Targeting TIM-3 to reverse immune exhaustion during chronic viral infection Open Access

Tieu, Roger (2015)

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Human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) have emerged as a major threat by being able to impair the immune system. Though the exact mechanism of disease pathogenesis is not clear, several inhibitory receptors involved in effector mechanisms are invoked by the chronic HIV infection leading to limited antiviral response. TIM-3 (T cell immunoglobulin and mucin domain 3) has been identified as an important albeit still understudied negative regulatory of immunity. In order to characterize TIM-3 in the context of chronic viral infection, we used SIV-infected rhesus macaques as our model system. We find there is a similar frequency of TIM-3 expressing dendritic cells (DCs) between humans and rhesus macaques. Further characterization of TIM-3 expressing DCs revealed they represent an activated population with increased CD86 expression and secreted tumor necrosis factor alpha (TNF-alpha) in response to the presence of dsRNA (double stranded RNA). We also find an increased concentration of soluble galectin-9, a TIM-3 ligand, in plasma samples after SIV infection. Staining for general TIM-3 ligands revealed their presence on various immune cell populations, including T cells, B cells, monocytes, and DCs. Finally, we created a fusion protein of extracellular TIM-3 and the Fc domain of an IgG2 antibody to generate TIM-3-Fc for blocking TIM-3 signaling. Treatment of peripheral blood mononuclear cells with TIM-3-Fc results in enhanced proliferation of antigen-specific CD4+ T cells in vitro. Overall, these findings reveal a need to further characterize TIM-3 since we establish TIM-3 as not only an inhibitory molecule for adaptive immunity, but also an activation molecule for innate immunity.

Table of Contents

Table of Contents

Introduction 1

Objectives 10

Methods 11

Results 20

Discussion 40

Future Directions 45

Conclusion 46

References 47

List of Figures

Figure 1. General origin and differentiation of cells from the immune system 2
Figure 2.
Proposed mechanism for TIM-3 in modulating T cell function 9
Figure 3.
Flow cytometry gating strategy for PBMCs 13
Figure 4.
Expression of TIM-3 on the various immune cell populations 21
Figure 5.
Expression of CD86 on TIM-3+ mDCs pre-infection and post-infection 22
Figure 6.
TLR agonist stimulation of the mDC populations 24
Figure 7.
Plasma levels of soluble galectin-9 in naive and SIV-infected rhesus macaques 25
Figure 8.
Intracellular staining of various immune cell populations for galectin-9 26
Figure 9.
Primer list for cloning and subcloning TIM-3 27
Figure 10.
Alignment of the amino acid sequences of human TIM-3 (huTIM-3) and rhesus TIM-3 (rhTIM-3) 28
Figure 11.
Construction of TIM-3 and its splice variants as Fc fusion proteins 29
Figure 12.
Sequences of TIM-3-Fc, IgV-Fc and Mucin-Fc 30
Figure 13.
Expression of TIM-3-Fc in S2 insect cells 31
Figure 14.
SDS-PAGE and Western blot analysis of the various Fc fusion proteins 33
Figure 15.
Binding activity of TIM-3-Fc, IgV-Fc, and mucin-Fc to immobilized galectin-9 34
Figure 16.
Staining for TIM-3 ligand on various immune cell populations using TIM-3-Fc/R-PE 37,38
Figure 17.
Proliferation of CD4+ and CD8+ T cells in response to in vitro blockade and SIV gag peptide stimulation 39

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