenous injection of PLD (10 mg/kg) plus 15-minute on-tumor pUH on Day 5 after tumor implantation and were then fed with CQ (50 mg/kg daily) thereafter. Prolonged suppression of tumor growth was attained with PLD + pUH + CQ treatment, whereas in PLD + pUH group tumors quickly recurred after an initial inhibition. Treatment with CQ monotherapy had no benefit compared to the control group. Immunohistochemical staining and Western blotting showed that autophagy of cancer cells was blocked for the mice receiving CQ. It indicates that PLD + pUH + CQ is a promising strategy to treat cancer for a long-term inhibition.

Reciprocal regulation of C-Maf tyrosine phosphorylation by Tec and Ptpn22.

C-Maf plays an important role in regulating cytokine production in TH cells. Its transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. In this study, we demonstrate that Tec kinase family member Tec, but not Rlk or Itk, is a tyrosine kinase of c-Maf and that Tec enhances c-Maf-dependent IL-4 promoter activity. This effect of Tec is counteracted by Ptpn22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by TH17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on TH cell-mediated immune responses.

PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

PTPN22, a protein tyrosine phosphatase expressed mainly in hematopoietic cells, has been linked to many autoimmune diseases. A C-to-T single nucleotide polymorphism (SNP) at position 1858 of human PTPN22 cDNA decreases the risk of Crohn's disease. However, the function of PTPN22 and the mechanism by which this SNP reduces the risk of Crohn's disease are poorly understood. We find that PTPN22 is expressed in macrophages. It suppresses M1 macrophage polarization and reciprocally promotes the expression of M2-associated genes. PTPN22-deficient mice develop severe colitis induced by dextran sulfate sodium, and their intestinal macrophages express higher levels of M1 genes but lower levels of M2-associated genes. Furthermore, the protective T allele of the C1858T SNP is associated with attenuated expression of inflammatory cytokines and a higher level of PTPN22 in human M1 macrophages. This T allele-associated aberrant expression of PTPN22 is partly attributed to an autoinhibition mechanism, in which PTPN22 suppresses its own expression in M1 but not M2 macrophages. Our data not only demonstrate a critical role of PTPN22 in regulating macrophage polarization but also provide a molecular explanation for the protective effect of the C1858T SNP in Crohn's disease.

Down-regulation of thymidylate synthase expression and its steady-state mRNA by oxaliplatin in colon cancer cells.

Recently, evidence has accumulated that weekly 24-h infusion of high-dose 5-fluorouracil (5-FU) with leucovorin (LV, folinic acid) biochemical modulation may improve the response rates compared with the bolus 5-FU regimens in colorectal cancer (CRC). Combining the infusional 5-FU/LV (iFL) regimens with oxaliplatin or irinotecan is widely adopted to further improve treatment efficacy. Either oxaliplatin-iFL or irinotecan-iFL may achieve an overall response rate of more than 50% in the first-line treatment. Intriguingly, in the salvage treatment for metastatic CRC patients who had failed iFL, only oxaliplatin-iFL may achieve a response rate of about 13-25%. In contrast, oxaliplatin alone or irinotecan-iFL had a very low response rate of 5% or less. To test if the oxaliplatin may reverse the iFL-related 5-FU resistance in CRC, we used DLD-1 colon adenocarcinoma cells as the in vitro study model. First, we revealed that oxaliplatin and 5-FU act synergistically on DLD-1 cells by MTT cytotoxicity assay and median drug effect analysis. Second, we treated the DLD-1 cells with serial concentrations of oxaliplatin (0.1-10 microM). Oxaliplatin treatment results in down-regulation of free thymidylate synthase (TS) protein expression by Western blotting. Further, we analyzed the TS mRNA level by reverse transcription and real-time quantitative polymerase chain reaction assay. Oxaliplatin treatment results in down-regulation of the TS mRNA level up to 40% (mean +/- SD of ratio to reference control = 0.60 +/- 0.21, range 0.42-0.84). In this study, our data provide important information explaining the reason why the combination of oxaliplatin and 5-FU results in a better objective response in 5-FU-resistant patients than oxaliplatin alone does. Our data also suggest that TS down-regulation happens at the transcriptional level. TS modulation and down-regulation had, thus, shed light on the useful potential strategy to achieve objective responses in 5-FU-resistant CRC patients.