In HIV infection a large frequency of HIV specific CD8+ and CD4+ T lymphocytes are dysfunctional andcoincidentally show an increased expression of the negative regulator of T cell activation PD-1, PD1 ligationby PD-L1 and PD-L2 leads to the inhibition of most T cell functions including proliferation, cytokineproduction and survival. However, the molecular mechanisms downstream of PD-1 remain unknown. Themajor objective of this project is to define the molecular mechanisms that render T cells dysfunctionalfollowing PD-1 ligation and to identify strategies that will allow the rescue of such dysfunction in HIV specificCD8 cells. We will test the hypothesis that PD1 interferes with upstream TCR signaling pathways in orderto affect all T cell functions. We have shown that PD-1 ligation leads to the phosphorylation of SHP-1 andSHP-2 phosphatases. Moreover PD1 crosslinking leads to the inhibition of p56Lck activation through thephosphorylation of the tyrosine Y505 by the inhibitory kinase Csk.
In Specific Aim 1 we will determine theproximal signaling defect in PD-1 hi exhausted cells from HIV infected patients and assess whether Csk isrecruited to the immunological synapse by PD1 ligation or whether it is released from Cbp/PAG by the SHP-2 phosphatase. We will determine by quantitative immunoprecipitation if the inhibitory kinase Csk and the inphosphatases SHP-1 and SHP-2 are recruited to the TCR complex. Fluorescence (Forster) resonanceenergy transfer (FRET) experiments will be carried out to identify the partners involved in the negativeregulation of p56Lck activity. The recruitment of these negative regulators will be assessed in cells showingvarying degrees of dysfunction . We will use siRNAs specific for Csk, SHP-1, SHP-2 and Fyn-T andactivated forms of Lck to rescue T cell function in T cells at the extremes of this gradient of dysfunction.
In Aim 2, live cell imaging using total internal reflection fluorescence microscopy (TIRFM) will be used tovisualize, (in collaboration with Core C) the dynamics of the immunological synapse formation upon PD-1crosslinking in HIV specific CD8+ T cells showing varying degrees of dysfunction. . We will assess whetherinhibition of the kinase Csk and the phosphatases SHP-112 will restore the integrity of the synapse asobserved in efficiently activated T cells. Results emanating from this project may lead to the identification ofnovel therapeutic targets that can allow immune reconstitution during HIV infection.
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