HIV-1 induced immunodeficiency is associated with severe impairment of both humoral and cellular adaptive immune responses that persists even under therapy. CD4 T cells, the main target cells for HIV-1 infection, execute important helper functions to both B and CD8 T cells in the context of antigen-specific immune responses, suggesting a connection between the functionality of CD4 T cells and adaptive immune dysfunction. A key step in generation of T cell help is antigen-specific interaction with DCs which activates and polarizes T cells towards a specialized effector phenotype, such as Tfh or Th1 cells, helping humoral or cellular immunity, respectively. Importantly, previous research demonstrated that HIV-1 directly interferes with CD4 T cell communication with B cells by action of the accessory Nef protein which impairs immune synapse (IS) function and thus, help to B cells, leading to B cell dysfunction in vivo. Concerning molecular architecture and dynamics, T cell IS formation with B cells is fundamentally different from T cell IS formation with DCs, the latter representing a crucial step in effector T helper cell generation and development of cellular immunity. Given the importance of T cell-DC interaction in determining the course of an immune response, I therefore aimed to investigate Nef’s role in CD4 T cell interaction with DCs in this study. By first using an antigen-specific mouse system in which Nef is expressed in CD4 T cells, I could demonstrate that Nef strongly interferes with early T cell-DC interaction dynamics and impairs effective activation of both CD4 T cells and DCs on a transcriptional and functional level ex vivo and in vivo. This dysfunction is in particular associated with inefficient Th1 polarization of CD4 T cells and establishment of a microenvironment which disfavors development of Th1 cells and cellular immunity. By using different Nef mutants together with a CRISPR/Cas9 KO approach, I could demonstrate that the inhibition exerted by Nef strongly depends on its CD4 downmodulation function, thus revealing an unknown role for this effector function in disruption of antigen-specific immune responses. In this context, I could observe that Nef had the capacity to limit CD4 T cell help to an antiviral CD8 T cell memory response in a preliminary LCMV infection experiment, suggesting Nef contributes to CD8 T cell dysfunction in vivo. In line with this, HIV-1 infection impaired CD4 T cell help to an EBVspecific memory CD8 T cell response in a complementary human ex vivo tonsil culture system for several, although not all donors with high variability. To mechanistically dissect adaptive immune cell interactions in the ex vivo tonsil culture system and to gain better understanding of the underlying variability, I lastly established a workflow that enables highly efficient and activation-neutral gene editing of tonsil CD4 T cells by CRISPR/Cas9 RNP nucleofection without affecting immunocompetency of the system. In summary, these findings provide evidence that HIV-1 Nef, by interfering with CD4 T cell interaction with and licensing of DCs, limits efficient CD4 T cell effector cell generation and in particular, Th1 responses in a murine cell context. As this was associated with suboptimal help to memory CD8 T cell responses in ex vivo human tonsil culture and in an in vivo LCMV infection model, Nef may in this way contribute to CD8 T cell dysfunction in the context of HIV infection. This study therefore identifies Nef as a global antagonist of adaptive immune responses and interesting viral therapeutic target.
Katharina Morath (Thu,) studied this question.