The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo , but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo , and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo . These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

A unique aspect of human monocytes, compared to monocytes from many other species, is that they express the CD4 molecule. However, the role of the CD4 molecule in human monocyte development and function is not known. We determined that the activation of CD4 via interaction with major histocompatibility complex class II (MHC-II) triggers cytokine expression and the differentiation of human monocytes into functional mature macrophages. Importantly, we determined that CD4 activation induces intracellular signaling in monocytes and that inhibition of the MAPK and Src family kinase pathways blocked the ability of CD4 ligation to trigger macrophage differentiation. We observed that ligation of CD4 by MHC-II on activated endothelial cells induced CD4-mediated macrophage differentiation of blood monocytes. Finally, CD4 ligation by MHC-II increases the susceptibility of blood-derived monocytes to HIV binding and subsequent infection. Altogether, our studies have identified a novel function for the CD4 molecule on peripheral monocytes and suggest that a unique set of events that lead to innate immune activation differ between humans and mice. Further, these events can have effects on HIV infection and persistence in the macrophage compartment. IMPORTANCEThe CD4 molecule, as the primary receptor for HIV, plays an important role in HIV pathogenesis. There are many cell types that express CD4 other than the primary HIV target, the CD4 ؉ T cell. Other than allowing HIV infection, the role of the CD4 molecule on human monocytes or macrophages is not known. We were interested in determining the role of CD4 in human monocyte/macrophage development and function and the potential effects of this on HIV infection. We identified a role for the CD4 molecule in triggering the activation and development of a monocyte into a macrophage following its ligation. Activation of the monocyte through the CD4 molecule in this manner increases the ability of monocytes to bind to and become infected with HIV. Our studies have identified a novel function for the CD4 molecule on peripheral monocytes in triggering macrophage development that has direct consequences for HIV infection.