T Cell Ig and Mucin Domain–Containing Protein 3 Is Recruited to the Immune Synapse, Disrupts Stable Synapse Formation, and Associates with Receptor Phosphatases

Abstract

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.

Too Much of a Good Thing? Tim-3 and TCR Signaling in T Cell Exhaustion

“…Importantly, the analogous tyrosines in human Tim-3 also scored as possible sites of phosphorylation by the same classes of kinases (not shown). We and others have demonstrated that the cytoplasmic tail of Tim-3 can indeed be phosphorylated on multiple tyrosine residues (35, 43, 44). While the identity of the kinases that phosphorylate the Tim-3 cytoplasmic tail have not been definitively shown in vivo, our data are consistent with such phosphorylation being carried out by the Src family kinases Fyn and/or Lck, at least in T cells (35).…”

“…Another recent study examined the interaction of Tim-3 with transmembrane proteins expressed on T cells, and reported that Tim-3 can associate with the transmembrane phosphatases CD45 and CD148, which the authors proposed might promote de-phosphorylation of downstream mediators of T cell activation (44). Furthermore, Tim-3, CD45 and CD148 were all found to be recruited into the immunological synapse.…”

Molecular and cellular insights into T cell exhaustion

“…3): first, by ectodomain competition, which refers to inhibitory receptors sequestering target receptors or ligands and/or preventing the optimal formation of microclusters and lipid rafts (for example, CTLA4 (REF. 31)); second, through modulation of intracellular mediators, which can cause local and transient intracellular attenuation of positive signals from activating receptors such as the TCR and co-stimulatory receptors 32–34 ; and third, through the induction of inhibitory genes 14 .…”

Galectin-9 Is Rapidly Released During Acute HIV-1 Infection and Remains Sustained at High Levels Despite Viral Suppression Even in Elite Controllers

“…45,[59][60][61] We and others have observed that Tim-3 + CD8 + T cells are increased in progressive HIV infection, and engagement presumably through a Gal-9 interaction leads to CD8 + T cell dysfunction. 33,60,62,63 During chronic HIV infection, Gal-9-expressing Treg cells are presumed to selectively suppress the proliferation of nonprotective HIVspecific CD8 + T cells with high Tim-3 levels, but not protective HIV-specific CD8 + T cells with low levels of Tim-3. 64 Soluble Gal-9 levels have been shown to be elevated in the plasma of HIV-infected subjects, [64][65][66] suggesting that Gal-9 may play an important role in HIV pathogenesis.…”