title: CD96: a new co-stimulatory receptor on primary human CD4+ T cells - Elucidating its downstream pathways and contribution to HIV-1 infection
Creators: name: Burkhard, Rabea
Publisher: name: Universität Tübingen
Year: 2027
Subjects: subject: HIV
subject: 500
subject: CD96
subject: CD4+ T cells
subject: Co-stimulatory
subject: Antiviral
subject: HIV-1 Nef
subject: HIV-1 Vpu

description: Die Dissertation ist gesperrt bis zum 04. Februar 2027 !
description: One strategy of HIV-1 to evade the host immune system is the manipulation of several receptors of the host cell plasma membrane. In this regard, CD96 (TACTILE = T cell activation, increased late expression) a member of the immunoglobulin superfamily, was identified in previous work by our group to be downregulated by HIV-1 accessory proteins Nef and Vpu. Although the mechanism of CD96 downregulation is well characterized, the biological relevance for HIV-1 pathogenesis is unclear. Together with the two receptors CD226 (DNAM-1 = DNAX accessory molecule-1) and TIGIT (T cell immunoglobulin and ITIM domain), as well as the commonly shared ligand CD155 (PVR = Poliovirus receptor), CD96 belongs to a complex receptor-ligand network. Whereas the function of CD226 and TIGIT is well characterized, the function of CD96, especially on primary human CD4+ T cells, is unclear. Thus, the biological function of CD96 on primary CD4+ T cells was investigated, as well as the contribution of its downregulation to HIV-1 replication. By performing co-immunoprecipitation experiments, it was observed that Grb2 and Lck differentially regulate CD96 expression. Co-expression of Grb2 and CD96 in HEK293T cells resulted in increased CD96 protein levels, whereas co-transfection of Lck resulted in decreased CD96 protein levels that could be restored if transfected cells were additionally treated with the lysosomal degradation inhibitor Bafilomycin A1. Yet, a direct interaction between CD96 and Grb2, or Lck, as well as previously suggested direct interaction partners MKK7 and Gab1, were not observed. Using primary CD4+ T cells it was possible to show that CD96 is phosphorylated and that this is independent of TCR stimulation. Finally, stimulation experiments with primary CD4+ T cells revealed a co-stimulatory function for CD96, resulting in increased numbers of CD69 and CD25 expressing cells, as well as enhanced secretion of pro-inflammatory cytokines, chemokines and growth factors. This effect was dependent on the activation of CD3 and CD28 signaling pathways in a time-dependent manner. Regarding HIV-1 infection, it was shown that primary HIV-1 isolates similar to lab-adapted HIV-1 strain NL4-3 downregulated CD96 from the cell surface in a Nef and Vpu dependent manner. Although it was previously shown by our group that CD96 is incorporated into viral particles if not removed from the cell surface, targeting virion incorporated CD96 with soluble CD155 did not impair particle infectivity. Moreover, co-stimulation experiments with HIV-1 infected resting and pre-stimulated CD4+ T cells suggest a positive effect of CD96 co-stimulation on HIV-1 replication. Altogether, further research on the contribution of CD96 downregulation to HIV-1 pathogenesis is required. Nevertheless, the obtained results show that CD96 functions as a co-stimulatory receptor on primary human CD4+ T cells, leading to increased numbers of activated T cells, enhanced secretion of pro-inflammatory cytokines and thus, enhanced T cell activation.