An Oligomeric Sulfated Hyaluronan and Silk-Elastinlike Polymer Combination Protects against Murine Radiation Induced Proctitis

Semisynthetic glycosaminoglycan ethers (SAGEs) are short, sulfated hyaluronans which combine the natural properties of hyaluronan with chemical sulfation. In a murine model, SAGEs provide protection against radiation induced proctitis (RIP), a side effect of lower abdominal radiotherapy for cancer. The anti-inflammatory effects of SAGE have been studied in inflammatory diseases at mucosal barrier sites; however, few mechanisms have been uncovered necessitating high throughput methods. SAGEs were combined with silk-elastinlike polymers (SELPs) to enhance rectal accumulation in mice. After high radiation exposure to the lower abdominal area, mice were followed for 3 days or until they met humane endpoints, before evaluation of behavioral pain responses and histological assessment of rectal inflammation. RNA sequencing was conducted on tissues from the 3-day cohort to determine molecular mechanisms of SAGE–SELP. After 3 days, mice receiving the SAGE–SELP combination yielded significantly lowered pain responses and amelioration of radiation-induced rectal inflammation. Mice receiving the drug–polymer combination survived 60% longer than other irradiated mice, with a fraction exhibiting long term survival. Sequencing reveals varied regulation of toll like receptors, antioxidant activities, T-cell signaling, and pathways associated with pain. This investigation elucidates several molecular mechanisms of SAGEs and exhibits promising measures for prevention of RIP.

DOK1 and DOK2 regulate CD8+ T cell signaling and memory formation without affecting tumor cell killing

Targeting intracellular inhibiting proteins is a promising strategy to improve CD8+ T cell anti-tumor efficacy. DOK1 and DOK2 are CD8+ T cell inhibitory proteins that are targeted in this study in order to improve the activation and cytotoxic capacities of these cells. To evaluate the role of DOK-1 and DOK-2 depletion in physiology and effector function of T CD8+ lymphocyte and in cancer progression, a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) was established. Depletion of both Dok1 and Dok2 did not affect the development, proliferation, mortality, activation and cytotoxic function of naive CD8+ T cells. However, after an in vitro pre-stimulation Dok1/Dok2 DKO CD8+ T cells had higher percentage of effector memory T cells and showed an increase in levels of pAKT and pERK upon TCR stimulation. Despite this improved TCR signaling, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxicity capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. In conclusion, DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.

Disc Large Homolog 1 Is Critical for Early T Cell Receptor Micro Cluster Formation and Activation in Human T Cells

T cell activation by antigen involves multiple sequential steps, including T cell receptor-microcluster TCR-(MC) formation, immunological synapse formation, and phosphorylation of mediators downstream of the TCR. The adaptor protein, Disc Large Homolog 1 (DLG1), is known to regulate proximal TCR signaling and, in turn, T cell activation, acting as a molecular chaperone that organizes specific kinases downstream of antigen recognition. In this study, we used knockdown and knockout technologies in human primary T cells and a human T cell line to demonstrate the role of DLG1 in proximal T cell signaling. High-end confocal microscopy was used for pictorial representation of T cell micro-clusters and colocalization studies. From all these studies, we could demonstrate that DLG1 functions even earlier than immunological synapse formation, to regulate T cell activation by promoting TCR-MC formation. Moreover, we found that DLG1 can act as a bridge between the TCR-ζ chain and ZAP70 while inhibiting binding of the phosphatase SHP1 to TCR-ζ. Together, these effects drive dysregulation of T cell activation in DLG1-deficient T cells. Overall, the activation and survival status of T cell is a critical determinant of effective vaccine response, and DLG1-mediated T cell signaling events can be a driving factor for improving vaccine-designing strategies.

A short hepatitis C virus NS5A peptide expression by AAV vector modulates human T cell activation and reduces vector immunogenicity

Viral vector-mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for the advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS protein 5A (NS5A) protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector-mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.

An evolutionary divergent thermodynamic brake in ZAP-70 finetunes the kinetic proofreading in T cell

T cell signaling starts with assembling several tyrosine kinases and adaptor proteins to the T cell receptor (TCR), following the antigen binding. The lifetime of the TCR: antigen complex and the time delay between the recruitment and activation of each kinase determines the T cell response. The mechanism by which the time delays are implemented in TCR signaling is not fully understood. Combining experiments and kinetic modeling, we here report a thermodynamic-brake in the regulatory module of ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation in TCR. Phylogenetic analysis revealed that the evolution of the thermodynamic-brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. Paralogous kinase Syk expressed in B cells, does not possess such a functional thermodynamic brake, which may explain higher basal activation and lack of ligand selectivity by Syk.

TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation

The conserved HIV-1 LTR cis elements RBE1/3 bind the factor RBF2, consisting of USF1/2 and TFII-I, and are essential for reactivation of HIV-1 by T cell signaling. We determined that TFII-I recruits the tripartite motif protein TRIM24 to the LTR, and this interaction is required for efficient reactivation of HIV-1 expression in response to T cell signaling, similar to the effect of TFII-I depletion. Knockout of TRIM24 did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9 to the LTR. These results demonstrate that TFII-I promotes transcriptional elongation in response to T cell activation through recruitment of the co-factor TRIM24, which is necessary for efficient recruitment of the elongation factor P-TEFb.

Immune Checkpoint Inhibitors in Hepatocellular Carcinoma: Current Progresses and Challenges

Hepatocellular carcinoma (HCC) is one of the most common malignant tumor in the world and its incidence is increasing in many countries. In recent years, with the deepening understanding of the immune and pathological mechanisms of HCC, immunotherapy based on the regulation of tumor immune microenvironment has become a new treatment choice for patients with HCC. Immune checkpoint inhibitors (ICIs) targeting programmed death protein-1, programmed death protein-ligand-1, or cytotoxic T-lymphocyte-associated antigen 4 are the most widely used. Instead of general immune-enhancing therapies, ICIs can reactivate anti-tumor immune responses by disrupting co-inhibitory T cell signaling. In this review, the research progress and existing problems of ICIs in the treatment of HCC in recent years are reviewed.