CD8+ cytotoxic T lymphocytes (CTL) are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g. IFN-γ) to aid this response. However, during cancer and chronic viral infections, such as with Human Immunodeficiency Virus, this CTL response is progressively impaired due to a process called T-cell exhaustion. Previous work has shown that the glycoprotein, T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) plays a functional role in establishing T-cell exhaustion. Tim-3 is highly upregulated on virus and tumor antigen-specific CD8+ T-cells and antagonizing Tim-3 helps restore function of CD8+ T cells. However, very little is known of how Tim-3 signals in CTLs. In this study, we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in primary human CD8+ T cells. Tim-3 was found withinCD8+T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted in a significantly greater number of stable synapses being formed between Tim-3hi CD8+T cells and target cells, suggesting that Tim-3 plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse and characterizing these interactions could aid in the development of therapeutics to restore Tim-3-mediated immune dysfunction.

Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.

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